Innovations in generation and analysis of 2D [(13)C,(1)H] COSY NMR spectra for metabolic flux analysis purposes.

2D [(13)C,(1)H] COSY NMR is used by the metabolic engineering community for determining (13)C-(13)C 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 [(13)C,(1)H] 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 (13)C-(13)C scalar couplings in 2D [(13)C,(1)H] COSY NMR spectra. Finally, the NMR resolution enhancement by dissolving amino acid derivatives in a nonpolar solvent is demonstrated.     

Structure of a quinobenzoxazine--G-quadruplex complex by REDOR NMR

Rotational-echo double resonance solid-state (31)P[(19)F] and (13)C[(19)F] NMR spectra have been used to locate the binding of a fluoroquinobenzoxazine to a DNA G-quadruplex labeled by phosphorothioation and [methyl-(13)C]thymidine.     

TCA cycle kinetics in the rat heart by analysis of (13)C isotopomers using indirect (1)H

This study was designed to test the hypothesis that indirect (1)H[(13)C] detection of tricarboxylic acid (TCA) cycle intermediates using heteronuclear multiple quantum correlation-total correlation spectroscopy (HMQC-TOCSY) nuclear magnetic resonance (NMR) spectroscopy provides additional (13)C isotopomer information that better describes the kinetic exchanges that occur between intracellular compartments than direct (13)C NMR detection. NMR data were collected on extracts of rat hearts perfused at various times with combinations of [2-(13)C]acetate, propionate, the transaminase inhibitor aminooxyacetate, and (13)C multiplet areas derived from spectra of tissue glutamate were fit to a standard kinetic model of the TCA cycle. Although the two NMR methods detect different populations of (13)C isotopomers, similar values were found for TCA cycle and exchange fluxes by analyzing the two data sets. Perfusion of hearts with unlabeled propionate in addition to [2-(13)C]acetate resulted in an increase in the pool size of all four-carbon TCA cycle intermediates. This allowed the addition of isotopomer data from aspartate and malate in addition to the more abundant glutamate. This study illustrates that metabolic inhibitors can provide new insights into metabolic transport processes in intact tissues.     

Solid-state NMR conformational studies of a melittin-inhibitor complex

Melittin is a cytolytic peptide whose biological activity is lost upon binding to a six-residue peptide, Ac-IVIFDC-NH(2), with which it forms a highly insoluble complex. As a result, the structural analysis of the interaction between the two peptides is difficult. Solid-state NMR spectroscopy was used to study the interaction between melittin and the peptide inhibitor. Location of the binding site in the melittin-inhibitor complex was determined using lanthanide ions, which quench NMR resonances from molecular sites that are in close proximity to the unique ion binding site. Our results indicated that the inhibitor binding site in melittin is near Leu13, Leu16 and Ile17, but not near Leu6 or Val8. On the basis of these data we propose that the inhibitor binds to melittin in the vicinity of Ala15 to Trp19 and prevents insertion of melittin into cell membranes by disrupting the helical structure. Supporting evidence for this model was produced by determining the distance, using rotational resonance NMR, between the [1-(13)C] of Leu13 in melittin and the [3-(13)C] of Phe4 in the inhibitor.     

Characterization of the complex of a trifluoromethyl-substituted shikimate-based bisubstrate inhibitor and 5-enolpyruvylshikimate-3-phosphate synthase by REDOR NMR

A combination of (15)N[(19)F], (31)P[(15)N], and (31)P[(19)F] rotational-echo double-resonance NMR has been used to characterize the conformation of a bound trifluoromethylketal, shikimate-based bisubstrate inhibitor of 5-enolpyruvylshikimate-3-phosphate synthase. The solid-state NMR experiments were performed on the complex formed in solution and then lyophilized at low temperatures in the presence of stabilizing lyoprotectants. The results of these experiments indicate that none of the side chains of the six arginines that surround the active site forms a compact salt bridge with the phosphate groups of the bound inhibitor.     

