Loss Of Deuterium Research Articles

Ion source parameters and hydrogen scrambling in the ECD of selectively deuterated peptides

Hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS) has become an important method to study protein dynamics in solution. Recently, electron-based fragmentation methods (ECD and ETD) have been utilized in HDX-MS/MS experiments as experimental tools to increase the spatial resolution (the ability to obtain deuterium levels of individual residues). An essential prerequisite for this approach is that the level of hydrogen scrambling is negligible. The occurrence of hydrogen scrambling depends critically on the extent of vibrational excitation in the mass spectrometer. In particular, the desolvation process in the electrospray ion source is likely to induce scrambling at standard operating conditions. Consequently, finding experimental conditions that minimize hydrogen scrambling to a negligible level is thus pivotal for the application of electron-based fragmentation in HDX-MS/MS experiments. In the present work, we investigate the occurrence of scrambling in the Apollo I electrospray ion source using ECD of selectively deuterium labeled peptides. The electrospray ion source settings leading to minimal scrambling were identified. Furthermore, an energy dependent loss of deuterium occurring in the ion source was also observed. This loss was critically dependent on the occurrence of scrambling.

Coupled Methyl Group Epimerization and Reduction by Polyketide Synthase Ketoreductase Domains. Ketoreductase-Catalyzed Equilibrium Isotope Exchange

Incubation of [2-(2)H]-(2S,3R)-2-methyl-3-hydroxypentanoyl-SACP ([2-(2)H]-1a) with the epimerizing ketoreductase domain EryKR1 in the presence of a catalytic amount NADP(+) (0.05 equiv) resulted in time- and cofactor-dependent washout of deuterium from 1a, as a result of equilibrium isotope exchange of transiently generated [2-(2)H]-2-methyl-3-ketopentanoyl-ACP. Incubations of [2-(2)H]-(2S,3S)-2-methyl-3-hydroxy-pentanoyl-SACP with RifKR7 and with NysKR1 also resulted in time-dependent loss of deuterium. By contrast, incubations of [2-(2)H]-(2R,3S)-2-methyl-3-hydroxypentanoyl-SACP and [2-(2)H]-(2R,3R)-2-methyl-3-hydroxypentanoyl-SACP with the non-epimerizing ketoreductase domains EryKR6 and TylKR1, respectively, did not result in any significant washout of deuterium. The isotope exchange assay directly establishes that specific polyketide synthase ketoreductase domains also have an intrinsic epimerase activity, thus enabling mechanistic analysis of a key determinant of polyketide stereocomplexity.

A Combined Experimental and Theoretical Study on the Gas‐Phase Synthesis of Toluene under Single Collision Conditions

physical-organic chemistry communities. Toluene portrays the simplest representative of an alkyl-substituted benzene molecule with the methyl group enhancing the reactivity toward radical and electrophile aromatic substitution compared to benzene. It is further considered as a crucial building block to form methyl-substituted polycyclic aromatic hydrocarbons (PAHs) such as 1and 2-methylnaphthalene. In combustion flames, the formation of phenylacetylene 2, styrene 3, and biphenyl 4 (Scheme 1) can be rationalized through phenyl addition. Atomic hydrogen is eliminated upon reaction of the phenyl radical with acetylene (C2H2), [14] ethylene (C2H4), [15] and benzene (C6H6). [16] The analogous reaction of the phenyl radical with methane (CH4) does not lead to toluene, but solely to benzene plus a methyl radical (CH3) through hydrogen abstraction. [17] Consequently, the formation routes of toluene in combustion flames and in the interstellar medium have not been unraveled to date. Here, we show that the reaction of the ethynyl radical (CCH; C2H) with isoprene (2-methyl-1,3-butadiene; C5H8) presents a facile, barrierless route to the toluene molecule in a single collision event in the gas phase through the reaction of two acyclic precursor molecules. This reaction is also of interest to the physical-organic chemistry community since it represents a benchmark system to untangle the formation of a methylsubstituted aromatic molecule through radical substitution reactions involving successive isomerizations through cyclization and hydrogen shifts. Reactive scattering signal from the reaction of [D1]ethynyl radical C2D(X S) with isoprene (C5H8; X A’) was monitored at mass-to-charge ratios (m/z) of 93 (C7H7D ), 92 (C7H6D /C7H8 ), and 91 (C7H5D /C7H7 ). The signal atm/ z= 93 originates from the C7H7D product(s) formed through atomic hydrogen loss, whereas ion counts at m/z= 92 could have two contributions: an atomic deuterium loss connected to the formation of C7H8 products or dissociative ionization of C7H7D products in the electron impact ionizer of the detector. The signal at m/z= 91 may depict two contributions from dissociative electron impact ionization of C7H7D and/or C7H6D product molecules. However, all data could be fit with the product mass combinations of 93 amu (C7H7D) plus 1 amu (H). This finding suggests the existence of a [D1]ethynyl (C2D, 26 amu) versus atomic hydrogen replacement channel and the gas-phase synthesis of a molecule with the molecular formula C7H7D (Figure 1). Considering that the [D1]ethynyl reactant does not have a hydrogen atom, the hydrogen atom is emitted from the isoprene molecule. The laboratory angular distribution (Figure 2) extends at least 408 within the scattering plane and peaks close to the center-of-mass angle at 41.1 1.28. These results suggest indirect scattering dynamics through C7H8D complex(es). Scheme 1. Toluene 1, phenylacetylene 2, styrene 3, and biphenyl 4.

