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.

In Tierstudien wurden von der Tierforschung Ethikrat an der McMaster University in Übereinstimmung mit Canadian Council on Animal Care (CCAC) Richtlinien zugelassen. 1. Die radioaktive Markierung von Tz-dreizähnigen Liganden mit 99m Tc ACHTUNG: Die folgenden Verfahren erfordern die Verwendung von radioaktiven Verbindungen. Die Arbeiten sollten nur in einem zugelassenen Labor mit der Einhaltung von Sicherheits- und Entsorgungsvorschriften erfolgen. Mikrowellen-Reaktionen sollten in einer Mikrowelle durchgeführt werden, die speziell für die chemische Synthese konstruiert. <ol><li> Synthese von [99m Tc (CO) 3 (H 2 O) 3] + 19, 20 <ol><li> In einem Mikrowellenfläschchen kombinieren 8 mg K 2 [BH 3 CO 2], 15 mg Na 2 CO 3, 20 mg Na 2 B 4 O 7 · 10H 2 O und 25 mg KOCO [CH (OH)]2 COONa · 4H 2 O Purge das Fläschchen für 10 Minuten mit Argongas. </li><li> 4 ml 99m TcO 4 - (~ 1,100 MBq, ~ 30 mCi) in 0,9% Kochsalzlösung in das Fläschchen. </li><li> Die Reaktion wird in einer Mikrowelle für 3,5 min bei 110 ° C nach 10 s Rühren eine gute Durchmischung der Reagenzien zu gewährleisten. </li><li> Der pH-Wert der Lösung auf 3,5-4 mittels ~ 400 &amp; mgr; l von 1 M HCl. Stellen Sie sicher, pH-Papier verwenden. </li></ol></li><li> Die radioaktive Markierung von Tetrazin - Liganden 1-5 <ol><li> Man löst 2 mg jedes Liganden (Verbindungen 1-5) in 250 &amp; mgr; l MeOH 21. </li><li> Nach Eintragen von 250 &amp; mgr; l [99m Tc (CO) 3 (H 2 O) 3] + (~ 74 MBq, ~ 2 mCi) zu jeder Lösung. </li><li> Das Reaktionsgemisch wird eine Mikrowelle für 20 min bei 60 ° C verwendet wird. Hinweis: dieser Schritt für alle 5 Tetrazinen identisch war. </li><li> Für Verbindungen 2- 5, verdampfe das Lösungsmittel und Wieder DISSOLve die resultierenden Produkte in 1 ml 1: 1 v / v DCM: TFA. </li><li> Wärme , die gelösten Reaktionsprodukte (2-5) bei 60 ° C in einer Mikrowelle für 6 min (2-4) oder 10 min (5). </li><li> Nach dem Abkühlen auf Raumtemperatur, dampfe das Lösungsmittel unter Verwendung eines Verdampfers (36 ° C, 8 mbar, 3 min, 6000 rpm) und lösen die getrocknete Verbindung in 1: 1 ACN: H 2 O oder 1: 1 MeOH: H 2 O, vor der HPLC-Reinigung. </li><li> Reinige den 99m Tc-markierten Verbindungen (1-5), einschließlich des markierten Produkts von unmarkiertem Liganden Tetrazin Trennung unter Verwendung von HPLC (C18 Umkehrphase). Typischerweise verwenden eines Elutionsgradienten von 30:70 ACN: H 2 O (beide mit 0,1% TFA) bis 40:60 ACN: H 2 O über 20 min (18 min) und ein C 18 analytisches 4,6 x 100 mm Säule. Verwenden sowohl UV (254 nm) und gamma-Detektion. <ol><li> Nehmen Sie eine kleine Probe jedes Produkts und vergleichen ihre HPLC-Retentionszeit, die der Co-injektiert, nicht-radioaktive, Re-markierten Standards (0,125 mg in 20% Methanol-H 2 O). Die Re-markiertem Standard wird in der UV - HPLC - Spur identifiziert, und zur gleichen Zeit wie die 99m Tc-markierte Verbindung , in der γ-HPLC - Spur eluieren. Diese Co-Injektion zeigt Peaks bei vergleichbaren Retentionszeiten, die Identität der 99m Tc-Verbindung bestätigt gekennzeichnet. </li></ol></li><li> Dampfe das Lösungsmittel von HPLC-Fraktionen unter Verwendung eines Verdampfers (36 ° C, 8 mbar, 3 min, 6.000 rpm). </li><li> Formulieren der gereinigten Verbindung in einer Konzentration von 7,4 kBq / &amp; mgr; l in PBS, enthaltend 0,5% BSA und 0,01% Tween-80. </li><li> Um die markierten Verbindungen sind stabil zu gewährleisten, führen Sie eine in - vitro - Stabilitätsstudie. Inkubieren der formulierten Verbindung bei 37 ° C für 1, 4 und 6 h, eine kleine Menge (3,7 MBq) der Mischung auf dem HPLC an jedem Zeitpunkt Injizieren Stabilität zu bewerten. </li></ol></li></ol> 2. Pre-gezielte Bio-Verteilung Studies <ol><li&gt; Vorbereitung der Tiere <ol><li> Mit 7-9 Wochen alt, weiblich Balb / c-Mäuse (n = 3), zu verwalten TCO-BP in Kochsalzlösung (20 mg / kg) (5 ug / ul) formuliert, über Schwanzveneninjektion. </li><li> Bewegen Sie die Maus in der physischen Rückhaltevorrichtung, und identifizieren die sich Venen an den