Deuterium-labeled Substrates Research Articles

De Novo Synthesis of Perdeuterated Phosphoinositide by Installing a Non-native Phospholipid Biopathway in E. coli.

Phosphatidylinositol (PI) and its phosphorylated derivatives are of paramount importance in cellular functions and diseases. Understanding their diverse roles is, however, challenged by difficulties in synthesis and labeling techniques. In this proof-of-concept study, we demonstrate that PI can be straightforwardly de novo-synthesized and deuterium (2H)-labeled in Escherichia coli by genomic insertion of PI synthase from Trypanosoma brucei under constitutive synthetic promoter proD. Insertion into loci atpi-gidB and ybb revealed PI accumulation of 41% and 34% (mol/mol), respectively, when cultivated with glycerol as the sole carbon source. Growth of the atpi-gidB-PIS strain in deuterium-labeled (2H) substrates D2O, D8-glycerol, and D6-myo-inositol achieved PI deuteration of 90%, PE deuteration of 95%, and total fatty acids|fatty acid (FA) deuteration of 97%. This study offers an alternative convenient route to chemical and enzymatic labeling synthesis of PI; more excitingly, this work also, in principle, opens a door for tailoring the FA profile of deuterated PI/PE for task-specific application by repurposing FA biosynthesis pathways.

Deuterium MR spectroscopy: potential applications in oncology research.

Metabolic imaging in clinical practice has long relied on PET with fluorodeoxyglucose (FDG), a radioactive tracer. However, this conventional method presents inherent limitations such as exposure to ionizing radiation and potential diagnostic uncertainties, particularly in organs with heightened glucose uptake like the brain. This review underscores the transformative potential of traditional deuterium MR spectroscopy (MRS) when integrated with gradient techniques, culminating in an advanced metabolic imaging modality known as deuterium MRI (DMRI). While recent advancements in hyperpolarized MRS hold promise for metabolic analysis, their widespread clinical usage is hindered by cost constraints and the availability of hyperpolarizer devices or facilities. DMRI, also denoted as deuterium metabolic imaging (DMI), represents a pioneering, single-shot, and noninvasive paradigm that fuses conventional MRS with nonradioactive deuterium-labelled substrates. Extensively tested in animal models and patient cohorts, particularly in cases of brain tumours, DMI's standout feature lies in its seamless integration into standard clinical MRI scanners, necessitating only minor adjustments such as radiofrequency coil tuning to the deuterium frequency. DMRI emerges as a versatile tool for quantifying crucial metabolites in clinical oncology, including glucose, lactate, glutamate, glutamine, and characterizing IDH mutations. Its potential applications in this domain are broad, spanning diagnostic profiling, treatment response monitoring, and the identification of novel therapeutic targets across diverse cancer subtypes.

An Iron-Mesoionic Carbene Complex for Catalytic Intramolecular C-H Amination Utilizing Organic Azides.

The synthesis of N-heterocycles is of paramount importance for the pharmaceutical industry. They are often synthesized through atom economic and environmentally unfriendly methods, generating significant waste. A less explored, but greener, alternative is the synthesis through the direct intramolecular C-H amination utilizing organic azides. Few examples exist by using this method, but many are limited due to the required use of stoichiometric amounts of Boc2O. Herein, we report a homoleptic C,O-chelating mesoionic carbene-iron complex, which is the first iron-based complex that does not require the addition of any protecting groups for this transformation and that is active also in strong donor solvents such as THF or even DMSO. The achieved turnover number is an order of magnitude higher than any other reported catalytic system. A variety of C-H bonds were activated, including benzylic, primary, secondary, and tertiary. By following the reaction over time, we determined the presence of an initiation period. Kinetic studies showed a first-order dependence on substrate concentration and half-order dependence on catalyst concentration. Intermolecular competition reactions with deuterated substrate showed no KIE, while separate reactions with deuterium-labeled substrate resulted in a KIE of 2.0. Moreover, utilizing deuterated substrate significantly decreased the initiation period of the catalysis. Preliminary mechanistic studies suggest a unique mechanism involving a dimeric iron species as the catalyst resting state.

