LowTempGAL: a highly responsive low temperature-inducible GAL system in Saccharomyces cerevisiae.

Temperature is an important control factor for biologics biomanufacturing in precision fermentation. Here, we explored a highly responsive low temperature-inducible genetic system (LowTempGAL) in the model yeast Saccharomyces cerevisiae. Two temperature biosensors, a heat-inducible degron and a heat-inducible protein aggregation domain, were used to regulate the GAL activator Gal4p, rendering the leaky LowTempGAL systems. Boolean-type induction was achieved by implementing a second-layer control through low-temperature-mediated repression on GAL repressor gene GAL80, but suffered delayed response to low-temperature triggers and a weak response at 30°C. Application potentials were validated for protein and small molecule production. Proteomics analysis suggested that residual Gal80p and Gal4p insufficiency caused suboptimal induction. 'Turbo' mechanisms were engineered through incorporating a basal Gal4p expression and a galactose-independent Gal80p-supressing Gal3p mutant (Gal3Cp). Varying Gal3Cp configurations, we deployed the LowTempGAL systems capable for a rapid stringent high-level induction upon the shift from a high temperature (37-33°C) to a low temperature (≤30°C). Overall, we present a synthetic biology procedure that leverages 'leaky' biosensors to deploy highly responsive Boolean-type genetic circuits. The key lies in optimisation of the intricate layout of the multi-factor system. The LowTempGAL systems may be applicable in non-conventional yeast platforms for precision biomanufacturing.

Flexibility of the petunia strigolactone receptor DAD2 promotes its interaction with signaling partners.

Strigolactones (SLs) are terpenoid-derived plant hormones that regulate various developmental processes, particularly shoot branching, root development, and leaf senescence. The SL receptor has an unusual mode of action. Upon binding SL, it hydrolyzes the hormone, and then covalently binds one of the hydrolytic products. These initial events enable the SL receptor DAD2 (in petunia) to interact with the F-box protein PhMAX2A of the Skp-Cullin-F-box (SCF) complex and/or a repressor of SL signaling, PhD53A. However, it remains unclear how binding and hydrolysis structurally alters the SL receptor to enable its engagement with signaling partners. Here, we used mutagenesis to alter DAD2 and affect SL hydrolysis or DAD2's ability to interact with its signaling partners. We identified three DAD2 variants whose hydrolytic activity had been separated from the receptor's interactions with PhMAX2A or PhD53A. Two variants, DAD2N242I and DAD2F135A, having substitutions in the core α/ß hydrolase-fold domain and the hairpin, exhibited hormone-independent interactions with PhMAX2A and PhD53A, respectively. Conversely, the DAD2D166A variant could not interact with PhMAX2A in the presence of SL, but its interaction with PhD53A remained unaffected. Structural analyses of DAD2N242I and DAD2D166A revealed only small differences compared with the structure of the WT receptor. Results of molecular dynamics simulations of the DAD2N242I structure suggested that increased flexibility is a likely cause for its SL-independent interaction with PhMAX2A. Our results suggest that PhMAX2A and PhD53A have distinct binding sites on the SL receptor and that its flexibility is a major determinant of its interactions with these two downstream regulators.

Synthesis and preliminary evaluation of novel 11C-labeled GluN2B-selective NMDA receptor negative allosteric modulators.

