Click conjugation of boron dipyrromethene (Bodipy) fluorophores to egfr-targeting linear and cyclic peptides

Article abstract of DOI:10.3390/molecules26030593

Through a simple 1,3-cycloaddition reaction, three BODIPY-peptide conjugates that target the extracellular domain of the epidermal growth factor receptor (EGFR) were prepared and their ability for binding to EGFR was investigated. The peptide ligands K(N₃)LARLLT and its cyclic analog cyclo(K(N₃)larllt, previously shown to have high affinity for binding to the extracellular domain of EGFR, were conjugated to alkynyl-functionalized BODIPY dyes 1 and 2 via a copper-catalyzed click reaction. This reaction produced conjugates 3, 4, and 5 in high yields (70–82%). In vitro studies using human carcinoma HEp2 cells that overexpress EGFR demonstrated high cellular uptake, particularly for the cyclic peptide conjugate 5, and low cytotoxicity in light (~1 J·cm^?2) and darkness. Surface plasmon resonance (SPR) results show binding affinity of the three BODIPY-peptide conjugates for EGFR, particularly for 5 bearing the cyclic peptide. Competitive binding studies using three cell lines with different expressions of EGFR show that 5 binds specifically to EGFR-overexpressing colon cancer cells. Among the three conjugates, 5 bearing the cyclic peptide exhibited the highest affinity for binding to the EGFR protein.

