Carbon Deuterium Research Articles

Incorporation of Heavy Water for Rapid Detection of Salmonella typhimurium by Raman Microspectroscopy

The presence of foodborne pathogens is one of the leading causes of food safety incidents; hence, early monitoring of the status of microbial contamination is a permanent concern. Here, a rapid and sensitive method for detection of Salmonella typhimurium was firstly developed by combining heavy water (D2O) labeling and Raman microspectroscopy to measure bacterial metabolic activity. It was based on the characteristics of Raman signals of carbon–deuterium (C–D) vibration as cells incorporate deuterium in place of hydrogen (H) during biosynthetic activity, and the live and dead Salmonella typhimurium cells could be distinguished by their metabolic activity within 1 h. The CD / (CD + CH)% of D2O-incorporated Salmonella typhimurium in logarithmic growth phase was linearly related to the log of the initial concentration, and Salmonella typhimurium with the initial concentration of 102–106 colony-forming unit (CFU)/mL could be quantified within 4–8 h. Furthermore, desirable recovery ranging from 95.41 to 106.6% was obtained to assess the practicality and stability of this method for rapid detection of Salmonella typhimurium in milk, indicating promising application for the detection of bacterial contamination in food.

The First Approved “Deuterated” Drug: A Short Review of the Concept

The first “deuterated” drug has recently been approved by the U.S. FDA (Food & Drug Administration). A “deuterated” drug is a drug in which the hydrogen atom in one or more of the carbon-hydrogen bonds in its chemical structure is replaced by deuterium (“heavy hydrogen”, a hydrogen isotope that has a neutron, i.e., one neutron instead of the usual no neutrons). A carbon-deuterium (C-D) bond is more stable in the body than a carbon-hydrogen (C-H) bond. If the deuterium is strategically located in a drug’s chemical structure, the extra stability of the bond will be more resistant to metabolic breakdown, and the duration of drug action will be prolonged. We review the general concept of deuterated drugs, historical examples of the classes of application, and the new approval.

Anomalous neutron yield in indirect-drive inertial-confinement-fusion due to the formation of collisionless shocks in the corona

Observations of anomalous neutron yield in the indirect-drive inertial confinement fusion implosion experiments conducted at SG-III prototype and SG-II upgrade laser facilities are interpreted. The anomalous mechanism results in a neutron yield which is 100-times higher than that predicted by 1D radiation-hydrodynamic simulations. 2D radiation-hydrodynamic simulations show that the supersonic, radially directed gold (Au) plasma jets arising from the laser-hohlraum interactions can collide with the carbon-deuterium (CD) corona plasma of the compressed pellet. It is found that in the interaction front of the high-Z jet with the low-Z corona, with low density ∼ and high temperature ∼keV, kinetic effects become important. Particle-in-cell simulations indicate that an electrostatic shock wave can be driven when the high-temperature Au jet expands into the low-temperature CD corona. Deuterium ions with an amount of ∼1015 can be accelerated to ∼25 keV by the collisionless shock wave, thus causing efficient neutron productions though the beam-target method by stopping these energetic ions in the corona. The evaluated neutron yield is consistent with the experiments conducted at SG laser facilities.

Transparent Window Vibrational Probes for the Characterization of Proteins With High Structural and Temporal Resolution.

