Name: Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry (Video) | JoVE Journal
Uploaded: 2017-02-04T15:00:00.000+00:00
Duration: 10 min 54 s
Description: Learn radiolabeling of tetrazine derivatives with technetium-99m for pre-targeted imaging using bioorthogonal chemistry. This method enables diagnostic imaging of organs and tissues with enhanced specificity.

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5 minutes

This work supported by research grant funding from the Natural Sciences and Engineering Research Council (NSERC) of Canada, the Ontario Institute for Cancer Research (OICR, #P.SI.015.8), and the Canadian Cancer Society (CCS, #703857). The authors acknowledge the contributions of Dr. Denis Snider who provided assistance in preparing the manuscript.

Animal studies were approved by the Animal Research Ethics Board at McMaster University in accordance with Canadian Council on Animal Care (CCAC) guidelines.

1. Radiolabeling of Tz-tridentate Ligands with 99mTc
CAUTION: The following procedures require the use of radioactive compounds. Work should only be done in a licensed laboratory with adherence to safety and disposal regulations. Microwave reactions should be performed in a microwave specifically designed for chemical synthesis.
<ol>
	<li>Synthesis of [99mTc(CO)3(H2O)3]+ 19,20
	<ol>
		<li>In a microwave vial, combine 8 mg K2[BH3CO2], 15 mg Na2CO3, 20 mg Na2B4O7·10H2O, and 25 mg KOCO[CH(OH)]2COONa·4H2O. Purge the vial for 10 min with argon gas.</li>
		<li>Add 4 mL of 99mTcO4- (~1,100 MBq, ~30 mCi) in 0.9% saline to the vial.</li>
		<li>Heat the reaction in a microwave for 3.5 min at 110 °C after 10 s of stirring to ensure thorough mixing of reagents.</li>
		<li>Adjust the pH of the solution to 3.5-4 using ~400 μL of 1 M HCl. Verify using pH paper.</li>
	</ol>
	</li>
	<li>Radiolabeling of Tetrazine ligands 1-5
	<ol>
		<li>Dissolve 2 mg of each ligand (compounds 1-5) in 250 μL MeOH21.</li>
		<li>Add 250 μL of [99mTc(CO)3(H2O)3]+ (~74 MBq, ~2 mCi) to each solution.</li>
		<li>Heat the reaction mixture using a microwave for 20 min at 60 °C. 
		NOTE: This step was identical for all 5 tetrazines.</li>
		<li>For compounds 2- 5, evaporate the solvent and re-dissolve the resulting products in 1 mL of 1:1 v/v DCM:TFA.</li>
		<li>Heat the dissolved reaction products (2-5) at 60 °C in a microwave for 6 min (2-4) or 10 min (5).</li>
		<li>After cooling to room temperature, evaporate the solvent using an evaporator (36 °C, 8 mbar, 3 min, 6,000 rpm) and dissolve the dried compound in 1:1 ACN:H2O or 1:1 MeOH:H2O, prior to HPLC purification.</li>
		<li>Purify the 99mTc-labeled compounds (1-5), including separating the labeled product from unlabeled tetrazine ligand, using HPLC (C18 reversed-phase). Typically, use an elution gradient of 30:70 ACN:H2O (both with 0.1% TFA) to 40:60 ACN:H2O over 20 min (18 min) and a C18 analytical 4.6 x 100 mm column. Use both UV (254 nm) and gamma detection.
		<ol>
			<li>Take a small sample of each labeled product and compare its HPLC retention time to that of a co-injected, non-radioactive, Re-labeled standard (0.125 mg in 20% methanol-H2O). The Re-labeled standard is identified in the UV HPLC trace, and will elute at the same time as the 99mTc-labeled compound in the γ-HPLC trace. This co-injection shows peaks at comparable retention times, confirming the identity of the 99mTc-labeled compound.</li>
		</ol>
		</li>
		<li>Evaporate the solvent from HPLC fractions using an evaporator (36 °C, 8 mbar, 3 min, 6,000 rpm).</li>
		<li>Formulate the purified compound at a concentration of 7.4 kBq/µL in PBS, containing 0.5% BSA and 0.01% Tween-80.</li>
		<li>To ensure the labelled compounds are stable, perform an in vitro stability study. Incubate the formulated compound at 37 °C for 1, 4 and 6 h, injecting a small amount (3.7 MBq) of the mixture on the HPLC at each time point to assess stability.</li>
	</ol>
	</li>
</ol>
2. Pre-targeted Bio-distribution Studies
<ol>
	<li>Preparation of animals
	<ol>
		<li>Using 7-9 week old, female Balb/c mice (n=3), administer TCO-BP formulated in saline (20 mg/kg) (5 μg/μL), via tail-vein injection.</li>
