A practical guide for the preparation of C1-labeled α-amino acids using aldehyde catalysis with isotopically labeled CO2.

Isotopically carbon-labeled α-amino acids are valuable synthetic targets that are increasingly needed in pharmacology and medical imaging. Existing preparations rely on early stage introduction of the isotopic label, which leads to prohibitive synthetic costs and time-intensive preparations. Here we describe a protocol for the preparation of C1-labeled α-amino acids using simple aldehyde catalysts in conjunction with [*C]CO2 (* = 14, 13, 11). This late-stage labeling strategy is enabled by the one-pot carboxylate exchange of unprotected α-amino acids with [*C]CO2. The protocol consists of three separate procedures, describing the syntheses of (±)-[1-13C]phenylalanine, (±)-[1-11C]phenylalanine and (±)-[1-14C]phenylalanine from unlabeled phenylalanine. Although the delivery of [*C]CO2 is operationally distinct for each experiment, each procedure relies on the same fundamental chemistry and can be executed by heating the reaction components at 50-90 °C under basic conditions in dimethylsulfoxide. Performed on scales of up to 0.5 mmol, this methodology is amenable to C1-labeling of many proteinogenic α-amino acids and nonnatural derivatives, which is a breakthrough from existing methods. The synthesis of (±)-[1-13C]phenylalanine requires ~2 d, with product typically obtained in a 60-80% isolated yield (n = 3, µ = 71, σ = 8.3) with an isotopic incorporation of 70-88% (n = 18, µ = 72, σ = 9.0). Starting from the preformed imino acid (~3 h preparation time), rapid synthesis of (±)-[1-11C]phenylalanine can be completed in ~1 h with an isolated radiochemical yield of 13%. Finally, (±)-[1-14C]phenylalanine can be accessed in ~2 d with a 51% isolated yield and 11% radiochemical yield.

The Future of (Radio)-Labeled Compounds in Research and Development within the Life Science Industry.

In this review the applications of isotopically labeled compounds are discussed and put into the context of their future impact in the life sciences. Especially discussing their use in the pharma and crop science industries to follow their fate in the environment, in vivo or in complex matrices to understand the potential harm of new chemical structures and to increase the safety of human society.

In situ Generated Iridium Nanoparticles as Hydride Donors in Photoredox-Catalyzed Hydrogen Isotope Exchange Reactions with Deuterium and Tritium Gas.

We have studied the photoredox-catalyzed hydrogen isotope exchange (HIE) reaction with deuterium or tritium gas as isotope sources and in situ formed transition metal nanoparticles as hydrogen atom transfer pre-catalysts. By this means we have found synergistic reactivities applying two different HIE mechanisms, namely photoredox-catalyzed and CH-functionalization HIE leading to the synthesis of highly deuterated complex molecules. Finally, we adopted these findings successfully to tritium chemistry.

Small-scale two-dimensional liquid chromatography for a preparative re-purification of a highly labile tritium-labeled compound.

Tritium-labeled compounds are generally less stable than their non-labeled counterparts. This requires storage at low temperatures, a constant workflow of quality checks, and subsequent re-purifications. As the amount of tritium-labeled material is typically purified in the µg range, repeated injections on analytical-scale ultra high-performance liquid chromatography systems can provide high-resolution re-purification results. Yet, degradants can be undesirably included in the compound isolation because the amount of decomposition can vary dramatically depending on the structure. We report a case of a sensitive molecule that could not be isolated in pure form even though the chromatographic separation was successful. In this case, the use of a small-scale two-dimensional preparative liquid chromatography approach with a direct transfer interface to a second (trapping) column resulted in a highly pure compound (>98% radiochemical purity). This approach combines high chromatographic resolution, accurate control over the re-purification process, minimal sample manipulation, and higher overall safety for the handling of radioactive samples.

Hydrogen Isotope Exchange by Homogeneous Iridium Catalysis in Aqueous Buffers with Deuterium or Tritium Gas.

We have studied the highly selective homogeneous iridium-catalyzed hydrogen isotope exchange (HIE) with deuterium or tritium gas as an isotope source in water and buffers. With an improved water-soluble Kerr-type catalyst, we have achieved the first insight into applying HIE reactions in aqueous media with varying pH. Density functional theory (DFT) calculations gave consistent insights in the calculated energies of transition states and coordination complexes, further explaining the observed reactivity and guidance on the scope and limitations for HIE reactions in water. Finally, we successfully adapted these findings to tritium chemistry.