Vibrational spectroscopy provides a direct route to the physicochemical characterization of molecules. While both IR and Raman spectroscopy have been used for decades to provide detailed characterizations of small molecules, similar studies with proteins are largely precluded due to spectral congestion. However, the vibrational spectra of proteins do include a "transparent window", between ∼1800 and ∼2500 cm-1, and progress is now being made to develop site-specifically incorporated carbon-deuterium (C-D), cyano (CN), thiocyanate (SCN), and azide (N3) "transparent window vibrational probes" that absorb within this window and report on their environment to facilitate the characterization of proteins with small molecule-like detail. This Review opens with a brief discussion of the advantages and limitations of conventional vibrational spectroscopy and then discusses the strengths and weaknesses of the different transparent window vibrational probes, methods by which they may be site-specifically incorporated into peptides and proteins, and the physicochemical properties they may be used to study, including electrostatics, stability and folding, hydrogen bonding, protonation, solvation, dynamics, and interactions with inhibitors. The use of the probes to vibrationally image proteins and other biomolecules within cells is also discussed. We then present four case studies, focused on ketosteroid isomerase, the SH3 domain, dihydrofolate reductase, and cytochrome c, where the transparent window vibrational probes have already been used to elucidate important aspects of protein structure and function. The Review concludes by highlighting the current challenges and future potential of using transparent window vibrational probes to understand the evolution and function of proteins and other biomolecules.

Proton pinhole imaging on the National Ignition Facility.

Pinhole imaging of large (mm scale) carbon-deuterium (CD) plasmas by proton self-emission has been used for the first time to study the microphysics of shock formation, which is of astrophysical relevance. The 3 MeV deuterium-deuterium (DD) fusion proton self-emission from these plasmas is imaged using a novel pinhole imaging system, with up to five different 1 mm diameter pinholes positioned 25 cm from target-chamber center. CR39 is used as the detector medium, positioned at 100 cm distance from the pinhole for a magnification of 4 ×. A Wiener deconvolution algorithm is numerically demonstrated and used to interpret the images. When the spatial morphology is known, this algorithm accurately reproduces the size of features larger than about half the pinhole diameter. For these astrophysical plasma experiments on the National Ignition Facility, this provides a strong constraint on simulation modeling of the experiment.

Methionine Ligand Interaction in a Blue Copper Protein Characterized by Site-Selective Infrared Spectroscopy.

The reactivity of metal sites in proteins is tuned by protein-based ligands. For example, in blue copper proteins such as plastocyanin (Pc), the structure imparts a highly elongated bond between the Cu and a methionine (Met) axial ligand to modulate its redox properties. Despite extensive study, a complete understanding of the contribution of the protein to redox activity is challenged by experimentally accessing both redox states of metalloproteins. Using infrared (IR) spectroscopy in combination with site-selective labeling with carbon-deuterium (C-D) vibrational probes, we characterized the localized changes at the Cu ligand Met97 in the oxidized and reduced states, as well as the Zn(II) or Co(II)-substituted, the pH-induced low-coordinate, the apoprotein, and the unfolded states. The IR absorptions of (d3-methyl)Met97 are highly sensitive to interaction of the sulfur-based orbitals with the metal center and are demonstrated to be useful reporters of its modulation in the different states. Unrestricted Kohn-Sham density functional theory calculations performed on a model of the Cu site of Pc confirm the observed dependence. IR spectroscopy was then applied to characterize the impact of binding to the physiological redox partner cytochrome (cyt) f. The spectral changes suggest a slightly stronger Cu-S(Met97) interaction in the complex with cyt f that has potential to modulate the electron transfer properties. Besides providing direct, molecular-level comparison of the oxidized and reduced states of Pc from the perspective of the axial Met ligand and evidence for perturbation of the Cu site properties by redox partner binding, this study demonstrates the localized spatial information afforded by IR spectroscopy of selectively incorporated C-D probes.

Detection of the iron impurity in CD x films formed in the T-10 tokamak and its influence on the sp 3 → sp 2 conversion

Experimental data on Fe-impurity detection in smooth carbon–deuterium (CD x , x ~ 0.5) films from the T-10 tokamak, which are obtained by means of X-ray diffraction, are presented. Additional analysis and systematization of the results of previous experiments are performed in order to clarify the iron charge state and estimate its influence on the sp 3 → sp 2 conversion.

