Optical annealing of peroxo-ferric intermediates in CYP17A1 and product formation.

Human cytochrome P450 CYP17A1 catalyzes the hydroxylation of pregnenolone and progesterone at the C17 position, with subsequent C17-C20 bond scission, to form dehydroepiandrosterone and androstenedione respectively. The first hydroxylation reaction is faster in H2O than in D2O, while the second carbon­carbon bond scission event demonstrates an inverse solvent isotope effect, which is more pronounced for 17-hydroxy pregnenolone. In order to better understand the cause of this difference, we compared the optical absorption spectra of oxygenated CYP17A1 with the four substrates (pregnenolone, progesterone, 17-hydroxy pregnenolone and 17-hydroxy progesterone) in both H2O and D2O. We also studied the temperature-dependent decay of the peroxo-ferric and hydroperoxo-ferric intermediates generated by cryoradiolysis of the corresponding oxygenated heme proteins at 77 K. For both pregnenolone and 17-hydroxypregnenolone, annealing of the peroxo-intermediates was observed at lower temperatures in H2O than in D2O. In contrast, no solvent isotope effect was detected when progesterone or 17-hydroxyprogesterone were used as substrates. These differences are attributed to their different positioning in the P450 active site with respect to the heme bound peroxo (Fe-OO-) moiety, which is in agreement with earlier structural and spectroscopic investigations. Analysis of the samples run in both H2O and in D2O, where 17-hydroxyprogesterone is the substrate, demonstrated significant (∼25%) yield of androstenedione product relative to the oxygenated starting material.

Hydrogen/Deuterium Exchange Reaction Rate of Cytochrome c Determined by Droplet Collision Atmospheric Pressure Infrared Laser Ablation Mass Spectrometry.

The hydrogen/deuterium (H/D) exchange rate is an optimal measure for studying the structures and dynamics of hydrogen bonding systems, as it reflects the molecular contact environment and the strength of the hydrogen bonds. A method for rapid measurement of the H/D exchange reaction rates is required to examine the intermolecular environments of molecules in solutions. We developed a droplet collision atmospheric pressure infrared laser ablation mass spectrometry technique for this purpose. We obtained the H/D exchange reaction rate of cytochrome c in a methanol/H2O·D2O solution. We revealed that the first hydration shell of the cytochrome c molecule hinders the penetration of D2O to the surface of the molecule from the rates, which provides a novel method to investigate solution structures by a mass-spectrometric method. The droplet-collision mass spectrometry method developed in the present study can be extended to research on the molecular interactions in solutions, such as the mutual interactions of protein molecules, which are of importance in living cells.

Effect of Solvent Properties on the Critical Solution Temperature of Thermoresponsive Polymers.

The ability of thermoresponsive polymers to respond to temperature with a reversible conformational change makes them promising 'smart' materials for solutions in medical and biotechnological applications. In this work, two such polymers and structural isomers were studied: poly(N-isopropyl acrylamide) (PNiPAm) and poly(2-isopropyl-2-oxazoline) (PiPOx). We compare the critical solution temperatures (CST) of these polymers in D2O and H2O in the presence of Hofmeister series salts, as results obtained under these different solvent conditions are often compared. D2O has a higher dipole moment and electronegativity than H2O, which could significantly alter the CST transition. We used two complementary methods to measure the CST, dynamic light scattering (DLS) and differential scanning calorimetry (DSC) and found that the CST decreased significantly in D2O compared to H2O. In the presence of highly concentrated kosmotropes, the CST of both polymers decreased in both solvents. The influence of the kosmotropic anions was smaller than the water isotope effect at low ionic strengths but considerably higher at physiological ionic strengths. However, the Hofmeister anion effect was quantitatively different in H2O than in D2O, with the largest relative differences observed for Cl-, where the CSTs in D2O decreased more than in H2O measured by DLS but less by DSC. PiPOx was more sensitive than PNiPAm to the presence of chaotropes. It exhibited much higher transition enthalpies and multistep transitions, especially in aqueous solutions. Our results highlight that measurements of thermoresponsive polymer properties in D2O cannot be compared directly or quantitatively to application conditions or even measurements performed in H2O.

Validating the utility of heavy water (Deuterium Oxide) as a potential Raman spectroscopic probe for identification of antibiotic resistance.

