Selective Deuterium Labeling Research Articles

H/D Exchange of Aromatic Sulfones via Base Promotion and Silver Catalysis.

Aromatic sulfones are the prevailing scaffolds in pharmaceutical and material sciences. However, compared to their widespread application, the selective deuterium labeling of these structures is restricted due to their electron-deficient properties. This study presents two comprehensive strategies for the deuteration of aromatic sulfones. The base-promoted deuteration uses DMSO-d6 as the deuterium source, resulting in a rapid H/D exchange within 2 h. Meanwhile, a silver-catalyzed protocol offers a much milder option by using economical D2O to furnish the labeled sulfones.

Electrochemical selective deuterium labelling of N-heteroarenes

Hydrogen-deuterium exchange (HIE) reaction is the most direct way to achieve the deuterium labeling as there is no need for extra pre-functionalization. Herein, we report an electrochemical selective deuterium labelling of N-heteroarenes using D2O. The formation of aromatic radicals has been directly identified by using the time-resolved electron paramagnetic resonance (EPR) technique under electrochemical conditions. Mechanistic studies revealed that the hydrogen/deuterium (H/D) exchange involved continuous redox of N-heteroarenes under paired electrolysis and the selectivity of deuteration was established by DFT calculations. This electrochemical synthesis method offers a promising avenue for deuterium incorporation at specific sites of aromatic compounds.

Organo‐Mediator Enabled Electrochemical Deuteration of Styrenes

AbstractDespite widespread use of the deuterium isotope effect, selective deuterium labeling of chemical molecules remains a major challenge. Herein, a facile and general electrochemically driven, organic mediator enabled deuteration of styrenes with deuterium oxide (D2O) as the economical deuterium source was reported. Importantly, this transformation could be suitable for various electron rich styrenes mediated by triphenylphosphine (TPP). The reaction proceeded under mild conditions without transition‐metal catalysts, affording the desired products in good yields with excellent D‐incorporation (D‐inc, up to >99 %). Mechanistic investigations by means of isotope labeling experiments and cyclic voltammetry tests provided sufficient support for this transformation. Notably, this method proved to be a powerful tool for late‐stage deuteration of biorelevant compounds.

Organo-Mediator Enabled Electrochemical Deuteration of Styrenes.

Despite widespread use of the deuterium isotope effect, selective deuterium labeling of chemical molecules remains a major challenge. Herein, a facile and general electrochemically driven, organic mediator enabled deuteration of styrenes with deuterium oxide (D2O) as the economical deuterium source was reported. Importantly, this transformation could be suitable for various electron rich styrenes mediated by triphenylphosphine (TPP). The reaction proceeded under mild conditions without transition-metal catalysts, affording the desired products in good yields with excellent D-incorporation (D-inc, up to > 99%). Mechanistic investigations by means of isotope labeling experiment and cyclic voltammetry tests provided sufficient support for this transformation. Notably, this method proved to be a powerful tool for late-stage deuteration of biorelevant compounds.

Recent Advances in Organocatalytic Methods for the Synthesis of Deuterated Aldehydes

Abstract: Formyl-selective deuteration of aldehydes is one of the important synthetic methods in the field of medicinal chemistry. Aldehyde-d is often used as an important building block for pharmaceutical and drug synthesis due to its versatile reactivity and applicability. Due to the recent interest and development in the use of deuterated pharma drugs, there is an urgent need for simple and practical synthetic methods that are effective in producing a broad range of highly deuterated (up to 99% D) functionalized aryl, heteroaryl, alkyl, and alkenyl aldehyde moieties. Organocatalytic processes mediated by NHC have recently been used to achieve selective deuterium labelling processes; this system is frequently used to analyze drug distribution, metabolism, absorption, and excretion (ADME). Moreover, deuterated pharmaceutical compounds are designed to develop therapeutic effectiveness and reduce significant side effects and toxicity by increasing the half-life of the isotope drug response. Remarkably, in 2019-2022, NHC-mediated various catalytic approaches have been dramatically developed. One such method is a practical and mild synthesis of functionalized deuterated aldehydes, drug molecules, therapeutic agents, small and complex natural products, and their analogues using a green method in the presence of water-d as a cheap reagent. These modern methods prepared deuterated drug scaffolds such as 3-formyl rifamycin, midecamycin, menthol, ibuprofen, naproxen, etc. In this concern, we could provide a succinct description of the NHC-organocatalyzed modern synthetic strategies, as well as a mild greener approach for the functional group-selective deuterium isotopic labeling of various formyl compounds using commercially available deuterium sources (D2O and CD3OD).