Synthesis and analysis of isotopically stable labeled nitrosamines as mass spectrometry standards for drug impurity quality control.

We describe a simple and easy pathway to synthesize nitrosamine mass spectrometry standards in good to moderate yields. N-alkylation of Boc-protected primary or secondary amines using stable isotope labeled alkyl halides yielded the key intermediates that were deprotected, and then, the nitrosamine was formed with sodium nitrite and sodium hydrogensulfate. Special attention to safety, disposal of waste, and surface cleaning was carried throughout.

Deuterated Drugs and Biomarkers in the COVID-19 Pandemic.

Coronavirus disease 2019 (COVID-19) is a highly contagious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Initially identified in Wuhan (China) in December 2019, COVID-19 rapidly spread globally, resulting in the COVID-19 pandemic. Carriers of the SARS-CoV-2 can experience symptoms ranging from mild to severe (or no symptoms whatsoever). Although vaccination provides extra immunity toward SARS-CoV-2, there has been an urgent need to develop treatments for COVID-19 to alleviate symptoms for carriers of the disease. In seeking a potential treatment, deuterated compounds have played a critical role either as therapeutic agents or as internal MS standards for studying the pharmacological properties of new drugs by quantifying the parent compounds and metabolites. We have identified >70 examples of deuterium-labeled compounds associated with treatment of COVID-19. Of these, we found 9 repurposed drugs and >20 novel drugs studied for potential therapeutic roles along with a total of 38 compounds (drugs, biomarkers, and lipids) explored as internal mass spectrometry standards. This review details the synthetic pathways and modes of action of these compounds (if known), and a brief analysis of each study.

Aldehyde-catalysed carboxylate exchange in α-amino acids with isotopically labelled CO2.

The isotopic labelling of small molecules is integral to drug development and for understanding biochemical processes. The preparation of carbon-labelled α-amino acids remains difficult and time consuming, with established methods involving label incorporation at an early stage of synthesis. This explains the high cost and scarcity of C-labelled products and presents a major challenge in 11C applications (11C t1/2 = 20 min). Here we report that aldehydes catalyse the isotopic carboxylate exchange of native α-amino acids with *CO2 (* = 14, 13, 11). Proteinogenic α-amino acids and many non-natural variants containing diverse functional groups undergo labelling. The reaction probably proceeds via the trapping of *CO2 by imine-carboxylate intermediates to generate iminomalonates that are prone to monodecarboxylation. Tempering catalyst electrophilicity was key to preventing irreversible aldehyde consumption. The pre-generation of the imine carboxylate intermediate allows for the rapid and late-stage 11C-radiolabelling of α-amino acids in the presence of [11C]CO2.

Molecular basis for inhibiting human glucose transporters by exofacial inhibitors.

Human glucose transporters (GLUTs) are responsible for cellular uptake of hexoses. Elevated expression of GLUTs, particularly GLUT1 and GLUT3, is required to fuel the hyperproliferation of cancer cells, making GLUT inhibitors potential anticancer therapeutics. Meanwhile, GLUT inhibitor-conjugated insulin is being explored to mitigate the hypoglycemia side effect of insulin therapy in type 1 diabetes. Reasoning that exofacial inhibitors of GLUT1/3 may be favored for therapeutic applications, we report here the engineering of a GLUT3 variant, designated GLUT3exo, that can be probed for screening and validating exofacial inhibitors. We identify an exofacial GLUT3 inhibitor SA47 and elucidate its mode of action by a 2.3 Å resolution crystal structure of SA47-bound GLUT3. Our studies serve as a framework for the discovery of GLUTs exofacial inhibitors for therapeutic development.

Remote Site-Selective Radical C(sp3 )-H Monodeuteration of Amides using D2 O.

