Monitoring allostery in D2O: a necessary control in studies using hydrogen/deuterium exchange to characterize allosteric regulation

There is currently a renewed focus aimed at understanding allosteric mechanisms at atomic resolution. This current interest seeks to understand how both changes in protein conformations and changes in protein dynamics contribute to relaying an allosteric signal between two ligand binding sites on a protein (e.g., active and allosteric sites). Both nuclear magnetic resonance (NMR), by monitoring protein dynamics directly, and hydrogen/deuterium exchange, by monitoring solvent accessibility of backbone amides, offer insights into protein dynamics. Unfortunately, many allosteric proteins exceed the size limitations of standard NMR techniques. Although hydrogen/deuterium exchange as detected by mass spectrometry (H/DX-MS) offers an alternative evaluation method, any application of hydrogen/deuterium exchange requires that the property being measured functions in both H₂O and D₂O. Due to the promising future H/DX-MS has in the evaluation of allosteric mechanisms in large proteins, we demonstrate an evaluation of allosteric regulation in D₂O. Exemplified using phenylalanine inhibition of rabbit muscle pyruvate kinase, we find that binding of the inhibitor is greatly reduced in D₂O, but the effector continues to elicit an allosteric response.     

Raman spectroscopic study of hydrogen bonding of N-alkyl-2-pyrrolidinones in heavy water

The Raman spectra of D₂O solutions of N-methyl-2-pyrrolidinone, N-ethyl-2-pyrrolidinone, and N-n-butyl-2-pyrrolidinone under diverse conditions were measured. Using a computer fitting of the band shape of the carbonyl stretching mode at various temperatures, an enthalpy difference for the inversion motion at the nitrogen atom due to hydrogen bonding with deuterium was estimated for these compounds. The enthalpy difference of hydrogen bond formation to the nitrogen atom of N-methyl-2-pyrrolidinone at 30 wt% in D₂O (mole fraction 0.080) was greater than that of N-methyl-2-pyrrolidinone in an aqueous solution at a mole fraction of 0.406. Furthermore, the enthalpy difference of N-alkyl-2-pyrrolidinones increased with the alkyl chain length. This is interepreted as a result of the change of the hydrophobic hydration of D₂O molecules around the solute molecules.     

Stereochemistry and the use of H2/D2 mixtures as probes into the mechanism of hydrogenations catalyzed by cationic rhodium (DIPHOS) complexes

The stereochemistry of the hydrogenation of 4-tert-butylmethylenecyclohexane (1) and the use of D₂ or D₂/H₂ mixtures in place of H₂ furnishes evidence that hydrogenations using the catalyst precursor [Rh(DIPHOS)(COD)]⁺BF ₄ ⁻ proceed via more than one mechanism. This evidence includes the effect of changes in pressure and added triethylamine upon the kinetics and isomerization of 1, as well as the distribution of the added deuterium in the products of the reaction of 1 or norbornene with either D₂ or H₂/D₂ mixtures. That an alkene necessarily causes the equilibration of H₂/D₂ mixtures, although it need not involve any of the alkenes’s hydrogen atoms (e.g., norbornene), provides a clue to the process by which the mononuclear mechanism proposed by Halpern, which is dominant near one atmosphere of H₂, merges into another with increasing pressure. It has been proposed that the cationic complex [Rh(DIPHOS)S₂]⁺ is transformed in the presence of an alkene and hydrogen into a binuclear hydrido complex, such as those described by Sivak and Muetterties (1979) and Fryzuk (1982), which represent a far more active catalyst than its mononuclear precursor. Such an intermediate should readily catalyze the H₂-D₂ equilibration and the isomerization of an alkene in the presence of D₂ without necessarily introducing deuterium into the product. Published in Russian in Kinetika i Kataliz, 2006, Vol. 47, No. 1, pp. 88–97. Deceased. The text was submitted by the authors in English. This work was conducted at the University of Arkansas.     

Diffuse Reflectance IR Spectra of Molecular Hydrogen and Deuterium Adsorbed on Zinc Oxide

Diffuse reflectance IR spectroscopy is used to study hydrogen and deuterium adsorption on zinc oxide at room temperature and 77 K. At room temperature, H₂ and D₂ molecules are dissociatively adsorbed with the formation of hydrides and hydroxy groups of three types. At 77 K, diffuse reflectance spectra reveal the bands from molecular hydrogen and deuterium in addition to the dissociatively adsorbed forms. The presence of several bands of stretching H–H and D–D vibrations points to the nonuniformity of adsorption sites. This nonuniformity is also confirmed by the fact that, after heating zinc oxide from 77 K to room temperature in an atmosphere of hydrogen, only an insignificant portion of adsorbed molecular hydrogen dissociates. Most of dissociatively adsorbed hydrogen is formed without a molecular precursor. The dissociation of H₂ and D₂ most likely occurs on very active adsorption species so rapidly that the molecular precursor is not observed. The bond energy in molecular deuterium precursors of dissociation estimated from the fundamental vibration frequency and the overtone of D–D vibrations suggests moderate excitation of the bond. This agrees well with the conclusion that the dissociative adsorption of hydrogen and deuterium occurs without a molecular precursor.     

