Name: synthesis and bioconjugation of thiol-reactive reagents for the creation of site-selectively modified immunoconjugates
Uploaded: 2019-03-06T14:30:00
Description: Watch this Scientific Journal Video about Synthesis and Bioconjugation of Thiol-Reactive Reagents for the Creation of Site-Selectively Modified Immunoconjugates at JoVE.com

作者感谢赛柯兰夏尔马博士的有益对话。

1. 4-[5-(甲基硫磷)-1, 3, 4-恶二唑-2-基]-苯胺的合成 (1)
注: 由于化合物的感光度, 请将所有反应保存在被箔覆盖的容器中。
<ol><li>在10毫升圆形底瓶中, 溶解100毫克 (0.517 mmol, 1 当量) 5-(4-氨基苯基)-1, 3, 4-二亚唑-2-硫醇在3毫升甲醇。</li>
	<li>在该溶液中, 加入360μl 的二异丙基乙胺 (dipea; 2.07 mmol; 4 当量; 无水) 和一个小磁搅拌杆。用橡胶塞子盖上烧瓶, 在室温下搅拌溶液10分钟。</li>
	<l...

<ol>
	<li>Wu, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibodies+and+antimatter:+The+resurgence+of+immuno-PET.">Antibodies and antimatter: The resurgence of immuno-PET.</a> Journal of Nuclear Medicine. 50 (1), 2-5 (2009).</li><li>Zeglis, B. M., Lewis, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+guide+to+the+construction+of+radiometallated+bioconjugates+for+positron+emission+tomography.">A practical guide to the construction of radiometallated bioconjugates for positron emission tomography.</a> Dalton Transactions. 40 (23), 6168-6195 (2011).</li><li>Agarwal, P., Bertozzi, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-specific+antibody-drug+conjugates:+the+nexus+of+bioorthogonal+chemistry,+protein+engineering,+and+drug+development.">Site-specific antibody-drug conjugates: the nexus of bioorthogonal chemistry, protein engineering, and drug development.</a> Bioconjugate Chemistry. 26 (2), 176-192 (2015).</li><li>Adumeau, P., Sharma, S. K., Brent, C., Zeglis, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-specifically+labeled+immunoconjugates+for+molecular+imaging-part+1:+Cysteine+residues+and+glycans.">Site-specifically labeled immunoconjugates for molecular imaging-part 1: Cysteine residues and glycans.</a> Molecular Imaging and Biology. 18 (1), 1-17 (2016).</li><li>Adumeau, P., Sharma, S. K., Brent, C., Zeglis, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-specifically+labeled+immunoconjugates+for+molecular+imaging-part+2:+Peptide+tags+and+unnatural+amino+acids.">Site-specifically labeled immunoconjugates for molecular imaging-part 2: Peptide tags and unnatural amino acids.</a> Molecular Imaging and Biology. 18 (1), 153-165 (2016).</li><li>Alley, S. 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=Contribution+of+linker+stability+to+the+activities+of+anticancer+immunoconjugates.">Contribution of linker stability to the activities of anticancer immunoconjugates.</a> Bioconjugate Chemistry. 19 (3), 759-765 (2008).</li><li>Baldwin, A. D., Kiick, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tunable+degradation+of+maleimide-thiol+adducts+in+reducing+environments.">Tunable degradation of maleimide-thiol adducts in reducing environments.</a> Bioconjugate Chemistry. 22 (10), 1946-1953 (2011).</li><li>Shen, B. -. Q., 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=Conjugation+site+modulates+the+in+vivo+stability+and+therapeutic+activity+of+antibody-drug+conjugates.">Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugates.</a> Nature Biotechnology. 30 (2), 184-189 (2012).</li><li>Jackson, 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=In+vitro+and+in+vivo+evaluation+of+cysteine+and+site+specific+conjugated+herceptin+antibody-drug+conjugates.">In vitro and in vivo evaluation of cysteine and site specific conjugated herceptin antibody-drug conjugates.</a> Plos One. 9 (1), (2014).</li><li>Ponte, J. 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=Understanding+how+the+stability+of+the+thiol-maleimide+linkage+impacts+the+pharmacokinetics+of+lysine-linked+antibody-maytansinoid+conjugates.">Understanding how the stability of the thiol-maleimide linkage impacts the pharmacokinetics of lysine-linked antibody-maytansinoid conjugates.</a> Bioconjugate Chemistry. 27 (7), 1588-1598 (2016).</li><li>Stimmel, J. B., 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=Site-specific+conjugation+on+serine+-&gt;+cysteine+variant+monoclonal+antibodies.">Site-specific conjugation on serine -&gt; cysteine variant monoclonal antibodies.</a> Journal of Biological Chemistry. 275 (39), 30445-30450 (2000).</li><li>Li, L., 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=Reduction+of+kidney+uptake+in+radiometal+labeled+peptide+linkers+conjugated+to+recombinant+antibody+fragments.+site-specific+conjugation+of+DOTA-peptides+to+a+cys-diabody.">Reduction of kidney uptake in radiometal labeled peptide linkers conjugated to recombinant antibody fragments. site-specific conjugation of DOTA-peptides to a cys-diabody.