Multiple Deuterium Research Articles

Synthesis of 13C Labeled Vitamin D Metabolites for Their Use in LC-MS/MS: Valuable Tools for Cancer Research and Other Applications.

Simultaneous assessment of various vitamin D metabolites in human biofluids by liquid chromatography tandem mass spectrometry (LC-MS/MS) represents a new promising tool for the differential diagnosis of vitamin D-related diseases. Particularly, besides 25(OH)VD2/3, low-abundant medicinally relevant vitamin D metabolites, such as 24,25(OH)2VD2/3, 1,25(OH)2VD2/3, and 1,24,25(OH)3VD2/3, along with their 3-epi-derivatives have to be considered. The assessment of these metabolites by LC-MS/MS requires the development of calibration and reference standards, that is, their labeling with multiple deuterium-, or even better, 13C- atoms. Overall, 10 13C-labeled vitamin D metabolites have been chemically synthesized and obtained in good yield and high purity. Access to a wide variety of 13C-labeled highly pure vitamin D metabolites enables the advancement of LC-MS/MS applications towards a better understanding of differential diagnosis of vitamin D-related diseases.

Study of runaway electron dynamics at the ASDEX Upgrade tokamak during impurity injection using fast hard x-ray spectrometry

To study the runaway electron (RE) dynamics during plasma discharge and develop scenarios for disruption mitigation, a hard x-ray (HXR) spectrometric system has been developed and commissioned at the ASDEX Upgrade tokamak (AUG). The diagnostic system consists of two high-performance spectrometers based on LaBr3(Ce) scintillation detectors supplied with advanced electronics and analysis algorithms. These spectrometers view the AUG tokamak chamber quasi-radially at the equatorial plane. The measurements were carried out in the RE beam generation regimes by injecting argon into a deuterium plasma. In the interaction of a developed RE beam with a heavy gas target, powerful bremsstrahlung flux is induced, reaching energy close to 20 MeV. The electron energy distributions were reconstructed from the measured HXR spectra by deconvolution methods. The experimentally obtained maximum RE energies at different discharge stages were compared with relativistic test particle simulations that include the effect of toroidal electric field, plasma collisional drag force, synchrotron deceleration force. It was observed that the electrons attain their maximum energies within 50–100 ms after the gas injection. It gradually decreases due to the drop in loop voltage, energy loss due to synchrotron radiation emission and collisions dissipation of energy with the background plasma. HXR measurements at the discharge with multiple deuterium pellet injections allowed observing the effects of plasma cooling and argon ion recombination after the pellet injections. Argon density in AUG plasma after massive gas injection was estimated using HXR measurements.

Dissecting the Mechanism of (R)-3-Hydroxybutyrate Dehydrogenase by Kinetic Isotope Effects, Protein Crystallography, and Computational Chemistry.

The enzyme (R)-3-hydroxybutyrate dehydrogenase (HBDH) catalyzes the enantioselective reduction of 3-oxocarboxylates to (R)-3-hydroxycarboxylates, the monomeric precursors of biodegradable polyesters. Despite its application in asymmetric reduction, which prompted several engineering attempts of this enzyme, the order of chemical events in the active site, their contributions to limit the reaction rate, and interactions between the enzyme and non-native 3-oxocarboxylates have not been explored. Here, a combination of kinetic isotope effects, protein crystallography, and quantum mechanics/molecular mechanics (QM/MM) calculations were employed to dissect the HBDH mechanism. Initial velocity patterns and primary deuterium kinetic isotope effects establish a steady-state ordered kinetic mechanism for acetoacetate reduction by a psychrophilic and a mesophilic HBDH, where hydride transfer is not rate limiting. Primary deuterium kinetic isotope effects on the reduction of 3-oxovalerate indicate that hydride transfer becomes more rate limiting with this non-native substrate. Solvent and multiple deuterium kinetic isotope effects suggest hydride and proton transfers occur in the same transition state. Crystal structures were solved for both enzymes complexed to NAD+:acetoacetate and NAD+:3-oxovalerate, illustrating the structural basis for the stereochemistry of the 3-hydroxycarboxylate products. QM/MM calculations using the crystal structures as a starting point predicted a higher activation energy for 3-oxovalerate reduction catalyzed by the mesophilic HBDH, in agreement with the higher reaction rate observed experimentally for the psychrophilic orthologue. Both transition states show concerted, albeit not synchronous, proton and hydride transfers to 3-oxovalerate. Setting the MM partial charges to zero results in identical reaction activation energies with both orthologues, suggesting the difference in activation energy between the reactions catalyzed by cold- and warm-adapted HBDHs arises from differential electrostatic stabilization of the transition state. Mutagenesis and phylogenetic analysis reveal the catalytic importance of His150 and Asn145 in the respective orthologues.

