Carbon Dioxide Radical Anion by Photoinduced Equilibration between Formate Salts and [11C, 13C, 14C]CO2: Application to Carbon Isotope Radiolabeling.

The need for carbon-labeled radiotracers is increasingly higher in drug discovery and development (carbon-14, ß-, t1/2 = 5730 years) as well as in positron emission tomography (PET) for in vivo molecular imaging applications (carbon-11, ß+, t1/2 = 20.4 min). However, the structural diversity of radiotracers is still systematically driven by the narrow available labeled sources and methodologies. In this context, the emergence of carbon dioxide radical anion chemistry might set forth potential unexplored opportunities. Based on a dynamic isotopic equilibration between formate salts and [13C, 14C, 11C]CO2, C-labeled radical anion CO2•- could be accessed under extremely mild conditions within seconds. This methodology was successfully applied to hydrocarboxylation and dicarboxylation reactions in late-stage carbon isotope labeling of pharmaceutically relevant compounds. The relevance of the method in applied radiochemistry was showcased by the whole-body PET biodistribution profile of [11C]oxaprozin in mice.

Transition-Metal-Free Carbon Isotope Exchange of Phenyl Acetic Acids.

A transition-metal-free carbon isotope exchange procedure on phenyl acetic acids is described. Utilizing the universal precursor CO2 , this protocol allows the carbon isotope to be inserted into the carboxylic acid position, with no need of precursor synthesis. This procedure enabled the labeling of 15 pharmaceuticals and was compatible with carbon isotopes [14 C] and [13 C]. A proof of concept with [11 C] was also obtained with low molar activity valuable for distribution studies.

Dynamic Carbon Isotope Exchange of Pharmaceuticals with Labeled CO2.

A copper-catalyzed procedure enabling dynamic carbon isotope exchange is described. Utilizing the universal precursor [14C]CO2, this protocol allows to insert, in one single step, the desired carbon tag into carboxylic acids with no need of structural modifications. Reducing synthetic costs and limiting the generation of radioactive waste, this procedure will facilitate the access to carboxylic acids containing drugs and accelerate early 14C-based ADME studies supporting drug development.

Late-Stage Isotopic Carbon Labeling of Pharmaceutically Relevant Cyclic Ureas Directly from CO2.

A robust, click-chemistry-inspired procedure for radiolabeling of cyclic ureas was developed. This protocol, suitable for all carbon isotopes (11 C, 13 C, 14 C), is based on the direct functionalization of carbon dioxide: the universal building block for carbon radiolabeling. The strategy is operationally simple and reproducible in different radiochemistry centers, exhibits remarkably wide substrate scope with short reaction times, and demonstrates superior reactivity as compared to previously reported systems. With this procedure, a variety of pharmaceuticals and an unprotected peptide were labeled with high radiochemical efficiency.

Palladium-catalyzed decarboxylative cyanation of aromatic carboxylic acids using [(13)C] and [(14)C]-KCN.

The development of robust and straightforward methods to efficiently label aromatic moieties starting from simple and convenient radio-synthetic sources still represents a considerable challenge. In this report, a new palladium-catalyzed decarboxylative cyanation protocol has been described. This procedure utilizes [(14)C]-labeled potassium cyanide, one of the simplest and commercially available sources of carbon-14. Under the optimized reaction conditions, a series of [(13)C] and [(14)C]-aromatic nitriles were easily prepared (12-74% yield starting from potassium cyanide). The usefulness of this methodology is highlighted by a rare example of a formal two-step [(12)C]-[(14)C] carbon isotope exchange. The current synthetic approach may represent a promising alternative to traditional preparations of relevant building blocks such as labeled aromatic nitriles.

A phosphine-catalyzed preparation of 4-arylidene-5-imidazolones.

A simple and efficient method for constructing 4-arylidene-5-imidazolones was developed using a phosphine-catalyzed tandem umpolung addition and intramolecular cyclization of amidine pronucleophiles on arylpropiolates. The reaction offers a robust route to heterocycle analogues of the fluorescent protein chromophores.