The impact of microbial infections is increasing over time, and it is one of the major reasons for death in both developed and developing countries. colistin is considered as the antibiotic of last choice for infections brought by major multidrug-resistant (MDR), gram-negative bacteria such as Enterobacter species, Acinetobacter species, and Pseudomonas aeruginosa. Existing approaches to diagnose these resistant species are relatively slow and take up to 2 to 3 days. In this work, we propose a novel interdisciplinary method based on Raman spectroscopy and heavy water to identify colistin-resistant microbes. Our hypothesis is based on the fact that resistant bacteria will be metabolically active in the culture medium containing antibiotics and heavy water, and these bacteria will take up deuterium instead of hydrogen to newly synthesized lipids and proteins. This effect will generate a 'C - D' bond-specific Raman spectral marker. Successful identification of this band in the spectral profile can confirm the presence of colistin-resistant bacteria. We have validated the efficacy of this approach in identifying colistin-resistant bacteria spiked in artificial urine and have compared sensitivity at different bacterial concentrations. Overall findings suggest that heavy water can potentially serve as a suitable Raman probe for identifying metabolically active colistin-resistant bacteria via urine under clinically implementable time and can be used in clinical settings after validation.

D2O as an Imperfect Replacement for H2O: Problem or Opportunity for Protein Research?

D2O is commonly used as a solvent instead of H2O in spectroscopic studies of proteins, in particular, in infrared and nuclear-magnetic-resonance spectroscopy. D2O is chemically equivalent to H2O, and the differences, particularly in hydrogen-bond strength, are often ignored. However, replacing solvent water with D2O can affect not only the kinetics but also the structure and stability of biomolecules. Recent experiments have shown that even the mesoscopic structures and the elastic properties of biomolecular assemblies, such as amyloids and protein networks, can be very different in D2O and H2O. We discuss these findings, which probably are just the tip of the iceberg, and which seem to call for obtaining a better understanding of the H2O/D2O-isotope effect on water-water and water-protein interactions. Such improved understanding may change the differences between H2O and D2O as biomolecular solvents from an elephant in the room to an opportunity for protein research.

Utilization of a kinetic isotope effect to decrease decomposition of ceftriaxone in a mixture of D2O/H2O.

The discovery of cephalosporin and demonstration of its improved stability in aqueous solution, as well as enhanced in vitro activity against penicillin-resistant organisms, were major breakthroughs in the development of ß-lactam antibiotics. Although cephalosporins are more stable with respect to hydrolytic degradation than penicillins, they still experience a variety of chemical transformations. The present study offers an insight into the rates and mechanisms of ceftriaxone degradation at the therapeutic concentration in water, a mixture of water and deuterium oxide, and deuterium oxide itself at the neutral pH. Specific ceftriaxone degradation products were observed in aged samples (including a previously unreported dimer-type species), and by comparing the degradation rates in H2O and D2O, the observation of a kinetic isotope effect provided some valuable insight as to the nature of the initial ceftriaxone degradation. The effect of protium to deuterium isotope change on the degradation kinetics of ceftriaxone was evaluated using the method of initial rates based on HPLC analysis as well as by quantitative 1H NMR spectroscopy. Moreover, computational analysis was utilized to get a molecular insight into chemical processes governing the ceftriaxone degradation and to rationalize the stabilizing effect of replacing H2O with D2O.

Effects of Water Deuteration on Thermodynamic and Structural Properties of Proteins and Biomembranes.

Light and heavy water are often used interchangeably in spectroscopic experiments with the tacit assumption that the structure of the investigated biomolecule does not depend too much on employing one or the other solvent. While this may often be a good approximation, we demonstrate here using molecular dynamics simulations incorporating nuclear quantum effects via modification of the interaction potential that there are small but significant differences. Namely, as quantified and discussed in the present study, both proteins and biomembranes tend to be slightly more compact and rigid in D2O than in H2O, which reflects the stronger hydrogen bonding in the former solvent.

Metabolic deuterium oxide (D2O) labeling in quantitative omics studies: A tutorial review.

