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&#183;10H2O, and 25 mg KOCO[CH(OH)]2COONa&#183;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 &#176;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 &#956;L 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 &#956;L MeOH21.</li>
		<li>Add 250 &#956;L 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 &#176;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 &#176;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 &#176;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 &#947;-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 &#176;C, 8 mbar, 3 min, 6,000 rpm).</li>
		<li>Formulate the purified compound at a concentration of 7.4 kBq/&#181;L 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 &#176;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 &#956;g/&#956;L), 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 &#956;Ci) of 99mTc-tetrazine formulated in 100 &#956;L 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&#39;s midline under the xiphoid process, at a 20&#176; 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 &#181;L) 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 &#181;L 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 &#181;L 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 &#181;Ci-1). 
		(1) <img alt="Equation 1" src="/files/ftp_upload/55188/55188eq1.jpg" /></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 used to improve the radiochemical yield and reduce reaction times which minimized degradation of the tetrazine21. The product was isolated and separated from unlabeled ligand and any radiochemical impurities by HPLC, resulting in radiochemical yields ranging from 31-83%, with all having &#62;99% radiochemical purity and a high specific activity of ~1.48 MBq/&#181;g. All compounds were shown to be stable in PBS containing 0.5% BSA and 0.01% Tween80 for up to 6 h (Figure 3).
Bisphosphonate compounds, like TCO-BP, localize to regions of active bone metabolism or injury, which include knee and shoulder joints in mice. TCO-BP therefore provides a simple means to assess the effectiveness of new radiolabeled tetrazines to deliver isotopes in vivo. Evaluation of the bio-distribution of all five 99mTc-tetrazines showed uptake in knee and shoulder joints 6 h post injection, demonstrating successful pre-targeting to bone in vivo (Figure 4). Previous studies confirmed that radiolabeled TCO-BP accumulates at the bone18, whereas the 99mTc-tetrazine construct (2) given alone does not (Figure 5). This allows one to conclude that bone uptake was due to the IEDDA reaction.
The more lipophilic constructs 1 and 2 had similar distribution data including high uptake in the knee (9.1 &#177; 1.9 (1); 7.6 &#177; 2.7 (2)) and the shoulder (4.6 &#177; 1.4 (1); 4.8 &#177; 1.9 (2)). High radioactivity concentrations were also seen in the gall bladder, liver and intestines, which is consistent with the distribution of the lipophilic 99mTc-tetrazine compound 2 in the absence of TCO-BP (Figure 5). Other non-target tissues and organs such as the skeletal muscle and spleen did not show any significant uptake (&#60;1%) when bio-distribution studies were performed on the 99mTc-tetrazines in the absence of the TCO-BP (Figure 5), so these organs were not taken for the pre-targeting experiments. Additionally, bio-distribution experiments with the 99mTc-tetrazines alone revealed good clearance from non-target tissues at 6 h post injection. Consequently, this time point, which is within one half-life of the isotope, was selected as the time point for comparing the different radiolabeled tetrazine ligands.
The more polar 99mTc-tetrazine compound 3 bearing a PEG5 linker showed very high knee and shoulder uptake (16.2 &#177; 4.8 and 20.7 &#177; 4.9 respectively). There was also lower activity observed in the liver and intestines. The corresponding PEG10 derivative also showed binding to the bone and reduced uptake in the liver compared to compounds 1 and 2. The most polar derivative 5, showed lower bone binding than all other constructs which is likely due to its rapid clearance.
The high bone uptake and bone:blood ratios (Table 1) particularly for compounds 3 and 4 demonstrate that pre-targeting and the IEDDA reaction can be used to localize 99mTc-labeled compounds in vivo. The methods reported here can be used to evaluate any radiolabeled tetrazine including next generation of Tc(I)-tetrazine ligands. It should be noted that for the class of ligands that were used in this study, the structures can be readily varied by changing the nature of the donor groups and linkers between the metal complex and the tetrazine, without significantly altering the ligand synthesis method21. Once a lead molecule is identified, an instant kit method, which will likely include solid phase purification methods, can be developed to support clinical translation.
The Tc(I) complexes reported here create the opportunity to prepare new 99mTc radiopharmaceuticals using a wide array of different TCO-derived targeting molecules including antibodies. Antibodies, despite their excellent targeting properties prior to the creation of technetium labeled tetrazines, would not typically be used with 99mTc because of their slow clearance (days), which is much longer than the half-life of the isotope (~6 h). An additional application of the chemistry reported here is that the same class of ligands can be prepared with the beta emitting radionuclides 186Re and 188Re. The isostructural Re(I) analogues of the Tc(I) agents when combined with the tumor seeking properties of TCO-BP can be used to treat bone metastases.

