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Monoalkoxy BODIPYs--a fluorophore class for bioimaging.

Courtis AM, Santos SA, Guan Y, Hendricks JA, Ghosh B, Szantai-Kis DM, Reis SA, Shah JV, Mazitschek R - Bioconjug. Chem. (2014)

Bottom Line: These novel fluorescent dyes, which we term MayaFluors, are characterized by good aqueous solubility and favorable in vitro physicochemical properties.MayaFluors are readily accessible in good yields in a one-pot, two-step approach starting from well-established BODIPY dyes, and allow for facile modification with functional groups of relevance to bioconjugate chemistry and bioorthogonal labeling.Biological profiling in living cells demonstrates excellent membrane permeability, low nonspecific binding, and lack of cytotoxicity.

View Article: PubMed Central - PubMed

Affiliation: Center for Systems Biology and ∥Center for Human Genetic Research, Massachusetts General Hospital , 185 Cambridge Street, Boston, Massachusetts 02114, United States.

ABSTRACT
Small molecule fluorophores are indispensable tools for modern biomedical imaging techniques. In this report, we present the development of a new class of BODIPY dyes based on an alkoxy-fluoro-boron-dipyrromethene core. These novel fluorescent dyes, which we term MayaFluors, are characterized by good aqueous solubility and favorable in vitro physicochemical properties. MayaFluors are readily accessible in good yields in a one-pot, two-step approach starting from well-established BODIPY dyes, and allow for facile modification with functional groups of relevance to bioconjugate chemistry and bioorthogonal labeling. Biological profiling in living cells demonstrates excellent membrane permeability, low nonspecific binding, and lack of cytotoxicity.

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Cellular uptake and intracellular distributionand wash-out kineticsof MayaFluors 12a and 12b in comparisonto difluoro BODIPY 9. MD-MBA 231 cells (expressing mCherrylabeled histone H2B) were treated for 30 min at 10 μM followedby media replacement every 5 min to remove excess dye. Both CMA-BODIPYsshow relatively homogeneous even intracellular distribution comparedto BODIPY 9, which seems to primarily locate to endosomalstructures. 12a and 12b are efficientlywashed out within 10 min in contrast to 9, which is retained.Each image was acquired from an independent replicate well, to ensureno confounding photobleaching effects. (High resolution images areprovided in SI Figure 5.) All images wereacquired with identical microscopy settings and have not been processeddifferently to allow for direct comparison. All images are to scale.
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fig4: Cellular uptake and intracellular distributionand wash-out kineticsof MayaFluors 12a and 12b in comparisonto difluoro BODIPY 9. MD-MBA 231 cells (expressing mCherrylabeled histone H2B) were treated for 30 min at 10 μM followedby media replacement every 5 min to remove excess dye. Both CMA-BODIPYsshow relatively homogeneous even intracellular distribution comparedto BODIPY 9, which seems to primarily locate to endosomalstructures. 12a and 12b are efficientlywashed out within 10 min in contrast to 9, which is retained.Each image was acquired from an independent replicate well, to ensureno confounding photobleaching effects. (High resolution images areprovided in SI Figure 5.) All images wereacquired with identical microscopy settings and have not been processeddifferently to allow for direct comparison. All images are to scale.

Mentions: To evaluate the applicability ofCMA-BODIPYs for live cell imaging, we interrogated representativederivatives for their ability to penetrate mammalian cell membranesand their propensity for nonspecific background staining. MD-MBA-231cells expressing mCherry-histone H2B were incubated with 10 μMof 12a, 12b, and 9, respectively.Following a 30 min incubation at 37 °C, the culture medium wasreplaced every 5 min to remove excess dye. Live cells were imagedby multichannel fluorescence microscopy to assess penetration efficiencyand subcellular staining patterns and estimate wash-out kinetics (Figure 4, SI Figures 6, 7). Alltested compounds exhibited efficient cell uptake. However, cellularuptake kinetics significantly differ for CMA-BODIPYs 12a–c compared to BODIPY 9. Quantitativeintracellular studies demonstrated that even at short incubation periods(5 min) and low concentrations (100 nM) 12a–c rapidly enter cells to a high degree and washed out efficiently,which is consistent with the favorable logD of these compounds. Incontrast, the highly lipophilic parent compound, BODIPY 9, showed minimal cellular uptake after short incubation (SI Figure 8).