Mass spectrometry (MS) is an invaluable tool for sensitive detection and characterization of individual biomolecules in omics studies. MS combined with stable isotope labeling enables the accurate and precise determination of quantitative changes occurring in biological samples. Metabolic isotope labeling, wherein isotopes are introduced into biomolecules through biosynthetic metabolism, is one of the main labeling strategies. Among the precursors employed in metabolic isotope labeling, deuterium oxide (D2O) is cost-effective and easy to implement in any biological systems. This tutorial review aims to explain the basic principle of D2O labeling and its applications in omics research. D2O labeling incorporates D into stable C-H bonds in various biomolecules, including nucleotides, proteins, lipids, and carbohydrates. Typically, D2O labeling is performed at low enrichment of 1%-10% D2O, which causes subtle changes in the isotopic distribution of a biomolecule, instead of the complete separation between labeled and unlabeled samples in a mass spectrum. D2O labeling has been employed in various omics studies to determine the metabolic flux, turnover rate, and relative quantification. Moreover, the advantages and challenges of D2O labeling and its future prospects in quantitative omics are discussed. The economy, versatility, and convenience of D2O labeling will be beneficial for the long-term omics studies for higher organisms.

Isotope effects observed in diluted D2O/H2O mixtures identify HOD-induced low-density structures in D2O but not H2O.

Normal and heavy water are solvents most commonly used to study the isotope effect. The isotope effect of a solvent significantly influences the behavior of a single molecule in a solution, especially when there are interactions between the solvent and the solute. The influence of the isotope effect becomes more significant in D2O/H2O since the hydrogen bond in H2O is slightly weaker than its counterpart (deuterium bond) in D2O. Herein, we characterize the isotope effect in a mixture of normal and heavy water on the solvation of a HOD molecule. We show that the HOD molecule affects the proximal solvent molecules, and these disturbances are much more significant in heavy water than in normal water. Moreover, in D2O, we observe the formation of low-density structures indicative of an ordering of the solvent around the HOD molecule. The qualitative differences between HOD interaction with D2O and H2O were consistently confirmed with Raman spectroscopy and NMR diffusometry.

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.

Three-Dimensional Confinement of Water: H2O Exhibits Long-Range (>50 nm) Structure while D2O Does Not.

Water is the liquid of life thanks to its three-dimensional adaptive hydrogen (H)-bond network. Confinement of this network may lead to dramatic structural changes influencing chemical and physical transformations. Although confinement effects occur on a <1 nm length scale, the upper length scale limit is unknown. Here, we investigate 3D-confinement over lengths scales ranging from 58-140 nm. By confining water in zwitterionic liposomes of different sizes and measuring the change in H-bond network conformation using second harmonic scattering (SHS), we determined long-range confinement effects in light and heavy water. D2O displays no detectable 3D-confinement effects <58 nm (<3 × 106 D2O molecules). H2O is distinctly different. The vesicle enclosed inner H-bond network has a different conformation compared to the outside network and the SHS response scales with the volume of the confining space. H2O displays confinement effects over distances >100 nm (>2 × 107 H2O molecules).

Research Progress in the Medical Application of Heavy Water, Especially in the Field of D2O-Raman Spectroscopy.

Heavy water is an ideal contrast agent for metabolic activity and can be adapted to a wide range of biological systems owing to its non-invasiveness, universal applicability, and cost-effectiveness. As a new type of probe, the heavy isotope of water has been widely used in the study of cell development, metabolism, tissue homeostasis, aging, and tumor heterogeneity. Herein, we review findings supporting the applications of and research on heavy water in monitoring of bacterial metabolism, rapid detection of drug sensitivity, identification of tumor cells, precision medicine, and evaluation of skin barrier function and promote the use of heavy water as a suitable marker for the development of detection and treatment methodologies.

Solvent-mediated isotope effects strongly influence the early stages of calcium carbonate formation: exploring D2O vs. H2O in a combined computational and experimental approach.

In experimental studies, heavy water (D2O) is employed, e.g., so as to shift the spectroscopic solvent background, but any potential effects of this solvent exchange on reaction pathways are often neglected. While the important role of light water (H2O) during the early stages of calcium carbonate formation has been realized, studies into the actual effects of aqueous solvent exchanges are scarce. Here, we present a combined computational and experimental approach to start to fill this gap. We extended a suitable force field for molecular dynamics (MD) simulations. Experimentally, we utilised advanced titration assays and time-resolved attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. We find distinct effects in various mixtures of the two aqueous solvents, and in pure H2O or D2O. Disagreements between the computational results and experimental data regarding the stabilities of ion associates might be due to the unexplored role of HDO, or an unprobed complex phase behaviour of the solvent mixtures in the simulations. Altogether, however, our data suggest that calcium carbonate formation might proceed "more classically" in D2O. Also, there are indications for the formation of new structures in amorphous and crystalline calcium carbonates. There is huge potential towards further improving the understanding of mineralization mechanisms by studying solvent-mediated isotope effects, also beyond calcium carbonate. Last, it must be appreciated that H2O and D2O have significant, distinct effects on mineralization mechanisms, and that care has to be taken when experimental data from D2O studies are used, e.g., for the development of H2O-based computer models.

