A spin-state-selective experiment for measuring heteronuclear one-bond and homonuclear two-bond couplings from an HSQC-type spectrum

Recently, a set of selective 1D experiments with spin-state-selective excitation for CH spin systems was introduced by Parella and Belloc (J. Magn. Reson., 148, 78–87 (2001)). We have expanded and generalized this concept further, and demonstrated that a very simple experiment utilizing spin-state-selective filtering can be used for simultaneous measurement of heteronuclear ¹ J _NH (or ¹ J _CH) and geminal ² J _HH couplings from two-dimensional ¹⁵N-¹H (or ¹³C-¹H) correlation spectrum. The experiment has very high sensitivity owing to the preservation of equivalent coherence transfer pathways analogous to the sensitivity and gradient enhanced HSQC experiment. However, overall length of the pulse sequence is 1/(2J) shorter than the gradient selected SE-HSQC experiment. Furthermore, the spin-state-selection can be utilized between NH and NH₂ (or CH and CH₂) moieties by changing the phase of only one pulse. The pulse scheme will be useful for the measurement of scalar and residual dipolar couplings in wide variety of samples, due to its high sensitivity and artifact suppression efficiency. The method is tested on NH₂ and CH₂ moieties in ¹⁵N- and ¹⁵N/¹³C–labeled ubiquitin samples.     

Innovations in Generation and Analysis of 2D [C,H] COSY NMR Spectra for Metabolic Flux Analysis Purposes

2D [¹³C,¹H] COSY NMR is used by the metabolic engineering community for determining ¹³C–¹³C connectivities in intracellular compounds that contain information regarding the steady-state fluxes in cellular metabolism. This paper proposes innovations in the generation and analysis of these specific NMR spectra. These include a computer tool that allows accurate determination of the relative peak areas and their complete covariance matrices even in very complex spectra. Additionally, a method is introduced for correcting the results for isotopic non-steady-state conditions. The proposed methods were applied to measured 2D [¹³C,¹H] COSY NMR spectra. Peak intensities in a one-dimensional section of the spectrum are frequently not representative for relative peak volumes in the two-dimensional spectrum. It is shown that for some spectra a significant amount of additional information can be gained from long-range ¹³C–¹³C scalar couplings in 2D [¹³C,¹H] COSY NMR spectra. Finally, the NMR resolution enhancement by dissolving amino acid derivatives in a nonpolar solvent is demonstrated.     

Ontogenetic Study of the Effects of Energetic Nutrients on Amino Acid Metabolism of Rat Cerebral Cortex

We studied the effect of various energetic nutrients on metabolism of l-[U-¹⁴C]leucine and [1–¹⁴C]glycine in cerebral cortex of rats at different ages. At gestational age, glucose and lactate stimulated protein synthesis from l-[U-¹⁴C]leucine and [1–¹⁴C]glycine and from l-[U-¹⁴C]leucine, respectively; glucose, -OH-butyrate and lactate stimulated lipid synthesis from l-[U-¹⁴C]leucine. At 10 days of age, glucose, mannose, and fructose stimulated protein synthesis, and glucose and mannose stimulated oxidation to CO₂ as well as lipid synthesis from l-[U-¹⁴C]leucine. In adult rats, glucose, mannose, and fructose stimulated protein synthesis from l-[U-¹⁴C]leucine and [1–¹⁴C]glycine; glutamine also markedly decreased the oxidation of l-[U-¹⁴C]leucine and [1–¹⁴C]glycine in 10–day-old and adult rats.     

Calmodulin tagging provides a general method of using lanthanide induced magnetic field orientation to observe residual dipolar couplings in proteins in solution

