Oligoethylene glycol-substituted aza-BODIPY dyes as red emitting ER-probes

Article abstract of DOI:10.1039/c5ob01104c

This study features aza-BODIPY (BF₂-chelated azadipyrromethene) dyes with two aromatic substituents linked by oligoethylene glycol fragments to increase hydrophilicity of aza-BODIPY for applications in intracellular imaging. To prepare these, t

Full text of DOI:10.1039/c5ob01104c

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Oligoethylene glycol-substituted aza-BODIPY dyes
as red emitting ER-probes†
Cite this: DOI: 10.1039/c5ob01104c
Anyanee Kamkaew,a Sopida Thavornpradit,a,b Thamon Puangsamlee,a,b
Dongyue Xin,a Nantanit Wanichachevab and Kevin Burgess*a,c
This study features aza-BODIPY (BF2-chelated azadipyrromethene) dyes with two aromatic substituents
linked by oligoethylene glycol fragments to increase hydrophilicity of aza-BODIPY for applications in
intracellular imaging. To prepare these, two chalcones were attached α,ω onto oligoethylene glycol frag-
ments, then reacted with nitromethane anion. Conjugate addition products from this reaction were then
subjected to typical conditions for synthesis of aza-BODIPY dyes (NH4OAc, nBuOH, 120 °C); formation of
boracycles in this reaction was concomitant with creation of macrocycles containing the oligoethylene
glycol fragments. Similar dyes with acyclic oligoelythene glycol substituents in the same position were
used to compare the efficiencies of the intra- and inter-molecular aza-BODIPY forming reactions, and
the characteristics of the products. All the fluors with oligoethylene glycol fragments, i.e. cyclic or acyclic,
localized in the endoplasmic reticulum of a fibroblast cell line (WEHI-13VAR), the human pancreatic
cancer cell line (PANC-1, rough ER predominates) and human liver cancer cell line (HepG2, smooth ER
prevalent). These fluors are potentially useful for near IR (λmax emis at 730 nm) ER staining probes.
Received 2nd June 2015,
Accepted 25th June 2015
DOI: 10.1039/c5ob01104c
www.rsc.org/obc
phobic dyes, such as aza-BODIPY dyes, are difficult to solubilize
for cell-based experiments, and tend to accumulate non-selec-
Introduction
The most prevalent applications of BODIPY dyes are for aspects tively in hydrophobic membranes. One way to counter this trend
of intracellular imaging of various organelles,1–3 but aza- is to attach oligoethylene glycol fragments to the aryl substitu-
BODIPY dyes have attracted far less attention as organelle- ents of aza-BODIPY dyes. Thus, last year, Perez-Inestrosa and co-
stains.4–7 There may be several reasons for this discrepancy, but workers reported aza-BODIPY systems with two, or with four,
one is that 1,3,5,7-tetraaryl aza-BODIPYs are much more accessi- acyclic triethylene glycol substituents; their dyes were water-
ble than non-aryl substituted analogues,8 and those aromatic
substituents tend to make the fluors very hydrophobic. Hydro-
aDepartment of Chemistry, Texas A & M University, Box 30012, College Station,
TX 77842, USA. E-mail: burgess@tamu.edu
bDepartment of Chemistry, Faculty of Science, Silpakorn University, Nakorn Pathom,
73000, Thailand
cDepartment of Pharmacology, Faculty of Medicine, University of Malaya,
50603 Kuala Lumpur, Malaysia
†Electronic supplementary information (ESI) available: Synthesis of oligoethyl-
ene glycol-strapped aza-BODIPY, relative yield experiments, fluorescence spectro-
scopy of fluors and metal complexes, structural optimization by DFT
calculations and Cartesian coordinates, live cell imaging of fluors. See DOI:
10.1039/c5ob01104c
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soluble, and permeated into the cytoplasm of HeLa cells.9 asked regarding fluors of this kind, but in the event the most
Overall, this approach should give probes that behave differently interesting ones concerned their roles as near-infrared (near-
in intracellular imaging relative to aza-BODIPY dyes that are IR) probes for intracellular imaging. Specifically, we report that
made more hydrophilic via introduction of charged groups.
