A large sample volume magic angle spinning nuclear magnetic resonance probe for in situ investigations with constant flow of reactants

A large-sample-volume constant-flow magic angle sample spinning (CF-MAS) NMR probe is reported for in situ studies of the reaction dynamics, stable intermediates/transition states, and mechanisms of catalytic reactions. In our approach, the reactants are introduced into the catalyst bed using a fixed tube at one end of the MAS rotor while a second fixed tube, linked to a vacuum pump, is attached at the other end of the rotor. The pressure difference between both ends of the catalyst bed inside the sample cell space forces the reactants flowing through the catalyst bed, which improves the diffusion of the reactants and products. This design allows the use of a large sample volume for enhanced sensitivity and thus permitting in situ(13)C CF-MAS studies at natural abundance. As an example of application, we show that reactants, products and reaction transition states associated with the 2-butanol dehydration reaction over a mesoporous silicalite supported heteropoly acid catalyst (HPA/meso-silicalite-1) can all be detected in a single (13)C CF-MAS NMR spectrum at natural abundance. Coke products can also be detected at natural (13)C abundance and under the stopped flow condition. Furthermore, (1)H CF-MAS NMR is used to identify the surface functional groups of HPA/meso-silicalite-1 under the condition of in situ drying. We also show that the reaction dynamics of 2-butanol dehydration using HPA/meso-silicalite-1 as a catalyst can be explored using (1)H CF-MAS NMR.     

Real‐Time Interrogation of Aspirin Reactivity,Biochemistry, and Biodistribution by Hyperpolarized Magnetic Resonance Spectroscopy

Hyperpolarized magnetic resonance spectroscopy enables quantitative, non‐radioactive, real‐time measurement of imaging probe biodistribution and metabolism in vivo. Here, we investigate and report on the development and characterization of hyperpolarized acetylsalicylic acid (aspirin) and its use as a nuclear magnetic resonance (NMR) probe. Aspirin derivatives were synthesized with single‐ and double‐¹³C labels and hyperpolarized by dynamic nuclear polarization with 4.7 % and 3 % polarization, respectively. The longitudinal relaxation constants (T₁) for the labeled acetyl and carboxyl carbonyls were approximately 30 seconds, supporting in vivo imaging and spectroscopy applications. In vitro hydrolysis, transacetylation, and albumin binding of hyperpolarized aspirin were readily monitored in real time by ¹³C‐NMR spectroscopy. Hyperpolarized, double‐labeled aspirin was well tolerated in mice and could be observed by both ¹³C‐MR imaging and ¹³C‐NMR spectroscopy in vivo.     

Real‐Time Interrogation of Aspirin Reactivity,Biochemistry, and Biodistribution by Hyperpolarized Magnetic Resonance Spectroscopy

Hyperpolarized magnetic resonance spectroscopy enables quantitative, non‐radioactive, real‐time measurement of imaging probe biodistribution and metabolism in vivo. Here, we investigate and report on the development and characterization of hyperpolarized acetylsalicylic acid (aspirin) and its use as a nuclear magnetic resonance (NMR) probe. Aspirin derivatives were synthesized with single‐ and double‐¹³C labels and hyperpolarized by dynamic nuclear polarization with 4.7 % and 3 % polarization, respectively. The longitudinal relaxation constants (T₁) for the labeled acetyl and carboxyl carbonyls were approximately 30 seconds, supporting in vivo imaging and spectroscopy applications. In vitro hydrolysis, transacetylation, and albumin binding of hyperpolarized aspirin were readily monitored in real time by ¹³C‐NMR spectroscopy. Hyperpolarized, double‐labeled aspirin was well tolerated in mice and could be observed by both ¹³C‐MR imaging and ¹³C‐NMR spectroscopy in vivo.     

