High Levels Of Deuteration Research Articles

Organophotocatalytic α-deuteration of unprotected primary amines via H/D exchange with D2O.

We report a straightforward H/D exchange method for the synthesis of α-deuterated primary amines from a diverse set of primary amines with high levels of deuteration and chemo- and site selectivity and preparative utility. This cost-effective strategy enables the direct conversion of primary amines to α-deuterated counterparts using D2O as the deuterium source under mild reaction conditions without requiring additional functionality manipulation and with minimal byproduct production.

Similar levels of deuteration in the pre-stellar core L1544 and the protostellar core HH211

Context. In the centre of pre-stellar cores, deuterium fractionation is enhanced due to low temperatures and high densities. Therefore, the chemistry of deuterated molecules can be used to probe the evolution and the kinematics in the earliest stages of star formation. Aims. We analyse the deuterium fractionation of simple molecules, comparing the level of deuteration in the envelopes of the prototypical pre-stellar core L1544 in Taurus and the young protostellar core HH211 in Perseus. Methods. We used single-dish observations of CCH, HCN, HNC, and HCO+ and their 13C-, 18O-, and D-bearing isotopologues, detected with the 20 m telescope at the Onsala Space Observatory. We derived the column densities, and subsequently the carbon isotopic ratios and deuterium fractions of the molecules. Additionally, we used radiative transfer simulations and results from chemical modelling to reproduce the observed molecular lines. We used new collisional rate coefficients for HNC, HN13C DNC, and DCN that consider the hyperfine structure of these molecules. Results. For CCH, we find high levels of deuteration (10%) in both sources, consistent with other carbon chains. We find moderate deuteration of HCN (5–7%), with a slight enhancement towards the protostellar core. Equal levels of deuteration for HNC towards both cores (~8%) indicate that HNC is tracing slightly different layers compared to HCN. We find that the deuterium fraction of HCO+ is enhanced towards HH211, most likely caused by isotope-selective photodissociation of C18O. With radiative transfer, we were able to reproduce the observed lines of CCH, HCN, H13CN HNC, HN13C and DNC towards L1544 as well as CCH, H13CN HN13C DNC, H13CO+ HC18O+ and DCO+ towards HH211. Conclusions. Similar levels of deuteration show that the deuterium fractionation is most probably equally efficient towards both cores, suggesting that the protostellar envelope still retains the chemical composition of the original pre-stellar core. The fact that the two cores are embedded in different molecular clouds also suggests that environmental conditions do not have a significant effect on the deuterium fractionation within dense cores. Our results highlight the uncertainties when dealing with 13C isotopologues and the influence of the applied carbon isotopic ratio. Radiative transfer modelling shows that it is crucial to include the effects of the hyperfine structure to reproduce the observed line shapes. In addition, to correctly model emission lines from pre-stellar cores, it is necessary to include the outer layers of the core to consider the effects of extended structures. In addition to HCO+ observations, HCN observations towards L1544 also require the presence of an outer diffuse layer where the molecules are relatively abundant.

Deuterium chemodynamics of massive pre-stellar cores

ABSTRACT High levels of deuterium fractionation of N2H+ (i.e. $\mathrm{D^{{\mathrm{N_2H^+}}}_{\text{frac}}}$≳ 0.1) are often observed in pre-stellar cores (PSCs) and detection of N2D+ is a promising method to identify elusive massive PSCs. However, the physical and chemical conditions required to reach such high levels of deuteration are still uncertain, as is the diagnostic utility of N2H+ and N2D+ observations of PSCs. We perform 3D magnetohydrodynamics simulations of a massive, turbulent, magnetized PSC, coupled with a sophisticated deuteration astrochemical network. Although the core has some magnetic/turbulent support, it collapses under gravity in about one free-fall time, which marks the end of the simulations. Our fiducial model achieves relatively low $\mathrm{D^{{\mathrm{N_2H^+}}}_{\text{frac}}}$∼0.002 during this time. We then investigate effects of initial ortho-para ratio of H2 ($\mathrm{OPR^{H_2}}$), temperature, cosmic ray (CR) ionization rate, CO and N-species depletion factors, and prior PSC chemical evolution. We find that high CR ionization rates and high depletion factors allow the simulated $\mathrm{D^{{\mathrm{N_2H^+}}}_{\text{frac}}}$ and absolute abundances to match observational values within one free-fall time. For $\mathrm{OPR^{H_2}}$, while a lower initial value helps the growth of $\mathrm{D^{{\mathrm{N_2H^+}}}_{\text{frac}}}$, the spatial structure of deuteration is too widespread compared to observed systems. For an example model with elevated CR ionization rates and significant heavy element depletion, we then study the kinematic and dynamic properties of the core as traced by its N2D+ emission. The core, undergoing quite rapid collapse, exhibits disturbed kinematics in its average velocity map. Still, because of magnetic support, the core often appears kinematically subvirial based on its N2D+ velocity dispersion.

