hRpn13 shapes the proteome and transcriptome through epigenetic factors HDAC8, PADI4, and transcription factor NF-κB p50.

The anti-cancer target hRpn13 is a proteasome substrate receptor. However, hRpn13-targeting molecules do not impair its interaction with proteasomes or ubiquitin, suggesting other critical cellular activities. We find that hRpn13 depletion causes correlated proteomic and transcriptomic changes, with pronounced effects in myeloma cells for cytoskeletal and immune response proteins and bone-marrow-specific arginine deiminase PADI4. Moreover, a PROTAC against hRpn13 co-depletes PADI4, histone deacetylase HDAC8, and DNA methyltransferase MGMT. PADI4 binds and citrullinates hRpn13 and proteasomes, and proteasomes from PADI4-inhibited myeloma cells exhibit reduced peptidase activity. When off proteasomes, hRpn13 can bind HDAC8, and this interaction inhibits HDAC8 activity. Further linking hRpn13 to transcription, its loss reduces nuclear factor κB (NF-κB) transcription factor p50, which proteasomes generate by cleaving its precursor protein. NF-κB inhibition depletes hRpn13 interactors PADI4 and HDAC8. Altogether, we find that hRpn13 acts dually in protein degradation and expression and that proteasome constituency and, in turn, regulation varies by cell type.

Methionine sulfoxide reductases and cholesterol transporter STARD3 constitute an efficient system for detoxification of cholesterol hydroperoxides.

Methionine sulfoxide reductases (MSRs) are key enzymes in the cellular oxidative defense system. Reactive oxygen species oxidize methionine residues to methionine sulfoxide, and the methionine sulfoxide reductases catalyze their reduction back to methionine. We previously identified the cholesterol transport protein STARD3 as an in vivo binding partner of MSRA (methionine sulfoxide reductase A), an enzyme that reduces methionine-S-sulfoxide back to methionine. We hypothesized that STARD3 would also bind the cytotoxic cholesterol hydroperoxides and that its two methionine residues, Met307 and Met427, could be oxidized, thus detoxifying cholesterol hydroperoxide. We now show that in addition to binding MSRA, STARD3 binds all three MSRB (methionine sulfoxide reductase B), enzymes that reduce methionine-R-sulfoxide back to methionine. Using pure 5, 6, and 7 positional isomers of cholesterol hydroperoxide, we found that both Met307 and Met427 on STARD3 are oxidized by 6α-hydroperoxy-3ß-hydroxycholest-4-ene (cholesterol-6α-hydroperoxide) and 7α-hydroperoxy-3ß-hydroxycholest-5-ene (cholesterol-7α-hydroperoxide). MSRs reduce the methionine sulfoxide back to methionine, restoring the ability of STARD3 to bind cholesterol. Thus, the cyclic oxidation and reduction of methionine residues in STARD3 provides a catalytically efficient mechanism to detoxify cholesterol hydroperoxide during cholesterol transport, protecting membrane contact sites and the entire cell against the toxicity of cholesterol hydroperoxide.

Perfluorinated Iridium Catalyst for Signal Amplification by Reversible Exchange Provides Metal-Free Aqueous Hyperpolarized [1-13C]-Pyruvate.

Hyperpolarized (HP) carbon-13 [13C] enables the specific investigation of dynamic metabolic and physiologic processes via in vivo MRI-based molecular imaging. As the leading HP metabolic agent, [1-13C]pyruvate plays a pivotal role due to its rapid tissue uptake and central role in cellular energetics. Dissolution dynamic nuclear polarization (d-DNP) is considered the gold standard method for the production of HP metabolic probes; however, development of a faster, less expensive technique could accelerate the translation of metabolic imaging via HP MRI to routine clinical use. Signal Amplification by Reversible Exchange in SHield Enabled Alignment Transfer (SABRE-SHEATH) achieves rapid hyperpolarization by using parahydrogen (p-H2) as the source of nuclear spin order. Currently, SABRE is clinically limited due to the toxicity of the iridium catalyst, which is crucial to the SABRE process. To mitigate Ir contamination, we introduce a novel iteration of the SABRE catalyst, incorporating bis(polyfluoroalkylated) imidazolium salts. This novel perfluorinated SABRE catalyst retained polarization properties while exhibiting an enhanced hydrophobicity. This modification allows the easy removal of the perfluorinated SABRE catalyst from HP [1-13C]-pyruvate after polarization in an aqueous solution, using the ReD-SABRE protocol. The residual Ir content after removal was measured via ICP-MS at 177 ppb, which is the lowest reported to date for pyruvate and is sufficiently safe for use in clinical investigations. Further improvement is anticipated once automated processes for delivery and recovery are initiated. SABRE-SHEATH using the perfluorinated SABRE catalyst can become an attractive low-cost alternative to d-DNP to prepare biocompatible HP [1-13C]-pyruvate formulations for in vivo applications in next-generation molecular imaging modalities.