		<li>Place mouse in physical restraint device, and identify the veins located on the lateral surfaces of the tail and wipe with an alcohol swab. At approximately 2 cm from the end of the tail, insert a 30-gauge needle at a shallow angle, parallel to the vein. Slowly depress the plunger to inject, remove needle and apply clean gauze sponge at injection site with slight pressure until bleeding stops.</li>
		<li>At 1 h post injection of TCO-BP, administer ~0.74 MBq (20 μCi) of 99mTc-tetrazine formulated in 100 μL of 0.5% BSA, 0.01% Tween-80 in PBS, via tail-vein injection.</li>
	</ol>
	</li>
	<li>Bio-distribution studies
	<ol>
		<li>At the desired time point (t = 6 h), anaesthetize the mice using 3% isoflurane and 2% oxygen gas mixture. Demonstrate a toe pinch withdrawal on the anesthetized mouse to ensure they are under surgical plane of anesthesia.</li>
		<li>Collect blood (1 mL) via cardiac puncture using a syringe pre-treated with heparin. Place mouse on its back with nose in the nose cone for continued anesthesia and locate the xiphoid process on the animal.
		<ol>
			<li>Insert a 25 G needle, slightly to the left of the animal's midline under the xiphoid process, at a 20° angle. Fully insert the needle, and slowly pull back on the plunger to see blood in the needle hub if the heart was punctured. Slightly readjust the needle while holding the plunger if necessary, to puncture the heart. Slowly draw blood into the syringe.</li>
		</ol>
		</li>
		<li>Euthanize the animal by cervical dislocation, while under anesthesia.</li>
		<li>Place each animal in a plastic bag and use a dose calibrator (99mTc setting) to measure the whole body activity level.</li>
		<li>Collect the following tissues and fluids in pre-weighed counting tubes: blood, bone (knee and shoulder), gall bladder, kidneys, liver, stomach (with contents), small intestines (with contents), large intestines and caecum (with contents), thyroid and trachea, urinary bladder with urine, and tail.</li>
		<li>Rinse appropriate tissues (excluding blood, gall bladder, and urinary bladder) in PBS to remove blood and blot dry before placing the tissues in appropriate counting tubes.</li>
		<li>Place animal carcass in a plastic bag and measure residual whole body activity using a dose calibrator.</li>
		<li>Weigh each tube containing a tissue sample. Subtract initial weight of the tube to obtain mass of the tissue.</li>
		<li>Use a dose calibrator (99mTc setting) to measure the amount of activity in a test sample (100 µL) at the time of injection for each mouse. 
		NOTE: This test sample is equal to the injection volume, thus giving the activity count at the time of injection.</li>
		<li>At the time of tissue measurement, aliquot 5 µL of the test sample used previously. Use a multi-detector gamma counter (99mTc setting) and count to obtain the count per minute (CPM) for the 5 µL test sample.</li>
		<li>Use the two values obtained in 2.2.9 and 2.2.10 to calculate the activity and CPM relationship using equation 1 to obtain a conversion factor (CPM µCi-1). 
		(1) <img alt="Radiotracer quantification formula, including count per minute, dose calibration, gamma counter." src="/files/ftp_upload/55188/55188eq1.jpg" data-altupdatedat="1788250015000" data-alt="Equation 1"></li>
		<li>Use the gamma counter to measure the amount of radioactivity in each tissue or fluid sample.</li>
		<li>Use equation 1 to calculate the amount of activity in each tissue or fluid at the time of measurement relative to the total injected dose. This value is then normalized by organ weight and reported as percent injected dose per gram (i.e., %ID/g) of tissue.</li>
		<li>Follow steps 2.1.2 to 2.2.13 to conduct a negative control experiment using the 99mTc-labeled tetrazine ligands in the absence of TCO-BP. Sacrifice mice (n = 3) at 0.5, 1, 4 and 6 h post injection and obtain tissue or fluid as described above.</li>
	</ol>
	</li>
</ol>

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The authors declare they have no competing financial interests.