all four compounds 1 and 2 that were investigated in this
Research described in this paper features the aza-BODIPY study accumulate quite selectively in rough and smooth endo-
structures 1 and 2 having cyclic or acyclic oligoethylene glycol plasmic reticula (ER) of several cell types.
substituents, respectively. Several obvious questions might be
Results and discussion
Syntheses of the strapped fluors 1 and controls 2
Scheme 1 describes syntheses of the strapped systems 1. Reac-
tion of the chalcone 3 with the ethylene glycol containing frag-
ments shown afforded the α,ω-functionalized systems 4. These
Michael electrophiles were reacted with the enolate of nitro-
methane, and the conjugate addition products 5 formed were
condensed with ammonium acetate to form the azadipyrro-
methenes, then complexation of those with boron trifluoride
gave the target molecules 1.
The non-macrocyclic control molecules 2 were prepared via a
route similar to Scheme 1, but featuring ω-methyl oligoethylene
glycol derivatives (Schemes S1 and S2†). Isolated yields for com-
pounds 2a and 2b were 83 and 74%, respectively; these are signifi-
cantly higher than those shown in Scheme 1 for 1a and 1b. A
HPLC study was performed to confirm that this observation was
not an artifact of the isolation procedure. Thus purified samples
of the intermediate conjugate addition products 5b (strapped) and
6b (acyclic control) were mixed in a 1 : 2 ratio with excess NH4OAc
then heated to 120 °C for 24 h. HPLC was used to compare the
relative intensities of the corresponding azadipyrromethenes with
purified authentic samples of those (Fig. S1 and S2†); the acyclic
azadipyrromethene was the dominant product indicating that the
two strap lengths examined were detrimental to the yield.
Scheme 1 Syntheses of the strapped fluors 1.
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Physiochemical characteristics of the strapped fluors 1 and the
acyclic controls 2
Acetonitrile dissolved all the compounds, as did dichloro-
methane except 1a which had limited solubility in that
solvent. None of the products 1 or 2 were significantly soluble
in pure water, water with approximately 0.1% Triton X, or
aqueous sodium chloride at various concentrations.
The fact that 1 and 2 were freely soluble in aqueous media
made it difficult to determine how they might complex
metal salts. When 1 and 2 (at micromolar concentrations) were
dissolved in acetonitrile or dichloromethane and treated with
any of a variety of metal salts (including NaCl, KCl, LiCl, ZnCl2,
CaCl2, CuCl2, MgCl2, HgCl2, CoCl2; at ∼33 μM, see ESI†) then
there was no significant effect on the fluorescence wavelengths
of the fluors. Micromolar concentrations of Cu(ClO4)2 dimini-
shed the fluorescence of compounds 1 but this is unlikely to be
due to strong complexation because the fluorescence of the
non-macrocyclic controls 2 were similarly decreased.
Even aside from solubility issues, the experiments
described above seem to indicate that the strapped aza-
BODIPY dyes 1a and 1b are not good chelating agents. Conse-
quently, some calculations were performed to try to under-
stand this behaviour. Thus the molecules were first minimized
via molecular mechanics in a continuous dielectric of 80, then
further optimized using density functional theory {B3LYP
density functional, and 6-31G(d’) basis set}. This routine was
first applied to 15-crown-5 then Na+·15-crown-5 as a control.
Without sodium the crown converged to irregular confor-
mations, but the expected complex was formed with the metal
(see ESI†). Application of this strategy to 1a and 1b led to the
calculated energy minimized conformations shown in Fig. 1.
Sodium cation complexation appears to be possible for both
compounds. Predictably, there is less space in the cavity of 1a
and, perhaps less obvious, the constraints caused by the aryl
groups in 1a do not allow so much movement in the ethylene
glycol-derived strap. Together these phenomena mean that if
the sodium were sequestered by 1a then the metal would be
drawn towards the aza-BODIPY nitrogen. For 1b however, four
of the oxygen atoms in the strap can orientate to complex the
Na+ atom, but the fifth oxygen is unsuitably aligned.
Fig. 1 Optimized minimum energy conformations of: (a) 1a and Na+·1a;
and, (b) 1b and Na+·1b. DFT B3LYP/6-31G(d’) throughout.