Deuterium tracing study of unsaturated aliphatics hydrogenation by supercritical water in upgrading heavy oil. Part I: Non-catalytic cracking

It is believed that the unique solubility and diffusivity of organics in supercritical water (SC-H2O) have a positive influence on upgrading of heavy oil; however, the important issue would be the chemical reactivity of SC-H2O. By applying isotope labeling technique, the present study has verified the chemical role of SC-H2O as the environmentally benign hydrogen-donor solvent in cracking the petroleum vacuum residue. The high pressure batch experiments were applied at several temperatures (350–450°C) and pressures (17–50MPa) keeping density constant at ca. 0.4g/ml, respectively. After 1h contact time between heavy water (D2O) and vacuum residue (VR) in a ratio of 80/3g/g, the hexane soluble product (maltene) was analyzed by attenuated total reflectance infrared (ATR-IR) spectroscopy. Substantial carbon–deuterium bonding (16% relative to overall hydrogenation by water and hydrocarbon) was detected in saturated compounds (alkanes) produced from the cracking process with water, higher than 425°C as the temperature beginning of considerable miscibility between water and oil. Accordingly, together with the modified physical properties (solvation and dispersion effects), hydrogen so formed from SC-H2O would be accessible to cover radical species formed during the cracking of asphaltenes and other oil species, and consequently reduced coke formation compared with the dry pyrolysis. This kind of in-situ hydrogen donation is in competition with hydrogen elaborated from condensation of polycyclic aromatics in the oily phase in which formation of coke is deduced. The higher portion of hydrogen by water relative to the hydrocarbon condensation causes reduction in radical addition reaction rate and suppression of coke, therefore allows the higher conversion of residues to desirable liquid products.

Advancements in the application of NanoSIMS and Raman microspectroscopy to investigate the activity of microbial cells in soils.

The combined approach of incubating environmental samples with stable isotope-labeled substrates followed by single-cell analyses through high-resolution secondary ion mass spectrometry (NanoSIMS) or Raman microspectroscopy provides insights into the in situ function of microorganisms. This approach has found limited application in soils presumably due to the dispersal of microbial cells in a large background of particles. We developed a pipeline for the efficient preparation of cell extracts from soils for subsequent single-cell methods by combining cell detachment with separation of cells and soil particles followed by cell concentration. The procedure was evaluated by examining its influence on cell recoveries and microbial community composition across two soils. This approach generated a cell fraction with considerably reduced soil particle load and of sufficient small size to allow single-cell analysis by NanoSIMS, as shown when detecting active N2-fixing and cellulose-responsive microorganisms via 15N2 and 13C-UL-cellulose incubations, respectively. The same procedure was also applicable for Raman microspectroscopic analyses of soil microorganisms, assessed via microcosm incubations with a 13C-labeled carbon source and deuterium oxide (D2O, a general activity marker). The described sample preparation procedure enables single-cell analysis of soil microorganisms using NanoSIMS and Raman microspectroscopy, but should also facilitate single-cell sorting and sequencing.

SERS imaging of cell-surface biomolecules metabolically labeled with bioorthogonal Raman reporters.

Live imaging of biomolecules with high specificity and sensitivity as well as minimal perturbation is essential for studying cellular processes. Here, we report the development of a bioorthogonal surface-enhanced Raman scattering (SERS) imaging approach that exploits small Raman reporters for visualizing cell-surface biomolecules. The cells were cultured and imaged by SERS microscopy on arrays of Raman-enhancing nanoparticles coated on silicon wafers or glass slides. The Raman reporters including azides, alkynes, and carbondeuterium bonds are small in size and spectroscopically bioorthogonal (background-free). We demonstrated that various cell-surface biomolecules including proteins, glycans, and lipids were metabolically incorporated with the corresponding precursors bearing a Raman reporter and visualized by SERS microscopy. The coupling of SERS microscopy with bioorthogonal Raman reporters expands the capabilities of live-cell microscopy beyond the modalities of fluorescence and label-free imaging.

