Correlation NMR Spectra Research Articles

Effects of Macromolecular Cosolutes on the Kinetics of Huntingtin Aggregation Monitored by NMR Spectroscopy.

The effects of two macromolecular cosolutes, specifically the polysaccharide dextran-20 and the protein lysozyme, on the aggregation kinetics of a pathogenic huntingtin exon-1 protein (hhtex1) with a 35 polyglutamine repeat, httex1Q35, are described. A unified kinetic model that establishes a direct connection between reversible tetramerization occurring on the microsecond time scale and irreversible fibril formation on a time scale of hours/days forms the basis for quantitative analysis of httex1Q35 aggregation, monitored by measuring cross-peak intensities in a series of 2D 1H-15N NMR correlation spectra acquired during the course of aggregation. The primary effects of the two cosolutes are associated with shifts in the prenucleation tetramerization equilibrium resulting in substantial changes in concentration of "preformed" httex1Q35 tetramers. Similar effects of the two cosolutes on the tetramerization equilibrium observed for a shorter, nonaggregating huntingtin variant with a 7-glutamine repeat, httex1Q7, lend confidence to the conclusions drawn from the fits to the httex1Q35 aggregation kinetics.

Labeling of methyl groups: a streamlined protocol and guidance for the selection of 2H precursors based on molecular weight.

A streamlined one-day protocol is described to produce isotopically methyl-labeled protein with high levels of deuterium for NMR studies. Using this protocol, the D2O and 2H-glucose content of the media and protonation level of ILV labeling precursors (ketobutyrate and ketovalerate) were varied. The relaxation rate of the multiple-quantum (MQ) state that is present during the HMQC-TROSY pulse sequence was measured for different labeling schemes and this rate was used to predict upper limits of molecular weights for various labeling schemes. The use of deuterated solvents (D2O) or deuterated glucose is not required to obtain 1H-13C correlated NMR spectra of a 50kDa homodimeric protein that are suitable for assignment by mutagenesis. High quality spectra of 100-150kDa proteins, suitable for most applications, can be obtained without the use of deuterated glucose. The proton on the β-position of ketovalerate appears to undergo partial exchange with deuterium under the growth conditions used in this study.

Ionic salt cocrystals studied via multinuclear solid-state magnetic resonance: a case study of lithium 4-methoxybenzoate:L-proline polymorphs

Lithium salts continue to find pharmaceutical applications, particularly as psychiatric medications. As with any active pharmaceutical ingredient, structural polymorphism is an important concern for lithium-based medications that can influence solubility and other physicochemical properties. Here we report a 13C, 1H, and 7Li magic-angle spinning solid-state nuclear magnetic resonance (MAS SSNMR) study of two 1:1 polymorphic ionic cocrystals of lithium 4-methoxybenzoate and L-proline (L4MPRO(α) and L4MPRO(β)). One-dimensional 13C cross-polarization MAS and two-dimensional heteronuclear correlation NMR spectra hint at differential mobilities of the proline and benzoate moieties for the two polymorphs. Five key resonances differ in 13C chemical shift by more than 1 ppm between the two polymorphs, clearly distinguishing between them. Gauge-including projector-augmented-wave density functional theory calculations of 13C and 1H magnetic shielding constants correlate strongly with the experimental chemical shifts for both polymorphs. R2 and root-mean-square deviation metrics are shown to be insufficient in the case of 13C, but sufficient in the case of 1H, for differentiating between the polymorphs. 7Li satellite-transition MAS NMR of both polymorphs are identical, as are the computed lithium magnetic shielding constants, demonstrating the insensitivity of 7Li NMR to polymorphism in these samples. This work highlights the utility of solid-state NMR spectroscopy for examining ionic salt cocrystals and also highlights some caveats in this regard.

Hydroxycinnamaldehyde-derived benzofuran components in lignins.