N-methyl-D-aspartate receptors (NMDARs) play critical roles in the physiological function of the mammalian central nervous system (CNS), including learning, memory, and synaptic plasticity, through modulating excitatory neurotransmission. Attributed to etiopathology of various CNS disorders and neurodegenerative diseases, GluN2B is one of the most well-studied subtypes in preclinical and clinical studies on NMDARs. Herein, we report the synthesis and preclinical evaluation of two 11C-labeled GluN2B-selective negative allosteric modulators (NAMs) containing N,N-dimethyl-2-(1H-pyrrolo[3,2-b]pyridin-1-yl)acetamides for positron emission tomography (PET) imaging. Two PET ligands, namely [11C]31 and [11C]37 (also called N2B-1810 and N2B-1903, respectively) were labeled with [11C]CH3I in good radiochemical yields (decay-corrected 28% and 32% relative to starting [11C]CO2, respectively), high radiochemical purity (>99%) and high molar activity (>74 GBq/µmol). In particular, PET ligand [11C]31 demonstrated moderate specific binding to GluN2B subtype by in vitro autoradiography studies. However, because in vivo PET imaging studies showed limited brain uptake of [11C]31 (up to 0.5 SUV), further medicinal chemistry and ADME optimization are necessary for this chemotype attributed to low binding specificity and rapid metabolism in vivo.

Illustration of a computational pipeline for evaluating cyclodextrin host-guest complex formation through conformational capture of bullvalene.

Cyclodextrins have a diverse range of applications, including as supramolecular hosts, as enzyme active-site analogs, in improving drug solubility and delivery, and in molecular selection. We have investigated their ability to form stable complexes with bullvalenes, unusual organic cage molecules that spontaneously interconvert between numerous degenerate isomers. The shape-shifting nature of substituted bullvalenes raises the potential for dynamic adaptive binding to biological targets. We tested whether ß- and γ-cyclodextrins can capture particular bullvalene isomers and whether the preferred binding mode(s) differ between isomers. We first applied our computational host-guest interaction potential energy profiling to determine the best binding mode(s) of unsubstituted bullvalene and each isomer of methylenehydroxybullvalene to ß- and γ-cyclodextrin. Subsequent molecular dynamics simulations of the predicted host-guest complexes showed that while unsubstituted bullvalene has a single, albeit ill-defined, binding mode with either cyclodextrin, each isomer of methylenehydroxybullvalene has two possible modes of binding to ß-cyclodextrin but only a single, nebulous mode of binding to γ-cyclodextrin. Experimental determination of the binding free energy of each methylenehydroxybullvalene-cyclodextrin complex showed that methylenehydroxybullvalene is more likely to bind to ß-cyclodextrin than to γ-cyclodextrin, despite its smaller cavity. Together, our results suggest that ß-cyclodextrin, but not γ-cyclodextrin, shows promise for conformational capture of mono-substituted bullvalenes. More broadly, our computational pipeline should prove useful for rapid characterization of cyclodextrin host-guest complexes, avoiding the need for costly synthesis of guest molecules that are unlikely to bind stably, as well as providing detailed atomic-level insight into the nature of complexation.

Site-specific glycation of Aß1-42 affects fibril formation and is neurotoxic.

Aß1-42 is involved in Alzheimer's disease (AD) pathogenesis and is prone to glycation, an irreversible process where proteins accumulate advanced glycated end products (AGEs). Nϵ-(Carboxyethyl)lysine (CEL) is a common AGE associated with AD patients and occurs at either Lys-16 or Lys-28 of Aß1-42. Methyglyoxal is commonly used for the unspecific glycation of Aß1-42, which results in a complex mixture of AGE-modified peptides and makes interpretation of a causative AGE at a specific amino acid residue difficult. We address this issue by chemically synthesizing defined CEL modifications on Aß1-42 at Lys-16 (Aß-CEL16), Lys-28 (Aß-CEL28), and Lys-16 and -28 (Aß-CEL16&28). We demonstrated that double-CEL glycations at Lys-16 and Lys-28 of Aß1-42 had the most profound impact on the ability to form amyloid fibrils. In silico predictions indicated that Aß-CEL16&28 had a substantial decrease in free energy change, which contributes to fibril destabilization, and a increased aggregation rate. Single-CEL glycations at Lys-28 of Aß1-42 had the least impact on fibril formation, whereas CEL glycations at Lys-16 of Aß1-42 delayed fibril formation. We also tested these peptides for neuronal toxicity and mitochondrial function on a retinoic acid-differentiated SH-SY5Y human neuroblastoma cell line (RA-differentiated SH-SY5Y). Only Aß-CEL16 and Aß-CEL28 were neurotoxic, possibly through a nonmitochondrial pathway, whereas Aß-CEL16&28 showed no neurotoxicity. Interestingly, Aß-CEL16&28 had depolarized the mitochondrial membrane potential, whereas Aß-CEL16 had increased mitochondrial respiration at complex II. These results may indicate mitophagy or an alternate route of metabolism, respectively. Therefore, our results provides insight into potential therapeutic approaches against neurotoxic CEL-glycated Aß1-42.

