Deuterium-labeled pyocyanin was prepared from deuterium-labeled phenazine methosulfate in gram scale by a simplified flow photosynthesis in water. The main product was the protonated red form of pyocyanin-d (Pyo-d-H) in 85 % yield. Quantum chemical calculations of NMR support that nitrogen-10 is protonated. The by-products of the photolysis and the stability of the photolysis mixture were carefully characterized by LC-MS and NMR. Four by-products were identified: An isomer of pyocyanin-d (9%), 8-hydroxypyocyanin-d (4%), 1-hydroxyphenazine (0.4%), and phenazine (1%). The Pyo-d-H product was stable in the photolysis solution after storage at 8°C for 2.5 years. Pure blue pyocyanin-d powder was isolated from the red photolysis solution by the Surrey method in 94 % yield. The addition of the red photolysis solution of Pyo-d-H (100 μM) and commercial pyocyanin (100 μM) to Pseudomonas aeruginosa cultures showed the same growth curves demonstrating that the minor impurities in the photolysis solution do not affect the growth behavior of the bacteria. The protonated deuterium-labeled pyocyanin may be used directly in biological experiments, which make the methodology extremely simple and useful for biologists.

DOTA-functionalized bisphosphonates can be useful tools for PET imaging of bone metastases when radiolabeled with Ga. Moreover, the versatility of DOTA allows the complexation of radiometals with therapeutic applications (e.g., Lu), positioning these bisphosphonates as attractive theranostic agents. Among these molecules, BPAMD is a compound whose radiolabeling with Ga has already been described, but only through manual methods. Thus, a fully automated protocol for Ga radiolabeling of BPAMD on the GAIA® ± LUNA® synthesis module was designed, and a thorough study of the radiolabeling conditions was undertaken. [Ga]Ga-BPAMD was produced in good radiochemical purity (> 93%) and high radiochemical yield (> 91%) using 0.3 M HEPES buffer. The nature of the reaction vessel showed no significant effect on the radiolabeling outcome. Similarly, addition of an antiradiolysis compound to the reaction medium did not significantly improve the already excellent stability of [Ga]Ga-BPAMD over time. The radiolabeled product obtained by automated synthesis was evaluated in vivo in healthy mice and confirmed high accumulation in the joints and along the backbone.

Radionuclide therapy employing alpha emitters holds great potential for personalized cancer treatment. However, certain challenges remain when designing alpha radiopharmaceuticals, including the lack of stability of used radioconjugates due to nuclear decay events. In this work, ultrasmall silver telluride nanoparticles with a core diameter of 2.1 nm were prepared and radiolabeled with lead-212 using a chelator-free method with a radiolabeling efficiency of 75%. The results from the in vitro radiochemical stability assay indicated a very high retention of bismuth-212 despite the internal conversion effects originating from the decay of Pb. To further evaluate the potential of the nanoparticles, they were radiolabeled with indium-111, and their cell uptake and subcellular distribution were determined in 2D U87 cells, showing accumulation in the nucleus. Although not intentional, it was observed that the indium-111-radiolabeled nanoparticles induced efficient tumor cell killing, which was attributed to the Auger electrons emitted by indium-111. Combining the results obtained in this work with other favorable properties such as fast renal clearance and the possibility to attach targeting vectors on the surface of the nanoparticles, all well-known from the literature, these ultra-small silver telluride nanoparticles provide exciting opportunities for the design of theragnostic radiopharmaceuticals.

Programmed death-ligand 1 (PD-L1) expression is related to the efficacy and prognosis in triple-negative breast cancer. This study employed an indirect labeling method to synthesize [I]PI-Atezolizumab. The in vitro stability of [I]PI-Atezolizumab was assessed through incubation in phosphate buffered saline and fetal bovine serum, revealing sustained stability. Specific binding of [I]PI-Atezolizumab to MDA-MB-231 cells expressing humanized PD-L1 was assessed through in vitro incubation, yielding a K value comparable to that of Atezolizumab. This suggests that the labeling process did not compromise the affinity of the Atezolizumab to PD-L1. Subsequently, pharmacokinetic studies were conducted in normal mice and biodistribution experiments in tumor-bearing mice. A comparison of the biodistribution results between [I]PI-Atezolizumab and I-labeled Atezolizumab indicated better in vivo stability for the former. Single photon emission computed tomography (SPECT)/CT imaging further confirmed the targeted specificity of [I]PI-Atezolizumab for PD-L1 in MDA-MB-231 xenografts, which were validated by immunohistochemistry staining. This research underscores the utility of [I]PI-Atezolizumab, prepared via indirect labeling, for monitoring PD-L1 in triple-negative breast cancer models.

