Synthesis and cellular properties of Near-IR BODIPY-PEG and carbohydrate conjugates

Article abstract of DOI:10.1016/j.tet.2013.03.082

A series of red and near-IR fluorescent BODIPY conjugates, containing either one or two indolylstyryl groups at the 3- and/or 5-positions and a low molecular weight PEG or carbohydrate group, were synthesized using a Cu(I)-catalyzed azide-alkyne Huisgen cycloaddition ('click' reaction). All BODIPY conjugates show emission and fluorescence quantum yields in the ranges of 642-732 nm and 0.24-0.56, respectively, and Stokes' shifts of ca. 30 nm. In vitro cellular investigations using human carcinoma HEp2 cells showed that all BODIPYs are non-toxic, both in the absence and presence of light (1 J/cm ²), up to 100 μM concentrations. The PEG and galactose conjugates were more efficient at cell internalization than the unconjugated BODIPYs. The mono-indolylstyryl-BODIPYs showed higher cellular uptake (about five-fold) compared with the di-indolylstyryl-BODIPYs, and higher fluorescence quantum yields.

Full text of DOI:10.1016/j.tet.2013.03.082

Accepted Manuscript
Synthesis and Cellular Properties of Near-IR BODIPY-PEG and Carbohydrate
Conjugates
Timsy Uppal, N.V.S. Dinesh, K. Bhupathiraju, M.Graç a H. Vicente
PII:
S0040-4020(13)00471-7
10.1016/j.tet.2013.03.082
TET 24173
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 15 January 2013
Revised Date: 18 March 2013
Accepted Date: 20 March 2013
Please cite this article as: Uppal T, Dinesh NVS, Bhupathiraju K, Vicente MGH, Synthesis and Cellular
Properties of Near-IR BODIPY-PEG and Carbohydrate Conjugates, Tetrahedron (2013), doi: 10.1016/
j.tet.2013.03.082.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Graphical Abstract
Leave this area blank for abstract info.
Syntheses and Cellular Properties of Near-
IR BODIPY-PEG and Carbohydrate
Conjugates
Timsy Uppal, N. V. S. Dinesh K. Bhupathiraju and M. Graça H. Vicente*
Department of Chemistry, Louisiana State University,
Baton Rouge, LA 70803, USA.
A series of novel BODIPY conjugates, containing either one or two indolylstyryl
moieties and a PEG or carbohydrate group were synthesized and investigated.
The conjugates show near-IR emissions (642-732 nm), quantum yields in the
range 0.24-0.56, no cytotoxicity and high cellular permeability in human
carcinoma HEp2 cells.

ACCEPTED MANUSCRIPT
Tetrahedron
1
TETRAHEDRON
Pergamon
Synthesis and Cellular Properties of Near-IR BODIPY-PEG and
Carbohydrate Conjugates
Timsy Uppal, N. V. S. Dinesh K. Bhupathiraju and M. Graça H. Vicente*
Department of Chemistry, Louisiana State University, Baton Rouge, LA 70803, USA.
Abstract—A series of red and near-IR fluorescent BODIPY conjugates, containing either one or two indolylstyryl groups at the 3- and/or-
5- positions and a low molecular weight PEG or carbohydrate group, were synthesized using a Cu(I)-catalyzed azide-alkyne Huisgen
cycloaddition (“click” reaction). All BODIPY-conjugates show emission and fluorescence quantum yields in the ranges of 642-732 nm and
0.24-0.56, respectively, and Stokes’ shifts of ca. 30 nm. In vitro cellular investigations using human carcinoma HEp2 cells showed that all
BODIPYs are non-toxic, both in the absence and presence of light (1 J/cm2), up to 100 µM concentrations. The PEG and galactose
conjugates were more efficient at cell internalization than the unconjugated BODIPYs. The mono-indolylstyryl-BODIPYs showed higher
cellular uptake (about 5-fold) compared with the di-indolylstyryl-BODIPYs, and higher fluorescence quantum yields.
Keywords: BODIPY, click chemistry, fluorescence, cytotoxicity, uptake, subcellular distribution. © 2013 Elsevier Science. All rights
reserved.
1. Introduction
stability, lengthy synthetic routes, poor solubility and
tendency for aggregation in aqueous media that surrounds a
biomolecule. Furthermore, there are few choices in dye sets
for multiplexed applications, and limited reactive
functional groups that would allow ready modification of
the fluorophore, which in turn results in reduced target
accumulation and biological efficacy of the dye. Therefore
intense research efforts have been directed towards the
synthesis of new near-IR emissive probes with desired
photophysical as well as biological properties. Recent
efforts have been focused on the development of 4,4-
Fluorescence-based in vivo imaging has emerged as a
promising real-time, non-invasive and high-resolution
technique that uses fluorescent probes to visualize normal
and abnormal biological processes.1-3 Among the currently
used technologies, near-IR fluorescence imaging in the
range 700-900 nm has attracted particular attention, due to
the optical sensitivity associated with the “biological
window”, including minimal interference from endogenous
chromophores, reduced light scattering, increased photon
penetration through tissue and low photodamage to the
cells or tissues under observation.4,5 As a result, cellular
and tissue imaging relies greatly on the development of
stable, highly specific and sensitive near-IR imaging
probes. A general and simple approach for improving the
target-specific accumulation of imaging probes is to
conjugate the dyes or dye-encapsulated nanoparticles with
specific ligands (such as amino acids, peptides, proteins,
carbohydrates and other small molecules) for targeting
imaging.6 Targets for the fluorescent imaging probes are
usually over-expressed cell membrane receptors (such as
the human epidermal growth factor receptor, vascular
endothelial growth factor receptor, cell-specific membrane
antigens, or molecules that are abundant in the extracellular
matrix (such as integrins and matrix metalloproteinases).7,8
Along with the target-specificity and stringent spectral
characteristics, including near-IR fluorescence emission
with high quantum yield, the fluorescent probes that are
small, amphiphilic and biocompatible are often potential
candidates for in vivo bioimaging.9 The major classes of
currently used synthetic fluorophores include the cyanines,
rhodamines and oxazines;10 While these dye systems
provide a host of functional groups that can be used for
covalent attachment to various target molecules, many have
limited biological utility due to poor photochemical
difluoro-4-bora-3a,4a-diaza-s-indacene
(abbreviated
hereafter as BODIPY)-based dyes due to their unique
spectral properties, which include intense absorption and
emission profiles, high molar absorption coefficients, high
fluorescence quantum yields, long fluorescence lifetimes
and moderate Stokes’ shift (ca. 30 nm).11-13 Their high
photostability, generally low toxicity and ease of synthetic
modifications make BODIPY-type dyes particularly
promising
candidates
for
biological
imaging
applications.14,15 We have recently reported the synthesis of
π-extended red-fluorescent BODIPY dyes (λem up to 686
nm) of low cytotoxicity and high cell membrane
permeability as promising fluorophores for biological
applications.16,17 We now report the synthesis and
investigation of
a new series of near-IR BODIPY
conjugates that emit in the range 642-732 nm, bearing a
stable 1,2,3-triazole ring linked to either a low molecular
weight PEG group or a carbohydrate moiety, for enhanced
cellular targeting and permeability. The Cu(I)-catalyzed
azide-alkyne cycloaddition or “click” reaction used for the
conjugations is a highly selective and versatile reaction,
which employs mild aqueous conditions compatible with a
variety of functional groups and the BODIPY scaffold.18,19