A general method is presented for magnetic field alignment of proteins in solution. By tagging a target protein with calmodulin saturated with paramagnetic lanthanide ions it is possible to measure substantial residual dipolar couplings (RDC) whilst minimising the effects of pseudocontact shifts on the target protein. A construct was made consisting of a calmodulin-binding peptide (M13 from sk-MLCK) attached to a target protein, dihydrofolate reductase in this case. The engineered protein binds tightly to calmodulin saturated with terbium, a paramagnetic lanthanide ion. By using only a short linker region between the M13 and the target protein, some of the magnetic field alignment induced in the CaM(Tb^{3 +})₄ is effectively transmitted to the target protein (DHFR). ¹H-¹⁵N HSQC IPAP experiments on the tagged complex containing ¹⁵N-labelled DHFR-M13 protein and unlabelled CaM(Tb^{3 +})₄ allow one to measure RDC contributions in the aligned complex. RDC values in the range +4.0 to –7.4 Hz were measured at 600 MHz. Comparisons of ¹H-¹⁵N HSQC spectra of ¹⁵N-DHFR-M13 alone and its complexes with CaM(Ca^{2 +})₄ and CaM(Tb³⁺)₄ indicated that (i) the structure of the target protein is not affected by the complex formation and (ii) the spectra of the target protein are not seriously perturbed by pseudocontact shifts. The use of a relatively large tagging group (CaM) allows us to use a lanthanide ion with a very high magnetic susceptibility anisotropy (such as Tb³⁺) to give large alignments while maintaining relatively long distances from the target protein nuclei (and hence giving only small pseudocontact shift contributions).     

A 4D HCC(CO)NNH experiment for the correlation of aliphatic side-chain and backbone resonances in 13C/15N-labelled proteins

Summary We recently proposed a novel four-dimensional (4D) NMR strategy for the assignment of backbone nuclei in spectra of ¹³C/¹⁵N-labelled proteins (Boucher et al. (1992) J. Am. Chem. Soc., 114, 2262–2264 and J. Biomol. NMR, 2, 631–637). In this paper we extend this approach with a new constant time 4D HCC(CO)NNH experiment that also correlates the chemical shifts of the aliphatic sidechain (¹H and ¹³C) and backbone (¹H, ¹³C and ¹⁵N) nuclei. It separates the sidechain resonances, which may heavily overlap in spectra of proteins with large numbers of similar residues, according to the backbone nitrogen and amide proton chemical shifts. When used in conjunction with a 4D HCANNH or HNCAHA experiment it allows, in principle, complete assignment of aliphatic sidechain and backbone resonances with just two 4D NMR experiments.     

Observation and measurement of internucleotide 2JNN coupling constants between 15N nuclei with widely separated chemical shifts

Scalar coupling correlations between hydrogen bonded 15N nuclei in non Watson–Crick base pairs is a critical step in the structure determination of unusual nucleic acids. For observing the 2JNN coupling constant between far upfield N2,N6 (amino) nitrogens and far downfield (N1,N3,N7) nitrogens (separated by 150–160 ppm), the HNN-COSY experiment (Dingley and Grzesiek, 1998) is rather insensitive, due to technical difficulties associated with simultaneous excitation of both extremes of the 15N spectrum. These nuclei may be correlated by treating them in a pseudo-heteronuclear manner, using 15N selective pulses. The wide chemical shift separation allows accurate measurement of the 2JNN coupling constant using spin-echo difference methods. Pulse sequences for observation and measurement of 2JNN coupling constants between amino and N7 nuclei are presented and demonstrated on an A-A mismatch segment of the uniformly (15N,13C) labelled DNA sample, d(GGAGGAT)2.     

Glial Metabolism of Valine

The three essential amino acids, valine, leucine and isoleucine, constitute the group of branched-chain amino acids (BCAAs). BCAAs are rapidly taken up into the brain parenchyma, where they serve several distinct functions including that as fuel material in brain energy metabolism. As one function of astrocytes is considered the production of fuel molecules that support the energy metabolism of adjacent neural cells in brain. Astroglia-rich primary cultures (APC) were shown to rapidly dispose of the BCAAs, including valine, contained in the culture medium. While the metabolisms of leucine and isoleucine by APC have already been studied in detail, some aspects of valine metabolism remained to be determined. Therefore, in the present study an NMR analysis was performed to identify the ¹³C-labelled metabolites that are generated by APC during catabolism of [U-¹³C]valine and that are subsequently released into the incubation medium. The results presented show that APC (1) are potently disposing of the valine contained in the incubation medium; (2) are capable of degrading valine to the tricarboxylic acid (TCA) cycle member succinyl-CoA; and (3) release into the extracellular milieu valine catabolites and compounds generated from them such as [U-¹³C]2-oxoisovalerate, [U-¹³C]3-hydroxyisobutyrate, [U-¹³C]2-methylmalonate, [U-¹³C]isobutyrate, and [U-¹³C]propionate as well as several TCA cycle-dependent metabolites including lactate. This article is dedicated to Dr. George DeVries.     