Mechanistic and computational studies of the reductive half-reaction of tyrosine to phenylalanine active site variants of D-arginine dehydrogenase.

The flavin-mediated enzymatic oxidation of a CN bond in amino acids can occur through hydride transfer, carbanion, or polar nucleophilic mechanisms. Previous results with D-arginine dehydrogenase from Pseudomonas aeruginosa (PaDADH) using multiple deuterium kinetic isotope effects (KIEs) and computational studies established preferred binding of the substrate protonated on the α-amino group, with cleavages of the NH and CH bonds occurring in asynchronous fashion, consistent with the three possible mechanisms. The hydroxyl groups of Y53 and Y249 are ≤4 Å from the imino and carboxylate groups of the reaction product iminoarginine, suggesting participation in binding and catalysis. In this study, we have investigated the reductive half-reactions of the Y53F and Y249F variants of PaDADH using substrate and solvent deuterium KIEs, solvent viscosity and pH effects, and quantum mechanical/molecular mechanical computational approaches to gain insights into the catalytic roles of the tyrosines and evaluate whether their mutations affect the transition state for substrate oxidation. Both Y53F and Y249F enzymes oxidized D-arginine with steady-state kinetic parameters similar to those of the wild-type enzyme. Rate constants for flavin reduction (k(red)) with D-leucine, a slow substrate amenable to rapid kinetics, were 3-fold smaller than the wild-type value with similar pKa values for an unprotonated group of ∼10.0. Similar pKa values were observed for (app)Kd in the variant and wild-type enzymes. However, cleavage of the substrate NH and CH bonds in the enzyme variants occurred in synchronous fashion, as suggested by multiple deuterium KIEs on k(red). These data can be reconciled with a hydride transfer mechanism, but not with carbanion and polar nucleophilic mechanisms.

Insights on the Mechanism of Amine Oxidation Catalyzed by d-Arginine Dehydrogenase Through pH and Kinetic Isotope Effects

The mechanism of amine oxidation catalyzed by D-arginine dehydrogenase (DADH) has been investigated using steady-state and rapid reaction kinetics, with pH, substrate and solvent deuterium kinetic isotope effects (KIE) as mechanistic probes, and computational studies. Previous results showed that 85-90% of the flavin reduction reaction occurs in the mixing time of the stopped-flow spectrophotometer when arginine is the substrate, precluding a mechanistic investigation. Consequently, leucine, with slower kinetics, has been used here as the flavin-reducing substrate. Free energy calculations and the pH profile of the K(d) are consistent with the enzyme preferentially binding the zwitterionic form of the substrate. Isomerization of the Michaelis complex, yielding an enzyme-substrate complex competent for flavin reduction, is established due to an inverse hyperbolic dependence of k(cat)/K(m) on solvent viscosity. Amine deprotonation triggers the oxidation reaction, with cleavage of the substrate NH and CH bonds occurring in an asynchronous fashion, as suggested by the multiple deuterium KIE on the rate constant for flavin reduction (k(red)). A pK(a) of 9.6 signifies the ionization of a group that facilitates flavin reduction in the unprotonated form. The previously reported high-resolution crystal structures of the iminoarginine and iminohistidine complexes of DADH allow us to propose that Tyr(53), on a mobile loop covering the active site, may participate in substrate binding and facilitate flavin reduction.