Seitenflächen des Schwanzes und wischen Sie mit einem Alkoholtupfer. Bei etwa 2 cm vom Ende des Schwanzes, legen Sie eine 30-Gauge-Nadel in einem flachen Winkel parallel zur Ader. Langsam drücken Sie den Kolben Nadel zu injizieren, entfernen und reinigen Gazeschwamm an der Injektionsstelle mit leichtem Druck, bis die Blutung aufhört gelten. </li><li> Bei 1 h nach der Injektion von TCO-BP, verabreichen ~ 0,74 MBq (20 uCi) von 99m Tc-Tetrazin in 100 ul 0,5% BSA formuliert, 0,01% Tween-80 in PBS, via Schwanzveneninjektion. </li></ol></li><li> Bio-Verteilungsstudien <ol><li> Bei dem gewünschten Zeitpunkt (t = 6 h), anästhesieren die Mäuse unter Verwendung von 3% Isofluran und 2% Sauerstoff-Gasgemisch. Demonstrieren Sie ein zue Prise Rückzug auf der narkotisierten Maus, um sicherzustellen, dass sie unter chirurgische Ebene der Anästhesie sind. </li><li> Sammeln Blut (1 ml) über eine Spritze Herzpunktur vorbehandelt mit Heparin verwendet wird. Bewegen Sie die Maus auf dem Rücken mit der Nase in die Nase Kegel für die weitere Anästhesie und suchen Sie den Xiphoidbasis auf das Tier. <ol><li> Legen Sie eine 25 G-Nadel, leicht nach links von der Mittellinie des Tieres unter dem Xiphoidbasis, bei einem Winkel von 20 °. Voll führen Sie die Nadel, und langsam auf den Kolben zurückziehen Blut in der Nadel-Ansatz, um zu sehen, ob das Herz durchbohrt wurde. Etwas die Nadel nachstellen, während der Kolben bei Bedarf halten, um das Herz zu durchbohren. ziehen langsam Blut in die Spritze. </li></ol></li><li> Euthanize das Tier durch Genickbruch, während der Narkose. </li><li> Legen Sie jedes Tier in einen Plastikbeutel und verwenden Sie einen Dosiskalibrators (99m Tc - Einstellung) , um den ganzen Körper Aktivitätsniveau zu messen. </li><li> Sammeln Sie die folgenden Gewebe und Flüssigkeiten in vorge wiegened Zählrohre: Blut, Knochen (Knie und Schulter), Gallenblase, Nieren, Leber, Magen (mit Inhalt), Dünndarm (mit Inhalt), Dickdarm und Blinddarm (mit Inhalt), der Schilddrüse und der Trachea, der Harnblase mit Urin und Schwanz. </li><li> Spülen Sie geeignete Gewebe (ohne Blut, Gallenblase und Harnblase) in PBS Blut zu entfernen und trocken tupfen, bevor das Gewebe in geeigneten Zählrohre platzieren. </li><li> Platzieren Sie Tierkadaver in eine Plastiktüte und messen Rest ganzen Körper Aktivität eine Dosis-Kalibrator verwenden. </li><li> Wiegen Sie jedes Röhrchen eine Gewebeprobe enthält. Subtract Anfangsgewicht des Rohrs Masse des Gewebes zu erhalten. </li><li> Verwenden , um eine Dosis - Kalibrator (99m Tc - Einstellung) die Menge an Aktivität in einer Testprobe (100 ul) zu der Zeit der Einspritzung für jede Maus gemessen. ANMERKUNG: Diese Testprobe ist gleich dem Injektionsvolumen, wodurch die Aktivitätszählung zum Zeitpunkt der Injektion geben. </li><li> Zum Zeitpunkt der Gewebemessung eineliquot 5 ul des zuvor verwendeten Testprobe. Verwenden , um einen Multidetektor - Gammazähler (99m Tc - Einstellung) und zähle die Zählung pro Minute (CPM) für die 5 &amp; mgr; l Testprobe zu erhalten. </li><li> Verwenden Sie die beiden erhaltenen Werte in 2.2.9 und 2.2.10 die Aktivität und CPM - Beziehung 1 unter Verwendung der Gleichung zu berechnen einen Umrechnungsfaktor zu erhalten (CPM uCi -1). (1) <img alt="Gleichung 1" src="/files/ftp_upload/55188/55188eq1.jpg" /></li><li> Verwenden, um die Gamma-Zähler die Menge an Radioaktivität in jedem Gewebe oder Flüssigkeitsprobe zu messen. </li><li> Verwenden der Gleichung 1 die Aktivitätsmenge in jedem Gewebe oder Flüssigkeit, die zum Zeitpunkt der Messung in Bezug auf die gesamten injizierten Dosis berechnet. Dieser Wert wird dann durch Organgewicht normalisiert und als Prozent injizierte Dosis pro Gramm (dh% ID / g) Gewebe berichtet. </li><li> Führen Sie die Schritte 2.1.2 bis 2.2.13 ein negatives Kontrollexperiment durchzuführen unter Verwendung der 99m Tc-markierten Tetrazin - Liganden in der Absence von TCO-BP. Opfer-Mäuse (n = 3) bei 0,5, 1, 4 und 6 h nach der Injektion und zu erhalten Gewebe oder Flüssigkeit, wie oben beschrieben. </li></ol></li></ol>

