Frequency selective heteronuclear dipolar recoupling in rotating solids: accurate (13)C-(15)N distance measurements in uniformly (13)C,(15)N-labeled peptides

We describe a magic-angle spinning NMR experiment for selective (13)C-(15)N distance measurements in uniformly (13)C,(15)N-labeled solids, where multiple (13)C-(15)N and (13)C-(13)C interactions complicate the accurate measurement of structurally interesting, weak (13)C-(15)N dipolar couplings. The new experiment, termed FSR (frequency selective REDOR), combines the REDOR pulse sequence with a frequency selective spin-echo to recouple a single (13)C-(15)N dipolar interaction in a multiple spin system. Concurrently the remaining (13)C-(15)N dipolar couplings and all (13)C-(13)C scalar couplings to the selected (13)C are suppressed. The (13)C-(15)N coupling of interest is extracted by a least-squares fit of the experimentally observed modulation of the (13)C spin-echo intensity to the analytical expression describing the dipolar dephasing in an isolated heteronuclear spin pair under conventional REDOR. The experiment is demonstrated in three uniformly (13)C,(15)N-labeled model systems: asparagine, N-acetyl-L-Val-L-Leu and N-formyl-L-Met-L-Leu-L-Phe; in N-formyl-[U-(13)C,(15)N]L-Met-L-Leu-L-Phe we have determined a total of 16 internuclear distances in the 2.5-6 A range.     

Boltzmann statistics rotational-echo double-resonance analysis

A new approach to rotational-echo double-resonance (REDOR) data analysis, analogous to Boltzmann maximum entropy statistics, is reported. This Boltzmann statistics REDOR (BS-REDOR) approach is useful for reconstructing an unbiased internuclear distance distribution for multiple internuclear distances from experimentally limited REDOR data sets on isolated spin pairs. The analysis is characterized by exploring reconstructions on model data and applied to both [1-(13)C,15N]-glycine and a long intramolecular distance in Abeta (16-22) peptide nanotubes. The approach also provides insight into the minimal number of REDOR data points required to allow faithful determination of dipolar couplings in systems with multiple internuclear distances.     

Revealing the configuration and crystal packing of organic compounds by solid-state NMR spectroscopy: methoxycarbonylurea, a case study

The molecular configuration and intermolecular arrangement of polycrystalline methoxycarbonylurea (MCU) has been studied by a combination of chemical editing, rotational echo double resonance (REDOR) spectroscopy and ab initio calculations. From the multispin IS(n) REDOR experiments several dipolar couplings were determined and converted into distance constraints. Intra- and intermolecular dipolar couplings were distinguished by isotope dilution. The configuration of the MCU molecule can be determined from three torsion angles Psi1, Psi2, and Psi3. Ab initio calculations showed that these angles are either 0 degrees or 180 degrees (Z or E). From the REDOR experiments, the E configuration was found for Psi1 and Psi2 and the Z configuration for Psi3. Thus the configuration of MCU in the solid state was determined to be EEZ. Distance constraints for the intermolecular arrangement of MCU were obtained by performing REDOR experiments on 13C15N2 MCU with different degrees of isotope dilution and on a cocrystallized 1:1 mixture of 13C(urea) MCU and 15N(amide) MCU. By combining these distance constraints with molecular modeling, three different possible packing motifs for MCU molecules were found. The molecules in these motifs are arranged as linear chains with methoxy groups at the borders of the chains. All the intermolecular hydrogen bond donors and acceptors in the interior of the chain are saturated.     

Plant cell-wall cross-links by REDOR NMR spectroscopy

We present a new method that integrates selective biosynthetic labeling and solid-state NMR detection to identify in situ important protein cross-links in plant cell walls. We have labeled soybean cells by growth in media containing l-[ring-d(4)]tyrosine and l-[ring-4-(13)C]tyrosine, compared whole-cell and cell-wall (13)C CPMAS spectra, and examined intact cell walls using (13)C{(2)H} rotational echo double-resonance (REDOR) solid-state NMR. The proximity of (13)C and (2)H labels shows that 25% of the tyrosines in soybean cell walls are part of isodityrosine cross-links between protein chains. We also used (15)N{(13)C} REDOR of intact cell walls labeled by l-[ε-(15)N,6-(13)C]lysine and depleted in natural-abundance (15)N to establish that the side chains of lysine are not significantly involved in covalent cross-links to proteins or sugars.     