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 &#8776; 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="Figure 1" src="/files/ftp_upload/55188/55188fig1.jpg" /> 
Figure 1: The bioorthogonal IEDDA reaction between tetrazine and trans-cyclooctene. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/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 border="0" cellpadding="0" cellspacing="0" width="1378">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Argon gas&#160;</td>
			<td style="width:255px;">Alphagaz</td>
			<td style="width:199px;">---</td>
			<td style="width:559px;">---</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Na2CO3</td>
			<td style="width:255px;">EMD Millipore</td>
			<td style="width:199px;">106395</td>
			<td style="width:559px;">---</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Na2B4O7.10H2O</td>
			<td style="width:255px;">Anachemia</td>
			<td style="width:199px;">S9640</td>
			<td style="width:559px;">---</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">KNaC4H4O6.4H2O</td>
			<td style="width:255px;">Anachemia</td>
			<td style="width:199px;">217255</td>
			<td style="width:559px;">---</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Technelite 99mTc generator</td>
			<td style="width:255px;">Lantheus medical imaging</td>
			<td style="width:199px;">---</td>
			<td>Source of 99mTcO4-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">0.9% Saline</td>
			<td style="width:255px;">Lantheus medical imaging</td>
			<td style="width:199px;">---</td>
			<td>To elute generator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 M HCl</td>
			<td style="width:255px;">Lab Chem</td>
			<td style="width:199px;">---</td>
			<td style="width:559px;">---</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MeOH</td>
			<td style="width:255px;">Caledon</td>
			<td style="width:199px;">---</td>
			<td style="width:559px;">---</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ACN</td>
			<td style="width:255px;">Caledon</td>
			<td style="width:199px;">---</td>
			<td>HPLC grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Millipore H2O</td>
			<td style="width:255px;">Thermo Fisher Scientific&#160;&#160;</td>
			<td style="width:199px;">Barnstead Nanopure</td>
			<td style="width:559px;">---</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">DCM</td>
			<td style="width:255px;">Caledon</td>
			<td style="width:199px;">---</td>
			<td style="width:559px;">---</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">TFA</td>
			<td style="width:255px;">Caledon</td>
			<td style="width:199px;">---</td>
			<td style="width:559px;">---</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">PBS</td>
			<td style="width:255px;">Thermo Fisher Scientific</td>
			<td style="width:199px;">10010023</td>
			<td>pH 7.4 1X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">BSA</td>
			<td style="width:255px;">Sigma Aldrich</td>
			<td style="width:199px;">A7906</td>
			<td style="width:559px;">---</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Tween80</td>
			<td style="width:255px;">Sigma Aldrich</td>
			<td style="width:199px;">P8047</td>
			<td style="width:559px;">---</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Isoflurane&#160;</td>
			<td style="width:255px;">CDMV</td>
			<td style="width:199px;">108737</td>
			<td>Supplier: Fresenius Kabi Animal Health&#160;</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:367px;">HPLC&#160;</td>
			<td style="width:255px;">Waters&#160;</td>
			<td style="width:199px;">1525 Binary Pump, 2998 Photodiodde Array Detector, E-SAT/IN, Bioscan Flowcount PMT detector (item # 15590)</td>
			<td style="width:559px;">---</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:367px;">HPLC column for analysis and purification of compounds 2-4</td>
			<td style="width:255px;">Phenomenex</td>
			<td style="width:199px;">00G-4435-E0</td>
			<td>Gemini&#174; 5 &#181;m C18 110 &#197;, LC Column 250 x 4.6 mm,</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:367px;">HPLC column for analysis and purification of compounds 1 and 5</td>
			<td style="width:255px;">Waters&#160;</td>
			<td style="width:199px;">186003115</td>
			<td>XBridge BEH C18 Column, 130 &#197;, 5 &#181;m, 4.6 mm X 100 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Microwave Reactor&#160;</td>
			<td style="width:255px;">Biotage&#160;</td>
			<td style="width:199px;">Initiator 8</td>
			<td style="width:559px;">---</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Biotage V10 Evaporator</td>
			<td style="width:255px;">Biotage&#160;</td>
			<td style="width:199px;">Serial # V1041</td>
			<td style="width:559px;">---</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Dose calibrator</td>
			<td style="width:255px;">Capintec, Inc.&#160;</td>
			<td style="width:199px;">CRC-25R</td>
			<td style="width:559px;">---</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:367px;">Gamma counter</td>
			<td style="width:255px;">Perkin Elmer</td>
			<td style="width:199px;">Wizard 1470 Automatic Gamma Counter</td>
			<td style="width:559px;">---</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Animal room scale&#160;</td>
			<td style="width:255px;">Mettler Toledo</td>
			<td style="width:199px;">XP105 Delta Range</td>
			<td style="width:559px;">---</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Microwave vials&#160;</td>
			<td style="width:255px;">Biotage&#160;</td>
			<td style="width:199px;">355629</td>
			<td>0.5-2 mL&#160;</td>
		</tr>
	</tbody>
</table>