The initial stages of solid acid-catalyzed reactions of adsorbed propane. A mechanistic study by in situ MAS NMR

In situ solid-state NMR spectroscopy was employed to study the kinetics of hydrogen/deuterium exchange and scrambling as well as (13)C scrambling reactions of labeled propane over Al(2)O(3)-promoted sulfated zirconia (SZA) catalyst under mild conditions (30-102 degrees C). Three competitive pathways of isotope redistribution were observed during the course of the reaction: (1) a regioselective H/D exchange between acidic protons of the solid surface and the deuterons of the methyl group of propane-1,1,1,3,3,3-d(6), monitored by in situ (1)H MAS NMR; (2) an intramolecular H/D scrambling between methyl deuterons and protons of the methylene group, without exchange with the catalyst surface, monitored by in situ (2)H MAS NMR; (3) a intramolecular (13)C scrambling, by skeletal rearrangement process, favored at higher temperatures, monitored by in situ (13)C MAS NMR. The activation energy of (13)C scrambling was estimated to be very close to that of (2)H scrambling, suggesting that these two processes imply a common transition state, responsible for both vicinal hydride migration and protonated cyclopropane formation. All pathways are consistent with a classical carbenium ion-type mechanism.     

Fluorescence probe for lysophospholipase C/NPP6 activity and a potent NPP6 inhibitor

Nucleotide pyrophosphatases/phosphodiesterases (NPPs) are ubiquitous membrane-associated or secreted ectoenzymes that have a role in regulating extracellular nucleotide and phospholipid metabolism. Among the members of the NPP family, NPP1 and -3 act on nucleotides such as ATP, while NPP2, -6, and -7 act on phospholipids such as lysophosphatidylcholine and sphingomyelin. NPP6, a recently characterized NPP family member, is a choline-specific glycerophosphodiester phosphodiesterase, but its functions remain to be analyzed, partly due to the lack of highly sensitive activity assay systems and practical inhibitors. Here we report synthesis of novel NPP6 fluorescence probes, TG-mPC and its analogues TG-mPC(3)C, TG-mPC(5)C, TG-mPENE, TG-mPEA, TG-mPhos, TG-mPA, TG-mPMe, and TG-mPPr. Among the seven NPPs, only NPP6 hydrolyzed TG-mPC, TG-mPC(3)C, and TG-mPENE. TG-mPC was hydrolyzed in the cell lysate from NPP6-transfected cells, but not control cells, showing that it is suitable for use in cell-based NPP6 assays. We also examined the usefulness of TG-mPC as a fluorescence imaging probe. We further applied TG-mPC to carry out high-throughput NPP6 inhibitor screening and found several NPP6-selective inhibitors in a library of about 80,000 compounds. Through structure-activity relationship (SAR) analysis, we identified a potent and selective NPP6 inhibitor with an IC(50) value of 0.21 μM. Our NPP6-selective fluorescence probe, TG-mPC, and the inhibitor are expected to be useful to elucidate the biological function of NPP6.     

Rapid upregulation of cytoprotective nitric oxide in breast tumor cells subjected to a photodynamic therapy-like oxidative challenge

Many tumor cells produce nitric oxide (NO) as an antiapoptotic/progrowth molecule which also promotes antiogenesis and tumor expansion. This study was designed to examine possible antagonistic effects of endogenous NO on tumor eradication by photodynamic therapy (PDT). Using COH-BR1 breast cancer cells sensitized in mitochondria with 5-aminolevulinic acid (ALA)-generated protoporphyrin IX as a model for ALA-based PDT, we found that caspase-9 activation and apoptotic death following irradiation were strongly enhanced by 1400W, an inhibitor of inducible nitric oxide synthase (iNOS). RT-PCR and Western analyses revealed a substantial upregulation of both iNOS mRNA and protein, beginning ca 4 h after irradiation and persisting for at least 20 h. Accompanying this was a strong 1400W-inhibitable increase in intracellular NO, as detected with the NO probe, DAF-2-DA. Short hairpin RNA-based iNOS knockdown in COH-BR1 cells dramatically reduced NO production under photostress while enhancing caspase-9 activation and apoptosis. These findings suggest that cytoprotective iNOS/NO induction in PDT-treated tumor cells could reduce treatment efficacy, and point to pharmacologic intervention with iNOS inhibitors for counteracting this.     