Styrene-Maleic Acid Copolymer Nanodiscs to Determine the Shape of Membrane Proteins.

Lipid nanodiscs can be used to solubilize functional membrane proteins (MPs) in nativelike environments. Thus, they are promising reagents that have been proven useful to characterize MPs. Both protein and non-protein molecular belts have shown promise to maintain the structural integrity of MPs in lipid nanodiscs. Small-angle neutron scattering (SANS) can be used to determine low-resolution structures of proteins in solution, which can be enhanced through the use of contrast variation methods. We present theoretical contrast variation SANS results for protein and styrene-maleic acid copolymer (SMA) belt 1,2-dimyristoyl-sn-glycero-3-phosphorylcholine (DMPC) nanodiscs with and without additional bound or transmembrane proteins. The predicted scattering properties are derived from atomistic molecular dynamics simulations to account for conformational fluctuations, and we determine deuterium-labeling conditions such that SANS intensity profiles only include contributions from the scattering of the MP of interest. We propose strategies to tune the neutron scattering length densities (SLDs) of the SMA and DMPC using selective deuterium labeling such that the SLD of the nanodisc becomes homogeneous and its scattering can essentially be eliminated in solvents containing an appropriate amount of D2O. These finely tuned labeled polymer-based nanodiscs are expected to be useful to extract the size and molecular shape information of MPs using SANS-based contrast variation experiments, and they can be used with MPs of any molecular weight.

Live-Cell Imaging and Quantification of PolyQ Aggregates by Stimulated Raman Scattering of Selective Deuterium Labeling.

Polyglutamine (polyQ) diseases are a group of neurodegenerative disorders, involving the deposition of aggregation-prone proteins with long polyQ expansions. However, the cytotoxic roles of these aggregates remain highly controversial, largely due to a lack of proper tools for quantitative and nonperturbative interrogations. Common methods including in vitro biochemical, spectroscopic assays, and live-cell fluorescence imaging all suffer from certain limitations. Here, we propose coupling stimulated Raman scattering microscopy with deuterium-labeled glutamine for live-cell imaging, quantification, and spectral analysis of native polyQ aggregates with subcellular resolution. First, through the enrichment of deuterated glutamine in the polyQ sequence of mutant Huntingtin (mHtt) exon1 proteins for Huntington’s disease, we achieved sensitive and specific stimulated Raman scattering (SRS) imaging of carbon–deuterium bonds (C–D) from aggregates without GFP labeling, which is commonly employed in fluorescence microscopy. We revealed that these aggregates became 1.8-fold denser compared to those with GFP. Second, we performed ratiometric quantifications, which indicate a surprising dependence of protein compositions on aggregation sizes. Our further calculations, for the first time, reported the absolute concentrations for sequestered mHtt and non-mHtt proteins within the same aggregates. Third, we adopted hyperspectral SRS for Raman spectroscopic studies of aggregate structures. By inducing a cellular heat shock response, a potential therapeutic approach for inhibiting aggregate formation, we found a possible aggregate intermediate state with changed solvation microenvironments. Our method may hence readily unveil new features and mechanistic insight of polyQ aggregates and pave the way for comprehensive in vivo investigations.

Coupled Methyl Group Rotation in FMN Radicals Revealed by Selective Deuterium Labeling.

Flavin semiquinones are common intermediate redox states in flavoproteins, and thus, knowledge of their electronic structure is essential for fully understanding their chemistry and chemical versatility. In this contribution, we use a combination of high-field electron nuclear double resonance spectroscopy and selective deuterium labeling of flavin mononucleotide (FMN) with subsequent incorporation as cofactor into a variant Avena sativa LOV domain to extract missing traits of the electronic structure of a protein-bound FMN radical. From these experiments, precise values of small proton hyperfine and deuterium nuclear quadrupole couplings could be extracted. Specifically, isotropic hyperfine couplings of -3.34, -0.11, and +0.91 MHz were obtained for the protons H(6), H(9), and H(7α), respectively. These values are discussed in the light of specific protein-cofactor interactions. Furthermore, the temperature behavior of the H(7α) methyl-group rotation elicited by its energy landscape was analyzed in greater detail. Pronounced interplay between the two methyl groups at C(7) and C(8) of FMN could be revealed. Most strikingly, this rotational behavior could be modulated by selective deuterium editing.

Small-angle neutron scattering studies suggest the mechanism of BinAB protein internalization.