Direct observation (NMR) of the efficacy of glucagon receptor antagonists in murine liver expressing the human glucagon receptor

The demonstration of pharmacodynamic efficacy of novel chemical entities represents a formidable challenge in the early exploration of synthetic lead classes. Here, we demonstrate a technique to validate the biological efficacy of novel antagonists of the human glucagon receptor (hGCGR) in the surgically removed perfused liver prior to the optimization of the pharmacokinetic properties of the compounds. The technique involves the direct observation by (13)C NMR of the biosynthesis of [(13)C]glycogen from [(13)C]pyruvate via the gluconeogenic pathway. The rapid breakdown of [(13)C]glycogen (glycogenolysis) following the addition of 50 pM exogenous glucagon is then monitored in real time in the perfused liver by (13)C NMR. The concentration-dependent inhibition of glucagon-mediated glycogenolysis is demonstrated for both the peptidyl glucagon receptor antagonist 1 and structurally diverse synthetic antagonists 2-7. Perfused livers were obtained from a transgenic mouse strain that exclusively expresses the functional human glucagon receptor, conferring human relevance to the activity observed with glucagon receptor antagonists. This technique does not provide adequate quantitative precision for the comparative ranking of active compounds, but does afford physiological evidence of efficacy in the early development of a chemical series of antagonists.     

Comparative structure analysis of tyrosine and valine residues in unprocessed silk fibroin (silk I) and in the processed silk fiber (silk II) from Bombyx mori using solid-state (13)C,(15)N, and (2)H NMR

The solid-state (13)C CP-MAS NMR spectra of biosynthetically labeled [(13)C(alpha)]Tyr, [(13)C(beta)]Tyr, and [(13)C(alpha)]Val silk fibroin samples of Bombyx mori, in silk I (the solid-state structure before spinning) and silk II (the solid-state structure after spinning) forms, have been examined to gain insight into the conformational preferences of the semicrystalline regions. To establish the relationship between the primary structure of B. mori silk fibroin and the "local" structure, the conformation-dependent (13)C chemical shift contour plots for Tyr C(alpha), Tyr C(beta), and Val C(alpha) carbons were generated from the atomic coordinates of high-resolution crystal structures of 40 proteins and their characteristic (13)C isotropic NMR chemical shifts. From comparison of the observed Tyr C(alpha) and Tyr C(beta) chemical shifts with those predicted by the contour plots, there is strong evidence in favor of an antiparallel beta-sheet structure of the Tyr residues in the silk fibroin fibers. On the other hand, Tyr residues take a random coil conformation in the fibroin film with a silk I form. The Val residues are likely to assume a structure similar to those of Tyr residues in silk fiber and film. Solid-state (2)H NMR measurements of [3,3-(2)H(2)]Tyr-labeled B. mori silk fibroin indicate that the local mobility of the backbone and the C(alpha)-C(beta) bond is essentially "static" in both silk I and silk II forms. The orientation-dependent (i.e., parallel and perpendicular to the magnetic field) solid-state (15)N NMR spectra of biosynthetically labeled [(15)N]Tyr and [(15)N]Val silk fibers reveal the presence of highly oriented semicrystalline regions.     

Mechanism of action of urocanase. Specific 13C-labelling of the prosthetic NAD+ and revision of the structure of its adduct with imidazolylpropionate

1. [4-13C]Nicotinate was synthesised and used to support the growth of a nicotinate auxotrophic mutant of Pseudomonas putida. 13C-NMR spectroscopy of the isolated urocanase confirmed the efficient incorporation of 13C into C4 of the nicotinamide ring of the tightly bound NAD+ cofactor. 2. beta-[( 2'-13C]Imidazol-4-yl)propionate was synthesised according to known procedures and used for inhibition of the 13C-labelled urocanase. An increase in the absorbance at 330 nm indicated adduct formation between enzyme-bound NAD+ and inhibitor. The adduct was stabilised by oxidation with phenazine methosulfate and isolated using a slight modification of the procedure of Matherly et al. [Matherly, L. H., DeBrosse, C. W. & Phillips, A. T. (1982) Biochemistry 21, 2789-2794]. 3. The 13C-NMR spectrum of the doubly labelled adduct, [4-13C]NAD-[2'-13C]imidazolylpropionate, showed no one-bond 13C-13C coupling between labelled sites. The 1H-NMR spectrum of this adduct in 2H2O showed only one imidazole signal, which appeared as a doublet (1JC-H = 212 Hz), confirming the presence of a proton at the labelled C2'. The lack of a C5' signal and further NMR data provide evidence for a C-C bond between C4 of the nicotinamide and C5' of the imidazole ring. 4. The revised structure for the enzymatically formed addition complex suggests a novel mechanism for the urocanase reaction which is not only chemically plausible but also explains the previously observed urocanase-catalysed exchange of the C5 proton of urocanate and of beta-(imidazol-4-yl)propionate.     