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 99mTc-radiolabeled compounds 1-5 were obtained in moderate to high radiochemical yield: 83% (1), 45% (2), 31% (3), 42% (4), and 54% (5). Following HPLC purification from unreacted ligand and evaporation using an evaporator, the compounds were formulated in PBS containing 0.5% BSA and 0.01% Tween80 prior to injection. The specific activity of the purified 99mTc-labeled tetrazine was ~1.48 MBq/&#181;g. Studies were conducted to assess the stability of the 99mTc-labeled tetrazine ligands prior to in vivo studies. The stability was monitored by HPLC at 1, 4 and 6 h with no visible degradation over 6 h (Rt= 14 min), as seen in Figure 3 for compound 4 as an example.
<img alt="Figure 2" src="/files/ftp_upload/55188/55188fig2.jpg" /> 
Figure 2: Compounds 1-5 were produced using different linkers (Y) and chelators (X) as shown (bottom). All compounds were radiolabeled with [99mTc(CO)3(H2O)3]+ using the same reaction conditions (top), with the exception of 1, which did not require step (ii).
<img alt="Figure 3" src="/files/ftp_upload/55188/55188fig3.jpg" /> 
Figure 3: Stability test results using compound 4. &#947;-HPLC traces of 4 incubated in PBS at 37 &#176;C for 1, 4 and 6 h.
For the in vivo testing, healthy Balb/c mice were used. Briefly, for each compound, groups of mice (n = 3) were injected with TCO-BP (100 &#956;L, 20 mg/kg), which was followed by administration of the 99mTc-labeled compounds 1 h later. At 6 h post-injection of the 99mTc complexes, the animals were sacrificed and the activity concentrations in various tissues and fluids determined. The resulting data is reported as percent injected dose per gram tissue (%ID/g) and is shown in Figure 4. Representative ratios of bone (knee or shoulder) to blood for each of the five 99mTc-labeled Tz compounds are shown on Table 1. These data indicate clearly that compound 3 provided optimal targeting combined with clearance from blood, and that there was substantial variation among the 99mTc-labeled compounds in regard to off-target tissue localization. A negative control study using CD1 mice (n = 3) was conducted, where mice were injected with 99mTc-tetrazine ligands in the absence of TCO-BP. Mice were sacrificed at 0.5, 1, 4 and 6 h and %ID/g was determined for all tissues and fluids. For all compounds tested, where data for compound 2 is presented in Figure 5, no significant uptake was seen in bone or other tissues (heart, lungs, spleen, skeletal muscle) not shown in Figure 4.
<img alt="Figure 4" src="/files/ftp_upload/55188/55188fig4.jpg" /> 
Figure 4. Bio-distribution results for 99mTc-labeled tetrazine derivatives 1-5 (bars indicated). Data shown were obtained from selected tissues and fluids taken 6 h post injection of the radiolabeled derivatives, and activity was normalized to tissue or fluid weight, as mean percent injected dose per gram of tissue or fluid (%ID/g) &#177; SEM. Bone targets are indicated by &#8226;. NOTE: All remaining tissues not shown had mean %ID/g that was less than 1%. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55188/55188fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/55188/55188fig5.jpg" /> 
Figure 5. Bio-distribution results for control study using 99mTc-labeled tetrazine (2) without prior injection of TCO-BP. Data shown were obtained from selected tissues and fluids taken from 3 mice at 0.5, 1, 4, and 6 h post injection of 2. Activity was normalized to tissue or fluid weight, as mean percent injected dose per gram of tissue or fluid (%ID/g) &#177; SEM. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55188/55188fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td colspan="5">Compound</td>
		</tr>
		<tr>
			<td>Ratio</td>
			<td>1</td>
			<td>2</td>
			<td>3</td>
			<td>4</td>
			<td>5</td>
		</tr>
		<tr>
			<td>Shoulder: Blood</td>
			<td>3.5 : 1</td>
			<td>3.5 : 1</td>
			<td>21 : 1</td>
			<td>7.8 : 1</td>
			<td>0.8 : 1</td>
		</tr>
		<tr>
			<td>Knee: Blood</td>
			<td>6.9 : 1</td>
			<td>5.6 : 1</td>
			<td>26 : 1</td>
			<td>12 : 1</td>
			<td>1.3 : 1</td>
		</tr>
	</tbody>
</table>
Table 1. Bone tissue: blood ratios determined from bio-distribution studies.

