Name: optimization of radiochemical reactions using droplet arrays
Uploaded: 2021-02-12T14:15:00
Description: Watch this Scientific Journal Video about Optimization of Radiochemical Reactions using Droplet Arrays at JoVE.com

We thank the UCLA Biomedical Cyclotron Facility and Dr. Roger Slavik and Dr. Giuseppe Carlucci for generously providing [18F]fluoride for these studies and the UCLA NanoLab for support with equipment for chip fabrication.

CAUTION: This protocol involves the handling of radioactive materials. Experiments should not be undertaken without the necessary training and personal protective equipment and approval from the radiation safety office at your organization. Experiments should be performed behind radiation shielding, preferably in a ventilated hot cell
1. Fabrication of multi-reaction chips
NOTE: Batches of multi-reaction microdroplet chips are fabricated from 4&#34; silicon wafers usi...

<ol>
	<li>Matthews, P. M., Rabiner, E. A., Passchier, J., Gunn, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positron+emission+tomography+molecular+imaging+for+drug+development.">Positron emission tomography molecular imaging for drug development.</a> British Journal of Clinical Pharmacology. 73 (2), 175-186 (2012).</li><li>Piel, M., Vernaleken, I., R&#246;sch, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positron+emission+tomography+in+CNS+drug+discovery+and+drug+monitoring.">Positron emission tomography in CNS drug discovery and drug monitoring.</a> Journal of Medicinal Chemistry. 57 (22), 9232-9258 (2014).</li><li>Cherry, S. R., Sorenson, J. A., Phelps, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Physics in Nuclear Medicine. , (2012).</li><li>Knapp, K. -. A., Nickels, M. L., Manning, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+current+role+of+microfluidics+in+radiofluorination+chemistry.">The current role of microfluidics in radiofluorination chemistry.</a> Molecular Imaging and Biology. 22 (3), 463-475 (2020).</li><li>Rensch, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics:+A+groundbreaking+technology+for+PET+tracer+production.">Microfluidics: A groundbreaking technology for PET tracer production.</a> Molecules. 18 (7), 7930-7956 (2013).</li><li>Pascali, G., Watts, P., Salvadori, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics+in+radiopharmaceutical+chemistry.">Microfluidics in radiopharmaceutical chemistry.</a> Nuclear Medicine and Biology. 40 (6), 776-787 (2013).</li><li>Keng, P. Y., van Dam, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+microfluidics:+A+new+paradigm+for+radiochemistry.">Digital microfluidics: A new paradigm for radiochemistry.</a> Molecular Imaging. 14, 579-594 (2015).</li><li>Wang, J., Chao, P. H., Janet, S., van Dam, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performing+multi-step+chemical+reactions+in+microliter-sized+droplets+by+leveraging+a+simple+passive+transport+mechanism.">Performing multi-step chemical reactions in microliter-sized droplets by leveraging a simple passive transport mechanism.</a> Lab on a Chip. 17 (24), 4342-4355 (2017).</li><li>Wang, J., Chao, P. H., van Dam, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultra-compact,+automated+microdroplet+radiosynthesizer.">Ultra-compact, automated microdroplet radiosynthesizer.</a> Lab on a Chip. (19), 2415-2424 (2019).</li><li>Rios, A., Wang, J., Chao, P. H., van Dam, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+multi-reaction+microdroplet+platform+for+rapid+radiochemistry+optimization.">A novel multi-reaction microdroplet platform for rapid radiochemistry optimization.</a> RSC Advances. 9 (35), 20370-20374 (2019).</li><li>Sergeev, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performing+radiosynthesis+in+microvolumes+to+maximize+molar+activity+of+tracers+for+positron+emission+tomography.">Performing radiosynthesis in microvolumes to maximize molar activity of tracers for positron emission tomography.</a> Communications Chemistry. 