Lignin is an abundant polymer in plant secondary cell walls. Prototypical lignins derive from the polymerization of monolignols (hydroxycinnamyl alcohols), mainly coniferyl and sinapyl alcohol, via combinatorial radical coupling reactions and primarily via the endwise coupling of a monomer with the phenolic end of the growing polymer. Hydroxycinnamaldehyde units have long been recognized as minor components of lignins. In plants deficient in cinnamyl alcohol dehydrogenase, the last enzyme in the monolignol biosynthesis pathway that reduces hydroxycinnamaldehydes to monolignols, chain-incorporated aldehyde unit levels are elevated. The nature and relative levels of aldehyde components in lignins can be determined from their distinct and dispersed correlations in 2D 1H-13C-correlated nuclear magnetic resonance (NMR) spectra. We recently became aware of aldehyde NMR peaks, well resolved from others, that had been overlooked. NMR of isolated low-molecular-weight oligomers from biomimetic radical coupling reactions involving coniferaldehyde revealed that the correlation peaks belonged to hydroxycinnamaldehyde-derived benzofuran moieties. Coniferaldehyde 8-5-coupling initially produces the expected phenylcoumaran structures, but the derived phenolic radicals undergo preferential disproportionation rather than radical coupling to extend the growing polymer. As a result, the hydroxycinnamaldehyde-derived phenylcoumaran units are difficult to detect in lignins, but the benzofurans are now readily observed by their distinct and dispersed correlations in the aldehyde region of NMR spectra from any lignin or monolignol dehydrogenation polymer. Hydroxycinnamaldehydes that are coupled to coniferaldehyde can be distinguished from those coupled with a generic guaiacyl end-unit. These benzofuran peaks may now be annotated and reported and their structural ramifications further studied.

Boron Adsorption Kinetics of Microcrystalline Cellulose and Polymer Resin

Tailoring boron-polysaccharide interactions is an important strategy for developing functional soft materials such as hydrogels, fire retardants, and sorbents for environmental remediation, for example, using lignocellulosic biomass. For such applications to be realized, it is paramount to understand the adsorption kinetics of borate anions on cellulose and their local structures. Here, the kinetic aspects of boron adsorption by microcrystalline cellulose, lignin, and polymeric resin are investigated and compared. Borate anions interact with the vicinal diols in the glucopyranoside moieties of cellulose to yield chemisorbed boron chelate complexes. In contrast to cellulose, technical lignin contains fewer cis-vicinal diols, and it does not have a tendency to form such chelate complexes upon treatment with the aqueous boric acid solution. The formation kinetics and stability of these chelate complexes strongly depend on nanoscale structures, as well as reaction conditions such as pH and concentration of the sorbate and sorbent. Specifically, insights into the distinct boron adsorption sites were obtained by solid-state one-dimensional (1D) 11B magic-angle spinning NMR and the local structures and intermolecular interactions in the vicinities of boron chelate complexes are elucidated by analyzing two-dimensional (2D) 1H-13C and 11B-1H heteronuclear correlation NMR spectra. The total boron adsorption capacity of cellulose is estimated to be in the 1.3-3.0 mg range per gram of sorbent, which is lower than the boron adsorption capacity of a polystyrene-based resin, ∼17.2 mg of boron per gram of Amberlite IRA 743. Our study demonstrates that the local backbone and side chain flexibility as well as the structures of polyol groups play a significant role in determining the kinetic and thermodynamic stability of chelate complexes, yielding to different boron adsorption capabilities of lignocellulosic polymers.

An Atomistic Picture of Boron Oxide Catalysts for Oxidative Dehydrogenation Revealed by Ultrahigh Field 11B-17O Solid-State NMR Spectroscopy.

Boron oxide/hydroxide supported on oxidized activated carbon (B/OAC) was shown to be an inexpensive catalyst for the oxidative dehydrogenation (ODH) of propane that offers activity and selectivity comparable to boron nitride. Here, we obtain an atomistic picture of the boron oxide/hydroxide layer in B/OAC by using 35.2 T 11B and 17O solid-state NMR experiments. NMR spectra measured at 35.2 T resolve the boron and oxygen sites due to narrowing of the central-transition powder patterns. A 35.2 T 2D 11B{17O} dipolar heteronuclear correlation NMR spectrum revealed the structural connectivity between boron and oxygen atoms. The approach outlined here should be generally applicable to determine atomistic structures of heterogeneous catalysts containing quadrupolar nuclei.