<ol>
	<li>Jurisson, S. S., Lydon, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+Technetium+Small+Molecule+Radiopharmaceuticals.">Potential Technetium Small Molecule Radiopharmaceuticals.</a> Chem. Rev. 99 (9), 2205-2218 (1999).</li><li>Kluba, C. A., Mindt, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Click-to-chelate:+Development+of+Technetium+and+Rhenium-Tricarbonyl+Labeled+Radiopharmaceuticals.">Click-to-chelate: Development of Technetium and Rhenium-Tricarbonyl Labeled Radiopharmaceuticals.</a> Molecules. 18, 3206-3226 (2013).</li><li>Amato, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+Medicines+Conundrum.">Nuclear Medicines Conundrum.</a> Chem. Eng. News. 87 (36), 58-70 (2009).</li><li>Hnatowich, D. J., Virzi, F., Rusckowski, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigations+of+Avidin+and+Biotin+for+Imaging+Applications.">Investigations of Avidin and Biotin for Imaging Applications.</a> J. Nucl. Med. 28 (8), 1294-1302 (1987).</li><li>Blackman, M. L., Royzen, M., Fox, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetrazine+Ligation:+Fast+Bioconjugation+Based+on+Inverse-Electron-Demand+Diels-Alder+Reactivity.">Tetrazine Ligation: Fast Bioconjugation Based on Inverse-Electron-Demand Diels-Alder Reactivity.</a> J. Am. Chem. Soc. 130 (41), 13518-13519 (2008).</li><li>Devaraj, N. K., Weissleder, R., Hilderbrand, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetrazine-Based+Cycloadditions:+Application+to+Pretargeted+Live+Cell+Imaging.">Tetrazine-Based Cycloadditions: Application to Pretargeted Live Cell Imaging.</a> Bioconjugate Chem. 19 (12), 2297-2299 (2008).</li><li>Rossin, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vivo+Chemistry+for+Pretargeted+Tumor+Imaging+in+Live+Mice.">In Vivo Chemistry for Pretargeted Tumor Imaging in Live Mice.</a> Angew. Chem., Int. Ed. 49 (19), 3375-3378 (2010).</li><li>Zeglis, B. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+a+Pretargeted+Strategy+for+the+PET+Imaging+of+Colorectal+Carcinoma+via+the+Modulation+of+Radioligand+Pharmacokinetics.">Optimization of a Pretargeted Strategy for the PET Imaging of Colorectal Carcinoma via the Modulation of Radioligand Pharmacokinetics.</a> Mol. Pharmaceutics. 12 (10), 3575-3587 (2015).</li><li>Rossin, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+Reactive+trans-Cyclooctene+Tags+with+Improved+Stability+for+Diels-Alder+Chemistry+in+Living+Systems.">Highly Reactive trans-Cyclooctene Tags with Improved Stability for Diels-Alder Chemistry in Living Systems.</a> Bioconjugate Chem. 24 (7), 1210-1217 (2013).</li><li>Rossin, R., Robillard, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pretargeted+Imaging+Using+Bioorthogonal+Chemistry+in+Mice.">Pretargeted Imaging Using Bioorthogonal Chemistry in Mice.</a> Curr. Opin. Chem. Biol. 21, 161-169 (2014).</li><li>Denk, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+18F-Labeled+Tetrazine+with+Favorable+Pharmacokinetics+for+Bioorthogonal+PET+Imaging.">Development of a 18F-Labeled Tetrazine with Favorable Pharmacokinetics for Bioorthogonal PET Imaging.</a> Angew. Chem., Int. Ed. 53 (36), 9655-9659 (2014).</li><li>Herth, M. M., Andersen, V. L., Lehel, S., Madsen, J., Knudsen, G. M., Kristensen, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+11C-labeled+Tetrazine+for+Rapid+Tetrazine-Trans-Cyclooctene+Ligation.">Development of a 11C-labeled Tetrazine for Rapid Tetrazine-Trans-Cyclooctene Ligation.</a> Chem. Commun. 49 (36), 3805-3807 (2013).