Hydrogen-deuterium Exchange Reaction Research Articles

Water distribution in human hair microstructure elucidated by spin contrast variation small-angle neutron scattering

Dynamic nuclear polarization (DNP) is effective for controlling the neutron scattering length of protons and can be utilized for contrast variation in small-angle neutron scattering (SANS). Using the TEMPOL solution soaking method as electron spin doping, the DNP–SANS technique was applied to human hair fiber for the first time. For dry and D2O-swollen hair samples, a drastic change in the SANS profile was observed at high polarization conditions (|P H P N| ∼ 60%, where P H and P N are the proton and neutron spin polarization, respectively). The SANS profile as a function of the magnitude of the scattering vector, q, was composed of a low-q upturn, a middle-q oscillation and a high-q flat region. The low-q upturn was assumed to be a combination of two power-law functions, q −4 due to a large structure interface (Porod's law) and q −2 due to random coil. The middle-q oscillation was well reproduced by numerical calculation based on the structure model of intermediate filaments (IFs) as proposed by Er Rafik et al. [Biophys. J. (2004), 86, 3893–3904]: one pair of keratin coiled-coils is located at the center and surrounded by seven pairs of keratin coiled-coils located in a circle (called the `7 + 1' model), and a collection of IFs is arranged in a quasi-hexagonal manner. For the observed SANS profiles for different P H P N, the IF term contribution maintained a constant q-dependent profile, despite significant changes in intensity. This indicates that the macrofibril is composed of two domains (keratin coiled-coils and matrix). In addition, D2O swelling enhanced the IF term intensity and shifted the polarization-dependent local minimum to higher P H P N. This behavior was reproduced by contrast factor calculation based on the two-domain model. Scattering length densities of keratin coiled-coil and surrounding matrix domains were calculated by use of the known amino acid composition, considering the hydrogen–deuterium exchange reaction during soaking with D2O solution of TEMPOL. As a result, it was found that for keratin coiled-coil domains, about 40% of the peptide backbone amide NH protons were replaced with deuterons. This means that 68% of the α-helix domain is rigid, but the rest is flexible to allow dynamic dissociation of the hydrogen bond. Furthermore, the local mass density of each domain was precisely evaluated. The obtained data are expected to be a guide for further detailed investigation of keratin and keratin-associated protein distribution. This approach is expected to be applied to a wide variety of bio-derived materials, which are water absorbing in general.

Separation of mass spectra of hydrogen–deuterium exchanged ions obtained by electrospray of solutions of biopolymers with unknown primary structure

The work is dedicated to further development of our described method for analyzing mass spectra of biomolecules acquired as a result of hydrogen-deuterium exchange reactions (HDXs). The modified method consists of separating HDX distributions via their approximations by aminimum number of components corresponding to independent H/D substitutions and independent charge carrier retentions in different spatial isoforms or conformations of biomolecules with unknown primary structures. In this case, neither the natural isotopic distribution nor the exact number of active sites involved in HDXs and H+ or D+ attachments can be determined in advance. Original H/D electrospray mass spectra of an apamin solution were taken from our previous work. In that work, taking into account the natural isotopic distribution of apamin molecules, three main conformations of apamin ions were found as a result of separating the H/D mass spectra of the apamin solution for the gas flow with theaddition of about 10% ND3 molecules. Using the proposed modified method that does not require knowledge of the primary structure of the biomolecules gave similar results with slight deviations of calculated HDX distributions of the apamin ions from those obtained earlier. The maximum difference between mean values of the calculated HDX distributions for ions of the same charge in both cases does not exceed a few percent. In addition, HDX mass spectra of the apamin complex with an adduct of unknown structure were processed. Such analysis gave also three main fractions of ions with relatively large contributions when ND3 was injected into a radio-frequency quadrupole. In the absence of ND3 flow, the results of calculations for apamin and its complex were close to each other too. The formation of the apamin complex most probably in solution was confirmed by performed calculations.

Rapid characterization of isotopic purity of deuterium-labeled organic compounds and monitoring of hydrogen-deuterium exchange reaction using electrospray ionization-high-resolution mass spectrometry.