Short polyglutamine peptide forms a high-affinity binding site for thioflavin-T at the N-terminus

Thioflavin-T is one of the most important amyloid specific dyes and has been used for more than 50 years; however, the molecular mechanism of staining is still not understood. Chemically synthesized short polyglutamine peptides (Q(n), n = 5-10) were subjected to the thioflavin-T (ThT) staining assay. It was found that the minimum Q(n) peptide that stained positive to ThT was Q(6). Two types of ThT-binding sites, a high-affinity site (k(d1) = 0.1-0.17 μM) and a low-affinity site (k(d2) = 5.7-7.4 μM), were observed in short polyQs (n = 6-9). (13)C{(2)H}REDOR NMR experiments were carried out to extract the local structure of ThT binding sites in Q(8) peptide aggregates by observing the intermolecular dipolar coupling between [3-Me-d(3)]ThT and natural abundance Q(8) or residue-specific [1,2-(13)C(2)] labeled Q(8)s. (13)C{(2)H}REDOR difference spectra of the [3-Me-d(3)]ThT/natural abundance Q(8) (1/9) complex indicated that all of the five carbons of the glutamine residue participated in the formation of ThT-binding sites. (13)C{(2)H}DQF-REDOR experiments of [3-Me-d(3)]ThT/residue-specific [1,2-(13)C(2)] labeled Q(8) (1/50) complexes demonstrated that the N-terminal glutamine residue had direct contact with the ThT molecule at the high-affinity ThT-binding sites.     

Cross-correlated relaxation enhanced 1H[bond]13C NMR spectroscopy of methyl groups in very high molecular weight proteins and protein complexes

A comparison of HSQC and HMQC pulse schemes for recording (1)H[bond](13)C correlation maps of protonated methyl groups in highly deuterated proteins is presented. It is shown that HMQC correlation maps can be as much as a factor of 3 more sensitive than their HSQC counterparts and that the sensitivity gains result from a TROSY effect that involves cancellation of intra-methyl dipolar relaxation interactions. (1)H[bond](13)C correlation spectra are recorded on U-[(15)N,(2)H], Ile delta 1-[(13)C,(1)H] samples of (i) malate synthase G, a 723 residue protein, at 37 and 5 degrees C, and of (ii) the protease ClpP, comprising 14 identical subunits, each with 193 residues (305 kDa), at 5 degrees C. The high quality of HMQC spectra obtained in short measuring times strongly suggests that methyl groups will be useful probes of structure and dynamics in supramolecular complexes.     

13C/15N distance determination by CPMAS NMR in uniformly 13C labeled molecules

The REDOR and CPMAS techniques are applied for measuring 13C-15N dipolar coupling constants in glycine. It is shown that the selective CP or SPECIFIC CP technique removes the coherent evolution of the spin system under homonuclear 13C-13C J couplings. While the large coupling constant (approximately 900 Hz) is readily determined because of the presence of large oscillations in the CPMAS dynamics, their absence precludes the measurement of the small coupling constant (approximately 200 Hz). The experimental results and numerical simulations demonstrate that the determination of 13C-15N coupling constants of medium size (<1 kHz) by the CPMAS technique is mainly limited by the strength of the 1H decoupling field and the size of the 13C and 15N chemical shift anisotropies.     