High-throughput hydrogen deuterium exchange mass spectrometry (HDX-MS) coupled with subzero-temperature ultrahigh pressure liquid chromatography (UPLC) separation for complex sample analysis

Hydrogen deuterium exchange coupled with mass spectrometry (HDX-MS) is a powerful technique for the characterization of protein dynamics and protein interactions. Recent technological developments in the HDX-MS field, such as sub-zero LC separations, large-scale data analysis tools, and efficient protein digestion methods, have allowed for the application of HDX-MS to the analysis of multi protein systems in addition to pure protein analysis. Still, high-throughput HDX-MS analysis of complex samples is not widespread because the co-elution of peptides combined with increased peak complexity after labeling makes peak de-convolution extremely difficult. Here, for the first time, we evaluated and optimized long gradient subzero-temperature ultra-high-pressure liquid chromatography (UPLC) separation conditions for the HDX-MS analysis of complex protein samples such as E. coli cell lysate digest. Under the optimized conditions, we identified 1419 deuterated peptides from 320 proteins at −10 °C, which is about 3-fold more when compared with a 15-min gradient separation under the same conditions. Interestingly, our results suggested that the peptides eluted late in the gradient are well-protected by peptide-column interactions at −10 °C so that peptides eluted even at the end of the gradient maintain high levels of deuteration. Overall, our study suggests that the optimized, sub-zero, long-gradient UPLC separation is capable of characterizing thousands of peptides in a single HDX-MS analysis with low back-exchange rates. As a result, this technique holds great potential for characterizing complex samples such as cell lysates using HDX-MS.

Submillimeter and Far-infrared Spectroscopy of Monodeuterated Amidogen Radical (NHD): Improved Rest Frequencies for Astrophysical Observations

Observations of ammonia in interstellar environments have revealed high levels of deuteration, and all its D-containing variants, including ND$_3$, have been detected in cold prestellar cores and around young protostars. The observation of these deuterated isotopologues is very useful to elucidate the chemical and physical processes taking place during the very early stages of star formation, as the abundance of deuterated molecules is highly enhanced in dense and cold gas. Nitrogen hydride radicals are key species lying at the very beginning of the reaction pathway leading to the formation of NH$_3$ and organic molecules of pre-biotic interest, but relatively little information is known about their D-bearing isotopologues. To date, only ND has been detected in the interstellar gas. To aid the identification of further deuterated nitrogen radicals, we have thoroughly re-investigated the rotational spectrum of NHD employing two different instruments: a frequency-modulation submillimetre spectrometer operating in the THz region and a synchrotron-based Fourier Transform infrared spectrometer operating in the 50-240 cm$^{-1}$ wavelength range. NHD was produced in a plasma of NH$_3$ and D$_2$. A wide range of rotational energy levels has been probed thanks to the observation of high $N$ (up to 15) and high $K_a$ (up to 9) transitions. A global analysis including our new data and those already available in the literature has provided a comprehensive set of very accurate spectroscopic parameters. A highly reliable line catalogue has been generated to assist archival data searches and future astronomical observations of NHD at submillimetre and THz regimes.