ACCEPTED MANUSCRIPT
2
Tetrahedron
2. Results and Discussion
glacial acetic acid in refluxing toluene, affording both the
mono- and di-styryl BODIPYs 4 and 5 in 36 and 55.6%
yield, respectively. The use of a Dean-Stark apparatus for
this condensation proved to be essential for ensuring the
removal of water formed in the reaction, thus shifting the
equilibrium in favor of the double condensation product,
BODIPY 5.22 The indole-3-carbaldehyde was used for π-
conjugation extension as it is anticipated to increase the
cellular uptake and biological efficacy of BODIPYs due to
its resemblance to tryptophan, a naturally occurring amino
acid and an important component of many enzymes,
peptides and proteins.23 On the other hand, the use of a
PEG group as carrier or covalently attached to a
The synthetic strategy to near-IR BODIPY conjugates 6
and 7 is based on the Knoevenagel condensation followed
by an azide/alkyne “click” reaction, as shown in Scheme 1.
The starting BODIPY 3 containing an alkyne group was
synthesized in 94% overall yield using a three-step one-pot
approach often used in BODIPY synthesis,11,20 starting
from p-propargyloxy-benzaldehyde 2 and two equiv. of 3-
ethyl-2,4-dimethylpyrrole 1. p-Propargyloxy-benzaldehyde
2
was prepared in quantitative yield from p-
hydroxybenzaldehyde via O-alkylation using propargyl
bromide and potassium carbonate in acetone, according to a
literature procedure.21
Table 1. Products and yields obtained in the “click” reaction.
Product
Yield
70 %
98 %
99 %
15 %
20 %
6a
6b
6c (R = Ac)
7a
7b
7c (R = H)
18 %
57 %
7c (R = Ac)
7d
23 %
biologically active molecule is a commonly used strategy
for increasing agent delivery to target tissues, by enhancing
aqueous solubility, serum life, and cellular permeability.24
Therefore, two low molecular weight PEG groups, tri- and
hexa-ethylene glycol were chosen for conjugation to
BODIPYs 4 and 5. The azide-terminated PEGs were
prepared from commercially available tri- or hexa-ethylene
glycol using a two-step reaction, involving selective
mesitylation in the presence of excess mesityl chloride,
followed by nucleophilic substitution by sodium azide at
room temperature (see Supplementary data).25,26 The
conjugation of the tri- and hexa-PEG groups to BODIPYs 4
and 5 was performed using a Cu(I)-catalyzed azide-alkyne
cycloaddition, known as “click” reaction.18,19 The mild
aqueous conditions required for this reaction offer distinct
advantages over other types of conjugations, such as ester
or amide bond formations. The conjugation of PEG-groups
to the BODIPY scaffold was performed using modified
conditions27 for solution-phase “click” reaction, in the
presence of copper(II) sulfate, copper(0) and sodium
ascorbate in THF/water (3:1). The elemental copper serves
as a convenient reductant for Cu(II) in aqueous solution to
generate Cu(I), which is a powerful catalyst for the
formation of 1,2,3-triazoles from terminal alkynes and
Scheme 1. Synthesis of near-IR BODIPY conjugates 6 and 7.
The introduction of two styryl groups on BODIPY 3 was
accomplished using electron-rich indole-3-carbaldehyde
under Knoevenagel reaction conditions, piperidine and

ACCEPTED MANUSCRIPT
Tetrahedron
3
azides.28 The reaction mixture was heated for about 6 h at
70 °C, under inert gas atmosphere. Once the reaction was
complete (upon disappearance of starting material), the
mixture was cooled to room temperature and partitioned
between ethyl acetate and water. The aqueous layer was
extracted with ethyl acetate to remove copper salts, and the
combined organic layers were dried over Na2SO4. The
solvent was evaporated under reduced pressure and the
pegylated BODIPYs were purified by silica gel preparative
TLC. The yields obtained in the “click” reaction are shown
in Table 1. The lower yields obtained for BODIPYs 7a,b
are probably due to the relatively higher hydrophobicity of
these BODIPYs compared with 6a,b, and their more
difficult purification and isolation.
as doublets in the aromatic region and the resonance
corresponding to the CH proton in the terminal position of
the propargyl group as a singlet at δ 2.52 ppm. In addition,
the two equivalent protons of the propargylic methylene
group were identified as a broad doublet centered at 4.72
1
ppm. On the other hand, the H NMR spectra of BODIPYs
4 and 5 showed several interesting features. For BODIPY
4, bearing a single indolylstyryl arm, characteristic proton
signals belonging to the NH (10.7 ppm), fused aryl and α-
pyrrolic (7.74 ppm) positions of the indole moiety were
observed. In addition, the vinylic protons in the 3-position
appeared at 7.91 and 7.65 ppm. In the case of the
symmetrically substituted distyryl-BODIPY 5, the vinylic
protons in the 3 and 5-positions appear as two pairs of trans
1
coupled protons, the most shielded at 8.05 ppm. The H
To demonstrate the functional group tolerance of azides
with BODIPYs 4 and 5, as well as to increase the solubility
and cellular targeting of the resulting conjugates,
commercially available azido-sugars, 1-azido-1-deoxy-β-D-
glucopyranoside and 1-azido-1-deoxy-β-D-lactopyranoside
were used for “click” ligation. Azido-sugars have
frequently been used for “click” labeling of surface
glycoproteins29 and of porphyrin-based photosensitizers.30
We anticipated that the targeted glucose- and lactose-
conjugated BODIPYs of low phototoxicity may find
application as fluorescent probes for galectin proteins, that
are often over-expressed in tumor cells.31 The galectins are
a family of small soluble proteins that contain an highly
conserved carbohydrate recognition domain (CRD).
Among the galectins, Galectin-1 (Gal-1) and Galectin-3
(Gal-3) have been the most studied because they are
abundant in tumor cells, promoting tumor growth and
development, angiogenesis and metastasis. Nevertheless,
since the mechanisms of action of galectins are still poorly
understood, carbohydrate-based fluorescent probes of low
phototoxicity can be particularly useful for detection and
investigation of galectin-mediated processes in tissues.32
Under similar “click” reaction conditions as described
above, the BODIPY cycloadducts 7c (R=H) and 7d were
obtained in 18 and 23% yields, respectively, and were
highly soluble in both polar organic and aqueous solvents,
which complicated their purification and resulted in low
yields of isolated product. To facilitate purification,
NMR analysis of the cycloadducts 6a-c (Supplementary
data, Figures S7, S9 and S11) displayed the most
characteristic signal for the proton on the triazole ring at
8.16 ppm as a broad singlet, signals of protons from the
PEG group between 3.50-3.94 ppm (6a and 6b) and singlet
peaks at 2.21, 2.05, 2.03 and 1.90 ppm due to four acetyl
groups on the sugar moiety (6c), respectively. Well-
resolved 1H NMR spectra of PEG- and carbohydrate-
functionalized cycloadducts 7a-d showed the characteristic
proton on the triazole ring at approximately 8.18 ppm
(Supplementary data, Figures S13, S15, S17, S19 and S20).
In addition, for 7a and 7b, the signals of the PEG protons
appeared between 3.51-4.61 ppm. For 7c (R = Ac), the four
acetyl groups appeared as four singlets at 2.21, 2.05, 2.03
and 1.96 ppm, respectively. The glucose and lactose units
in cycloadducts 7c (R = H) and 7d were found to resonate
in a very crowded region of the NMR spectrum, between
3.31-4.26 ppm and 3.49-4.67 ppm, respectively.
The absorbance and fluorescence emission spectra of
BODIPYs 3, 4, 5, 6a-c and 7a-d were recorded in DMSO
at room temperature and the results obtained are shown in
Figure 1 and summarized in Table 2. In the absorption
spectra, a strong band centered at 525, 613 and 703 nm for
BODIPYs 3, 4 and 5, respectively, is assigned to the S0-S1
transition. In the wavelength region below the absorption
maximum, there is a “shoulder” due to the 0-1 vibrational
transition, and a second higher energy absorption maximum
peak due to the S0-S2 transition. These three peaks
constitute the main spectral characteristics of the absorption
spectrum of the BODIPY system, as previously
reported.17,33 All BODIPYs showed absorptions with high
molar extinction coefficients (log ε = 4.1-4.5) and narrow
fluorescence emission bands. Upon excitation at 480, 575
and 660 nm, BODIPYs 3, 4 and 5 showed fluorescence
emissions centered at 534, 644 and 732 nm, respectively,
and fluorescence quantum yields in the range of 0.21-
0.70.34 The quantum yields decreased with the successive
replacement of the methyl groups in the 3,5-positions of
BODIPY 3 with the indolylstyryl moieties; therefore the
mono-indolylstyryl BODIPY derivatives showed higher
quantum yields (in the order of 0.6) than the di-
indolylstyryl derivatives (in the order of 0.3), due to the
higher flexibility of the styryl arms compared with the
methyl group. In addition, and as expected, styryl-
derivatization caused a large bathochromic shift in the
commercially
available
1-azido-1-deoxy-β-D-
galactopyranoside tetraacetate was used in the “click”
reaction. The acetyl groups protect the reactive hydroxyl
groups and facilitate product purification, and are
subsequently cleaved by carboxyesterase enzymes within
cells. The BODIPY-carbohydrate cycloadduct 6c (R= Ac)
was obtained in quantitative yield, while 7c (R = Ac) was
isolated in 57% yield, using the optimized reaction
conditions described above. These results further show the
compatibility and efficiency of the “click” reaction for
conjugation to the BODIPY platform.
The structures of all BODIPYs and their cycloadducts were
confirmed by 1H and 13C NMR, HRMS (ESI-TOF) or
MALDI-TOF mass spectrometry and UV-Vis and
fluorescence spectroscopies (see Table
2
and
Supplementary data). The 1H NMR spectrum of BODIPY 3
showed the characteristic ortho- and meta-phenyl protons