Fully Automated Cassette-Based Synthesis of 2-Deoxy-2-[18F]Fluorocellobiose Using Trasis AllInOne Module.

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-[18F]fluorocellobiose ([18F]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-[18F]fluoroglucose ([18F]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 [18F]FCB for clinical PET imaging.

Isolation and Identification of a Urinary Biomarker for Lung Cancer: 27-Nor-5ß-Cholestane-3α,7α,12α,24R,25S Pentol Glucuronide and Its Deuterated Analog.

An untargeted metabolomic study identified four potential lung cancer diagnostic biomarkers in human urine. One of the potential biomarkers was an unidentified feature possessing a m/z value of 561+. "561+" was isolated from human urine and tentatively identified as 27-nor-5ß-cholestane-3α,7α,12α,24,25 pentol glucuronide with unknown C24,25 stereochemistry using 1H NMR and mass spectrometry. In a prior report, the C24,25 stereochemistry of the aglycone, 27-nor-5ß-cholestane-3α,7α,12α,24,25 pentol, was found to be 24S,25R through GC analysis of the acetonide-TMS derivative. An authentic sample was prepared and found not to have the same stereochemistry as "561+". To identify the C24,25 stereochemistry, four C24,C25 diastereoisomeric alcohols of 27-nor-5ß-cholestane-3α,7α,12α,24,25 pentol were prepared from chiral amino acids. Using an LCMS method, the C24,C25 stereochemistry of the "561+" aglycone was determined to be 24R,25S. With the correct aglycone in hand, it was coupled with glucuronic acid to complete the first reported synthesis of 27-nor-5ß-cholestane-3α,7α,12α,24R,25S pentol glucuronide. Deuterium labeled 27-nor-5ß-cholestane-3α,7α,12α,24R,25S pentol was also synthesized for use as an internal standard for MS quantitation.

Orthogonal Enzyme-Substrate Design Strategy for Discovery of Human Protein Palmitoyltransferase Substrates.

Protein palmitoylation, with more than 5000 substrates, is the most prevalent form of protein lipidation. Palmitoylated proteins participate in almost all areas of cellular physiology and have been linked to several human diseases. Twenty-three zDHHC enzymes catalyze protein palmitoylation with extensive overlap among the substrates of each zDHHC member. Currently, there is no global strategy to delineate the physiological substrates of individual zDHHC enzymes without perturbing the natural cellular pool. Here, we outline a general approach to accomplish this on the basis of synthetic orthogonal substrates that are only compatible with engineered zDHHC enzymes. We demonstrate the utility of this strategy by validating known substrates and use it to identify novel substrates of two human zDHHC enzymes. Finally, we employ this method to discover and explore conserved palmitoylation in a family of host restriction factors against pathogenic viruses, including SARS-CoV-2.

Synthesis and Evaluation of Fluorine-18-Labeled L-Rhamnose Derivatives.

The use of radiolabeled glucose for PET imaging resulted in the most commonly used tracer in the clinic, 2-deoxy-2-[18F]fluoroglucose (FDG). More recently, other radiolabeled sugars have been reported for various applications, including imaging tumors and infections. Therefore, in this study, we developed a series of fluorine-18-labeled L-rhamnose derivatives as potential PET tracers of various fungal and bacterial strains. Acetyl-protected triflate precursors of rhamnose were prepared and radiolabeled with fluorine-18 followed by hydrolysis to produce L-deoxy [18F]fluororhamnose. The overall radiochemical yield was 7-27% in a 90 min synthesis time with a radiochemical purity of 95%. In vivo biodistribution of the ligands using PET imaging showed that 2-deoxy-2-[18F]fluoro-L-rhamnose is stable for at least up to 60 min in mice and eliminated via renal clearance. The tracer also exhibited minimal tissue or skeletal uptake in healthy mice resulting in a low background signal.