Analysis of Conformational Changes during Activation of Protein Kinase Pak2 by Amide Hydrogen/Deuterium Exchange

During apoptotic stress, protein kinase Pak2 is cleaved by caspase 3 to form a heterotetramer that is constitutively activated following autophosphorylation. The active protein kinase migrates slightly slower than the inactive holoenzyme when analyzed by gel filtration, suggesting an expanded conformation. Activation of Pak2 comprises a series of structural changes resulting from caspase cleavage, ATP binding, and autophosphorylation of Pak2. Changes at each step were individually analyzed by amide hydrogen/deuterium exchange coupled with mass spectrometry and compared with inactive Pak2. The auto-inhibited form was shown to bind ATP in the active site, with minor changes in the glycine loop and the autoinhibitory domain (AID). Caspase cleavage produced significant changes in solvent accessibility in the AID and upper lobe of the catalytic domain. Cleavage of ATP-bound Pak2 relaxes the allosteric inhibition, as shown by increased solvent accessibility in the upper and lower lobes, including the G-helix, facilitating the autophosphorylation of two sites required for activation, Ser-141 in the regulatory domain and Thr-402 in the catalytic domain. Autophosphorylation increased the amide hydrogen/deuterium exchange solvent accessibility of the contact region between the AID and the G-helix, the E-F loop, and the N terminus. Thus, activation of Pak2 via caspase cleavage is associated with structural relaxation of Pak2 that allows for complete auto-phosphorylation, resulting in a more comprehensive solvent-exposed and conformationally dynamic enzyme.

Isomerisation of ω-hydroxyalkenes under hydroxycarbonylation conditions in palladium catalysed aqueous phase systems

The ω-hydroxyolefins 3-buten-1-ol, 3-buten-1-methyl-1-ol and 4-penten-1-ol were subjected to hydroxycarbonylation conditions in water in the presence of PdCl 2(PhCN) 2 and 4–8 equiv. of water soluble tris(3-sodiumsulfonatophenyl)phosphine (TPPTS), or N-bis( N′, N′-diethyl-2-aminoethyl)-4-aminomethylphenyl-diphenylphosphine (N3P). Under conditions of high conversion, the olefins primarily undergo isomerisation through a chain walking mechanism with selectivities for aldehyde ranging from 65% to 98%, with the lower values for longer chain alcohols. The lactones formed as the minor product are almost exclusively branched, indicating that in the first step 2,1-insertion is strongly favoured over 1,2-insertion. In the N3P system also linear lactone is produced at lower conversion. Running the reaction in D 2O produces multiple deuterium incorporation in all positions of the carbon chain. A mechanism is discussed.

Kinetic and Chemical Mechanisms of the fabG-Encoded Streptococcus pneumoniae β-Ketoacyl-ACP Reductase

Beta-ketoacyl-acyl carrier protein reductase (KACPR) catalyzes the NADPH-dependent reduction of beta-ketoacyl-acyl carrier protein (AcAc-ACP) to generate (3S)-beta-hydroxyacyl-ACP during the chain-elongation reaction of bacterial fatty acid biosynthesis. We report the evaluation of the kinetic and chemical mechanisms of KACPR using acetoacetyl-CoA (AcAc-CoA) as a substrate. Initial velocity, product inhibition, and deuterium kinetic isotope effect studies were consistent with a random bi-bi rapid-equilibrium kinetic mechanism of KACPR with formation of an enzyme-NADP(+)-AcAc-CoA dead-end complex. Plots of log V/K(NADPH) and log V/K(AcAc)(-)(CoA) indicated the presence of a single basic group (pK = 5.0-5.8) and a single acidic group (pK = 8.0-8.8) involved in catalysis, while the plot of log V vs pH indicated that at high pH an unprotonated form of the ternary enzyme complex was able to undergo catalysis. Significant and identical primary deuterium kinetic isotope effects were observed for V (2.6 +/- 0.4), V/K(NADPH) (2.6 +/- 0.1), and V/K(AcAc)(-)(CoA) (2.6 +/- 0.1) at pH 7.6, but all three values attenuated to values of near unity (1.1 +/- 0.03 or 0.91 +/- 0.02) at pH 10. Similarly, the large alpha-secondary deuterium kinetic isotope effect of 1.15 +/- 0.02 observed for [4R-(2)H]NADPH on V/K(AcAc)(-)(CoA) at pH 7.6 was reduced to a value of unity (1.00 +/- 0.04) at high pH. The complete analysis of the pH profiles and the solvent, primary, secondary, and multiple deuterium isotope effects were most consistent with a chemical mechanism of KACPR that is stepwise, wherein the hydride-transfer step is followed by protonation of the enolate intermediate. Estimations of the intrinsic primary and secondary deuterium isotope effects ((D)k = 2.7, (alpha)(-D)k = 1.16) and the correspondingly negligible commitment factors suggest a nearly full expression of the intrinsic isotope effects on (D)V/K and (alpha)(-D)V/K, and are consistent with a late transition state for the hydride transfer step. Conversely, the estimated intrinsic solvent effect ((D)2(O)k) of 5.3 was poorly expressed in the experimentally derived parameters (D)2(O)V/K and (D)2(O)V (both = 1.2 +/- 0.1), in agreement with the estimation that the catalytic commitment factor for proton transfer to the enolate intermediate is large. Such detailed knowledge of the chemical mechanism of KAPCR may now help guide the rational design of, or inform screening assay-design strategies for, potent inhibitors of this and related enzymes of the short chain dehydrogenase enzyme class.