ACCEPTED MANUSCRIPT
4
Tetrahedron
absorption and emission spectra, of about 100 nm per styryl
arm. For instance, the single Knoevenagel condensation of
BODIPY 3 to produce mono-indolylstyryl-BODIPY 4 lead
to nearly a 90 nm bathochromic shift in the absorption and
emission bands, whereas double Knoevenagel condensation
to generate di-indolylstyryl BODIPY 5 led to another
approximate 100 nm red-shift. The spectral properties of
BODIPY conjugates 6a-c and 7a-d in DMSO showed small
differences (1-3 ppm) among each other and the
corresponding precursor BODIPYs 4 and 5, demonstrating
that the incorporation of PEG and carbohydrate moieties
via the cycloaddition reaction does not alter the spectral
properties of the BODIPY conjugates.27 All the BODIPYs
(except 3) showed relatively high Stokes’ shifts, in the
range of 27-31 nm.
(Promega), at concentrations up to 100 µM for each
BODIPY and conjugate. Higher concentrations were not
evaluated due to compound precipitation upon dilution of
stock solutions of BODIPYs in 1% Cremophor EL (a non-
ionic solubilizer)/DMSO into aqueous solutions. The
results obtained are shown in Figures 2 and 3, and in
Supplementary data, Figures S21 and S22. All BODIPYs
showed no cytotoxicity up to 100 µM concentrations (IC50
> 100 µM), both in the presence or the absence of light (1
J/cm2). The low dark and phototoxicity observed for all
BODIPYs
is
in
agreement
with
previous
investigations,16,17,35 and warrants further investigations of
BODIPY-based dyes as imaging probes.
Table 2. Spectral properties of BODIPYS in DMSO at room temperature.
[a]. The fluorescence quantum yields for BODIPYs 3 (λexc = 490 nm), 4,
6a-c (λexc = 575 nm), 5 and 7a-d (λexc = 660 nm) were calculated using,
respectively, rhodamine 6G (0.80 in methanol), cresyl violet perchlorate
(0.54 in ethanol), and ZnPc (0.28 in DMF) as the references32 and a PTI
Quantum Master4/2006SE spectrofluorimeter.
BODIPY
Abs
Emissiona
Stokes’
shift
(nm)
Φfb
Log ε
λmax (nm)
λmax (nm)
(1M-
1.cm-1)
4.44
3
4
525
613
613
613
613
702
704
702
703
703
703
534
643
642
644
644
732
731
730
732
732
731
0.75
0.56
0.55
0.55
0.56
0.28
0.24
0.25
0.26
0.26
0.26
9
30
29
31
31
30
27
28
29
29
28
4.53
4.49
4.45
4.77
4.38
4.15
4.15
4.56
4.56
4.56
6a
6b
6c(Ac)
5
7a
7b
7c(H)
7c(Ac)
7d
Figure 1: Normalized (a) absorption (1.5 x 10-5 M), and (b) emission (1.5
x 10-6 M) spectra of BODIPYs 3 (black), 4 (red), 5 (dark yellow), 6a (dark
cyan), 6b (magenta), 6c (R=Ac, blue), 7a (olive), 7b (royal), 7c (R = Ac,
navy), 7c (R = H, wine) and 7d (orange) in DMSO at room temperature.
The in vitro biological properties of BODIPYs 4, 5, 6a-c
and 7a-d including the time-dependent cellular uptake,
cytotoxicity and intracellular localization, were investigated
in human carcinoma HEp2 cells. The cytotoxicity was
evaluated using the Cell Titer Blue viability assay

ACCEPTED MANUSCRIPT
Tetrahedron
5
3 shows the fluorescent patterns observed for conjugates
6b,c and 7b,c (R = Ac) (see also Supplementary data,
Figures S23-27). Co-localization experiments were
conducted using several organelle tracers, however, all
BODIPYs were found to localize mainly in the cell ER, as
seen in the overlays of Figure 3. This result is also in
agreement with previous observations, showing that
BODIPY-based molecules tend to localize within the
ER.17,36
Figure 2: Time-dependent uptake of BODIPYs 4 (grey), 5 (light green),
6a (red), 6b (orange), 6c (R = Ac, brown), 7a (purple), 7b (black), 7c (R =
Ac, dark green), 7c (R = H, light blue) and 7d (blue), at 10 µM by HEp2
cells.
Significant differences were also observed in the cellular
uptake of the BODIPYs and their conjugates, as shown in
Figure 2. In general, the mono-indolylstyryl BODIPYs 4
and 6a-c accumulated within the cells about 5 times more
than the corresponding di-indolylstyryl-BODIPYs, at all
the time points investigated. This may be attributed to the
lower molecular weight, increased amphiphilicity and
enhanced solubility of conjugates 6a-c compared with 7a-
d. Additionally, the PEG-functionalized BODIPYs showed
significantly higher cellular uptake compared with the
carbohydrate-functionalized BODIPYs, for both series,
except for conjugate 7c (R = H) bearing a glucose
functionality, which showed similar uptake as the PEG-
conjugates 7a,b. These results are in good agreement with
previous studies indicating that the use of PEG groups
enhance the hydrophobic/hydrophilic interactions of the
conjugates with the cell membrane and enhance their
cellular uptake.17,25,35 For the BODIPYs 4 and 6a-c series,
all conjugates accumulated within cells to a higher extent
than the unconjugated BODIPY, at all times. While the
uptake of 6a and 6c was similar in the first 4 h, after 24 h
the amount of the PEG conjugate 6a within cells was about
25% higher than that for the carbohydrate conjugate 6c. For
the BODIPYs 5 and 7a-d series, the uptake followed the
order 7b > 7c (R = OH) > 7a > 7d > 7c (R = Ac). The
highest uptake in this series was observed for the hexa-PEG
conjugate 7b, at times > 2h. On the other hand, the tri-PEG
conjugate 7a accumulated the fastest during the first 2h
after which it reached a plateau. Interestingly, conjugate 7c
containing the tetraacetate-protected galactose accumulated
the least within cells, about 5 times less than 7c (R = H),
suggesting that the unprotected carbohydrate moiety is
more efficient at cell internalization, probably due to
increased amphiphilicity, and of receptor-mediated
recognition processes.
Figure 3: Subcellular distribution of BODIPYs 6b,c and 7b,c in HEp2
cells at 10 µM for 6 h. (a) 6b fluorescence, (b) 6c (R = Ac) fluorescence,
(c) 7b fluorescence, (d) 7c (R = Ac) fluorescence, (e-h) ER Tracker Green
fluorescence, (i-l) overlays of ER Tracker and BODIPYs fluorescence.
Scale bar: 12 µm.
3. Conclusions
In this report, we describe the synthesis of a new series of
red and near-IR fluorescent BODIPY-PEG and BODIPY-
carbohydrate conjugates, containing either one or two
indolylstyryl- arms at the 3- and/or 5-positions, using a
Knoevenagel condensation followed by a rapid and
convenient
Cu(I)-catalyzed
azide-alkyne
Huisgen
cycloaddition (i.e. “click” reaction). All the new BODIPY-
1
based conjugates were fully characterized using H NMR,
13C NMR, high-resolution ESI-TOF or MALDI-TOF mass
spectrometry, UV-Vis and fluorescence spectroscopies.
The BODIPY-PEG and BODIPY-carbohydrate conjugates
display strong absorptions and emissions in the ranges of
613-704 and 642-732 nm, respectively, high molar
The main sites of subcellular localization of the BODIPYs
were investigated by fluorescence microscopy, upon
exposure of HEp2 cells to 10 µM BODIPY for 6 h. Figure