</a> Bioconjugate Chemistry. 13 (5), 985-995 (2002).</li><li>Li, J., Wang, X. H., Wang, X. M., Chen, Z. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-specific+conjugation+of+bifunctional+chelator+BAT+to+mouse+IgG(1)+Fab'+fragment.">Site-specific conjugation of bifunctional chelator BAT to mouse IgG(1) Fab' fragment.</a> Acta Pharmacologica Sinica. 27 (2), 237-241 (2006).</li><li>Tinianow, J. N., 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=Site-specifically+Zr-89-labeled+monoclonal+antibodies+for+ImmunoPET.">Site-specifically Zr-89-labeled monoclonal antibodies for ImmunoPET.</a> Nuclear Medicine and Biology. 37 (3), 289-297 (2010).</li><li>Li, L., 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=Site-specific+conjugation+of+monodispersed+DOTA-PEGn+to+a+thiolated+diabody+reveals+the+effect+of+increasing+PEG+size+on+kidney+clearance+and+tumor+uptake+with+improved+64-copper+PET+imaging.">Site-specific conjugation of monodispersed DOTA-PEGn to a thiolated diabody reveals the effect of increasing PEG size on kidney clearance and tumor uptake with improved 64-copper PET imaging.</a> Bioconjugate Chemistry. 22 (4), 709-716 (2011).</li><li>Khalili, H., Godwin, A., Choi, J. -. w., Lever, R., Brocchini, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+binding+of+disulfide-bridged+PEG-Fabs.">Comparative binding of disulfide-bridged PEG-Fabs.</a> Bioconjugate Chemistry. 23 (11), 2262-2277 (2012).</li><li>Koniev, O., Wagner, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developments+and+recent+advancements+in+the+field+of+endogenous+amino+acid+selective+bond+forming+reactions+for+bioconjugation.">Developments and recent advancements in the field of endogenous amino acid selective bond forming reactions for bioconjugation.</a> Chemical Society Reviews. 44 (15), 5495-5551 (2015).</li><li>Patterson, J. T., Asano, S., Li, X., Rader, C., Barbas, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+the+serum+stability+of+site-specific+antibody+conjugates+with+sulfone+linkers.">Improving the serum stability of site-specific antibody conjugates with sulfone linkers.</a> Bioconjugate Chemistry. 25 (8), 1402-1407 (2014).</li><li>Toda, N., Asano, S., Barbas, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid,+stable,+chemoselective+labeling+of+thiols+with+Julia-Kocienski-like+reagents:+A+serum-stable+alternative+to+maleimide-based+protein+conjugation.">Rapid, stable, chemoselective labeling of thiols with Julia-Kocienski-like reagents: A serum-stable alternative to maleimide-based protein conjugation.</a> Angewandte Chemie-International Edition. 52 (48), 12592-12596 (2013).</li><li>Zhang, Q., 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=Last-step+enzymatic+F-18-fluorination+of+cysteine-tethered+RGD+peptides+using+modified+Barbas+linkers.">Last-step enzymatic F-18-fluorination of cysteine-tethered RGD peptides using modified Barbas linkers.</a> Chemistry-a European Journal. 22 (31), 10998-11004 (2016).</li><li>Chiotellis, 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=Novel+chemoselective+F-18-radiolabeling+of+thiol-containing+biomolecules+under+mild+aqueous+conditions.">Novel chemoselective F-18-radiolabeling of thiol-containing biomolecules under mild aqueous conditions.</a> Chemical Communications. 52 (36), 6083-6086 (2016).</li><li>Adumeau, P., Davydova, M., Zeglis, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thiol-reactive+bifunctional+chelators+for+the+creation+of+site-selectively+modified+radioimmunoconjugates+with+improved+stability.">Thiol-reactive bifunctional chelators for the creation of site-selectively modified radioimmunoconjugates with improved stability.</a> Bioconjugate Chemistry. 29, 1364-1372 (2018).</li><li>Sakamoto, J., Kojima, H., Kato, J., Hamashima, H., Suzuki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ-specific+expression+of+the+intestinal+epithelium-related+antigen+A33,+a+cell+surface+target+for+antibody-based+imaging+and+treatment+in+gastrointestinal+cancer.">Organ-specific expression of the intestinal epithelium-related antigen A33, a cell surface target for antibody-based imaging and treatment in gastrointestinal cancer.</a> Cancer Chemotherapy and Pharmacology. 46, S27-S32 (2000).</li><li>Sakamoto, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+phase+I+radioimmunolocalization+trial+of+humanized+monoclonal+antibody+huA33+in+patients+with+gastric+carcinoma.">A phase I radioimmunolocalization trial of humanized monoclonal antibody huA33 in patients with gastric carcinoma.</a> Cancer Science. 97 (11), 1248-1254 (2006).