Deuterium-labeled organic compounds, reagents, and drugs are widely used in many scientific research fields. Isotopic purity as the feature parameter of deuterated compounds is of great importance. In this article, we used electrospray ionization with high-resolution mass spectrometry (ESI-HRMS) to study isotopic purity of deuterium-labeled organic compounds based on assigning and distinguishing the corresponding H/D (hydrogen-deuterium) isotopolog ions of deuterated compounds. We systematically considered the specific requirements of accuracy and resolution of ESI-HRMS when measuring isotopic purity and demonstrated some actual cases using ESI-HRMS and ultraperformance liquid chromatography (UPLC)-HRMS. Meanwhile, ESI-HRMS/MS of deuterated compounds was performed to obtain more information on deuterium-labeled position characterization. Two isotopic purity calculation methods based on the relative abundance in ESI-HRMS and UPLC-HRMS of H/D isotopolog ions (D0 -Dn ) were compared, which gave consistent isotopic purity values and were in good agreement with the certified isotopic purity values. We further studied and monitored the H/D exchange reaction of ethyl 3-(4-bromophenyl)-3-oxopropanoate (EBPO) by evaluating the dynamic isotopic purity changes in EBPO-D2 in the H/D exchange reaction in situ. The isotopic purity characterization methods using ESI-HRMS discussed in this article have some outstanding advantages: rapid, highly sensitive, very low sample consumption (even below nanogram level), and deuterated solvent-free. Thus, this low-impact analytical method requires less time and is cost effective and might have good application potential for in-situ isotopic purity analysis and for monitoring the H/D exchange reactions.

Evaluating the role of hydroxyl keto-hydroperoxide in the low temperature oxidation of alkenes

The low temperature oxidation of alkenes exhibits some distinct reaction pathways that may form different kinds of keto-hydroperoxides to start the chain-branching by further decomposition. This is mainly due to the existence of the double bond that the OH radical could add to it. This work explores that phenomenon by investigating the low temperature oxidation of three alkenes (1-hexene, 1-heptene and 1-decene) in a jet-stirred reactor (JSR) at atmospheric pressure. The synchrotron vacuum ultraviolet photoionization mass spectrometry (SVUV-PIMS) experiments combined with hydrogen-deuterium (H-D) exchange reactions were used to evaluate the chain branching pathways. Alkenyl keto-hydroperoxide (AnKHP) and hydroxyl keto-hydroperoxide (HyKHP) are identified. They represented two different chain branching reaction networks that are initiated from the H-abstraction of the primary and secondary CHs and via OH addition to the double bond. The experimental estimated and model predicted ratios of AnKHP to HyKHP are compared, and the result indicates that the literature model underpredicts the chain branching reactions via the formation of AnKHP, but overpredicts the chain branching reactions via HyKHP.

Hydrogen-deuterium (H/D) exchange reaction of acebutolol hydrochloride in D₂O and CD₃OD solution.

H/D exchange reactions can be observed by NMR spectroscopy of acebutolol (ACE). The results obtained showed deuterium incorporation at α-posi t ion of the carbonyl group of acebutolol, when using deuterium oxide or deuterated methanol as deuterium source and solvent. The spontaneous deuteration is proceeded by the following pathway CH₃→CH₂D→CHD→CD₃, through a keto-enol tautomerization reaction. Furthermore, LC-MS / QTOF analyses have confirmed the proposed H/D exchange. In order to reduce the time of total deuteration observed at the acetyl group alkaline catalysts were employed.

Preparation of Deuterium-Labeled Armodafinil by Hydrogen-Deuterium Exchange and Its Application in Quantitative Analysis by LC-MS.