Straightforward preparation of labeled potassium cyanate by ozonation and application to the synthesis of [13C] or [14C]ureidocarboxylic acids.

The development of new efficient syntheses of labeled reagents is a great challenge. Avoidance of overcomplicated procedures, availability and cost of starting materials are important considerations in choosing the synthetic route. In this report, we describe a facile and rapid preparation of labeled cyanate by ozonation of cyanide, a basic precursor. The crude cyanate was used without purification for the synthesis of various [(13)C] or [(14)C]ureidocarboxylic acids (20-68% yield from potassium cyanide). According to these results, cyanide ozonation may prove to be a promising alternative to traditional preparations of labeled cyanate.

Unlocking full and fast conversion in photocatalytic carbon dioxide reduction for applications in radio-carbonylation.

Harvesting sunlight to drive carbon dioxide (CO2) valorisation represents an ideal concept to support a sustainable and carbon-neutral economy. While the photochemical reduction of CO2 to carbon monoxide (CO) has emerged as a hot research topic, the full CO2-to-CO conversion remains an often-overlooked criterion that prevents a productive and direct valorisation of CO into high-value-added chemicals. Herein, we report a photocatalytic process that unlocks full and fast CO2-to-CO conversion (<10 min) and its straightforward valorisation into human health related field of radiochemistry with carbon isotopes. Guided by reaction-model-based kinetic simulations to rationalize reaction optimisations, this manifold opens new opportunities for the direct access to 11C- and 14C-labeled pharmaceuticals from their primary isotopic sources [11C]CO2 and [14C]CO2.

A general procedure for carbon isotope labeling of linear urea derivatives with carbon dioxide.

Carbon isotope labeling is a traceless technology, which allows tracking the fate of organic compounds either in the environment or in living organisms. This article reports on a general approach to label urea derivatives with all carbon isotopes, including 14C and 11C, based on a Staudinger aza-Wittig sequence. It provides access to all aliphatic/aromatic urea combinations.

Enhanced drug loading in polymerized micellar cargo.

A new drug carrier system based on self-assembly and polymerization of polydiacetylenic amphiphiles is described. Although classical amphiphiles can help in solubilizing hydrophobic molecules upon self-arrangement into a variety of nanometric structures, a greater effect on drug loading was observed for our polymerized micelles as compared to the non-polymerized analogues. This permitted higher aqueous solubilization of lipophilic drugs with low micelle concentration. (14)C labeling of a model drug on one side and of the amphiphile on the other side permitted assessment, after intravenous injection, of biodistribution and excretion profiles of the drug cargo.

Nanotoxicology at the particle/micelle frontier: influence of core-polymerization on the intracellular distribution, cytotoxicity and genotoxicity of polydiacetylene micelles.

The understanding of the cellular uptake and the intracellular fate of nanoparticles and their subsequent influence on cell viability is challenging as far as micelles are concerned. Such systems are dynamic by nature, existing as unimers under their critical micelle concentration (CMC), and as micelles in equilibrium with unimers above the CMC, making canonical dose-response relationships difficult to establish. The purpose of this study was to investigate the in vitro cytotoxicity and uptake of two micellar sytems that are relevant for drug delivery. The two micelles incorporate a poly(ethylene glycol) coating and a pentacosadiynoic core which is either polymerized (pDA-PEG micelles) or non-polymerized (DA-PEG micelles), with the aim of evaluating the influence of the micelles status ("particle-like" or "dynamic", respectively) on their toxicological profile. Intracellular distribution and cytotoxicity of polymerized and non-polymerized micelles were investigated on RAW 264.7 macrophages in order to compare any different interactions with cells. Non-polymerized micelles showed significantly higher cytotoxicity than polymerized micelles, especially in terms of cell permeabilization, correlated to a higher accumulation in cell membranes. Other potential toxicity endpoints of polymerized micelles were then thoroughly studied in order to assess possible responses resulting from their endocytosis. No specific mechanisms of cytotoxicity were observed, neither in terms of apoptosis induction, cell membrane damage, release of inflammatory mediators nor genotoxicity. These data indicate that non-polymerized micelles accumulate in the cell membrane and induce cell membrane permeabilization, resulting in significant toxicity, whereas polymerized, stable micelles are internalized by cells but exert no or very low toxicity.