</li><li>Junutula, J. 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=Site-specific+conjugation+of+a+cytotoxic+drug+to+an+antibody+improves+the+therapeutic+index.">Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index.</a> Nature Biotechnology. 26 (8), 925-932 (2008).</li><li>Pillow, T. H., 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=Site-specific+trastuzumab+maytansinoid+antibody-drug+conjugates+with+improved+therapeutic+activity+through+linker+and+antibody+engineering.">Site-specific trastuzumab maytansinoid antibody-drug conjugates with improved therapeutic activity through linker and antibody engineering.</a> Journal of Medicinal Chemistry. 57 (19), 7890-7899 (2014).</li><li>Boswell, C. 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=Enhanced+tumor+retention+of+a+radiohalogen+label+for+site-specific+modification+of+antibodies.">Enhanced tumor retention of a radiohalogen label for site-specific modification of antibodies.</a> Journal of Medicinal Chemistry. 56 (23), 9418-9426 (2013).</li><li>Boswell, C. 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=Impact+of+drug+conjugation+on+pharmacokinetics+and+tissue+distribution+of+anti-STEAP1+antibody-drug+conjugates+in+rats.">Impact of drug conjugation on pharmacokinetics and tissue distribution of anti-STEAP1 antibody-drug conjugates in rats.</a> Bioconjugate Chemistry. 22 (10), 1994-2004 (2011).</li><li>Alvarez, V. L., 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=Site-specifically+modified+111In+labelled+antibodies+give+low+liver+backgrounds+and+improved+radioimmunoscintigraphy.">Site-specifically modified 111In labelled antibodies give low liver backgrounds and improved radioimmunoscintigraphy.</a> Nuclear Medicine and Biology. 13 (4), 347-352 (1986).</li><li>Strop, 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=Location+matters:+SIte+of+conjugation+modulates+stability+and+pharmacokinetics+of+antibody+drug+conjugates.">Location matters: SIte of conjugation modulates stability and pharmacokinetics of antibody drug conjugates.</a> Chemistry, Biology. 20 (2), 161-167 (2013).</li><li>Hallam, T. J., Wold, E., Wahl, A., Smider, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody+conjugates+with+unnatural+amino+acids.">Antibody conjugates with unnatural amino acids.</a> Molecular Pharmaceutics. 12 (6), 1848-1862 (2015).</li><li>Axup, J. Y., 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+of+site-specific+antibody-drug+conjugates+using+unnatural+amino+acids.">Synthesis of site-specific antibody-drug conjugates using unnatural amino acids.</a> Proceedings of the National Academy of Sciences. 109 (40), 16101-16106 (2012).</li><li>Lang, K., Chin, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+incorporation+of+unnatural+amino+acids+and+bioorthogonal+labeling+of+proteins.">Cellular incorporation of unnatural amino acids and bioorthogonal labeling of proteins.</a> Chemical Reviews. 114 (9), 4764-4806 (2014).</li><li>Yamasaki, R. B., Osuga, D. T., Feeney, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Periodate+oxidation+of+methionine+in+proteines.">Periodate oxidation of methionine in proteines.</a> Analytical Biochemistry. 126 (1), 183-189 (1982).</li><li>Wang, W., 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=Impact+of+methionine+oxidation+in+human+IgG1+Fc+on+serum+half-life+of+monoclonal+antibodies.">Impact of methionine oxidation in human IgG1 Fc on serum half-life of monoclonal antibodies.</a> Molecular Immunology. 48 (6-7), 860-866 (2011).</li><li>O'Shannessy, D. J., Dobersen, M. J., Quarles, R. H. <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+procedure+for+labeling+immunoglobulins+by+conjugation+to+oligosaccharide+moieties.">A novel procedure for labeling immunoglobulins by conjugation to oligosaccharide moieties.</a> Immunology Letters. 8 (5), 273-277 (1984).</li><li>Panowski, S., Bhakta, S., Raab, H., Polakis, P., Junutula, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-specific+antibody+drug+conjugates+for+cancer+therapy.">Site-specific antibody drug conjugates for cancer therapy.</a> Mabs. 6 (1), 34-45 (2014).</li><li>Hu, 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=Site-specific+conjugation+of+HIV-1+tat+peptides+to+IgG:+a+potential+route+to+construct+radioimmunoconjugates+for+targeting+intracellular+and+nuclear+epitopes+in+cancer.">Site-specific conjugation of HIV-1 tat peptides to IgG: a potential route to construct radioimmunoconjugates for targeting intracellular and nuclear epitopes in cancer.</a> European Journal of Nuclear Medicine and Molecular Imaging. 33 (3), 301-310 (2006).</li></ol>