Complete resonance assignment of a pharmaceutical drug at natural isotopic abundance from DNP-Enhanced solid-state NMR

Solid-state dynamic nuclear polarization enhanced magic angle spinning (DNP-MAS) NMR measurements coupled with density functional theory (DFT) calculations enable the full resonance assignment of a complex pharmaceutical drug molecule without the need for isotopic enrichment. DNP dramatically enhances the NMR signals, thereby making possible previously intractable two-dimensional correlation NMR spectra at natural abundance. Using inputs from DFT calculations, herein we describe a significant improvement to the structure elucidation process for complex organic molecules. Further, we demonstrate that a series of two-dimensional correlation experiments, including 15N–13C TEDOR, 13C–13C INADEQUATE/SARCOSY, 19F–13C HETCOR, and 1H–13C HETCOR, can be obtained at natural isotopic abundance within reasonable experiment times, thus enabling a complete resonance assignment of sitagliptin, a pharmaceutical used for the treatment of type 2 diabetes.

Pulse induced resonance with angular dependent total enhancement of multi-dimensional solid-state NMR correlation spectra

We demonstrate a new resonance condition that obeys the relation Δδ=nνR/2, where Δδ is the chemical shift difference between two homonuclear-coupled spins, νR is the magic-angle spinning speed and n is an integer. This modulation on the rotational resonance recoupling condition is obtained by the application of rotor-synchronous 1H pulses when at least one proton is dipolar-coupled to one of the homonuclear spins. We suggest a new experimental scheme entitled ‘pulse induced resonance with angular dependent total enhancement’ (PIRATE) that can enhance proton-driven spin diffusion by the application of a single 1H pulse every rotor period. Experimental evidence is demonstrated on the two carbon spins of glycine and on the Y21M mutant of fd bacteriophage virus. Numerical simulations reveal the existence of the resonances and report on the important interactions governing these phenomena.

Metabolic composition and authenticity evaluation of bergamot essential oil assessed by nuclear magnetic resonance spectroscopy.

In this work, a sample of pure and certified bergamot essential oil (BEO) was extensively studied for the first time directly by NMR spectroscopy with the aim of investigating its metabolic composition, quantifying the main components of this complex natural matrix and simultaneously assessing whether the NMR technique is able to highlight possible frauds to which this high-cost product may be subjected. Eleven low molecular weight compounds have been identified by using 1D 1H and 13C-{1H} NMR experiments, 2D homo- and heteronuclear correlation NMR spectra, and 2D 1H DOSY experiments; the most abundant of them, i.e., about 90% of the sample analyzed, has been quantified by employing benzoic acid as an internal standard by 1H NMR spectrum. Moreover, since the commercial fraud of this precious oil is often due to the addition of less expensive oils, we have simulated a possible adulteration through the preparation of BEO samples to which different percentages of orange essential oil (OEO) were added. The results, obtained by combining the 1H NMR spectra collected on the adulterated samples and on pure BEO, with chemometric analysis, principal component analysis (PCA), indicate that it is possible to distinguish the sample of pure BEO from the adulterated ones and also, among them, to differentiate between the degrees of adulteration.

Compositions, Structures, and Properties of Mesoporous-Fe,N-Containing Carbon Materials for Oxygen- and Sulfur-Reduction Electrocatalysts