Rapid 13C Hyperpolarization of the TCA Cycle Intermediate α-Ketoglutarate via SABRE-SHEATH.

α-Ketoglutarate is a key biomolecule involved in a number of metabolic pathways─most notably the TCA cycle. Abnormal α-ketoglutarate metabolism has also been linked with cancer. Here, isotopic labeling was employed to synthesize [1-13C,5-12C,D4]α-ketoglutarate with the future goal of utilizing its [1-13C]-hyperpolarized state for real-time metabolic imaging of α-ketoglutarate analytes and its downstream metabolites in vivo. The signal amplification by reversible exchange in shield enables alignment transfer to heteronuclei (SABRE-SHEATH) hyperpolarization technique was used to create 9.7% [1-13C] polarization in 1 minute in this isotopologue. The efficient 13C hyperpolarization, which utilizes parahydrogen as the source of nuclear spin order, is also supported by favorable relaxation dynamics at 0.4 µT field (the optimal polarization transfer field): the exponential 13C polarization buildup constant Tb is 11.0 ± 0.4 s whereas the 13C polarization decay constant T1 is 18.5 ± 0.7 s. An even higher 13C polarization value of 17.3% was achieved using natural-abundance α-ketoglutarate disodium salt, with overall similar relaxation dynamics at 0.4 µT field, indicating that substrate deuteration leads only to a slight increase (∼1.2-fold) in the relaxation rates for 13C nuclei separated by three chemical bonds. Instead, the gain in polarization (natural abundance versus [1-13C]-labeled) is rationalized through the smaller heat capacity of the "spin bath" comprising available 13C spins that must be hyperpolarized by the same number of parahydrogen present in each sample, in line with previous 15N SABRE-SHEATH studies. Remarkably, the C-2 carbon was not hyperpolarized in both α-ketoglutarate isotopologues studied; this observation is in sharp contrast with previously reported SABRE-SHEATH pyruvate studies, indicating that the catalyst-binding dynamics of C-2 in α-ketoglutarate differ from that in pyruvate. We also demonstrate that 13C spectroscopic characterization of α-ketoglutarate and pyruvate analytes can be performed at natural 13C abundance with an estimated detection limit of 80 micromolar concentration × *%P13C. All in all, the fundamental studies reported here enable a wide range of research communities with a new hyperpolarized contrast agent potentially useful for metabolic imaging of brain function, cancer, and other metabolically challenging diseases.

Order-Unity 13 C Nuclear Polarization of [1-13 C]Pyruvate in Seconds and the Interplay of Water and SABRE Enhancement.

Signal Amplification By Reversible Exchange in SHield Enabled Alignment Transfer (SABRE-SHEATH) is investigated to achieve rapid hyperpolarization of 13 C1 spins of [1-13 C]pyruvate, using parahydrogen as the source of nuclear spin order. Pyruvate exchange with an iridium polarization transfer complex can be modulated via a sensitive interplay between temperature and co-ligation of DMSO and H2 O. Order-unity 13 C (>50 %) polarization of catalyst-bound [1-13 C]pyruvate is achieved in less than 30 s by restricting the chemical exchange of [1-13 C]pyruvate at lower temperatures. On the catalyst bound pyruvate, 39 % polarization is measured using a 1.4 T NMR spectrometer, and extrapolated to >50 % at the end of build-up in situ. The highest measured polarization of a 30-mM pyruvate sample, including free and bound pyruvate is 13 % when using 20 mM DMSO and 0.5 M water in CD3 OD. Efficient 13 C polarization is also enabled by favorable relaxation dynamics in sub-microtesla magnetic fields, as indicated by fast polarization buildup rates compared to the T1 spin-relaxation rates (e. g., ∼0.2 s-1 versus ∼0.1 s-1 , respectively, for a 6 mM catalyst-[1-13 C]pyruvate sample). Finally, the catalyst-bound hyperpolarized [1-13 C]pyruvate can be released rapidly by cycling the temperature and/or by optimizing the amount of water, paving the way to future biomedical applications of hyperpolarized [1-13 C]pyruvate produced via comparatively fast and simple SABRE-SHEATH-based approaches.

General Method to Increase Carboxylic Acid Content on Nanodiamonds.