在本报告中, 我们选择不包括任何用于放射性标记或体内实验的协议。我们的理由很简单。关于前者, 基于 pod 的免疫共轭体的放射性标记与使用其他生物共轭策略合成的免疫共轭的放射标记一点不同, 这些程序已在其他地方进行了全面的审查 2.关于后者, 临床前体内实验的具体细节 (即小鼠模型、剂量等) 可能因应用和抗体抗原系统的不同而有很大差异。
我们...

长期以来, 放射性药物化学家一直在利用疾病生物标志物抗体的选择性和特异性用于核成像和靶向放射治疗1。抗体的放射标记最常见的方法是基于放射性标记的假体群或放射性螯合剂不加区别地附着在免疫球蛋白结构中的氨基酸--通常是赖氨酸 (图 1a)2。虽然这种策略肯定是有效的, 但其随机的、非特定地点的性质会造成问题。具体而言, 传统的生物共轭方法产生定义差和异质性的免疫共轭, 由成千上万种不同的区域异构体的混合物组成, 每个物质具有自己的生物和药理特性集 3。此外, 如果将货物附加到免疫球蛋白的抗原结合域, 随机生物共轭会阻碍抗体的免疫反应。
多年来, 为解决这些问题, 制定了各种特定地点和场地选择性的生物结合战略4、5.其中最常见的方法是将含马来酰亚胺探针连接到半胱氨酸的磺基 (图 1b)。igg1 抗体自然含有4个链间二硫基桥, 这些连接可以选择性地减少, 产生自由的硫醇, 能够与马来酰亚胺发生迈克尔加成反应, 形成琥珀酰硫醚键。与传统方法相比, 硫醇和马来酰亚胺的使用无疑是一种改进, 目前有各种各样的含苹果胺的合成器和双功能螯合剂。然而, 必须指出, 这种方法也有严重的局限性。基于马来酰的免疫共轭物在体内表现出有限的稳定性, 因为硫醚链可以经历迈克尔的反应 (图 2)6,7,8,9,10. 当然, 这可能导致放射性有效载荷的释放或与循环中的带有硫醇的生物分子 (例如谷胱甘肽或血清白蛋白) 的交换。这两种过程都可以增加健康器官中的活性浓度, 并降低目标组织中的活性浓度, 从而降低影像学对比度和治疗率。为了规避这些问题, 开发了几种替代硫醇反应试剂, 包括托基酸盐、溴化和碘乙酰, 以及11、12、13乙烯基磺酸.14,15,16,17. 然而, 所有这些办法都有其局限性, 妨碍了其广泛适用。
大约五年前, 斯克里普斯研究所已故卡洛斯·巴巴三世的实验室率先使用苯氧基二唑基甲基磺酸作为试剂, 有选择地与硫醇形成高度稳定的联系 (图1c 和图 3)18,19. 作者使用了一种含苯氧基甲基磺酸的荧光素变种来修饰几种抗体, 这些抗体设计成含有游离半胱氨酸残基, 最终产生的免疫共轭具有比类似的更高的稳定性。使用基于恶意的探测器创建的构造。看到这项很有前途的工作, 我们感到有些惊讶, 因为这项技术只在放射化学中很少使用, 还没有被用于双功能螯合剂或放射免疫共轭 20,21的合成.然而, 由于应用不足, 很快就开始变得更加有意义: 几次试图从 sigma-aldrich 采购试剂的努力, 结果收到了复杂的降解产物混合物, 并将所需化合物的 lt;15%。此外, 合成所报告的试剂本身也不是一个现实的选择, 因为公布的合成路线有些繁琐, 需要精密的有机化学设备, 而大多数放射化学和分子成像实验室--包括我们的实验室--根本不拥有。
针对这些障碍, 我们着手创建一种易于获取且高度稳定的苯氧基甲基磺酸试剂, 可通过坚固且合理、简单的合成路线获得。今年早些时候, 我们报告了一种模块化、稳定且易于获得的苯氧基甲基磺酸试剂 (称为 "pods"), 作为基于硫醇的生物共轭平台 (图1c 和图 3)22。peds 与 barbas 等人报告的试剂之间的主要区别是, 前者使用的是附着在苯氧基磺酸的苯胺环上, 而后者的特点是处于同一位置的苯酚 (图 4)。这一变化促进了更直接、更容易获得的合成路线, 以及--如果我们在商业上可获得的化合物方面的经验具有代表性--更稳定的最终试剂。在这项工作中, 我们还合成了一对含 pods 双功能螯合剂--pods-dfo 和 pods-chx-a '-dtpa--分别用于创建89zr 标记和177lu-taid 免疫共轭。正如我们将要讨论的, 我们已经证明, 基于 pod 的位选择生物共轭可重复和强大地创建均匀, 明确的, 高度免疫反应, 和高度稳定的放射免疫共轭。此外, 大肠癌小鼠模型的临床前实验表明, 与通过马来所合成的放射性标记抗体相比, 这些位点选择性标记的放射免疫共轭在体内表现出优越的性能。共轭。
这项工作的首要目标是促进创建定义明确、均匀、高度稳定和高度免疫反应的免疫共轭, 用于体外和体内应用。合成方法很简单, 几乎可以在任何实验室进行, 并且母 pds 试剂可以用大量不同的螯合剂、荧光剂或货物进行改性。在本协议和随附的视频中, 我们将描述简单的四步合成 peds (图 5);创建一个携带 dods 的 dota 变种, 这是一种广泛使用的螯合剂, 用于协调64cu、 68ga 、111 in、 177lu 和225ac (图 6);以及 dods-dota 与模型抗体 her2 靶向 igg1 trastuzumab 的生物结合 (图 7)。