1 (1), 10 (2018).</li><li>Pascali, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+nucleophilic+18F+radiofluorinations+using+a+microfluidic+reaction+approach.">Optimization of nucleophilic 18F radiofluorinations using a microfluidic reaction approach.</a> Nature Protocols. 9 (9), 2017-2029 (2014).</li><li>Lisova, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscale+radiosynthesis,+preclinical+imaging+and+dosimetry+study+of+[18F]AMBF3-TATE:+A+potential+PET+tracer+for+clinical+imaging+of+somatostatin+receptors.">Microscale radiosynthesis, preclinical imaging and dosimetry study of [18F]AMBF3-TATE: A potential PET tracer for clinical imaging of somatostatin receptors.</a> Nuclear Medicine and Biology. 61, 36-44 (2018).</li><li>Wang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+radio-TLC+analysis.">High-throughput radio-TLC analysis.</a> Nuclear Medicine and Biology. 82-83, 41-48 (2020).</li><li>Dooraghi, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+microfluidic+PET+tracer+synthesis+with+Cerenkov+imaging.">Optimization of microfluidic PET tracer synthesis with Cerenkov imaging.</a> Analyst. 138 (19), 5654-5664 (2013).</li><li>Collins, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+diverse+PET+probes+with+limited+resources:+24+18F-labeled+compounds+prepared+with+a+single+radiosynthesizer.">Production of diverse PET probes with limited resources: 24 18F-labeled compounds prepared with a single radiosynthesizer.</a> Proceedings of the National Academy of Sciences. 114 (43), 11309-11314 (2017).</li><li>Lazari, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fully+automated+production+of+diverse+18F-labeled+PET+tracers+on+the+ELIXYS+multireactor+radiosynthesizer+without+hardware+modification.">Fully automated production of diverse 18F-labeled PET tracers on the ELIXYS multireactor radiosynthesizer without hardware modification.</a> Journal of Nuclear Medicine Technology. 42 (3), 203-210 (2014).</li><li>Lisova, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid,+efficient,+and+economical+synthesis+of+PET+tracers+in+a+droplet+microreactor:+application+to+O-(2-[18F]fluoroethyl)-L-tyrosine+([18F]FET).">Rapid, efficient, and economical synthesis of PET tracers in a droplet microreactor: application to O-(2-[18F]fluoroethyl)-L-tyrosine ([18F]FET).</a> EJNMMI Radiopharmacy and Chemistry. 5 (1), 1 (2019).</li><li>Wang, J., Holloway, T., Lisova, K., van Dam, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Green+and+efficient+synthesis+of+the+radiopharmaceutical+[18F]FDOPA+using+a+microdroplet+reactor.">Green and efficient synthesis of the radiopharmaceutical [18F]FDOPA using a microdroplet reactor.</a> Reaction Chemistry &amp; Engineering. 5 (2), 320-329 (2020).</li><li>Lisova, K., Wang, J., Rios, A., van Dam, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptation+and+optimization+of+[F-18]+Florbetaben+([F-18]+FBB)+radiosynthesis+to+a+microdroplet+reactor.">Adaptation and optimization of [F-18] Florbetaben ([F-18] FBB) radiosynthesis to a microdroplet reactor.</a> Journal of Labelled Compounds and Radiopharmaceuticals. 62, 353-354 (2019).</li><li>Wang, J., Chao, P. H., Slavik, R., van Dam, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-GBq+production+of+the+radiotracer+[18F]fallypride+in+a+droplet+microreactor.">Multi-GBq production of the radiotracer [18F]fallypride in a droplet microreactor.</a> RSC Advances. 10 (13), 7828-7838 (2020).</li></ol>

Due to limitations of conventional radiochemistry systems that allow only one or a small number of reactions per day and consume a significant quantity of reagents per data point, only a tiny portion of the overall reaction parameter space can be explored in practice, and many times results are reported with no repeats (n=1). Compared to conventional systems, this multi-reaction droplet radiosynthesis platform makes it practical to accomplish more comprehensive and rigorous studies of radiosynthesis conditions while cons...