Mesoionic Carbene-Iridium Complex Catalyzed Ortho-Selective Hydrogen Isotope Exchange of Anilines with High Functional Group Tolerance.

Mesoionic carbene-iridium complex 1a has been investigated in the hydrogen isotope exchange (HIE) reaction of anilines. By employing 1 mol % of 1a as catalyst, anilines were selectively deuterated at the ortho-position with high deuteration levels. High ortho-selectivity was observed for anilines with various competing directing groups, which is in contrast with catalytic results of Kerr's catalysts.

Structural Aspects of Glycosylated Protein–Glycan Interactions by NMR

Characterization of glycosylated proteins and their response to interaction with glycans is challenging for any method, because of the heterogeneity of glycosylation and the extensive conformational sampling by most glycans. For NMR there are extra challenges because expression in mammalian cell cultures, which can preserve near‐native glycosylation, requires expensive substrates to achieve uniform isotopic labeling with 15N or 13C, and mammalian cells do not tolerate high levels of deuteration (something required to improve spectral resolution). We have developed a sparse‐labeling approach that preserves high resolution without deuteration, proves to be economical in most applications, and returns enough structural data to define geometries of multiple domain proteins and ligand protein complexes. The approach is based on labeling proteins with a single type of amino acid isotopically labeled in methyl groups with 13C (13C‐valine or 13C alanine). NMR signals from methyl groups are special in terms of the sensitivity and resolution that they provide. The addition of paramagnetic tags to specific sites in a protein provides long range distance and angular information on the location of labeled methyl groups. In combination with a newly developed approach to assign methyl resonances to specific sites in a protein, this information proves adequate to define relative domain orientations and facilitate the identification of ligand binding sites. Application to terminal domains from a cell surface signaling molecule, ROBO1, is used to illustrate the approach. It also provides an example of how binding of a heparan sulfate oligomer alters the relative orientation of domains.Support or Funding InformationThis work was supported by grants from the National Institutes of Health, P41GM103390 and R01GM033225.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

AN ORDERED BIPOLAR OUTFLOW FROM A MASSIVE EARLY-STAGE CORE

ABSTRACT We present ALMA follow-up observations of two massive, early-stage core candidates, C1-N and C1-S, in IRDC G028.37+00.07, that were previously identified by their N 2 D + (3-2) emission, and show high levels of deuteration of this species. The cores are also dark at far-infrared wavelengths up to ∼ 100 μ m . We detect 12CO(2-1) from a narrow, highly collimated bipolar outflow that is being launched from near the center of the C1-S core, which is also the location of the peak 1.3 mm dust continuum emission. This protostar, C1-Sa, has associated dense gas traced by C 18 O (2-1) and DCN(3-2), from which we estimate that it has a radial velocity that is near the center of the range exhibited by the C1-S massive core. A second outflow-driving source is also detected within the projected boundary of C1-S, but it appears to be at a different radial velocity. After considering the properties of the outflows, we conclude that C1-Sa is a promising candidate for an early-stage massive protostar and as such it shows that these early phases of massive star formation can involve highly ordered outflow, and thus accretion, processes, similar to models developed to explain low-mass protostars.

Optimized Phospholipid Bilayer Nanodiscs Facilitate High-Resolution Structure Determination of Membrane Proteins

Structural studies of membrane proteins are still hampered by difficulties of finding appropriate membrane-mimicking media that maintain protein structure and function. Phospholipid nanodiscs seem promising to overcome the intrinsic problems of detergent-containing environments. While nanodiscs can offer a near-native environment, the large particle size complicates their routine use in the structural analysis of membrane proteins by solution NMR. Here, we introduce nanodiscs assembled from shorter ApoA-I protein variants that are of markedly smaller diameter and show that the resulting discs provide critical improvements for the structure determination of membrane proteins by NMR. Using the bacterial outer-membrane protein OmpX as an example, we demonstrate that the combination of small nanodisc size, high deuteration levels of protein and lipids, and the use of advanced non-uniform NMR sampling methods enable the NMR resonance assignment as well as the high-resolution structure determination of polytopic membrane proteins in a detergent-free, near-native lipid bilayer setting. By applying this method to bacteriorhodopsin, we show that our smaller nanodiscs can also be beneficial for the structural characterization of the important class of seven-transmembrane helical proteins. Our set of engineered nanodiscs of subsequently smaller diameters can be used to screen for optimal NMR spectral quality for small to medium-sized membrane proteins while still providing a functional environment. In addition to their key improvements for de novo structure determination, due to their smaller size these nanodiscs enable the investigation of interactions between membrane proteins and their (soluble) partner proteins, unbiased by the presence of detergents that might disrupt biologically relevant interactions.

