Unusual deuterium isotope effects in 13C NMR spectra of trans-stilbene

A detailed analysis of the ¹³C NMR spectra of trans-stilbene and ten deuteriated trans-stilbenes has been undertaken. Some unusual deuterium isotope effects on carbon–hydrogen spin–spin coupling constants could not be explained by the ordinary primary and secondary isotope effects. The positive and negative changes of ⁿJ(CH) were interpreted in terms of a steric effect, the vibrational influence of the C? D bond and the para-effect induced by deuterium. In this respect, deuterium behaves as a real substituent with electronic properties different from those of hydrogen. The deuterium isotope effects on ¹³C NMR chemical shifts and carbon–deuterium coupling constants have also been determined.     

Deuterium isotope effects on 13C nuclear shielding as a measure of tautomeric equilibria

Large deuterium isotope effects of both signs have been observed on the ¹³C nuclear shielding in proton-chelating tautomeric forms of β-thioxoketones, and small isotope effects of both signs have been found in related forms of Schiff's bases of salicylaldehydes. These effects are interpreted in terms of shifts in the tautomeric equilibria on deuterium substitution of the enolic proton. The observation of deuterium isotope effects is suggested as a useful method for the detection of fast tautomeric equilibria in systems of the following type. .     

On the models for deuterium long-range isotope effects in13C NMR spectroscopy

The long-range deuterium isotope effects on¹³C nuclear shielding are physically not yet completely understood. Two existing models for explaining these effects, vibrational and substituent, are compared here. The vibrational model is based on the Born-Oppenheimer approximation, but it can explain only one-bond deuterium effects. To the contrary, the substituent model may explain many long-range isotope effects, but it is controversial due to the assumption of some distinct electronic properties of isotopes. We explain how long-range deuterium isotope effects may be rationalized by the subtle electronic changes induced by isotope substitution, which does not violate the Born-Oppenheimer approximation.     

Factor analysis of deuterium isotope effects on 13C NMR chemical shifts in Schiff bases

We have analyzed deuterium isotope effects on (13)C chemical shifts in a series of o-hydroxy Schiff bases by applying factor analysis. Two orthogonal factors were obtained that explain about 80 and 10 % of the variance of the data. The numerical values of these factors can be related to 1H NMR chemical shifts of the proton involved in the intramolecular bonds delta(XH) (X = O or N). Such a relation allows one to identify clusters of compounds with different tautomeric forms of hydrogen bonding. Application of a similar approach to solution 13C NMR chemical shifts produces three important factors, which have a different structure to factors describing isotope effects. This illustrates well the different nature of chemical shifts and isotope effects. The three factors explain about 54, 15, and 13 % of variance. They can be rationalized and are strongly related to the electronic properties and location of substituents.     

pH and pK determinations by high-resolution solid-state 13C NMR: acid-base and tautomeric equilibria of lyophilized L-histidine

Acid-base properties of lyophilized powders of L-histidine have been systematically investigated using parent solutions at pH varying from 1.8 to 10. For the first time, high-resolution solid-state 13C NMR was shown to allow separate observation of all three acid-base pairs in the successive deprotonations of the carboxylic end, the imidazolium cation, and the terminal ammonio group of histidine. 1H CRAMPS NMR spectra directly visualize the absence of the N3-H(pi) tautomer in neutral and anionic species. Solid-state titration shifts are enlarged by approximately 1-4 ppm with respect to those measured in solution, permitting unambiguous observation of conjugate acid-base pairs. Calculated pK's from solid-state acid-to-base ratios r are found equal to those classically measured in solution at 0 degrees C with a similar ionic strength of 0.1 mol x dm(-3). This proves that natural-abundance 13C solid-state determinations of r can be used to measure pK's in parent solutions without recourse to full titration curves and subsequent curve-fitting procedures. Such an approach also leads to noninvasive characterizations of the acidity of lyophilized powders, i.e., to the prediction of in situ pH of products obtained after rehydration and solubilization of powders. These results show the possibility of measuring the pK of nonvolatile acidic substrates dissolved in any sublimable solvent through lyophilization of the investigated solutions; this leads the way to pH and pK determinations when electrochemical or spectrophotometric measurements are impossible or ambiguous, e.g., for concentrated solutions, polyacids, or mixtures of acidic solutes, and possibly to the establishment of pK scales in nonaqueous solvents and in melts.     