Positron-emission tomography (PET) radiopharmaceuticals are widely used as research tools to monitor specific in vivo biochemical processes and study diseases, and for the development of new drugs and therapies. Moreover, PET is a critical tool for diagnosing or staging disease and monitoring a patient&#39;s response to therapy1,2,3. Due to the short half-life of PET radioisotopes (e.g., 110 min for fluorine-18-labeled radiopharmaceuticals) and radiation hazard, these compounds are prepared using specialized automated systems operating behind radiation shielding and must be prepared just before use.
Current systems used to synthesize radiopharmaceuticals are designed to produce large batches that are divided up into many individual doses to share the production cost. While current systems are suitable for the production of widely used radiotracers like [18F]FDG (because multiple patient scans and research experiments can be scheduled in a single day), these systems can be wasteful for the production of novel radiotracers during early-stage development, or less commonly used radiotracers. Volumes that conventional systems use are typically in the 1-5 mL range, and the reactions require precursor amounts in the 1-10 mg range. Furthermore, using conventional radiosynthesizers is generally cumbersome during optimization studies since the apparatus becomes contaminated after use and the user must wait for radioactivity to decay before performing the next experiment. Aside from equipment cost, the cost of the radioisotope and reagents can, therefore, become very substantial for studies requiring production of multiple batches. This can occur, for example, during the optimization of synthesis protocols for novel radiotracers to achieve sufficient yield and reliability for initial in vivo imaging studies.
Microfluidic technologies have been increasingly used in radiochemistry to capitalize on several advantages over conventional systems4,5,6. Microfluidic platforms, including those based on 1-10 &#181;L reaction volumes7,8,9, have shown a significant reduction of reagent volumes and consumption of expensive precursors, as well as short reaction times. These reductions lead to lower costs, faster heating and evaporation steps, shorter and more straightforward downstream purification, an overall &#34;greener&#34; chemistry process10, and higher molar activity of the produced radiotracers11. These improvements make it more practical to perform more extensive optimization studies by lowering the reagent cost of each synthesis. Further benefits can be achieved by performing multiple experiments from a single batch of radioisotope in a single day. For example, microfluidic flow chemistry radiosynthesizers operating in &#34;discovery mode&#34; can sequentially perform dozens of reactions, each using only 10s of &#181;L reaction volume12.
Inspired by these advantages, a multi-reaction droplet array chip in which microvolume reactions are confined to an array of surface-tension traps on a silicon surface, created using a patterned Teflon coating, was developed. These chips enable multiple reactions at the 1-20 &#181;L scale to be performed simultaneously, opening the possibility to explore 10s of different reaction conditions per day, each with multiple replicates. In this paper, the utility of this new high-throughput approach for performing rapid and low-cost radiochemistry optimizations is demonstrated. Using multi-reaction droplet chips allows for convenient exploration of the impact of reagent concentrations and reaction solvent, and the use of multiple chips could enable the study of reaction temperature and time, all while consuming very low amounts of precursor.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2,3-dimethyl-2-butanol (thexyl alcohol)</td>
			<td>Sigma-Aldrich</td>
			<td>594-60-5</td>
			<td>98%</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>KMG Chemicals</td>
			<td></td>
			<td>Cleanroom LP grade</td>
		</tr>
		<tr>
			<td>Ammonium formate (NH4HCO2)</td>
			<td>Sigma-Aldrich</td>
			<td>540-69-2</td>
			<td>97%</td>
		</tr>
		<tr>
			<td>Anhydrous acetonitrile (MeCN)</td>
			<td>Sigma-Aldrich</td>
			<td>75-05-8</td>
			<td>99.80%</td>
		</tr>
		<tr>
			<td>Ceramic heater</td>
			<td>Watlow</td>
			<td>Utramic CER-1-01-0093</td>
			<td>25 mm x 25 mm</td>
		</tr>
		<tr>
			<td>Cerenkov imaging chamber</td>