Watch this Scientific Journal Video about Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry at JoVE.com

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

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

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8.1K Views.  McMaster University. Here, we describe a protocol for radiolabeling and in vivo testing of tridentate 99mTc(I) chelate-tetrazine derivatives for pre-targeting and bioorthogonal chemistry. 

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

Preparation and Characterization of SDF-1&#945;-Chitosan-Dextran Sulfate Nanoparticles

Chitosan (CS) and dextran sulfate (DS) are charged polysaccharides (glycans), which form polyelectrolyte complex-based nanoparticles when mixed under appropriate conditions. The glycan nanoparticles are useful carriers for protein factors, which facilitate the in vivo delivery of the proteins and sustain their retention in the targeted tissue. The glycan polyelectrolyte complexes are also ideal for protein delivery, as the incorporation is carried out in aqueous solution, which reduces the likelihood of inactivation of the proteins. Proteins with a heparin-binding site adhere to dextran sulfate readily, and are, in turn, stabilized by the binding. These particles are also less inflammatory and toxic when delivered in vivo. In the protocol described below, SDF-1&#945; (Stromal cell-derived factor-1&#945;), a stem cell homing factor, is first mixed and incubated with dextran sulfate. Chitosan is added to the mixture to form polyelectrolyte complexes, followed by zinc sulfate to stabilize the complexes with zinc bridges. The resultant SDF-1&#945;-DS-CS particles are measured for size (diameter) and surface charge (zeta potential). The amount of the incorporated SDF-1&#945; is determined, followed by measurements of its in vitro release rate and its chemotactic activity in a particle-bound form.

