Site-Selective Approaches to Attain Fluorescent Human Insulin Conjugates: Balancing the Site of Labeling and the In Vivo Activity

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 in vivo. To this end, monolabeled insulin at the N-terminal of A chain (GlyA1-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 (PheB1-N-FITC-insulin), di-tert-butyl decarbonate, known as Boc anhydride, was used before FITC labeling. The attained insulin conjugates, namely, GlyA1-N-FITC-insulin and PheB1-N-FITC-insulin, were characterized using protein mass spectroscopy and peptide analysis. A third fluorescent conjugate was prepared using alpha-haloacetyl-based chemistry. This chemistry's advantage is maintaining the chain A N-terminal amine basicity, which was essential for its activity. Using alpha-haloacetyl-based chemistry, azide group-functionalized insulin was prepared, which was further clicked with fluorescent dye affording GlyA1-N-Cy5-insulin. According to the in vivo efficacy study of the three insulin conjugates, both fluorescent GlyA1-N-FITC-insulin and GlyA1-N-Cy5-insulin retained the insulin biological activity, suggesting no structural alteration upon the conjugation conditions. Hence, both GlyA1-N-FITC-insulin and GlyA1-N-Cy5-insulin are effective in labeling and, more importantly, maintaining the in vivo activity of insulin. Lastly, in vitro binding of GlyA1-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 in vivo biological activity.