Alkhawaja Bayan, AlDabet Ghayda', Alkhawaja Nour, Ghanim Bayan Y, Al-Khatib Khaled, Reeksting Shaun, Michael Andreas, Abuarqoub Duaa, Mohammad Marwa, Watts Andrew G, Qinna Nidal A
Faculty of Pharmacy and Medical Sciences, University of Petra, Amman 11196, Jordan.
Department of Life Sciences, University of Bath, Claverton Down, BA2 7AY Bath, U.K.
ACS Omega. 2025 Feb 17;10(8):8140-8151. doi: 10.1021/acsomega.4c09498. eCollection 2025 Mar 4.
Fluorescent insulin is commonly used for a range of detection and imaging purposes. Achieving site-selective insulin labeling affords superior labeling yield while retaining its biological activity. Insulin labeling is usually achieved using commercial kits with minimal emphasis on the site and degree of labeling. To bridge this gap, this work highlights the essential parameters concerning the development of fluorescent insulin and reflects them on the biological activity of insulin . To this end, monolabeled insulin at the N-terminal of A chain (Gly-N-FITC-insulin) was prepared using the minimal equivalents of fluorescein isothiocyanate (FITC) dye. In our hands, temperature and pH control were the main parameters affecting the reaction yield, with no dilabeled insulin being attained. To label the N-terminal of the B chain (Phe-N-FITC-insulin), di-butyl decarbonate, known as Boc anhydride, was used before FITC labeling. The attained insulin conjugates, namely, Gly-N-FITC-insulin and Phe-N-FITC-insulin, were characterized using protein mass spectroscopy and peptide analysis. A third fluorescent conjugate was prepared using α-haloacetyl-based chemistry. This chemistry's advantage is maintaining the chain A N-terminal amine basicity, which was essential for its activity. Using α-haloacetyl-based chemistry, azide group-functionalized insulin was prepared, which was further clicked with fluorescent dye affording Gly-N-Cy5-insulin. According to the efficacy study of the three insulin conjugates, both fluorescent Gly-N-FITC-insulin and Gly-N-Cy5-insulin retained the insulin biological activity, suggesting no structural alteration upon the conjugation conditions. Hence, both Gly-N-FITC-insulin and Gly-N-Cy5-insulin are effective in labeling and, more importantly, maintaining the activity of insulin. Lastly, binding of Gly-N-FITC-insulin was successful when it was assayed in NIH/3T3 fibroblast cells. This work has provided facile conjugation approaches for site-specific insulin labeling with dyes or clickable chemistry in conjunction with insulin's biological activity.
荧光胰岛素通常用于一系列检测和成像目的。实现位点选择性胰岛素标记可在保留其生物活性的同时提供更高的标记产率。胰岛素标记通常使用商业试剂盒完成，而对标记位点和标记程度的关注较少。为了弥补这一差距，本研究突出了与荧光胰岛素开发相关的关键参数，并将其与胰岛素的生物活性相关联。为此，使用最少当量的异硫氰酸荧光素（FITC）染料制备了A链N端的单标记胰岛素（Gly-N-FITC-胰岛素）。在我们的实验中，温度和pH控制是影响反应产率的主要参数，未得到双标记胰岛素。为了标记B链的N端（Phe-N-FITC-胰岛素），在FITC标记之前使用了二叔丁基焦碳酸酯（即Boc酸酐）。所得的胰岛素偶联物，即Gly-N-FITC-胰岛素和Phe-N-FITC-胰岛素，通过蛋白质质谱和肽分析进行了表征。使用基于α-卤代乙酰基的化学方法制备了第三种荧光偶联物。这种化学方法的优点是保持了A链N端胺的碱性，这对其活性至关重要。使用基于α-卤代乙酰基的化学方法，制备了叠氮基功能化的胰岛素，其进一步与荧光染料反应得到Gly-N-Cy5-胰岛素。根据对三种胰岛素偶联物的功效研究，荧光Gly-N-FITC-胰岛素和Gly-N-Cy5-胰岛素均保留了胰岛素的生物活性，表明在偶联条件下结构未发生改变。因此，Gly-N-FITC-胰岛素和Gly-N-Cy5-胰岛素在标记方面均有效，更重要的是，它们保持了胰岛素的活性。最后，在NIH/3T3成纤维细胞中检测时，Gly-N-FITC-胰岛素的结合成功。本研究提供了简便的偶联方法，可通过染料或点击化学对胰岛素进行位点特异性标记，并结合胰岛素的生物活性。