Experimental Characterization of Electrostatic and Conformational Heterogeneity in an SH3 Domain

Electrostatic and conformational heterogeneity make central contributions to protein function, but their experimental characterization requires a combination of spatial and temporal resolution that is challenging to achieve. Src homology 3 (SH3) domains mediate protein-protein interactions, and NMR studies have demonstrated that most possess conformational heterogeneity, which could be critical for their function. Here, we use the IR absorptions of carbon-deuterium (C-D) bonds site-selectively incorporated throughout the N-terminal SH3 domain from the murine adapter protein Crk-II to characterize its different microenvironments with high spatial and temporal resolution. The C-D absorptions are only differentiated in the folded state of the protein where they show evidence of significant environmental heterogeneity. However, the spectra of the folded state are independent of temperature, and upon thermal denaturation the protein undergoes a single, global unfolding transition. While some evidence of conformational heterogeneity is found within the peptide backbone, the majority of the environmental heterogeneity appears to result from electrostatics.

Vibrational imaging of newly synthesized proteins in live cells by stimulated Raman scattering microscopy

Synthesis of new proteins, a key step in the central dogma of molecular biology, has been a major biological process by which cells respond rapidly to environmental cues in both physiological and pathological conditions. However, the selective visualization of a newly synthesized proteome in living systems with subcellular resolution has proven to be rather challenging, despite the extensive efforts along the lines of fluorescence staining, autoradiography, and mass spectrometry. Herein, we report an imaging technique to visualize nascent proteins by harnessing the emerging stimulated Raman scattering (SRS) microscopy coupled with metabolic incorporation of deuterium-labeled amino acids. As a first demonstration, we imaged newly synthesized proteins in live mammalian cells with high spatial-temporal resolution without fixation or staining. Subcellular compartments with fast protein turnover in HeLa and HEK293T cells, and newly grown neurites in differentiating neuron-like N2A cells, are clearly identified via this imaging technique. Technically, incorporation of deuterium-labeled amino acids is minimally perturbative to live cells, whereas SRS imaging of exogenous carbon-deuterium bonds (C-D) in the cell-silent Raman region is highly sensitive, specific, and compatible with living systems. Moreover, coupled with label-free SRS imaging of the total proteome, our method can readily generate spatial maps of the quantitative ratio between new and total proteomes. Thus, this technique of nonlinear vibrational imaging of stable isotope incorporation will be a valuable tool to advance our understanding of the complex spatial and temporal dynamics of newly synthesized proteome in vivo.

Deuterium inventory in Tore Supra: Coupled carbon–deuterium balance

This paper presents an analysis of the carbon–deuterium circulation and the resulting balance in Tore Supra over the period 2002–2007. Carbon balance combines the estimation of carbon gross erosion from spectroscopy, net erosion and deposition using confocal microscopy, lock-in thermography and SEM, and a measure of the amount of deposits collected in the vacuum chamber. Fuel retention is determined from post-mortem (PM) analyses and gas balance (GB) measurements. Special attention was paid to the deuterium outgassed during the nights and weekends of the experimental campaign (vessel under vacuum, Plasma Facing Components at 120°C) and during vents (vessel at atmospheric pressure, PFCs at room temperature). It is shown that this outgassing is the main process reconciling the PM and GB estimations of fuel retention, closing the coupled carbon–deuterium balance. In particular, it explains why the deuterium concentration in deposits decreases with increasing depth.

Lipid Bilayers: The Effect of Force Field on Ordering and Dynamics.