<table>
	<tbody>
		<tr>
			<td>5-(4-aminophenyl)-1,3,4-oxadiazole-2-thiol</td>
			<td>Sigma-Aldrich</td>
			<td>675024</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL LoBind Microcentrifugal Tube</td>
			<td>Eppendorf</td>
			<td>925000090</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL Microcentrifugal Tube</td>
			<td>Fisherbrand</td>
			<td>05-408-129</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Fisher Scientific</td>
			<td>A998-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra-2 Centrifugal Filter Unit</td>
			<td>EMD Millipore</td>
			<td>EN300000141G</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyclohexane</td>
			<td>Fisher Scientific</td>
			<td>C556-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Dichloromethane</td>
			<td>Fisher Scientific</td>
			<td>AC383780010</td>
			<td></td>
		</tr>
		<tr>
			<td>Diisopropylethylamine</td>
			<td>MP Biomedicals, LLC</td>
			<td>150915</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethylsulfoxide</td>
			<td>Fisher Scientific</td>
			<td>31-727-5100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl Acetate</td>
			<td>Fisher Scientific</td>
			<td>E145 4</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric Acid</td>
			<td>Fisher Scientific</td>
			<td>A144-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Iodomethane</td>
			<td>Sigma-Aldrich</td>
			<td>289566-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulfate</td>
			<td>Acros Organics</td>
			<td>413485000</td>
			<td></td>
		</tr>
		<tr>
			<td>m-chloroperbenzoic acid</td>
			<td>Sigma-Aldrich</td>
			<td>273031</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher Scientific</td>
			<td>A412 1</td>
			<td></td>
		</tr>
		<tr>
			<td>NBoc-N&#8242;-succinyl-4,7,10-trioxa-1,13-tridecanediamine</td>
			<td>Sigma-Aldrich</td>
			<td>671401</td>
			<td>Store at -80 &#176;C</td>
		</tr>
		<tr>
			<td>N-ethyl-N&#8242;- [3- (dimethylamino)propyl] carbodiimide hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>3450</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Sigma-Aldrich</td>
			<td>P5493</td>
			<td>10&#215; Concentration</td>
		</tr>
		<tr>
			<td>p-SCN-Bn-DOTA</td>
			<td>Macrocyclics</td>
			<td>B-205</td>
			<td>Store at -80 &#176;C</td>
		</tr>
		<tr>
			<td>Sephadex G-25 in PD-10 Desalting Columns</td>
			<td>GE Healthcare</td>
			<td>17085101</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Carbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S7795</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Fisher Scientific</td>
			<td>S318-1</td>
			<td></td>
		</tr>
		<tr>
			<td>TCEP</td>
			<td>ThermoFischer Scientific</td>
			<td>20490</td>
			<td></td>
		</tr>
		<tr>
			<td>Triethylamine</td>
			<td>Fisher Scientific</td>
			<td>AC157911000</td>
			<td></td>
		</tr>
		<tr>
			<td>Trifluoroacetic Acid</td>
			<td>Fisher Scientific</td>
			<td>A116-50</td>
			<td></td>
		</tr>
	</tbody>
</table>