Mesoporous N- and Fe,N-carbon materials, prepared by polymerization of small molecule precursors and subsequent pyrolysis, exhibit high and accessible surface areas, high electrical conductivities, and high electrocatalytic activities that are comparable to more expensive activated-carbon-supported precious-metal (Pt) fuel cell electrocatalysts. Their favorable properties are due to a combination of factors, including high nitrogen contents that increase the number of active moieties, selective formation of certain types of N moieties at surface sites, relatively large (3 nm) mesopores to promote facile diffusion, and sufficient electron conductivity to surface N environments where the reactions occur. The resulting materials exhibit electrocatalytic properties that are comparable to a standard catalyst containing 20 wt% Pt supported on activated carbon. This is evident in Figure 1a, which shows current-voltage plots associated with the reduction of oxygen for 20 wt% Pt-C and for various N- and Fe,N-mesoporous carbon materials that were prepared identically except for the use of different mesostructured porogen templates. These results show that the oxygen reduction activities of the mesoporous N-carbons are significantly higher when a ZnCl2/NaCl salt mixture is used to produce mesoporosity, compared to mesoporous silica. In addition, the incorporation of a small amount (0.5 wt%) of Fe in the mesoporous N-carbon greatly improves oxygen reduction reactivity, which is shown to be superior to the commercial 20 wt% Pt-C. Similarly favorable activities and trends are observed for these materials as electrocatalysts for sulfur reduction.While the inclusion of N heteroatoms has previously been demonstrated to improve the reduction activities of carbon-based electrocatalysts,1 the atomic-level origins of such properties have remained elusive. This has been due to the multicomponent and non-stoichiometric compositions of these porous graphitic solids, their complicated distributions of disordered and ordered regions, and their paramagnetic and/or conductive properties. Nevertheless, advanced solid-state NMR techniques, in combination with Raman spectroscopy, X-ray diffraction, electron microscopy, and DFT modeling, yield detailed new insights on the atomic environments and interactions of N heteroatoms in mesoporous N-carbon electrocatalysts.2 Most importantly, two-dimensional (2D) 13C-15N correlation NMR spectra resolve distinct signals that enable the types and distributions of different N heteroatom environments to be established and correlated with electrocatalytic properties. For example, as shown in Figure 1b, the distributions of correlated of 15N and 13C signals in the 2D contour-plot spectrum of the salt-templated mesoporous N-carbon material manifest at least four distinct types of N-heteroatom environments: pyrrolic (purple), graphitic (blue), edge/isolated pyridinic (yellow), and pyrazinic/pyridinic (red) moieties. Green-shaded regions indicate correlated signals that arise from atomically proximate (<1 nm) moieties. These assignments are corroborated by DFT calculations to determine the chemical shieldings of different N-carbon moieties, which are shown as points overlaying the experimental 2D spectrum. Similar 2D spectra acquired for silica-templated mesoporous N-carbons yield different quantities and distributions of these N-carbon moieties, which account for the significantly different (lower) macroscopic oxygen reduction activities (Figure 1a). Furthermore, 1D 15N{1H} CP-MAS NMR spectra enable surface N species to be selectively distinguished from interior moieties (e.g., graphitic N) by the formers’ interactions with adsorbed water. In combination, these powerful multi-nuclear NMR analyses unambiguously establish that certain types of N-carbon moieties are more important to electrocatalytic performance than others.Incorporation of non-precious transition metals, such as Fe, significantly increases electrocatalytic activity.3,4 This is similarly the case for mesoporous N-carbon materials investigated here that were synthesized under otherwise identical conditions, except for the addition of a small (0.5 wt%) Fe (Figure 1a). The important role(s) of Fe, however, are challenging to establish, due to its generally dilute loading and its paramagnetic (and possibly ferromagnetic) properties. Nevertheless, 57Fe Mössbauer spectra, solid-state 15N NMR spectra, and 15N spin-lattice (T 1) relaxation time analysesresolve 15N signals from 15N species that are proximate to paramagnetic Fe heteroatoms, from which 15N-Fe distances are estimated. These analyses provide detailed new insights into the types, atomic environments, and distributions of 15N heteroatoms in mesoporous N- and Fe,N-carbon materials, which, until now, have been infeasible to distinguish by scattering or other spectroscopic techniques. Understanding the roles of these moieties in the catalytic reduction of oxygen or sulfur is expected to yield new design criteria for syntheses of high performance non-precious-metal electrocatalysts for diverse fuel cell and battery applications. References (1) Gong, K.; et al. Science 2009, 323, 760–764.(2) Becwar, S. M.; et al. submitted.(3) Donghun, K.; et al. ACS Appl. Mater. Interfaces 2018, 10, 25337−25349.(3) Al-Zoubi, T.; et al. J. Am. Chem. Soc. 2020, 142, 5477–5481. Figure 1

Structure Determination of Boron-Based Oxidative Dehydrogenation Heterogeneous Catalysts with Ultra-High Field 35.2 T 11B Solid-State NMR Spectroscopy.