Time course of hepatic gluconeogenesis during hindlimb suspension unloading

The goal of this work was to determine the time-dependent changes in fractional hepatic gluconeogenesis (GNG) during conditions of hindlimb suspension unloading (HSU), a 'ground-based' method for inducing muscular atrophy to simulate space flight. We hypothesized that GNG would increase in HSU conditions as a result of metabolic shifts in the liver and skeletal muscle. A significant and progressive atrophy was observed in the soleus (30, 47 and 55%) and gastrocnemius muscles (0, 15 and 17%) after 3, 7 and 14 days of HSU, respectively. Fractional hepatic GNG was determined following the incorporation of deuterium from deuterated water ((2)H(2)O) into C-H bonds of newly synthesized glucose after an 8 h fast. Enrichment of plasma glucose with (2)H was measured using the classic method of Landau et al. (the 'hexamethylenetetramine (HMT) method'), based on specific (2)H labelling of glucose carbons, and the novel method of Chacko et al. ('average method'), based on the assumption of equal (2)H enrichment on all glucose carbons (except C2). After 3 days of HSU, fractional GNG was significantly elevated in the HSU group, as determined by either method (∼13%, P < 0.05). After 7 and 14 days of HSU, gluconeogenesis was not significantly different. Both analytical methods yielded similar time-dependent trends in gluconeogenic rates, but GNG values determined using the average method were consistently lower (∼30%) than those found by the HMT method. To compare and validate the average method against the HMT method further, we starved animals for 13 h to allow for hepatic GNG to contribute 100% to endogenous glucose production. The HMT method yielded 100% GNG, while the average method yielded GNG of ∼70%. As both methods used the same values of precursor enrichment, we postulated that the underestimation of gluconeogenic rate was as a result of differences in the measurements of product enrichment ((2)H labelling of plasma glucose). This could be explained by the following factors: (i) loss of deuterium via exchange between acetate and glucose; (ii) interference caused by fragment m/z 169, representing multiple isobaric species; and (iii) interference from other sugars at m/z 169. In conclusion, HSU caused a time-dependent increase in hepatic gluconeogenesis, irrespective of the analytical methods used.

Spatially Resolved Protein Hydrogen Exchange Measured by Subzero-Cooled Chip-Based Nanoelectrospray Ionization Tandem Mass Spectrometry

Mass spectrometry has become a valuable method for studying structural dynamics of proteins in solution by measuring their backbone amide hydrogen/deuterium exchange (HDX) kinetics. In a typical exchange experiment one or more proteins are incubated in deuterated buffer at physiological conditions. After a given period of deuteration, the exchange reaction is quenched by acidification (pH 2.5) and cooling (0 °C) and the deuterated protein (or a digest thereof) is analyzed by mass spectrometry. The unavoidable loss of deuterium (back-exchange) that occurs under quench conditions is undesired as it leads to loss of information. Here we describe the successful application of a chip-based nanoelectrospray ionization mass spectrometry top-down fragmentation approach based on cooling to subzero temperature (-15 °C) which reduces the back-exchange at quench conditions to very low levels. For example, only 4% and 6% deuterium loss for fully deuterated ubiquitin and β(2)-microglobulin were observed after 10 min of back-exchange. The practical value of our subzero-cooled setup for top-down fragmentation HDX analyses is demonstrated by electron-transfer dissociation of ubiquitin ions under carefully optimized mass spectrometric conditions where gas-phase hydrogen scrambling is negligible. Our results show that the known dynamic behavior of ubiquitin in solution is accurately reflected in the deuterium contents of the fragment ions.