13C NMR spectra of benzo[b]thiophene and 1-(X-benzo[b]thienyl)ethyl acetate derivatives

Carbon-13 chemical shift assignments are reported for benzo[b]thiophene and 1-(X-benzo[b]thienyl)ethyl acetate derivatives, where X=? CH(OAc)CH₃ substituted at positions 2-7. Substituent chemical shift (SCS) effects for the ethyl acetate group are additive at all positions. A substantial upfield shift was observed at C-3, arising from the peri interaction of H-3 and the 4-ethyl acetate substituent. Carbon-13 relaxation times (T₁) and nuclear Overhauser enhancements (η) have been measured for benzo[b]thiophene and its derivatives, and the contributions of dipolar, T^DD₁, and spin rotation, T^SR₁, relaxation have been determined. Intramolecular dipole–dipole interactions are found to provide by far the most important spin-lattice relaxation mechanism whenever protons are bound directly to the carbons under investigation. Nonprotonated ring carbons are relaxed by both DD and SR mechanisms. Anisotropic motion has an easily observable effect on the DD contribution to T₁, and can form the basis for spectral assignments, as in 1-phenylethyl acetate. Long-range ¹³C? ¹H coupling constants were observed both between ring carbons and between ring carbons with ring side-chain hydrogens. These results have been used for the structure determination of the title compounds.     

Conformational equilibrium isotope effects in glucose by (13)C NMR spectroscopy and computational studies.

Anomeric equilibrium isotope effects for dissolved sugars are required preludes to understanding isotope effects for these molecules bound to enzymes. This paper presents a full molecule study of the alpha- and beta-anomeric forms of D-glucopyranose in water using deuterium conformational equilibrium isotope effects (CEIE). Using 1D (13)C NMR, we have found deuterium isotope effects of 1.043 +/- 0.004, 1.027 +/- 0.005, 1.027 +/- 0.004, 1.001 +/- 0.003, 1.036 +/- 0.004, and 0.998 +/- 0.004 on the equilibrium constant, (H/D)K(beta/alpha), in [1-(2)H]-, [2-(2)H]-, [3-(2)H]-, [4-(2)H]-, [5-(2)H]-, and [6,6'-(2)H(2)]-labeled sugars, respectively. A computational study of the anomeric equilibrium in glucose using semiempirical and ab initio methods yields values that correlate well with experiment. Natural bond orbital (NBO) analysis of glucose and dihedral rotational equilibrium isotope effects in 2-propanol strongly imply a hyperconjugative mechanism for the isotope effects at H1 and H2. We conclude that the isotope effect at H1 is due to n(p) --> sigma* hyperconjugative transfer from O5 to the axial C1--H1 bond in beta-glucose, while this transfer makes no contribution to the isotope effect at H5. The isotope effect at H2 is due to rotational restriction of OH2 at 160 degrees in the alpha form and 60 degrees in the beta-sugar, with concomitant differences in n --> sigma* hyperconjugative transfer from O2 to CH2. The isotope effects on H3 and H5 result primarily from syn-diaxial steric repulsion between these and the axial anomeric hydroxyl oxygen in alpha-glucose. Therefore, intramolecular effects play an important role in isotopic perturbation of the anomeric equilibrium. The possible role of intermolecular effects is discussed in the context of recent molecular dynamics studies on aqueous glucose.     

Unusually large deuterium isotope effects on one-bond 13C, 1H coupling constants in trans-stilbene challenged

The reported apparent large decrease in ¹J(C-α, H-α) by 1.75±0.20 Hz on replacement of one of the olefinic protons by deuterium in trans-stilbene is due to improper first-order analysis of the ¹H-coupled ¹³C spectrum of the parent compound. Consequently, the implied conformational difference between trans-stilbene and α-deuterio-trans-stilbene, which was used to explain the result, is not substantiated.     

Application of biosynthetic 13C-enrichment using [1-13C]-, [2-13C]- and [1,2-13C2]-acetate as precursor for 13C NMR spectral assignment of higher plant metabolites. The assignments of some bryonolic acid derivatives