All compounds in the series 1 and 2 have very similar
absorption and fluorescence maxima. Moreover, the quantum
yields of all the compounds were similar (in the 0.26–0.29
range), hence restriction of the 1,7-aryl substituents with an
ethylene glycol linker did not increase the quantum yields of
1 over 2. We were somewhat surprised by this observation, so
checked it by making solutions of all the compounds with
almost identical absorbance at the λmax wavelength (∼688 nm)
and measuring their fluorescence intensities (Fig. 2a). In the
event, these solutions had almost the same fluorescence inten-
sities, consistent with their having almost identical quantum
yields as asserted above.
Fig. 2b shows UV absorbance and fluorescence spectra for
equimolar solutions of the four compounds 1 and 2, where
fluor 1b is significantly more brilliant than the others. The
Fig. 2 (a) Quantum yields of all four compounds 1 and 2 are very
similar; however, (b) the strapped system 1b is more brilliant because it
has a higher molar extinction coefficient. CH2Cl2 solutions throughout.
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probe is sold by Life Technologies who also market a BODIPY
based ER-tracker that emits around 615 nm (https://www.
lifetechnologies.com/order/catalog/product/E34250).10 All four
aza-BODIPY dyes in this study localize in the same organelle
as that ER-tracker, and it is potentially useful that they emit at
a complementary λmax emis (ca. 730 nm) that could be observed
simultaneously.
Incubation of probes 1 and 2 with two other cell lines was
investigated to explore the generality of their affinity for the
ER. PANC-1 human pancreatic cancer cells are known to have
a prevalent rough ER,11 whereas smooth ER predominates for
human liver cancer HepG2 cells.12,13 Fig. 4 shows both these
cell lines were stained just as the fibroblast cells by illustrative
probe 1b (the other three probes gave similar images,
Fig. S11†), except there was one difference. The smooth ER of
the HepG2 liver cells appeared as just punctates, just as others
have observed for the smooth ER.12
Table 1 Electronic spectra of compounds 1 and 2 in dichloromethane
(9.2 × 10−6 M): salient characteristics
a
λmax abs
(nm)
εmax
λmax emis
(nm)
Compound
(104 M−1 cm−1
)
Φfluor
b
1a
1b
2a
2b
688
686
690
688
2.55
8.22
2.51
5.75
725
725
725
725
0.26 0.003
0.29 0.001
0.29 0.004
0.28 0.004
a Excited at 650 nm. b Zn-phthalocyanine is a standard (Φfluor = 0.30).
quantum yields of all the compounds are almost the same (see
above) so the relative brilliance of 1b can be attributed to its
higher extinction coefficient (Table 1).
Behaviour of fluors 1 and 2 as near-IR ER-stains
All the probes 1 and 2 were tested in all three cell types
(the fibroblasts, pancreatic cancer, and liver cancer cells) for
colocalization with LysoTracker Green, and with MitoTracker
Green, but these did not colocalize. Moreover, there was
no significant accumulation of the probes in the cell
membranes.
Localization of fluors 1 and 2 in WEHI-13VAR fibroblast cells
was investigated first. Incubation with 10 μM of each sample
for 3 h, for all the fluors, showed the fluorescence localized in
the ER, and co-localized with a blue ER-tracker dye (Fig. 3 for
1b and S10† for all the other probes). That blue standard
Fig. 3 Images of WEHI-13VAR cells. (a) Under a bright-field (DIC image); (b) with ER tracker; (c) with fluor 1b; and, (d) overlay of images (a–c).
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Fig. 4 Parallel images for staining PANC-1 (left) and HepG2 cells (right) with: (a and b) ER tracker; (c and d) with fluor 1b. (e and f) Shows the
overlay of images for each cell type.
et al.4 for (positively charged) ammonium salts of aza-BODIPY
Conclusion
dyes that stained cell membranes, the cytoplasm, and the ER.
Oligoethylene glycol substituents on the aza-BODIPY probes 1 It is also different to the observations of Perez-Inestrosa’s tri-
and 2 probably increase their hydrophobicity enough to over- ethylene glycol substituted aza-BODIPY dyes that are neutral
come non-selective accumulation in all hydrophobic mem- like the ones reported here, but that localize in the cytoplasm
branes, and promote accumulation in the rough and smooth of HeLa cells.9 Overall, these comparisons between different
ER. This observation is different to those made by X. Chen aza-BODPY dyes reflect their potential to be directed to
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different organelles via relatively small changes to the peri-
phery of the dye core. Probes 1 and 2 are complementary to ER
stains that fluoresce in the blue and green region,11–14 and
they should facilitate clear observations of blue- and green-
labeled molecules in the ER without significant cross-talk.