An Enzymatic Preparation of UDP (U-13C) Glucose

Uniformly labeled uridine diphosphoglucose (UDP(U-¹³C)G) was prepared by a two-step enzymatic synthesis. (U-¹³C) G-6-P was prepared quantitatively by incubating (U-¹³C) glucose, ATP, MgS0₄, and hexokinase. UDP(U-¹³C) Glucose was prepared by incubation of (U-¹³C)G-6-P with UDPG pyrophosphorylase, phosphoglucomutase, inorganic pyrophosphatase, UTP, and glucose-1, 6-diphosphate in pH 7.5, 100 mM Tris-HCl buffer. After purification over Biogel P-2 and subsequent preparative HPLC, UDP (U-¹³C)G was obtained in 50% yield. UDP(U-¹³C)G was characterized by ¹³C NMR and FAB-MS.     

Evidence for Transaldolase Activity in the Isolated Heart Supplied with [U-13C3]Glycerol

Studies of glycerol metabolism in the heart have largely emphasized its role in triglyceride synthesis. However, glycerol may also be oxidized in the citric acid cycle, and glycogen synthesis from glycerol has been reported in the nonmammalian myocardium. The intent of this study was to test the hypothesis that glycerol may be metabolized to glycogen in mammalian heart. Isolated rat hearts were supplied with a mixture of substrates including glucose, lactate, pyruvate, octanoate, [U-¹³C₃]glycerol, and ²H₂O to probe various metabolic pathways including glycerol oxidation, glycolysis, the pentose phosphate pathway, and carbon sources of stored glycogen. NMR analysis confirmed that glycogen production from the level of the citric acid cycle did not occur and that the glycerol contribution to oxidation in the citric acid cycle was negligible in the presence of alternative substrates. Quite unexpectedly, ¹³C from [U-¹³C₃]glycerol appeared in glycogen in carbon positions 4–6 of glucosyl units but none in positions 1–3. The extent of [4,5,6-¹³C₃]glucosyl unit enrichment in glycogen was enhanced by insulin but decreased by H₂O₂. Given that triose phosphate isomerase is generally assumed to fully equilibrate carbon tracers in the triose pool, the marked ¹³C asymmetry in glycogen can only be attributed to conversion of [U-¹³C₃]glycerol to [U-¹³C₃]dihydroxyacetone phosphate and [U-¹³C₃]glyceraldehyde 3-phosphate followed by rearrangements in the nonoxidative branch of the pentose phosphate pathway involving transaldolase that places this ¹³C-enriched 3-carbon unit only in the bottom half of hexose phosphate molecules contributing to glycogen.     

A set of HNCO-based experiments for measurement of residual dipolar couplings in 15N, 13C, (2H)-labeled proteins

Several HNCO-based three-dimensional experiments are described for the measurement of ¹³C(i–1)-¹³C(i–1), ¹⁵N(i)-¹³C(i–1), ¹⁵N(i)-¹³C(i), ¹⁵N(i)-¹³C(i–1), ¹H^N(i)-¹³C(i), ¹H^N(i)-¹³C(i–1), and ¹³C(i–1)-¹³C(i–1) scalar and dipolar couplings in ¹⁵N, ¹³C, (²H)-labelled protein samples. These pulse sequences produce spin-state edited spectra superficially resembling an HNCO correlation spectrum, allowing accurate and simple measurement of couplings without introducing additional spectral crowding. Scalar and dipolar couplings are measured with good sensitivity from relatively large proteins, as demonstrated with three proteins: cardiac Troponin C, calerythrin and ubiquitin. Measurement of several dipolar couplings between spin-1/2 nuclei using spin-state selective 3D HNCO spectra provides a wealth of structural information.     

Heteronuclear relayed E.COSY applied to the determination of accurate 3J(HN,C′) and 3J(Hβ,C′) coupling constants in Desulfovibrio vulgaris flavodoxin