Dynamic Control of Photocatalytic Proton Reduction through the Mechanical Actuation of a Hydrogel Host Matrix.

This paper describes a photocatalytic hydrogen evolution system that is dynamically and reversibly responsive to the pH of the surrounding solution through the actuation of a microhydrogel (microgel) matrix that hosts the photocatalysts (CdSe/CdS nanorods). The reversible actuation occurs within 0.58 (swelling) and 1.7 s (contraction). ΔpH = 0.01 relative to the pKa of the tertiary amine on the microgel polymer (7.27) results in a reversible change in the average diameter of the microgel hosts by a factor of 2.4 and a change in the photocatalytic turnover frequency (TOF) by a factor of 5. Kinetic isotope effect and photoluminescence quenching experiments reveal that the scavenging of the photoexcited hole by sulfite ions is the rate-limiting step and leads to the observed response of the TOF to pH through the actuation of the microgel. Molecular dynamics simulations quantify a greater local concentration of sulfite hole scavengers for pH < pKa.

Synergistic Properties of Arabinogalactan (AG) and Hyaluronic Acid (HA) Sodium Salt Mixtures.

The properties of mixtures of two polysaccharides, arabinogalactan (AG) and hyaluronic acid (HA), were investigated in solution by the measurement of diffusion coefficients D of water protons by DOSY (Diffusion Ordered SpectroscopY), by the determination of viscosity and by the investigation of the affinity of a small molecule molecular probe versus AG/HA mixtures in the presence of bovine submaxillary mucin (BSM) by 1HNMR spectroscopy. Enhanced mucoadhesive properties, decreased mobility of water and decreased viscosity were observed at the increase of AG/HA ratio and of total concentration of AG. This unusual combination of properties can lead to more effective and long-lasting hydration of certain tissues (inflamed skin, dry eye corneal surface, etc.) and can be useful in the preparation of new formulations of cosmetics and of drug release systems, with the advantage of reducing the viscosity of the solutions.

Zero-Field NMR of Urea: Spin-Topology Engineering by Chemical Exchange.

Well-resolved and information-rich J-spectra are the foundation for chemical detection in zero-field NMR. However, even for relatively small molecules, spectra exhibit complexity, hindering the analysis. To address this problem, we investigate an example biomolecule with a complex J-coupling network─urea, a key metabolite in protein catabolism─and demonstrate ways of simplifying its zero-field spectra by modifying spin topology. This goal is achieved by controlling pH-dependent chemical exchange rates of 1H nuclei and varying the composition of the D2O/H2O mixture used as a solvent. Specifically, we demonstrate that by increasing the proton exchange rate in the [13C,15N2]-urea solution, the spin system simplifies, manifesting through a single narrow spectral peak. Additionally, we show that the spectra of 1H/D isotopologues of [15N2]-urea can be understood easily by analyzing isolated spin subsystems. This study paves the way for zero-field NMR detection of complex biomolecules, particularly in biofluids with a high concentration of water.

Structure-Activity Relationships in Nonenzymatic Template-Directed RNA Synthesis.

The template-directed synthesis of RNA played an important role in the transition from prebiotic chemistry to the beginnings of RNA based life, but the mechanism of RNA copying chemistry is incompletely understood. We measured the kinetics of template copying with a set of primers with modified 3'-nucleotides and determined the crystal structures of these modified nucleotides in the context of a primer/template/substrate-analog complex. pH-rate profiles and solvent isotope effects show that deprotonation of the primer 3'-hydroxyl occurs prior to the rate limiting step, the attack of the alkoxide on the activated phosphate of the incoming nucleotide. The analogs with a 3 E ribose conformation show the fastest formation of 3'-5' phosphodiester bonds. Among those derivatives, the reaction rate is strongly correlated with the electronegativity of the 2'-substituent. We interpret our results in terms of differences in steric bulk and charge distribution in the ground vs. transition states.

Use of deuterium oxide (2H2O) to assess muscle protein synthesis in juvenile red drum (Sciaenops ocellatus) fed complete, and valine-deficient diets.