The objective of this protocol is to incorporate SDF-1&#945;, a stem cell homing factor, into dextran sulfate-chitosan nanoparticles. The resultant particles are measured for their size and zeta potential, as well as the content, activity, and in vitro release rate of SDF-1&#945; from the nanoparticles.

Chitosan (CS) and dextran sulfate (DS) are charged polysaccharides (glycans), which form polyelectrolyte complex-based nanoparticles when mixed under ...

Preparation of Mica and Silicon Substrates for DNA Origami Analysis and Experimentation

The designed nature and controlled, one-pot synthesis of DNA origami provides exciting opportunities in many fields, particularly nanoelectronics. Many of these applications require interaction with and adhesion of DNA nanostructures to a substrate. Due to its atomically flat and easily cleaned nature, mica has been the substrate of choice for DNA origami experiments. However, the practical applications of mica are relatively limited compared to those of semiconductor substrates. For this reason, a straightforward, stable, and repeatable process for DNA origami adhesion on derivatized silicon oxide is presented here. To promote the adhesion of DNA nanostructures to silicon oxide surface, a self-assembled monolayer of 3-aminopropyltriethoxysilane (APTES) is deposited from an aqueous solution that is compatible with many photoresists. The substrate must be cleaned of all organic and metal contaminants using Radio Corporation of America (RCA) cleaning processes and the native oxide layer must be etched to ensure a flat, functionalizable surface. Cleanrooms are equipped with facilities for silicon cleaning, however many components of DNA origami buffers and solutions are often not allowed in them due to contamination concerns. This manuscript describes the set-up and protocol for in-lab, small-scale silicon cleaning for researchers who do not have access to a cleanroom or would like to incorporate processes that could cause contamination of a cleanroom CMOS clean bench. Additionally, variables for regulating coverage are discussed and how to recognize and avoid common sample preparation problems is described.

Reproducible cleaning processes for substrates used in DNA origami research are described, including bench-top RCA cleaning and derivatization of silicon oxide. Protocols for surface preparation, DNA origami deposition, drying parameters, and simple experimental set-ups are illustrated.

The designed nature and controlled, one-pot synthesis of DNA origami provides exciting opportunities in many fields, particularly nanoelectronics. Many of ...

Preparation of Biopolymer Aerogels Using Green Solvents

Although the first reports on aerogels made by Kistler1 in the 1930s dealt with aerogels from both inorganic oxides (silica and others) and biopolymers (gelatin, agar, cellulose), only recently have biomasses been recognized as an abundant source of chemically diverse macromolecules for functional aerogel materials. Biopolymer aerogels (pectin, alginate, chitosan, cellulose, etc.) exhibit both specific inheritable functions of starting biopolymers and distinctive features of aerogels (80-99% porosity and specific surface up to 800 m2/g). This synergy of properties makes biopolymer aerogels promising candidates for a wide gamut of applications such as thermal insulation, tissue engineering and regenerative medicine, drug delivery systems, functional foods, catalysts, adsorbents and sensors. This work demonstrates the use of pressurized carbon dioxide (5 MPa) for the ionic cross linking of amidated pectin into hydrogels. Initially a biopolymer/salt dispersion is prepared in water. Under pressurized CO2 conditions, the pH of the biopolymer solution is lowered to 3 which releases the crosslinking cations from the salt to bind with the biopolymer yielding hydrogels. Solvent exchange to ethanol and further supercritical CO2 drying (10 - 12 MPa) yield aerogels. Obtained aerogels are ultra-porous with low density (as low as 0.02 g/cm3), high specific surface area (350 - 500 m2/g) and pore volume (3 - 7 cm3/g for pore sizes less than 150 nm).