Summary A simple constant-time 3D heteronuclear NMR pulse sequence has been developed to quantitatively determine the heteronuclear three-bond couplings ³J(H^N,C) and ³J(H,C) in uniformly ¹³C-enriched proteins. The protocols for measuring accurate coupling constants are based on ¹H,¹³C-heteronuclear relayed E.COSY [Schmidt, J.M., Ernst, R.R., Aimoto, S. and Kainosho, M. (1995) J. Biomol. NMR, 6, 95–105] in combination with numerical least-squares spectrum evaluation. Accurate coupling constants are extracted from 2D spectrum projections using 2D multiplet simulation. Confidence intervals for the obtained three-bond coupling constants are calculated from F-statistics. The three-bond couplings are relevant to the determination of and X ₁ dihedral-angle conformations in the amino acid backbone and side chain. The methods are demonstrated on the recombinant ¹³C, ¹⁵N-doubly enriched 147-amino acid protein Desulfovibrio vulgaris flavodoxin with bound flavin mononucleotide in its oxidized form. In total, 109 ³J(H^N,C) and 100 ³J(H,C) coupling constants are obtained from a single spectrum.Abbreviations ANOVA analysis of variances - COSY correlated spectroscopy - E.COSY exclusive correlation spectroscopy - FMN flavin mononucleotide - HMQC heteronuclear multiple-quantum coherence - HSQC heteronuclear single-quantum coherence     

<Superscript>13</Superscript>C-detected NMR experiments for measuring chemical shifts and coupling constants in nucleic acid bases

The paper presents a set of two-dimensional experiments that utilize direct ¹³C detection to provide proton–carbon, carbon–carbon and carbon–nitrogen correlations in the bases of nucleic acids. The set includes a ¹³C-detected proton–carbon correlation experiment for the measurement of ¹³C–¹³C couplings, the CaCb experiment for correlating two quaternary carbons, the HCaCb experiment for the ¹³C–¹³C correlations in cases where one of the carbons has a proton attached, the HCC-TOCSY experiment for correlating a proton with a network of coupled carbons, and a ¹³C-detected ¹³C–¹⁵N correlation experiment for detecting the nitrogen nuclei that cannot be detected via protons. The IPAP procedure is used for extracting the carbon–carbon couplings and/or carbon decoupling in the direct dimension, while the S³E procedure is preferred in the indirect dimension of the carbon–nitrogen experiment to obtain the value of the coupling constant. The experiments supply accurate values of ¹³C and ¹⁵N chemical shifts and carbon–carbon and carbon–nitrogen coupling constants. These values can help to reveal structural features of nucleic acids either directly or via induced changes when the sample is dissolved in oriented media. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A 2D NMR study of the internal flexibility of the antifungal peptide stendomycin

A 2-D ¹H NMR study (NOESY, COSY, HOHAHA and ROESY experiments) of the antifungal peptide stendomycin is presented. The variation of the NOESY cross peak intensities is measured as a function of temperature in order to discriminate between constant and fluctuating interproton distances. It is shown that among 71 NOESY cross peaks, only 12 correspond to well defined interproton distances and their correlation time is determined. The other cross peaks cannot be translated accurately in terms of distances owing to internal molecular motions. ¹H}–¹³C nOe measurements confirm the internal mobility of the molecule. Finally a flexibility map of stendomycin can be established. Offprint requests to: J. P. Simorre     

Characterization of particulate D-apiosyl-and D-xylosyltransferase from Lemna minor.

A particulate enzyme preparation capable of catalyzing the transfer of d-[U-¹⁴C]apiose and d-[U-¹⁴C]xylose from uridine 5′-(α-d-[U-¹⁴C]apio-d-furanosyl pyrophosphate) (UDP[U-¹⁴C]Api) and uridine 5′-(α-d-[U-¹⁴C]xylopyranosyl pyrophosphate) (UDP[U-¹⁴C]Xyl) to endogenous acceptor molecules was isolated from Lemna minor. The two enzymes were named UDP-d-apiose:acceptor d-apiosyltransferase and UDP-d-xylose:acceptor d-xylosyltransferase and were associated with particulate material sedimenting between 480 and 34,800g. The rate of d-[U-¹⁴C]apiose or d-[U-¹⁴C]xylose incorporation was proportional to the quantity of enzyme preparation used and was constant with time to 1.5 min. Both enzymes showed a pH optimum of 5.7 in citrate-phosphate buffer. The d-apiosyltransferase has a K_m for UDP[U-¹⁴C]Api of 4.9 μm. Bovine serum albumin and sucrose stimulated the rate of incorporation of both pentoses. Both enzymes rapidly lost activity; with our best conditions, approximately 50% of each enzyme activity was lost in 6 min at 25 °C or in 3 h at 4 °C. Incorporation of d-[U-¹⁴C]apiose was obtained in the absence of added uridine 5′-(α-d-galactopyranosyluronic acid pyrophosphate) (UDPGalUA); however, the addition of UDPGalUA not only almost doubled the rate of incorporation, but also increased the total incorporation of d-[U-^l4C]apiose and extended the proportional range of incorporation at 25 °C from 1.5 to 2 min.     