A collection of tetrazine-linked tridentate chelates of varying polarities was prepared, and the utility of their 99mTc complexes in the IEDDA reaction with a TCO derivative in vivo was assessed. An effective and reproducible 99mTc labeling method was developed for five tetrazine-chelates, where the ligand concentration was 10-3 M. The labeling step was followed by deprotection of t-butyl groups (for compounds 2-5). The high concentration of ligand was u...

99mTc remains the dominant radioisotope used in diagnostic nuclear medicine, with over 50 million imaging procedures conducted per year worldwide1,2,3. The majority of 99mTc agents used clinically are perfusion type radiopharmaceuticals. There are a limited number of actively targeted compounds in which 99mTc is directed to bind a specific biomarker through ligation to a targeting construct. The creation of targeted 99mTc radiopharmaceuticals is often hindered by the influence of 99mTc-ligand complexes on the ability of the targeting molecule to bind the biomarker of interest, or the isotopes half-life is not long enough for use with higher molecular weight biomolecules such as antibodies. The latter typically requires several days before images are acquired in order for the biomolecule to clear from non-target tissues. Pre-targeting offers an alternative approach to overcome these challenges.
Pre-targeting combined with bioorthogonal chemistry has been shown to be an effective way to develop new molecular imaging probes for both fluorescence and radio-imaging4,5,6,7,8. The inverse electron demand Diels-alder (IEDDA) reaction between 1,2,4,5-tetrazine (Tz) and trans-cyclooctene (TCO) derivatives, as shown in Figure 1, has been shown to be particularly effective6. The IEDDA reaction with these components can exhibit fast kinetics in PBS (k2 ≈ 6,000 M-1 s-1) and high selectivity, making it ideal for in vivo pre-targeting applications9,10.
The most common approach used involves administering a TCO-derived targeting vector and following a sufficient delay period, a radiolabeled tetrazine is administered. Radiolabeled tetrazines based on 11C, 18F, 64Cu, 89Zr, and 111In have been reported11,12,13,14,15. In contrast, there is only one report of a 99mTc-labeled Tz, which was prepared using a HYNIC type ligand requiring the use of co-ligands to prevent protein binding and degradation in vivo16. As an alternative, we report here the synthesis of 99mTc(I) labeled tetrazines using a family of ligands which form stable tridentate complexes with a [99mTc(CO)3]+ core.
<img alt="Chemical reaction diagram; azide-alkyne cycloaddition, nitrogen release." src="/files/ftp_upload/55188/55188fig1.jpg" data-altupdatedat="1788250015000" data-alt="Figure 1"> 
Figure 1: The bioorthogonal IEDDA reaction between tetrazine and trans-cyclooctene. <a href="http://cloudflare.jove.com/files/ftp_upload/55188/55188fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The family of ligands prepared contain tridentate chelates that vary in polarity and the nature of the linker group between the metal binding region and the Tz (Figure 2). The goal was to identify a 99mTc-Tetrazine construct that could effectively localize and react with TCO-labeled sites in vivo and rapidly clear when not bound, in order to yield high target-to-non-target ratios. To test the ligands, a TCO-derivative of a bisphosphonate (TCO-BP) was used17. We have shown previously that TCO-BP localizes to areas of active bone metabolism and can react with radiolabeled tetrazines in vivo18. It is a convenient reagent to test new tetrazines, because it can be prepared in a single step and experiments can be performed in normal mice where localization occurs primarily in the joints (knees and shoulders).