Armodafinil, the R enantiomer of modafinil, was approved in 2007 by the US Food and Drug Administration as a wake-promoting agent for excessive sleepiness treatment. Due to its abuse by students and athletes, there is a need of its quantification. Quantitative analysis by liquid chromatography-mass spectrometry, however, though very common and sensitive, frequently cannot be performed without isotopically labeled standards which usually have to be specially synthesized. Here we reported our investigation on the preparation of deuterated standard of armodafinil based on the simple and inexpensive hydrogen–deuterium exchange reaction at the carbon centers. The obtained results clearly indicate the possibility of introduction of three deuterons into the armodafinil molecule. The introduced deuterons do not undergo back exchange under neutral and acidic conditions. Moreover, the deuterated and non-deuterated armodafinil isotopologues revealed co-elution during the chromatographic analysis. The ability to control the degree of deuteration using different reaction conditions was determined. The proposed method of deuterated armodafinil standard preparation is rapid, cost-efficient and may be successfully used in its quantitative analysis by LC-MS.

Decoding reactive structures in dilute alloy catalysts

Rational catalyst design is crucial toward achieving more energy-efficient and sustainable catalytic processes. Understanding and modeling catalytic reaction pathways and kinetics require atomic level knowledge of the active sites. These structures often change dynamically during reactions and are difficult to decipher. A prototypical example is the hydrogen-deuterium exchange reaction catalyzed by dilute Pd-in-Au alloy nanoparticles. From a combination of catalytic activity measurements, machine learning-enabled spectroscopic analysis, and first-principles based kinetic modeling, we demonstrate that the active species are surface Pd ensembles containing only a few (from 1 to 3) Pd atoms. These species simultaneously explain the observed X-ray spectra and equate the experimental and theoretical values of the apparent activation energy. Remarkably, we find that the catalytic activity can be tuned on demand by controlling the size of the Pd ensembles through catalyst pretreatment. Our data-driven multimodal approach enables decoding of reactive structures in complex and dynamic alloy catalysts.

Catalytic Method for the Synthesis of Deuterium-Labeled N-Heterocyclic Carbenes Enabled by a Coordinatively Unsaturated Ruthenium N-Heterocyclic Carbene Catalyst.

The wide usage of N-heterocyclic carbenes (NHCs) has raised the quest for their deuterated molecules. Effective synthesis method to obtain them, however, has remained elusive. We present here a catalytic method for the preparation of deuterated NHCs, namely, the catalytic hydrogen-deuterium exchange reaction between NHCs and deuterated benzene using a coordinatively unsaturated Ru NHC catalyst. The catalytic system enables selective deuteration of the C(sp3)-H bonds of the alkyl groups on N-substituents, as well as the sterically nonhindered C(sp2)-H bonds of NHCs as demonstrated by the preparation of 16 deuterium-labeled NHCs that have a deuteration ratio on specified sites higher than 90%. The gram-scale synthesis of deuterated IMes indicated the applicability of this catalytic method. Mechanistic studies revealed that the high regio-selectivity toward those C(sp3)-H bonds on NHCs originates from the regio-selectivity of cyclometalation reactions of coordinatively unsaturated Ru NHC species.

Rerouting the Organocatalytic Benzoin Reaction toward Aldehyde Deuteration

Reactive intermediates are key to halting and promoting chemical transformations; however, due to their elusive nature, they are not straightforwardly harnessed for reaction design. Herein, we describe studies aimed at stabilizing reactive intermediates in the N-heterocyclic carbene (NHC) catalytic cycle, which enabled the full shutdown of the known benzoin coupling pathway, while rerouting its intermediates toward deuteration. The reversible nature of NHC catalysis and the selective stabilization of reaction intermediates facilitated clean hydrogen–deuterium exchange reactions of aromatic aldehydes by D2O, even for challenging electron-withdrawing substrates. In several cases, the addition of catalytic amounts of phenyl boronic acid was used to further stabilize highly reactive intermediates and mitigate the formation of benzoin coupling byproducts. The mechanistic understanding at the foundation of this work resulted in unprecedented mild conditions with base and catalyst loadings as low as 0.1 mol %, and a scalable deuteration reaction applicable to a broad substrate scope with outstanding functional group tolerance. More importantly, adopting this approach enabled the construction of a guideline for identifying the appropriate catalyst and conditions for different substrates. Experimental studies combined with machine learning and computational methods shed light on the nontrivial mechanistic underpinnings of this reaction.

Synthesis of Deuterated (E)-Alkene through Xanthate-Mediated Hydrogen-Deuterium Exchange Reactions.