Site-selective incorporation of deuterium into biologically active compounds is of high interest in pharmaceutical industry. We present a mild and environmentally benign metal-free method for the remote selective radical C-H monodeuteration of aliphatic C-H bonds in various amides with inexpensive heavy water (D2 O) as the deuterium source. The method uses the easily installed N-allylsulfonyl moiety as an N-radical precursor that generates the remote C-radical via site-selective 1,5- or 1,6-hydrogen atom transfer (HAT). Methyl thioglycolate, that readily exchanges its proton with D2 O, serves as the radical deuteration reagent and as a chain-carrier. The highly site-selective monodeuteration has been applied to different types of unactivated sp3 -C-H bonds and also to the deuteration of C-H bonds next to heteroatoms. The potential utility of this method is further demonstrated by the site-selective incorporation of deuterium into natural product derivatives and drugs.

Easy access to drug building-blocks through benzylic C-H functionalization of phenolic ethers by photoredox catalysis.

A visible light-mediated photocatalyzed C-C-bond forming method for the benzylic C-H functionalization of phenolether containing synthetic building blocks based on a radical-cation/deprotonation strategy is reported. This method allows the mild, selective generation of benzyl radicals in phenolic complex molecules and drug-like compounds, providing new entries in synthetic and medicinal chemistry.

Carbon-13 synthesis and NMR spectroscopic geometric isomer evaluation to support the filing of teriflunomide.

The two isotopomers of teriflunomide were synthesized starting from isotopically stable-labeled stocks of [13 C]potassium cyanide and [1-13 C]ethyl bromoacetate. The two 13 C-labeled compounds 1a, b were applied in several NMR studies to study the E/Z ratio in different matrices. In a solution, such as dimethyl sulfoxide (DMSO), a dynamic equilibrium between E/Z-isomers (ratio of 8:92) was determined by initial 13 C-carbon NMR experiments. To get insights into the E/Z ratio of teriflunomide under in vivo conditions, advanced heteronuclear NMR (heteronuclear Overhauser effect spectroscopy [HOESY]) in D2 O and mixtures of D2 O/plasma were performed. Whereas NMR experiments in mixtures of water and plasma failed owing to extreme line broadening, NMR spectra in water at pH 7.4 showed only the Z-isomer.

The magic of small structure differences in a sodium-glucose cotransporter drug discovery project-14 C-labelled drug candidates in a key-differentiating study.

We describe the dramatic differences in the synthesis and physiological and pharmacokinetical profiling of two sodium-glucose cotransporter (SGLT) drug candidates AVE2268 and AVE8887 with very similar chemical structures. It is a classic example of how a radioactive study was able to spare resources in preclinical development prior to entering a costly clinical program. It also demonstrated that radioactive compounds can be used to study differences between two very similar compounds in vivo.

Triantennary GalNAc Molecular Imaging Probes for Monitoring Hepatocyte Function in a Rat Model of Nonalcoholic Steatohepatitis.

Nonalcoholic steatohepatitis (NASH) is a progressive form of nonalcoholic fatty liver disease that can lead to irreversible liver cirrhosis and cancer. Early diagnosis of NASH is vital to detect disease before it becomes life-threatening, yet noninvasively differentiating NASH from simple steatosis is challenging. Herein, bifunctional probes have been developed that target the hepatocyte-specific asialoglycoprotein receptor (ASGPR), the expression of which decreases during NASH progression. The results show that the probes allow longitudinal, noninvasive monitoring of ASGPR levels by positron emission tomography in the newly developed rat model of NASH. The probes open new possibilities for research into early diagnosis of NASH and development of drugs to slow or reverse its progression.

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 (D2 O or T2 O) 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.

C-H Functionalization-Prediction of Selectivity in Iridium(I)-Catalyzed Hydrogen Isotope Exchange Competition Reactions.

An assessment of the C-H activation catalyst [(COD)Ir(IMes)(PPh3 )]PF6 (COD=1,5-cyclooctadiene, IMes=1,3-bis(2,4,6-trimethylphenyl)imidazol-2-ylidene) in the deuteration of phenyl rings containing different functional directing groups is divulged. Competition experiments have revealed a clear order of the directing groups in the hydrogen isotope exchange (HIE) with an iridium (I) catalyst. Through DFT calculations the iridium-substrate coordination complex has been identified to be the main trigger for reactivity and selectivity in the competition situation with two or more directing groups. We postulate that the competition concept found in this HIE reaction can be used to explain regioselectivities in other transition-metal-catalyzed functionalization reactions of complex drug-type molecules as long as a C-H activation mechanism is involved.