原位制备8-氘代奥美拉唑钠

奥美拉唑分子中的氮原子质子化,对邻近的碳核产生有效的电四极矩驰豫,使邻近碳核信号加宽或消失,以致奥美拉唑在DMSO-d6或DMSO-d6/D2O溶液中的碳谱(13CNMR、DEPT-90、DEPT-135)及碳-氢相关谱(HMQC、HMBC)具有非典型的特征,即部分碳原子不出峰且峰形很差,以致无法确定具体的碳数,延长脉冲重复时间无改观。本实验通过将奥美拉唑与NaOH的D2O溶液反应原位制备8-氘代奥美拉唑钠,使吡啶氮原子去质子化,从而使部分不出峰的碳核信号变窄或重又出现,顺利地完成了8-氘代奥美拉唑钠上述谱图的采集,间接地解决了奥美拉唑的碳谱分析,因而提供了一种可通用的测试苯骈咪唑类质子泵抑制剂碳谱及其二维相关谱的方法。     

Mechanism studies of the conversion of 13C-labeled n-butane on zeolite H-ZSM-5 by using 13C magic angle spinning NMR spectroscopy and GC-MS analysis

By using 13C MAS NMR spectroscopy (MAS = magic angle spinning), the conversion of selectively 13C-labeled n-butane on zeolite H-ZSM-5 at 430-470 K has been demonstrated to proceed through two pathways: 1) scrambling of the selective 13C-label in the n-butane molecule, and 2) oligomerization-cracking and conjunct polymerization. The latter processes (2) produce isobutane and propane simultaneously with alkyl-substituted cyclopentenyl cations and condensed aromatic compounds. In situ 13C MAS NMR and complementary ex situ GC-MS data provided evidence for a monomolecular mechanism of the 13C-label scrambling, whereas both isobutane and propane are formed through intermolecular pathways. According to 13C MAS NMR kinetic measurements, both pathways proceed with nearly the same activation energies (E(a) = 75 kJ mol(-1) for the scrambling and 71 kJ mol(-1) for isobutane and propane formation). This can be rationalized by considering the intermolecular hydride transfer between a primarily initiated carbenium ion and n-butane as being the rate-determining stage of the n-butane conversion on zeolite H-ZSM-5.     

Glucosylation of isatin-3-oxime followed by 2D in situ NMR in plant cells at highest magnetic field without labelling

The glucosylation of isatin-3-oxime (1) was monitored by in situ 2D 1H-13C inverse correlated gradient assisted NMR spectroscopy in plant cell suspension cultures of Rauvolfia serpentina without labelling. The applied high magnetic field of 800 MHz allowed measurements within 20 min at concentrations of 1 of 5.76 mM. Complete glucosylation of 1 occurs inside the cells within 72 hours. During this time isatin-3-oxime-glucoside (2) accumulates without further metabolism.     

六氟磷酸二(4,4'-二甲基-2,2'-联吡啶)·(2,2'-联吡啶-4,4'-二羧酸)合钌(Ⅱ)的二维核磁共振

用1H NMR和13C NMR谱研究了新型电化学发光探针六氟磷酸二(4,4'-二甲基-2,2'-联吡啶)·(4,4'-二羧酸-2,2'-联吡啶)合钌(Ⅱ)的立体结构,通过1H-1H COSY、13C-1H HETCOR谱对其氢谱和碳谱中的各谱峰进行了归属,并给出了氢谱和碳谱峰的化学位移值.     

Direct voltammetric specific recognition of dopamine using AlIII-DA complexes at the hanging mercury drop electrode.