This research presents the development of positron emission tomography (PET) radiotracers for detecting Mycobacterium tuberculosis (MTB) for the diagnosis and monitoring of tuberculosis. Two phage display-derived peptides with proven selective binding to MTB were identified for development into PET radiopharmaceuticals: H8 (linear peptide) and PH1 (cyclic peptide). We sought to functionalize H8/PH1 with NODASA, a bifunctional chelator that allows complexation of PET-compatible radiometals such as gallium-68. Herein, we report on the chelator functionalization, optimized radiosynthesis, and assessment of the radiopharmaceutical properties of [Ga]Ga-NODASA-H8 and [Ga]Ga-NODASA-PH1. Robust radiolabeling was achieved using the established routine method, indicating consistent production of a radiochemically pure product (RCP ≥ 99.6%). For respective [Ga]Ga-NODASA-H8 and [Ga]Ga-NODASA-PH1, relatively high levels of decay-corrected radiochemical yield (91.2% ± 2.3%, 86.7% ± 4.0%) and apparent molar activity (A, 3.9 ± 0.8 and 34.0 ± 5.3 GBq/μmol) were reliably achieved within 42 min, suitable for imaging purposes. Notably, [Ga]Ga-NODASA-PH1 remained stable in blood plasma for up to 2 h, while [Ga]Ga-NODASA-H8 degraded within 30 min. For both Ga peptides, minimal whole-blood cell binding and plasma protein binding were observed, indicating a favorable pharmaceutical behavior. [Ga]Ga-NODASA-PH1 is a promising candidate for further in vitro/in vivo evaluation as a tuberculosis-specific infection imaging agent.

Radioimmunoconjugates (RICs) composed of tumor-targeting monoclonal antibodies and radionuclides have been developed for diagnostic and therapeutic application. A new radiolabeling method using microfluidic devices is expected to facilitate simpler and more rapid synthesis of RICs. In the microfluidic method, microfluidic chips can promote the reaction between reactants by mixing them efficiently, and pumping systems enable automated synthesis. In this study, we synthesized RICs by the pre-labeling method, in which the radiometal is coordinated to the chelator and then the radiolabeled chelator is incorporated into the antibodies, using microfluidic devices for the first time. As a result of examining the reaction parameters including the material of mixing units, reaction temperature, and flow rate, RICs with radiochemical purity (RCP) exceeding 90% were obtained. These high-purity RICs were successfully synthesized without any purification simply by pumping three solutions of a chelating agent, radiometal, and antibody into microfluidic devices. Under the same conditions, the RCP of RICs labeled by conventional methods was below 50%. These findings indicate the utility of microfluidic devices for automatic and rapid synthesis of high-quality RICs.

Recently, the folate receptor (FR) has become an exciting target for the diagnosis of FR-positive malignancies. Nevertheless, suboptimal in vivo pharmacokinetic properties, particularly high uptake in the renal and hepatobiliary systems, are important limiting factors for the clinical translation of most FR-based radiotracers. In this study, we developed a novel F-labeled FR-targeted positron emission tomography (PET) tracer [F]AlF-NOTA-Asp-PEG-Folate modified with a hydrophilic linker (-Asp-PEG) to optimize its pharmacokinetic properties and conducted a comprehensive preclinical assessment. The [F]AlF-NOTA-Asp-PEG-Folate was manually synthesized within 30 min with a non-decay-corrected radiochemical yield of 16.3 ± 2.0% (n = 5). Among KB cells, [F]AlF-NOTA-Asp-PEG-Folate exhibited high specificity and affinity for FR. PET/CT imaging and biodistribution experiments in KB tumor-bearing mice showed decent tumor uptake (1.7 ± 0.3% ID/g) and significantly decreased uptake in kidneys and liver (22.2 ± 2.1 and 0.3 ± 0.1% ID/g at 60 min p.i., respectively) of [F]AlF-NOTA-Asp-PEG-Folate, compared to the known tracer [F]AlF-NOTA-Folate (78.6 ± 5.1 and 5.3 ± 0.5 % ID/g at 90 min p.i., respectively). The favorable properties of [F]AlF-NOTA-Asp-PEG-Folate, including its efficient synthesis, decent tumor uptake, relatively low renal uptake, and rapid clearance from most normal organs, portray it as a promising PET tracer for FR-positive tumors.