Water–Protein Hydrogen Exchange in the Micro-Crystalline Protein Crh as Observed by Solid State NMR Spectroscopy

We report site-resolved observation of hydrogen exchange in the micro-crystalline protein Crh. Our approach is based on the use of proton T₂^′ -selective ¹H–¹³C–¹³C correlation spectra for site-specific assignments of carbons nearby labile protein protons. We compare the proton T₂^′ selective scheme to frequency selective water observation in deuterated proteins, and discuss the impacts of deuteration on ¹³C linewidths in Crh. We observe that in micro-crystalline proteins, solvent accessible hydroxyl and amino protons show comparable exchange rates with water protons as for proteins in solution, and that structural constraints, such as hydrogen bonding or solvent accessibility, more significantly reduce exchange rates.     

13C-2H correlation NMR spectroscopy studies of the in vivo transformations of natural products from Artemisia annua

¹³C-²H correlation NMR spectroscopy (¹³C-²H COSY) permits the identification of ¹³C and ²H nuclei which are connected to one another by a single chemical bond via the sizeable ¹J_CD coupling constant. The practical development of this technique is described using a ¹³C-²H COSY pulse sequence which is derived from the classical ¹³C-¹H correlation experiment. An example is given of the application of ¹³C-²H COSY to the study of the biogenesis of natural products from the anti-malarial plant Artemisia annua, using a doubly-labelled precursor molecule. Although the biogenesis of artemisinin, the anti-malarial principle from this species, has been extensively studied over the past twenty years there is still no consensus as to the true biosynthetic route to this important natural product – indeed, some published experimental results are directly contradictory. One possible reason for this confusion may be the ease with which some of the metabolites from A. annua undergo spontaneous autoxidation, as exemplified by our recent in vitro studies of the spontaneous autoxidation of dihydroartemisinic acid, and the application of ¹³C-²H COSY to this biosynthetic problem has been important in helping to mitigate against such processes. In this in vivo application of ¹³C-²H COSY, [15-¹³C²H₃]-dihydroartemisinic acid (the doubly-labelled analogue of the natural product from this species which was obtained through synthesis) was fed to A. annua plants and was shown to be converted into several natural products which have been described previously, including artemisinin. It is proposed that all of these transformations occurred via a tertiary hydroperoxide intermediate, which is derived from dihyroartemisinic acid. This intermediate was observed directly in this feeding experiment by the ¹³C-²H COSY technique; its observation by more traditional procedures (e.g., chromatographic separation, followed by spectroscopic analysis of the purified product) would have been difficult owing to the instability of the hydroperoxide group (as had been established previously by our in vitro studies of the spontaneous autoxidation of dihydroartemisinic acid). This same hydroperoxide has been reported as the initial product of the spontaneous autoxidation of dihydroartemisinic acid in our previous in vitro studies. Its observation in this feeding experiment by the ¹³C-²H COSY technique, a procedure which requires the minimum of sample manipulation in order to achieve a reliable identification of metabolites (based on both ¹³C and ²H chemical shifts at the 15-position), provides the best possible evidence for its status as a genuine biosynthetic intermediate, rather than merely as an artifact of the experimental procedure.     

A new glycation product ‘norpronyl-lysine,’ and direct characterization of cross linking and other glycation adducts: NMR of model compounds and collagen

NMR is ideal for characterizing non-enzymatic protein glycation, including AGEs (advanced glycation endproducts) underlying tissue pathologies in diabetes and ageing. Ribose, R5P (ribose-5-phosphate) and ADPR (ADP-ribose), could be significant and underinvestigated biological glycating agents especially in chronic inflammation. Using [U-¹³C]ribose we have identified a novel glycoxidation adduct, 5-deoxy-5-desmethylpronyl-lysine, ‘norpronyl-lysine’, as well as numerous free ketones, acids and amino group reaction products. Glycation by R5P and ADPR proceeds rapidly with R5P generating a brown precipitate with PLL (poly-L-lysine) within hours. ssNMR (solid-state NMR) ¹³C–¹³C COSY identifies several crosslinking adducts such as the newly identified norpronyl-lysine, in situ, from the glycating reaction of ¹³C₅-ribose with collagen. The same adducts are also identifiable after reaction of collagen with R5P. We also demonstrate for the first time bio-amine (spermidine, N-acetyl lysine, PLL) catalysed ribose 2-epimerization to arabinose at physiological pH. This work raises the prospect of advancing understanding of the mechanisms and consequences of glycation in actual tissues, in vitro or even ex vivo, using NMR isotope-labelled glycating agents, without analyses requiring chemical or enzymatic degradations, or prior assumptions about glycation products.     