In this paper, we firstly report the direct voltammetric recognition and determination of dopamine (DA) by using Al(III)-DA complexes at the hanging mercury drop electrode (HMDE). A new sensitive cathodic peak of Al(III)-DA can be detected at -900 mV (vs. SCE) in 0.1 M NH(4)Cl-NH(3).H(2)O-0.1 M KCl buffer solution at pH 8.5. This unique -900 mV cathodic peak arises from the specific interaction between Al(III) and DA on the HMDE, whereas other substances with similar structures, such as L-dopa, epinephrine (EP), norepinephrine (NE), catechols, caffeic acid (CA), trihydric phenols and tiron, do not yield any new peak on the voltammograms in the potential range from -100 to -1200 mV when Al(III) is added. The distinct voltammetric characteristic of the recognition of DA can effectively inhibit the interferences of both ascorbic acid and uric acid in the DA determination by the direct electrochemistry, which is a major difficulty when a solid electrode is used. The proposed method can be anticipated as an effective means for the recognition of DA in the elucidation of the mechanisms of Parkinson's disease (PD) and Alzheimer's disease (AD) in the presence of Al(III).     

Site-directed 13C solid-state NMR studies on membrane proteins: strategy and goals toward revealing conformation and dynamics as illustrated for bacteriorhodopsin labeled with [1-13C]amino acid residues

We have so far demonstrated that well-resolved and site-specifically assigned (13)C peaks as recorded by site-directed NMR study on (13)C-labeled membrane proteins can serve as a convenient probe to reveal their local conformation and dynamics. We attempted here to clarify the extent to which (13)C NMR spectra of (13)C-labeled fully hydrated bacteriorhodopsin (bR) as a typical membrane protein are visible or well resolved in the presence of inherent fluctuation motions with frequency of 10(2)-10(8) Hz, especially at the membrane surfaces. Accordingly, we estimated the relative proportion of (13)C NMR signals from the surface areas with and without peak suppression by the accelerated transverse relaxation effect by surface-bound Mn(2+) ions, which could be effective for residues within 8.7 angstroms of the membrane surface. It turned out that the experimental findings are consistent with the predicted amount of amino acid residues under consideration located within 8.7 angstroms of the surface for [1-(13)C]Val- and Ile-labeled bR and also [3-(13)C]Ala-bR. In contrast, (13)C NMR peaks from such surfaces area are almost completely or partially suppressed for [1-(13)C]Gly-, Ala-, Leu-, Phe- and Trp-labeled bR, as a result of plausible interference of the fluctuation frequency with frequency of magic angle spinning (10(4) Hz). We further assigned several (13)C NMR signals of [1-(13)C] Val-, Trp- and Ile-labeled bR on the basis of a variety of site-directed mutants with reference to those of the wild type. Further, we recorded the (13)C NMR of bR in lipid bilayers to search for the optimal conditions to be able to obtain signals with the highest peak intensities and spectral resolution. Backbone dynamics turn out to be essential for recording (13)C NMR spectra so as to escape from motional frequencies of the order of 10(4)-10(5) Hz, either in the direction of accelerated fluctuation or slowed motions in the direction of forming the 2D array.     

NMR studies of the dynamics of a bifunctional rhodamine probe attached to troponin C

Fluorescence polarization measurements of bifunctional rhodamine (BR) probes provide a powerful approach to determine the in situ orientation of proteins within ordered complexes such as muscle fibers. For accurate interpretation of fluorescence measurements, it is important to understand the probe dynamics relative to the protein to which it is attached. We previously determined the structure of the N-domain of chicken skeletal troponin C, BR-labeled on the C helix, in complex with the switch region of troponin I, and demonstrated that the probe does not perturb the structure or dynamics of the protein. In this study, the motion of the fluorescence label relative to the protein has been characterized using NMR relaxation measurements of 13C-labeled methyl groups on the BR probe and 15N-labeled backbone amides of the protein. Probe dynamics were monitored using off-resonance 13C-R(1rho), 13C-R(1) and {1H}-13C NOE at magnetic field strengths of 500, 600, and 800 MHz. Relaxation data were interpreted in terms of the overall rotational correlation time of the protein and a two-time scale model for internal motion of the BR methyl groups, using a numerical optimization with Monte Carlo parameter error estimation. The analysis yields a 1.5 +/- 0.4 ps correlation time for rotation around the three-fold methyl symmetry axis, and a 0.8 +/- 0.4 ns rotational correlation time for reorientation of the 13C-14N bond with an associated S2s of 0.79 +/- 0.03. Order parameters of the backbone NH vectors in the helix to which the probe is attached average S2 approximately 0.85, implying that the amplitude of independent reorientation of the BR probe is small in magnitude, consistent with results from fluorescence polarization measurements in reconstituted muscle fibers.     