Rhodium(I)-Catalyzed Enantioselective Cyclization of Enynes through Site-Selective C(sp3)–H Bond Activation Triggered by Formation of Rhodacycle

AbstractRhodium(I)-catalyzed enantioselective cyclization of enynes through C(sp3)–H bond activation was investigated. It was found that the cyclization of enynes having a tert-butyl moiety on the alkene afforded a spirocyclic compound (up to 92% ee), while the cyclization of enynes having an isopropyl or an ethyl group on the alkene gave a cyclic diene (up to 98% ee). Furthermore, an intermolecular competition reaction using a deuterium-labeled substrate revealed that C(sp3)–H bond activation was one of the key steps, having a high energy barrier, in this cyclization.

Mechanistic investigation of Rh(i)-catalysed asymmetric Suzuki–Miyaura coupling with racemic allyl halides

Understanding how catalytic asymmetric reactions with racemic starting materials can operate would enable new enantioselective cross-coupling reactions that give chiral products. Here we propose a catalytic cycle for the highly enantioselective Rh(i)-catalysed Suzuki–Miyaura coupling of boronic acids and racemic allyl halides. Natural abundance 13C kinetic isotope effects provide quantitative information about the transition-state structures of two key elementary steps in the catalytic cycle, transmetallation and oxidative addition. Experiments with configurationally stable, deuterium-labelled substrates revealed that oxidative addition can happen via syn- or anti-pathways, which control diastereoselectivity. Density functional theory calculations attribute the extremely high enantioselectivity to reductive elimination from a common Rh complex formed from both allyl halide enantiomers. Our conclusions are supported by analysis of the reaction kinetics. These insights into the sequence of bond-forming steps and their transition-state structures will contribute to our understanding of asymmetric Rh–allyl chemistry and enable the discovery and application of asymmetric reactions with racemic substrates. A major drive in current chemistry research is to develop asymmetric versions of widely used carbon–carbon bond-forming reactions, such as Suzuki-Miyaura cross-couplings. Now, the origins of diastereo- and enantioselectivity in a Rh-catalysed cross-coupling of boronic acid and racemic allyl halides have been established.

The Sesquiterpene Synthase PtTPS5 Produces (1S,5S,7R,10R)-Guaia-4(15)-en-11-ol and (1S,7R,10R)-Guaia-4-en-11-ol in Oomycete-Infected Poplar Roots.

Pathogen infection often leads to the enhanced formation of specialized plant metabolites that act as defensive barriers against microbial attackers. In this study, we investigated the formation of potential defense compounds in roots of the Western balsam poplar (Populus trichocarpa) upon infection with the generalist root pathogen Phytophthora cactorum (Oomycetes). P. cactorum infection led to an induced accumulation of terpenes, aromatic compounds, and fatty acids in poplar roots. Transcriptome analysis of uninfected and P. cactorum-infected roots revealed a terpene synthase gene PtTPS5 that was significantly induced upon pathogen infection. PtTPS5 had been previously reported as a sesquiterpene synthase producing two unidentified sesquiterpene alcohols as major products and hedycaryol as a minor product. Using heterologous expression in Escherichia coli, enzyme assays with deuterium-labeled substrates, and NMR analysis of reaction products, we could identify the major PtTPS5 products as (1S,5S,7R,10R)-guaia-4(15)-en-11-ol and (1S,7R,10R)-guaia-4-en-11-ol, with the former being a novel compound. The transcript accumulation of PtTPS5 in uninfected and P. cactorum-infected poplar roots matched the accumulation of (1S,5S,7R,10R)-guaia-4(15)-en-11-ol, (1S,7R,10R)-guaia-4-en-11-ol, and hedycaryol in this tissue, suggesting that PtTPS5 likely contributes to the pathogen-induced formation of these compounds in planta.

Enantioselective Intramolecular Allylic Substitution via Synergistic Palladium/Chiral Phosphoric Acid Catalysis: Insight into Stereoinduction through Statistical Modeling.