			<td>Custom built</td>
			<td></td>
			<td>Other instruments can be used for TLC plate readout including: small animal in vivo optical imaging system, 2D radio-TLC scanner, 1D radio-TLC scanner</td>
		</tr>
		<tr>
			<td>DI water</td>
			<td>Sigma-Aldrich</td>
			<td>7732-18-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable transfer pipets, 3 mL</td>
			<td>Falcon</td>
			<td>13-680-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Dose calibrator</td>
			<td>Capintec, Inc.</td>
			<td>CRC-25 PET</td>
			<td></td>
		</tr>
		<tr>
			<td>Fallypride</td>
			<td>ABX Advanced Biochemical Compounds</td>
			<td>1560.0010.000</td>
			<td>Fallypride reference standard, &#62;95%</td>
		</tr>
		<tr>
			<td>[18F]fluoride in [18O]H2O</td>
			<td>UCLA Ahmanson Biomedical Cyclotron Facility</td>
			<td></td>
			<td>Due to short half-life this must be obtained from local radiochemistry lab or commercial radiopharmacy</td>
		</tr>
		<tr>
			<td>Glass cover plates (76.2 mm x 50.8 mm x 1 mm thick)</td>
			<td>C&#38;A Scientific</td>
			<td>6101</td>
			<td></td>
		</tr>
		<tr>
			<td>Headway spin coater</td>
			<td>Headway Research, Inc.</td>
			<td>PWM50-PS-R790 Sipinner system</td>
			<td>PWM50-control box, PS-motor, R790-bowl</td>
		</tr>
		<tr>
			<td>High temperature oven</td>
			<td>Carbolite</td>
			<td>HTCR 6 28</td>
			<td></td>
		</tr>
		<tr>
			<td>Hot plate</td>
			<td>Thermo Scientific</td>
			<td>Super-Nuova HP133425</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol (IPA)</td>
			<td>KMG Chemicals</td>
			<td></td>
			<td>Cleanroom LP grade</td>
		</tr>
		<tr>
			<td>Mask aligner</td>
			<td>Karl Suss</td>
			<td>MA/BA6</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol (MeOH)</td>
			<td>Sigma-Aldrich</td>
			<td>67-56-1</td>
			<td>&#8805;99.9%</td>
		</tr>
		<tr>
			<td>Microcentrifuge tube</td>
			<td>Eppendorf</td>
			<td>0030 123.301</td>
			<td>500 &#181;L, colorless, polypropylene</td>
		</tr>
		<tr>
			<td>Micropipette (0.5-10 &#181;L)</td>
			<td>Labnet</td>
			<td>BioPette P3940-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette (100-1000 &#181;L)</td>
			<td>Labnet</td>
			<td>BioPette P3940-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette (10-100 &#181;L)</td>
			<td>Labnet</td>
			<td>BioPette P3940-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette tips (0.1-10 &#181;L)</td>
			<td>USA Scientific Inc Tips</td>
			<td>11113810</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette tips (2-200 &#181;L)</td>
			<td>BrandTech</td>
			<td>13-889-143</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette tips (50-1000 &#181;L)</td>
			<td>BrandTech</td>
			<td>13-889-145</td>
			<td></td>
		</tr>
		<tr>
			<td>Photoresist developer solution</td>
			<td>MicroChem</td>
			<td>MEGAPOSIT MF-26A</td>
			<td></td>
		</tr>
		<tr>
			<td>Positive photoresist</td>
			<td>MicroChem</td>
			<td>MEGAPOSIT 220-7.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Reactive-ion etcher (RIE)</td>
			<td>Oxford Instruments</td>
			<td>Plasma Lab 80 Plus</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon wafer cutter</td>
			<td>Euro Tool</td>
			<td>CSCB-431.00</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon wafer; 4&#34; diameter</td>
			<td>Silicon Valley Microelectronics Inc.&#160;</td>
			<td>0017227-048</td>
			<td>P type, boron doped, thickness 525 &#177; 25 &#181;m</td>
		</tr>
		<tr>
			<td>Teflon AF 2400</td>
			<td>Chemours&#160;</td>
			<td>D14896765</td>
			<td>1% solids</td>
		</tr>
		<tr>
			<td>Tetrabutylammonium bicarbonate (TBAHCO3)</td>
			<td>ABX Advanced Biochemical Compounds</td>
			<td>808</td>
			<td>Aqueous solution stabilized with ethanol, 0.075 M</td>
		</tr>
		<tr>
			<td>Themal conducting paste</td>
			<td>OMEGA</td>
			<td>OT-201-2</td>
			<td></td>
		</tr>
		<tr>
			<td>TLC plates</td>
			<td>Merck KGaA</td>
			<td>1.05554.0001</td>
			<td>Silica gel 60 F254, 50 mm x 60 mm, aluminum back</td>
		</tr>
		<tr>
			<td>Tosyl-fallypride</td>
			<td>ABX Advanced Biochemical Compounds</td>
			<td>1550.004.000</td>
			<td>Fallypride precursor, &#62;90%</td>
		</tr>
		<tr>
			<td>Trimethylamine (TEA)</td>
			<td>Sigma-Aldrich</td>
			<td>75-50-3</td>
			<td>&#8805; 99%</td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>Cole-Parmer</td>
			<td>UX-07387-08</td>
			<td>Stainless steel, fine tip</td>
		</tr>
	</tbody>
</table>