In this work the oligoethylene glycol substituents used con-
tained four or less repeats. This was not enough to render the
fluors significantly water-soluble, and the size and shape of
the crown-like cavities was not conducive to complexation of
metals, especially relative to conventional crown ethers
(cf. stability constant of Na+·15-crown-5 is ca. 6.2).15 However,
since the organelle staining properties of fluorophores tend
to be governed by lipophilicity and charge characteristics,
we predict variations on organelle-staining properties
could be induced by varying the length of the ethylene
glycol substituents, and conceivably by their acyclic/cyclic
characteristics.
Notes and references
1 R. Ziessel, G. Ulrich and A. Harriman, New J. Chem., 2007,
31, 496–501.
2 A. Loudet and K. Burgess, in Handbook of Porphyrin Science:
With Applications to Chemistry, Physics, Materials Science,
Engineering, Biology and Medicine, ed. K. Kadish, K. Smith
and R. Guilard, World Scientific, 2010, p. 203.
3 F. Csende, F. Miklos and G. Stajer, Curr. Org. Chem., 2012,
16, 1005–1050.
4 X.-X. Zhang, Z. Wang, X. Yue, Y. Ma, D. O. Kiesewetter and
X. Chen, Mol. Pharmaceutics, 2013, 10, 1910–1917.
5 J. Murtagh, D. O. Frimannsson and D. F. O’Shea, Org. Lett.,
2009, 11, 5386–5389.
6 M. Tasior, J. Murtagh, D. O. Frimannsson, S. O. McDonnell
and D. F. O’Shea, Org. Biomol. Chem., 2010, 8, 522–525.
7 M. J. Hall, L. T. Allen and D. F. O’Shea, Org. Biomol. Chem.,
2006, 4, 776–780.
The authors declare no competing financial interests.
8 M. J. Hall, S. O. McDonnell, J. Killoran and D. F. O’Shea,
J. Org. Chem., 2005, 70, 5571–5578.
9 D. Collado, Y. Vida, F. Najera and E. Perez-Inestrosa, RSC
Adv., 2014, 4, 2306–2309.
Acknowledgements
10 L. Cole, D. Davies, G. J. Hyde and A. E. Ashford, J. Microsc.,
2000, 197, 239–248.
Financial support for this project was provided by the National
Institutes of Health (GM087981), and the Robert A. Welch
Foundation (A-1121) and the High Impact Research (HIR
(UM.C/625/1/HIR/MOHE/MED/17 & UM.C/625/1/HIR/MOHE/
MED/33) from the Ministry of Higher Education, Malaysia and
partial support from Grant RSA 5680034 from the Thailand
Research Fund (TRF) and Faculty of Science, Silpakorn Univer-
sity, Thailand. TAMU/LBMS-Applications Laboratory provided
mass spectrometric support. The NMR instrumentation at
Texas A&M University was supported by a grant from the
National Science Foundation (DBI-9970232) and the Texas
A&M University System. The Olympus FV1000 confocal micro-
scope acquisition was supported by the Office of the Vice Presi-
dent for Research at Texas A&M University.
11 X.-Q. Chen, M. D. Sans, J. R. Strahler, A. Karnovsky,
S. A. Ernst, G. Michailidis, P. C. Andrews and
J. A. Williams, J. Proteome Res., 2010, 9, 885–896.
12 C. Zuber, J. H. Cormier, B. Guhl, R. Santimaria,
D. N. Hebert and J. Roth, Proc. Natl. Acad. Sci. U. S. A.,
2007, 104, 4407–4412.
13 M. M. Galteau, B. Antoine and H. Reggio, EMBO J., 1985, 4,
2793–2800.
14 S. Arai, S.-C. Lee, D. Zhai, M. Suzuki and Y. T. Chang, Sci.
Rep., 2014, 4, 6701.
15 D. M. Dishong and G. W. Gokel, J. Org. Chem., 1982, 47,
147–148.
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