<table><tbody><tr><td>Argon gas</td><td>Alphagaz</td><td>---</td><td>---</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;CO&lt;sub&gt;3&lt;/sub&gt;</td><td>EMD Millipore</td><td>106395</td><td>---</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;B&lt;sub&gt;4&lt;/sub&gt;O&lt;sub&gt;7&lt;/sub&gt;&amp;#183;10H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Anachemia</td><td>S9640</td><td>---</td></tr><tr><td>KNaC&lt;sub&gt;4&lt;/sub&gt;H&lt;sub&gt;4&lt;/sub&gt;O&lt;sub&gt;6&lt;/sub&gt;&amp;#183;4H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Anachemia</td><td>217255</td><td>---</td></tr><tr><td>Technelite &lt;sup&gt;99m&lt;/sup&gt;Tc generator</td><td>Lantheus medical imaging</td><td>---</td><td>Source of &lt;sup&gt;99m&lt;/sup&gt;TcO&lt;sup&gt;4-&lt;/sup&gt;</td></tr><tr><td>0.9% Saline</td><td>Lantheus medical imaging</td><td>---</td><td>To elute generator</td></tr><tr><td>1 M HCl</td><td>Lab Chem</td><td>---</td><td>---</td></tr><tr><td>MeOH</td><td>Caledon</td><td>---</td><td>---</td></tr><tr><td>ACN</td><td>Caledon</td><td>---</td><td>HPLC grade</td></tr><tr><td>Millipore H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Thermo Fisher Scientific</td><td>Barnstead Nanopure</td><td>---</td></tr><tr><td>DCM</td><td>Caledon</td><td>---</td><td>---</td></tr><tr><td>TFA</td><td>Caledon</td><td>---</td><td>---</td></tr><tr><td>PBS</td><td>Thermo Fisher Scientific</td><td>10010023</td><td>pH 7.4 1x</td></tr><tr><td>BSA</td><td>Sigma Aldrich</td><td>A7906</td><td>---</td></tr><tr><td>Tween80</td><td>Sigma Aldrich</td><td>P8047</td><td>---</td></tr><tr><td>Isoflurane</td><td>CDMV</td><td>108737</td><td>Supplier: Fresenius Kabi Animal Health</td></tr><tr><td>HPLC</td><td>Waters</td><td>1525 Binary Pump, 2998 Photodiodde Array Detector, E-SAT/IN, Bioscan Flowcount PMT detector (item # 15590)</td><td>---</td></tr><tr><td>HPLC column for analysis and purification of compounds 2-4</td><td>Phenomenex</td><td>00G-4435-E0</td><td>Gemini&amp;reg; 5 &amp;micro;m C18 110&amp;nbsp;&amp;Aring;, LC Column 250 x 4.6 mm</td></tr><tr><td>HPLC column for analysis and purification of compounds 1 and 5</td><td>Waters&amp;nbsp;</td><td>186003115</td><td>XBridge BEH C18 Column, 130 &amp;Aring;, 5 &amp;micro;m, 4.6 mm x 100 mm</td></tr><tr><td>Microwave Reactor&amp;nbsp;</td><td>Biotage&amp;nbsp;</td><td>Initiator 8</td><td>---</td></tr><tr><td>Biotage V10 Evaporator</td><td>Biotage&amp;nbsp;</td><td>Serial # V1041</td><td>---</td></tr><tr><td>Dose calibrator</td><td>Capintec, Inc.&amp;nbsp;</td><td>CRC-25R</td><td>---</td></tr><tr><td>Gamma counter</td><td>Perkin Elmer</td><td>Wizard 1470 Automatic Gamma Counter</td><td>---</td></tr><tr><td>Animal room scale&amp;nbsp;</td><td>Mettler Toledo</td><td>XP105 Delta Range</td><td>---</td></tr><tr><td>Microwave vials&amp;nbsp;</td><td>Biotage&amp;nbsp;</td><td>355629</td><td>0.5-2 mL&amp;nbsp;</td></tr></tbody></table>

preparation and evaluation of 99mtc-labeled tridentate chelates for pre-targeting using bioorthogonal chemistry

Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry

Pre-targeting combined with bioorthogonal chemistry is emerging as an effective way to create new radiopharmaceuticals. Of the methods available, the inverse electron demand Diels-Alder (IEDDA) cycloaddition between a radiolabeled tetrazines and trans-cyclooctene (TCO) linked to a biomolecule has proven to be a highly effective bioorthogonal approach to imaging specific biological targets. Despite the fact that technetium-99m remains the most widely used isotope in diagnostic nuclear medicine, there is a scarcity of methods for preparing 99mTc-labeled tetrazines. Herein we report the preparation of a family of tridentate-chelate-tetrazine derivatives and their Tc(I) complexes. These hitherto unknown compounds were radiolabeled with 99mTc using a microwave-assisted method in 31% to 83% radiochemical yield. The products are stable in saline and PBS and react rapidly with TCO derivatives in vitro. Their in vivo pre-targeting abilities were demonstrated using a TCO-bisphosphonate (TCO-BP) derivative that localizes to regions of active bone metabolism or injury. In murine studies, the 99mTc-tetrazines showed high activity concentrations in knees and shoulder joints, which was not observed when experiments were performed in the absence of TCO-BP. The overall uptake in non-target organs and pharmacokinetics varied greatly depending on the nature of the linker and polarity of the chelate.