ACCEPTED MANUSCRIPT
6
Tetrahedron
extinction coefficients (4.1-4.6), fluorescence quantum
yields (0.24-0.56) and Stokes’ shifts (28-31 nm).
Furthermore, the cycloaddition strategy had negligible
effects on the spectral characteristics of the BODIPY-PEG
and BODIPY-carbohydrate conjugates. In vitro cellular
investigations using human carcinoma HEp2 cells indicate
that all BODIPY conjugates are cell permeable, localize
preferentially in the cell ER, and are non-cytotoxic, both in
the absence and presence of light (1 J/cm2) up to 100 µM
concentrations investigated. The monoindolylstyryl-
BODIPY conjugates (6a-c) have the highest quantum
yields (0.6) and were the most efficiently taken up by the
HEp2 cells, accumulating nearly 5 times more than the di-
indolylstyryl BODIPY conjugates (7a-d). Since a variety of
azido-PEGs and carbohydrate azides are readily available,
this flexible modular synthesis is expected to provide a
versatile platform for vast arrays of functionalized
BODIPYs possessing potential utility in in vivo
fluorescence imaging.
mmole, 1.2 equiv.) in dry dichloromethane (10 mL) was
added via syringe at 0 C. After stirring for 20 min at room
°
temperature, triethylamine (4 mL, 7 equiv.) and BF3•Et2O
°
(5.6 mL, 11 equiv.) were added dropwise at 0 C over a
period of 10 min and the mixture was stirred for 10 h at
room temperature. Once TLC indicated reaction
completion, the mixture was passed through a short pad of
silica gel to remove oxidized dipyrromethene derivatives
and starting materials. The solvent was removed under
reduced pressure and the residue was dissolved in
dichloromethane (30 mL) and kept for overnight stirring at
room temperature to decompose any unreacted BF3•Et2O.
The reaction mixture was extracted with 0.1 M HCl (2 x 30
mL) to remove excess DDQ, brine (1 x 30 mL) and dried
over anhydrous Na2SO4. The organic extracts were
collected, dried over Na2SO4 and the solvent was
evaporated. The crude product was purified by column
chromatography using petroleum ether/dichloromethane
(1/1, v/v) for elution. The orange-green fluorescent
fraction containing the desired BODIPY 3 was collected
and recrystallized from dichloromethane/hexane mixtures
to yield the title compound as an orange-red solid, yield:
1.656 g, 94%. 1H NMR (400 MHz, CDCl3) δ; 7.19 (d, J =
8.8 Hz, 2H), 7.08 (d, J = 8.8 Hz, 2H), 4.76 (d, J = 2.0 Hz,
2H), 2.56 (d, J = 2.0 Hz, 1H), 2.53 (s, 6H), 2.29 (q, J = 7.6
Hz, 4H), 1.33 (s, 6H), 0.98 (t, J = 7.6 Hz, 6H); 13C NMR
(100 MHz, CDCl3) δ; 157.9, 153.6, 139.9, 138.4, 132.7,
129.5, 128.8, 115.5, 78.1, 75.8, 56.0, 17.0, 14.6, 12.4, 11.8;
MALDI-TOF m/z 434.331; calcd. for C26H29BF2N2O:
434.322.
4. Experimental
4.1. Syntheses. Commercially available reagents were
purchased from Sigma-Aldrich Co. Canada, Alfa Aesar Co.
or TCI America Co. and were used without further
purification. All solvents were purchased from Fisher
Scientific (HPLC grade) and used directly. All air and
moisture sensitive reactions were performed under argon
atmosphere in oven-dried glassware. Melting points were
measured on an Electrothermal MEL-TEMP instrument.
Analytical
thin-layer-chromatography
(TLC)
was
performed on polyester backed TLC plates 254 (pre-coated,
200 µm, Sorbent Technologies). Column chromatography
was performed on silica gel (Sorbent Technologies, 60 Å,
BODIPYs 4 and 5: BODIPY 3 (0.3123 g, 0.7190 mmol)
and indole-3-carbaldehyde (0.3131 g, 2.157 mmol, 3
equiv.) were added to a 150 mL round bottom flask
containing a mixture of dry toluene (50 mL), piperidine
(0.4 mL) and glacial acetic acid (0.4 mL). The mixture was
refluxed and the water formed during the reaction was
removed azeotropically using a Dean-Stark trap. After
completion of the reaction (monitored via TLC), the
mixture was cooled to room temperature and the solvent
removed under reduced pressure. The residue was purified
1
40-63 µm) slurry packed into glass columns. H NMR and
13C NMR spectra were recorded using a Bruker DPX-400
1
spectrometer (operating at 400MHz for H NMR and 100
MHz for 13C NMR) in CDCl3 (7.26 ppm, 1H and 77.0
ppm,13C), (CD3)2CO (2.05 ppm, 1H and 29.84 ppm and
1
206.26 ppm, 13C) and (CD3)2SO (2.50 ppm, H and 39.52
ppm,13C). Chemical shifts are given in parts per millions
(ppm) relative to tetramethylsilane (TMS,
0 ppm);
multiplicities are indicated as br (broad), s (singlet), d
(doublet), t (triplet), q (quartet) and m (multiplet). All
spectra were recorded at room temperature and coupling
constants (J values) are given in Hz. Mass spectra were
obtained at the LSU Department of Chemistry Mass
Spectrometry Facility using either a Bruker Omniflex
MALDI-TOF or an Applied Biosystems QSTAR XL ESI-
by
silica
gel
column
chromatography
using
dichloromethane/petroleum ether (2/1, v/v) for elution. The
first fraction with red fluorescence was dried in vacuo,
affording BODIPY 4 as a red-blue solid (145 mg, yield
1
36%); H NMR (400 MHz, (CD3)2CO): δ; 10.72 (s, NH),
8.16 (t, J = 5.4 Hz, 1H), 7.91 (d, J = 8.1 Hz, 1H), 7.74 (d, J
= 8.1 Hz, 1H), 7.65 (d, J = 8.1 Hz, 1H), 7.49–7.51 (m, 2H),
7.20-7.32 (m, 6H), 4.89 (d, J = 2.0 Hz, 2H), 3.13 (t, J = 2.0
Hz, 1H), 2.69 (q, J = 7.6 Hz, 2H), 2.56 (s, 3H), 2.36 (q, J =
7.6 Hz, 2H), 1.41 (s, 3H), 1.38 (s, 3H), 1.19 (t, J = 7.6 Hz,
3H), 0.99 (t, J = 7.6 Hz, 3H); 13C NMR (100 MHz,
(CD3)2CO): δ; 158.3, 152.1, 151.4, 129.8, 128.7, 128.2,
125.3, 122.5, 120.9, 120.7, 120.2, 115.8, 115.7, 115.6,
112.0, 78.5, 76.4 55.7, 18.0, 16.6, 14.1, 13.5, 11.8, 11.1,
10.9; HRMS (ESI-TOF) m/z [M+Na]+ 584.2656 calc for
C35H33BF2N3ONa: 583.2661. The second fraction with
green fluorescence was dried under vacuum, affording
BODIPY 5 as a green solid (0.275 g, yield 55.6%). 1H
TOF.
4-(Prop-2-ynyloxy)-benzaldehyde
(2)
was
synthesized as previously described.21 Melting points were
determined using a MEL-TEMP electrothermal instrument.
BODIPY 3: Dimethylethylpyrrole (1.0 g, 8.12 mmole, 2
equiv.) and benzaldehyde 2 (0.649 g, 4.06 mmol, 1 equiv.)
were dissolved in dry dichloromethane (80 mL) under an
inert gas atmosphere. Trifluoroacetic acid (0.2 mL) was
added and the reaction mixture was stirred overnight at
room temperature. When the aldehyde was consumed
(monitored by TLC), a solution of DDQ (0.921 g, 4.06