Carboxylic acid is a commonly utilized functional group for covalent surface conjugation of carbon nanoparticles that is typically generated by acid oxidation. However, acid oxidation generates additional oxygen containing groups, including epoxides, ketones, aldehydes, lactones, and alcohols. We present a method to specifically enrich the carboxylic acid content on fluorescent nanodiamond (FND) surfaces. Lithium aluminum hydride is used to reduce oxygen containing surface groups to alcohols. The alcohols are then converted to carboxylic acids through a rhodium (II) acetate catalyzed carbene insertion reaction with tert-butyl diazoacetate and subsequent ester cleavage with trifluoroacetic acid. This carboxylic acid enrichment process significantly enhanced nanodiamond homogeneity and improved the efficiency of functionalizing the FND surface. Biotin functionalized fluorescent nanodiamonds were demonstrated to be robust and stable single-molecule fluorescence and optical trapping probes.

Low-Cost High-Pressure Clinical-Scale 50% Parahydrogen Generator Using Liquid Nitrogen at 77 K.

We report on a robust and low-cost parahydrogen generator design employing liquid nitrogen as a coolant. The core of the generator consists of catalyst-filled spiral copper tubing, which can be pressurized to 35 atm. Parahydrogen fraction >48% was obtained at 77 K with three nearly identical generators using paramagnetic hydrated iron oxide catalysts. Parahydrogen quantification was performed on the fly via benchtop NMR spectroscopy to monitor the signal from residual orthohydrogen-parahydrogen is NMR silent. This real-time quantification approach was also used to evaluate catalyst activation at up to 1.0 standard liter per minute flow rate. The reported inexpensive device can be employed for a wide range of studies employing parahydrogen as a source of nuclear spin hyperpolarization. To this end, we demonstrate the utility of this parahydrogen generator for hyperpolarization of concentrated sodium [1-13C]pyruvate, a metabolic contrast agent under investigation in numerous clinical trials. The reported pilot optimization of SABRE-SHEATH (signal amplification by reversible exchange-shield enables alignment transfer to heteronuclei) hyperpolarization yielded 13C signal enhancement of over 14,000-fold at a clinically relevant magnetic field of 1 T corresponding to approximately 1.2% 13C polarization-if near 100% parahydrogen would have been employed, the reported value would be tripled to 13C polarization of 3.5%.

Synthesis of [1-13 C-5-12 C]-alpha-ketoglutarate enables noninvasive detection of 2-hydroxyglutarate.

Isocitrate dehydrogenase 1 (IDH1) mutations that generate the oncometabolite 2-hydroxyglutarate (2-HG) from α-ketoglutarate (α-KG) have been identified in many types of tumors and are an important prognostic factor in gliomas. 2-HG production can be determined by hyperpolarized carbon-13 magnetic resonance spectroscopy (HP-13 C-MRS) using [1-13 C]-α-KG as a probe, but peak contamination from naturally occurring [5-13 C]-α-KG overlaps with the [1-13 C]-2-HG peak. Via a newly developed oxidative-Stetter reaction, [1-13 C-5-12 C]-α-KG was synthesized. α-KG metabolism was measured via HP-13 C-MRS using [1-13 C-5-12 C]-α-KG as a probe. [1-13 C-5-12 C]-α-KG was synthesized in high yields, and successfully eliminated the signal from C5 of α-KG in the HP-13 C-MRS spectra. In HCT116 IDH1 R132H cells, [1-13 C-5-12 C]-α-KG allowed for unimpeded detection of [1-13 C]-2-HG. 12 C-enrichment represents a novel method to circumvent spectral overlap, and [1-13 C-5-12 C]-α-KG shows promise as a probe to study IDH1 mutant tumors and α-KG metabolism.

Repurposing the Pummerer Rearrangement: Determination of Methionine Sulfoxides in Peptides.

The reversible oxidation of methionine residues in proteins has emerged as a biologically important post-translational modification. However, detection and quantitation of methionine sulfoxide in proteins is difficult. Our aim is to develop a method for specifically derivatizing methionine sulfoxide residues. We report a Pummerer rearrangement of methionine sulfoxide treated sequentially with trimethylsilyl chloride and then 2-mercaptoimidazole or pyridine-2-thiol to produce a dithioacetal product. This derivative is stable to standard mass spectrometry conditions, and its formation identified oxidized methionine residues. The scope and requirements of dithioacetal formation are reported for methionine sulfoxide and model substrates. The reaction intermediates have been investigated by computational techniques and by 13 C NMR spectroscopy. These provide evidence for an α-chlorinated intermediate. The derivatization allows for detection and quantitation of methionine sulfoxide in proteins by mass spectrometry and potentially by immunochemical methods.