synthesis and bioconjugation of thiol-reactive reagents for the creation of site-selectively modified immunoconjugates

含锂双功能探针已被用于生物分子中硫醇的现场选择性修饰, 特别是抗体。然而, 基于马来酰亚胺的共轭在体内表现出有限的稳定性, 因为琥珀酰硫醚结合可以经历迈克尔的反性反应。当然, 这可能导致放射性有效载荷的释放或与流通中的带有硫醇的生物分子的交换。这两种过程都能在健康器官中产生较高的活性浓度, 并降低目标组织中的活性浓度, 从而降低影像学对比度, 降低治疗率。2018年, 我们报告了一种模块化、稳定且易于获得的苯氧基甲基磺酸试剂 (称为 "pods"), 作为基于硫醇的生物共轭平台。我们已经清楚地证明, 基于 pod 的位点选择性生物共轭可重现性和强大地创建均匀性, 明确定义, 高度免疫反应, 和高度稳定的放射免疫共轭。此外, 在大肠癌小鼠模型的临床前实验表明, 这些位点选择性标记的放射免疫共轭物在体内表现出的性能远远优于通过马来化症合成的放射性标记抗体共轭。在该协议中, 我们将描述 dods 的四步合成, 制备的双功能的 dods 轴承变种的无处不在的螯合物 dota (dods-dota), 以及 dods-dota 与 her2 靶向抗体 trastuzumab 的共轭。

该协议的前四个步骤----pods 的合成----被设计为具有鲁棒性和可靠性。5-(4-氨基苯基)-1, 3, 4-恶二唑-2-硫醇的分解和替代, 形成所需的硫醚产品, 仅45分钟后就能使硫醚的产率达到 gt;99%。其次, 通过标准的肽偶联过程, 实现了 1和 n-bc-n '-琥珀-4、7、10-三氧分析-1, 13 三苯胺的结扎, 从而以55% 的收率收集了产品 (2)。然后, 用广泛使用的氧化剂--m-氯前氧苯甲?...

Watch this Scientific Journal Video about Synthesis and Bioconjugation of Thiol-Reactive Reagents for the Creation of Site-Selectively Modified Immunoconjugates at JoVE.com

Synthesis and Bioconjugation of Thiol-Reactive Reagents for the Creation of Site-Selectively Modified Immunoconjugates

硫醇反应试剂的合成与生物共轭作用--用于原位选择性修饰免疫共轭的生成

在本议定书中, 我们将描述 pods 的合成, 这是一种基于苯氧二唑基甲基磺酸试剂的合成, 用于将货物选择性地附着在生物分子的硫醇中, 特别是抗体中。此外, 我们将描述含有 pod 的双官能螯合剂的合成和表征及其与模型抗体的结合。

Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. ...