Stoichiometric and catalytic H/D incorporation by cationic iridium complexes: a common monohydrido-iridium intermediate.

A mechanistic study of the stoichiometric and catalytic H/D exchange reactions involving cationic iridium complexes is presented. Strong evidence suggests that both stoichiometric and catalytic reactions proceed via a monohydrido-iridium species. Stoichiometric deuterium incorporation reactions introduce multiple deuterium atoms into the organic products when aryliridium compounds CpPMe(3)Ir(C(6)H(4)X)(OTf) (X = H, o-CH(3), m-CH(3), p-CH(3)) react with D(2). Multiple deuteration occurs at the unhindered positions (para and meta) of toluene, when X = CH(3). The multiple-deuteration pathway is suppressed in the presence of an excess of the coordinating ligand, CH(3)CN. The compound CpPMe(3)IrH(OTf) (1-OTf) is observed in low-temperature, stoichiometric experiments to support a monohydrido-iridium intermediate that is responsible for catalyzing multiple deuteration in the stoichiometric system. When paired with acetone-d(6)(), [CpPMe(3)IrH(3)][OTf] (4) catalytically deuterates a wide range of substrates with a variety of functional groups. Catalyst 4 decomposes to [CpPMe(3)Ir(eta(3)-CH(2)C(OH)CH(2))][OTf] (19) in acetone and to [CpPMe(3)IrH(CO)][OTf] (1-CO) in CH(3)OH. The catalytic H/D exchange reaction is not catalyzed by simple H(+) transfer, but instead proceeds by a reversible C-H bond activation mechanism.

Fluoride-facilitated deuterium exchange from DMSO-d6 to polyamide-based cryptands.

Multiple deuterium exchange between DMSO-d6 and amide hydrogens in two hexaamido cryptand fluoride receptors has been verified by 19F and 2H NMR and FAB mass spectral studies. Structural results for one of the complexes indicate a tricapped trigonal prism hydrogen bond coordination geometry around an encapsulated fluoride, with hydrogen bonds from fluoride to six amide and three phenyl hydrogens.

The mechanism of protonolysis of phenylplatinum(II) bonds in complexes with phenyl trans to nitrogen or carbon donors

Addition of acids of the form HX (X = Cl, BF4, CF3SO3, CF3CO2) to complexes [PtPh2(NN)] (NN = bu2bpy = 4,4'-di-tert-butyl-2,2'-bipyridine) and [PtPh(NCN)] (NCN = 2,6-C6H3(CH2NMe2)2) at –78 °C gave the corresponding phenyl(hydrido)platinum(IV) complexes [PtX(H)Ph2(NN)] and [PtX(H)Ph(NCN)], which decomposed by reductive elimination of benzene at about –20 °C to give the platinum(II) complexes [PtXPh(NN)] and [PtX(NCN)]. Further addition of HCl to [PtClPh(NN)] at low temperature gave [PtHCl2Ph(NN)], which decomposed above –10 °C, to give benzene and [PtCl2(NN)]. The reaction of DBF4 in the presence of excess CD3OD with [PtPh2(NN)] led to formation of C6H5D and C6H4D2 but with [PtPh(NCN)] no multiple deuterium incorporation was observed in the product benzene. The mechanisms of these reactions are discussed. Key words: platinum, phenyl, benzene, protonolysis.