In Situ Hydrogenation and Crystal Chemistry Studies of Co2Si Type Compounds MgPd2 and Pd2Zn

The hydrogenation properties of the intermetallic compounds MgPd₂ and Pd₂Zn, crystallizing in the Co₂Si type, were studied by in situ thermal analysis (DSC) under hydrogen pressure. Pd₂Zn does not show any reaction with hydrogen while MgPd₂ reversibly forms the hydride MgPd₂H. Neutron diffraction on the deuterides reveals the compositions MgPd₂D_0.861(6) (ambient) and MgPd₂D_0.97(1) [308(2) K, 2.56(5) MPa deuterium] with hydrogen (deuterium) occupying distorted [MgPd₅] octahedral voids. Quantum mechanical calculations support the structure models and show the hydrogenation to be exergonic for MgPd₂ and endergonic for Pd₂Zn. MgPd₂H releases hydrogen under normal conditions or vacuum. Heating under hydrogen pressure leads first reversibly to MgPd₂H_≈0.2 and subsequently irreversibly to MgPd₃H_≈1 and MgH₂. MgPd₂, Pd₂Zn, and MgPd₂H were classified in a structure map. Trends of axial ratio changes upon hydrogenation of TiNiSi type and ZrBeSi type compounds are discussed.     

Primary and secondary kinetic isotope effects in E2 elimination reactions

Primary and secondary deuterium kinetic isotope effects have been measured for elimination of LCl (L = H or D) from some substituted 1,2-diaryl-1-chloroethanes. Although changes in these effects are in agreement with theoretical predictions, the high values of (E_H-E_D)_β and the significantly low A_H/A_D may suggest that either proton tunnelling or an internal return mechanism is complicating this E2 elimination.     

Methyl guanine isomer distinction by hydrogen / deuterium exchange using a fourier transform mass spectrometer

Analytical Chemistry Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA Differentiation of the seven isomers of methyl guanine has been accomplished by monitoring gas-phase hydrogen/deuterium (H/D) exchange reactions of the protonated molecular ions with deuterium oxide (D₂O) in a Fourier transform mass spectrometer. In each case a distinctive reaction rate for the first H/D exchange was observed, and exchanges of up to three deuterium atoms occurred with characteristic ion abundances that could be used to differentiate the isomers. O⁶-Methyl guanine, for example, showed only one slow H/D exchange with D₂O, whereas l-methyl guanine exchanged two hydrogen atoms at a significantly faster rate. On comparison of the possible resonance structures of each protonated isomer with the experimental information about the number and rate of H/D exchanges observed, a reaction mechanism involving a concerted proton abstraction-deuterium cation donation was proposed.     

The bimolecular hydrogen-deuterium exchange behavior of protonated alkyl dipeptides in the gas phase

As part of an ongoing characterization of the intrinsic chemical properties of peptides, thermal hydrogen-deuterium exchange has been studied for a series of fast-atom-bombardment-generated protonated alkyldipeptides and related model compounds in the reaction with D₂O, CH₃OD, and ND₃ in a Fourier transform ion cyclotron resonance mass spectrometer. Despite the very large basicity difference between the dipeptides and the D₂O and CH₃OD exchange reagents, efficient exchange of all active hydrogen atoms occurs. From the kinetic data it appears that exchange of the amino, amide, and hydroxyl hydrogens proceeds with different efficiencies, which implies that the proton in thermal protonated dipeptides is immobile. The selectivity of the exchange at the different basic sites is governed by the nature of both the dipeptide and the exchange reagent. The results indicate that reversible proton transfer in the reaction complexes, which effectuates the deuterium incorporation, is assisted by formation of multiple hydrogen bonds between the reagents. Exchange is considered to proceed via the intermediacy of different competing intermediate complexes, each of which specifically leads to deuterium incorporation at different basic sites. The relative stabilization of the competing intermediate complexes can be related to the relative efficiencies of deuterium incorporation at different basic sites in the dipeptide. For all protonated dipeptides studied, the exchange in the reaction with ND₃ proceeds with unit efficiency, whereas all active hydrogen atoms are exchanged equally efficiently. Evidently specific multiple hydrogen bond formations are far less important in the reversible proton transfers with the relatively basic ammonia, which allows effective randomization of all active hydrogen atoms in the reaction complexes.     

