Development of photoactivatable formaldehyde donors

with fluorescence monitoring for biological studies

Nicholas W. Pino, Lukas P. Smaga, Jefferson Chan
Department of Chemistry, School of Chemical Sciences, University of Illinois at Urbana-Champaign

INTRODUCTION SYNTHESIS IN VITRO STUDIES ACKNOWLEDGM

• Controlled light mediated release of analytes has emerged
• Analyte-linked biological effects are usually dependent on
Figure 4. (a) Absorbance
and emission of photoFAD-
ENTS
concentration and location 3 and dye after cleaveage
• We have developed a formaldehyde donors that allow controlled (4) (b) Fluorescence
release with concentration and location monitoring by fluorescence 760 nm enhancement with
• Accurate concentration monitoring was achieved through highly increased irradiation time
photostable design with UV light (c) Non-
• Application of photoFAD-3 uncovered the concentration range spectral representation of
necessary for arresting wound healing in live cells (b) (d) normalized cell
• Formaldehyde is a critical compound in biology that is toxic at high viability assayed via trypan
concentrations but involved in nucleobase synthesis at lower blue assay indicating both
the donor and turnover
concentrations
product are not toxic
Nature 548, 549–554 (2017).

Figure 2 Synthesis of photoFAD-3. Modular approach allows

synthesis of a variety of dye platforms for optimization Tissue Microenvironment
Traineeship UIUC (NIH T32)

• Modular synthesis allows for a variety of aryl Dr. Robert and Mrs. Carolyn
Springborn Graduate Fellowship
halides (bottom) pieces and xanthone (top)
pieces

• Chlorinated ethers an easily be coupled to the

dye to open up delivered aldehyde CELL STUDIES
Figure 1 One-carbon cycle in which formaldehyde is diverted into opportunities
nucleobase synthesis • Developed
calibration curve
protocol
TRIGGER AND PLATFORM DESIGN • Compatible with IVIS
(full well) and
epifluorescence
(frame of cells)
• Used a wound
healing assay to
asses HEK293
resistance to
Formaldehyde
• 2.2 μM
formaldehyde
Figure 5 (a) Epifluorescence imaging at varied times of UV inhibits wound Figure 6 (a) Wound closure with a non-releasing
irradiation (b) IVIS imaging of full wells at variet times of
UV irradiation (c) Fluorescence from IVIS compared to healing control dye and photoFAD-3 (b) Quantification of (a)
(c) wound closure (%) vs fluorescence of lysate
fluorescence of lysate (d) calibration curve of dye

FUTURE STUDIES
Figure 3 (a) Panel of TokyoMagenta used to select for a design with suitable photostability and cellular retention. (b) Mechanism of photo uncaging
• Future donors will deliver more elaborate aldehydes
and formaldehyde release to produce a fluorescent reporter

• Tokyo Magenta platform was used as it is compatible with a large majority of flagship probes for other • Later generations can also be delivered to specific
analytes and fluorescent proteins (typically green) organielles
• CF3 group (photoFAD-2 and photoFAD-3) allowed for enhanced photostability and chloros (photoFAD-3)
reduced the pKa of the uncapped Tokyo Magenta, preventing leakage of donor in the cell after release • Redder dyes coupled with upconverting nanoparticles will
• Acetal protection of the aldehyde is well-established and generalizable when elaborating to more complex allow for in vivo aldehyde delivery Figure 7 Further donors will have varied R groups for
the study of a variety of aldehydes in cellular systems
aldehydes