Chemical dynamics of the CH(X2Π) + C2H4(X1A1g), CH(X2Π) + C2D4(X1A1g), and CD(X2Π) + C2H4(X1A1g) reactions studied under single collision conditions

The crossed beam reactions of the methylidyne radical with ethylene (CH(X(2)Π) + C(2)H(4)(X(1)A(1g))), methylidyne with D4-ethylene (CH(X(2)Π) + C(2)D(4)(X(1)A(1g))), and D1-methylidyne with ethylene (CD(X(2)Π) + C(2)H(4)(X(1)A(1g))) were conducted at nominal collision energies of 17-18 kJ mol(-1) to untangle the chemical dynamics involved in the formation of distinct C(3)H(4) isomers methylacetylene (CH(3)CCH), allene (H(2)CCCH(2)), and cyclopropene (c-C(3)H(4)) via C(3)H(5) intermediates. By tracing the atomic hydrogen and deuterium loss pathways, our experimental data suggest indirect scattering dynamics and an initial addition of the (D1)-methylidyne radical to the carbon-carbon double bond of the (D4)-ethylene reactant forming a cyclopropyl radical intermediate (c-C(3)H(5)/c-C(3)D(4)H/c-C(3)H(4)D). The latter was found to ring-open to the allyl radical (H(2)CCHCH(2)/D(2)CCHCD(2)/H(2)CCDCH(2)). This intermediate was found to be long lived with life times of at least five times its rotational period and decomposed via atomic hydrogen/deuterium loss from the central carbon atom (C2) to form allene via a rather loose exit transition state in an overall strongly exoergic reaction. Based on the experiments with partially deuterated reactants, no compelling evidence could be provided to support the formation of the cyclopropene and methylacetylene isomers under single collision conditions. Likewise, hydrogen/deuterium shifts in the allyl radical intermediates or an initial insertion of the (D1)-methylidyne radical into the carbon-hydrogen/deuterium bond of the (D4)-ethylene reactant were found to be-if at all-of minor importance. Our experiments propose that in hydrocarbon-rich atmospheres of planets and their moons such as Saturn's satellite Titan, the reaction of methylidyne radicals should lead predominantly to the hitherto elusive allene molecule in these reducing environments.

Experimental Evidence of Phosphoenolpyruvate Resynthesis from Pyruvate in Illuminated Leaves

Day respiration is the cornerstone of nitrogen assimilation since it provides carbon skeletons to primary metabolism for glutamate (Glu) and glutamine synthesis. However, recent studies have suggested that the tricarboxylic acid pathway is rate limiting and mitochondrial pyruvate dehydrogenation is partly inhibited in the light. Pyruvate may serve as a carbon source for amino acid (e.g. alanine) or fatty acid synthesis, but pyruvate metabolism is not well documented, and neither is the possible resynthesis of phosphoenolpyruvate (PEP). Here, we examined the capacity of pyruvate to convert back to PEP using (13)C and (2)H labeling in illuminated cocklebur (Xanthium strumarium) leaves. We show that the intramolecular labeling pattern in Glu, 2-oxoglutarate, and malate after (13)C-3-pyruvate feeding was consistent with (13)C redistribution from PEP via the PEP-carboxylase reaction. Furthermore, the deuterium loss in Glu after (2)H(3)-(13)C-3-pyruvate feeding suggests that conversion to PEP and back to pyruvate washed out (2)H atoms to the solvent. Our results demonstrate that in cocklebur leaves, PEP resynthesis occurred as a flux from pyruvate, approximately 0.5‰ of the net CO(2) assimilation rate. This is likely to involve pyruvate inorganic phosphate dikinase and the fundamental importance of this flux for PEP and inorganic phosphate homeostasis is discussed.