Automated Synthesis of Fluorine-18 Labeled CXCR4 Ligand via the Conjugation with Nicotinic Acid N-Hydroxysuccinimide Ester (6-[18F]SFPy).

The C-X-C motif chemokine receptor 4 (CXCR4) is a seven-transmembrane G protein-coupled receptor that is overexpressed in numerous diseases, particularly in various cancers and is a powerful chemokine, attracting cells to the bone marrow niche. Therefore, CXCR4 is an attractive target for imaging and therapeutic purposes. The goal of this study is to develop an efficient, reproducible, and straightforward method to prepare a fluorine-18 labeled CXCR4 ligand. 6-[18F]Fluoronicotinic acid-2,3,5,6-tetrafluorophenyl ester (6-[18F]FPy-TFP) and nicotinic acid N-hydroxysuccinimide ester (6-[18F]SFPy) have been prepared using 'fluorination on the Sep-Pak' method. Conjugation of 6-[18F]SFPy or 6-[18F]FPy-TFP with the alpha-amino group at the N terminus of the protected T140 precursor followed by deprotection, yielded the final product 6-[18F]FPy-T140. The overall radiochemical yields were 6-17% (n = 15, decay-corrected) in a 90-min radiolabeling time with a radiochemical purity >99%. 6-[18F]FPy-T140 exhibited high specific binding and nanomolar affinity for CXCR4 in vitro, indicating that the biological activity of the peptide was preserved. For the first time, [18F]SFPy has been prepared using 'fluorination on the Sep-Pak' method that allows rapid automated synthesis of 6-[18F]FPy-T140. In addition to increased synthetic efficiency, this construct binds with CXCR4 in high affinity and may have potential as an in vivo positron emission tomography (PET) imaging agent. This radiosynthesis method should encourage wider use of this PET agent to quantify CXCR4 in both research and clinical settings.

Immuno-PET Imaging of the Programmed Cell Death-1 Ligand (PD-L1) Using a Zirconium-89 Labeled Therapeutic Antibody, Avelumab.

OBJECTIVE: The goal is to evaluate avelumab, an anti-PD-L1 monoclonal immunoglobulin G antibody labeled with zirconium-89 in human PD-L1-expressing cancer cells and mouse xenografts for clinical translation. METHODS: [89Zr]Zr-DFO-PD-L1 monoclonal antibody (mAb) was synthesized using avelumab conjugated to desferrioxamine. In vitro binding studies and biodistribution studies were performed with PD-L1+MDA-MB231 cells and MDA-MB231 xenograft mouse models, respectively. Biodistributions were determined at 1, 2, 3, 5, and 7 days post coinjection of [89Zr]Zr-DFO-PD-L1 mAb without or with unlabeled avelumab (10, 20, 40, and 400 µg). RESULTS: [89Zr]Zr-DFO-PD-L1 mAb exhibited high affinity (Kd ∼ 0.3 nM) and detected moderate PD-L1 expression levels in MDA-MB231 cells. The spleen and lymph nodes exhibited the highest [89Zr]Zr-DFO-PD-L1 mAb uptakes in all time points, while MDA-MB231 tumor uptakes were lower but highly retained. In the unlabeled avelumab dose escalation studies, spleen tissue-muscle ratios decreased in a dose-dependent manner indicating specific [89Zr]Zr-DFO-PD-L1 mAb binding to PD-L1. In contrast, lymph node and tumor tissue-muscle ratios increased 4- to 5-fold at 20 and 40 µg avelumab doses. CONCLUSIONS: [89Zr]Zr-DFO-PD-L1 mAb exhibited specific and high affinity for PD-L1 in vitro and had target tissue uptakes correlating with PD-L1 expression levels in vivo. [89Zr]Zr-DFO-PD-L1 mAb uptake in PD-L1+tumors increased with escalating doses of avelumab.

Photoinducible Oncometabolite Detection.