Herein, we demonstrate an efficient method for multi-deuterium labelling of pirtobrutinib-a Bruton's tyrosine kinase inhibitor recently approved by the FDA-using a straightforward hydrogen isotope exchange (HIE) reaction. A remarkably high level of deuterium incorporation was achieved using an excess of a Kerr-type iridium catalyst. The key factor in the significant deuterium labelling was the decision to employ a deuterium uniformly labelled solvent, chlorobenzene-d, at an elevated temperature. Virtually, no d-d species were detected, with only traces of d-d isotopomers (< 5%) observable in the mass spectrum of pirtobrutinib-d, fulfilling requirements for stable isotope-labelled internal standard. The labelled compound-mainly consisting of isotopomers d-d at 82.4% of the total abundance-was isolated in a high yield (73%) and purity (99%). Noteworthy, fluorine group acting as a directing group was observed for the first time. Significant incorporation of deuterium in ortho-positions, exceeding 87%, was observed. Interestingly, chlorinated solvent used in the HIE reactions was non-specifically deuterated yielding up to 0.42 deuterium per chlorobenzene molecule even at an exceptionally low iridium catalyst loading of 4.17 × 10 mol%.

A new automated radiosynthesis of [C]2-(2,6-difluoro-4-((2-(N-methylphenylsulfonamido)ethyl)thio)phenoxy)acetamide ([C]K2), a radiopharmaceutical for the glutamate α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor, is reported. Although manual syntheses have been described, these are unsuitable for routine production of larger batches of [C]K2 for (pre)clinical PET imaging applications. To meet demands for the imaging agent from our functional neuroimaging collaborators, herein, we report a current good manufacturing practice (cGMP)-compliant synthesis of [C]K2 using a commercial synthesis module. The new synthesis is fully automated and has been validated for clinical use. The total synthesis time is 33 min from end of bombardment, and the production method provides 2.66 ± 0.3 GBq (71.9 ± 8.6 mCi) of [C]K2 in 97.7 ± 0.5% radiochemical purity and 754.1 ± 231.5 TBq/mmol (20,382.7 ± 6256.1 Ci/mmol) molar activity (n = 3). Batches passed all requisite quality control testing confirming suitability for clinical use.

Due to the continuous rise in global incidence and severity of invasive fungal infections (IFIs), particularly among immunocompromised and immunodeficient patients, there is an urgent demand for swift and accurate fungal pathogen diagnosis. Therefore, the need for fungal-specific positron emission tomography (PET) imaging agents that can detect the infection in the early stages is increasing. Cellobiose, a disaccharide, is readily metabolized by fungal pathogens such as Aspergillus species. Recently, our group reported fluorine-18 labeled cellobiose, 2-deoxy-2-[F]fluorocellobiose ([F]FCB), for specific imaging of Aspergillus infection. The positive imaging findings with very low background signal on delayed imaging make this ligand a promising fungal-specific imaging ligand. Inspired by this result, the decision was made to automate the radiolabeling procedure for better reproducibility and to facilitate clinical translation. A Trasis AllInOne (Trasis AIO) automated module was used for this purpose. The reagent vials contain commercially available 2-deoxy-2-[F]fluoroglucose ([F]FDG), glucose-1-phosphate, and enzyme (cellobiose phosphorylase). A Sep-Pak cartridge was used to purify the tracer. The overall radiochemical yield was 50%-70% (n = 6, decay corrected) in 75-min synthesis time with a radiochemical purity of > 98%. This is a highly reliable protocol to produce current good manufacturing practice (cGMP)-compliant [F]FCB for clinical PET imaging.

Mitochondrial membrane translocator protein 18 kDa (TSPO) expression is increased in activated microglia, established as a plausible target of neuroinflammation imaging. [C]ER176, specifically binding to TSPO, has been developed as the third generation of radioligand for PET imaging of TSPO, which showed the potential in better quantifying neuroinflammation than its predecessors. In the current study, we developed an automated radiosynthesis with an improved HPLC purification method for [C]ER176 clinical production. The improved HPLC separation was integrated into the automated production of [C]ER176 using a reverse phase semi-preparative HPLC column with an isocratic pump and the mixture of methanol and 50 mM ammonium acetate as the mobile phase. The fraction corresponding to [C]ER176 was collected around 8.5-9.0 min without the risk of getting contaminations from nearby impurities. The automated production process took about 30 min after end of bombardment (EOB) and the quality of the final product [C]ER176 met all specifications for clinical use based on current US Pharmacopeia and FDA CGMP requirements.