optimization of radiochemical reactions using droplet arrays

Current automated radiosynthesizers are designed to produce large clinical batches of radiopharmaceuticals. They are not well suited for reaction optimization or novel radiopharmaceutical development since each data point involves significant reagent consumption, and contamination of the apparatus requires time for radioactive decay before the next use. To address these limitations, a platform for performing arrays of miniature droplet-based reactions in parallel, each confined within a surface-tension trap on a patterned polytetrafluoroethylene-coated silicon "chip", was developed. These chips enable rapid and convenient studies of reaction parameters including reagent concentrations, reaction solvent, reaction temperature and time. This platform permits the completion of hundreds of reactions in a few days with minimal reagent consumption, instead of taking months using a conventional radiosynthesizer.

A representative experiment was performed to illustrate this method. Using 16 reactions, optimization studies of the radiopharmaceutical [18F]fallypride were performed by varying precursor concentration (77, 39, 19, 9.6, 4.8, 2.4, 1.2, and 0.6 mM) in thexyl alcohol:MeCN (1:1, v/v) as the reaction solvent. Reactions were performed at 110 &#176;C for 7 min. Collection efficiency, sample composition (i.e., proportions of [18F]fallypride product, unreacted [18...

Watch this Scientific Journal Video about Optimization of Radiochemical Reactions using Droplet Arrays at JoVE.com

Optimization of Radiochemical Reactions using Droplet Arrays

This method describes the use of a novel high-throughput methodology, based on droplet chemical reactions, for the rapid and economical optimization of radiopharmaceuticals using nanomole amounts of reagents.

Current automated radiosynthesizers are designed to produce large clinical batches of radiopharmaceuticals. They are not well suited for reaction ...