Lamellar structure in poly(ala-gly) determined by solid-state NMR and statistical mechanical calculations

Lamellar structure of poly(Ala-Gly) or (AG)n in the solid was examined using 13C solid-state NMR and statistical mechanical approaches. Two doubly labeled versions, [1-13C]Gly14[1-13C]Ala15- and [1-13C]Gly18[1-13C]Ala19 of (AG)15 were examined by two-dimensional (2D) 13C spin diffusion NMR in the solid state. In addition five doubly labeled [15N,13C]-versions of the same peptide, (AG) 15 and 15 versions labeled [3-13C] in each of the successive Ala residues were utilized for REDOR and 13C CP/MAS NMR measurements, respectively. The observed spin diffusion NMR spectra were consistent with a structure containing a combination of distorted beta-turns with a large distribution of the torsion angles and antiparallel beta-sheets. The relative proportion of the distorted beta-turn form was evaluated by examination of 13C CP/MAS NMR spectra of [3-13C]Ala-(AG)15. In addition, REDOR determinations showed five kinds of atomic distances between doubly labeled 13C and 15N nuclei which were also interpreted in terms of a combination of beta-sheets and beta-turns. Our statistical mechanical analysis is in excellent agreement with our Ala Cbeta 13C CP/MAS NMR data strongly suggesting that (AG)15 has a lamellar structure.     

The economical synthesis of [2'-(13)C, 1,3-(15)N2]uridine; preliminary conformational studies by solid state NMR

The synthesis of [2'-(13)C, 1,3-(15)N2]uridine 11 was achieved as follows. An epimeric mixture of D-[1-(13)C]ribose 3 and D-[1-(13)C]arabinose 4 was obtained in excellent yield by condensation of K13CN with D-erythrose 2 using a modification of the Kiliani-Fischer synthesis. Efficient separation of the two aldose epimers was pivotally achieved by a novel ion-exchange (Sm3+) chromatography method. D-[2-(13)C]Ribose 5 was obtained from D-[1-(13)C]arabinose 4 using a Ni(II) diamine complex (nickel chloride plus TEMED). Combination of these procedures in a general cycling manner can lead to the very efficient preparation of specifically labelled 13C-monosaccharides of particular chirality. 15N-labelling was introduced in the preparation of [2'-(13)C, 1,3-(15)N2]uridine 11 via [15N2]urea. Cross polarisation magic angle spinning (CP-MAS) solid-state NMR experiments using rotational echo double resonance (REDOR) were carried out on crystals of the labelled uridine to show that the inter-atomic distance between C-2' and N-1 is closely similar to that calculated from X-ray crystallographic data. The REDOR method will be used now to determine the conformation of bound substrates in the bacterial nucleoside transporters NupC and NupG.     

1H‐19F REDOR‐filtered NMR spin diffusion measurements of domain size in heterogeneous polymers

Solid state NMR spectroscopy is inherently sensitive to chemical structure and composition and thus makes an ideal method to probe the heterogeneity of multicomponent polymers. Specifically, NMR spin diffusion experiments can be used to extract reliable information about spatial domain sizes on multiple length scales, provided that magnetization selection of one domain can be achieved. In this paper, we demonstrate the preferential filtering of protons in fluorinated domains during NMR spin diffusion experiments using ¹H‐¹⁹F heteronuclear dipolar dephasing based on rotational echo double resonance (REDOR) MAS NMR techniques. Three pulse sequence variations are demonstrated based on the different nuclei detected: direct ¹H detection, plus both ¹H?¹³C cross polarization and ¹H?¹⁹F cross polarization detection schemes. This ¹H‐¹⁹F REDOR‐filtered spin diffusion method was used to measure fluorinated domain sizes for a complex polymer blend. The efficacy of the REDOR‐based spin filter does not rely on spin relaxation behavior or chemical shift differences and thus is applicable for performing NMR spin diffusion experiments in samples where traditional magnetization filters may prove unsuccessful. This REDOR‐filtered NMR spin diffusion method can also be extended to other samples where a heteronuclear spin pair exists that is unique to the domain of interest.     

