Background:
Herein we report the multigram-scale synthesis, characterization and
application of a rhodamine B-based fluorophore (ROSA) suitable for fluorescent studies in
biological applications. This fluorophore is devoid of rhodamine spirolactone formation and
furthermore characterized by a high molar extinction coefficient (ϵ=87250 ± 1630 M-1cm-1) and
quantum yield (φ) of 0.589 ± 0.070 in water. Reported here is also the application of ROSA
towards synthesis of a ROSA-PEG-GRGDS-NH2 fluorescent probe suitable for live cell imaging of
αvβ3 integrins for in vitro assays.
Objective:
The main objective of this study is to efficiently prepare rhodamine B derivative,
devoid of spirolactone formation that would be suitable for bioconjugation and subsequent
bioimaging.
Methods:
Rhodamine B was transformed into rhodamine B succinimide ester (RhoB-OSu) using
N-hydroxysuccinimide. RhoB-OSu was further coupled to sarcosine to obtain rhodamine Bsarcosine
dye (ROSA) in good yield. The ROSA dye was then coupled to a αvβ3 integrin binding
sequence using standard solid-phase conditions. Resulting ROSA-PEG-GRGDS-NH2 probe was
used to image integrins on cancer cells.
Results:
The rhodamine B-sarcosine dye (ROSA) was obtained in multigram scale in good total
yield of 47%. Unlike rhodamine B, the ROSA dye does not undergo pH-dependent
spirolactone/spirolactam formation as compared with rhodamine B-glycine. It is also characterized
by excellent quantum yield (φ) of 0.589 ± 0.070 in water and high molar extinction coefficient of
87250 ± 1630 M-1cm-1. ROSA coupling to the RGD-like peptide was proved to be efficient and
straightforward. Imaging using standard filters on multimode plate reader and confocal microscope
was performed. The αvβ3 integrins present on the surface of live WM-266-4 (melanoma) and MCF-
7 (breast cancer) cells were successfully imaged.
Conclusion:
We successfully derivatized rhodamine B to create an inexpensive, stable and
convenient to use fluorescent probe. The obtained derivative has excellent photochemical properties
and it is suitable for bioconjugation and many imaging applications.
A polyamine-modified near-infrared fluorescent probe for selective staining of live cancer cells