A Combined Experimental and Density Functional Theory Investigation of Hydrocarbon Activation at a Cationic Platinum(II) Diimine Aqua Complex under Mild Conditions in a Hydroxylic Solvent

Controlled protonolysis of (Nf-Nf)Pt(CH3)2 (1; Nf-Nf = ArNCMeCMeNAr, Ar = 3,5-(CF3)2C6H3) with HBF4·Et2O in dichloromethane in the presence of small quantities of water gives the BF4- salt of the aqua complex (Nf-Nf)Pt(CH3)(H2O)+ (6). When dissolved in trifluoroethanol (TFE), 6(BF4-) effects the activation of methane and benzene C−H bonds under very mild conditions. Thus, 6 reacted with benzene in TFE-d3 at ambient temperature to quantitatively yield (Nf-Nf)Pt(C6H5)(H2O)+ and methane after 2−3 h. The use of C6D6 led to multiple incorporation of deuterium into the methane produced and suggests the involvement of methane σ-complex and benzene σ- or π-complex intermediates. When the solution of 6(BF4-) was exposed to 13CH4, an exchange reaction produced ca. 50% of (Nf-Nf)Pt(13CH3)(H2O)+ and CH4 after ca. 48 h at 45 °C. The reaction was inhibited by added water, suggesting that water is reversibly lost from 6 before C−H activation takes place. The use of CD4 resulted in multiple deuterium incorporation into t...

Analysis of Rates of Multiple Enzymes in Cell Lysates by Electrospray Ionization Mass Spectrometry

Analysis of Rates of Multiple Enzymes in Cell Lysates by Electrospray Ionization Mass Spectrometry

High-field fourier transform ion cyclotron resonance mass spectrometry for simultaneous trapping and gas-phase hydrogen/deuterium exchange of peptide ions

Gas-phase hydrogen/deuterium exchange of D 2O with [M + H] + ions of angiotensin II, angiotensin I, [Sar 1]-angiotensin II, bradykinin, des-Arg 1-bradykinin, des-Arg 9-bradykinin, luteinizing hormone releasing hormone (LH-RH), and substance P has been examined by Fourier transform ion cyclotron resonance mass spectrometry at 9.4 tesla. Because the FTICR dynamic range increases quadratically with magnetic field, parent ions from a mixture of several peptides may be confined simultaneously for long periods at high pressure (e.g., 1 h at 1 × 10 −5 torr) without quadrupolar axialization (and its attendant ion heating), for faster data acquisition and better controlled comparisons between different peptides. A high magnetic field also facilitates stored waveform inverse Fourier transform (SWIFT) isolation of monoisotopic [M + H] + parent ions, so that deuterium incorporation patterns may be determined directly without the need for isotopic distribution deconvolution. Finally, a higher magnetic field provides for a greatly extending trapping period, for measurement of much slower rates. Angiotensin I, angiotensin II, and [Sar 1]-angiotensin II are found to undergo a rapid exchange. Angiotensin II and [Sar 1]-angiotensin II exhibit multiple deuterium uptake distributions, corresponding to multiple gas-phase conformations. In contrast, substance P exchanges slowly and LH-RH displays no observable exchange. Comparison of the relative H/D exchange rates for bradykinin and its des-Arg-derivatives supports the hypothesis that bradykinin adopts a folded gas-phase conformation that unfolds upon removal of either terminal arginine residue.

Synthesis of the microbial elicitor syringolide 2 multiply labelled with deuterium

Multiply-deuterated syringolide 2 3 was prepared from D-xylulose and 8-bromooctanoic acid by a convergent route based on our previously reported biomimetic synthesis of this microbial elicitor. Key steps were the thermal acylation of the anisylidene-protected sugar 6 with the C-octenoyl Meldrum's acid derivative 7b, the one-pot triple-cyclisation of the xylulose β-ketoester 9b to 9,10-dehydrosyringolide 2 (10), and the introduction of multiple deuterium labels in the side chain by catalytic exchange and reduction. The route is applicable to the synthesis of tritium labelled syringolide 2 4.