Boron-based heterogenous catalysts, such as hexagonal boron nitride (h-BN) as well as supported boron oxides, are highly selective catalysts for the oxidative dehydrogenation (ODH) of light alkanes to olefins. Previous catalytic measurements and molecular characterization of boron-based catalysts by 11B solid-state NMR spectroscopy and other techniques suggests that oxidized/hydrolyzed boron clusters are the catalytically active sites for ODH. However, 11B solid-state NMR spectroscopy often suffers from limited resolution because boron-11 is an I = 3/2 half-integer quadrupolar nucleus. Here, ultra-high magnetic field (B 0 = 35.2 T) is used to enhance the resolution of 11B solid-state NMR spectra and unambiguously determine the local structure and connectivity of boron species in h-BN nanotubes used as a ODH catalyst (spent h-BNNT), boron substituted MCM-22 zeolite [B-MWW] and silica supported boron oxide [B/SiO2] before and after use as an ODH catalyst. One-dimensional direct excitation 11B NMR spectra recorded at B 0 = 35.2 T are near isotropic in nature, allowing for the easy identification of all boron species. Two-dimensional 1H-11B heteronuclear correlation NMR spectra aid in the identification of boron species with B-OH functionality. Most importantly, 2D 11B dipolar double-quantum single-quantum homonuclear correlation NMR experiments were used to unambiguously probe boron-boron connectivity within all heterogeneous catalysts. These experiments are practically infeasible at lower, more conventional magnetic fields due to a lack of resolution and reduced NMR sensitivity. The detailed molecular structures determined for the amorphous oxidized/hydrolyzed boron layers on these heterogenous catalysts will aid in the future development of next generation ODH catalysts.

Extraction of Individual Spectra from Mixture Data Based on High-Resolution 13C-13C NMR Correlation Spectrum and DECODE Procedure.

In NMR analysis of complex organic molecules, low natural abundance of 13C and the low resolution of two-dimensional (2D) experiments are significant difficulties. Also challenging is the analysis of a mixture spectrum without separation, which has been limited to simple molecules. Through nonuniform sampling using modified heteronuclear multiple bond correlationcombined with indirect covariance, a high-resolution13C-13C correlation spectrum was obtained with 1H sensitivity. Built on the thus-obtained 13C-13C connectivities, deconvolution of the mixture spectra was achieved through a new signal-processing procedure, termed DECODE, tailored to the indirect covariance eigendecomposition. When applied to a complex natural product mixture of rotenone and brucine with many quaternary carbons, the method resolved very close carbon peaks and extracted clean individual spectra. Essentially providing molecule-wide 13C connectivities for complex molecules from 1H-detected 2D spectra, our approach should prove useful in many areas of NMR analysis.

Best Practices in Utilization of 2D-NMR Spectral Data as the Input for Chemometric Analysis in Biopharmaceutical Applications.

Quality attributes (QAs) are measureable parameters of a biologic that impact product safety and efficacy and are essential characteristics that are linked to positive patient health outcomes. One QA, higher order structure (HOS), is directly coupled to the function of protein biologics, and deviations in this QA may cause adverse effects. To address the critical need for HOS assessment, methods for analyzing structural fingerprints from 2D nuclear magnetic resonance spectroscopy (2D-NMR) spectra have been established for drug substances as large as monoclonal antibody therapeutics. Here, chemometric analyses have been applied to 2D 1H,13C-methyl NMR correlation spectra of the IgG1κ NIST monoclonal antibody (NISTmAb), recorded at natural isotopic abundance, to benchmark the performance and robustness of the methods. In particular, a variety of possible spectral input schemes (e.g., chemical shift, peak intensity, and total spectral matrix) into chemometric algorithms are examined using two case studies: (1) a large global 2D-NMR interlaboratory study and (2) a blended series of enzymatically glycan-remodeled NISTmAb isoforms. These case studies demonstrate that the performance of chemometric algorithms using either peak positions or total spectral matrix as the input will depend on the study design and likely be product-specific. In general, peak positions are found to be a more robust spectral parameter for input into chemometric algorithms, whereas the total spectral matrix approach lends itself to easier automation and requires less user intervention. Analysis with different input data also shows differences in sensitivity to certain changes in HOS, highlighting that product knowledge will further guide appropriate method selection based on the fit-for-purpose application in the context of biopharmaceutical development, production, and quality control.

Hadamard acquisition of 13 C-13 C 2-D correlation NMR spectra.