Cyanoglycosylation products of 17-O-acetyl-testosterone

17-O-Acetyl testosterone, which has no susceptible hydroxyl or carboxyl group for glycosylation, was glycosylated with 2,3,4,6-tetra-O-acetyl-α- -glucopyranosyl bromide in the presence of a mixed catalyst, Hg(CN)₂ and HgBr₂, in benzene–nitromethane. Reaction occurred on the α,β-unsaturated ketone on the six–membered A-ring to give six 3-O-glycosides, each bearing a cyano group at the 3- or 5-position of the aglycon, and a 3-O-glycoside bearing a CONH₂ group at the 3-position. Structural analyses of these products were carried out by various NMR (¹H, ¹³C NMR, ¹H–¹H and ¹H–¹³C COSY, HMBC, and DEPT), FABMS and X-ray analyses. The mechanisms of the formations of the products are discussed. It was determined that mercuric cyanide was essential as a catalyst for the progress of the cyanoglycosylation.     

Phase labeling of C−H and C−C spin-system topologies: Application in PFG-HACANH and PFG-HACA(CO)NH triple-resonance experiments for determining backbone resonance assignments in proteins

Summary Triple-resonance experiments can be designed to provide useful information on spin-system topologies. In this paper we demonstrate optimized proton and carbon versions of PFG-CT-HACANH and PFG-CT-HACA(CO)NH straight-through triple-resonance experiments that allow rapid and almost complete assignments of backbone H, ¹³C, ¹⁵N and H^N resonances in small proteins. This work provides a practical guide to using these experiments for determining resonance assignments in proteins, and for identifying both intraresidue and sequential connections involving glycine residues. Two types of delay tunings within these pulse sequences provide phase discrimination of backbone Gly C and H resonances: (i) C–H phase discrimination by tuning of the refocusing period _{a_f}; (ii) C–C phase discrimination by tuning of the ¹³C constant-time evolution period 2T_c. For small proteins, C–C phase tuning provides better S/N ratios in PFG-CT-HACANH experiments while C–H phase tuning provides better S/N ratios in PFG-CT-HACA(CO)NH. These same principles can also be applied to triple-resonance experiments utilizing ¹³C-¹³C COSY and TOCSY transfer from peripheral side-chain atoms with detection of backbone amide protons for classification of side-chain spin-system topologies. Such data are valuable in algorithms for automated analysis of resonance assignments in proteins.     

(3, 2)D <Superscript>1</Superscript>H, <Superscript>13</Superscript>C BIRD<Superscript>r,X</Superscript>-HSQC-TOCSY for NMR structure elucidation of mixtures: application to complex carbohydrates

Overlap of NMR signals is the major cause of difficulties associated with NMR structure elucidation of molecules contained in complex mixtures. A 2D homonuclear correlation spectroscopy in particular suffers from low dispersion of ¹H chemical shifts; larger dispersion of ¹³C chemical shifts is often used to reduce this overlap, while still providing the proton–proton correlation information e.g. in the form of a 2D ¹H, ¹³C HSQC-TOCSY experiment. For this methodology to work, ¹³C chemical shift must be resolved. In case of ¹³C chemical shifts overlap, ¹H chemical shifts can be used to achieve the desired resolution. The proposed (3, 2)D ¹H, ¹³C BIRD^r,X-HSQC-TOCSY experiment achieves this while preserving singlet character of cross peaks in the F₁ dimension. The required high-resolution in the ¹³C dimension is thus retained, while the cross peak overlap occurring in a regular HSQC-TOCSY experiment is eliminated. The method is illustrated on the analysis of a complex carbohydrate mixture obtained by depolymerisation of a fucosylated chondroitin sulfate isolated from the body wall of the sea cucumber Holothuria forskali.