Herein we have developed a reversible hydrogen-deuterium exchange reaction of nonactivated olefins. By using EtOCS2K as a mediator, the H/D exchange reaction was realized through repeated addition and elimination reactions, demonstrating reversible H/D exchange between ordinary olefins and deuterated olefins. Using the lowest cost D2O without precious metal catalysts and ligands, a broad spectrum of compatibility of functional groups was achieved.

Synthesis of deuterium-labeled cinnamic acids: Understanding the volatile benzenoid pathway in the flowers of the Japanese loquat Eriobotrya japonica.

Cinnamic acids are widely distributed in plants, including crops for human use, and exhibit a variety of activities that are beneficial to human health. They also occupy a pivotal position in the biosynthesis of phenylpropanoids such as lignins, anthocyanins, flavonoids, and coumarins. In this context, deuterium-labeled cinnamic acids have been used as tracers and internal standards in food and medicinal chemistry as well as plant biochemistry. Therefore, a concise synthesis of deuterium-labeled cinnamic acids would be highly desirable. In this study, we synthesized deuterium-labeled cinnamic acids using readily available deuterium sources. We also investigated a hydrogen-deuterium exchange reaction in an ethanol-d1 /Et3 N system. This method can introduce deuterium atoms at the ortho and para positions of the phenolic hydroxy groups as well as at the C-2 position of alkyl cinnamates and is applicable to various phenolic compounds. Using the synthesized labeled compounds, we demonstrated that the benzenoid volatiles, such as 4-methoxybenzaldehyde, in the scent of the flowers of the Japanese loquat Eriobotrya japonica are biosynthesized from phenylalanine via cinnamic and 4-coumaric acids. This study provides easy access to a variety of deuterium-labeled (poly)phenols, as well as to useful tools for studies of the metabolism of cinnamic acids in living systems.

Capillary Electrophoresis-based Hydrogen/Deuterium Exchange for Conformational Characterization of Proteins with Top-down Mass Spectrometry.

Resolving conformational heterogeneity of multiple protein states that coexist in solution remains one of the main obstacles in the characterization of protein therapeutics and the determination of the conformational transition pathways critical for biological functions, ranging from molecular recognition to enzymatic catalysis. Hydrogen/deuterium exchange (HDX) reaction coupled with top-down mass spectrometric (MS) analysis provides a means to characterize protein higher-order structures and dynamics in a conformer-specific manner. The conformational resolving power of this technique is highly dependent on the efficiencies of separating protein states at the intact protein level and minimizing the residual non-deuterated protic content during the HDX reactions. Here we describe a capillary electrophoresis (CE)-based variant of the HDX MS approach that aims to improve the conformational resolution. In this approach, proteins undergo HDX reactions while migrating through a deuterated background electrolyte solution (BGE) during the capillary electrophoretic separation. Different protein states or proteoforms that coexist in solution can be efficiently separated based on their differing charge-to-size ratios. The difference in electrophoretic mobility between proteins and protic solvent molecules minimizes the residual non-deuterated solvent, resulting in a nearly complete deuterating environment during the HDX process. The flow-through microvial CE-MS interface allows efficient electrospray ionization of the eluted protein species following a rapid mixing with the quenching and denaturing modifier solution at the outlet of the sprayer. The online top-down MS analysis measures the global deuteration level of the eluted intact protein species, and subsequently, the deuteration of their gas-phase fragments. This paper demonstrates this approach in differential HDX for systems, including the natural protein variants coexisting in milk.

Helium-Plasma-Ionization Mass Spectrometry of Metallocenes and Their Derivatives.