Small-angle neutron scattering (SANS) is one of the most widely used neutron-based approaches to study the solution structure of biological macromolecular systems. The selective deuterium labelling of different protein components of a complex provides a means to probe conformational changes in multiprotein complexes. The Lysinibacillus sphaericus mosquito-larvicidal BinAB proteins exert toxicity through interaction with the receptor Cqm1 protein; however, the nature of the complex is not known. Rationally engineered deuterated BinB (dBinB) protein from the L. sphaericus ISPC-8 species was synthesized using an Escherichia coli-based protein-expression system in M9 medium in D2O for 'contrast-matched' SANS experiments. SANS data were independently analysed by ab initio indirect Fourier transform-based modelling and using crystal structures. These studies confirm the dimeric status of Cqm1 in 100% D2O with a longest intramolecular vector (D max) of ∼94 Å and a radius of gyration (R g) of ∼31 Å. Notably, BinB binds to Cqm1, forming a heterodimeric complex (D max of ∼129 Å and R g of ∼40 Å) and alters its oligomeric status from a dimer to a monomer, as confirmed by matched-out Cqm1-dBinB (D max of ∼70 Å and R g of ∼22 Å). The present study thus provides the first insight into the events involved in the internalization of larvicidal proteins, likely by raft-dependent endocytosis.

A Biphasic Medium Slows Down the Transfer Hydrogenation and Allows a Selective Catalytic Deuterium Labeling of Amines from Imines Mediated by a Ru−H/D+ Exchange in D2O

AbstractThe transfer hydrogenation (TH) of several aldimines has been studied using [RuCl(p‐cymene)(dmbpy)]BF4, 1, (dmbpy=4,4′‐dimethyl‐2,2′‐bipyridine) as a precatalyst. Both neat water and a biphasic water/toluene mixture (w/t) have been successfully used as solvents. In the w/t medium the corresponding precursors, amine and aldehyde, were also used as substrates for a transfer hydrogenative reductive amination. Selective deuterium labeling of the resulting alkylated amines was the main goal of this work. On using D2O the D‐content in the amine was negligible but a high level of D‐incorporation was achieved in D2O/toluene. According to calculations, this is due to the effect of the relative rates of the hydride transfer and that of the RuH/D+ exchange. The incorporation of deuterium increases with time as a consequence of the reduction in the hydride transfer rate as the substrate concentration diminishes. The recyclability assays performed reflect the importance of pH in the selectivity of the TH towards the imine or the aldehyde resulting from imine hydrolysis.

Changes in lipid bilayer structure caused by the helix-to-sheet transition of an HIV-1 gp41 fusion peptide derivative

HIV-1, like other enveloped viruses, undergoes fusion with the cell membrane to infect it. Viral coat proteins are thought to bind the virus to the membrane and actively fuse the viral and cellular membranes together. The actual molecular mechanism of fusion is challenging to visualize, resulting in the use of model systems. Here, the bilayer curvature modifying properties of a synthetic variant of the HIV-1 gp41 fusion peptide with lipid bilayer vesicles composed of a mixture of dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylserine (DMPS) were studied. In 7:3 DMPC:DMPS vesicles made with deuterium-labeled DMPC, the peptide was observed to undergo a concentration-dependent conformational transition between an α-helix and an antiparallel β-sheet. Through the use of small-angle neutron scattering (SANS) and selective deuterium labeling, it was revealed that conformational transition of the peptide is also accompanied by a transition in the structure of the lipid bilayer. In addition to changes in the distribution of the lipid between the leaflets of the vesicle, the SANS data are consistent with two regions having different thicknesses. Of the two different bilayer structures, the one corresponding to the smaller area fraction, being ∼8% of the vesicle area, is much thicker than the remainder of the vesicle, which suggests that there are regions of localized negative curvature similar to what takes place at the point of contact between two membranes immediately preceding fusion.

Li2(NH2BH3)(BH4)/LiNH2BH3: The first metal amidoborane borohydride complex with inseparable amidoborane precursor for hydrogen storage

A novel lithium amidoborane borohydride complex, Li2(NH2BH3)(BH4), was synthesized using mechanochemical method and its crystal structure was successfully determined by a combination of X-ray diffraction (XRD) analysis and first-principles calculations. Interestingly, this compound does not exist as a pure phase, but requires almost equivalent amount of amorphous LiAB as a stabilizing agent. In this paper, we report a careful study of the structure, property, and dehydrogenation mechanism of the 1:1 Li2(NH2BH3)(BH4)/LiAB composite. This composite can release ∼8 wt% H2 at 100 °C via a two-step dehydrogenation process, with dehydrogenation kinetics better than the parenting phases. The composite and its dehydrogenation products were characterized by the combined XRD, Fourier transformation infrared (FTIR) spectroscopy, and solid-state 11B MAS NMR techniques. Selective deuterium labeling was performed to elucidate a reaction sequence for the hydrogen release by analyzing the released gases.