A new way for the production of biopolymer-based aerogels by carbon dioxide (CO2) induced gelation is shown. The technique utilizes pressurized carbon dioxide (5 MPa) for the production of biopolymer hydrogels and supercritical CO2 (12 MPa) to convert gels into aerogels. The only solvents needed besides CO2 are water and ethanol.

Although the first reports on aerogels made by Kistler1 in the 1930s dealt with aerogels from both inorganic oxides (silica and others) and biopolymers ...

The Preparation and Properties of Thermo-reversibly Cross-linked Rubber Via Diels-Alder Chemistry

A method for using Diels Alder thermo-reversible chemistry as cross-linking tool for rubber products is demonstrated. In this work, a commercial ethylene-propylene rubber, grafted with maleic anhydride, is thermo-reversibly cross-linked in two steps. The pending anhydride moieties are first modified with furfurylamine to graft furan groups to the rubber backbone. These pendant furan groups are then cross-linked with a bis-maleimide via a Diels-Alder coupling reaction. Both reactions can be performed under a broad range of experimental conditions and can easily be applied on a large scale. The material properties of the resulting Diels-Alder cross-linked rubbers are similar to a peroxide-cured ethylene/propylene/diene rubber (EPDM) reference. The cross-links break at elevated temperatures (&#62; 150 &#176;C) via the retro-Diels-Alder reaction and can be reformed by thermal annealing at lower temperatures (50-70 &#176;C). Reversibility of the system was proven with infrared spectroscopy, solubility tests and mechanical properties. Recyclability of the material was also shown in a practical way, i.e., by cutting a cross-linked sample into small parts and compression molding them into new samples displaying comparable mechanical properties, which is not possible for conventionally cross-linked rubbers.

A simple two-step approach involving rubber modification and cross-linking yields fully reworkable, elastic rubber products.

A method for using Diels Alder thermo-reversible chemistry as cross-linking tool for rubber products is demonstrated. In this work, a commercial ...

Simple Methods for the Preparation of Non-noble Metal Bulk-electrodes for Electrocatalytic Applications

The rock material pentlandite with the composition Fe4.5Ni4.5S8 was synthesized via high temperature synthesis from the elements. The structure and composition of the material was characterized via powder X-ray diffraction (PXRD), M&#246;ssbauer spectroscopy (MB), scanning electron microscopy (SEM), differential scanning calorimetry (DSC) and energy dispersive X-ray spectroscopy (EDX). Two preparation methods of pentlandite bulk electrodes are presented. In the first approach a piece of synthetic pentlandite rock is directly contacted via a wire ferrule. The second approach utilizes pentlandite pellets, pressed from finely ground powder, which is immobilized in a Teflon casing. Both electrodes, whilst being prepared by an additive-free method, reveal high durability during electrocatalytic conversions in comparison to common drop-coating methods. We herein showcase the striking performance of such electrodes to accomplish the hydrogen evolution reaction (HER) and present a standardized method to evaluate the electrocatalytic performance by electrochemical and gas chromatographic methods. Furthermore, we report stability tests via potentiostatic methods at an overpotential of 0.6 V to explore the material limitations of the electrodes during electrolysis under industrial relevant conditions.

A facile preparation method of electrodes using the bulk material Fe4.5Ni4.5S8 is presented. This method provides an alternative technique to conventional electrode fabrication and describes prerequisites for unconventional electrode materials including a straightforward electrocatalytic testing method.

The rock material pentlandite with the composition Fe4.5Ni4.5S8 was synthesized via high temperature synthesis from the elements. The structure and ...

Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and reactive moieties onto proteins directly in the live cell. Each ncAA is incorporated by a dedicated orthogonal suppressor-tRNA/amino-acyl-tRNA-synthetase (AARS) pair that is imported into the host organism. The incorporation efficiency of different ncAAs can greatly differ, and be unsatisfactory in some cases. Orthogonal pairs can be improved by manipulating either the AARS or the tRNA. However, directed evolution of tRNA or AARS using large libraries and dead/alive selection methods are not feasible in mammalian cells. Here, a facile and robust fluorescence-based assay to evaluate the efficiency of orthogonal pairs in mammalian cells is presented. The assay allows screening tens to hundreds of AARS/tRNA variants with a moderate effort and within a reasonable time. Use of this assay to generate new tRNAs that significantly improve the efficiency of the pyrrolysine orthogonal system is described, along with the application of ncAAs to the study of G-protein coupled receptors (GPCRs), which are challenging objects for ncAA mutagenesis. First, by systematically incorporating a photo-crosslinking ncAA throughout the extracellular surface of a receptor, binding sites of different ligands on the intact receptor are mapped directly in the live cell. Second, by incorporating last-generation ncAAs into a GPCR, ultrafast catalyst-free receptor labeling with a fluorescent dye is demonstrated, which exploits bioorthogonal strain-promoted inverse Diels Alder cycloaddition (SPIEDAC) on the live cell. As ncAAs can be generally applied to any protein independently on its size, the method is of general interest for a number of applications. In addition, ncAA incorporation does not require any special equipment and is easily performed in standard biochemistry labs.

A facile fluorescence assay is presented to evaluate the efficiency of amino-acyl-tRNA-synthetase/tRNA pairs incorporating non-canonical amino-acids (ncAAs) into proteins expressed in mammalian cells. The application of ncAAs to study G-protein coupled receptors (GPCRs) is described, including photo-crosslinking mapping of binding sites and bioorthogonal GPCR labeling on live cells.

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and ...

Preparation and Characterization of C60/Graphene Hybrid Nanostructures

Physical thermal deposition in a high vacuum environment is a clean and controllable method for fabricating novel molecular nanostructures on graphene. We present methods for depositing and passively manipulating C60 molecules on rippled graphene that advance the pursuit of realizing applications involving 1D C60/graphene hybrid structures. The techniques applied in this exposition are geared towards high vacuum systems with preparation areas capable of supporting molecular deposition as well as thermal annealing of the samples. We focus on C60 deposition at low pressure using a homemade Knudsen cell connected to a scanning tunneling microscopy (STM) system. The number of molecules deposited is regulated by controlling the temperature of the Knudsen cell and the deposition time. One-dimensional (1D) C60 chain structures with widths of two to three molecules can be prepared via tuning of the experimental conditions. The surface mobility of the C60 molecules increases with annealing temperature allowing them to move within the periodic potential of the rippled graphene. Using this mechanism, it is possible to control the transition of 1D C60 chain structures to a hexagonal close packed quasi-1D stripe structure.

Preparation and Characterization of C60/Graphene Hybrid Nanostructures

Here we present a protocol for the fabrication of C60/graphene hybrid nanostructures by physical thermal evaporation. Particularly, the proper manipulation of deposition and annealing conditions allow the control over the creation of 1D and quasi 1D C60 structures on rippled graphene.

Physical thermal deposition in a high vacuum environment is a clean and controllable method for fabricating novel molecular nanostructures on graphene. We ...

Preparation of Stable Bicyclic Aziridinium Ions and Their Ring-Opening for the Synthesis of Azaheterocycles

Bicyclic aziridinium ions were generated by the removal of an appropriate leaving group through internal nucleophilic attack by nitrogen atom in the aziridine ring. The utility of bicyclic aziridinium ions, specifically 1-azoniabicyclo[3.1.0]hexane and 1-azoniabicyclo[4.1.0]heptane tosylate highlighted in the aziridine ring openings by the nucleophile with the release of the ring strain to yield the corresponding ring-expanded azaheterocycles such as pyrrolidine, piperidine and azepane with diverse substituents on the ring in regio- and stereospecific manner. Herein, we report a simple and convenient method for the preparation of the stable 1-azabicyclo[4.1.0]heptane tosylate followed by selective ring opening via a nucleophilic attack either at the bridge or at the bridgehead carbon to yield piperidine and azepane rings, respectively. This synthetic strategy allowed us to prepare biologically active natural products containing piperidine and azepane motif including sedamine, allosedamine, fagomine and balanol in highly efficient manner.