We show that a multiselective excitation with Hadamard encoding is a powerful tool for 2-D acquisition of 13 C─13 C homonuclear correlations. This method is not designed to improve the sensitivity, but rather to reduce the experiment time, provided there is sufficient sensitivity. Therefore, it allows fast acquisition of such 2-D spectra in labeled molecules. The technique has been demonstrated using a U─13 C─15 N histidine hydrochloride monohydrate sample allowing each point of the build-up curves of the 13 C─13 C cross-peaks to be recorded within 4 min 35 s, which is very difficult with conventional methods. Using the U─13 C─15 N f-MLF sample, we have demonstrated that the method can be applied to molecules with 14 13 C resonances with a minimum frequency separation of 240 Hz.

Crystallization of Mordenite Platelets using Cooperative Organic Structure-Directing Agents.

Organic structure-directing agents (OSDAs) are exploited in the crystallization of microporous materials to tailor the physicochemical properties of the resulting zeolite for applications ranging from separations to catalysis. The rational design of these OSDAs often entails the identification of molecules with a geometry that is commensurate with the channels and cages of the target zeolite structure. Syntheses tend to employ only a single OSDA, but there are a few examples where two or more organics operate synergistically to yield a desired product. Using a combination of state-of-the-art characterization techniques and molecular modeling, we show that the coupling of N,N,N-trimethyl-1,1-adamantammonium and 1,2-hexanediol, each yielding distinct zeolites when used alone, results in the cooperative direction of a third structure, HOU-4, with the mordenite framework type (MOR). Rietveld refinement using synchrotron X-ray diffraction data reveals the spatial arrangement of the organics in the HOU-4 crystals, with amines located in the large channels and alcohols oriented in the side pockets lining the one-dimensional pores. These results are in excellent agreement with molecular dynamics calculations, which predict similar spatial distributions of organics with an energetically favorable packing density that agrees with experimental measurements of OSDA loading, as well as with solid-state two-dimensional 27Al{29Si}, 27Al{1H}, and 13C{1H} NMR correlation spectra, which establish the proximities and interactions of occluded OSDAs. A combination of high-resolution transmission electron microscopy and atomic force microscopy is used to quantify the size of the HOU-4 crystals, which exhibit a platelike morphology, and to index the crystal facets. Our findings reveal that the combined OSDAs work in tandem to produce ultrathin, nonfaulted HOU-4 crystals that exhibit improved catalytic activity for cumene cracking in comparison to mordenite crystals prepared via conventional syntheses. This novel demonstration of cooperativity highlights the potential possibilities for expanding the use of dual structure-directing agents in zeolite synthesis.

Investigating the Microstructure of Poly(cyclosilane) by 29Si Solid-State NMR Spectroscopy and DFT Calculations

Using high-resolution magic angle spinning (MAS) solid-state NMR spectroscopy and density-functional theory (DFT) calculations, we determine the microstructure of the silicon-based functional polymer poly(1,4Si6) arising from the dehydrocoupling polymerization of cyclosilane 1,4Si6. 1H–29Si refocused-INEPT solid-state NMR experiments allow the unambiguous determination of the number of attached protons to a silicon atom for each 29Si NMR signal in 1,4Si6 and poly(1,4Si6). One-dimensional 1H → 29Si cross-polarization MAS (CPMAS) NMR spectra of poly(1,4Si6) show the development of SiH resonances upon polymerization, and peak integration indicates an average degree of polymerization of 20. The 1H → 29Si CPMAS spectrum of poly(1,4Si6) also shows two sets of isotropic signals, suggesting the presence of at least two distinct species. Two-dimensional 29Si dipolar double-quantum–single-quantum and single-quantum–single-quantum homonuclear correlation NMR spectra reveal similar connectivity in the two species, po...

Synthesis and Photoelectrical Properties of 3-(Diphenylamino)Carbazolyl-Functionalized DMABI Derivatives

A modular approach was used in the synthesis of the 3-(diphenylamino)carbazolyl- functionalized 2-(4-(dimethylamino)benzylidene)-1H-indene-1,3(2H)-dione (DMABI) derivatives. For this purpose, carboxyl functional group was introduced into the framework of DMABI molecule. Additionally, a regioselective method for substitution of carbonyl group of DMABI with dicyanomethylene group was investigated and verified using the heteronuclear multiple bond correlation NMR spectra. Steglich esterification method was used to connect the 3‑(diphenylamino)carbazolyl moiety via an unconjugated spacer. The UV-Vis absorption spectra and molecular energy level data were obtained for the evaluation of the synthesized compounds as donor materials in bulk-heterojunction solar cells.