</li><li>Li, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetrazine-Trans-Cyclooctene+Ligation+for+the+Rapid+Construction+of+18F+Labeled+Probes.">Tetrazine-Trans-Cyclooctene Ligation for the Rapid Construction of 18F Labeled Probes.</a> Chem. Commun. 46 (42), 8043 (2010).</li><li>Nichols, B., Qin, Z., Yang, J., Vera, D. R., Devaraj, N. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=68Ga+Chelating+Bioorthogonal+Tetrazine+Polymers+for+the+Multistep+Labeling+of+Cancer+Biomarkers.">68Ga Chelating Bioorthogonal Tetrazine Polymers for the Multistep Labeling of Cancer Biomarkers.</a> Chem. Commun. 50 (40), 5215-5217 (2014).</li><li>Zeglis, B. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Pretargeted+PET+Imaging+Strategy+Based+on+Bioorthogonal+Diels-Alder+Click+Chemistry.">A Pretargeted PET Imaging Strategy Based on Bioorthogonal Diels-Alder Click Chemistry.</a> J. Nucl. Med. 54 (8), 1389-1396 (2013).</li><li>Garc&#237;a, M. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=99mTc-Bioorthogonal+Click+Chemistry+Reagent+for+In+Vivo+Pretargeted+Imaging.">99mTc-Bioorthogonal Click Chemistry Reagent for In Vivo Pretargeted Imaging.</a> Bioorg. Med. Chem. 24 (6), 1209-1215 (2016).</li><li>Russell, R. G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bisphosphonates:+The+First+40+Years.">Bisphosphonates: The First 40 Years.</a> Bone. 49 (1), 2-19 (2011).</li><li>Yazdani, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Bone-Seeking+Trans-Cyclooctene+for+Pretargeting+and+Bioorthogonal+Chemistry:+A+Proof+of+Concept+Study+Using+99mTc+and+177Lu-Labeled+Tetrazines.">A Bone-Seeking Trans-Cyclooctene for Pretargeting and Bioorthogonal Chemistry: A Proof of Concept Study Using 99mTc and 177Lu-Labeled Tetrazines.</a> J. Med. Chem. , (2016).</li><li>Alberto, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Organometallic+Aqua+Complex+of+Technetium+for+the+Labeling+of+Biomolecules:+Synthesis+of+[99mTc(OH2)3(CO)3]++from+[99mTcO4]-+in+Aqueous+Solution+and+its+Reaction+with+a+Bifunctional+Ligand.">A Novel Organometallic Aqua Complex of Technetium for the Labeling of Biomolecules: Synthesis of [99mTc(OH2)3(CO)3]+ from [99mTcO4]- in Aqueous Solution and its Reaction with a Bifunctional Ligand.</a> J. Am. Chem. Soc. 120 (31), 7987-7988 (1998).</li><li>Alberto, R., Ortner, K., Wheatley, N., Schibli, R., Schubiger, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+properties+of+boranocarbonate:+A+convenient+in+situ+CO+source+for+the+aqueous+preparation+of+[99mTc(OH2)3(CO)3.">Synthesis and properties of boranocarbonate: A convenient in situ CO source for the aqueous preparation of [99mTc(OH2)3(CO)3.</a> J. Am. Chem. Soc. 123 (13), 3135-3136 (2001).</li><li>Lu, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+SAR+of+99mTc/Re-labeled+Small+Molecule+Prostate+Specific+Membrane+Antigen+Inhibitors+with+Novel+Polar+Chelates.">Synthesis and SAR of 99mTc/Re-labeled Small Molecule Prostate Specific Membrane Antigen Inhibitors with Novel Polar Chelates.</a> Bioorg. Med. Chem. Lett. 23 (5), 1557-1563 (2013).</li><li>Maresca, K. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+Molecule+Inhibitors+of+PSMA+Incorporating+Technetium-99m+for+Imaging+Prostate+Cancer:+Effects+of+Chelate+Design+on+Pharmacokinetics.">Small Molecule Inhibitors of PSMA Incorporating Technetium-99m for Imaging Prostate Cancer: Effects of Chelate Design on Pharmacokinetics.</a> Inorg. Chim. Acta. 389, 168-175 (2012).</li><li>Bartholom&#228;, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insight+into+the+Mode+of+Action+of+Re(CO)3+Thymidine+Complexes.">Insight into the Mode of Action of Re(CO)3 Thymidine Complexes.</a> ChemMedChem. 5 (9), 1513-1529 (2010).</li></ol>