Reductive N-C-methylation using [C]formaldehyde and amines has been used to prepare N-C-methylated compounds. However, the yields of the N-C-methylated compounds are often insufficient. In this study, we developed an efficient method for base-free reductive N-C-methylation that is applicable to a wide variety of substrates, including arylamines bearing electron-withdrawing and electron-donating substituents. A 2-picoline borane complex, which is a stable and mild reductant, was used. Dimethyl sulfoxide was used as the primary reaction solvent, and glacial acetic acid or aqueous acetic acid was used as a cosolvent. While reductive N-C-methylation efficiently proceeded under anhydrous conditions in most cases, the addition of water to the reductive N-C-methylation generally increased the yield of the N-C-methylated compounds. Substrates with hydroxy, carboxyl, nitrile, nitro, ester, amide, and phenone moieties and amine salts were applicable to the reaction. This proposed method for reductive N-C-methylation should be applicable to a wide variety of substrates, including thermo-labile and base-sensitive compounds because the reaction was performed under relatively mild conditions (70°C) without the need for a base.

Rimsulfuron is a sulfonylurea herbicide that controls grass and broadleaf weeds in maize, potatoes, fruits, nuts, and other crops. It can also be used as a burndown herbicide to clear invasive weed species along roadsides and other nonagricultural land. Rimsulfuron acts as an acetolactase synthase (ALS) inhibitor, blocking the synthesis of essential amino acids required for plant growth. As is common practice, rimsulfuron has been subject to periodic reviews by regulatory agencies for reregistration since its introduction into the market in the early 1990s. The goal of these reviews is to ensure that the herbicide carries out its intended use without creating adverse side effects to humans and the environment. Since scientific methods are continually evolving and being developed, global regulatory agencies can require additional studies to address data gaps for pesticide renewals. During this reregistration process for rimsulfuron, a new confined rotational crop study was required to address a data gap requested by the European Food Safety Authority (EFSA). Consequently, the corresponding pyridine and pyrimidine radiolabeled [C]rimsulfuron and [M + 3] stable isotopes of rimsulfuron were synthesized for this study to support the reregistration process.

A key aspect for the applicability of Zr-radioimmunoconjugates is inert modification and radiolabeling. The two commercially available bifunctional variants of the siderophore desferrioxamine (DFO), Fe-DFO-N-suc-TFP-ester and p-NCS-Bz-DFO, are most often used for clinical Zr-immuno-PET. The use of Fe-DFO-N-suc-TFP-ester is advantageous with regard to higher radiolysis stability and more facile assessment of radiochemical purity as well as chelator-to-mAb ratio. However, not all mAbs withstand the Fe-removal step at relatively low pH (4-4.5) using EDTA, which is needed after conjugation to allow Zr labeling. In this study, it was investigated whether hydroxybenzyl ethylenediamine (HBED) or the clinically approved deferiprone (DFP) can serve as an alternative for EDTA to establish a pH-independent mild method for Fe-removal and thereby broaden the applicability of Fe-DFO-N-suc-TFP-ester. Carrier-added [Fe]Fe-DFO-N-suc-TFP-ester was used for mAb modification to enable direct tracking of the Fe-removal efficiency under various conditions. Whereas incomplete Fe-removal with HBED was observed at pH 5 or higher, Fe-removal with DFP was possible at a broad pH range (4-9). This provides a mild, pH-independent method for Fe-removal, improving the applicability and attractiveness of Fe-DFO-N-suc-TFP-ester for Zr-mAb preparation.

Carbon-14 labeling synthesis of RORγt inhibitor JNJ-61803534 (1) was accomplished in four steps with the C14 label located at the thiazole-2-carboxamide carbon. The synthesis featured a highly efficient conversion of nitrile [C]-12 to ester [C]-17 under mild conditions via an imidate intermediate, overcoming the unsuccessful direct hydrolysis of nitrile 12 under either acidic or basic conditions. Since carbon-14 labeling via [C]-nitrile installation and subsequent conversion to [C]-carboxylic acid derivatives is a common labeling strategy, an efficient conversion of a nitrile to an ester under mild conditions could be of use for the future C14 labeling syntheses.