Monoalkoxy BODIPYs--a fluorophore class for bioimaging.

Courtis AM, Santos SA, Guan Y, Hendricks JA, Ghosh B, Szantai-Kis DM, Reis SA, Shah JV, Mazitschek R - Bioconjug. Chem. (2014)

Cellular uptake and intracellular distributionand wash-out kineticsof MayaFluors 12a and 12b in comparisonto difluoro BODIPY 9. MD-MBA 231 cells (expressing mCherrylabeled histone H2B) were treated for 30 min at 10 μM followedby media replacement every 5 min to remove excess dye. Both CMA-BODIPYsshow relatively homogeneous even intracellular distribution comparedto BODIPY 9, which seems to primarily locate to endosomalstructures. 12a and 12b are efficientlywashed out within 10 min in contrast to 9, which is retained.Each image was acquired from an independent replicate well, to ensureno confounding photobleaching effects. (High resolution images areprovided in SI Figure 5.) All images wereacquired with identical microscopy settings and have not been processeddifferently to allow for direct comparison. All images are to scale.
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fig4: Cellular uptake and intracellular distributionand wash-out kineticsof MayaFluors 12a and 12b in comparisonto difluoro BODIPY 9. MD-MBA 231 cells (expressing mCherrylabeled histone H2B) were treated for 30 min at 10 μM followedby media replacement every 5 min to remove excess dye. Both CMA-BODIPYsshow relatively homogeneous even intracellular distribution comparedto BODIPY 9, which seems to primarily locate to endosomalstructures. 12a and 12b are efficientlywashed out within 10 min in contrast to 9, which is retained.Each image was acquired from an independent replicate well, to ensureno confounding photobleaching effects. (High resolution images areprovided in SI Figure 5.) All images wereacquired with identical microscopy settings and have not been processeddifferently to allow for direct comparison. All images are to scale.
Mentions: To evaluate the applicability ofCMA-BODIPYs for live cell imaging, we interrogated representativederivatives for their ability to penetrate mammalian cell membranesand their propensity for nonspecific background staining. MD-MBA-231cells expressing mCherry-histone H2B were incubated with 10 μMof 12a, 12b, and 9, respectively.Following a 30 min incubation at 37 °C, the culture medium wasreplaced every 5 min to remove excess dye. Live cells were imagedby multichannel fluorescence microscopy to assess penetration efficiencyand subcellular staining patterns and estimate wash-out kinetics (Figure 4, SI Figures 6, 7). Alltested compounds exhibited efficient cell uptake. However, cellularuptake kinetics significantly differ for CMA-BODIPYs 12a–c compared to BODIPY 9. Quantitativeintracellular studies demonstrated that even at short incubation periods(5 min) and low concentrations (100 nM) 12a–c rapidly enter cells to a high degree and washed out efficiently,which is consistent with the favorable logD of these compounds. Incontrast, the highly lipophilic parent compound, BODIPY 9, showed minimal cellular uptake after short incubation (SI Figure 8).

Bottom Line: These novel fluorescent dyes, which we term MayaFluors, are characterized by good aqueous solubility and favorable in vitro physicochemical properties.MayaFluors are readily accessible in good yields in a one-pot, two-step approach starting from well-established BODIPY dyes, and allow for facile modification with functional groups of relevance to bioconjugate chemistry and bioorthogonal labeling.Biological profiling in living cells demonstrates excellent membrane permeability, low nonspecific binding, and lack of cytotoxicity.

View Article: PubMed Central - PubMed

Affiliation: Center for Systems Biology and ∥Center for Human Genetic Research, Massachusetts General Hospital , 185 Cambridge Street, Boston, Massachusetts 02114, United States.

ABSTRACT
Small molecule fluorophores are indispensable tools for modern biomedical imaging techniques. In this report, we present the development of a new class of BODIPY dyes based on an alkoxy-fluoro-boron-dipyrromethene core. These novel fluorescent dyes, which we term MayaFluors, are characterized by good aqueous solubility and favorable in vitro physicochemical properties. MayaFluors are readily accessible in good yields in a one-pot, two-step approach starting from well-established BODIPY dyes, and allow for facile modification with functional groups of relevance to bioconjugate chemistry and bioorthogonal labeling. Biological profiling in living cells demonstrates excellent membrane permeability, low nonspecific binding, and lack of cytotoxicity.

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