Dysregulated metabolism can fuel cancer by altering the production of bioenergetic building blocks and directly stimulating oncogenic gene-expression programs. However, relatively few optical methods for the direct study of metabolites in cells exist. To address this need and facilitate new approaches to cancer treatment and diagnosis, herein we report an optimized chemical approach to detect the oncometabolite fumarate. Our strategy employs diaryl tetrazoles as cell-permeable photoinducible precursors to nitrileimines. Uncaging these species in cells and cell extracts enables them to undergo 1,3-dipolar cycloadditions with endogenous dipolarophile metabolites such as fumarate to form pyrazoline cycloadducts that can be readily detected by their intrinsic fluorescence. The ability to photolytically uncage diaryl tetrazoles provides greatly improved sensitivity relative to previous methods, and enables the facile detection of dysregulated fumarate metabolism through biochemical activity assays, intracellular imaging, and flow cytometry. Our studies showcase an intersection of bioorthogonal chemistry and metabolite reactivity that can be applied for biological profiling, imaging, and diagnostics.

Synthesis and Characterization of Bodipy-FL-Cyclosporine A as a Substrate for Multidrug Resistance-Linked P-Glycoprotein (ABCB1).

Fluorescent conjugates of drugs can be used to study cellular functions and pharmacology. These compounds interact with proteins as substrates or inhibitors, helping in the development of unique fluorescence-based methods to study in vivo localization and molecular mechanisms. P-glycoprotein (P-gp, ABCB1) is an ATP-binding cassette (ABC) transporter that effluxes most anticancer drugs from cells, contributing to the development of drug resistance. To study the transport function of P-gp, we synthesized a Bodipy-labeled fluorescent conjugate of cyclosporine A (BD-CsA). After synthesis and characterization of its chemical purity, BD-CsA was compared with the commonly used 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-CsA probe. In flow cytometry assays, the fluorescence intensity of BD-CsA was almost 10 times greater than that of NBD-CsA, enabling us to use significantly lower concentrations of BD-CsA to achieve the same fluorescence levels. We found that BD-CsA is recognized as a transport substrate by both human and mouse P-gp. The rate of efflux of BD-CsA by human P-gp is comparable to that of NBD-CsA. The transport of BD-CsA was inhibited by tariquidar, with similar IC50 values to those for NBD-CsA. BD-CsA and NBD-CsA both partially inhibited the ATPase activity of P-gp with similar IC50 values. In silico docking of BD-CsA and NBD-CsA to the human P-gp structure indicates that they both bind in the drug-binding pocket with similar docking scores and possibly interact with similar residues. Thus, we demonstrate that BD-CsA is a sensitive fluorescent substrate of P-gp that can be used to efficiently study the transporter's localization and function in vitro and in vivo. SIGNIFICANCE STATEMENT: The goal of this study was to develop an effective probe to study drug transport by P-glycoprotein (P-gp). Fluorophore-conjugated substrates are useful to study the P-gp transport mechanism, structural characteristics, and development of its inhibitors. Cyclosporine A (CsA), a cyclic peptide comprising 11 amino acids, is a known substrate of P-gp. P-gp affects CsA pharmacokinetics and interactions with other coadministered drugs, especially during transplant surgeries and treatment of autoimmune disorders, when CsA is given as an immunosuppressive agent. We synthesized and characterized Bodipy-FL-CsA as an avid fluorescent substrate that can be used to study the function of P-gp both in vitro and in vivo. We demonstrate that Bodipy-FL-conjugation does not affect the properties of CsA as a P-gp substrate.

Comparison of Solution Properties of Polymethylated DOTA-like Lanthanide Complexes with Opposite Chirality of the Pendant Arms.