Cerebral dicarboxylate transport and metabolism studied with isotopically labelled fumarate,malate and malonate

Transport and metabolism of dicarboxylates may be important in the glial-neuronal metabolic interplay. Further, exogenous dicarboxylates have been suggested as cerebral energy substrates. After intrastriatal injection of [(14) C]fumarate or [(14) C]malate, glutamine attained a specific activity 4.1 and 2.6 times higher than that of glutamate, respectively, indicating predominantly glial uptake of these four-carbon dicarboxylates. In contrast, the three-carbon dicarboxylate [(14) C]malonate gave a specific activity in glutamate which was approximately five times higher than that of glutamine, indicating neuronal uptake of malonate. Therefore, neurones and glia take up different types of dicarboxylates, probably by different transport mechanisms. Labelling of alanine from [(14) C]fumarate and [(14) C]malate demonstrated extensive malate decarboxylation, presumably in glia. Intravenous injection of 75 micromol [U-(13) C]fumarate rapidly led to high concentrations of [U-(13) C]fumarate and [U-(13) C]malate in serum, but neither substrate labelled cerebral metabolites as determined by (13) C NMR spectroscopy. Only after conversion of [U-(13) C]fumarate into serum glucose was there (13) C-labelling of cerebral metabolites, and only at <10% of that obtained with 75 micromol [3-(13) C]lactate or [2-(13) C]acetate. These findings suggest a very low transport capacity for four-carbon dicarboxylates across the blood-brain barrier and rule out a role for exogenous fumarate as a cerebral energy substrate.     

Biosynthesis of porphyrins and corrins. 2. Isolation, purification, and NMR investigations of the porphobilinogen-deaminase covalent complex

The procedures for the generation of enzyme-substrate complexes from labeled porphobilinogens [(2,11-13C]PBG and [2,6,11-3H]PBG) with deaminase and the methods employed for their purification are described. Use of 13C NMR failed to detect the substrate bound to the enzyme, suggesting that the line width must be inordinately large. The complex was found to disproportionate with time when stored at 25 degrees C. However, enzyme-bound uroporphyrinogen I (uro'gen I) was detected, both in the intact protein and in the oligopeptides from tryptic digestion and peptide mapping. The first detection of an enzyme-substrate complex by 3H NMR is described for [3H]PBG and deaminase. The line widths of the observed resonances were found to be extremely large and dependent upon temperature, giving chemical shifts that suggest the involvement of a sulfhydryl group as the nucleophilic enzyme group that binds the substrate. The catalytic competence of this complex was also demonstrated by displacing bound [3H]PBG with unlabeled PBG. During the resultant formation of [3H]uro'gen I, a transient low-intensity signal was detected that has been tentatively assigned to the highly reactive azafulvene species, proposed in several mechanistic schemes for porphyrin biosynthesis.     

Evidence for a tetrahedral intermediate complex during serpin-proteinase interactions

Proteinase inhibitors in the serpin family form complexes with serine proteinases by interactions between the gamma-OH group at serine 195 of the enzyme and a specific peptide bond within the reactive site loop of the inhibitor. However, the type of complex formed (i.e. Michaelis, acyl, or tetrahedral) is unknown. Until now, 13C NMR spectroscopy studies have only been useful in examining complexes formed with either peptide-related or small protein inhibitors, where 13C-labeled amino acids can be inserted semi-synthetically. Recombinant DNA technology has, however, made it possible to specifically enrich larger proteins with 13C. In the case of serpins we have examined the structure of the complex formed between human alpha 1-proteinase inhibitor uniformally labeled with [13C]methionine and porcine pancreatic elastase. 13C NMR spectroscopic studies revealed a large upfield chemical shift of the carbonyl signal of Met-358 upon complex formation suggesting for the first time that a tetrahedral adduct is formed between a serpin inhibitor and a serine proteinase.     

