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 specifical...

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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 &#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="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 border="0" cellpadding="0" cellspacing="0" width="1378">
	<tbody>
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			<td height="21" style="height:21px;width:367px;">Argon gas&#160;</td>
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			<td style="width:199px;">---</td>
			<td style="width:559px;">---</td>
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			<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>
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			<td style="width:199px;">217255</td>
			<td style="width:559px;">---</td>
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			<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>
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			<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>
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			<td style="width:559px;">---</td>
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			<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>
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			<td height="21" style="height:21px;width:367px;">Millipore H2O</td>
			<td style="width:255px;">Thermo Fisher Scientific&#160;&#160;</td>
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			<td style="width:559px;">---</td>
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		<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>
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			<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>
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			<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>
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			<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>
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			<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>
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			<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>
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			<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>
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			<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>
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			<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>
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			<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>
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			<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>
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			<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>
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			<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 99m</su...

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 ...

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.

preparation-evaluation-99mtc-labeled-tridentate-chelates-for-pre

Research

JoVE Journal

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.

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

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 ...

Synthesis and Evaluation of a Ruthenium-based Mitochondrial Calcium Uptake Inhibitor

We detail the synthesis and purification of a mitochondrial calcium uptake inhibitor, [(OH2)(NH3)4Ru(&#181;-O)Ru(NH3)4(OH2)]5+. The optimized synthesis of this compound commences from [Ru(NH3)5Cl]Cl2 in 1 M NH4OH in a closed container, yielding a green solution. Purification is accomplished with cation-exchange chromatography. This compound is characterized and verified to be pure by UV-vis and IR spectroscopy. The mitochondrial calcium uptake inhibitory properties are assessed in permeabilized HeLa cells by fluorescence spectroscopy.

A protocol for the synthesis, purification, and characterization of a ruthenium-based inhibitor of mitochondrial calcium uptake is presented. A procedure to evaluate its efficacy in permeabilized mammalian cells is demonstrated.

We detail the synthesis and purification of a mitochondrial calcium uptake inhibitor, [(OH2)(NH3)4Ru(&#181;-O)Ru(NH3)4(OH2)]5+. The optimized synthesis of ...

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 ...