Fractional deuteration applied to biomolecular solid-state NMR spectroscopy

Solid-state Nuclear Magnetic Resonance can provide detailed insight into structural and dynamical aspects of complex biomolecules. With increasing molecular size, advanced approaches for spectral simplification and the detection of medium to long-range contacts become of critical relevance. We have analyzed the protonation pattern of a membrane-embedded ion channel that was obtained from bacterial expression using protonated precursors and D2O medium. We find an overall reduction of 50% in protein protonation. High levels of deuteration at Hα and Hβ positions reduce spectral congestion in (1H,13C,15N) correlation experiments and generate a transfer profile in longitudinal mixing schemes that can be tuned to specific resonance frequencies. At the same time, residual protons are predominantly found at amino-acid side-chain positions enhancing the prospects for obtaining side-chain resonance assignments and for detecting medium to long-range contacts. Fractional deuteration thus provides a powerful means to aid the structural analysis of complex biomolecules by solid-state NMR.Electronic supplementary materialThe online version of this article (doi:10.1007/s10858-011-9585-2) contains supplementary material, which is available to authorized users.

On the Solution Conformation and Dynamics of the HIV-1 Viral Infectivity Factor

Human immunodeficiency virus-1 (HIV-1) has evolved a cunning mechanism to circumvent the antiviral activity of the APOBEC3 family of host cell enzymes. HIV-1 Vif [ viral (also called virion) infectivity factor], one of several HIV accessory proteins, targets APOBEC3 proteins for proteasomal degradation and downregulates their expression at the mRNA level. Despite the importance of Vif for HIV-1 infection, there is little conformational data on Vif alone or in complex with other cellular factors due to incompatibilities with many structural techniques and difficulties in producing suitable quantities of the protein for biophysical analysis. As an alternative, we have turned to hydrogen exchange mass spectrometry (HX MS), a conformational analysis method that is well suited for proteins that are difficult to study using X-ray crystallography and/or NMR. HX MS was used to probe the solution conformation of recombinant full-length HIV-1 Vif. Vif specifically interacted with the previously identified binding partner Hck and was able to cause kinase activation, suggesting that the Vif studied by HX MS retained a biochemically competent conformation relevant to Hck interaction. HX MS analysis of Vif alone revealed low deuteration levels in the N-terminal portion, indicating that this region contained structured or otherwise protected elements. In contrast, high deuteration levels in the C-terminal portion of Vif indicated that this region was likely unstructured in the absence of cellular interacting proteins. Several regions within Vif displayed conformational heterogeneity in solution, including the APOBEC3G/F binding site and the HCCH zinc finger. Taken together, these HX MS results provide new insights into the solution conformation of Vif.

Cell-free protein synthesis of perdeuterated proteins for NMR studies

Cell-free protein synthesis protocols for uniformly deuterated proteins typically yield low, non-uniform deuteration levels. This paper introduces an E. coli cell-extract, D-S30, which enables efficient production of proteins with high deuteration levels for all non-labile hydrogen atom positions. Potential applications of the new protocol may include production of proteins with selective isotope-labeling of selected amino acid residues on a perdeuterated background for studies of enzyme active sites or for ligand screening in drug discovery projects, as well as the synthesis of perdeuterated polypeptides for NMR spectroscopy with large supra-molecular structures. As an illustration, it is demonstrated that the 800-kDa chaperonine GroEL synthesized with the D-S30 cell-free system had a uniform deuteration level of about 95% and assembled into its biologically active oligomeric form.