Cathepsin B (CTSB) is a lysosomal protease that is overexpressed in tumor cells. Radioimmunoconjugates (RICs) composed of CTSB-recognizing chelating agents are expected to increase the molecular weights of their radiometabolites by forming conjugates with CTSB in cells, resulting in their improved retention in tumor cells. We designed a novel CTSB-recognizing trifunctional chelating agent, azide-[In]In-DOTA-CTSB-substrate ([In]In-ADCS), to synthesize a RIC, trastuzumab-[In]In-ADCS ([In]In-TADCS), and evaluated its utility to improve tumor retention of the RIC. [In]In-ADCS and [In]In-TADCS were synthesized with satisfactory yield and purity. [In]In-ADCS was markedly stable in murine plasma until 96 h postincubation. [In]In-ADCS showed binding to CTSB in vitro, and the conjugation was blocked by the addition of CTSB inhibitor. In the internalization assay, [In]In-TADCS exhibited high-level retention in SK-OV-3 cells, indicating the in vitro utility of the CTSB-recognizing unit. In the biodistribution assay, [In]In-TADCS showed high-level tumor accumulation, but the retention was hardly improved. In the first attempt to combine a CTSB-recognizing unit and RIC, these findings show the fundamental properties of the CTSB-recognizing trifunctional chelating agent to improve tumor retention of RICs.

Malaria continues to be a serious and debilitating disease. The emergence and spread of high-level resistance to multiple antimalarial drugs by Plasmodium falciparum has brought about an urgent need for new treatments that will be active against multidrug resistant malaria infections. One such treatment, ELQ-331 (MMV-167), an alkoxy carbonate prodrug of 4(1H)-quinolone ELQ-300, is currently in preclinical development with the Medicines for Malaria Venture. Clinical development of ELQ-331 or similar compounds will require the availability of isotopically labeled analogs. Unfortunately, a suitable method for the deuteration of these important compounds was not found in the literature. Here, we describe a facile and scalable method for the deuteration of 4(1H)-quinolone ELQ-300, its alkoxycarbonate prodrug ELQ-331, and their respective N-oxides using deuterated acetic acid.

The rise of nucleic acid-based therapeutics continues apace. At the same time, the need for radiolabelled oligonucleotides for determination of spatial distribution is increasing. Complex molecular structures with mostly multiple charges and low solubility in organic solvents increase the challenge of integrating radionuclides. In preclinical research, it is important to understand the fate of new drug candidates in biodistribution studies, target binding or biotransformation studies. Depending on a specific question, the selection of a respective radiolabelling strategy is crucial. Radiometals for molecular imaging with positron emission tomography or single-photon computed tomography generally require an attached chelating agent for stable complexation of the metal with the oligonucleotide, whereas labelling using carbon-11/-14 or tritium allows incorporation of the radioisotope into the native structure without altering it. Moreover, the suitability of direct radiolabelling of the oligonucleotide of interest or indirect radiolabelling, for example, by a two-step pretargeting approach, for the study design requires consideration. This review focuses on the challenges of radiolabelling nucleic acid-based molecules with beta-plus, gamma and beta-minus emitters and their use for tracking and monitoring.

Immune checkpoint therapy has emerged as an effective treatment option for various types of cancers. Key immune checkpoint molecules, such as cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), and lymphocyte activation gene 3 (LAG-3), have become pivotal targets in cancer immunotherapy. Antibodies designed to inhibit these molecules have demonstrated significant clinical efficacy. Nevertheless, the ability to monitor changes in the immune status of tumors and predict treatment response remains limited. Conventional methods, such as assessing lymphocytes in peripheral blood or conducting tumor biopsies, are inadequate for providing real-time, spatial information about T-cell distributions within heterogeneous tumors. Positron emission tomography (PET) using T-cell specific probes represents a promising and noninvasive approach to monitor both systemic and intratumoral immune changes during treatment. This technique holds substantial clinical significance and potential utility. In this paper, we review the applications of PET probes that target immune cells in molecular imaging.

Herein, the successful syntheses of D- and C-N-methyl and D-tert-butyl Hoechst dyes are presented. This includes the preparation of the labelled D- and C-N-methyl piperazines and D-tert-butylated hydroxytoluene precursors. The tert-butyl Hoechst dye is known to bind a specific RNA aptamer. Spectroscopic NMR studies of the labelled Hoechst dye-aptamer complexes allowed for the unambiguous assignment of chemical shifts, as well as the dynamics of the bound dye.