The sensitivity of the structure and dynamics of a fully hydrated pure bilayer of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in molecular dynamics simulations to changes in force-field and simulation parameters has been assessed. Three related force fields (the Gromos 54A7 force field, a Gromos 53A6-derived parameter set and a variant of the Berger parameters) in combination with either particle-mesh Ewald (PME) or a reaction field (RF) were compared. Structural properties such as the area per lipid, carbon-deuterium order parameters, electron density profile and bilayer thicknesses, are reproduced by all the parameter sets within the uncertainty of the available experimental data. However, there are clear differences in the ordering of the glycerol backbone and choline headgroup, and the orientation of the headgroup dipole. In some cases, the degree of ordering was reminiscent of a liquid-ordered phase. It is also shown that, although the lateral diffusion of the lipids in the plane of the bilayer is often used to validate lipid force fields, because of the uncertainty in the experimental measurements and the fact that the lateral diffusion is dependent on the choice of the simulation conditions, it should not be employed as a measure of quality. Finally, the simulations show that the effect of small changes in force-field parameters on the structure and dynamics of a bilayer is more significant than the treatment of the long-range electrostatic interactions using RF or PME. Overall, the Gromos 54A7 best reproduced the range of experimental data examined.

Monitoring Intramolecular Proton Transfer with Two-Dimensional Infrared Spectroscopy: A Computational Prediction.

Proton transfer processes are ubiquitous and play a vital role in a broad range of chemical and biochemical phenomena. The ability of two-dimensional infrared (2D IR) spectroscopy with a carbon-deuterium (C-D) reporter to monitor the kinetics of proton transfer in the model compound malonaldehyde was demonstrated computationally. One of the two carbonyl/enol carbon atoms in malonaldehyde was labeled with a C-D bond. The C-D stretch vibrational frequency provides ∼150 cm(-1) of sensitivity to the two tautomers of malonaldehyde. Mixed quantum mechanics/molecular mechanics simulations employing the self-consistent-charge density functional tight binding (SCC-DFTB) method were used to compute 2D IR line shapes for the C-D stretch of labeled malonaldehyde in aqueous solution. The 2D IR spectra reveal cross peaks from the chemical exchange of the proton. The kinetics for the growth of the cross-peaks (and the decay of the diagonal peaks) precisely match the proton transfer rate observed in the SCC-DFTB simulations.

Chiral Phosphoric Acid Catalyzed Enantioselective Transfer Deuteration of Ketimines by Use of Benzothiazoline As a Deuterium Donor: Synthesis of Optically Active Deuterated Amines

By use of 2-deuterated benzothiazoline as a deuterium donor in combination with a chiral phosphoric acid, the transfer deuteration of ketimine and α-iminoester took place smoothly to give α-deuterated amines in high yields with excellent enantioselectivities. The remarkable kinetic isotope effect suggests that carbon-deuterium bond cleavage is the rate-determining step.

Carbon–Deuterium Bonds as Probes of Protein Thermal Unfolding

We report a residue-specific characterization of the thermal unfolding mechanism of ferric horse heart cytochrome c using C-D bonds site-specifically incorporated at residues dispersed throughout three different structural elements within the protein. As the temperature increases, Met80 first dissociates from the heme center, and the protein populates a folding intermediate before transitioning to a solvent exposed state. With further increases in temperature, the C-terminal helix frays and then loses structure along with the core of the protein. Interestingly, the data also reveal that the state populated at high temperature retains some structure and possibly represents a molten globule. Elucidation of the detailed unfolding mechanism and the structure of the associated molten globule, both of which represent challenges to conventional techniques, highlights the utility of the C-D technique.

Deuterium Retention and Desorption Behavior of Co-Deposited Carbon Film Produced in Gap

Co-deposition of deuterium with carbon in an opening on a plasma-facing surface, a so-called ‘gap’, was simulated by using a deuterium arc discharge with carbon electrodes. The carbon deposition distribution and deuterium retention/desorption behavior of the carbon film were investigated. The amount of deposited carbon decreased exponentially with an increase of the distance from the gap entrance and more rapidly decreased with an increase in discharge gas pressure. The deuterium concentration in the carbon film increased with discharge gas pressure. At a high discharge gas pressure of 36 Pa, the atomic ratio of D/C in the carbon film reached as high as 0.9. Deuterium retained in the film desorbed mainly in the forms of D2, HD, CD4 and C2D4. The desorption behavior of retained deuterium depended on D/C. In a film with a high D/C ratio, desorption of D2 started at lower temperatures. The amount of desorbed hydrocarbons (CD4 and C2D4) increased with D/C. Carbon film with high D/C tended to contain a polymer-like structure, which could be related to the desorption behavior of the retained deuterium. c