Current automated radio synthesizers are designed to produce large batches of widely used radiopharmaceuticals, such as FDG. Due to the limited number of syntheses possible per day, and relatively high reagent consumption, these systems, however, are not well-suited for performing synthesis optimization studies. With this technique, throughput is significantly increased by performing up to 16 simultaneous reactions in parallel and reagent consumption is reduced a hundred-fold.Furthermore, by performing reactions in parallel, fair batches of radioisotope are needed to complete a study. Increased throughput enables wider exploration of reaction conditions with greater number of replicates each compared to the use of conventional instruments. While this protocol shows the optimization of precursor concentration in the synthesis of fallypride, the technique can be used to optimize other conditions and other radiopharmaceuticals.Begin by fabricating batches of multi-reaction microdroplet chips from four inch silicon wafers using standard photolithography techniques. In this protocol, the high-throughput optimization of precursor concentration is demonstrated with the synthesis of the radiopharmaceutical Fallypride. 16 simultaneous reactions can be performed on a single chip.The conditions to be compared are mapped to the reaction sites. Prepare a stock solution of the reaction solvent consisting of thexyl alcohol and acetonitrile in a one-to-one by volume mixture. Ensure that the volume is enough to create the planned dilution series.Prepare a 30 microliters stock solution of precursor in the reaction solvent with the maximum concentration to be explored. From the precursor stock solution and reaction solvent perform two times serial dilutions in a set of microcentrifuge tubes to prepare the different concentrations of the precursor solution. Prepare another set of microcentrifuge tubes to collect each crude reaction product using a permanent marker to label each tube with a unique number.Ensure that the total number of microcentrifuge tubes matches the number of conditions multiplied by the number of replicates. Prepare a 10 milliliter stock of collection solution comprising of nine to one methanol to deionized water. Aliquot 50 microliters of each into an additional set of 16 microcentrifuge tubes labeled as collection solution.Prepare a Fluoride stock solution by mixing around seven millicuries of the Fluoride source with 56 microliters of 75 millimolar tetrabutylammonium bicarbonate and diluting with DI water up to 140 microliters. Using a micropipette, load an eight microliter droplet of Fluoride stock solution on the first reaction spot of a multi-reaction chip. Measure the activity of the chip by placing it in a dose calibrator and record the time at which measurement is conducted.Remove the chip from the dose calibrator and load an eight microliter droplet of Fluoride stock solution on the second reaction spot. Measure the activity on the chip and record the time at which measurement is conducted. Repeat this process for all other reaction sites.Calculate the activity loaded per reaction spot by taking the activity measurement after loading the radioisotope and subtracting the previous measurement before that site was loaded. To align the multi-reaction chip on the heater, add a thin layer of thermal paste on top of the ceramic heater. Carefully place the chip on top of the heater using tweezers aligning the reference corner of the chip with the reference corner of the heater.The chip should overhang the heater by a small amount. Heat the chip for one minute by setting the heater to 105 degrees Celsius in the control program to evaporate the droplets, leaving a dried residue of Fluoride and tetrabutylammonium bicarbonate. Then, cooled the chip by setting the heater to 30 degrees Celsius and turning on the cooling fan with the control program.Using a micropipette, add a six microliters solution of fallypride precursor on top of the dried residue on the first reaction site. Repeat this for all other reaction sites on the chip. Use the optimization plan to determine which concentration of the dilution series is used for each reaction site.Heat the chip to 110 degrees Celsius for seven minutes using the control program to perform the radiofluorination reaction, then cool the chip by setting the heater to 30 degrees Celsius and turning on the cooling fan with the control program. Collect the crude product at the first reaction site by adding 10 microliters of collection solution from the designated microcentrifuge tube. After waiting for five seconds, aspirate the diluted crude product and transfer it to the corresponding collection microcentrifuge tube.Repeat this process a total of four times using the same pipette tip and then close the microcentrifuge tube. Repeat these steps to collect the crude product from all other reaction sites on the chip. To determine the collection efficiency for the first reaction on the chip, place the microcentrifuge tube with the collected crude product of the first reaction spot in the dose calibrator to measure the activity.Record the measurement and time of the measurement. Repeat this process for each of the collected crude products. Calculate the collection efficiency by dividing the activity of the collected crude product by the starting activity measured for the same reaction site.Repeat this for all other reaction sites on the chip. Next, analyze the composition of each collected crude product. With a pencil, draw a line 15 millimeters away from the bottom edge of the TLC plate and another line 50 millimeters away from the same edge.The first line is the origin line and the second is the solvent frontline. Draw eight small X's along the origin line at five millimeter spacing to define the sample spotting position for each of the eight lanes. Using a micropipette, transfer 0.5 microliters of the first crude product onto the TLC plate at the X for the first lane.Repeat this for additional crude products, then wait for the spots to dry. Develop each TLC plate using a mobile phase of 60%of acetonitrile in 25 millimolar ammonium formate with 1%TEA until the solvent front reaches the solvent frontline. At that time, remove the TLC plate from the chamber and wait for the solvent on the TLC plate to dry, then placed the TLC plate in the Cerenkov imaging system and cover it with a glass microscope slide.Obtain a radioactivity image of each TLC plate by setting the Cerenkov imaging system to a five minute exposure, then select the produced file of the image to TLC plate and perform standard image corrections. Use region of interest analysis for the first lane of the first TLC plate. Draw regions around each band visible in the lane, then compute the fraction of integrated intensity of each region compared to the total integrated intensity of all regions.Determine the fluorination efficiency as the fraction of activity in the Fallypride band. Repeat this analysis for all other lanes on all TLC plates. Then, compute the crude radiochemical yield for each reaction and choose the optimal precursor concentration by examining the crude radiochemical yield as a function of precursor concentration.Optimization studies of the radiopharmaceutical Fallypride were performed by varying precursor concentration in thexyl alcohol acetonitrile. Reactions were performed at 110 degrees Celsius per seven minutes. Collection efficiency and sample composition are shown here.The fluorination efficiency increased with increasing precursor concentration and the concentration of unreacted Fluoride decreased. There was a small amount of radioactive side product at low precursor concentrations, which was not formed at the higher precursor concentrations. The collection efficiency was nearly quantitative for most conditions, though it dropped slightly at low precursor concentrations.The highest crude radiochemical yield was achieved with a 39 millimolar precursor concentration. At this condition, the fluorination efficiency was 96%The crude RCY was 87%and there was no observed radioactive side product formation. It is critical to have a map plan of what reaction condition corresponds to which reaction droplet on the chip and to have appropriately labeled reagent tubes and product collection tubes that can be double-checked during the experiment.The procedure can be used for the optimization of other reaction conditions, such as amount of base, type of solvent, or reaction volume. It can also be used to optimize the synthesis of other radiopharmaceuticals.