The ¹³C NMR spectra of some derivatives of bryonolic acid (1) (D:C-friedoolean-8-en-3β-ol-29-oic acid) were assigned by means of ¹³C-enrichment, lanthanide-induced shifts (LIS) and comparison of chemical shift data between derivatives. The ¹³C-enriched species of 1, i.e., 1a, 1b and 1c were biosynthesized by Luffa cylindrica (Cucurbitaceae) callus fed with [1-¹³C]-, [2-¹³C]- or [1,2-¹³C₂]-acetate, respectively. Methyl acetylbryonolates 2, 2a, 2b and 2c, methyl bryonolates 3, 3a, 3b and 3c, methyl bryononates 4 and 4a, diacetyl-3β,29-diols (3,29-diacetyl-D:C-friedoolean-8-en-β,29-diol) 5, 5a, 5b and 5c, and 3-acetyl-3β,29-diols 6, 6a and 6b were prepared from 1, 1a, 1b and 1c, and their ¹³C NMR spectra were recorded. The ¹³C concentration of the ¹³C-enriched species was high enough to exhibit the satellite peaks clearly, and the analysed data were very useful for this study. Thus, total assignments for 2, 3, 4, 5 and 6 were established. It was found that conversion of the methoxycarbonyl group at C-29 into an acetoxymethyl group caused complex changes in the chemical shifts of the C, D- and E-ring carbons and those of the methyl carbons linked to these rings.     

[13C]Dynamic NMR and Molecular Modeling of 2,4-Bis(N-Pyrrolidinyl)-6-chloro-s-triazine

The room temperature [¹³C]NMR spectrum of 2,4-bis(N-pyrrolidinyl)-6-chloro-s-triazine shows doubled signals for the pyrrolidine rings, which suggests restricted rotation about the Ar-C—N bond. The rotational barrier around this bond was determined by [¹³C]dynamic NMR (DNMR) spectra run at different increasing temperatures and also by the PM3 Hamiltonian contained in the MOPAC package. The values thus obtained, 16.6 and 13.6 kcal mol^–1, respectively, are in good agreement.     

Biosynthesis of the antibiotic verrucarin E use of [1-13C]-, [2-13C]-, [1,2-13C]- and [2-13C, 2-2H3]-acetates. Verrucarins and roridins, 37th communication [1]

The origin of the carbon skeleton of verrucarin E (1) from acetate as precursor is confirmed. Incorporation studies with [1,2-¹³C]-acetate have demonstrated that two acetoacetate units couple together as shown in pattern A (Scheme 2) and not as in B . Analysis of the deuterium distribution in both verrucarin E (1) isolated after the incorporation of [2-¹³C,2-²H₃]-acetate and in sodium acetate obtained after Kuhn-Roth oxidation of the metabolite demonstrated that C(7) is derived from the starter unit of one of the acetoacetate moieties. The deuterium exchange in verrucarin E (1) occurring during fermentation was investigated.     

Deuterium isotope effects on 13C NMR chemical shifts of some α-(2-hydroxyaryl)-N-phenylnitrones (Schiff base N-oxides)

The deuterium isotope effect on the ¹³C NMR chemical shifts of some α-2-hydroxyaryl-N-phenylnitrones (Schiff base N-oxides) was studied. The existence of an intramolecular hydrogen bond with the proton localized on the phenolic oxygen atom was evidenced. Exceptionally large isotope effects ΔC-2(D) and ΔC-α(D) suggest that the substitution of the proton of the OH group by deuterium leads to a weakening of the hydrogen bond and some conformational changes in the molecule. This conclusion was drawn on the basis of a comparison of the deuterium isotope effects of Schiff base N-oxides and parent Schiff bases. Copyright © 2001 John Wiley & Sons, Ltd.     

Probing lactate dehydrogenase activity in tumors by measuring hydrogen/deuterium exchange in hyperpolarized l-[1-(13)C,U-(2)H]lactate

(13)C magnetic resonance spectroscopy and spectroscopic imaging measurements of hyperpolarized (13)C label exchange between exogenously administered [1-(13)C]pyruvate and endogenous lactate, catalyzed by lactate dehydrogenase (LDH), has proved to be a powerful approach for probing tissue metabolism in vivo. This experiment has clinical potential, particularly in oncology, where it could be used to assess tumor grade and response to treatment. A limitation of the method is that pyruvate must be administered in vivo at supra-physiological concentrations. This problem can be avoided by using hyperpolarized [1-(13)C]lactate, which can be used at physiological concentrations. However, sensitivity is limited in this case by the relatively small pyruvate pool size, which would result in only low levels of labeled pyruvate being observed even if there was complete label equilibration between the lactate and pyruvate pools. We demonstrate here a more sensitive method in which a doubly labeled lactate species can be used to measure LDH-catalyzed exchange in vivo. In this experiment exchange of the C2 deuterium label between injected hyperpolarized l-[1-(13)C,U-(2)H]lactate and endogenous unlabeled lactate is observed indirectly by monitoring phase modulation of the spin-coupled hyperpolarized (13)C signal in a heteronuclear (1)H/(13)C spin-echo experiment.     