Measurements of carbon to amide-proton distances by C-H dipolar recoupling with 15N NMR detection

A new magic-angle spinning NMR method for measuring internuclear distances between a 13C-labeled site and amide protons is described. The magnetization of the protons evolves under homonuclear decoupling and the recoupled 13C-1H dipolar interaction, which provides simple spin-pair REDOR curves if only one 13C-labeled site is present. The modulation of the amide proton HN is detected via short 1H-15N cross polarization followed by 15N detection. The method is demonstrated on two specifically 13C- and 15N-labeled peptides, with 13C-HN distances from 2.2 to ca. 6 A. This technique promises to be particularly useful for measuring distances between 13C=O and H-15N groups, to identify hydrogen bonds in peptides and proteins.     

Pyrrole-appended derivatives of O-confused oxaporphyrins and their complexes with nickel(II), palladium(II), and silver(III)

Condensation of 2,4-bis(phenylhydroxymethyl)furan with pyrrole and p-toluylaldehyde formed, instead of the expected 5,20-diphenyl-10,15-di(p-tolyl)-2-oxa-21-carbaporphyrin, a pyrrole addition product [(H,pyr)OCPH]H(2); this product can formally be considered as an effect of hydrogenation of 3-(2'-pyrrolyl)-5,20-diphenyl-10,15-di(p-tolyl)-2-oxa-21-carbaporphyrin ([(pyr)OCPH]H). The new oxacarbaporphyrinoid presents the (1)H NMR spectroscopy features of an aromatic molecule, including the upfield shift of the inner H21 atom. Insertion of NiCl(2) or PdCl(2) into [(H,pyr)OCPH]H(2) gave two structurally related organometallic complexes, [(pyr)OCP]Ni(II)] and [(pyr)OCP]Pd(II)], in which the metal ions are bound by three pyrrolic nitrogens and the trigonally hybridized C21 atom of the inverted furan. The reaction of [(H,pyr)OCPH]H(2) with silver(I) acetate yields a stable Ag(III) complex [(C(2)H(5)O,pyr)OCP]Ag(III)] substituted at the C3 position by the ethoxy and pyrrole moieties. The macrocyclic frame of [(H,pyr)OCPH]H(2) is conserved. Addition of trifluoroacetic acid to [(C(2)H(5)O,pyr)OCP]Ag(III)] yielded a new aromatic complex [(pyr)OCP]Ag(III)](+). The structures of [(pyr)OCP]Ni(II)] and [(C(2)H(5)O,pyr)OCP]Ag(III)] have been determined by X-ray crystallography. In both molecules the macrocycles are only slightly distorted from planarity and the nickel(II) and silver(III) are located in the NNNC plane. The dihedral angle between the macrocyclic and appended-pyrrole planes of [(pyr)OCP]Ni(II)] reflects the biphenyl-like arrangement with the NH group pointing out toward the adjacent phenyl ring on the C5 position. Tetrahedral geometry around the C3 atom was detected for [(C(2)H(5)O,pyr)OCP]Ag(III)]. The Ni[bond]C and Ag[bond]C bond lengths are similar to other nickel(II) or silver(III) carbaporphyrinoids where the trigonal carbon atom coordinates the metal ion. The trend detected in the (13)C chemical shifts for the appended-pyrrole resonances has been rationalized by the extent of effective conjugation between the macrocycle and the appended pyrrole moiety controlled by the hybridization of the C3 atom and the metal ion oxidation state. The dianionic or trianionic macrocyclic core of the pyrrole-appended derivatives is favored to match the oxidation state of nickel(II), palladium(II), or silver(III), respectively.     

A streamlined approach to the analysis of volatile fatty acids and its application to the measurement of whole-body flux