Ultrafast Single-Scan 2D NMR Spectroscopic Detection of a PHIP-Hyperpolarized Protease Inhibitor.

Two-dimensional NMR spectroscopy is one of the most important spectroscopic tools for the investigation of biological macromolecules. However, due to the low sensitivity of NMR spectroscopy, it takes usually from several minutes to many hours to record such spectra. Here, the possibility of detecting a bioactive derivative of the sunflower trypsin inhibitor-1 (SFTI-1), a tetradecapeptide, by combining parahydrogen-induced polarization (PHIP) and ultrafast 2D NMR spectroscopy is shown. The PHIP activity of the inhibitor was achieved by labeling with O-propargyl-l-tyrosine. In 1D PHIP experiments a signal enhancement of a factor of approximately 1200 compared to standard NMR was found. This enhancement permits measurement of 2D NMR correlation spectra of low-concentrated SFTI-1 in less than 10 seconds, employing ultrafast single-scan 2D NMR detection. As experimental examples PHIP-assisted ultrafast single-scan TOCSY spectra of SFTI-1 are shown.

Probing O-H Bonding through Proton Detected 1H-17O Double Resonance Solid-State NMR Spectroscopy.

The ubiquity of oxygen in organic, inorganic, and biological systems has stimulated the application and development of 17O solid-state NMR spectroscopy as a probe of molecular structure and dynamics. Unfortunately, 17O solid-state NMR experiments are often hindered by a combination of broad NMR signals and low sensitivity. Here, it is demonstrated that fast MAS and proton detection with the D-RINEPT pulse sequence can be generally applied to enhance the sensitivity and resolution of 17O solid-state NMR experiments. Complete 2D 17O → 1H D-RINEPT correlation NMR spectra were typically obtained in less than 10 h from less than 10 mg of material, with low to moderate 17O enrichment (less than 20%). Two-dimensional 1H-17O correlation solid-state NMR spectra allow overlapping oxygen sites to be resolved on the basis of proton chemical shifts or by varying the mixing time used for 1H-17O magnetization transfer. In addition, J-resolved or separated local field (SLF) blocks can be incorporated into the D-RINEPT pulse sequence to allow the direct measurement of one-bond 1H-17O scalar coupling constants (1 JOH) or 1H-17O dipolar couplings ( DOH), respectively, the latter of which can be used to infer 1H-17O bond lengths. 1 JOH and DOH calculated from plane-wave density functional theory (DFT) show very good agreement with experimental values. Therefore, the 2D 1H-17O correlation experiments, 1H-17O scalar and dipolar couplings, and plane-wave DFT calculations provide a method to precisely determine proton positions relative to oxygen atoms. This capability opens new opportunities to probe interactions between oxygen and hydrogen in a variety of chemical systems.

Determining Cholesterol Binding to Membrane Proteins by Cholesterol 13C Labeling in Yeast and Dynamic Nuclear Polarization NMR.

We present a general strategy for determining the cholesterol-binding site of eukaryotic membrane proteins in native-like lipid membranes by NMR spectroscopy. The strategy combines yeast biosynthetic 13C enrichment of cholesterol with detection of protein-cholesterol 13C-13C cross peaks in 2D correlation NMR spectra under the dynamic nuclear polarization (DNP) condition. Low-temperature DNP not only allows high-sensitivity detection of weak protein-cholesterol cross peaks in 2D spectra but also immobilizes cholesterol and protein to enable intermolecular distance measurements. We demonstrate this approach on the influenza M2 protein, which utilizes cholesterol to conduct membrane scission in the last step of virus budding and release from the host cell plasma membrane. A 13C-13C double-quantum filter was employed to significantly simplify the 2D 13C-13C correlation spectra and facilitate the identification of protein-cholesterol cross peaks. A number of cross peaks between the M2 transmembrane residues' side chains and the cholesterol sterol group were detected, which complement recently measured protein contacts to the isooctyl tail of cholesterol to define an extended binding interface. These results provide atomic-level evidence of M2-cholesterol interaction to cause membrane curvature and scission, and the approach is generally applicable to other eukaryotic membrane proteins for understanding the influence of cholesterol on membrane protein function.