Arene Ruthenium Complexes as Versatile Catalysts in Water in both Transfer Hydrogenation of Ketones and Oxidation of Alcohols. Selective Deuterium Labeling of rac-1-Phenylethanol

The preparation of three series of arene Ru(II) half-sandwich compounds with the functional ligand 4,4′-dimethoxy-2,2′-bipyridine (dmobpy) is described. The new cationic derivatives have the general formula [(η6-arene)RuCl(κ2-N,N-dmobpy)]X (arene = benzene, X = Cl– ([1]Cl), BF4– ([1][BF4]), TsO– ([1]TsO), PF6– ([1][PF6]); arene = p-cymene (p-cym), X = Cl– ([2]Cl), BF4– ([2][BF4]), TsO– ([2]TsO), PF6– ([2][PF6]); arene = 2-phenoxy-1-ethanol (phoxet), X = Cl– ([3]Cl), BF4– ([3][BF4]), TsO– ([3]TsO), PF6– ([3][PF6])). The structures of [1]Cl, [1]TsO, [2]TsO, [2][BF4], and [2][PF6] were determined by X-ray crystallography. All of the complexes except the PF6– salts were water-soluble, and they behaved as active catalysts in two different processes: the transfer hydrogenation of water-soluble and -insoluble ketones to the corresponding alcohols, using HCOONa as the hydrogen source at pH 4, and the oxidation of rac-1-phenylethanol to acetophenone with tBuOOH at pH 7, both in aqueous solution. For the transfer h...

Isotope-Labeling of the Fibril Binding Compound FSB via a Pd-Catalyzed Double Alkoxycarbonylation

We have synthesized two isotopically labeled variants of the β-amyloid binding compound FSB possessing (13)C-labels on the two terminal aryl carboxylic acid moieties. One of these was also fully deuterated on the olefinic spacers. The (13)C-isotope labeling was achieved applying a Pd-catalyzed methoxycarbonylation of the corresponding aryl chlorides with externally (ex situ) generated (13)C-labeled CO. Application of the Shirakawa-Hayashi protocol for the Pd-catalyzed reduction of a dialkyne intermediate using D(2)O allowed for the selective deuterium labeling of the two trans-C,C double bonds of FSB.

Study of the Influence of the Penetration Enhancer Isopropyl Myristate on the Nanostructure of Stratum Corneum Lipid Model Membranes Using Neutron Diffraction and Deuterium Labelling

In order to elucidate the mode of action of the lipophilic penetration enhancer isopropyl myristate (IPM) on a molecular scale, we investigated oriented quaternary stratum corneum (SC) lipid model membranes based on ceramide AP, cholesterol, palmitic acid and cholesterol sulfate containing 10 wt% IPM by means of neutron diffraction. Our results indicate that IPM affects the lamellar lipid assembly in terms of bilayer perturbation and disordering. Phase segregation occurred, indicating that IPM is not likely to mix properly with the other SC lipids due to its branched structure. We used selective deuterium labelling to localize the penetration enhancer, and could successfully prove the presence of IPM in the two coexisting lamellar phases. We conclude that IPM’s mode of action as penetration promoter is presumably based on incorporation into the SC lipid matrix, extraction of certain SC lipids into a separate phase and perturbation of the multilamellar lipid assembly.

Peptide-Induced Asymmetric Distribution of Charged Lipids in a Vesicle Bilayer Revealed by Small-Angle Neutron Scattering

Cellular membranes are complex mixtures of lipids, proteins, and other small molecules that provide functional, dynamic barriers between the cell and its environment, as well as between environments within the cell. The lipid composition of the membrane is highly specific and controlled in terms of both content and lipid localization. The membrane structure results from the complex interplay between the wide varieties of molecules present. Here, small-angle neutron scattering and selective deuterium labeling were used to probe the impact of the membrane-active peptides melittin and alamethicin on the structure of lipid bilayers composed of a mixture of the lipids dimyristoyl phosphatidylglycerol (DMPG) and chain-perdeuterated dimyristoyl phosphatidylcholine (DMPC). We found that both peptides enriched the outer leaflet of the bilayer with the negatively charged DMPG, creating an asymmetric distribution of lipids. The level of enrichment is peptide concentration-dependent and is stronger for melittin than it is for alamethicin. The enrichment between the inner and outer bilayer leaflets occurs at very low peptide concentrations and increases with peptide concentration, including when the peptide adopts a membrane-spanning, pore-forming state. The results suggest that these membrane-active peptides may have a secondary stressful effect on target cells at low concentrations that results from a disruption of the lipid distribution between the inner and outer leaflets of the bilayer that is independent of the formation of transmembrane pores.