Microenvironmental and conformational structure of triblock copolymers in aqueous solution by 1H and 13C NMR spectroscopy

1H and 13C nuclear magnetic resonance (NMR) spectra of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) triblock copolymers in D2O solutions have been systematically investigated. The detailed assignments of various 1H and 13C NMR signals are presented. The hyperfine structure of PO -CH2- protons was clearly assigned, the arising reason of this hyperfine structure was attributed to the influence of the chiral center of -CHCH3- groups and the direct coupling between the PO -CH2- and -CH3 protons. The external standard 2,2-dimethyl-2-silapentane-5-sulfonate sodium salt (DSS) was firstly applied in this system. Accurate chemical shift values referenced to the external standard DSS were obtained. 1H NMR chemical shift of PO -CH2- and -CH3 signals shows a larger decrease in ppm values than that of EO -CH2- signal with the increase of PPO/PEO ratio or temperature indicating that PO segments exist in a more hydrophobic microenvironment. A new resonance signal assigned to the PO -CH2- protons appeared when the temperature is above the CMT, which is attributed to the breakdown of the intra-molecular (C-H)...O hydrogen bond between the PO -CH2- protons and the ester oxygens. The breakdown of this intra-molecular hydrogen bond may result in a decrease of gauche conformers of the PPO chain. The increase of 13C NMR chemical shift of block copolymers validates this conformational change assumption. It can be inferred that the amount of gauche conformers decreases whereas that of trans conformers increases in both PO and EO chains when elevating the PPO/PEO ratio or temperature. The observed 13C NMR chemical shifts of PO segments show a bigger increase than those of EO segments, supporting the formation of a nonpolar microenvironment around PO segments.     

13C-labeled N-acetyl-neuraminic acid in aqueous solution: detection and quantification of acyclic keto, keto hydrate, and enol forms by 13C NMR spectroscopy

Aqueous solutions of N-acetyl-neuraminic acid (Neu5Ac, 1) labeled with (13)C at C1, C2, and/or C3 were analyzed by (13)C NMR spectroscopy to detect and quantify the acyclic forms (keto, keto hydrate, enol) present at varying pHs. In addition to pyranoses, solutions contained the keto form, based on the detection of C2 signals at approximately 198 ppm (approximately 0.7% at pH 2). Spectra of [2-(13)C] and [3-(13)C] isotopomers contained signals arising from labeled carbons at approximately 143 and approximately 120 ppm, respectively, which were attributed to enol forms. Solution studies of [1,2,3-(13)C3] 1 substantiated the presence of enol (approximately 0.5% at pH 2). Enol was not detected at pH > 6.0. A C2 signal observed at approximately 94 ppm was identified as C2 of the keto hydrate (approximately 1.9% at pH 2), based partly on its abundance as a function of solution pH. Density functional theory (DFT) calculations were used to study the effect of enol and hydrate structure on J(CH) and J(CC) values involving C2 and C3 of these forms. Solvated DFT calculations showed that (2)J(C2,H3) in cis and trans enols have similar magnitudes but opposite signs, making this J-coupling potentially useful to distinguish enol configurations. Solvent deuterium exchange studies of 1 showed rapid incorporation of (2)H from (2)H2O at H3 axial in the pyranoses at p(2)H 8.0, followed by slower exchange at H3 equatorial. The acyclic keto form, which presumably participates in this reaction, must assume a pseudo-cyclic conformation in solution in order to account for the exchange selectivity. Weak (13)C signals arising from labeled species were also observed consistently and reproducibly in aqueous solutions of (13)C-labeled 1, possibly arising from products of lactonization or intermolecular esterification.     

Long-range deuterium isotope effects on (13)C chemical shifts of intramolecularly hydrogen-bonded N-substituted 3-(cycloamine)thiopropionamides or amides: a case of electric field effects

A series of intramolecularly hydrogen-bonded N-substituted 3-(piperidine, morpholine, N-methylpiperazine)thiopropionamides and some corresponding amides have been studied with special emphasis on hydrogen bonding. The compounds have been selected in order to vary and to minimize the N...N distance. Geometries, charge distributions, and chemical shifts of these compounds are obtained from DFT-type BP3LYP calculations. 1H and 13C 1D and 2D NMR experiments were performed to obtain H,H coupling constants, 13C chemical shifts assignments, and deuterium isotope effects on13C chemical shifts. Variable-temperature NMR studies and 2D exchange NMR spectra have been used to describe the rather complicated conformational behavior mainly governed by the ring flipping of the piperidine (morpholine) rings and intramolecular hydrogen bonding. Unusual long-range deuterium isotope effects on 13C chemical shifts are observed over as far as eight bonds away from the site of deuteriation. The isotope effects are related to the N...N distances, thus being related to the hydrogen bonding and polarization of the N-H bond. Arguments are presented showing that the deuterium isotope effects on 13C chemical shifts originate in electric field effects.     