The authors declare they have no competing financial interests.

Eine Sammlung von Tetrazin-linked dreizähnigen Chelate verschiedener Polaritäten wurde hergestellt, und die Nützlichkeit der 99m Tc - Komplexe in der IEDDA Reaktion mit einer TCO - Derivat in vivo bewertet. Eine effektive und reproduzierbare 99m Tc wurde Markierungsverfahren für fünf Tetrazin-Chelate entwickelt, wobei der Ligand - Konzentration 10 -3 M. war der Markierungsschritt durch Entfernung der Schutzgruppe von t- Butyl - Gruppen (für die Verbindungen <stron...

99m Tc bleibt die dominierende Radioisotop in der diagnostischen Nuklearmedizin verwendet wird , mit mehr als 50 Millionen Bildgebungsverfahren pro Jahr weltweit durchgeführt 1, 2, 3. Die Mehrheit der 99m Tc Mittel werden klinisch Perfusion Radiopharmaka. Es gibt eine begrenzte Anzahl von aktiv gezielte Verbindungen , in denen 99m Tc gerichtet ist eine spezifische Biomarker durch Ligation an ein Targeting - Konstrukt zu binden. Die Erzeugung gezielter 99m Tc - Radiopharmaka wird häufig durch den Einfluß von 99m Tc-Ligand - Komplexe auf die Fähigkeit des dirigierenden Moleküls zu binden , um die Biomarker von Interesse gehinderte oder die Isotopen - Halbwertszeit nicht lang genug ist für die Verwendung mit höherem Molekulargewicht Biomoleküle wie Antikörper. Letzteres erfordert üblicherweise mehrere Tage vor Bildern, um für das Biomolekül erworben werden von Nicht-Ziel zu löschen tiss<html> ues. Pre-Targeting bietet einen alternativen Ansatz, diese Herausforderungen zu überwinden. Pre-Targeting mit bioorthogonale Chemie kombiniert wurde ein effektiver Weg gezeigt werden , für die Entwicklung neuer molekularer Bildgebungssonden sowohl für Fluoreszenz und Radio-Imaging - 4, 5, 6, 7, 8. Die inversem Elektronenbedarf Diels-Alder (IEDDA) Reaktion zwischen 1,2,4,5-Tetrazin (Tz) und trans -Cycloocten (TCO) -Derivate, wie in Figur 1 gezeigt ist , hat sich als besonders wirksam 6 erwiesen. Die IEDDA Reaktion mit diesen Komponenten können schnell Kinetik in PBS (k 2 ≈ 6000 M -1 s -1) und eine hohe Selektivität aufweisen, wodurch es ideal für in - vivo - Pre-Targeting - Anwendungen macht 9, 10. e_content &quot;&gt; Die am häufigsten verwendete Ansatz umfasst ein TCO-derived Targeting - Vektor Verwaltung und eine ausreichende Verzögerung Folgezeit ein radioaktiv markiertes Tetrazin verabreicht. Radiolabeled Tetrazinen basierend auf 11 C, 18 F, 64 Cu, 89 Zr und 111 In gewesen 11 berichtete, 12, 13, 14, 15. im Gegensatz dazu gibt es nur einen Bericht eines 99m ist Tz-Tc markiert, die eine HYNIC - Typ - Liganden wurde unter Verwendung von der Verwendung von Co-Liganden erfordern Protein zu verhindern Bindung und den Abbau in vivo 16. Alternativ berichten wir hier die Synthese von 99m Tc (I) markiertem Tetrazinen eine Familie von Liganden , die mit einem [99m Tc (CO) 3] + Kern stabil dreizähnige Komplexe bilden. Abbildung 1: Die bioorthogonale IEDDA Reaktion zwischen Tetrazin und trans -Cycloocten. <a href="http://ecsource.jove.com/files/ftp_upload/55188/55188fig1large.jpg" target="_blank">Bitte klicken Sie hier</a> , um <a href="http://ecsource.jove.com/files/ftp_upload/55188/55188fig1large.jpg" target="_blank">eine größere Version dieser Figur zu sehen.