Photoredox-Mediated Hydrogen Isotope Exchange Reactions of Amino-Acids,Peptides, and Peptide-Derived Drugs

Hydrogen isotopically labelled compounds are essential diagnostic tools in drug research and development, as they provide vital information about the biological metabolism of drug candidates and their metabolites. Herein we report a photoredox-initiated hydrogen atom transfer (HAT) protocol which efficiently and selectively introduces deuterium or tritium at C(sp³)−H bonds, utilizing heavy water (D₂O or T₂O) as the hydrogen isotope source, and a guanidine base. This protocol has been successfully applied to the incorporation of deuterium in several amino acids (lysine, glycine and proline) and small peptides. Finally, the method has been applied to tritium, because tritium-labelled peptides are essential for application in biological experiments, such as ligand-binding assays, or absorption, distribution, metabolism, and excretion (ADME) studies.     

Volumetric and transport properties of aerosol-OT reversed micelles containing light and heavy water

Densities and viscosities of sodium bis(2-ethylhexyl) sulfosuccinate (AOT in-heptane system containing light and heavy water, as a function of the molar ratio R (R=[H₂O or D₂O]/[AOT]) were measured at 0, 5, 25 and 40°C. At low R values, the apparent molar volume of deuterium oxide is smaller than that of light water. The difference is related to the strength of the hydrogen bonding H₂O and D₂O. The viscosities of both H₂O-AOT-n-heptane and D₂O-AOT-n-heptane systems were explained in terms of intermicellar interactions mainly governed by hydration of the head groups of AOT.     

Measurement of neutron emission from a SiO2−D2 system

As a new fact, the neutron emission from the d-d nuclear fusion reaction in a SiO₂–D₂ system has been confirmed as well as that in the Ti–D₂ system. By using a liquid scintillation detector, the neutron emissions from 8 sample materials consisted of SiO₂, in which a small amount of deuterium was adsorbed chemically on the surface layer, were measured in the range of temperature between liquid nitrogen temperature and 400 °C. As a result, it was demonstrated that the neutron-emission reactions take place predominantly on the surface layer of sample material.     

NHC-Stabilized Iridium Nanoparticles as Catalysts in Hydrogen Isotope Exchange Reactions of Anilines

The preparation of N-heterocyclic carbene-stabilized iridium nanoparticles and their application in hydrogen isotope exchange reactions is reported. These air-stable and easy-to-handle iridium nanoparticles showed a unique catalytic activity, allowing selective and efficient hydrogen isotope incorporation on anilines using D₂ or T₂ as isotopic source. The usefulness of this transformation has been demonstrated by the deuterium and tritium labeling of diverse complex pharmaceuticals.     

Base catalyzed enolization and hydrogen exchange of trifluoroacetone. A comparison to acetone

The kinetics of the pyridine catalyzed hydrogen exchange of 1,1,1-trifluoroacetone in 50% D₂O-dioxane have been measured using ¹H-NMR. Rates of hydrogen exchange of acetone were also measured under comparable conditions and the rate of deuterium uptake by trifluoroacetone was found to exceed that of acetone by a factor of 1700 at 25°C. However trifluoroacetone is known to be extensively hydrated under these conditions. The hydrogen exchange of trifluoroacetone is interpreted as most probably proceeding through proton abstraction by pyridine from the free ketone to form the enolate followed by deuteration on carbon, with the rate of proton abstraction from trifluoroacetone exceeding that of acetone by a factor of 10⁵ to 10⁶. Other possibilities are also considered.     

Radiolysis in H2O/D2O mixtures. Hydrogen production under LOCA simulation

The hydrogen isotope radiolytic yields, G(H₂), G(HD) and G(D₂) were determined in H₂O/D₂O mixtures under chemical conditions close to a LOCA in a PHWR like Atucha I Nuclear Station, that is 2·10^–3 MH₃BO₃ and p(H+D)=8.5±0.2. The total hydrogen radiolytic yield G(H₂+HD+D₂) as a function of the deuterium atom fraction goes through a flat maximum at about 0.58. This result in dicates that the 4% flammability limit for hydrogen in the reactor's containment with be reached sooner than what is expected assuming a linear combination of pure H₂ and D₂ radiolytic yields. Hydrogen radiolytic production in 10^–3 M KBr in H₂O/D₂O mixtures gives the same results as in the boric solutions suggesting a bimolecular B(OH) ₄ ^– +OH reaction. Identical isotope concentration factors were calculated for both solutions.     

On the mechanism of reaction of tert-butoxyl radicals with dialkylphosphites

It has been shown by ESR spectroscopy that the title reaction involves abstraction of hydrogen from the phosphite, since at ?10°C the reaction has a kinetic deuterium isotope effect, k_H/k_D, or ～3. The rate constant for hydrogen abstraction is c. 2 × 10⁴ M^?1 s^?1. There is no significant addition of alkoxyl radicals to the phosphite.     