Polymethylated lanthanide 4S4R-M4DOTMA complexes, bearing the ring methyl groups oriented in the SSSS position and the arm methyl groups in the RRRR configuration, exist exclusively as the SAP [Λ(δδδδ)] isomer in solution throughout the lanthanide series. This observation is in contrast to Ln-8S-M4DOTMA, which was recently reported to adopt the SAP [Λ(δδδδ)] isomer in the early lanthanides, while the late lanthanides adopt the TSAP [Δ(δδδδ)] isomer. The methyl groups on the ring and the arm are both oriented in the SSSS configuration for Ln-8S-M4DOTMA ( Dalton Trans. 2016 , 45 , 4673 - 4687 , DOI: 10.1039/C5DT03210E ). Quantum chemical calculations for Pr- and Yb-4S4R-M4DOTMA indicate that the SAP isomer is significantly more stable. The luminescence profiles of Eu-8S-M4DOTMA and Eu-4S4R-M4DOTMA showed similar profiles signifying identical coordination environments. The hydration state, q, of the Eu(III) complexes was q = 0.91-0.95, while Tb-8S-M4DOTMA had q = 0.86. A much lower q value was obtained for Tb-4S4R-M4DOTMA (q = 0.67), which indicates an elongation of the Ln-Ow bond. At 400 MHz, the relaxivity of Gd-8S-M4DOTMA is 5.1 ± 0.1 mM-1 s-1 and 3.9 ± 0.1 mM-1 s-1 at 25 and 37 °C, respectively, whereas the relaxivity of Gd-4S4R-M4DOTMA is 4.6 ± 0.1 mM-1 s-1 at 25 °C and 3.6 ± 0.1 mM-1 s-1 at 37 °C. At 45 MHz, the relaxivity of Gd-8S-M4DOTMA is 5.4 ± 0.1 mM-1 s-1, and the relaxivity of Gd-4S4R-M4DOTMA is 4.5 ± 0.1 mM-1 s-1 at 25 °C. The temperature dependence of the 17O NMR transverse relaxation rate of the Gd complexes revealed a 7-fold increase in the bound water residence lifetime of Gd-8S-M4DOTMA (1/kex = τM = 9.0 ± 0.5 ns and 1/kex = τM = 60 ± 3 ns). The Pr(III) complex of 8S-M4DOTMA crystallized as TSAP isomer with an apical water. The presence of the apical water for the TSAP of Pr-8S-M4DOTMA was further confirmed with the observation that the fluoride ion replaces the bound water from the TSAP isomer of Pr-8S-M4DOTMA. This was shown by the disappearance of the TSAP peaks and appearance of a new set of less shifted resonances, which exchange with the SAP isomer as confirmed by NMR exchange spectroscopy (EXSY).

An azeotropic drying-free approach for copper-mediated radiofluorination without addition of base.

Copper-mediated radiofluorination provides a quick and versatile approach for 18 F-labeling of arenes and heteroarenes. However, this method is known to be base sensitive, which has been a barrier for preparative scale radiosynthesis. In this report, we provide an approach for copper-mediated radiofluorination without azeotropic drying or adding a base. [18 F]Fluoride trapped on a PS-HCO3 Sep-Pak was quantitatively eluted with a solution of 4-dimethylaminopyridinium trifluoromethanesulfonate (DMAP·OTf) in anhydrous N,N-dimethylformamide (DMF). The eluted solution was directly used for copper-mediated radiofluorination. Twelve boronic ester substrates were tested, yielding fluorinated products in 27% to 83% radiochemical yield based on HPLC analysis. This approach was successfully applied to the radiosynthesis of [18 F]flumazenil, a well-known positron emission tomography (PET) tracer for imaging central benzodiazepine receptors, with a radiochemical yield of 47%. This highly efficient protocol significantly augments the powerful copper-mediated radiofluorination approach.

Fluorine-18 Labeled Fluorofuranylnorprogesterone ([18F]FFNP) and Dihydrotestosterone ([18F]FDHT) Prepared by "Fluorination on Sep-Pak" Method.

To further explore the scope of our recently developed "fluorination on Sep-Pak" method, we prepared two well-known positron emission tomography (PET) tracers 21-[18F]fluoro-16α,17α-[(R)-(1'-α-furylmethylidene)dioxy]-19-norpregn-4-ene-3,20-dione furanyl norprogesterone ([18F]FFNP) and 16ß-[18F]fluoro-5α-dihydrotestosterone ([18F]FDHT). Following the "fluorination on Sep-Pak" method, over 70% elution efficiency was observed with 3 mg of triflate precursor of [18F]FFNP. The overall yield of [18F]FFNP was 64-72% (decay corrected) in 40 min synthesis time with a molar activity of 37-81 GBq/µmol (1000-2200 Ci/mmol). Slightly lower elution efficiency (~55%) was observed with the triflate precursor of [18F]FDHT. Fluorine-18 labeling, reduction, and deprotection to prepare [18F]FDHT were performed on Sep-Pak cartridges (PS-HCO3 and Sep-Pak plus C-18). The overall yield of [18F]FDHT was 25-32% (decay corrected) in 70 min. The molar activity determined by using mass spectrometry was 63-148 GBq/µmol (1700-4000 Ci/mmol). Applying this quantitative measure of molar activity to in vitro assays [18F]FDHT exhibited high-affinity binding to androgen receptors (Kd~2.5 nM) providing biological validation of this method.