Research

JoVE Journal

Chemistry

Microwave-assisted Intramolecular Dehydrogenative Diels-Alder Reactions for the Synthesis of Functionalized Naphthalenes/Solvatochromic Dyes

Functionalized naphthalenes have applications in a variety of research fields ranging from the synthesis of natural or biologically active molecules to ...

Retropinacol/Cross-pinacol Coupling Reactions - A Catalytic Access to 1,2-Unsymmetrical Diols

Unsymmetrical 1,2-diols are hardly accessible by reductive pinacol coupling processes. A successful execution of such a transformation is bound to a clear ...

Hot Biological Catalysis: Isothermal Titration Calorimetry to Characterize Enzymatic Reactions

Isothermal titration calorimetry (ITC) is a well-described technique that measures the heat released or absorbed during a chemical reaction, using it as ...

Real-time Monitoring of Reactions Performed Using Continuous-flow Processing: The Preparation of 3-Acetylcoumarin as an Example

By using inline monitoring, it is possible to optimize reactions performed using continuous-flow processing in a simple and rapid way. It is also possible ...

Optimization of Synthetic Proteins: Identification of Interpositional Dependencies Indicating Structurally and/or Functionally Linked Residues

Protein alignments are commonly used to evaluate the similarity of protein residues, and the derived consensus sequence used for identifying functional ...

Curtain Flow Column: Optimization of Efficiency and Sensitivity

Active Flow Technology (AFT) is a form of column technology that increases the separation performance of a HPLC column through the use of a specially ...

A Protocol for Safe Lithiation Reactions Using Organolithium Reagents

Organolithium reagents are powerful tools in the synthetic chemist&#39;s toolbox. However, the extreme pyrophoric nature of the most reactive reagents ...

Synthesis of Platinum-nickel Nanowires and Optimization for Oxygen Reduction Performance

Platinum-nickel (Pt-Ni) nanowires were developed as fuel cell electrocatalysts, and were optimized for the performance and durability in the oxygen ...

Enzymatic Cascade Reactions for the Synthesis of Chiral Amino Alcohols from L-lysine

Amino alcohols are versatile compounds with a wide range of applications. For instance, they have been used as chiral scaffolds in organic synthesis. ...