Rapid 1H[13C]-resolved diffusion and spin-relaxation measurements by NMR spectroscopy

Hadamard-encoded heteronuclear-resolved NMR diffusion and relaxation measurements allow overlapping signal decays to be resolved with substantially shorter measuring times than are generally associated with 2D heteronuclear cross-correlation experiments. Overall measuring time requirements can be reduced by approximately an order of magnitude, compared to typical 2D heteronuclear single-quantum correlation-resolved diffusion or relaxation measurements. Specifically, in cases where chemical shift correlation information provides enhanced spectral resolution, the use of Hadamard encoding can be used to overcome uniqueness challenges that are associated with the analysis of concurrent dynamic processes and the extraction of time constants from overlapping exponential signal decays. This leads to substantially improved resolution of similar time constants than can be achieved solely through the use of post-acquisition processing techniques. In the ideal case of complete spectral separation of the signal decays, the usual constraint that time constants must be sufficiently different to resolve by exponential analysis can be circumvented entirely. Hadamard-based pulse sequences have been used to determine 1H[13C]-resolved diffusion coefficients and spin-relaxation time constants for the chemically similar components of an aqueous solution of ethanol, glycerol, and poly(ethylene glycol), and a dye-containing block-copolymer solution, which exhibit significant spectral overlap in their 1H NMR spectra.     

NMR study on the tautomeric equilibria between the hydrazone imine and diazenyl enamine forms in side chained quinoxalines: Solvent effects and temperature dependence

The reaction of 7-chloro-4-ethoxycarbonylmethylene-4,5-dihydro-1,2,4-triazolo[4,3-a]quinoxaline 6 with 4-ethoxycarbonyl-1-methyl-1H-pyrazole-5-diazonium chloride or 4-cyano-1,3-dimethyl-1H-pyrazole-5-diazonium chloride gave 7-chloro-4-[α-(4-ethoxycarbonyl-1-methyl-1H-pyrazol-5-ylhydrazono)-ethoxycarbonylmethyl]-1,2,4-triazolo[4,3-a]quinoxaline 8a or 7-chloro-4-[α-(4-cyano-1,3-dimethyl-1H-pyrazol-5-ylhydrazono)ethoxycarbonylmethyl]-1,2,4-triazolo[4,3-a]quinoxaline 8b , respectively, while the reaction of 7-chloro-4-ethoxycarbonylmethylene-4,5-dihydrotetrazolo[1,5-a]quinoxaline 7 with 4-ethoxycarbonyl-1-methyl-1H-pyrazole-5-diazonium chloride or 4-cyano-1,3-dimethyl-1H-pyrazole-5-diazomum chloride provided 7-chloro-4-[α-(4-ethoxycarbonyl-1-methyl-1H-pyrazol-5-ylhydrazono)ethoxycarbonylmethyl]tetrazolo[1,5-a]quinoxaline 9a or 7-chloro-4-[α-(4-cyano-1,3-dimethyl-1H-pyrazol-5-ylhydrazono)ethoxycarbonylmethyl]tetrazolo[1,5-a]quinoxaline 9b , respectively. Compounds 8a,b and 9a,b showed the tautomeric equilibria between the hydrazone imine C and diazenyl enamine D forms in dimethyl sulfoxide and/or trifluoroacetic acid, and the effects of solvent and temperature on the tautomer ratios of C to D were studied by the nmr measurements in a series of mixed trifluoroacetic acid/dimethyl sulfoxide media (compounds 8a,b and 9a,b ) and at various temperatures (compounds 8a,b ).     

An NMR relaxation study of hydrogen exchange and its deuterium isotope effects in aqueous carboxylic acid solutions

Exchange-rate measurements of water protons and deuterons in aqueous solutions of acetic, malonic and glutaric acid by the NMR T₂ Carr-Purcell method, employing oxygen-17-enriched water, are reported. In contrast to previous results on acetic-acid solutions, intermolecular proton exchange via solvent between COOH and COO^? groups is found to contribute considerably to the exchange rate for all three acids investigated. The rate constants necessary for the calculation of the proton residence time in the COOH groups have been determined. Observed deuterium isotope effects on the rate constants for hydrogen-ion and acetic-acid-catalyzed exchange are discussed in terms of fractionation-factor theory.