Order and disorder in Ca 2ND 0.90H 0.10–A structural and thermal study

The structure of calcium nitride hydride and its deuterided form has been re-examined at room temperature and studied at high temperature using neutron powder diffraction and thermal analysis. When synthesised at 600 °C, a mixture of both ordered and disordered Ca 2ND 0.90H 0.10 phases results. The disordered phase is the minor component and has a primitive rocksalt structure (spacegroup Fm3 m) with no ordering of D/N on the anion sites and the ordered phase is best described using the rhombohedral spacegroup R-3 m with D and N arranged in alternate layers in (111) planes. This mixture of ordered and disordered phases exists up to 580 °C, at which the loss of deuterium yields Ca 2ND 0.85 with the disappearance of the disordered phase. In the new ordered phase there exists a similar content of vacancies on both anion sites; to achieve this balance, a little N transfers onto the D site, whereas there is no indication of D transferring onto the N-sites. These observations are thought to indicate that the D/N ordering is difficult to achieve with fully occupied anion sites. It has previously been reported that Ca 2ND has an ordered cubic cell with alternating D and N sites in the [100] directions [1]; however, for the samples studied herein, there were clearly two coexisting phases with apparent broadening/splitting of the primitive peaks but not for the ordered peaks. The rhombohedral phase was in fact metrically cubic; however, all the observed peaks were consistent with the rhombohedral unit cell with no peaks requiring the larger ordered cubic unit cell to be utilised. Furthermore this rhombohedral cell displays the same form of N–D ordering as the Sr and Ba analogues, which are metrically rhombohedral.

DNA Binding Restricts the Intrinsic Conformational Flexibility of Methyl CpG Binding Protein 2 (MeCP2)

Mass spectrometry-based hydrogen/deuterium exchange (H/DX) has been used to define the polypeptide backbone dynamics of full-length methyl CpG binding protein 2 (MeCP2) when free in solution and when bound to unmethylated and methylated DNA. Essentially the entire MeCP2 polypeptide chain underwent H/DX at rates faster than could be measured (i.e. complete exchange in ≤10 s), with the exception of the methyl DNA binding domain (MBD). Even the H/DX of the MBD was rapid compared with that of a typical globular protein. Thus, there is no single tertiary structure of MeCP2. Rather, the full-length protein rapidly samples many different conformations when free in solution. When MeCP2 binds to unmethylated DNA, H/DX is slowed several orders of magnitude throughout the MBD. Binding of MeCP2 to methylated DNA led to additional minor H/DX protection, and only locally within the N-terminal portion of the MBD. H/DX also was used to examine the structural dynamics of the isolated MBD carrying three frequent mutations associated with Rett syndrome. The effects of the mutations ranged from very little (R106W) to a substantial increase in conformational sampling (F155S). Our H/DX results have yielded fine resolution mapping of the structure of full-length MeCP2 in the absence and presence of DNA, provided a biochemical basis for understanding MeCP2 function in normal cells, and predicted potential approaches for the treatment of a subset of RTT cases caused by point mutations that destabilize the MBD.

Stereochemistry and Mechanism of the Brønsted Acid Catalyzed Intramolecular Hydrofunctionalization of an Unactivated Cyclic Alkene

Through employment of deuterium-labeled substrates, the triflic acid catalyzed intramolecular exo addition of the X-H(D) (X=N, O) bond of a sulfonamide, alcohol, or carboxylic acid across the C=C bond of a pendant cyclohexene moiety was found to occur, in each case, with exclusive formation (≥90%) of the anti-addition product without loss or scrambling of deuterium as determined by (1)H and (2)H NMR spectroscopy and mass spectrometry analysis. Kinetic analysis of the triflic-acid-catalyzed intramolecular hydroamination of N-(2-cyclohex-2'-enyl-2,2-diphenylethyl)-p-toluenesulfonamide (1a) established the second-order rate law: rate=k(2)[HOTf][1a] and the activation parameters ΔH(++)=(9.7±0.5) kcal mol(-1) and ΔS(++)=(-35±5) cal K(-1) mol(-1). An inverse α-secondary kinetic isotope effect of k(D)/k(H) =(1.15±0.03) was observed upon deuteration of the C2' position of 1a, consistent with partial C-N bond formation in the highest energy transition state of catalytic hydroamination. The results of these studies were consistent with a mechanism for the intramolecular hydroamination of 1a involving concerted, intermolecular proton transfer from an N-protonated sulfonamide to the alkenyl C3' position of 1a coupled with intramolecular anti addition of the pendant sulfonamide nitrogen atom to the alkenyl C2' position.