Dynamic Propagation of Long-Range Allosteric Signals by Nanoscale Protein Domain Motion Revealed by Neutron Spin Echo Spectroscopy

NHERF1 is a multi-domain scaffolding protein that assembles signaling complexes, and regulates the cell surface expression and endocytic recycling of a variety of membrane proteins. The ability of the two PDZ domains in NHERF1 to assemble protein complexes is allosterically modulated by the membrane-cytoskeleton linker protein ezrin, whose binding site is located as far as 110 angstroms away from the PDZ domains. Here, using neutron spin echo (NSE) spectroscopy, selective deuterium labeling, and theoretical analyses, we reveal the activation of interdomain motion in NHERF1 on nanometer length scales and on sub-microsecond time scales upon forming a complex with ezrin. We show that a much simplified coarse-grained model suffices to describe inter-domain motion of a multi-domain protein or protein complex. We expect that future NSE experiments will benefit by exploiting our approach of selective deuteration to resolve the specific domain motions of interest from a plethora of global translational and rotational motions. Our results demonstrate that the dynamic propagation of allosteric signals to distal sites involves changes in long-range coupled domain motions on submicrosecond time scales, and that these coupled motions can be distinguished and characterized by NSE.

Activation of Nanoscale Allosteric Protein Domain Motion Revealed by Neutron Spin Echo Spectroscopy

NHERF1 is a multidomain scaffolding protein that assembles signaling complexes, and regulates the cell surface expression and endocytic recycling of a variety of membrane proteins. The ability of the two PDZ domains in NHERF1 to assemble protein complexes is allosterically modulated by the membrane-cytoskeleton linker protein ezrin, whose binding site is located as far as 110 Ångstroms away from the PDZ domains. Here, using neutron spin echo (NSE) spectroscopy, selective deuterium labeling, and theoretical analyses, we reveal the activation of interdomain motion in NHERF1 on nanometer length-scales and on submicrosecond timescales upon forming a complex with ezrin. We show that a much-simplified coarse-grained model suffices to describe interdomain motion of a multidomain protein or protein complex. We expect that future NSE experiments will benefit by exploiting our approach of selective deuteration to resolve the specific domain motions of interest from a plethora of global translational and rotational motions. Our results demonstrate that the dynamic propagation of allosteric signals to distal sites involves changes in long-range coupled domain motions on submicrosecond timescales, and that these coupled motions can be distinguished and characterized by NSE.

ChemInform Abstract: GIFIV Oxidation of Some Indolic Alkaloids.

The title oxidation was performed on four indolic alkaloids, β-carboline (1), reserpine (6), ajmaline (10) and ibogaine (11), leading to alicyclic hydroxylation of the starting materials. The numerous side products characterised during this reaction (due to reduction or coupling with solvent) as well as selective deuterium labelling experiments enabled us apart from the oxidation sites to study the Gif reaction mechanism

The Reaction of Synechocystis Catalase–Peroxidase (KatG) with Isoniazid Investigated by Multifrequency (9–285 GHz) EPR Spectroscopy

Three distinct electron paramagnetic resonance (EPR) spectra of radical intermediates formed as reactive intermediates in the catalytic cycle of Synechocystis catalase–peroxidase were identified. Multifrequency EPR spectroscopy, combined with site-directed mutagenesis and selective deuterium labeling of Trp and Tyr residues, allowed us to unequivocally assign such intermediates to an [Fe(IV) = O Por·+] species, the first committed intermediate in monofunctional peroxidases and two protein-based radicals, identified as Trp106· and a Tyr·, formed subsequently to the [Fe(IV) = O Por·+] species by intramolecular electron transfer. Our recent characterization of the Mycobacterium tuberculosis catalase–peroxidase showed that the Trp· sites differ among these enzymes, and that the [Fe(IV) = O Trp·] species was the reactive intermediate with the prodrug isoniazid. Accordingly, the question to address was whether the dissimilarity in the sites for the formation of the Trp· intermediates and in the geometry of the distal side was reflected by differences in the peroxidase-like reaction of Synechocystis and Mycobacterium tuberculosis catalase–peroxidases with the prodrug isoniazid. Our findings show that in the Synechocystis enzyme, the isoniazid substrate can get closer to the heme distal side and can react readily with the [Fe(IV) = O Por·+] species, at variance to the situation in the M. tuberculosis catalase–peroxidase. These results indicate that, as in the case of monofunctional peroxidases, the difference in the sites for the formation of the Trp· as alternative reactive intermediates to the [Fe(IV) = O Por·+] species is correlated to differences in substrate binding sites.