Volatile fatty acids (VFAs) are produced in the human colon by the bacterial breakdown of carbohydrates that escape digestion and absorption in the small intestine. They have important local and systemic effects on gastrointestinal and nutritional functions. Measuring their production is difficult because of inaccessibility of sampling sites and low circulating concentrations. Stable isotope tracer techniques are a way to measure VFA production but require measurement of isotope dilution in blood and other biological fluids. We have developed a streamlined and robust method to measure the concentration and enrichment of [(2)H]-labelled VFAs by gas chromatography/mass spectrometry (GC/MS) and [(13)C]-labelled VFAs by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). Both types of analysis were carried out on the same samples allowing multiple tracer studies to be conducted. Good accuracy and repeatability were found for GC/MS analysis of [(2)H]-labelled VFAs. Careful handling of the background contribution, especially acetate, allowed quantitation of concentration and enrichment within the analysis. GC/C/IRMS analysis of [(13)C] VFAs was also achieved with good accuracy and repeatability. This methodology was used to determine whole-body acetate production in two subjects using multiple tracers ([(2)H(3)]- and [1-(13)C]acetate) and blood and urine sampling. Whole-body acetate flux was similar when measured either with [(2)H(3)]- or [1-(13)C]acetate, and when flux was determined from plasma or urine tracer enrichment. This new method will permit rapid and accurate measurement of VFA flux using [(2)H]- and/or [(13)C]-labelled VFAs as tracers. Measurements of the contribution of colonic VFA production to whole-body VFA flux are now possible.     

Determining dihedral angles and local structure in silk peptide by 13C-2H REDOR

13C-2H REDOR NMR experiments were performed on 30-residue (AlaGly)15 silk I mimics of Bombyx mori silk fibroin to gain structural details about the elusive structure of the silk I conformation. 13C,2H-labeling strategies are illustrated for measuring individual dihedral angles in peptides and for determining local structure by REDOR. A major turn of type II character is found in the region Gly(14)-Ala(17).     

Synthesis and testing of a p-H2 hyperpolarized 13C probe based on the pyrazolo[1,5-a]pyrimidineacetamide DPA-713, an MRI vector to target the peripheral benzodiazepine receptors

DPA-713 is the lead compound of a recently developed 2-phenylpyrazolo[1,5-a]pyrimidineacetamide series that has been shown to display a good targeting capability toward peripheral benzodiazepine receptors, recently renamed translocator protein (18 kDa) or in short TSPO. On the basis of this structure, a novel derivative bearing a [(13)C]butynoate moiety has been designed and synthesized (three steps-42% overall yield) providing, upon rapid and quantitative para-hydrogenation, the corresponding hyperpolarized [(13)C]alkene. Para-hydrogen-induced polarization effects have been detected in both (1)H and (13)C-NMR spectra. Upon applying a field cycling procedure, the spin order of para-H(2) added hydrogens is transferred on the (13)C carboxylate moiety yielding a signal enhancement of approximately 4500 times. T(1) of the carboxylate carbon atom is approximately 21.9 s (at 9.37 T). A (13)C-MR image has been acquired by using the (13)C RARE (Rapid Acquisition by Relaxation Enhancement) acquisition protocol on a 10-mM solution. The main limitation to the in vivo use of this novel para-hydrogenated [(13)C]derivative is its relatively low solubility in aqueous systems.     

Metallaboranes of the Early Transition Metals: Direct Synthesis and Characterization of [{(eta5-C5Me5)Ta}2BnHm] (n=4, m=10; n=5, m=11), [{(eta5-C5Me5)Ta}2B5H10(C6H4CH3)], and [{(eta5-C5Me5)TaCl}2B5H11

Reaction of [Cp*TaCl4] (Cp*=eta5-C5Me5) with a sixfold excess of LiBH(4)thf followed by BH3thf in toluene at 100 degrees C led to the isolation of hydrogen-rich metallaboranes [(Cp*Ta)2B4H10] (1), [(Cp*Ta)2B5H11] (2), [(Cp*Ta)2B5H10(C6H4CH3)] (3), and [(Cp*TaCl)2B5H11] (4) in modest yield. Compounds 1-3 are air- and moisture-sensitive but 4 is reasonably stable in air. Their structures are predicted by the electron-counting rules to be a bicapped tetrahedron (1), bicapped trigonal bipyramids (2, 3), and a nido structure based on a closo dodecahedron 4. Yellow tantalaborane 1 has a nido geometry with C2v symmetry and is isostructural with [(Cp*M)2B4H8] (M=Cr and Re); whereas 2 and 3 are C3v-symmetric and isostructural with [(Cp*M)2B5H9] (M=Cr, Mo, W) and [(Cp*ReH)2B5Cl5]. The most remarkable feature of 4 is the presence of a hydride ligand bridging the ditantalum center to form a symmetrical tantalaborane cluster with a long Ta--Ta bond (3.22 A). Cluster 4 is a rare example of electronically unsaturated metallaborane containing four TaHB bonds. All these new metallaboranes have been characterized by mass spectrometry, 1H, 11B, and 13C NMR spectroscopy, and elemental analysis, and the structural types were unequivocally established by crystallographic analysis of 1-4.     