该协议描述了PODS的合成和应用，PODS是一种用于蛋白质和肽的特异性生物结合的新试剂。这项技术比传统的雄胺-硫醇基生物结合技术有了明显的提高。此过程的最大优点是试剂的制造简单性。该方法允许几乎在任何实验室合成试剂。在10毫升圆底烧瓶中，将100毫克的氨基苯丙胺氧化二醇溶解在三毫升甲醇中。在溶液中，加入360微升二乙基胺和磁搅拌棒。用橡胶塞密封烧瓶，并在室温下搅拌溶液 10 分钟。使用一毫升玻璃注射器，在橡胶塞上戳一个洞，并迅速在混合物中加入32微升的碘烷。让混合物在室温下反应45分钟。在25毫升的圆底烧瓶中，将387毫克的Bobo保护的二氯甲烷溶解在10毫升的二氯甲烷中。在溶液中，加入480微升二乙基乙胺、264毫克EDCI、200毫克苯胺和磁搅拌棒。用玻璃塞子密封烧瓶，让反应在室温下搅拌五天。反应完成后，将反应混合物转移到分离漏斗中，用五毫升一摩尔盐酸洗涤三次。收集有机相并转移到分离漏斗。然后用一摩尔碳酸钠洗净有机相，然后用水。接下来，收集有机相并添加硫酸镁，以去除任何水的痕迹。然后使用中玻璃褶皱过滤混合物。使用旋转蒸发器，在减压下去除挥发性溶剂，以提供非白色固体。在此之后，在10毫升乙酸乙酯中重新溶解分离的固体。然后通过逐渐添加30毫升环氧烷沉淀产品。使用中玻璃褶皱过滤溶液，以获得作为白色粉末的卡巴马特产品。在10毫升的圆底烧瓶中，将30毫克的甲壳素溶解在4毫升二氯甲烷中。慢慢地将49毫克70%m-氯苯酚酸和磁搅拌棒加入混合物中，用玻璃塞子密封烧瓶。在室温下隔夜搅拌溶液，最终产生黄色混合物。第二天，将混合物转移到分离漏斗。然后用0.1摩尔氢氧化钠先用水清洗混合物。收集有机相并添加硫酸镁，以去除任何水的痕迹。然后使用中玻璃褶皱过滤混合物。在25毫升的圆底烧瓶中，将30毫克的甲酸酯溶解在两毫升二氯甲烷中。在混合物中加入磁搅拌棒。400微升三氟乙酸，用玻璃塞子封住烧瓶。然后在室温下搅拌反应混合物三小时。反应完成后，使用旋转蒸发器在室温下降低压力下去除挥发性，以获得油性残留物。将油性残留物溶解在7毫升水中。并转移到分离漏斗。用四毫升乙酸乙酯洗三次水溶液。在1.5毫升的微离心管中，溶解10毫克PODS和300微升二甲基硫化物。然后加入26微升的N，二二丙乙胺到溶液中。将 15.2 毫克 DOTA NCS 溶解在 100 微升二甲基硫化物中，并将溶液与 PODS 溶液结合使用。密封微离心管，让反应在室温下孵育过夜。接下来，在低蛋白结合的1.5毫升微离心管中，用859微升PBS稀释61微升的trastuzumab库存溶液。在这种混合物中，在水中加入6.7微升新鲜制造的10毫升TCEP溶液。在反应混合物中加入73微升每毫升1毫克PODS-DOTA溶液。最后，密封微离心管，在室温下孵育溶液两小时。PODS 合成是稳健可靠的。脱蛋白和替代氨基苯二甲酸酯硫醇在定量产量上给予硫醚一剂。硫化一和bobo保护的碳氧酸的连结在55%的收益率中产生化合物二。氧化提供化合物三在90%的屈服。去除boss保护组，产生的PODS在98%的收益率。通过质子 NMR、碳 NMR 和高分辨率 MS 确认每种产品的身份。切合器DTA的同位素含物变种与PODS的吊坠胺耦合，获得75%的双功能溷合器。通过质子 NMR、碳 NMR 和高分辨率 MS 确认产品的身份。此处显示了 PODS-DOTA 与 HER2 靶向抗体的位点选择性生物结合。抗体铰链区域的二硫化物链接被选择性地降低，然后用PODS-DOTA孵育抗体，使带有DTA的免疫结合得到80%的产生。MALDI-TOF 分析显示每个抗体的标签度为 1.8 DOTA。在一系列人类、人性化和嵌合性IgG1抗体中保持一致。然而，同样的条件产生免疫结合，对于MurineIgG1抗体，其标记度为1.5。由于这种化合物的光敏性，重要的是保持所有反应在铝箔覆盖的容器。这将增加产品的整体产量。虽然该协议描述了PODS-DOTA的合成，但类似的程序可以适用于各种潜在的货物，包括荧光团、毒素和其他溷合剂。这种方法将促进定义明确、同质和高度稳定的免疫结合的构建，这些免疫结合可用于各种领域。重要的是要记住，在这个实验的第一步中添加的碘烷可以产生有害影响，因此应该用在烟罩中。