Synthesis and pharmacokinetic study of a 11C-labeled cholesterol 24-hydroxylase inhibitor using 'in-loop' [11C]CO2 fixation method.

Cholesterol 24-hydroxylase, also known as CYP46A1 (EC 1.14.13.98), is a monooxygenase and a member of the cytochrome P450 family. CYP46A1 is specifically expressed in the brain where it controls cholesterol elimination by producing 24S-hydroxylcholesterol (24-HC) as the major metabolite. Modulation of CYP46A1 activity may affect Aß deposition and p-tau accumulation by changing 24-HC formation, which thereafter serves as potential therapeutic pathway for Alzheimer's disease. In this work, we showcase the efficient synthesis and preliminary pharmacokinetic evaluation of a novel cholesterol 24-hydroxylase inhibitor 1 for use in positron emission tomography.

A concisely automated synthesis of TSPO radiotracer [18 F]FDPA based on spirocyclic iodonium ylide method and validation for human use.

Fluorine-18 labeled N,N-diethyl-2-(2-(4-(2-fluoroethoxy)phenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)acetamide ([18 F]FDPA) is a potent and selective radiotracer for positron-emission tomography (PET) imaging of the translocator protein 18 kDa (TSPO). Our previous in vitro and in vivo evaluations have proven that this tracer is promising for further human translation. Our study addresses the need to streamline the automatic synthesis of this radiotracer to make it more accessible for widespread clinical evaluation and application. Here, we successfully demonstrate a one-step radiolabeling of [18 F]FDPA based on a novel spirocyclic iodonium ylide (SCIDY) precursor using tetra-n-butyl ammonium methanesulfonate (TBAOMs), which has demonstrated the highest radiochemical yields and molar activity from readily available [18 F]fluoride ion. The nucleophilic radiofluorination was completed on a GE TRACERlab FX2 N synthesis module, and the formulated [18 F]FDPA was obtained in nondecay corrected (n.d.c) radiochemical yields of 15.6 ± 4.2%, with molar activities of 529.2 ± 22.5 GBq/µmol (14.3 ± 0.6 Ci/µmol) at the end of synthesis (60 minutes, n = 3) and validated for human use. This methodology facilitates efficient synthesis of [18 F]FDPA in a commercially available synthesis module, which would be broadly applicable for routine production and widespread clinical PET imaging studies.

Metal Protein-Attenuating Compound for PET Neuroimaging: Synthesis and Preclinical Evaluation of [11C]PBT2.

Dyshomeostasis or abnormal accumulation of metal ions such as copper, zinc, and iron have been linked to the pathogenesis of multiple neurodegenerative disorders including Alzheimer's disease (AD) and Huntington's disease (HD). 5,7-Dichloro-2-((dimethylamino)methyl)quinolin-8-ol, PBT2, is a second generation metal protein-attenuating compound that has recently advanced in Phase II clinical trials for the treatment of AD and HD based on promising preclinical efficacy data. Herein, we report the first radiosynthesis and preclinical positron emission tomography (PET) neuroimaging evaluation of [11C]PBT2 in rodents and nonhuman primates. Carbon-11 labeled PBT2 was synthesized in 4.8 ± 0.5% (nondecay corrected) radiochemical yield (RCY) at end-of-synthesis, based upon [11C]CH3I (n = 6), with >99% radiochemical purity and 80-90 GBq/µmol molar activity (Am) from the corresponding normethyl precursor. In the nonhuman primate brain, [11C]PBT2 uptake was extensive with peak concentration SUVpeak of 3.2-5.2 within 2.5-4.5 min postinjection in all cortical and subcortical gray matter regions (putamen > caudate > cortex ≫ white matter) followed by rapid washout from normal brain tissues. Furthermore, it is shown that [11C]PBT2 binds specifically in AD human brain tissue in vitro. The results presented here, combined with the clinical data available for PBT2, warrant the evaluation of [11C]PBT2 as an exploratory PET radiotracer in humans.