The Manufacture of Deuterium and Its Compounds

THE NEWLY DISCOVERED isotope of hydrogen, deuterium, which has caused so much interest among scientists is rapidly being made available to interested purchasers. After six months of research, the writer has developed a commercially practical method for obtaining deuterium oxide of high purity. He has organized The California Isotope Co., which will be devoted to the manufacture of deuterium and its compounds. Starting with the electrolytic method first used successfully by G. N. Lewis and Ronald MacDonald, 1 the author has evolved an efficient and rapid process for separating the isotope. Heavy water that has been partially concentrated in the electrolytic cells of concerns making hydrogen gas by electrolysis is used as the raw product. This is put into a battery of cells and by a process of continual fractional electrolysis the concentration of H 2 2 O is increased continuously until the ultimate of nearly pure isotopic water is reached. Although the loss of deuterium in the process ...

Investigation of amide hydrogen back-exchange in Asp and His repeats measured by hydrogen (1H/2H) exchange mass spectrometry

Hydrogen/deuterium exchange (HDX) monitored by mass spectrometry (HDX-MS) has become a valuable tool in studies of protein dynamics and protein interactions. Isotopic exchange is typically initiated by diluting a protein solution into deuterated buffer at physiological conditions. For MS analysis, the exchange reaction is quenched by acidification and cooling and the labeled protein (or a digest thereof) is analyzed by mass spectrometry. An inevitable deuterium loss occurs during quench conditions (i.e., back-exchange). The primary structure of the peptide influences the back-exchange rate due to steric hindrance by bulky side groups and by inductive and charge effects. Here we show that the back-exchange in histidine repeats (His6) at HDX-MS quench conditions is complete within a few seconds using either acetic acid or formic acid in the quench solution, while aspartic acid repeats (Asp6) retain deuterons for several minutes using formic acid. We employ electron transfer dissociation to obtain residue-specific deuterium levels of the Asp repeat in K2D6IIKIIK using a hybrid linear ion trap-Orbitrap mass spectrometer. Our results show an unexpected uneven distribution of deuterium in the Asp repeat. The implication of the rapid back-exchange of His repeats for HDX-MS protein hydrogen exchange studies is discussed. We also discuss the implications of retained deuterons in the Asp repeat of K2D6IIKIIK when this peptide is used as a probe for the occurrence of hydrogen scrambling.

ChemInform Abstract: On the Mechanism of the Ruthenium-Catalyzed Reconstitutive Condensation of Allylic Alcohols and Terminal Alkynes.

The ruthenium complex ({eta}{sup 5}-C{sub 5}H{sub 5})(PPh{sub 3}){sub 2}RuCl (1) catalyzes the addition of allytic alcohols to terminal alkynes, yielding {Beta}{gamma}-unsaturated ketones. The intermediacy of a ruthenium vinylidene complex is indicated by the synthesis of this proposed intermediate and the demonstration of the same reaction profile as with catalyst 1. Loss of terminal deuterium in labeled alkynes supports this conclusion. Ligand substitution studies demonstrate the necessity of phosphine loss and precoordination of the allylic alcohol. Deuterium labeling of allyl alcohol demonstrates that the two allylic termini do not become equivalent and that the olefin geometry does not scramble. In contrast to these observations, 3-buten-2-ol shows complete regioselectivity in the condensation but randomization of olefin geometry as determined by deuterium labeling. A cohesive mechanistic rationale accommodates these seemingly disparate observations. 24 refs., 4 figs.