Experimental studies on the hydrogen isotope recovery using low-pressure palladium membrane diffuser

In this paper, we introduce a set of low-pressure palladium membrane diffuser designed to recover hydrogen isotopes from inert mixture gases. Several gaseous mixtures (D₂/Ar and D₂/He) with different deuterium concentration have been used for cleanup test of the low-pressure palladium membrane diffuser at 723 K. Effect of the composition of feed gas on the pressure of permeate side has been observed by gas chromatography (GC) and pressure sensor. With the feed flow rate of the mixture gases increasing, the D₂ permeate pressure is increasing as well. Decontamination factor (DF) of more than 1000 and recovery efficiency greater than 99.9% have been obtained by controlling the feed gas flow rate. The same palladium membrane diffuser was used to process helium-3 gas with more than 10% hydrogen isotope and about 0.3% tritium gas. The pure helium-3 (above 99.4%) with low content of hydrogen isotopes (about 0.084%) has been obtained. Recovery efficiency of all hydrogen isotopes is 99.5% above.     

Hydrolyse acide du p-nitrophényl-diazométhane dans les mélanges H2O?D2O: analyse des effets isotopiques

The velocity of the hydrogen ion catalysed hydrolysis of p-nitrophenyl-diazo-methane (I) has been measured in H₂O? D₂O mixtures, giving an isotopic α_i = 0.49. The product isotope effect r = 5.1, determined from product analyses, combined with the (overall) solvent isotope effect k_H/k_D = 2.81, yields the primary kinetic isotope effect (k_H/k_D)^I = 3.8, and the secondary kinetic isotope effect (k_H/k_D)^II = 0.75. The CICH₂COOH-catalysed hydrolysis of I in H₂O? D₂O mixtures gave a straight-line plot of k_n/k_H versus the atomic fraction n of deuterium. With four carboxylic acids, as catalysts, values of about 4.3 for the kinetic (overall) isotope effects were observed.     

Kinetic isotope effect for hydrogen/deuterium abstraction by chlorine atoms from (CH3)2NNO2 and (CD3)2NNO2

The kinetic isotope effect for the abstraction of hydrogen/deuterium from dimethylnitramine and dimethylnitramine-d₆ by chlorine atoms has been studied in the temperature range 273–353 K. The rate constant ratio k_H0/k_D is given by the Arrhenius expression, k_H/k_D=(0.92 ± 0.07)exp(286 ± 250/RT), where R is expressed in cal mol^?1 K^?1. The absolute rate constant for the deuterium abstraction reaction is extrapolated as k_D=(1.50 ± 0.90) × 10^?10 exp(?1,486 ± 370/RT) cm³ molecule^?1 s^?1. The temperature dependence of the kinetic isotope effect was calculated using the conventional transition-state theory, and the obtained values for k_H/k_D and ΔE_{H, D} are in good agreement with the experimental value for a bent transition state geometry, with two new vibrational frequencies of 340 cm^?1 (272 cm^?1) corresponding to the in-plane and out-of-plane motions of hydrogen (deuterium) atoms in the Cl…H…C arrangement. © 1993 John Wiley & Sons, Inc.     

NHC‐Stabilized Iridium Nanoparticles as Catalysts in Hydrogen Isotope Exchange Reactions of Anilines

The preparation of N‐heterocyclic carbene‐stabilized iridium nanoparticles and their application in hydrogen isotope exchange reactions is reported. These air‐stable and easy‐to‐handle iridium nanoparticles showed a unique catalytic activity, allowing selective and efficient hydrogen isotope incorporation on anilines using D₂ or T₂ as isotopic source. The usefulness of this transformation has been demonstrated by the deuterium and tritium labeling of diverse complex pharmaceuticals.     

Formyl-selective deuteration of aldehydes with D2O via synergistic organic and photoredox catalysis

Formyl-selective deuteration of aldehydes is of high interest for labeling purposes and for optimizing properties of drug candidates. Herein, we report a mild general method for formyl-selective deuterium labeling of aldehydes with D₂O, an inexpensive deuterium source, via a synergistic combination of light-driven, polyoxometalate-facilitated hydrogen atom transfer and thiol catalysis. This highly efficient, scalable reaction showed excellent deuterium incorporation, a broad substrate scope, and excellent functional group tolerance and selectivity and is therefore a practical method for late-stage modification of synthetic intermediates in medicinal chemistry and for generating libraries of deuterated compounds.

Formyl-selective deuteration of aldehydes with D₂O mediated by the synergistic combination of light-driven, polyoxometalate-facilitated HAT and thiol catalysis is reported.