Cerebral metabolism of [1,2-13C2]acetate as detected by in vivo and in vitro 13C NMR

The metabolism of [1,2-13C2]acetate in rat brain was studied by in vivo and in vitro 13C NMR spectroscopy, in particular by taking advantage of the homonuclear 13C-13C spin coupling patterns. Well nourished rats were infused with [1,2-13C2]acetate or [1-13C]acetate in the jugular vein, and the in situ kinetics of 13C labeling during the infusion period was followed by 13C NMR techniques. The in vivo 13C NMR spectra showed signals from (i) the C-1 carbon of [1,2-13C2] acetate or [1-13C]acetate, (ii) 13CO3H-, and (iii) the natural abundance 13C carbons of sufficiently mobile fatty acids. Methanol/HCl/perchloric acid extracts of the brains were prepared and were further analyzed by high resolution 13C NMR. The homonuclear 13C-13C spin coupling patterns after infusion of [1,2-13C2]acetate showed very different isotopomer populations in glutamate, glutamine, and gamma-aminobutyric acid. Analyzing the relative proportions of these isotopomers revealed (i) two different glutamate compartments in the rat brain characterized by the presence and absence, respectively, of glutamine synthase activity, (ii) two different tricarboxylic acid cycles, one preferentially metabolizing [(1,2-13C2]acetate, the other mainly using unlabeled acetyl-coenzyme A, (iii) a hitherto unknown cerebral pyruvate recycling system associated with the tricarboxylic acid cycle, metabolizing primarily unlabeled acetyl-coenzyme A, and (iv) a predominant production of gamma-aminobutyric acid in the glutamate compartment lacking glutamine synthase.     

13C NMR studies of methylene and methine carbons of substrate bound to a 280,000-dalton protein, porphobilinogen synthase

13C NMR has been used to observe the equilibrium complex of [5,5-2H,5-13C]-5-aminolevulinate [( 5,5-2H,5-13C]ALA) bound to porphobilinogen (PBG) synthase (5-aminolevulinate dehydratase), a 280,000-dalton protein. [5,5-2H,5-13C]ALA (chemical shift 46.9 ppm in D2O) was prepared from [5-13C]ALA through enolization in deuteriated neutral potassium phosphate buffer. In the PBG synthase reaction [5,5-2H,5-13C]ALA forms [2,11,11-2H,2,11-13C]PBG (chemical shifts 116.2 ppm for C2 and 34.2 ppm for C11 in D2O). For the complex formed between [5,5-2H,5-13C]ALA and methyl methanethiosulfonate (MMTS) modified PBG synthase, which does not catalyze PBG formation but can form a Schiff base adduct, the chemical shift of 44.2 ppm (line width 92 Hz) identifies an imine structure as the predominant tautomeric form of the Schiff base. By comparison to model compounds, the stereochemistry of the imine has been deduced; however, the protonation state of the imine nitrogen remains unresolved. Reconstitution of the MMTS-modified enzyme-Schiff base complex with Zn(II) and 2-mercaptoethanol results in the holoenzyme-bound equilibrium complex; this complex contains predominantly enzyme-bound PBG, and spectra reveal two peaks from bound PBG and two from free PBG. For bound PBG, C2 is -2.8 ppm from the free signal and C11 is +2.6 ppm from the free signal; the line widths of the bound signals are 55 and 75 Hz, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Utilization of glycine and serine as nitrogen sources in the roots of Zea mays and Chamaegigas intrepidus

Glycine and serine are potential sources of nitrogen for the aquatic resurrection plant Chamaegigas intrepidus Dinter in the rock pools that provide its natural habitat. The pathways by which these amino acids might be utilized were investigated by incubating C. intrepidus roots and maize (Zea mays) root tips with [(15)N]glycine, [(15)N]serine and [2-(13)C]glycine. The metabolic fate of the label was followed using in vivo NMR spectroscopy, and the results were consistent with the involvement of the glycine decarboxylase complex (GDC) and serine hydroxymethyltransferase (SHMT) in the utilization of glycine. In contrast, the labelling patterns provided no evidence for the involvement of serine:glyoxylate aminotransferase in the metabolism of glycine by the root tissues. The key observations were: (i) the release of [(15)N]ammonium during [(15)N]-labelling experiments; and (ii) the detection of a characteristic set of serine isotopomers in the [2-(13)C]glycine experiments. The effects of aminoacetonitrile, amino-oxyacetate, and isonicotinic acid hydrazide, all of which inhibit GDC and SHMT to some extent, and of methionine sulphoximine, which inhibited the reassimilation of the ammonium, supported the conclusion that GDC and SHMT were essential for the metabolism of glycine. C. intrepidus was observed to metabolize serine more readily than the maize root tips and this may be an adaptation to its nitrogen-deficient habitat. Overall, the results support the emerging view that GDC is an essential component of glycine catabolism in non-photosynthetic tissues.     