ACCEPTED MANUSCRIPT
Tetrahedron
7
NMR (400 MHz, (CD3)2CO): δ; 10.70 (br, 2H), 8.30 (d, J =
8.1 Hz, 2H), 8.05 (d, J = 8.1 Hz, 2H), 7.69 (d, J = 8.1 Hz,
4H), 7.51 (d, J = 2.0 Hz, 2H), 7.20–7.35 (m, 8H), 4.88 (d, J
= 2.0 Hz, 2H), 3.09 (s, 1H), 2.73 (q, J = 7.6 Hz, 4H), 1.42
(s, 6H), 1.22 (t, J = 7.6 Hz, 6H); 13C NMR (100 MHz,
(CD3)2CO) δ; 184.3, 137.7, 137.0, 132.7, 130.2, 129.9,
128.4, 125.3, 124.6, 123.5, 122.5, 122.1, 121.3, 121.0,
120.5, 116.3, 116.0, 115.5, 112.0, 76.3, 55.7, 18.1, 13.5,
10.9; HRMS (ESI-TOF) m/z [M+Na]+ 711.3066; calcd. for
C44H38BF2N4NaO: 711.3085.
8.1 Hz, 1H), 7.50–7.52 (m, 2H), 7.22-7.33 (m, 6H), 6.25 (d,
J = 9.2 Hz, 1H), 5.75 (t, J = 9.2 Hz, 1H), 5.57 (d, J = 2.4
Hz, 1H), 5.46 (d, J = 2.4 Hz, 1H), 5.32 (s, 2H), 4.63 (t, J =
5.7 Hz, 1H), 4.12-4.27 (m, 2H), 2.72 (q, J = 7.6 Hz, 2H),
2.36 (q, J = 7.6 Hz, 2H), 2.21 (s, 3H), 2.05 (s, 3H), 2.03 (s,
3H), 1.90 (s, 3H), 1.80 (s,3H), 1.44 (s, 3H), 1.40 (s, 3H),
1.19 (t, J = 7.6 Hz, 3H), 1.00 (t, J = 7.6 Hz, 3H); 13C NMR
(100 MHz, (CD3)2CO): δ 169.8, 169.6, 169.2, 168.4, 159.1,
151.4, 143.9, 137.8, 137.0, 132.6, 130.3, 129.9, 128.4,
128.1, 125.3, 122.8, 122.5, 120.6, 120.2, 115.8, 115.7,
115.4, 112.0, 85.5, 73.7, 70.9, 68.1, 67.3, 61.6, 61.4, 19.6,
19.5, 19.3, 18.0, 16.6, 14.1, 13.5, 11.8, 11.1, 11.0; HRMS
(ESI-TOF) m/z [M+Na]+ 957.3785, calcd. for
C49H52BF2N6NaO10: 957.3785.
General procedure for click chemistry: meso-
Alkynylphenyl-BODIPY (40 mg), azido-compound (2.6
equiv.) and Cu(0) (1.0 equiv.) were dissolved in a mixture
of THF:water (8 mL, 3:1), under inert gas atmosphere. A
solution of CuSO4.5H2O (0.20 equiv.) and Na-ascorbate
(0.5 equiv.) in THF:water (8 mL, 3:1) was added (after
sonication for 10 min) and the reaction mixture was heated
for 6 h at 70 °C. Once the reaction was complete, the
mixture was cooled to room temperature and partitioned
between ethyl acetate (30 mL) and water (30 mL). The
combined organic layers were dried over Na2SO4 and the
solvent was evaporated under reduced pressure. The crude
product was then purified by preparative thin layer
chromatography to obtain the desired product.
BODIPY 7a: Eluent: acetone/ethyl acetate (1/1, v/v); Yield:
1
8.6 mg, 15%. H NMR (400 MHz, (CD3)2CO): δ 10.7 (br,
2H), 8.30 (d, J = 8.1 Hz, 2H), 8.18 (s, 1H), 8.04 (d, J = 8.1
Hz, 2H), 7.76 (d, J = 8.1 Hz, 2H), 7.69 (d, J = 8.1 Hz, 2H),
7.53 (d, J = 2.4 Hz, 2H), 7.23–7.36 (m, 8H), 5.30 (s, 2H),
4.62-3.65 (m, 12H), 2.75 (q, J = 7.6 Hz, 4H), 1.45 (s, 6H),
1.21 (t, J = 7.6 Hz, 6H); 13C NMR (100 MHz, (CD3)2CO):
δ 138.0, 137.8, 132.7, 130.2, 129.9, 128.6, 128.4, 125.3,
124.4, 122.4, 121.0, 120.5, 116.3, 116.0, 115.4, 112.0, 72.6,
70.1, 69.2, 61.7, 61.1, 49.8, 18.1, 13.5, 10.9; HRMS (ESI-
TOF)
m/z
[M+Na]+
885.4916;
calcd.
for
BODIPY 6a: Eluent, dichloromethane/ethyl acetate (10/1,
v/v); Yield: 36.7 mg, 70%. 1H NMR (400 MHz,
(CD3)2CO): δ; 10.72 (s, NH), 8.16 (d, J = 8.1 Hz, 2H), 7.92
(d, J = 8.1 Hz, 1H), 7.75 (s, 1H), 7.65 (d, J = 8.1 Hz, 1H),
7.50–7.52 (m, 2H), 7.22-7.33 (m, 6H), 4.60 (d, J = 2.0 Hz,
2H), 3.50-3.94 (m, 13H), 2.69 (q, J = 7.6 Hz, 2H), 2.55 (s,
3H), 2.36 (q, J = 7.6 Hz, 2H), 2.05 (s, 3H), 1.43 (s, 3H),
1.39 (s, 3H), 1.20 (t, J = 7.6 Hz, 3H), 1.00 (t, J = 7.6 Hz,
3H); 13C NMR (100 MHz, (CD3)2CO): δ 159.3, 152.1,
151.4, 142.9, 139.0, 138.7, 137.8, 137.1, 132.7, 132.4,
132.2, 131.4, 130.3, 129.8, 128.2, 128.1, 125.3, 124.6,
122.5, 120.6, 120.2, 115.8, 115.7, 115.5, 112.0, 72.6, 70.1,
69.1, 61.7, 61.1, 49.8, 18.0, 16.6, 14.1, 13.5, 11.8, 11.1,
11.0; HRMS (ESI-TOF) m/z [M+Na]+759.3611; calcd. for
C41H46BF2N6NaO4: 759.3581.
C50H51BF2N7NaO4: 885.4894.
BODIPY 7b: Eluent: acetone/ethyl acetate (1/1, v/v); Yield:
13.2 mg, 20%. 1H NMR (400 MHz, (CD3)2CO): δ 10.7 (br,
2H), 8.30 (d, J = 8.1 Hz, 2H), 8.18 (s, 1H), 7.69 (d, J = 8.1
Hz, 2H), 7.23–7.54 (m, 14H), 5.30 (s, 2H), 3.51-4.61 (m,
24H), 2.73 (q, J = 7.6 Hz, 4H), 1.38 (s, 6H), 1.15 (t, J =
7.6 Hz, 6H); 13C NMR (100 MHz, (CD3)2CO): δ 150.3,
138.0, 137.7, 132.7, 130.2, 129.9, 128.4, 125.3, 124.4,
122.4, 121.0, 120.5, 116.3, 116.0, 115.4, 112.0, 72.5, 70.1,
69.2, 61.8, 61.0, 49.9, 18.1, 13.5, 10.9; HRMS (ESI-TOF)
m/z [M+Na]+1018.4836; calcd. for C56H63BF2N7NaO7:
1018.4835.
BODIPY 7c: For R = Ac, eluent: dichloromethane/ethyl
acetate (1/1, v/v); Yield: 40.8 mg, 57.3%. H NMR (400
1
BODIPY 6b: Eluent: 12% acetone in ethyl acetate; Yield:
60.5 mg, 98%. 1H NMR (400 MHz, (CD3)2CO): δ; 10.72 (s,
NH), 8.16 (d, J = 8.1 Hz, 2H), 7.88 (d, J = 8.1 Hz, 1H),
7.75 (s, 1H), 7.67 (d, J = 8.1 Hz, 1H), 7.50–7.52 (m, 2H),
7.22-7.31 (m, 6H), 4.61 (d, J = 2.0 Hz, 2H), 3.50-3.94 (m,
25H), 2.69 (q, J = 7.6 Hz, 2H), 2.55 (s, 3H), 2.35 (q, J = 7.6
Hz, 2H), 2.05 (s, 3H), 1.43 (s, 3H), 1.39 (s, 3H), 1.19 (t, J =
7.6 Hz, 3H), 1.00 (t, J = 7.6 Hz, 3H); 13C NMR (100 MHz,
(CD3)2CO): δ 159.3, 152.1, 151.4, 142.8, 139.0, 138.7,
137.8, 137.1, 132.7, 132.4, 132.3, 130.4, 129.8, 128.2,
125.3, 124.6, 122.5, 120.6, 120.2, 115.8, 115.6, 115.5,
112.1, 72.5, 70.2, 70.1, 69.2, 61.8, 61.0, 49.9, 18.0, 16.6,
14.1, 13.5, 11.8, 11.1, 11.0; HRMS (ESI-TOF) m/z
[M+Na]+ 891.4407; for C47H58BF2N6NaO7: 891.4407.
MHz, (CD3)2CO): δ 10.70 (br s, 2H), 8.32 (d, J = 8.1 Hz,
2H), 8.17 (s, 1H), 8.05 (d, J = 8.1 Hz, 2H), 7.77 (d, J = 2.4
Hz, 2H), 7.69 (d, J = 2.4 Hz, 2H), 7.51-7.64 (m, 2H), 7.23–
7.32-7.40 (m, 8H), 6.25 (d, J = 9.1 Hz, 1H), 5.77 (t, J = 9.2
Hz, 1H), 5.57 (t, J = 5.4 Hz, 1H), 5.42-5.50 (m, 1H), 5.31
(s, 2H), 4.63 (t, J = 5.4 Hz, 1H), 4.13-4.28 (m, 2H), 2.73 (q,
J = 7.6 Hz, 4H), 2.21 (s, 3H), 2.05 (s, 3H), 2.03 (s, 3H),
1.96 (s, 3H), 1.44 (s, 6H), 1.20 (t, J = 7.6 Hz, 6H); 13C
NMR (100 MHz, (CD3)2CO): δ 184.4, 169.8, 169.7, 169.2,
168.4, 159.1, 150.3, 143.9, 138.0, 137.8, 137.1, 136.1,
132.7, 130.3, 129.8, 128.8, 128.5, 128.3, 125.3, 123.6,
122.9, 122.5, 122.1, 121.3, 121.07, 120.5, 116.3, 116.07,
116.02, 115.4, 112.1, 85.5, 73.7, 70.9, 68.1, 67.4, 61.6,
61.4, 19.69, 19.61, 19.3, 18.1, 13.5, 10.9; HRMS (ESI-
TOF)
m/z
[M+Na]+1083.4225;
calcd.
for
BODIPY 6c (R = Ac): Eluent: 12% acetone in ethyl acetate;
Yield: 65.0 mg, quantitative. 1H NMR (400 MHz,
(CD3)2CO): δ; 10.72 (s, NH), 8.34 (s, 1H), 8.17 (d, J = 8.1
Hz, 1H), 7.90 (d, J = 8.1 Hz, 1H), 7.75 (s, 1H), 7.65 (d, J =
C58H58BF2N7O10Na: 1083.4208. For
R = H, Eluent:
1
acetone/ethyl acetate (1/1, v/v); Yield: 10.8 mg, 18.2%. H
NMR (400 MHz, (CD3)2SO): δ 10.7 (br s, 2H), 8.48 (s,
1H), 8.08 (d, J = 8.1 Hz, 2H), 7.83 (s, 2H), 7.75 (s, 2H),

