Synthesis and Characterization of ROSA Dye - A Rhodamine B-type Fluorophore, Suitable for Bioconjugation and Fluorescence Studies in Live Cells.
Protein Pept Lett
; 26(10): 758-767, 2019.
Article
in En
| MEDLINE
| ID: mdl-31215362
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 αvß3 integrins for in vitro assays. OBJECTIVES: 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 αvß3 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 αvß3 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.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Rhodamines
/
Integrin alphaVbeta3
/
Optical Imaging
/
Fluorescent Dyes
Limits:
Humans
Language:
En
Journal:
Protein Pept Lett
Journal subject:
BIOQUIMICA
Year:
2019
Document type:
Article
Affiliation country:
United States
Country of publication:
Netherlands