The use of 2H2O in tank water to assess protein synthesis rates in fish is a relatively novel methodology that could allow for a better understanding of the effects of particular nutritional and environmental variables on rates of protein accretion. As such, this study involved an assessment and comparison of protein synthesis rates in the muscle of juvenile red drum fed a control diet (nutritionally complete) versus a valine (Val)-deficient diet. Six groups of 12 juvenile red drum, initially weighing ~ 4.5 g/fish, were stocked in six separate 38-L aquaria operating as a recirculating system. Fish were acclimatized to experimental conditions for 2 weeks while being fed the control diet. Just prior to initiating the protein synthesis assay, one aquarium of fish was fed the control diet while a second aquarium of fish was fed the Val-deficient diet. Immediately after consuming the experimental diets, each group of fish was moved to an independent aquarium containing 2H2O, and the fractional synthetic rate (FSR) of protein synthesis was obtained at 12, 24, 36 and 48 h after feeding by collecting two fish per treatment at each time point. This protein synthesis assay procedure was performed in three separate sessions, and considered as replicates over time (n = 3) for fish fed the control or Val-deficient diets immediately before initiating the session. Results indicated that a one-time feeding of a diet deficient in Val significantly reduced protein synthesis rates in the muscle of red drum. In addition, a significant effect of time after feeding was found, where observed FSR values peaked at 12 h after feeding and decreased as time progressed. In conclusion, deuterium methodologies were applicable to red drum, and this approach had the sensitivity to assess differences in protein synthesis rates when dietary perturbations were introduced.

A Water-Stable Tb-MOF As a Rapid, Accurate, and Highly Sensitive Ratiometric Luminescent Sensor for the Discriminative Sensing of Antibiotics and D2O in H2O.

The design and development of self-calibrating ratiometric luminescent sensors for the fast, accurate, and sensitive discrimination and determination of pollutants in wastewater is highly desirable for public and environmental health. Herein, a 3D porous Tb(III)-based metal-organic framework (MOF), {[Tb(HL)(H2O)2]·x(solv)}n (1), was facilely synthesized using a urea-functionalized tetracarboxylate ligand, 5,5'-(((1,4-phenylenebis(azanediyl))bis(carbonyl))bis(azanediyl))diisophthalic acid (H4L). The activated framework showed a good water stability in both aqueous solutions at a wide pH range of 2-14 and simulated antibiotic wastewaters. Interestingly, this Tb-MOF exhibited dual luminescence owing to the partial energy transfer from the antenna H4L to Tb3+. More importantly, activated 1 (1a) that was dispersed in water showed a fast, accurate, and highly sensitive discrimination ability toward antibiotics with a good recyclability, discriminating three different classes of antibiotics from each other via the quenching or enhancement of the luminescence and tuning the emission intensity ratio between the H4L ligand and the Tb3+ center for the first time. Simultaneously, 1a is a ratiometric luminescent sensor for the rapid, accurate, and quantitative discrimination of D2O from H2O. Furthermore, this complex was successfully used for the effective determination of antibiotics and D2O in real water samples. This work indicates that 1a represents the first ever MOF material for the discriminative sensing of antibiotics and D2O in H2O and promotes the practical application of Ln-MOF-based ratiometric luminescent sensors in monitoring water quality and avoiding any major leak situation.

Light-driven decarboxylative deuteration enabled by a divergently engineered photodecarboxylase.

Despite the well-established chemical processes for C-D bond formation, the toolbox of enzymatic methodologies for deuterium incorporation has remained underdeveloped. Here we describe a photodecarboxylase from Chlorella variabilis NC64A (CvFAP)-catalyzed approach for the decarboxylative deuteration of various carboxylic acids by employing D2O as a cheap and readily available deuterium source. Divergent protein engineering of WT-CvFAP is implemented using Focused Rational Iterative Site-specific Mutagenesis (FRISM) as a strategy for expanding the substrate scope. Using specific mutants, several series of substrates including different chain length acids, racemic substrates as well as bulky cyclic acids are successfully converted into the deuterated products (>40 examples). In many cases WT-CvFAP fails completely. This approach also enables the enantiocomplementary kinetic resolution of racemic acids to afford chiral deuterated products, which can hardly be accomplished by existing methods. MD simulations explain the results of improved catalytic activity and stereoselectivity of WT CvFAP and mutants.