Design of a Hyperpolarized Molecular Probe for Detection of Aminopeptidase N Activity

Aminopeptidase N (APN) is an important enzyme that is involved in tumor angiogenesis. Detection of APN activity can thus lead to early diagnosis and elucidation of tumor development. Although some molecular probes for APN have been developed, the detection of APN activity in opaque biological samples remains a challenge. To this end, we designed a hyperpolarized NMR probe [1‐¹³C]Ala‐NH₂ which satisfies the prerequisites for APN detection, namely, sufficient retention of the hyperpolarized state, a high reactivity to APN, and an APN‐induced chemical shift change. The [1‐¹³C]Ala‐NH₂ probe allowed sensitive detection of APN activity using ¹³C NMR spectroscopy.     

The use of DSC curves to determine the acetylation degree of chitin/chitosan samples

The use of DSC curves is proposed as an alternative method to determine the degree of N-acetylation (DA) in chitin/chitosan samples, based in both peak area and height of the decomposition signal. Samples with DA from 74 to 16% were prepared from a chitin commercial sample and the DA was determined by ¹H NMR, ¹³C CP/MAS NMR and IR spectra. The effect of water content, heating rate, sample mass and gas flow on the DSC peaks were evaluated and optimized. Using optimized conditions a linear relationship between peak area and height with the DA could be achieved with linear correlation coefficients of −0.998 and −0.999 (n = 7), respectively. The calibration graphs were used to determine the DA of a commercial chitosan sample with relative errors ranging from 2 to 3% for both peak area and peak height, when compared with the DA determined by ¹H NMR method.     

含萘三氮唑甲烷骨架的硫代乙酸类尿酸转运体1(URAT1)抑制剂的合成及其构效关系

分别以1-溴萘和酮或1-萘甲醛及有机金属试剂为原料,经12步反应合成了8个含萘三氮唑甲烷骨架的硫代乙酸类尿酸转运体1(URAT1)抑制剂(1h~1o),其结构经¹H NMR, ¹³C NMR和MS（ESI）表征。体外活性测试结果显示：对URAT1的抑制活性最强的是1k,是阳性对照药lesinurad的133倍［IC₅₀=0.054 μmol·L^-1(1k), 7.18 μmol·L^-1(lesinurad)］。     

High-resolution solid-state (13)C NMR studies of chemisorbed organometallics. Chemisorptive formation of cation-like and alkylidene organotantalum complexes on high surface area inorganic oxides