Recent applications of a single quadrupole mass spectrometer in 11C, 18F and radiometal chemistry.

Mass spectrometry (MS) has longstanding applications in radiochemistry laboratories, stemming from carbon-dating. However, research on the development of radiotracers for molecular imaging with either positron emission tomography (PET) or single photon emission computed tomography has yet to take full advantage of MS. This inertia has been attributed to the relatively low concentrations of radiopharmaceutical formulations and lack of access to the required MS equipment due to the high costs for purchase and maintenance of specialized MS systems. To date, single quadrupole (SQ)-MS coupled to liquid chromatography (LC) systems is the main form of MS that has been used in radiochemistry laboratories. These LC/MS systems are primarily used for assessing the chemical purity of radiolabeling precursor or standard molecules but also have applications in the determination of metabolites. Herein, we highlight personal experiences using a compact SQ-MS in our PET radiochemistry laboratories, to monitor the small amounts of carrier observed in most radiotracer preparations, even at high molar activities. The use of a SQ-MS in the observation of the low mass associated with non-radioactive species which are formed along with the radiotracer from the trace amounts of carrier found is demonstrated. Herein, we describe a pre-concentration system to detect dilute radiopharmaceutical formulations and metabolite analyses by SQ-MS. Selected examples where SQ-MS was critical for optimization of radiochemical reactions and for unequivocal characterization of radiotracers are showcased. We also illustrate examples where SQ-MS can be applied in identification of radiometal complexes and development of a new purification methodology for Pd-catalyzed radiofluorination reactions, shedding light on the identity of metal complexes present in the labelling solution.

"In-loop" [11 C]CO2 fixation: Prototype and proof of concept.

Carbon-11-labeled carbon dioxide is the most common feedstock for the synthesis of positron emission tomography radiotracers and can be directly used for 11 C-carbonylation. Herein, we report the development of an apparatus that takes advantage of "in-loop" technologies to facilitate robust and reproducible syntheses of 11 C-carbonyl-based radiotracers by [11 C]CO2 -fixation. Our "in-loop" [11 C]CO2 -fixation method is simple, efficient, and proceeds smoothly at ambient pressure and temperature. We selected model 11 C-carbonyl-labeled carbamates as well as symmetrical and unsymmetrical ureas based on their widespread use in radiotracer design and our clinical research interests for proof of concept. Utility of this method is demonstrated by the synthesis of a reversible radiopharmaceutical for monoamine oxidase B, [11 C]SL25.1188, and 2 novel fatty acid amide hydrolase inhibitors. These radiotracers were isolated and formulated (>3.5 GBq; 100 mCi) with radiochemical purities (>99%) and molar radioactivity (≥80 GBq/µmol; ≥2162 mCi/µmol).

Discovery of PET radiopharmaceuticals at the academia-industry interface.

Project-specific collaborations between academia and pharmaceutical partners are a growing phenomenon within molecular imaging and in particular in the positron emission tomography (PET) radiopharmaceutical community. This cultural shift can be attributed in part to decreased public funding in academia in conjunction with the increased reliance on outsourcing of chemistry, radiochemistry, pharmacology and molecular imaging studies by the pharmaceutical industry. This account highlights some of our personal experiences working with industrial partners to develop new PET radiochemistry methodologies for drug discovery and neuro-PET research studies. These symbiotic academic-industrial partnerships have not only led to novel radiotracers for new targets but also to the application of new carbon-11 and fluorine-18 labeling methodologies and technologies to label previously unprecedented compounds for in vivo evaluations.

cGMP production of the radiopharmaceutical [18 F]MK-6240 for PET imaging of human neurofibrillary tangles.

