Jacob Schaefer

Magic-angle spinning and polarization transfer in proton-enhanced NMR

Abstract High-speed spinning at the magic angle can significantly modify the rate of polarization transfer from abundant to rare spins in proton-enhanced NMR experiments on solids, if the spinning speed is greater than or comparable to the static dipole-dipole interaction among abundant spins in the rotating frame. In adamantane, this fact can be strikingly demonstrated experimentally. When the spinning speed is much less than dipolar coupling among abundant spins, the effect of spinning on polarization transfer is not dramatic, regardless of the nature of the static dipolar coupling between rare and abundant spins. A semiquantitative theoretical analysis is presented which describes the principal features of these phenomena in terms of the amplitude and frequency modulation of the dipolar interactions between and among the two spin systems. This analysis of the effect of coherent motion (spinning) on polarization transfer also provides some qualitative insight into how incoherent random molecular motion affects transfer in cross-polarization experiments.

Prospects for carbon-13 nuclear magnetic resonance analysis of solid fossil-fuel materials

Abstract Because of excessive line broadening in solids due to magnetic dipole—dipole interactions and chemical shift anisotropies, and because of long spin-lattice relaxation times, standard continuous wave (CW) or pulse Fourier transform techniques do not normally yield structurally informative 13 C n.m.r. spectra from solid samples. However, the techniques of dipolar decoupling, cross polarization and magic-angle spinning show great promise for routine 13 C n.m.r. studies of solids. Applications of these techniques to analysis of anthracite, lignite and algal coal samples, and to oil shale and kerogen samples, are discussed. It is shown that cross-polarization spectra obtained with dipolar decoupling display chemical shift anisotropy which interferes with attempts to distinguish the resonances of aromatic and aliphatic carbons. However, with magic-angle spinning the distinction can be made. Prospects and potential difficulties with applications of these techniques are discussed.

Determination of CN internuclear distances by rotational-echo double-resonance NMR of solids

Abstract Rotational-echo, double-resonance (REDOR) 15 N 13 C NMR has been performed on an alanine cocrystallized from five-component alanines, isotopically enriched in 13 C, 15 N, or 12 C. REDOR 15 N 13 C NMR involves the dephasing of carbon magnetization by 15 N 180° pulses synchronized with magic-angle spinning. The CN dipolar coupling determines the extent of dephasing. The results of these experiments on alanine show that it is practical to use REDOR to measure the CN dipolar coupling of 5 μmol of a 13 C 15 N-labeled pair having an Internuclear separation of the order of 4.5 A.

Comparisons of quadrature and single-phase fourier transform NMR

Abstract Reflections of lines in time-averaged Fourier transform NMR spectra obtained by imperfect quadrature detection can be removed either by 90° rf phase shifting of the excitation pulse together with simple data routing, or by phase and amplitude manipulations of the two imperfect free induction decays after completion of data accumulation. The sensitivity of the resulting reflection-free spectra is comparable to that of spectra obtained by single-phase detection using a rf crystal filter to remove aliased noise. However, since the quadrature experiment involves data sampling rates half as fast as the comparable single-phase experiment, distortions associated with the finiterecovery time of the spectrometer areless severe. Even for fast-recovery spectrometers, asymmetric spectral distortions in Fourier transform experiments involving moderately broad lines can occur. In quadrature detection schemes, these distortions are less severe since they are not folded through the entire spectral width as occurs in single-phase experiments.

Oritavancin Exhibits Dual Mode of Action to Inhibit Cell-Wall Biosynthesis in Staphylococcus aureus

Solid-state NMR measurements performed on intact whole cells of Staphylococcus aureus labeled selectively in vivo have established that des-N-methylleucyl oritavancin (which has antimicrobial activity) binds to the cell-wall peptidoglycan, even though removal of the terminal N-methylleucyl residue destroys the D-Ala-D-Ala binding pocket. By contrast, the des-N-methylleucyl form of vancomycin (which has no antimicrobial activity) does not bind to the cell wall. Solid-state NMR has also determined that oritavancin and vancomycin are comparable inhibitors of transglycosylation, but that oritavancin is a more potent inhibitor of transpeptidation. This combination of effects on cell-wall binding and biosynthesis is interpreted in terms of a recent proposal that oritavancin-like glycopeptides have two cell-wall binding sites: the well-known peptidoglycan D-Ala-D-Ala pentapeptide stem terminus and the pentaglycyl bridging segment. The resulting dual mode of action provides a structural framework for coordinated cell-wall assembly that accounts for the enhanced potency of oritavancin and oritavancin-like analogues against vancomycin-resistant organisms.