Carbon-13 nuclear magnetic resonance studies of the selectively isotope-labeled reactive site peptide bond of the basic pancreatic trypsin inhibitor in the complexes with trypsin, trypsinogen, and anhydrotrypsin

A previously characterized modification of the basic pancreatic trypsin inhibitor (BPTI), with the carbonyl carbon atom of Lys-15 selectively enriched in 13C, the peptide bond Arg-39--Ala-40 cleaved, and Arg-39 removed, was used for 13C NMR studies of the reactive site peptide bond Lys-15--Ala-16 in the complexes with trypsin, trypsinogen, and anhydrotrypsin. The chemical shift of [1-13C]Lys-15 was 175.7 ppm in the free inhibitor, 176.4 ppm in the complexes with trypsin and anhydrotrypsin and the ternary complex with trypsinogen and H-Ile-Val-OH, and 175.7 ppm in a neutral solution containing the inhibitor and trypsinogen. These data show that the trypsin--BPTI complex does not contain a covalent tetrahedral carbon atom in the position of the reactive site peptide carbonyl of the inhibitor. They would be consistent with the formation of a noncovalent complex but cannot at present be used to further characterize the degree of a possible pyramidalization of the carbonyl carbon of Lys-15 in such a complex. The identical chemical shifts in the complexes with trypsin and anhydrotrypsin indicate that the gamma-hydroxyl group of Ser-195 of trypsin does not have an important role in the binding of the inhibitor. The previously described [Perkins, S. J. & Wüthrich, K. (1980) J. Mol. Biol. 138, 43--64] stepwise transition from the trypsinogen conformation to an intermediate conformational state in the trypsinogen--BPTI complex and a trypsin-like conformation in the ternary complex trypsinogen--BPTI--H-Ile-Val-OH appears to be manifested also in the chemical shift of [1-13C]Lys-15 of labeled BPTI.     

A comparison of the metabolism of eighteen-carbon 13C-unsaturated fatty acids in healthy women

Altered use of different dietary fatty acids may contribute to several chronic diseases, including obesity, noninsulin-dependent diabetes mellitus, and cardiovascular disease. However, few comparative data are available to support this link, so the goal of the present study was to compare the metabolism of [(13)C]oleate, [(13)C]alpha-linolenate, [(13)C]elaidate, and [(13)C]linoleate through oxidation and incorporation into plasma lipid fractions and adipose tissue. Each tracer was given as a single oral bolus to six healthy women. Samples were collected over 8 days, and (13)C was analyzed using isotope ratio mass spectrometry. At 9 h postdose, cumulative oxidation was similar for [(13)C]elaidate, [(13)C]oleate, and [(13)C]alpha-linolenate (19 +/- 1%, 20 +/- 4%, and 19 +/- 3% dose, respectively). Significantly lower oxidation of [(13)C]linoleate (12 +/- 4% dose; P < 0.05) was accompanied by its higher incorporation into plasma phospholipids and cholesteryl esters. Abdominal adipose tissue was enriched with [(13)C]alpha-linolenate, [(13)C]elaidate, or [(13)C]linoleate within 6 h. The percentage linoleate in plasma phospholipids correlated positively with [(13)C]linoleate and [(13)C]elaidate oxidation, indicating a potential role of background diet. Conversion of [(13)C]linoleate and [(13)C]alpha-linolenate to longer chain polyunsaturates was a quantitatively minor route of utilization.     

Synthesis and Structure Assignment of 1-(2-Acetoxyethoxy)methyl Derivatives of 5-Chloro-6-azauracil and 5-Bromo-6-azaisocytosine

Abstract

1-[(2-Acetoxyethoxy)methyl]-5-chloro-6-azauracil has been prepared and its unambiguous assignment of ¹H and ¹³C peaks through the ¹H-¹³C heteronuclear correlation (HETCOR) NMR experiments is described. The isosteric 1-[(2-acetoxyethoxy)methyl]-5-bromo-6-azaisocytosine has also been synthesized. The X-Ray crystallographic analysis reveals unambiguously the site of glycosylation at N¹. Deacetylation of both acyclonucleosides provided 5-chloro-1-[(2-hydroxyethoxy)methyl]-6-azauracil and 5-bromo-1-[(2-hydroxyethoxy)methyl]-6-azaisocytosine respectively. Their structures have been well established by the NMR spectra and the elemental analyses.     