The mode of asymmetric induction in an enantioselective intramolecular allylic substitution reaction catalyzed by a combination of palladium and a chiral phosphoric acid was investigated by a combined experimental and statistical modeling approach. Experiments to probe nonlinear effects, the reactivity of deuterium-labeled substrates, and control experiments revealed that nucleophilic attack to the π-allylpalladium intermediate is the enantio-determining step, in which the chiral phosphate anion is involved in stereoinduction. Using multivariable linear regression analysis, we determined that multiple noncovalent interactions with the chiral environment of the phosphate anion are integral to enantiocontrol in the transition state. The synthetic protocol to form chiral pyrrolidines was further applied to the asymmetric construction of C-O bonds at fully substituted carbon centers in the synthesis of chiral 2,2-disubstituted benzomorpholines.

Cationic Iridium/Chiral Bisphosphine-Catalyzed Enantioselective Hydroacylation of Ketones.

A facile and convenient synthesis of the chiral phthalide framework catalyzed by cationic iridium was developed. The method utilized cationic iridium/bisphosphine-catalyzed asymmetric intramolecular carbonyl hydroacylation of 2-keto benzaldehydes to furnish the corresponding optically active phthalide products in good to excellent enantioselectivities (up to 98% ee). The mechanistic studies using a deuterium-labelled substrate suggested that the reaction involved an intramolecular carbonyl insertion mechanism to iridium hydride intermediate. In addition, we investigated the kinetic isotope effect (KIE) of intramolecular hydroacylation with deuterated substrate and determined that the C-H activation step is not included in the turnover-limiting step.

Limitations of Deuterium-Labelled Substrates for Quantifying NADPH Metabolism in Heterotrophic Arabidopsis Cell Cultures.

NADPH is the primary source of cellular reductant for biosynthesis, and strategies for increasing productivity via metabolic engineering need to take account of the requirement for reducing power. In plants, while the oxidative pentose phosphate pathway is the most direct route for NADPH production in heterotrophic tissues, there is increasing evidence that other pathways make significant contributions to redox balance. Deuterium-based isotopic labelling strategies have recently been developed to quantify the relative production of NADPH from different pathways in mammalian cells, but the application of these methods to plants has not been critically evaluated. In this study, LC-MS was used to measure deuterium incorporation into metabolites extracted from heterotrophic Arabidopsis cell cultures grown on [1-2H]glucose or D2O. The results show that a high rate of flavin-enzyme-catalysed water exchange obscures labelling of NADPH from deuterated substrates and that this exchange cannot be accurately accounted for due to exchange between triose- and hexose-phosphates. In addition, the duplication of NADPH generating reactions between subcellular compartments can confound analysis based on whole cell extracts. Understanding how the structure of the metabolic network affects the applicability of deuterium labelling methods is a prerequisite for development of more effective flux determination strategies, ensuring data are both quantitative and representative of endogenous biological processes.

Nickel-Catalyzed Directed C6-Selective C-H Alkylation of 2-Pyridones with Dienes and Activated Alkenes.

A pyridine-directed, C6-selective nickel-catalyzed ring-contracting C-H alkylation of 2-pyridones with 1,5-cyclooctadiene (cod) has been developed. The reaction proceeds smoothly under external-ligand-free conditions and is accelerated uniquely by a K3PO4 base. Preliminary mechanistic investigations with deuterium-labeled substrates and related reactions with some alkenes are also disclosed.

Simultaneous Measurement of Glucose-6-phosphate 3-Dehydrogenase (NtdC) Catalysis and the Nonenzymatic Reaction of Its Product: Kinetics and Isotope Effects on the First Step in Kanosamine Biosynthesis.