Research

JoVE Journal

Chemistry

Synthesis of Hypervalent Iodonium Alkynyl Triflates for the Application of Generating Cyanocarbenes

高价碘鎓炔基三氟甲磺酸酯的生成Cyanocarbenes的合成应用

The procedures described in this article involve the synthesis and isolation of hypervalent iodonium alkynyl triflates (HIATs) and their subsequent ...

科研

化学

Split-and-pool Synthesis and Characterization of Peptide Tertiary Amide Library

分裂和池合成及肽叔酰胺图书馆表征

Peptidomimetics are great sources of protein ligands. The oligomeric nature of these compounds enables us to access large synthetic libraries on solid ...

The Bioconjugation and Radiosynthesis of 89Zr-DFO-labeled Antibodies

The Bioconjugation and Radiosynthesis of 89Zr-DFO-labeled Antibodies

该生物耦合和重新布置<sup&gt; 89</sup&gt;锆DFO标记的抗体

The exceptional affinity, specificity, and selectivity of antibodies make them extraordinarily attractive vectors for tumor-targeted PET ...

Protocol for the Synthesis of Ortho-trifluoromethoxylated Aniline Derivatives

Protocol for the Synthesis of Ortho-trifluoromethoxylated Aniline Derivatives

协议的合成<em&gt;邻</em&gt; -trifluoromethoxylated苯胺类化合物

Molecules bearing trifluoromethoxy (OCF3) group often show desired pharmacological and biological properties. However, facile synthesis of ...

Synthesis and Characterization of Supramolecular Colloids

超分子胶体的合成与表征

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical ...

HKUST-1 as a Heterogeneous Catalyst for the Synthesis of Vanillin

科大-1作为香兰素的合成的非均相催化剂

Vanillin (4-hydoxy-3-methoxybenzaldehyde) is the main component of the extract of vanilla bean. The natural vanilla scent is a mixture of approximately ...

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

蛋白质生物缀合物的合成<em&gt;通过</em&gt;半胱氨酸马来酰亚胺化学

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

Facile Synthesis of Worm-like Micelles by Visible Light Mediated Dispersion Polymerization Using Photoredox Catalyst

使用Photoredox催化剂蠕虫状胶束的简便合成可见光介导的分散聚合

Presented herein is a protocol for the facile synthesis of worm-like micelles by visible light mediated dispersion polymerization. This approach begins ...

Synthesis and Mass Spectrometry Analysis of Oligo-peptoids

寡 peptoids 的合成及质谱分析

Peptoids are sequence-controlled peptide-mimicking oligomers consisting of N-alkylated glycine units. Among many potential applications, peptoids have ...

Chemical Precipitation Method for the Synthesis of Nb2O5 Modified Bulk Nickel Catalysts with High Specific Surface Area

Chemical Precipitation Method for the Synthesis of Nb2O5 Modified Bulk Nickel Catalysts with High Specific Surface Area

化学沉淀法合成铌2O5改性高比表面积镍催化剂

We demonstrate a method for the synthesis of NixNb1-xO catalysts with sponge-like and fold-like nanostructures. By varying the Nb:Ni ratio, a series of ...