Homonuclear and heteronuclear NMR studies of a statherin fragment bound to hydroxyapatite crystals

Acidic proteins found in mineralized tissues act as nature's crystal engineers, where they play a key role in promoting or inhibiting the growth of minerals such as hydroxyapatite (HAP), Ca10(PO4)6(OH)2, the main mineral component of bone and teeth. Key to understanding the structural basis of protein-crystal recognition and protein control of hard tissue growth is the nature of interactions between the protein side chains and the crystal surface. In an earlier work we have measured the proximity of the lysine (K6) side chain in an SN-15 peptide fragment of the salivary protein statherin adsorbed to the Phosphorus-rich surface of HAP using solid-state NMR recoupling experiments. 15N{31P} rotational echo double resonance (REDOR) NMR data on the side-chain nitrogen in K6 gave rise to three different models of protein-surface interaction to explain the experimental data acquired. In this work we extend the analysis of the REDOR data by examining the contribution of interactions between surface phosphorus atoms to the observed 15N REDOR decay. We performed 31P-31P recoupling experiments in HAP and (NH4)2HPO4 (DHP) to explore the nature of dipolar coupled 31P spin networks. These studies indicate that extensive networks of dipolar coupled 31P spins can be represented as stronger effective dipolar couplings, the existence of which must be included in the analysis of REDOR data. We carried out 15N{31P} REDOR in the case of DHP to determine how the size of the dephasing spin network influences the interpretation of the REDOR data. Although use of an extended 31P coupled spin network simulates the REDOR data well, a simplified 31P dephasing system composed of two spins with a larger dipolar coupling also simulates the REDOR data and only perturbs the heteronuclear couplings very slightly. The 31P-31P dipolar couplings between phosphorus nuclei in HAP can be replaced by an effective dipolar interaction of 600 Hz between two 31P spins. We incorporated this coupling and applied the above approach to reanalyze the 15N{31P} REDOR of the lysine side chain approaching the HAP surface and have refined the binding models proposed earlier. We obtain 15N-31P distances between 3.3 and 5 A from these models that are indicative of the possibility of a lysine-phosphate hydrogen bond.     

X–H rotational-echo double-resonance NMR for torsion angle determination of peptides

C–H and N–H rotational-echo double-resonance (REDOR) NMR is developed for determining torsion angles in peptides. The distance between an X spin such as ¹³C or ¹⁵N and a proton is measured by evolving the proton magnetization under REDOR-recoupled X–H dipolar interaction. The proton of interest is selected through its directly bonded heteronuclear spin Y. The sidechain torsion angle χ₁ is extracted from a ¹³Cβ-detected Hβ–N distance, while the backbone torsion angle φ is extracted from an ¹⁵N-detected H^N–C^′ distance. The approach is demonstrated on three model peptides with known crystal structures to illustrate its utility.     