Glucose-6-phosphate 3-dehydrogenase (NtdC) is an NAD-dependent oxidoreductase encoded in the NTD operon of Bacillus subtilis. The oxidation of glucose 6-phosphate by NtdC is the first step in kanosamine biosynthesis. The product, 3-oxo-d-glucose 6-phosphate (3oG6P), has never been synthesized or isolated. The NtdC-catalyzed reaction is very slow at low and neutral pH, and its rate increases to a maximum near pH 9.5. However, under alkaline conditions, the product is not stable because of ring opening followed by deprotonation of the 1,3-dicarbonyl compound. The absorbance band due to this enolate at 310 nm overlaps with that of the other enzymatic product, NADH, complicating kinetic measurements. We report the deconvolution of the resulting spectra of the reaction to determine the rate constants and likely kinetic mechanism. In doing so, we were able to determine the extinction coefficient of the enolate of 3oG6P (23000 M-1 cm-1), which allowed the measurement of the first-order rate constant (5.51 × 10-3 s-1) and activation energy (93 kJ mol-1) of nonenzymatic enolate formation. Using deuterium-labeled substrates, we show that hydride transfer from carbon 3 is partially rate-limiting in the enzymatic reaction, and deuterium substitution on carbon 2 has no significant effect on the enzymatic reaction but lowers the rate of deprotonation of 3oG6P 4-fold. These experiments clearly establish the regiochemistry of the reactions. Coupling of the NtdC reaction with the subsequent step in the pathway, NtdA-catalyzed glutamate-dependent amino transfer, has a small but significant effect on the rate of NAD reduction, consistent with these enzymes working together to process the unstable metabolite.

Copper-catalyzed [1,2]-rearrangements of allylic iodides and aryl α-diazoacetates

The [1,2]- and [2,3]-rearrangements of iodonium ylides are synthetically useful reactions for the generation of functionalized α-iodoesters. Allylic iodides are coupled with α-diazoesters in the presence of a copper catalyst and a ligand to generate iodonium ylides, which undergo metal-mediated rearrangements. By fine-tuning the structure of the ligand, we have reversed the regioselectivity of copper-catalyzed reactions of iodonium ylides from [2,3]- to [1,2]-rearrangements with the use of alternate bipyridine ligands. The preference for [1,2]-rearrangements was further improved by using bulky aryl α-diazoester substrates. Several α-iodoesters with a diverse range of functional groups were generated in good yields (up to 88% yield) and high regioselectivities (up to >95:5 regioisomeric ratio). A deuterium-labeled substrate was utilized to gain insight into the mechanism of the reaction.

Spectroscopic Evidence for the Two C-H-Cleaving Intermediates of Aspergillus nidulans Isopenicillin N Synthase.

The enzyme isopenicillin N synthase (IPNS) installs the β-lactam and thiazolidine rings of the penicillin core into the linear tripeptide l-δ-aminoadipoyl-l-Cys-d-Val (ACV) on the pathways to a number of important antibacterial drugs. A classic set of enzymological and crystallographic studies by Baldwin and co-workers established that this overall four-electron oxidation occurs by a sequence of two oxidative cyclizations, with the β-lactam ring being installed first and the thiazolidine ring second. Each phase requires cleavage of an aliphatic C-H bond of the substrate: the pro-S-CCys,β-H bond for closure of the β-lactam ring, and the CVal,β-H bond for installation of the thiazolidine ring. IPNS uses a mononuclear non-heme-iron(II) cofactor and dioxygen as cosubstrate to cleave these C-H bonds and direct the ring closures. Despite the intense scrutiny to which the enzyme has been subjected, the identities of the oxidized iron intermediates that cleave the C-H bonds have been addressed only computationally; no experimental insight into their geometric or electronic structures has been reported. In this work, we have employed a combination of transient-state-kinetic and spectroscopic methods, together with the specifically deuterium-labeled substrates, A[d2-C]V and AC[d8-V], to identify both C-H-cleaving intermediates. The results show that they are high-spin Fe(III)-superoxo and high-spin Fe(IV)-oxo complexes, respectively, in agreement with published mechanistic proposals derived computationally from Baldwin's founding work.

Mechanistic Studies on Garratt-Braverman Cyclization: The Diradical-Cycloaddition Puzzle.

In this work, we present the results of extensive multiprong studies involving the fate of deuterium-labeled substrates, EPR, trapping experiments, and LA-LDI mass spectrometry to sort out the controversies relating to the mechanism of Garratt-Braverman cyclization in two systems, namely bis-propargyl sulfones and ethers. The results are in conformity with a diradical mechamism for the sulfone, while for the ether, the anionic [4 + 2] appears to be the preferred pathway. This shows that the mechanistic pathway toward GB cyclization is dependent upon the nature of heteroatom (O or S in sulfone) bridging the propargyl arms.