The ligands were synthesized using different linkers and chelators via a simple reductive amination strategy (Figure 2), followed by coupling of the product to a commercially available tetrazine22,23. Radiolabeling was performed using the same method for all compounds and was highly reproducible. The process was optimized by varying the pH, amount of ligand, reaction time and temperature whereupon the 99m</su...

Here, we describe a protocol for radiolabeling and in vivo testing of tridentate 99mTc(I) chelate-tetrazine derivatives for pre-targeting and bioorthogonal chemistry.

Pre-targeting combined with bioorthogonal chemistry is emerging as an effective way to create new radiopharmaceuticals. Of the methods available, the ...

The overall goal of this procedure is to produce technetium-99m labeled tetrazines for pre-targeted imaging using bioorthogonal chemistry with trans-cyclooctene modified biomolecules as targeting vectors. This method provides an important new platform for the development of novel radio pharmaceuticals. Through pre-targeting the technetium complexes described here can be used to image a wide array of organs, tissues, and bio-markers.This technique is advantageous as it provides a robust step-by-step method to produce tetrazines radio-labeled with technetium-99m, the most widely used medical isotope in diagnostic medicine. This technique can be used with different targeting vectors, including small molecules and antibodies. It can also be used for radio therapy by exchanging technetium with beta-emitting isotopes of rhenium.Demonstrating part of the animal procedures will be Nancy Janzen, biology technician in our lab. To begin the radio-labeling procedure, in a microwave vial, combine eight milligrams of potassium boranocabonate, 15 milligrams of sodium carbonate, 20 milligrams of sodium tetraborate decahydrate, and 25 milligrams of potassium sodium tartrate tetrahydrate. Purge the vial with argon gas for 10 minutes.Then, add four milliliters of technetium-99m pertechnetate in 0.9%saline with an activity of 1, 110 megabecquerels to the vial. Load the reaction mixture into a microwave reactor, and stir for 10 seconds. Then, heat the mixture at 110 degrees Celsius for 3.5 minutes to obtain the triaqua tricarbonyl technetium-99m cation.Add about 400 microliters of one molar hydrochloric acid to adjust the solution pH to 3.5 to four. Next, for each tetrazine ligand, dissolve two milligrams of ligand in 250 microliters of methanol. Add 250 microliters of the technetium-99m complex with an activity of 74 megabecquerels to each ligand solution.Heat mixture in a microwave reactor at 60 degrees Celsius for 20 minutes. For tetrazine ligands with t-Butyl protecting groups, transfer the solution from a microwave vial to a 20-milliliter scintillation vial, and then, evaporate the solvent and redissolve each complex in one milliliter of a one-to-one by volume solution of dichloromethane and trifluoroacetic acid. Then, transfer the solution to a microwave vial, and heat those complexes in a microwave reactor at 60 degrees Celsius for six to 10 minutes to de-protect the ligands.Cool the complexes to room temperature, and then, evaporate the solvent. Redissolve the residues in 500 microliters of a one-to-one aqueous solution of either acetonitrile or methanol and water before proceeding to HPLC purification. Use C18 reversed phase HPLC to purify each radio-labeled tetrazine complex with an elution gradient of 30 to 70 to 40 to 60 acetonitrile to water over 20 minutes with 0.1%trifluoroacetic acid present in the eluent throughout.Collect the purified product when shown eluding in the gamma trace. Then, co-inject each labeled complex with a solution of 0.125 milligrams of the corresponding radium-tetrazine complex in 20%methanol in water. Compare the retention times of the rhenium complex peak in the UV trace and the technetium complex peak in the gamma trace to verify the identity of the gamma peak.Remove the solvent from each purified technetium-tetrazine complex. Formulate each complex to a concentration of 7.4 kilobecquerels per microliter in phosphate-buffered saline with 0.5%bovine serum albumin and 0.01%polysorbate 80. For each mouse subject, draw into a syringe 100 microliters of a five microgram per microliter solution of the TCOPB derivative in saline to prepare a 20 milligram per kilogram dose.Connect a 30-gauge needle to the dosing syringe. Place the mouse subject in a physical restraint device. Identify the veins on the lateral surface of the tail approximately two centimeters from the end of the tail, and wipe the area with an alcohol swab.Insert the syringe needle at a shallow angle parallel to the vein. Slowly