Applications of Solids NMR to the Analysis of Insect Sclerotized Structures

Abstract This article reviews the solids NMR research conducted on insect sclerotized structures in the last 10 years and previews some of the experiments that will be conducted in the future. Solids NMR has been used as a noninvasive approach to investigate the chemical compositions of, and some covalent interactions that occur in, several types of sclerotized structures that are otherwise highly intractable to conventional chemical analyses. Sclerotization is a complex process used by insects to confer stability and mechanical versatility to their cuticular exoskeletons and certain other proteinaceous structures. Samples analyzed include cuticular exoskeletons, egg cases, egg shells, cocoons and peritrophic membranes. Cross polarization, dipolar decoupling, magic angle spinning, magnetization dephasing, and isotropic enrichment were used to obtain high resolution spectra that provide information about the types and relative concentrations of carbon atoms as well as internuclear distances and covalent bonds between carbon and nitrogen atoms. Relative amounts of protein, chitin, catechols, lipids, pigment, and oxalate were estimated. Covalent interactions between protein nitrogens and catechol carbons were detected in the stiff brown pupal cuticle of the tobacco hornworm, Manduca sexta . The results of these solids NMR studies support the hypothesis that sclerotization of insect structures occurs primarily when quinones derived from N -acylcatecholamines form cross-links and adducts with functional groups of proteins deposited in the structures. Future applications of solids NMR will utilize advanced techniques for further probing the covalent interactions of 13 C, 15 N and 17 O-labeled catechols, chitin and protein in sclerotized structures.

High-resolution NMR of biological solids.

Solid-state NMR experiments have recently provided a number of biochemical insights: motionally averaged 2H lineshapes have shown that the motion of a backbone loop protecting a protein binding site is not ligand gated; isotropic 13C chemical shifts of freeze-quenched enzyme-ligand intermediates have revealed mechanistic details of reaction pathways; multiple heteronuclear distance determinations have characterized the binding-site geometry of a 46 kDa noncrystalline enzyme complex; and homonuclear recoupling experiments have established that insoluble amyloid fibrils form a pleated beta sheet.

Quantitative determination of the concentrations of 13C15N chemical bonds by double cross-polarization NMR

Methode de determination des liaisons chimiques entre 15 N et 13 C dans des composes doublement marques par ces isotopes en utilisant la double polarisation croisee en RMN

Vancomycin derivative with damaged D-Ala-D-Ala binding cleft binds to cross-linked peptidoglycan in the cell wall of Staphylococcus aureus

Des-N-methylleucyl-4-(4-fluorophenyl)benzyl-vancomycin (DFPBV) retains activity against vancomycin-resistant pathogens despite its damaged d-Ala-d-Ala binding cleft. Using solid-state nuclear magnetic resonance (NMR), a DFPBV binding site in the cell walls of whole cells of Staphylococcus aureus has been identified. The cell walls were labeled with d-[1-(13)C]alanine, [1-(13)C]glycine, and l-[epsilon-(15)N]lysine. Internuclear distances from (19)F of the DFPBV to the (13)C and (15)N labels of the cell-wall peptidoglycan were determined by rotational-echo double-resonance (REDOR) NMR. The (13)C{(19)F} and (15)N{(19)F} REDOR spectra show that, in situ, DFPBV binds to the peptidoglycan as a monomer with its vancosamine hydrophobic side chain positioned near a pentaglycyl bridge. This result suggests that the antimicrobial activity of other vancosamine-modified glycopeptides depends upon both d-Ala-d-Ala stem-terminus recognition (primary binding site) and stem-bridge recognition (secondary binding site).

Vancomycin and Oritavancin Have Different Modes of Action in Enterococcus faecium

The increasing frequency of Enterococcus faecium isolates with multidrug resistance is a serious clinical problem given the severely limited number of therapeutic options available to treat these infections. Oritavancin is a promising new alternative in clinical development that has potent antimicrobial activity against both staphylococcal and enterococcal vancomycin-resistant pathogens. Using solid-state NMR to detect changes in the cell-wall structure and peptidoglycan precursors of whole cells after antibiotic-induced stress, we report that vancomycin and oritavancin have different modes of action in E. faecium. Our results show the accumulation of peptidoglycan precursors after vancomycin treatment, consistent with transglycosylase inhibition, but no measurable difference in cross-linking. In contrast, after oritavancin exposure, we did not observe the accumulation of peptidoglycan precursors. Instead, the number of cross-links is significantly reduced, showing that oritavancin primarily inhibits transpeptidation. We propose that the activity of oritavancin is the result of a secondary binding interaction with the E. faecium peptidoglycan. The hypothesis is supported by results from (13)C{(19)F} rotational-echo double-resonance (REDOR) experiments on whole cells enriched with l-[1-(13)C]lysine and complexed with desleucyl [(19)F]oritavancin. These experiments establish that an oritavancin derivative with a damaged d-Ala-d-Ala binding pocket still binds to E. faecium peptidoglycan. The (13)C{(19)F} REDOR dephasing maximum indicates that the secondary binding site of oritavancin is specific to nascent and template peptidoglycan. We conclude that the inhibition of transpeptidation by oritavancin in E. faecium is the result of the large number of secondary binding sites relative to the number of primary binding sites.