The abundances of nitrogen-containing molecules during pre-protostellar collapse

Aims. We have studied the chemistry of nitrogen-bearing species during the initial stages of protostellar collapse, with a view to explaining the observed longevity of N 2 H + and NH 3 and the high levels of deuteration of these species. Methods. We followed the chemical evolution of a medium comprising gas and dust as it underwent free-fall gravitational collapse. Chemical processes which determine the relative populations of the nuclear spin states of molecules and molecular ions were included explicitly, as were reactions which lead ultimately to the deuteration of the nitrogen-containing species N 2 H + and NH 3 . The freeze-out of heavy molecules on to dust grains was taken into account. Results. We found that the timescale required for the nitrogen-containing species to attain their steady-state values was much larger than the free-fall time and even comparable with the probable lifetime of the precursor molecular cloud. However, it transpires that the chemical evolution of the gas during gravitational collapse is insensitive to its initial composition. If we suppose that the grain-sticking probabilities of atomic nitrogen and atomic oxygen are both less than unity (S ≤ 0.3), we find that the observed differential freeze-out of nitrogen- and carbon-bearing species can be reproduced by the model of free-fall collapse when a sufficiently large grain radius (a g ≈ 0.50 μm) is adopted. Furthermore, the results of our collapse model are consistent with the high levels of deuteration of N 2 H + and NH 3 which have been observed in L1544, for example, providing that 0.5 ≤ ag ≤ 1.0 μm. We note that the ortho:para H 2 D + ratio, and fractional abundance of ortho-H 2 D + , which is the observed form of H 2 D + , should be largest where ND 3 is most abundant.

Ultrahigh Resolution in Proton Solid‐State NMR Spectroscopy at High Levels of Deuteration

Ultrahigh Resolution in Proton Solid‐State NMR Spectroscopy at High Levels of Deuteration

The importance of the ortho:para H2 ratio for the deuteration of molecules during pre-protostellar collapse

We have studied the evolution of molecular gas during the early stages of protostellar collapse. In addition to allowing for the freeze out of `heavy' species on to grains, we have computed the variation of population densities of the different nuclear spin states of ``tracer'' molecular ions such as H2D+ and D2H+. Processes which determine the relative populations of the nuclear spin states of molecules and molecular ions have received much less attention in the literature than those involved in their deuteration; but in fact the former processes are as significant as the latter and often involve the same reactants. We find that the ortho:para ratio of some species, e.g. H2D+, vary considerably as the density increases. Because the dynamical timescale is much shorter than some of the chemical timescales, there can be large departures of the predictions of the free--fall model from the steady state solution at the same density and temperature. In the case of H2, it seems unlikely that the steady state value of the ortho:para ratio is attained before protostellar collapse from the progenitor molecular cloud commences. Values of the ortho:para H2 ratio much higher than in steady state, which would prevail in ``young'' molecular clouds, are found to be inconsistent with high levels of deuteration of the gas. The internal energy of ortho--H2 acts as a reservoir of chemical energy which inhibits the deuteration of H3+ and hence of other species, such as N2H+ and NH3. In essence, the deuteration of molecular ions and molecules is sensitive to the ortho:para H2 ratio and hence to the chemical and thermal history of the parent molecular cloud.

Utilization of Methyl Proton Resonances in Cross-Saturation Measurement for Determining the Interfaces of Large Protein–Protein Complexes

Cross-saturation experiments allow the identification of the contact residues of large protein complexes (MW>50 K) more rigorously than conventional NMR approaches which involve chemical shift perturbations and hydrogen-deuterium exchange experiments [Takahashi et al. (2000) Nat. Struct. Biol., 7, 220-223]. In the amide proton-based cross-saturation experiment, the combined use of high deuteration levels for non-exchangeable protons of the ligand protein and a solvent with a low concentration of (1)H(2)O greatly enhanced the selectivity of the intermolecular cross-saturation phenomenon. Unfortunately, experimental limitations caused losses in sensitivity. Furthermore, since main chain amide protons are not generally exposed to solvent, the efficiency of the saturation transfer directed to the main chain amide protons is not very high. Here we propose an alternative cross-saturation experiment which utilizes the methyl protons of the side chains of the ligand protein. Owing to the fast internal rotation along the methyl axis, we theoretically and experimentally demonstrated the enhanced efficiency of this approach. The methyl-utilizing cross-saturation experiment has clear advantages in sensitivity and saturation transfer efficiency over the amide proton-based approach.