Progress in 13C and 1H solid-state nuclear magnetic resonance for paramagnetic systems under very fast magic angle spinning

High-resolution solid-state NMR (SSNMR) of paramagnetic systems has been largely unexplored because of various technical difficulties due to large hyperfine shifts, which have limited the success of previous studies through depressed sensitivity/resolution and lack of suitable assignment methods. Our group recently introduced an approach using "very fast" magic angle spinning (VFMAS) for SSNMR of paramagnetic systems, which opened an avenue toward routine analyses of small paramagnetic systems by (13)C and (1)H SSNMR [Y. Ishii et al., J. Am. Chem. Soc. 125, 3438 (2003); N. P. Wickramasinghe et al., ibid. 127, 5796 (2005)]. In this review, we discuss our recent progress in establishing this approach, which offers solutions to a series of problems associated with large hyperfine shifts. First, we demonstrate that MAS at a spinning speed of 20 kHz or higher greatly improves sensitivity and resolution in both (1)H and (13)C SSNMR for paramagnetic systems such as Cu(II)(DL-alanine)(2)H(2)O (Cu(DL-Ala)(2)) and Mn(acac)(3), for which the spectral dispersions due to (1)H hyperfine shifts reach 200 and 700 ppm, respectively. Then, we introduce polarization transfer methods from (1)H spins to (13)C spins with high-power cross polarization and dipolar insensitive nuclei enhanced by polarization transfer (INEPT) in order to attain further sensitivity enhancement and to correlate (1)H and (13)C spins in two-dimensional (2D) SSNMR for the paramagnetic systems. Comparison of (13)C VFMAS SSNMR spectra with (13)C solution NMR spectra revealed superior sensitivity in SSNMR for Cu(DL-Ala)(2), Cu(Gly)(2), and V(acac)(3). We discuss signal assignment methods using one-dimensional (1D) (13)C SSNMR (13)C-(1)H rotational echo double resonance (REDOR) and dipolar INEPT methods and 2D (13)C(1)H correlation SSNMR under VFMAS, which yield reliable assignments of (1)H and (13)C resonances for Cu(Ala-Thr). Based on the excellent sensitivity/resolution and signal assignments attained in the VFMAS approach, we discuss methods of elucidating multiple distance constraints in unlabeled paramagnetic systems by combing simple measurements of (13)C T(1) values and anisotropic hyperfine shifts. Comparison of experimental (13)C hyperfine shifts and ab initio calculated shifts for alpha- and beta-forms of Cu(8-quinolinol)(2) demonstrates that (13)C hyperfine shifts are parameters exceptionally sensitive to small structural difference between the two polymorphs. Finally, we discuss sensitivity enhancement with paramagnetic ion doping in (13)C SSNMR of nonparamagnetic proteins in microcrystals. Fast recycling with exceptionally short recycle delays matched to short (1)H T(1) of approximately 60 ms in the presence of Cu(II) doping accelerated 1D (13)C SSNMR for ubiquitin and lysozyme by a factor of 7.3-8.4 under fast MAS at a spinning speed of 40 kHz. It is likely that the VFMAS approach and use of paramagnetic interactions are applicable to a variety of paramagnetic systems and nonparamagnetic biomolecules.     

Isotope effect-mapping of the ionization of glucose demonstrates unusual charge sharing

Isotopic substitution is known to affect kinetic rate constants and equilibrium constants in chemistry. In this study, we have used tritium substitution and high pH to probe the glucose left harpoon over right harpoon glucose(-) + H(+) equilibrium. Passing partially ionized mixtures of [(3)H]- and [(14)C]glucose over anionic exchange resin has permitted the detection of subtle differences in pK(a). We have found that, at pH 11.7 in an anionic exchange system, [(3)H]glucose always elutes ahead of the [(14)C]glucose, and we report isotope effects of 1.051 +/- 0.0007, 1.012 +/- 1.0005, 1.014 +/- 0.0004, 1.024 +/- 0.0003, 1.014 +/- 0.0004, and 1.015 +/- 0.0014 for [1-(3)H]-, [2-(3)H]-, [3-(3)H]-, [4-(3)H]-, [5-(3)H]-, and [6,6-(3)H(2)]glucose, respectively, as compared to either [2-(14)C]-or [6-(14)C]glucose. The elevated isotope effects at H1 and H4 imply unusual charge sharing in anionic aqueous glucose. Base titration of (13)C-chemical shift changes demonstrates the dominance of pyranose forms at elevated pH, and ab initio isotope effect computations on gas-phase glucose anions are presented.