(13)C CPMAS NMR spectroscopy has been employed to investigate the surface chemistry of the organotantalum hydrocarbyl/alkylidene complexes, Cp'Ta((13)CH(3))(4) (1*), Cp(2)Ta((13)CH(3))(3) (2*), Cp(2)Ta((13)CH(2))((13)CH(3)) (3*), and Ta((13)CH(t)Bu)((13)CH(2)(t)Bu)(3) (4*) [Cp' = eta(5)-(CH(3))(5)C(5), Cp = eta(5)-C(5)H(5)] supported on partially dehydroxylated silica (PDS), dehydroxylated silica (DS), or dehydroxylated gamma-alumina (DA). Mono-Cp tantalum hydrocarbyl 1* undergoes chemisorption to form Cp'Ta((13)CH(3))(3)O-Si mu-oxo species on silica, and "cation-like" Cp'Ta((13)CH(3))(3)(+) and Cp'Ta((13)CH(3))(3)O-Al mu-oxo species on DA, via pathways analogous to those established for organo-group 4 and actinide complexes. When supported on DA, bis-Cp tantalum hydrocarbyl 2* follows the same chemisorption mode as 1*. However, when 2* is chemisorbed on PDS and DS, a "cation-like" Cp(2)Ta((13)CH(3))(2)(+) species is the major adsorbate product. On PDS, bis-Cp tantalum alkylidene complex 3* is converted predominantly to a stable "cation-like" Cp(2)Ta((13)CH(3))(2)(+) species, presumably via electrophilic addition of a proton from the PDS surface. In contrast to 3*, Ta alkylidene complex 4* forms predominantly a Ta((13)CH(t)Bu)((13)CH(2)(t)Bu)(2)O-Si, mu-oxo-alkylidene species on PDS.     

Reactivity of C1 surface species formed in methane activation on Zn-modified H-ZSM-5 zeolite

Solid-state (13)C magic angle spinning (MAS) NMR spectroscopy investigations identified zinc methyl species, formate species, and methoxy species as C(1) surface species formed in methane activation on the zeolite Zn/H-ZSM-5 catalyst at T≤573 K. These C(1) surface species, which are possible intermediates in further transformations of methane, were prepared separately by adsorption of (13)C-enriched methane, carbon monoxide, and methanol onto zinc-containing catalysts, respectively. Successful isolation of each surface species allowed convenient investigations into their chemical nature on the working catalyst by solid-state (13)C MAS NMR spectroscopy. The reactivity of zinc methyl species with diverse probe molecules (i.e., water, methanol, hydrochloride, oxygen, or carbon dioxide) is correlated with that of organozinc compounds in organometallic chemistry. Moreover, surface formate and surface methoxy species possess distinct reactivity towards water, hydrochloride, ammonia, or hydrogen as probe molecules. To explain these and other observations, we propose that the C(1) surface species interconvert on zeolite Zn/H-ZSM-5. As implied by the reactivity information, potential applications of methane co-conversion on zinc-containing zeolites might, therefore, be possible by further transformation of these C(1) surface species with rationally designed co-reactants (i.e., probe molecules) under optimized reaction conditions.     

Two-dimensional spectroscopy with parallel acquisition of 1H-X and 19F-X correlations

Two-dimensional NMR spectra correlating both (1)H and (19)F nuclei with either (13)C or (15)N, are recorded at the same time, using a 600-MHz broadband radio frequency probe feeding independent (1)H and (19)F receiver channels. This technique, known as parallel acquisition NMR spectroscopy (PANSY), speeds up multidimensional NMR and is compatible with other fast-acquisition schemes. The method is illustrated with single-bond (HSQC) and multiple-bond (HMBC) experiments on 2-bromophenyl-3-trifluoromethyl-5-methylpyrazole, giving simultaneous (1)H-X and (19)F-X correlation spectra (X = (13) C or (15)N).     

13C NMR spectroscopy of 13C1-labeled octanethiol-protected Au nanoparticles: shifts,relaxations, and particle-size effect

We report here an investigation of metal-ligand interactions in nanoparticles with 13C NMR, using a labeled 13C1-octanethiol, a protecting ligand for self-assembled monolayer (SAM) systems, in which close proximity of the 13C1 to the metal surface serves as an effective probe for the changing electronic environment. Several remarkable results have been obtained: as the metal core size increases from 1.6 to 4.0 nm, the 13C1 spectrum is downshifted from 40.5 to 53 ppm, and the spin-spin relaxation rate, T2-1, increases while the spin-lattice relaxation ratio decreases. Although the spin-lattice relaxation may be due to particle tumbling and ligand motion in the liquid state, the main source of the spin-spin relaxation, and NMR shift, is most possibly due to the changing electronic properties of the metal core.