Carbon isotope labeling of carbamates by late-stage [11C], [13C] and [14C]carbon dioxide incorporation.

A general procedure for the late-stage [11C], [13C] and [14C]carbon isotope labeling of cyclic carbamates is reported. This protocol allows the incorporation of carbon dioxide, the primary source of carbon-14 and carbon-11 radioisotopes, in a direct, cost-effective and sustainable manner. A disconnection/reconnection strategy, involving ring opening/isotopic closure, was also implemented.

Phosphine-catalyzed construction of sulfur heterocycles.

A simple and efficient method for constructing sulfur heterocycles was developed using a phosphine-catalyzed tandem umpolung addition and intramolecular cyclization of bifunctional sulfur pronucleophiles on arylpropiolates. The reaction offers a promising route to synthetically useful as well as biologically active heterocycles under neutral conditions.

A Synthetic Alternative to Canonical One-Carbon Metabolism.

One-carbon metabolism is an ubiquitous metabolic pathway that encompasses the reactions transferring formyl-, hydroxymethyl- and methyl-groups bound to tetrahydrofolate for the synthesis of purine nucleotides, thymidylate, methionine and dehydropantoate, the precursor of coenzyme A. An alternative cyclic pathway was designed that substitutes 4-hydroxy-2-oxobutanoic acid (HOB), a compound absent from known metabolism, for the amino acids serine and glycine as one-carbon donors. It involves two novel reactions, the transamination of l-homoserine and the transfer of a one-carbon unit from HOB to tetrahydrofolate releasing pyruvate as coproduct. Since canonical reactions regenerate l-homoserine from pyruvate by carboxylation and subsequent reduction, every one-carbon moiety made available for anabolic reactions originates from CO2. The HOB-dependent pathway was established in an Escherichia coli auxotroph selected for prototrophy using long-term cultivation protocols. Genetic, metabolic and biochemical evidence support the emergence of a functional HOB-dependent one-carbon pathway achieved with the recruitment of the two enzymes l-homoserine transaminase and HOB-hydroxymethyltransferase and of HOB as an essential metabolic intermediate. Escherichia coli biochemical reprogramming was achieved by minimally altering canonical metabolism and leveraging on natural selection mechanisms, thereby launching the resulting strain on an evolutionary trajectory diverging from all known extant species.

Conjugated linoleic acid isomers and their conjugated derivatives inhibit growth of human cancer cell lines.

Conjugated linoleic acid (CLA), mainly c9,t11- and t10,c12-isomers, and polyunsaturated n-3 fatty acids (n-3 PUFA) have been shown to reduce tumor growth. This study compared, on a set of human tumor cells (breast, lung, colon, prostate and melanoma), the antiproliferative effects of: i) trans monounsaturated fatty acids (MUFA) vs. cis MUFA and MUFA vs. PUFA, ii) individual isomers of CLA vs. linoleic acid, iii) CLA-conjugated derivatives vs. their non-conjugated homologues and vs. CLA isomers. Tumor cells were exposed to medium containing individual FA (100 microM) for 48 h and their proliferation was determined by measuring the cellular DNA content (fluorescent Hoechst 33342 dye). The antiproliferative effects of FA varied with the type of cells and were mainly dependent on the degree of unsaturation and on the position and configuration of their double bonds. One isomer of CLA (t9,t11-18:2) and CLA-conjugated derivatives exhibited the strongest growth-inhibitory effect against cancer cells. These results suggest that ruminant products contain active compounds against human tumor cell proliferation.

Vaccenic acid metabolism in the liver of rat and bovine.