Microwave measurements of the rotational spectrum and structure of butadieneiron tricarbonyl

The rotational spectra or 10 isotopomers or (butadiene)iron tricarbonyl were measured using a Flygare-Balle-type microwave spectrometer operating in the 5-17-GHz frequency range. The 30 measured rotational constants were used to determine 12 gas-phase structural parameters describing the cis-butadiene conformation and the locations and orientations or the butadiene and CO ligands relative to the iron atom. Kraitchman analyses or these microwave measurements, with single and multiple deuterium substitution, provide accurate data on the hydrogen atom coordinates in the complex. The terminal CH 2 groups or butadiene are found to be rotated by 28 o out or the butadiene plane, and the CH 2 plane is folded away from the C 1 -C 2 axis by 27 o in a direction away from the ion atom

Attainment of high plasma densities in TFTR with injection of multiple deuterium pellets

Sequential injection of multiple deuterium pellets combined with high power neutral beam heating in TFTR has produced stable plasmas with very high central densities, up to 5×1020 m-3. The line averaged densities achieved in these experiments correspond to Murakami parameters neR/BT up to 12×1019 m-2.T-1. This value, which does not appear to represent an intrinsic density limit, surpasses the values previously obtained in TFTR and exceeds, by more than a factor of two, the density limit predicted by the scaling of Greenwald et al. (Nuclear Fusion 28 (1988) 2199). The highest densities obtained with both pellet and gas fuelling have been achieved since boronization was applied to the first wall in TFTR. The characteristics and energy balance of the highest density plasma are discussed

Multiple deuterium occupancy of vacancies in Pd and related metals.

The binding energies of up to six deuterium atoms into monovacancies of Pd, Ni, Fe, Cu, Nb, and Mo have been calculated using the effective-medium theory. For all metals it is found that at least six D atoms can be accommodated in the vacancy. Of the systems studied the deuterium-deuterium interaction is found to be least repulsive for Pd. This multiple occupancy of the vacancy defect can have potentially significant implications for the recently proposed fusion of D atoms in Pd.

Results of pellet fuelled discharges on the Doublet III tokamak

Results are presented for multiple deuterium pellet injection into both the Ohmic phase and the beam heated phase of Doublet III discharges. Pellet injection experiments were carried out using an injector, built at Oak Ridge National Laboratory, which has the capability of firing up to 40 hydrogen or deuterium pellets per second at a speed of 800 m·s−1. For the deuterium pellets used in this experiment, one pellet raises the line average density to 1.3 × 1013 cm−3. In the Ohmic phase, the pellets penetrate deep into the plasma, but with 2.2 MW of neutral beam heating they penetrate only half as deep. This difference is mostly due to the increased ablation in the outer 25% of the plasma during beam heating. Ablation calculations using the neutral shielding model agree well with experimental results during Ohmic heating, but they predict a shallower pellet penetration than is actually observed during neutral beam heating. About 90–100% of the pellet mass can be accounted for after injection. Electron temperature profile relaxation accompanies pellet injection, but on a time-scale that is shorter than the pellet lifetime. A transient increase in the energy confinement time τE is possible, provided pellet penetration is sufficient to sustain a peaked density profile. In discharges with one or more multifractured pellets, which ablate rapidly at the plasma edge and thus broaden the density profile, no increase in τE is seen. These results indicate that continuous τE improvement may be possible if a peaked density profile can be maintained by sustained deep pellet penetration.

Multiple hydrogen occupancy of vacancies in Fe

The binding of multiple deuterium (D) atoms to vacancies in iron (Fe) is investigated both experimentally, by use of ion-beam techniques, and theoretically, by use of the effective-medium scheme. The experimental D-release stages are accounted for by trapping at vacancies with a binding enthalpy that depends on occupancy. It is found that for 1–2 D in a vacancy, the trap strength is 0.63 eV, whereas the binding enthalpy for 3–6 D in a vacancy is 0.43 eV. These results are in good agreement with predictions from the effective-medium theory.