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

## TL;DR

This paper presents new methods to label insulin with fluorescent dyes at specific sites without affecting its biological activity in the body.

## Contribution

The study introduces site-selective labeling approaches that preserve insulin's in vivo activity, using controlled chemistry and clickable methods.

## Key findings

- Monolabeled insulin at the A chain N-terminal (GlyA1-N-FITC-insulin) was successfully prepared with controlled reaction parameters.
- Fluorescent conjugates GlyA1-N-FITC-insulin and GlyA1-N-Cy5-insulin retained insulin's biological activity in vivo.
- In vitro binding of GlyA1-N-FITC-insulin was confirmed in NIH/3T3 fibroblast cells.

## Abstract

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
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 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.