Fluorine-18-labelled 6-(fluoro)-3-(1H-pyrrolo[2,3-c]pyridin-1-yl)isoquinolin-5-amine ([18 F]MK-6240) is a novel potent and selective positron emission tomography (PET) radiopharmaceutical for detecting human neurofibrillary tangles, which are made up of aggregated tau protein. Herein, we report the fully automated 2-step radiosynthesis of [18 F]MK-6240 using a commercially available radiosynthesis module, GE Healthcare TRACERlab FXFN . Nucleophilic fluorination of the 5-diBoc-6-nitro precursor with potassium cryptand [18 F]fluoride (K[18 F]/K222 ) was performed by conventional heating, followed by acid deprotection and semipreparative high-performance liquid chromatography under isocratic conditions. The isolated product was diluted with formulation solution and sterile filtered under Current Good Manufacturing Practices, and quality control procedures were established to validate this radiopharmaceutical for human use. At the end of synthesis, 6.3 to 9.3 GBq (170-250 mCi) of [18 F]MK-6240 was formulated and ready for injection, in an uncorrected radiochemical yield of 7.5% ± 1.9% (relative to starting [18 F]fluoride) with a specific activity of 222 ± 67 GBq/µmol (6.0 ± 1.8 Ci/µmol) at the end of synthesis (90 minutes; n = 3). [18 F]MK-6240 was successfully validated for human PET studies meeting all Food and Drug Administration and United States Pharmacopeia requirements for a PET radiopharmaceutical. The present method can be easily adopted for use with other radiofluorination modules for widespread clinical research use.

Microfluidic radiosynthesis of [18F]FEMPT, a high affinity PET radiotracer for imaging serotonin receptors.

Continuous-flow microfluidics has shown increased applications in radiochemistry over the last decade, particularly for both pre-clinical and clinical production of fluorine-18 labeled radiotracers. The main advantages of microfluidics are the reduction in reaction times and consumption of reagents that often result in increased radiochemical yields and rapid optimization of reaction parameters for 18F-labeling. In this paper, we report on the two-step microfluidic radiosynthesis of the high affinity partial agonist of the serotonin 1A receptor, [18F]FEMPT (pKi = 9. 79; Ki = 0.16 nM) by microfluidic radiochemistry. [18F]FEMPT was obtained in ≈7% isolated radiochemical yield and in >98% radiochemical and chemical purity. The molar activity of the final product was determined to be >148 GBq/µmol (>4 Ci/µmol).

Preclinical PET Neuroimaging of [11C]Bexarotene.

Activation of retinoid X receptors (RXRs) has been proposed as a therapeutic mechanism for the treatment of neurodegeneration, including Alzheimer's and Parkinson's diseases. We previously reported radiolabeling of a Food and Drug Administration-approved RXR agonist, bexarotene, by copper-mediated [(11)C]CO2 fixation and preliminary positron emission tomography (PET) neuroimaging that demonstrated brain permeability in nonhuman primate with regional binding distribution consistent with RXRs. In this study, the brain uptake and saturability of [(11)C]bexarotene were studied in rats and nonhuman primates by PET imaging under baseline and greater target occupancy conditions. [(11)C]Bexarotene displays a high proportion of nonsaturable uptake in the brain and is unsuitable for RXR occupancy measurements in the central nervous system.

Synthesis of 18F-Arenes from Spirocyclic Iodonium(III) Ylides via Continuous-Flow Microfluidics.