Unusual base-catalyzed exchange in the synthesis of deuterated PF-2413873

The preparation of deuterated PF-2413873 (4-[3-cyclopropyl-1-(methanesulfonylmethyl)-5-methyl-1H-pyrazol-4-yl]oxy-2,6-dimethylbenzonitrile, 1) is described for use as a bioanalytical standard in clinical trials. Two strategies were investigated. The sulfone-containing substituent was labelled by base-catalyzed exchange, but unacceptable deuterium loss was noted under assay conditions. Alternatively, labelling 4-cyano-3,5-dimethylphenol was achieved by heating with deuterium oxide over platinum oxide. After building up the pyrazole ring we discovered that, during the subsequent alkylation to attach the methylthiomethyl group, the base, potassium t-butoxide, caused unwanted scrambling of deuteriums on the aromatic portion and the methylthiomethyl group. Thus, it was necessary to remove all base-labile hydrogens to prevent their exchange. This was accomplished by alkylating the pyrazole with per-deuterated chloromethyl methylsulfide, oxidation to the sulfone, and selective removal of its deuteriums by treatment with sodium hydroxide. The unusual sensitivity and selectivity of these base-promoted exchange reactions are discussed. Thus, 4-[3-cyclopropyl-1-(methanesulfonylmethyl)-5-methyl-1H-pyrazol-4-yl]oxy-[2H6]2,6-dimethyl-[3,5-2H]benzonitrile (17) was obtained, labelled with eight deuterium atoms and an acceptable D0/D8 ratio. Copyright © 2009 John Wiley & Sons, Ltd.

Conformational Changes during Nucleotide Selection by Sulfolobus solfataricus DNA Polymerase Dpo4

The mechanism of nucleotide selection by Y-family DNA polymerases has been the subject of intense study, but significant structural contacts and/or conformational changes that relate to polymerase fidelity have been difficult to identify. Here we report on the conformational dynamics of a model Y-family polymerase Dpo4 from Sulfolobus solfataricus. Hydrogen-deuterium exchange in tandem with mass spectrometry was used to monitor changes in Dpo4 structure as a function of time and the presence or absence of specific substrates and ligands. Analysis of the data revealed previously unrecognized structural changes that accompany steps in the catalytic cycle leading up to phosphoryl transfer. For example, the solvent accessibility of the alphaB-loop-alphaC region in the finger domain decreased in the presence of all four dNTP insertion events, but the rate of deuterium exchange, an indicator of conformational flexibility, only decreased during an accurate insertion event. Of particular note is a change in the region surrounding the H-helix of the thumb domain. Upon binding DNA and Mg2+, the H-helix showed a decrease in solvent accessibility and flexibility that was relaxed only upon addition of dCTP, which forms a Watson-Crick base pair with template dG and not during mispairing events. The current study expands upon a previous report from our group that used a fluorescent probe located near the thumb domain to measure the kinetic properties of Dpo4 conformational changes. We now present a model for nucleotide selection by Dpo4 that arises from a synthesis of both structural and kinetic data.

On the existence of the hypervalent H 3O, H 2DO, HD 2O, and D 3O radicals

The existence of oxonium radicals on the microsecond time scale has been up for much debate, and strong isotope effects have been reported. Here we show that H 3O, H 2DO, HD 2O, and D 3O all have subnanosecond lifetimes when they are formed in charge-transfer collisions between oxonium cations and cesium. A kinetic isotope effect on hydrogen versus deuterium loss from HD 2O was found to be 2.4, similar to that found in dissociative recombination of HD 2O + and for dissociation after electron transfer to HD 2O + from potassium.

Biosynthesis of scopoletin and scopolin in cassava roots during post-harvest physiological deterioration: The E- Z-isomerisation stage

Two to three days after harvesting, cassava ( Manihot esculenta Crantz) roots suffer from post-harvest physiological deterioration (PPD) when secondary metabolites are accumulated. Amongst these are hydroxycoumarins (e.g. scopoletin and its glucoside scopolin) which play roles in plant defence and have pharmacological activities. Some steps in the biosynthesis of these molecules are still unknown in cassava and in other plants. We exploit the accumulation of these coumarins during PPD to investigate the E-Z-isomerisation step in their biosynthesis. Feeding cubed cassava roots with E-cinnamic-3,2′,3′,4′,5′,6′- d 5 acid gave scopoletin- d 2. However, feeding with E-cinnamic-3,2′,3′,4′,5′,6′- d 6 and E-cinnamic-2,3,2′,3′,4′,5′,6′- d 7 acids, both gave scopoletin- d 3, the latter not affording the expected scopoletin- d 4. We therefore synthesised and fed with E-cinnamic-2- d 1 when unlabelled scopoletin was biosynthesised. Solely the hydrogen (or deuterium) at C2 of cinnamic acid is exchanged in the biosynthesis of hydroxycoumarins. If the mechanism of E-Z-cinnamic acid isomerisation were photochemical, we would not expect to see the loss of deuterium which we observed. Therefore, a possible mechanism is an enzyme catalysed 1,4-Michael addition, followed by σ-bond rotation and hydrogen (or deuterium) elimination to yield the Z-isomer. Feeding the roots under light and dark conditions with E-cinnamic-2,3,2′,3′,4′,5′,6′- d 7 acid gave scopoletin- d 3 with no significant difference in the yields. We conclude that the E-Z-isomerisation stage in the biosynthesis of scopoletin and scopolin, in cassava roots during PPD, is not photochemical, but could be catalysed by an isomerase which is independent of light.