Ferrocene and its derivatives and nickelocene undergo facile ionization when exposed directly to the ionizing plasma of a helium-plasma ionization (HePI) source. Mass spectra recorded from such samples under ambient positive-ion-generating conditions show intense peaks for the respective molecular ions [M+•] and protonated species [(M + H)+]. The protonation process occurs most efficiently when traces of water are present in the heated nitrogen used as the "heating gas." In fact, the relative population of the two categories of ions generated in this way can be manipulated by regulating the heating-gas flow. Moreover, rapid and highly efficient gas-phase hydrogen-deuterium exchange (HDX) reactions can be performed in the ion source by passing the heating gas through a vial with D2O before it reaches the HePI source. Moreover, the ionized species generated in this way can be subjected to in-source CID fragmentation in the QDa-HePI source very efficiently by varying the sampling-cone voltage. By this procedure, ions generated from ferrocene and nickelocene could be stripped so far as to ultimately generate the bare-metal cation. Other typical fragment-ions produced from protonated metallocenes included the M(cp)1+ ions (M = Fe or Ni), by elimination of a cyclopentadiene molecule, or the molecular cation, by loss of a H• radical. Moreover, H/D exchanges and subsequent tandem mass spectrometric analysis indicated that the central metal core participates in the initial protonation process of ferrocene under HePI conditions. However, in compounds such as ferrocene carboxaldehyde and ferrocene boronic acid, the protonation takes place at the peripheral functional group.

Activation of Ligand Reaction on an Iron Complex: H/D Exchange Reaction of a Low-Spin Bis[2-(Pyridylmethylidene)-1-(2-pyridyl)methylamine]iron(II) Complex.

With the aim of shedding some light on the still scarcely investigated mechanism of transformation of imines in metal complexes, this study describes the investigation of the hydrogen-deuterium (H/D) exchange reaction of a bis[2-(pyridylmethylidene)-1-(2-pyridylmethylamine]iron(II) complex ([Fe(PMAP)2]2+), following our previous work on a low-spin iron(II) complex bearing two molecules of S-2-pyridylmethylidene-1-(2-pyridyl)ethylamine. This complex has been proven to undergo successive transiminations in acetonitrile, yielding a bis[1-(2-pyridyl)ethylidene-2-pyridylmethylamine]iron(II) complex. In the analogous [Fe(PMAP)2]2+ complex, a 1,3-hydrogen rearrangement occurs in a 10% deuterium oxide-acetonitrile-d3 (D2O-CD3CN) solution. The H/D exchange reaction of [Fe(PMAP)2]2+ was examined in the presence of various concentrations of 2,6-dimethylpyridine as a base in a 10% D2O-CD3CN solution at 45 °C, and the reaction mechanism was investigated.

Development of Software for the In-Depth Analysis of Protein Dynamics as Determined by MALDI Mass Spectrometry-Based Hydrogen/Deuterium Exchange.

Hydrogen/deuterium exchange (HDX) coupled with pepsin digestion is useful for rapidly analyzing the kinetic properties of small amounts of protein. However, the analysis of HDX by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) is time-consuming due to a lack of dedicated software. Currently available software programs mainly calculate average mass shifts, even though the isotopic distribution width contains information regarding multiple protein conformations. Moreover, HDX reaction samples are typically composed of peptides that contain various numbers of deuterium atoms, which also hinders the rapid and comprehensive analysis of protein dynamics. We report here on the development of a software program “Scipas DX” that can be used to automatically analyze the hydrogen–deuterium isotopic distribution in peaks in HDX spectra and calculate the average number of atoms exchanged, the average deuteration ratio, the abundance ratio for exchanged atoms, and their fitted spectra with a high degree of accuracy within a few minutes. Analysis of the abundance ratio for exchanged atoms of a model protein, adenylate kinase 1, using Scipas DX indicate that the local structure at residues 83–106 and 107–117 are in a slow equilibrium, suggesting that these regions adopt multiple conformations that are involved in the stability and in switching between the active and inactive forms. Furthermore, precise HDX kinetics of the average deuteration ratio both confirmed the known induced conformations of two regions (residues 46–75 and 131–165) that are responsible for ligand binding and verified the novel structural dynamics of residues 107–117 and 166–196 following ligand binding to ligand-binding pockets 1 and 2, respectively. Collectively, these results highlight the usefulness and versatility of Scipas DX in MALDI-MS HDX-based analyses of protein dynamics.

Visible light driven deuteration of formyl C-H and hydridic C(sp3)-H bonds in feedstock chemicals and pharmaceutical molecules.