</a> Die Familie von Liganden hergestellt enthalten dreizähnigen Chelate , die in der Polarität und der Natur der Linkergruppe zwischen der Metallbindungsbereich und dem Tz (Abbildung 2) variieren. Das Ziel war es, ein 99m zu identifizieren Tc-Tetrazin , dass effektiv lokalisieren und reagieren mit TCO-markierten Stellen in vivo konstruieren konnte und schnell klar , wenn sie nicht gebunden, um eine hohe Ziel-zu-Nichtziel - Verhältnisse zu erhalten. Um die Liganden zu testen, ein TCO-Derivat einer Bisphosphonat (TCO-BP) wurde 17 verwendet. Wir haben bereits gezeigt, dass TCO-BP zu den Bereichen der aktiven Knochenmetabolismus lokalisiert und reagieren mitradiomarkierten Tetrazinen in vivo 18. Es ist eine bequeme Reagenz neue Tetrazinen zu testen, da es in einem einzigen Schritt hergestellt werden können, und Experimente können in normalen Mäusen durchgeführt werden, in denen die Lokalisierung in erster Linie in den Gelenken (Knie und Schultern) auftritt.

<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>
			<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.

Die Liganden wurden unter Verwendung von verschiedenen Linkern synthetisiert und Chelatoren über eine einfache reduktive Aminierung Strategie (Abbildung 2), gefolgt von Kupplung des Produktes mit einem handelsüblichen Tetrazin 22, 23. Die radioaktive Markierung wurde mit der gleichen Methode für alle Verbindungen durchgeführt und war in hohem Maße reproduzierbar. Das Verfahren wurde durch Variieren des pH, d...

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.

Herstellung und Auswertung der<sup&gt; 99m</sup&gt; Tc-markierte Dreizähnige Chelate für Pre-Targeting Bioorthogonale Chemie

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

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Chemistry

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

8.1K Aufrufe.  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. 

Serie: 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.

Herstellung und Charakterisierung von SDF-1α-Chitosan-Nanopartikel Dextransulfat

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

Forschung

Chemie

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.

Herstellung von Mica und Siliziumsubstraten für die DNA-Origami-Analyse und Experimentieren

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.

Herstellung von Biopolymer Aerogele unter Verwendung der grünen Lösungsmittel

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.

Die Herstellung und Eigenschaften von Thermo reversibel vernetzten Kautschuk Via Diels-Alder-Chemie

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.

Einfache Methoden zur Herstellung von nicht edlen Metall-Bulk-Elektroden für elektrokatalytische Anwendungen

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.

Optimierung der genetischen Einbeziehung der chemischen Sonden in GPCRs für Foto-Vernetzung-Mapping und Bioorthogonal Chemie in Live Säugerzellen

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.

Herstellung und Charakterisierung von 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 ...

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.

Vorbereitung der stabile bicyclische Aziridinium Ionen und ihre Ring-Öffnung für die Synthese von Azaheterocycles

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.

Vorbereitung der funktionalen Kieselsäure mit einer Muskelmodelle Methode

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.

Durch eine schnelle Bioorthogonal Reaktion Konjugate genetischen Codierung einer nicht-kanonische Aminosäure für die Erzeugung von Antikörper-Wirkstoff

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

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