ACCEPTED MANUSCRIPT
8
Tetrahedron
7.57 (d, J = 8.1 Hz, 2H), 7.50 (d, J = 8.1 Hz, 2H), 7.13-7.32
(m, 8H), 5.56 (d, J = 5.4 Hz, 2H), 5.23 (s, 2H), 4.45 (s,
1H), 4.13-4.28 (m, 2H), 3.31 (br s, 2H), 2.66 (br s, 2H),
1.38 (s, 6H), 1.15 (t, J = 7.6 Hz, 6H); 13C NMR (100 MHz,
(CD3)2SO): δ 143.9, 138.0, 137.8, 132.7, 131.2, 130.9,
128.5, 127.4, 124.3, 123.4, 122.4, 121.0, 120.5, 116.3,
116.0, 115.4, 112.0, 72.6, 70.1, 69.2, 61.7, 61.3, 50.4, 18.1,
13.4, 12.9, 11.2; HRMS (ESI-TOF) m/z [M+Na]+
915.4071; calcd. for C50H49BF2N7NaO6: 915.4067.
concentration of 1% and Cremophor concentration of 0.1%.
For the uptake test, the compounds were diluted to 20 µM
(2 x stock solution) and added to the 96-well plate to give a
final concentration of 10 µM at 0, 1, 2, 4, 8, 12 and 24h
interval. The uptake was terminated by removing the
loading medium and washing the wells with phosphate
buffer saline (PBS). The cells were then solubilized using
100 µL of 0.25% Triton X-100 (Calbiochem) in PBS. The
concentration of the BODIPYs was measured using
intrinsic fluorescence as measured with a BMG FLUOstar
plate reader equipped with a 355 nm excitation and a 650
nm emission filter. The cell numbers were quantified using
a CyQuant cell proliferation assay (Invitrogen) as per
manufactures’ instructions, and the uptake expressed in
terms of nM compound per cell.
BODIPY 7d: Eluent: acetone/ethyl acetate (3/2, v/v); Yield:
1
15.8 mg, 22.5%. H NMR (400 MHz, (CD3)2SO): 11.7 (br,
2H), 8.49 (s, 1H), 8.08 (d, J = 8.1 Hz, 2H), 7.80 (s, 2H),
7.54 (d, J = 8.1 Hz, 2H), 7.50 (d, J = 8.1 Hz, 2H), 7.20-7.34
(m, 8H), 5.62 (d, J = 5.4 Hz, 2H), 5.23 (s, 2H), 4.94 (s,
2H), 3.49-4.67 (m, 17H), 2.66 (br s, 4H), 1.22 (s, 6H), 1.15
(t, J = 7.6 Hz, 6H); 13C NMR (100 MHz, (CD3)2SO): δ
145.7, 138.3, 137.2, 136.7, 135.2, 132.5, 130.5, 129.7,
128.3, 128.2, 125.9, 124.9, 122.9, 121.5, 120.9, 118.9,
116.3, 116.0, 115.4, 112.0, 72.6, 70.1, 69.2, 67.8, 61.7,
61.5, 61.1, 50.2, 49.8, 18.1, 13.5, 10.9; HRMS (ESI-TOF)
m/z [M+Na]+ 1077.4413; calcd. for C56H59BF2N7NaO11:
1077.4415.
Cytotoxicity: For the dark toxicity, the HEp2 cells were
plated as described above for the uptake experiment and
allowed 36-48 h to attach. The cells were exposed to
increasing concentrations of BODIPYs up to 100 µM and
incubated overnight. The loading medium was removed
and the medium containing Cell Titer Blue (Promega) was
added as per manufacturer’s instructions. The cytotoxicity
was measured and compared with untreated cells
considered as 100% viable and cells treated with 0.2%
saponin as 0% viable. The IC50 values were determined
from dose-response curves obtained using GraphPad Prism
software. For the phototoxicity, the HEp2 cells were
prepared as described above for the dark cytotoxicity assay
with BODIPY concentration range from 6.25 to 100 µM.
After loading overnight, the medium was removed and
replaced with medium containing 50 mM HEPES pH 7.4.
The cells were exposed to a 175 W halogen lamp filtered
through a 610 nm long pass filter to provide approximately
1 Jcm-2 light dose. The cells were kept cool by placing the
4.2. Steady-state Absorption and Fluorescence
Spectroscopy. The absorption measurements were carried
out on a Varian Cary 50 UV/Vis spectrophotometer and the
steady-state fluorescence spectroscopic studies were
performed in DMSO on a PTI Quantum Master4/2006SE
spectrofluorometer. For the fluorescence quantum yield
experiments, only dilute solutions with the optical density
between 0.04-0.06 at the excitation wavelength were used
to minimize the reabsorption effects. The slit width was 5
nm for both the excitation and emission. The fluorescence
quantum yield of BODIPY 3 was obtained by comparing
the area under the corrected emission spectrum of the test
sample with that of rhodamine 6G (0.80 in methanol). For
BODIPYs 4 and 6a-c, cresyl violet perchlorate (0.54 in
ethanol) and for BODIPYs 5 and 7a-d, Zn-phthalocyanine
(0.28 in DMF) were used as external standards,
respectively. All spectra were recorded at 298 K within 2h
of solution preparation, using non-degassed samples,
spectroscopic grade solvents and a 10 mm quartz cuvette.
In all the cases, the correction for the refractive index was
applied. The results are shown in Table 2.
°
culture on a 5 C Echotherm chilling plate (Torrey Pines
Scientific, Inc.). After exposure to light for 20 min, the
°
plate was incubated overnight in a 37 C incubator and
assayed for viability using CellTiter Blue Viability Assay
Kit, as described above for the dark cytotoxicity
experiment. The results are shown in Figures S21 and S22
of Supplementary data.
Microscopy: The HEp2 cells were incubated in a glass
bottom 6-well plate (MatTek) and allowed to grow for 48
h. The cells were then exposed to 10 ꢀM of each BODIPY
conjugate for 6 h. Organelle tracers were obtained from In-
vitrogen and used at the following concentrations:
LysoSensor Green 50 nm, MitoTracker Green 250 nm,
BODIPY FL C5 Ceramide 1 mm, ER Tracker Blue/white
100 nm and ER Tracker green 100 nm. The organelle
tracers were diluted in medium and the cells were
incubated concurrently with BODIPY conjugate and tracers
for 30 min before washing 3 times with PBS. Images were
acquired using a Leica DM RXA2 upright microscope with
40 X NA 0.8dip objective lens and DAPI, GFP and Texas
Red filter cubes (Chroma Technologies) and a Leica TCS
SP2 spectral confocal DM IRE2 inverted microscope with
40 X NA objectives and DIC optics using Ar, He/Ne
(green), and He/Ne (red) lasers with laser lines at 488, 543,
and 633 nm.
4.3 Cell Culture. Human carcinoma HEp2 cells were
maintained in
a 50:50 mixture of DMEM:AMEM
(Invitrogen) supplemented with 5% FBS (Invitrogen),
Primocin antibiotic (Invitrogen) and 5% CO2 at 37 oC. The
cells were sub-cultured twice weekly to maintain
subconfluent stocks. The 4th to 15th passage cells were used
for all - experiments.
Time-Dependent Cellular Uptake: The HEp2 cells were
plated at 7500 cells per well in a Costar 96 well plate and
allowed to grow for 48h. BODIPY stock solutions were
prepared at 32 mM in DMSO with Cremophor (1% of
Cremophor EL in DMSO) and then diluted into medium to
give a final concentration of 10 mM with maximum DMSO