Doubly deuterated formaldehyde in star-forming regions: an observational approach

We present the results of a search for the doubly deuterated form of formaldehyde (D 2CO) in a large sample of objects associated to star-formation. D 2CO was detected in nearly 20 low-mass protostars, a success rate of 100%, with [D 2CO]/[H 2CO] abundance ratios of 2–40%. On the other hand, no detection was obtained towards more massive protostars (where [D 2CO]/[H 2CO]<0.5%) nor towards the shocks along the molecular outflows powered by protostars (where [D 2CO]/[H 2CO]<0.5–1%). The results reported here multiply the number of D 2CO detections by a factor of 6. Since low temperatures and high densities are needed to obtain high levels of multiple deuteration, the D 2CO detected towards low-mass protostars is most likely currently desorbed from dust grain mantles where it has accumulated and/or formed during the earlier and colder pre-stellar stage. The lack of detection towards molecular outflows is naturally explained in this scheme since the high-velocity gas there is mostly previously ambient material, that never went through the cold phase required to attain high deuteration levels. The lack of detection towards higher mass protostars indicates that the chemistry in low-mass protostars is significantly different from that in higher mass objects, and that much caution should be used when generalizing chemical results obtained in high-mass sources to low-mass objects.

Study of Degradation on Deuteration of a Diblock Copolymer of Polystyrene and Poly(ethylene-alt-propylene)

This paper reports a method of selectively deuterating the aromatic ring in a diblock copolymer of polystyrene and poly(ethylene-alt-propylene). The molecular weight distribution and molecular weight of the copolymer were determined by size exclusion chromatography and the extent of deuteration by 1H nuclear magnetic resonance spectroscopy. High levels of deuteration were sought with minimal changes in molecular weight distribution. A range of deuteration experiments were performed, some of which produced significant changes in molecular weight of the diblock copolymer. Changes in molecular weight are discussed with reference to groups in the copolymer chain.

Stable Isotopes Labeling of Drugs in Pediatric Clinical Pharmacology

Stable isotope labeling (SIL) still has not been used very much in pediatric and perinatal clinical pharmacology. However, this method has numerous advantages. It allows one to determine the concentration of drugs with great sensitivity, thereby allowing quantification in small amounts of biologic samples. It also allows quantification with great specificity, eliminating interference by drug metabolites found in some radioactive tracer methods. SIL is safe because stable isotopes are not radioactive. The only possible toxicity that may be related to an isotope effect is a slowing of biochemical reactions because of the greater mass of the stable isotope. Because of the great difference between deuterium and hydrogen, a significant isotope effect occurs only with deuterium. However, toxicity related to the isotope effect of deuterium can only be produced by very high levels of deuteration (>15% of body water), far higher than the amount of deuterium in a typical tracer dose of drug.1 SIL also allows noninvasive in vivo studies such as the co2 breath test (CBT). All of these advantages explain the great potential interest in SIL in pediatric pharmacology. The costs of stable isotopes and limited facilities available for sample analysis are undoubtedly the greatest limitations to their increased use. SIL is useful in different types of studies,2 such as studies on the fate of drugs (drug metabolic pathways and pharmacokinetics), determination of compliance, and assessment of therapeutic and unwanted effects of drugs. The use of SIL of drugs in positron emission tomography investigations is not addressed in this article. ### Definition In the periodic table, the elements are ranked according to their atomic number, ie, the number of protons contained in the nucleus. All elements located at the same place in the table are isotopes of the same family, for example, the family of carbon. All members …