Deuterium labelled compounds are of significant importance in chemical mechanism investigations, mass spectrometric studies, diagnoses of drug metabolisms, and pharmaceutical discovery. Herein, we report an efficient hydrogen deuterium exchange reaction using deuterium oxide (D2O) as the deuterium source, enabled by merging a tetra-n-butylammonium decatungstate (TBADT) hydrogen atom transfer photocatalyst and a thiol catalyst under light irradiation at 390 nm. This deuteration protocol is effective with formyl C–H bonds and a wide range of hydridic C(sp3)–H bonds (e.g. α-oxy, α-thioxy, α-amino, benzylic, and unactivated tertiary C(sp3)–H bonds). It has been successfully applied to the high incorporation of deuterium in 38 feedstock chemicals, 15 pharmaceutical compounds, and 6 drug precursors. Sequential deuteration between formyl C–H bonds of aldehydes and other activated hydridic C(sp3)–H bonds can be achieved in a selective manner.

From heavy water to heavy aldehydes

Simple methods to incorporate deuterium into organic compounds are highly sought after as deuteration can enable mechanistic studies or improve the metabolic stability of pharmaceuticals. Now, a catalytic hydrogen–deuterium exchange reaction using deuterated water allows convenient access to deuterated aldehyde building blocks.

Practical Synthesis of C-1 Deuterated Aldehydes Enabled by NHC Catalysis.

The recent surge in applications of deuterated pharmaceutical agents has created an urgent demand for synthetic methods that efficiently generate deuterated building blocks. Here we show that N-heterocyclic carbenes (NHC) promote a reversible hydrogen-deuterium exchange (HDE) reaction with simple aldehydes, which leads to a practical approach to synthetically valuable C-1 deuterated aldehydes. The reactivity of the well-established NHC catalysed formation of Breslow intermediates from aldehydes is reengineered to overcome the overwhelmingly kinetically favorable, irreversible benzoin condensation reaction and achieve the critical reversibility to drive the formation of desired deuterated products when an excess of D2O is employed. Notably, this operationally simple and cost-effective protocol serves as a general and truly practical approach to all types of 1-D-aldehydes including aryl, -alkyl and -alkenyl aldehydes and enables chemoselective late-stage deuterium incorporation into complex, native therapeutic agents and natural products with uniformly high levels (>95%) of deuterium incorporation for a total of 104 substrates tested.

Metal-organic frameworks tailor the properties of aluminum nanocrystals.

Metal-organic frameworks (MOFs) and metal nanoparticles are two classes of materials that have received considerable recent attention, each for controlling chemical reactivities, albeit in very different ways. Here, we report the growth of MOF shell layers surrounding aluminum nanocrystals (Al NCs), an Earth-abundant metal with energetic, plasmonic, and photocatalytic properties. The MOF shell growth proceeds by means of dissolution-and-growth chemistry that uses the intrinsic surface oxide of the NC to obtain the Al3+ ions accommodated into the MOF nodes. Changes in the Al NC plasmon resonance provide an intrinsic optical probe of its dissolution and growth kinetics. This same chemistry enables a highly controlled oxidation of the Al NCs, providing a precise method for reducing NC size in a shape-preserving manner. The MOF shell encapsulation of the Al NCs results in increased efficiencies for plasmon-enhanced photocatalysis, which is observed for the hydrogen-deuterium exchange and reverse water-gas shift reactions.

Catalysis of Hydrogen-Deuterium Exchange Reactions by 4-Substituted Proline Derivatives.

The identification and understanding of structure-activity relationships is vital for rational catalyst design. A kinetic study of the hydrogen-deuterium exchange reaction of cyclohexanone in aqueous solution, as catalyzed by proline derivatives, has revealed valuable structure-activity relationships. In phosphate-buffered solution, cis-4-fluoroproline is more active than the trans isomer, a distinction that appears to originate from a destabilizing interaction between the fluorine atom and phosphate anion during general acid-catalyzed dehydration of the carbinolamine intermediate. trans-4-Ammoniumprolines are exceptionally active catalysts owing to favorable Coulombic interactions involving the ammonium group and the alkoxide moiety formed upon 1,2-addition of the proline derivative to the ketone. These results could be used for the optimization of proline catalysts, especially in transformations where the formation of the putative iminium ion is rate-limiting.