Cumulative bondomers: a new concept in flux analysis from 2D [13C,1H] COSY NMR data

A well-established way of determining metabolic fluxes is to measure 2D [(13)C,(1)H] COSY NMR spectra of components of biomass grown on uniformly (13)C-labeled carbon sources. When using the entire set of measured data to simultaneously determine all fluxes in a proposed metabolic network model, the (13)C-labeling distribution in all measured compounds has to be simulated. This requires very large sets of isotopomer or cumomer balances. This article introduces the new concept of bondomers; entities that only vary in the numbers and positions of C-C bonds that have remained intact since the medium substrate molecule entered the metabolism. Bondomers are shown to have many analogies to isotopomers. One of these is that bondomers can be transformed to cumulative bondomers, just like isotopomers can be transformed to cumomers. Similarly to cumomers, cumulative bondomers allow an analytical solution of the entire set of balances describing a metabolic network. The main difference is that cumulative bondomer models are considerably smaller than corresponding cumomer models. This saves computational time, allows easier identifiability analysis, and yields new insights in the information content of 2D [(13)C,(1)H] COSY NMR data. We illustrate the theoretical concepts by means of a realistic example of the glycolytic and pentose phosphate pathways. The combinations of 2D [(13)C,(1)H] COSY NMR data that allow identification of all metabolic fluxes in these pathways are analyzed, and it is found that the NMR data contain less information than was previously expected.     

Nuclear magnetic resonance study of the state of protonation of inhibitors bound to mutant dihydrofolate reductase lacking the active-site carboxyl

13C nuclear magnetic resonance spectra have been obtained for complexes of [2-13C]methotrexate and [2-13C]trimethoprim with wild-type dihydrofolate reductase (DHFR) from Escherichia coli and with two mutant enzymes in which aspartic acid-27 is replaced by asparagine and by serine, respectively. In both the wild-type and mutated enzymes, exchange between the free inhibitor and the enzyme-complexed inhibitor is slow on the NMR time scale; hence, despite the considerably increased dissociation constants for binary complexes with the enzymes, the dissociation rate remains small relative to the frequency separation of the resonances. In all cases but one, the pKa of an inhibitor that is complexed to enzyme differs greatly from that of the free inhibitor. However, while the pKa of both inhibitors in complexes with the wild-type enzyme is elevated to above 10, the pKa of the inhibitors complexed with the Asn-27 and Ser-27 enzymes is lowered to a value below 4. Exact determinations of bound pKa values are limited by the solubility of the enzyme and the dissociation constants of the complexes. The single exception to these general conclusions is the ternary complex of the Ser-27 DHFR with trimethoprim and NADPH. In this complex, both free and enzyme-complexed trimethoprim exhibit similar pKa values (approximately equal to 7.6). However, both the exchange between free and enzyme-complexed inhibitor and the protonation of the enzyme-complexed inhibitor are slow in the NMR time scale, so that the spectra reveal three resonances corresponding to free inhibitor, to protonated enzyme-complexed inhibitor, and to unprotonated enzyme-complexed inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Assignment of the 13C nuclear magnetic resonance spectrum of a short DNA-duplex with 1H-detected two-dimensional heteronuclear correlation spectroscopy.

Proton-detected 1H-13C heteronuclear correlated spectroscopy [( 1H,13C]-COSY) was used to establish relations between the carbon-13 and proton nuclear magnetic resonance chemical shifts in the hexadeoxynucleoside pentaphosphate d-(GCATGC)2. Using the previously established sequence-specific proton NMR assignments, sequence-specific assignments were thus obtained for nearly all proton-bearing carbons. This approach offers a new criterion for distinguishing between the proton NMR lines of purines and pyrimidines, based on the different proton-carbon-13 coupling constants. Furthermore, the adenine ring carbon 2 has a unique carbon-13 chemical shift, which enables a straightforward identification of the adenine C2H resonances by [1H,13C]-COSY.