Spirocyclic hypervalent iodine(III) ylides have proven to be synthetically versatile precursors for efficient radiolabelling of a diverse range of non-activated (hetero)arenes, highly functionalised small molecules, building blocks and radiopharmaceuticals from [18F]fluoride ion. Herein, we report the implementation of these reactions onto a continuous-flow microfluidic platform, thereby offering an alterative and automated synthetic procedure of a radiopharmaceutical, 3-[18F]fluoro-5-[(pyridin-3-yl)ethynyl]benzonitrile ([18F]FPEB) and a routinely used building block for click-radiochemistry, 4-[18F]fluorobenzyl azide. This new protocol was applied to the synthesis of [18F]FPEB (radiochemical conversion (RCC) = 68 ± 5%) and 4-[18F]fluorobenzyl azide (RCC=68 ± 5%; isolated radiochemical yield = 24±0%). We anticipate that the high throughput microfluidic platform will accelerate the discovery and applications of 18F-labelled building blocks and labelled compounds prepared by iodonium ylide precursors as well as the production of radiotracers for preclinical imaging studies.

(18)F-Labeling of Aryl-SCF3, -OCF3 and -OCHF2 with [(18)F]Fluoride.

We report that halogenophilic silver(I) triflate permits halogen exchange (halex) nucleophilic (18)F-fluorination of aryl-OCHFCl, -OCF2Br and -SCF2Br precursors under mild conditions. This Ag(I)-mediated process allows for the first time access to a range of (18)F-labeled aryl-OCHF2, -OCF3 and -SCF3 derivatives, inclusive of [(18)F]riluzole. The (18)F-labeling of these medicinally important motifs expands the radiochemical space available for PET applications.

First human use of a radiopharmaceutical prepared by continuous-flow microfluidic radiofluorination: proof of concept with the tau imaging agent [18F]T807.

Despite extensive preclinical imaging with radiotracers developed by continuous-flow microfluidics, a positron emission tomographic (PET) radiopharmaceutical has not been reported for human imaging studies by this technology. The goal of this study was to validate the synthesis of the tau radiopharmaceutical 7-(6-fluoropyridin-3-yl)-5H-pyrido[4,3-b]indole ([18F]T807) and perform first-in-human PET scanning enabled by microfluidic flow chemistry. [18F]T807 was synthesized by our modified one-step method and adapted to suit a commercial microfluidic flow chemistry module. For this proof of concept, the flow system was integrated to a GE Tracerlab FXFN unit for high-performance liquid chromatography purification and formulation. Three consecutive productions of [18F]T807 were conducted to validate this radiopharmaceutical. Uncorrected radiochemical yields of 17 ± 1% of crude [18F]T807 (≈ 500 mCi, radiochemical purity 95%) were obtained from the microfluidic device. The crude material was then purified, and > 100 mCi of the final product was obtained in an overall uncorrected radiochemical yield of 5 ± 1% (n  =  3), relative to starting [18F]fluoride (end of bombardment), with high radiochemical purity (≥ 99%) and high specific activities (6 Ci/µmol) in 100 minutes. A clinical research study was carried out with [18F]T807, representing the first reported human imaging study with a radiopharmaceutical prepared by this technology.

Alternative approaches for PET radiotracer development in Alzheimer's disease: imaging beyond plaque.

Alzheimer's disease (AD) and related dementias show increasing clinical prevalence, yet our understanding of the etiology and pathobiology of disease-related neurodegeneration remains limited. In this regard, noninvasive imaging with radiotracers for positron emission tomography (PET) presents a unique tool for quantifying spatial and temporal changes in characteristic biological markers of brain disease and for assessing potential drug efficacy. PET radiotracers targeting different protein markers are being developed to address questions pertaining to the molecular and/or genetic heterogeneity of AD and related dementias. For example, radiotracers including [(11) C]-PiB and [(18) F]-AV-45 (Florbetapir) are being used to measure the density of Aß-plaques in AD patients and to interrogate the biological mechanisms of disease initiation and progression. Our focus is on the development of novel PET imaging agents, targeting proteins beyond Aß-plaques, which can be used to investigate the broader mechanism of AD pathogenesis. Here, we present the chemical basis of various radiotracers which show promise in preclinical or clinical studies for use in evaluating the phenotypic or biochemical characteristics of AD. Radiotracers for PET imaging neuroinflammation, metal ion association with Aß-plaques, tau protein, cholinergic and cannabinoid receptors, and enzymes including glycogen-synthase kinase-3ß and monoamine oxidase B amongst others, and their connection to AD are highlighted.