ACCEPTED MANUSCRIPT
Tetrahedron
9
Acknowledgments
20. Olivier, G.; Cecile, D.-V.; Rachel, M.-R. and Gilles, C.
Org. Biomol. Chem. 2010, 8, 4546–4553.
The work described was supported by the National Science
Foundation, grant number CHE-0611629.
21. Giguère, J.-B.; Thibeault, D.; Cronier, F.; Marois, J.-
S.; Auger, M.; Morin, J.-F. Tetrahedron Lett. 2009, 50,
5497-5500.
22. Deniz, E.; Isbasar, G. C.; Bozdemir, O. A.; Yildirim,
L. T.; Siemiarczuk, A.; Akkaya, E. U. Organic Lett.
2008, 10, 3401-3403.
23. Sharma, V.; Kumar, P.; Pathak, D. J. Heterocyclic
Chem. 2010, 47, 491-502.
24. Greenwald, R. B. J. Control Release 2001, 74, 159–
171.
25. Sibrian-Vazquez, M.; Jensen, T. J.; Vicente, M. G. H.
J. Photochem. Photobiol. B: Biol. 2007, 86, 9-21.
26. Bakleh, M. E.; Sol, V.; Estieu-Gionnet, K.; Granet, R.;
Déléris, G.; Krausz, P. Tetrahedron 2009, 65, 7385-
7392.
27. Murtagh, J.; Frimannsson, D. O.; O'Shea, D. F.
Organic Lett. 2009, 11, 5386-5389.
28. Wang, Q.; Chan, T. R.; Hilgraf, R.; Fokin, V. V.;
Sharpless, K. B.; Finn, M. G. J. Am. Chem. Soc. 2003,
125, 3192-3193.
29. Shao, F.; Weissleder, R.; Hilderbrand, S. A.
Bioconjugate Chem. 2008, 19, 2487-2491.
30. Hao, E.; Jensen, T. J.; Vicente, M. G. H. J. Porphyrins
Phthalocyanines 2009, 13, 51-59.
31. Leffler, H.; Carlsson, S.; Hedlund, M.; Qian, Y.;
Poirier, F. Glycoconj. J. 2004, 19 , 433-440.
32. van Scherpenzeel, M.; Moret, E. E.; Ballell, L.;
Liskamp, R. M. J.; Nilsson, U. J.; Leffler, H.; Pieters,
R. J. ChemBioChem 2009, 10 , 1724-1733.
33. Kim, K.; Jo, C.; Easwaramoorthi, S.; Sung, J.; Kim, D.
H.; Churchill, D. G. Inorganic Chem. 2010, 49, 4881-
4894.
34. Olmsted, J. J. Phys. Chem. 1979, 83, 2581-2584.
35. Lim, S. H.; Thivierge, C.; Nowak-Sliwinska, P.; Han,
J.; van den Bergh, H.; Wagnieres, G.; Burgess, K.;
Lee, H. B. J. Med. Chem. 2010, 53, 2865-2874.
36. Zheng, Q., Xu, G., Prasad, P. N. Chem. Eur. J. 2008,
14, 5812-5819.
Supplementary data
Supplementary data associated with this article can be
found online at doi: XXXXXXX.
References
1. Kelloff, G. J.; Krohn, K. A.; Larson, S. M.;
Weissleder, R.; Mankoff, D. A.; Hoffman, J. M.; Link,
J. M.; Guyton, K. Z.; Eckelman, W. C.; Scher, H. I.;
O’Shaughnessy, J.; Cheson, B. D.; Sigman, C. C.;
Tatum, J. L.; Mills, G. Q.; Sullivan, D. C.; Woodcock,
J. Clin Cancer Res. 2005, 11, 7967-7985.
2. He, X.; Wang, K.; Cheng, Z. Nanomed. Nanobiotechn.
2010, 2, 349-66.
3. Rao, J.; Dragulescu-Andrasi, A.; Yao, H. Curr. Op.
Biotechn. 2007, 18, 17-25.
4. Adams, K. E.; K. E.; Kwon, S.; Liang, F.; Fan, Z.;
Hirschi, K.; Mawad, M. E.; Barry, M. A.; Sevick-
Muraca, E. M. J. Biomed. Opt. 2007, 12, 024017.
5. Luo, S.; Zhang, E.; Su, Y.; Cheng, T.; Shi, C.
Biomaterials 2011, 32, 7127-38.
6. Joshi, B. P.; Wang, T. D. Cancers 2010, 2, 1251-1287.
7. Alexis, F.; Pridgen, E.; Molnar, L. K.; Farokhzad, O.
C. Mol. Pharm. 2008, 5, 505-15.
8. Giuntini, F.; Alonso, C. M. A.; Boyle, R. W.
Photochem. Photobiol. Sci. 2011, 10, 759-791.
9. Kobayashi, H.; Ogawa, M.; Alford, R.; Choyke, P. L.;
Urano, Y. Chem. Rev. 2010, 110, 2620-40.
10. Gomez-Hens, A.; Aguilar-Caballos, M. P. Trac-Trends
Anal. Chem. 2004, 23, 127-136.
11. Loudet, A.; Burgess, K. Chem. Rev. 2007, 107, 4891-
4932.
12. Ulrich, G.; Ziessel, R.; Harriman, A. Angew. Chem.
Int. Ed. 2008, 47, 1184-1201.
13. Descalzo, A. B.; Xu, H.-J.; Shen, Z.; Rurack, K. Ann.
N.Y. Acad. Sci. 2008, 1130, 164–171.
14. Benstead, M.; Mehl, G. H.; Boyle, R. W. Tetrahedron
2011, 67, 3573-3601.
15. Boens, N.; Volker Leen, V.; Dehaen, W. Chem. Soc.
Rev. 2012, 41, 1130–1172.
16. Jiao, L.; Yu, C.; Uppal, T.; Liu, M.; Li, Y.; Zhou, Y.;
Hao, E.; Hu, X.; Vicente, M. G. H. Org. Biomol.
Chem. 2010, 8, 2517-2519.
17. Uppal, T.; Hu, X.; Fronczek, F. R.; Maschek, S.;
Bobadova-Parvanova, P.; Vicente, M. G. H. Chem.
Eur. J. 2012, 18, 3893-3905.
18. Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew.
Chem. Int. Ed. 2001, 40, 2004-2021.
19. Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.;
Sharpless, K. B. Angew. Chem. Int. Ed. 2002, 41,
2596-2599.