ChemInform Abstract: Pd(0)‐Catalyzed Atom Transfer Radical Cyclization of N‐Allyl‐α‐chloroamides: Highly Stereoselective Synthesis of Substituted γ‐Lactam.

Abstract A catalysis system comprised of Pd(cod)Cl2 precursor and IMes ligand has been developed, which is efficient to catalyze the atom transfer radical cyclization of N-allyl-α-chloroamides with the help of NaI additive. This reaction provides a facile access to β-iodomethyl-γ-lactam with high stereoselectivity. Moreover, the results from the reaction of deuterium-labeled substrate and TEMPO-trapping experiment clearly indicate that radical intermediate(s) should be involved in the reaction.

Synthesis of deuterium-labelled substrates for the study of oleuropein biosynthesis in Olea europaea callus cultures

We propose the cell culture approach to investigate oleuropein (1) biogenesis in Olea europaea L. We suggest employing olive callus cultures to identify the iridoidic precursor of oleuropein. In fact, we confirmed that callus cells from olive shoot explants are able to produce key secoiridoid as 1. To enable this approach, we synthesised and characterised deuterium-labelled iridoidic precursors belonging both to the loganin and the 8-epiloganin series. These iridoids are [7,8-2H2]-7-deoxy-8-epi-loganin (2D), [8,10-2H2]-8-epi-loganin (4D) and [7,8-2H2]-7-deoxy-loganin (3D).

Intramolecular hydrogen transfer reactions catalyzed by pentamethylcyclopentadienyl rhodium and cobalt olefin complexes: Mechanistic studies

The mechanism of intramolecular transfer dehydrogenation catalyzed by [Cp∗M(VTMS)2] (1, M=Rh, 2, M=Co, Cp∗=C5Me5, VTMS=vinyltrimethylsilane) complexes has been studied using vinyl silane protected alcohols as substrates. Deuterium-labeled substrates have been synthesized and the regioselectivity of H/D transfers investigated using 1H and 2H NMR spectroscopy. The labeling studies establish a regioselective pathway consisting of alkene directed α C–H activation, 2,1 alkene insertion, and finally β-hydride elimination to give silyl enol ether products.

Pd(0)-catalyzed atom transfer radical cyclization of N-allyl-α-chloroamides: highly stereoselective synthesis of substituted γ-lactam

A catalysis system comprised of Pd(cod)Cl2 precursor and IMes ligand has been developed, which is efficient to catalyze the atom transfer radical cyclization of N-allyl-α-chloroamides with the help of NaI additive. This reaction provides a facile access to β-iodomethyl-γ-lactam with high stereoselectivity. Moreover, the results from the reaction of deuterium-labeled substrate and TEMPO-trapping experiment clearly indicate that radical intermediate(s) should be involved in the reaction.

Rhodium-catalyzed dehydrogenative coupling of phenylheteroarenes with alkynes or alkenes.

Benzo-fused tri- to heptacyclic heteroarenes were effectively constructed by the rhodium-catalyzed dehydrogenative coupling of phenylheteroarenes with alkynes. Using alkenes as coupling partners, dehydrogenative alkenylation took place selectively on the phenyl moiety of phenylheteroarenes. Several experiments with deuterium-labeled substrates indicated that double C-H bond cleavages take place even in the reaction with alkenes.

Investigating the nature of palladium chain-walking in the enantioselective redox-relay Heck reaction of alkenyl alcohols.

The mechanism of the redox-relay Heck reaction was investigated using deuterium-labeled substrates. Results support a pathway through a low energy palladium–alkyl intermediate that immediately precedes product formation, ruling out a tautomerization mechanism. DFT calculations of the relevant transition structures at the M06/LAN2DZ+f/6-31+G* level of theory show that the former pathway is favored by 5.8 kcal/mol. Palladium chain-walking toward the alcohol, following successive β-hydride eliminations and migratory insertions, is also supported in this study. The stereochemistry of deuterium labels is determined, lending support that the catalyst remains bound to the substrate during the relay process and that both cis- and trans-alkenes form from β-hydride elimination.