Hepatic metabolism of vaccenic acid (VA), especially its conversion into CLA, was studied in the bovine (ruminant species that synthesizes CLA) and in the rat (model for non-ruminant) by using the in vitro technique of liver explants. Liver tissue samples were collected from fed animals (5 male Wistar rats and 5 Charolais steers) and incubated at 37 degrees C for 17 h under an atmosphere of 95% O2/5% CO2 in medium supplemented with 0.75 mM of FA mixture and with 55 microM [1-14C]VA. VA uptake was about sixfold lower in bovine than in rat liver slices (P< 0.01). For both species, VA that was oxidized to partial oxidation products represented about 20% of VA incorporated by cells. The chemical structure of VA was not modified in bovine liver cells, whereas in rat liver cells, 3.2% of VA was converted into 16:0 and only 0.33% into CLA. The extent of esterification of VA was similar for both animal species (70-80% of incorporated VA). Secretion of VA as part of VLDL particles was very low and similar in rat and bovine liver (around 0.07% of incorporated VA). In conclusion, characteristics of the hepatic metabolism of VA were similar for rat and bovine animals, the liver not being involved in tissue VA conversion into CLA in spite of its high capacity for FA desaturation especially in the rat. This indicates that endogenous synthesis of CLA should take place exclusively in peripheral tissues.

Pharmacokinetics, biodistribution and metabolism of squalenoyl adenosine nanoparticles in mice using dual radio-labeling and radio-HPLC analysis.

Adenosine is a pleiotropic endogenous nucleoside with potential neuroprotective pharmacological activity. However, clinical use of adenosine is hampered by its extremely fast metabolization. To overcome this limitation, we recently developed a new squalenoyl nanomedicine of adenosine [Squalenoyl-Adenosine (SQAd)] by covalent linkage of this nucleoside to the squalene, a natural lipid. The resulting nanoassemblies (NAs) displayed a dramatic pharmacological activity both in cerebral ischemia and spinal cord injury pre-clinical models. The aim of the present study was to investigate the plasma profile and tissue distribution of SQAd NAs using both Squalenoyl-[(3)H]-Adenosine NAs and [(14)C]-Squalenoyl-Adenosine NAs as respective tracers of adenosine and squalene moieties of the SQAd bioconjugate. This study was completed by radio-HPLC analysis allowing to determine the metabolization profile of SQAd. We report here that SQAd NAs allowed a sustained circulation of adenosine under its prodrug form (SQAd) for at least 1h after intravenous administration, when free adenosine was metabolized within seconds after injection. Moreover, the squalenoylation of adenosine and its formulation as NAs also significantly modified biodistribution, as SQAd NAs were mainly captured by the liver and spleen, allowing a significant release of adenosine in the liver parenchyma. Altogether, these results suggest that SQAd NAs provided a reservoir of adenosine into the bloodstream which may explain the previously observed neuroprotective efficacy of SQAd NAs against cerebral ischemia and spinal cord injury.

In vitro comparison of hepatic metabolism of 9cis-11 trans and 10trans-12cis isomers of CLA in the rat.