A Stepwise Solvent-Promoted SNi Reaction of α-d-Glucopyranosyl Fluoride: Mechanistic Implications for Retaining Glycosyltransferases

The solvolysis of α-d-glucopyranosyl fluoride in hexafluoro-2-propanol gives two products, 1,1,1,3,3,3-hexafluoropropan-2-yl α-d-glucopyranoside and 1,6-anhydro-β-D-glucopyranose. The ratio of these two products is essentially unchanged for reactions that are performed between 56 and 100 °C. The activation parameters for the solvolysis reaction are as follows: ΔH(++) = 81.4 ± 1.7 kJ mol(-1), and ΔS(++) = -90.3 ± 4.6 J mol(-1) K(-1). To characterize, by use of multiple kinetic isotope effect (KIE) measurements, the TS for the solvolysis reaction in hexafluoro-2-propanol, we synthesized a series of isotopically labeled α-d-glucopyranosyl fluorides. The measured KIEs for the C1 deuterium, C2 deuterium, C5 deuterium, anomeric carbon, ring oxygen, O6, and solvent deuterium are 1.185 ± 0.006, 1.080 ± 0.010, 0.987 ± 0.007, 1.008 ± 0.007, 0.997 ± 0.006, 1.003 ± 0.007, and 1.68 ± 0.07, respectively. The transition state for the solvolysis reaction was modeled computationally using the experimental KIE values as constraints. Taken together, the reported data are consistent with the retained solvolysis product being formed in an S(N)i (D(N)(++)*A(Nss)) reaction with a late transition state in which cleavage of the glycosidic bond is coupled to the transfer of a proton from a solvating hexafluoro-2-propanol molecule. In comparison, the inverted product, 1,6-anhydro-β-D-glucopyranose, is formed by intramolecular capture of a solvent-equilibrated glucopyranosylium ion, which results from dissociation of the solvent-separated ion pair formed in the rate-limiting ionization reaction (D(N)(++) + A(N)). The implications that this model reaction have for the mode of action of retaining glycosyltransferases are discussed.

A B3LYP investigation of the conformational and environmental sensitivity of carbon–deuterium frequencies of aryl-perdeuterated phenylalanine and tryptophan

Carbon–deuterium labeled amino acids can serve as sensitive probes for biophysical characterization. Although multiple research groups have used infrared spectroscopy in conjunction with alkyl backbone or side-chain deuterated amino acids for the biophysical characterization of conformational and/or environmental changes, it was not entirely clear to the authors that perdeuterated aryl rings would demonstrate a similar sensitivity toward conformational or environmental changes. In an effort to evaluate the sensitivity of aryl carbon–deuterium (C–D) IR frequencies, a B3LYP investigation of the sensitivity of aryl C–D frequencies toward conformational and environmental changes was conducted for phenylalanine (Phe) and tryptophan (Trp). To compensate for the low molar absorptivity of C–D frequencies, perdeuterated aryl rings were investigated, which are commercially available and can be readily compared to experimental data. B3LYP results suggest that aryl-deuterated Phe and Trp will exhibit moderate sensitivities toward conformational and environmental changes with frequency shifts upward of 13 and 26 cm−1 for Phe and Trp, respectively. B3LYP predicts that conformational sensitivity arises from dipole changes and not orbital alignment changes. In an effort to mimic what might be observed experimentally, simulated IR spectra were created and show absorption band changes with conformational and environmental changes, which indicate that IR characterization of perdeuterated aryl rings in amino acids could serve as a biophysical tool.