Bicyclic aziridinium ions such as 1-azoniabicyclo[4.1.0]heptane tosylate were generated from 2-[4-tolenesulfonyloxybutyl]aziridine, which was utilized for the preparation of substituted piperidines and azepanes via regio- and stereospecific ring-expansion with various nucleophiles. This highly efficient protocol allowed us to prepare diverse azaheterocycles including natural products such as fagomine, febrifugine analogue and balanol.

Bicyclic aziridinium ions were generated by the removal of an appropriate leaving group through internal nucleophilic attack by nitrogen atom in the ...

Preparation of Functional Silica Using a Bioinspired Method

The goal of the protocols described herein is to synthesize bioinspired silica materials, perform enzyme encapsulation therein, and partially or totally purify the same by acid elution. By combining sodium silicate with a polyfunctional bioinspired additive, silica is rapidly formed at ambient conditions upon neutralization. 
The effect of neutralization rate and biomolecule addition point on silica yield are investigated, and biomolecule immobilization efficiency is reported for varying addition point. In contrast to other porous silica synthesis methods, it is shown that the mild conditions required for bioinspired silica synthesis are fully compatible with the encapsulation of delicate biomolecules. Additionally, mild conditions are used across all synthesis and modification steps, making bioinspired silica a promising target for the scale-up and commercialization as both a bare material and active support medium.
The synthesis is shown to be highly sensitive to conditions, i.e., the neutralization rate and final synthesis pH, however tight control over these parameters is demonstrated through the use of auto titration methods, leading to high reproducibility in reaction progression pathway and yield. 
Therefore, bioinspired silica is an excellent active material support choice, showing versatility towards many current applications, not limited to those demonstrated here, and potency in future applications.

Here, we present a protocol to synthesize bioinspired silica materials and immobilize enzymes therein. Silica is synthesized by combining sodium silicate and an amine &#39;additive&#39;, which neutralize at a controlled rate. Material properties and function can be altered either by in situ enzyme immobilization or post-synthetic acid elution of encapsulated additives.

The goal of the protocols described herein is to synthesize bioinspired silica materials, perform enzyme encapsulation therein, and partially or totally ...

Genetic Encoding of a Non-Canonical Amino Acid for the Generation of Antibody-Drug Conjugates Through a Fast Bioorthogonal Reaction

Antibody-drug conjugates (ADCs) used nowadays in clinical practice are mixtures of antibody molecules linked to a varying number of toxins at different positions. Preclinical studies have shown that the therapeutic index of these traditional ADCs can be improved by the site-specific linkage of toxins. However, current approaches to produce homogeneous ADCs have several limitations, such as low protein expression and slow reaction kinetics. In this protocol we describe how to set up an expression system to incorporate a cyclopropene derivative of lysine (CypK) into antibodies using genetic code expansion. This minimal bioorthogonal handle allows rapid conjugation of tetrazine derivatives through an inverse-demand Diels-Alder cycloaddition. The expression system here reported enables the facile production and purification of trastuzumab bearing CypK in each of the heavy chains. We explain how to link the antibody to the toxin monomethyl auristatin E and characterize the immunoconjugate by hydrophobic interaction chromatography and mass spectrometry. Finally, we describe assays to assess the stability in human serum of the dihydropyridazine linkage resulting from the conjugation and to test the selective cytotoxicity of the ADC for breast cancer cells with high levels of HER2 receptor.

Incorporating a cyclopropene derivative of lysine into antibodies allows the site-specific, rapid and efficient linkage of tetrazine-bearing molecules to generate antibody-drug conjugates.

Antibody-drug conjugates (ADCs) used nowadays in clinical practice are mixtures of antibody molecules linked to a varying number of toxins at different ...