Hepatic metabolism of the two main isomers of CLA (9cis-11trans, 10trans-12cis C18:2) was compared to that of oleic acid (representative of the main plasma FA) in 16 rats by using the in vitro method of incubated liver slices. Liver tissue samples were incubated at 37 degrees C for 17 h under an atmosphere of 95% O2/5% CO2 in a medium supplemented with 0.75 mM of FA mixture (representative of circulating nonesterified FA) and with 55 microM [1-(14)C]9cis-11trans C18:2,11-(14)C]10trans-12cis C18:2, or 11-(14)C]oleate. The uptake of CLA by hepatocytes was similar for both isomers (9%) and was three times higher (P < 0.01) than for oleate (2.6%). The rate of CLA isomer oxidation was two times higher (49 and 40% of incorporated amounts of 9cis-11 trans and 10trans-12cis, respectively) than that of oleate (P < 0.01). Total oxidation of oleate and CLA isomers into [14CO2] was low (2 to 7% of total oxidized FA) compared to the partial oxidation (93 to 98%) leading to the production of [14C] acid-soluble products. CLA isomers escaping from catabolism were both highly desaturated (26.7 and 26.8%) into conjugated 18:3. Oleate and CLA isomers were mainly esterified into neutral lipids (70% of esterifled FA) and, to a lesser extent, into polar lipids (30%). They were slowly secreted as parts of VLDL particles (< 0.4% of FA incorporated into cells), the extent of secretion of oleate and of 10trans-12cis being 2.2-fold higher than that of 9cis-11trans (P < 0.02). In conclusion, this study clearly showed that both CLA isomers were highly catabolized by hepatocytes, reducing their availability for peripheral tissues. Moreover, more than 25% of CLA escaping from catabolism was converted into conjugated 18:3, the biological properties of which remain to be elucidated.

Squalenoyl adenosine nanoparticles provide neuroprotection after stroke and spinal cord injury.

There is an urgent need to develop new therapeutic approaches for the treatment of severe neurological trauma, such as stroke and spinal cord injuries. However, many drugs with potential neuropharmacological activity, such as adenosine, are inefficient upon systemic administration because of their fast metabolization and rapid clearance from the bloodstream. Here, we show that conjugation of adenosine to the lipid squalene and the subsequent formation of nanoassemblies allows prolonged circulation of this nucleoside, providing neuroprotection in mouse stroke and rat spinal cord injury models. The animals receiving systemic administration of squalenoyl adenosine nanoassemblies showed a significant improvement of their neurologic deficit score in the case of cerebral ischaemia, and an early motor recovery of the hindlimbs in the case of spinal cord injury. Moreover, in vitro and in vivo studies demonstrated that the nanoassemblies were able to extend adenosine circulation and its interaction with the neurovascular unit. This Article shows, for the first time, that a hydrophilic and rapidly metabolized molecule such as adenosine may become pharmacologically efficient owing to a single conjugation with the lipid squalene.

Trans-10, cis-12 conjugated linoleic acid decreases de novo lipid synthesis in human adipocytes.

Conjugated linoleic acid (CLA) reduces adiposity in vivo. However, mechanisms mediating these changes are unclear. Therefore, we treated cultures of human adipocytes with trans-10, cis-12 (10,12) CLA, cis-9, trans-11 (9,11) CLA or other trans fatty acids (FA), and measured indices of lipid metabolism. The lipid-lowering effects of 10,12 CLA were unique, as other trans FA did not reduce TG content to the same extent. Using low levels of [(14)C]-CLA isomers, it was shown that both isomers were readily incorporated into acylglycerols and phospholipids, albeit at lower levels than [(14)C]-oleic or [(14)C]-linoleic acids. When using [(14)C]-acetic acid and [(14)C]-pyruvic acid as substrates, 30 µM 10,12 CLA, but not 9,11 CLA, decreased de novo synthesis of triglyceride, free FA, diacylglycerol, cholesterol esters, cardiolipin, phospholipids and ceramides within 3-24 h. Treatment with 30 µM 10,12 CLA, but not 9,11 CLA, decreased total cellular lipids within 3 days and the ratio of monounsaturated FA (MUFA) to saturated FA, and increased C18:0 acyl-CoA levels within 24 h. Consistent with these data, stearoyl-CoA desaturase (SCD)-1 mRNA and protein levels were down-regulated by 10,12 CLA within 7-12 h, respectively. The mRNA levels of liver X receptor (LXR)α and sterol regulatory element binding protein (SREBP)-1c, transcription factors that regulate SCD-1, were decreased by 10,12 CLA within 5 h. These data suggest that the isomer-specific decrease in de novo lipid synthesis by 10,12 CLA is due, in part, to the rapid repression of lipogenic transcription factors that regulate MUFA synthesis, suggesting an anti-obesity mechanism unique to this trans FA.