inject the TCOBP derivative. Remove the needle and apply a clean gauze sponge with slight pressure to the injection site until bleeding stops.One hour after injection of the TCOBP derivative, administer a 100-microliter dose of the desired technetium-99m tetrazine complex with an activity of approximately 0.74 megabecquerel by tail vein injection. Use a dose calibrator to measure the activity of a 100-microliter test sample of the technetium complex at time of injection. Save the test sample for later measurements.Anesthetize the mouse with a 3%isoflurane and 2%oxygen gas mixture six hours after injection. Place the mouse on its back with its nose in the nose cone for continued anesthesia, and locate the xiphoid process. Connect a 25-gauge needle to a syringe pre-treated with heparin.Insert the needle at a 20 degree angle under the xiphoid process, slightly to the left of the midline. Once the needle is fully inserted, slowly withdraw the plunger to check for blood in the needle hub, confirming a successful heart puncture. Readjust the needle as needed.Once the heart has been punctured, slowly draw one milliliter of blood into the syringe. Transfer the blood sample to a pre-weighed counting tube. Euthanize the mouse by cervical dislocation while under anesthesia.Place the euthanized animal in a plastic bag and measure the whole body activity with a dose calibrator. Then, collect the knee and shoulder bones, the gallbladder, the kidneys, the liver, the thyroid and trachea, and the tail. Along with their contents, collect the stomach, the small intestines, the large intestines and cecum, and the urinary bladder.Rinse the blood from all collected tissues except the gall and urinary bladders in phosphate-buffered saline. Blot dry the tissues, and place each tissue in a pre-weighed counting tube. Measure the residual activity of the carcass.Weigh each tissue sample, and calculate the mass of the tissue alone. Use a multi-detector gamma counter to measure the activity of a five-microliter aliquot of the technetium complex test sample. Using the obtained value in counts per minute and the activity measured at time of injection, calculate a conversion factor.Measure the activity of each tissue and fluid sample with the gamma detector. Convert the values to microcurie, and normalize each value by organ weight to determine the percent injected does per gram of tissue. Repeat this process with additional mice without the injection of the TCOPB derivative as a negative control series.Using this method, five tetrazine complexes differing in their linker and key-leader combinations were labeled with technetium-99m. A TCOPB derivative, which localizes to regions of high calcium turnover in bone, was used to pre-target the shoulder and knee joints of mice. The ratios of percent injected dose per gram of tissue for bone to blood were calculated for each tetrazine compound.All five compounds cleared the blood rapidly and targeted bone, indicating that the selective bioorthogonal coupling reaction between the tetrazine compounds and the TCOBP derivative had occurred in vivo. Compound three showed the best combination of targeting and clearance from blood. Negative control experiments indicated that the radio-labeled tetrazine complexes did not significantly localize to bone in the absence of the TCOBP derivative.For instance, when the TCOBP derivative was not administered, compound two had high levels in the gallbladder and intestines but very little in bone. Wide variation was observed in tissue localization in mice receiving the TCOBP derivative among the five compounds tested. For example, very little of compound three was seen in the large intestine, liver, or urine and bladder compared to other complexes, whereas much more of compound three was seen in bone.Once mastered, the labeling technique described here can be performed from start to finish in less than two hours if performed correctly. While attempting this technique, it's important to ensure all reagents are weighed out accurately and that the salts are purged with argon before addition to the technetium solution. It is also critical that the pH of the technetium solution is adjusted to 3.5 to four before addition of the tetrazine ligand.After watching this video, you should have a good understanding on how to prepare and purify technetium-labeled tetrazines and how they can be used to create new radio pharmaceuticals using pre-targeting and bioorthogonal chemistry. Don't forget to take appropriate precautions when working with radioactivity, such as understanding the type of radiation emitted and how to best shield and handle samples.

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