Integration of Yeast Episomal/Integrative Plasmid Causes Genotypic and Phenotypic Diversity and Improved Sesquiterpene Production in Metabolically Engineered Saccharomyces cerevisiae.

The variability in phenotypic outcomes among biological replicates in engineered microbial factories presents a captivating mystery. Establishing the association between phenotypic variability and genetic drivers is important to solve this intricate puzzle. We applied a previously developed auxin-inducible depletion of hexokinase 2 as a metabolic engineering strategy for improved nerolidol production in Saccharomyces cerevisiae, and biological replicates exhibit a dichotomy in nerolidol production of either 3.5 or 2.5 g L-1 nerolidol. Harnessing Oxford Nanopore's long-read genomic sequencing, we reveal a potential genetic cause─the chromosome integration of a 2µ sequence-based yeast episomal plasmid, encoding the expression cassettes for nerolidol synthetic enzymes. This finding was reinforced through chromosome integration revalidation, engineering nerolidol and valencene production strains, and generating a diverse pool of yeast clones, each uniquely fingerprinted by gene copy numbers, plasmid integrations, other genomic rearrangements, protein expression levels, growth rate, and target product productivities. Τhe best clone in two strains produced 3.5 g L-1 nerolidol and ∼0.96 g L-1 valencene. Comparable genotypic and phenotypic variations were also generated through the integration of a yeast integrative plasmid lacking 2µ sequences. Our work shows that multiple factors, including plasmid integration status, subchromosomal location, gene copy number, sesquiterpene synthase expression level, and genome rearrangement, together play a complicated determinant role on the productivities of sesquiterpene product. Integration of yeast episomal/integrative plasmids may be used as a versatile method for increasing the diversity and optimizing the efficiency of yeast cell factories, thereby uncovering metabolic control mechanisms.

Microfluidic radiosynthesis and biodistribution of [18 F] 2-(5-fluoro-pentyl)-2-methyl malonic acid.

Microfluidics technology has emerged as a powerful tool for the radiosynthesis of positron emission tomography (PET) and single-photon emission computed tomography radiolabeled compounds. In this work, we have exploited a continuous flow microfluidic system (Advion, Inc., USA) for the [(18) F]-fluorine radiolabeling of the malonic acid derivative, [(18) F] 2-(5-fluoro-pentyl)-2-methyl malonic acid ([(18) F]-FPMA), also known as [(18) F]-ML-10, a radiotracer proposed as a potential apoptosis PET imaging agent. The radiosynthesis was developed using a new tosylated precursor. Radiofluorination was initially optimized by manual synthesis and served as a basis to optimize reaction parameters for the microfluidic radiosynthesis. Under optimized conditions, radio-thin-layer chromatography analysis showed 79% [(18) F]-fluorine incorporation prior to hydrolysis and purification. Following hydrolysis, the [(18) F]-FPMA was purified by C18 Sep-Pak, and the final product was analyzed by radio-HPLC (high-performance liquid chromatography). This resulted in a decay-corrected 60% radiochemical yield and ≥98% radiochemical purity. Biodistribution data demonstrated rapid blood clearance with less than 2% of intact [(18) F]-FPMA radioactivity remaining in the circulation 60 min post-injection. Most organs showed low accumulation of the radiotracer, and radioactivity was predominately cleared through kidneys (95% in 1 h). Radio-HPLC analysis of plasma and urine samples showed a stable radiotracer at least up to 60 min post-injection.