ACCEPTED MANUSCRIPT
Supplementary Data
Synthesis and Cellular Properties of Near-IR BODIPY-PEG and
Carbohydrate Conjugates
Timsy Uppal, N. V. S. Dinesh K. Bhupathiraju and M. Graça H. Vicente*
Department of Chemistry, Louisiana State University, Baton Rouge, LA 70803, USA.
Table of Contents:
Synthesis and characterization of azido-PEG compounds .........................................2
1H NMR and 13C NMR Spectra of BODIPYs.................................………...............4
Dark and Phototoxicity of BODIPYs.........................................................................24
Subcellular localization of BODIPYs........................................................................25
1

ACCEPTED MANUSCRIPT
a) MsCl/Ag2O, CH2Cl2, 48 h, rt, 51%.
b) NaN3, DMF, 1100C/3 h, 96%.
O
O
HO
OH
N3
OH
n
n
n= 2 8a
n= 5 8b
Azido-terminated triethylene glycol (a):
To a mixture of Ag2O (0.902 g, 3.89 mmol) and triethylene glycol (0.531 g, 3.54 mmol) in 8
ml of CH2Cl2 at room temperature was added methane sulfonyl chloride (0.487 g, 4.25
mmol ) in 2 ml of CH2Cl2. The reaction mixture was stirred for 48 h under argon. Then the
mixture was passed through celite, the solvent evaporated and the residue purified by flash
column chromatography using ethyl acetate for elution. The target product was obtained as a
colorless oil in 51% yield, 0.411 g. 1H NMR (CDCl3, 400 MHz): δ 4.17-4.20 (2H, m), 3.38-
3.53 (10H, m), 3.11 (1H, s), 2.91 (3H, s). 13C NMR (CDCl3, 100 MHz): δ 72.01, 69.95, 69.90,
69.67, 69.02, 68.37, 60.91, 37.03. HRMS (ESI) m/z (M + Na+) 251.5432, calculated for
C7H16O6SNa 251.2531.
To the above product (0.411 g, 1.635 mmol) in 4 ml of dry DMF was added NaN3 (0.16 g,
2.44 mmol) under argon. The reaction mixture was heated at 110 0C for 3 h and then cooled
0
to room temperature. The reaction mixture was co-evaporated with toluene at 50 C and the
residue purified by flash chromatography using CH2Cl2/ethyl acetate 9:1 for elution. The
1
product was isolated as colorless oil in 98% yield, 0.52 g. H NMR (CDCl3, 400 MHz): δ
13
3.47-3.56 (10H, m), 3.26 (2H, t, J = 4.99 Hz), 3.07 (1H, s). C NMR (CDCl3, 100 MHz): δ
72.88, 70.98, 70.86, 70.75, 70.73, 70.66, 70.41, 70.34, 70.31, 70.28, 70.25, 70.22, 61.71,
50.79, 36.65, 31.55. HRMS (ESI) m/z (M + Na+) 198.2654, calculated for C6H13N3O3Na
198.1775.
2

ACCEPTED MANUSCRIPT
Azido-terminated hexaethylene glycol (b):
To a mixture of Ag2O (0.451 g, 1.945 mmol) and hexaethylene glycol (0.5 g, 1.77 mmol) in 4
ml of CH2Cl2 at room temperature was added methane sulfonyl chloride (0.2435 g, 2.125
mmol) in 1 ml of CH2Cl2. The above reaction mixture was stirred for 48 h under argon. Then
the mixture was passed through celite, the solvent evaporated and the residue purified by
flash column chromatography using ethyl acetate/methanol 10:1 for elution. The target
1
product was obtained as colorless oil in 50% yield, 0.311 g. H NMR (CDCl3, 400 MHz): δ
13
4.17-4.20 (2H, m), 3.38-3.59 (22H, m), 3.21 (1H, s), 2.91 (3H, s). C NMR (CDCl3, 100
MHz): δ 72.44, 70.35, 70.31, 70.22, 70.06, 69.46, 68.79, 61.31, 61.28, 57.61, 37.45. HRMS
(ESI) m/z (M + Na+) 383.1331, calculated for C13H28O9SNa 383.1346.
To the above product (0.311 g, 0.863 mmol) in 2 ml of dry DMF was added NaN3 (0.084 g,
1.29 mmol) under argon. The reaction mixture was heated at 110 0C for 3 h and then cooled
0
to room temperature. The reaction mixture was co-evaporated with toluene at 50 C and the
residue purified by flash column chromatography using dichloromethane/methanol 9.7:0.3
1
for elution. The product was isolated as pale yellow oil in 98% yield, 0.26 g. H NMR
13
(CDCl3, 400 MHz): δ 3.57-3.63 (22H, m), 3.30 (2H, t, J = 4.0 Hz), 3.09 (1H, s). C NMR
(CDCl3, 100 MHz): δ 77.54, 77.22, 76.90, 72.52, 70.57, 70.19, 69.93, 61.50, 61.33, 50.58,
36.42, 31.33. LRMS (ESI)m/z (M + Na+) 330.1643, calculated for C12H25N3O6Na 330.1636.
3

ACCEPTED MANUSCRIPT
Figure S1: 1H-NMR spectrum of BODIPY 3 in CDCl3 at 400 MHz
4

ACCEPTED MANUSCRIPT
Figure S2: 13C-NMR spectrum of BODIPY 3 in CDCl3 at 400 MHz
5

ACCEPTED MANUSCRIPT
Figure S3: 1H-NMR spectrum of BODIPY 4 in deuterated acetone at 400 MHz
6

ACCEPTED MANUSCRIPT
Figure S4: 13C-NMR spectrum of BODIPY 4 in deuterated acetone at 400 MHz
7

ACCEPTED MANUSCRIPT
Figure S5: 1H-NMR spectrum of BODIPY 5 in deuterated acetone at 400 MHz
8

ACCEPTED MANUSCRIPT
Figure S6: 13C-NMR spectrum of BODIPY 5 in deuterated acetone at 400 MHz
9

ACCEPTED MANUSCRIPT
Figure S7: 1H-NMR spectrum of BODIPY 6a in deuterated acetone at 400 MHz
10

ACCEPTED MANUSCRIPT
Figure S8: 13C-NMR spectrum of BODIPY 6a in deuterated acetone at 400 MHz
11

ACCEPTED MANUSCRIPT
Figure S9: 1H-NMR spectrum of BODIPY 6b in deuterated acetone at 400 MHz
12

ACCEPTED MANUSCRIPT
Figure S10: 13C-NMR spectrum of BODIPY 6b in deuterated acetone at 400 MHz
13

ACCEPTED MANUSCRIPT
Figure S11: 1H-NMR spectrum of BODIPY 6c (Ac) in deuterated acetone at 400 MHz
14

ACCEPTED MANUSCRIPT
Figure S12: 13C-NMR spectrum of BODIPY 6c (Ac) in deuterated acetone at 400 MHz
15

ACCEPTED MANUSCRIPT
Figure S13: 1H-NMR spectrum of BODIPY 7a in deuterated acetone at 400 MHz
16

ACCEPTED MANUSCRIPT
Figure S14: 13C-NMR spectrum of BODIPY 7a in deuterated acetone at 400 MHz
17

ACCEPTED MANUSCRIPT
Figure S15: 1H-NMR spectrum of BODIPY 7b in deuterated acetone at 400 MHz
18

ACCEPTED MANUSCRIPT
Figure S16: 13C-NMR spectrum of BODIPY 7b in deuterated acetone at 400 MHz
19