Synthesis of 19‐trideuterated ent‐testosterone and the GABAA receptor potentiators ent‐androsterone and ent‐etiocholanolone

19-Trideuteromethyl enantiomers of androgens namely ent-testosterone, ent-androsterone and ent-etiocholanolone were prepared by total synthesis. The isotope labeling at the carbon-19 angular methyl group was achieved by using deuterated methyl iodide (99.5% d(3)) for introduction of C-19 before closure of the steroid A-ring. This method yields 19,19,19-trideuterated steroids without increasing the number of steps involved in the total synthesis of ent-androgens. Analysis by mass spectrometry showed no loss of deuterium during incorporation of C-19 into ent-testosterone. The availability of the compounds will enable these ent-androgens to be distinguished by mass spectrometry from their natural enantiomers in future pharmacokinetic and metabolic studies.

Ion mobility adds an additional dimension to mass spectrometric analysis of solution‐phase hydrogen/deuterium exchange

The goal of this study was to determine the utility of adding ion mobility spectrometry to studies probing the solution-phase hydrogen/deuterium exchange (HX) of proteins. The HX profile of the Hck SH3 domain was measured at both the intact protein and the peptic peptide levels in the Waters Synapt HDMS system which uses a traveling wave to accomplish ion mobility separation prior to time-of-flight (Tof) m/z analysis. The results indicated a similar loss of deuterium with or without use of mobility in the Synapt and a level of deuterium loss comparable with a non-mobility Q-Tof instrument. The drift time of this small protein and its peptic peptides did not noticeably change due to solution-based deuterium incorporation. Importantly, ion mobility separations provided an orthogonal dimension of separation in addition to the reversed-phase high-performance liquid chromatography (RP-HPLC). The additional dimension of separation allowed for the deconvolution of overlapping isotopic patterns for co-eluting peptides and extraction of valuable deuterium incorporation data for those peptides. Taken together, these results indicate that including ion mobility separation in HX MS analyses further improves the mass spectrometry portion of such experiments.

Crystal Structure of a Prolactin Receptor Antagonist Bound to the Extracellular Domain of the Prolactin Receptor

The crystal structure of the complex between an N-terminally truncated G129R human prolactin (PRL) variant and the extracellular domain of the human prolactin receptor (PRLR) was determined at 2.5A resolution by x-ray crystallography. This structure represents the first experimental structure reported for a PRL variant bound to its cognate receptor. The binding of PRL variants to the PRLR extracellular domain was furthermore characterized by the solution state techniques, hydrogen exchange mass spectrometry, and NMR spectroscopy. Compared with the binding interface derived from mutagenesis studies, the structural data imply that the definition of PRL binding site 1 should be extended to include residues situated in the N-terminal part of loop 1 and in the C terminus. Comparison of the structure of the receptor-bound PRL variant with the structure reported for the unbound form of a similar analogue ( Jomain, J. B., Tallet, E., Broutin, I., Hoos, S., van Agthoven, J., Ducruix, A., Kelly, P. A., Kragelund, B. B., England, P., and Goffin, V. (2007) J. Biol. Chem. 282, 33118-33131 ) demonstrates that receptor-induced changes in the backbone of the four-helix bundle are subtle, whereas large scale rearrangements and structuring occur in the flexible N-terminal part of loop 1. Hydrogen exchange mass spectrometry data imply that the dynamics of the four-helix bundle in solution generally become stabilized upon receptor interaction at binding site 1.