Full text of DOI:10.3390/molecules26030593

molecules
Article
Click Conjugation of Boron Dipyrromethene (BODIPY)
Fluorophores to EGFR-Targeting Linear and Cyclic Peptides
Tyrslai M. Williams 1, Nichole E. M. Kaufman 1, Zehua Zhou 1, Sitanshu S. Singh 2 , Seetharama D. Jois 2 and
Maria da Graça H. Vicente 1,
*
1
Department of Chemistry, Louisiana State University, Baton Rouge, LA 70803, USA;
twil161@lsu.edu (T.M.W.); nkaufm1@lsu.edu (N.E.M.K.); zzhou2@lsu.edu (Z.Z.)
School of Basic Pharmaceutical and Toxicological Sciences, College of Pharmacy, University of Louisiana at
Monroe, Monroe, LA 71201, USA; singhss@warhawks.ulm.edu (S.S.S.); jois@ulm.edu (S.D.J.)
Correspondence: vicente@lsu.edu; Tel.: +1-225-578-7405; Fax: +1-225-578-3458
2
*
Abstract: Through a simple 1,3-cycloaddition reaction, three BODIPY-peptide conjugates that target
the extracellular domain of the epidermal growth factor receptor (EGFR) were prepared and their
ability for binding to EGFR was investigated. The peptide ligands K(N3)LARLLT and its cyclic analog
cyclo(K(N3)larllt, previously shown to have high affinity for binding to the extracellular domain of
EGFR, were conjugated to alkynyl-functionalized BODIPY dyes
reaction. This reaction produced conjugates , and in high yields (70–82%). In vitro studies using
human carcinoma HEp2 cells that overexpress EGFR demonstrated high cellular uptake, particularly
for the cyclic peptide conjugate , and low cytotoxicity in light (~1 J
cm−2) and darkness. Surface
plasmon resonance (SPR) results show binding affinity of the three BODIPY-peptide conjugates for
EGFR, particularly for bearing the cyclic peptide. Competitive binding studies using three cell
lines with different expressions of EGFR show that binds specifically to EGFR-overexpressing colon
bearing the cyclic peptide exhibited the highest affinity
1 and 2 via a copper-catalyzed click
3,
4
5
5
·
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
5
Citation: Williams, T.M.; Kaufman,
N.E.M.; Zhou, Z.; Singh, S.S.; Jois,
S.D.; Vicente, M.d.G.H. Click
Conjugation of Boron
5
cancer cells. Among the three conjugates,
for binding to the EGFR protein.
5
Dipyrromethene (BODIPY)
Fluorophores to EGFR-Targeting
Linear and Cyclic Peptides. Molecules
2021, 26, 593. https://doi.org/
10.3390/molecules26030593
Keywords: BODIPY; EGFR; peptide; click reaction
1. Introduction
Academic Editors: Mário J. F. Calvete
and Mariette M. Pereira
The study of protein–protein interactions (PPIs) is very useful when investigating mu-
tagenic behaviors that lead to diseases and oncogenic mutations [
insight on the behavior of PPIs in human diseases, in particularly oncogenic mutations of
the epidermal growth factor receptor (EGFR) in colorectal cancer (CRC) [ ]. This mutation
1–3]. Several studies offer
Received: 30 December 2020
Accepted: 20 January 2021
Published: 23 January 2021
4
makes targeting EGFR for CRC detection very attractive. To capitalize on this strategy,
recent studies have targeted EGFR, as a method to increase tumor cell specificity for cancer
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
therapy [
4
–
7
]. Practices including FDA-approved anti-EGFR antibody cetuximab [
12], anti-EGFR affibody [13 14], accessible small
17] have all been employed to understand
8,9],
single-chain anti-EGFR ScFvEGFR [10
–
,
peptides [15], and tyrosine kinase inhibitors [16
targeting EGFR.
,
EGFR is a ligand-stimulated receptor that holds a crucial role in regulating cellular
functions, more directly comprising cell proliferation and survival [ 18]. In addition,
4–7,
EGFR houses a single polypeptide backbone chain that is understood to be an important
component in exploring effective treatments and screening of cancers, such as CRC. While
the overexpression of EGFR on the surface of CRC is due to mutations in the gene, and
its upregulation, the activation of EGFR can also occur through mutation in the kinase
domain [17,18].
While current research has identified several practices for targeting EGFR, our group
has previously prepared and investigated EGFR-targeted porphyrin-peptide [19], phthaloc-
yanine-peptide [20], and BODIPY-peptide conjugates [21–23]. In those studies, two small
Copyright:
© 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Molecules 2021, 26, 593. https://doi.org/10.3390/molecules26030593
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 593
2 of 14
peptides reported in the literature, designated EGFR-L1 (LARLLT) [24] and EGFR-L2 (YH-
WYGYTPQNVI) [25], showed to have high EGFR specificity both in in vitro and in vivo
studies, were used. The conjugates linked to EGFR-L1 via a short PEG linker showed
enhanced water solubility when compared with the conjugates attached to EGFR-L2, which
bears a longer hydrophobic peptide sequence [20,21]. Additionally, these peptide-bearing
conjugates showed enhanced EGFR-targeting ability, and up to 90-fold increased accumu-
lation in EGFR-overexpressed cells compared with unconjugated fluorophore [21]. These
results suggest that fluorophores conjugated to EGFR-L1 and EGFR-L2 have substantially
increased EGFR- targeting ability and may be very useful for the early detection and
diagnosis of CRC and other EGFR-overexpressing cancers. While EGFR-L1 is known to
bind in domain I of EGFR, away from the EGF binding pocket, EGFR-L2 binds to the
EGF binding pocket on the extracellular domain of EGFR. Furthermore, previous studies
suggest that EGFR-L1 conjugates tend to bind to EGFR with higher affinity compared
with the EGFR-L2-based conjugates [19,20,26]. This might be a result of EGFR-L1 binding
away from the EGF binding pocket, allowing for interactions with various conformations
of EGFR, as the binding site for EGFR-L1 in domain I is not affected by conformational
changes in EGFR [19].
Designing a fluorophore that is robust enough to handle the biological framework
of CRC requires the scaffold to be robust, easily assembled, and visible enough to ensure
proper screening. One must consider the development of these types of compounds
to ensure stability and biocompatibility. Peptides bear an amine or carboxyl terminus
available for conjugation to fluorophores such as porphyrins, phthalocyanines and boron
dipyrromethenes (BODIPYs). BODIPYs are extremely useful fluorophores because of their
tunable core structure and excellent photophysical properties [27–29]. Research shows
that BODIPYs maintain a highly spectroscopic tunable core that is stable under various
conditions. Increasing efficient approaches for conjugating BODIPY dyes to peptides
requires that a clear position for attachment be available at each molecule. In peptide
synthesis, the most evident attachment point is the exposed free amino group at the N-
terminus of the sequence that can be used in traditional conjugation through amidation.
Over time research has recognized the simplicity in forming attachment points during
peptide synthesis (amines), however, the required carboxyl component on BODIPY dyes is
not as easily attained, and the conjugation yields are moderate to low [23]. Therefore, a
simple and high-yielding conjugation method, such as a so-called “click” reaction [30
has become more attractive in the last decade.
,31]
The copper(I)-catalyzed azide-alkyne cycloadditions, or click reactions, have a multi-
tude of suitable features, including mild reaction conditions, short completion times, simple
to no purification required, and high stability of the resulting triazoles under various re-
action conditions, such as to air and moisture. Because of these benefits, the 1,3-dipolar
cycloaddition is very attractive to researchers performing porphyrinoid conjugations for
use in imaging and photodynamic therapy [32–35]. Even more striking is that this cy-
cloaddition is especially fitting for sensitive constituents such as peptides, carbohydrates,
and nucleotides. The overall attraction of click chemistry is deep-rooted in its nature to
be selective, mild, and adaptable [36–38]. Other key components of click reactions lie in
their ability to be nearly quantitative through the robust behavior and insensitivity of the
resulting triazoles to various conditions [37]. The orthogonal ligation that occurs is suitable
for various biomolecular pairing and in vivo labeling. The 1,2,3-triazoles formed in this
reaction are stable under acidic and basic conditions, reductive and oxidative environments,
and are resistant to proteolytic cleavages [38]. In addition, triazoles are also resilient to
metabolic degradation. Overall, the steady nature of click compounds creates an ideal
platform for developing stable fluorophore conjugates for bioimaging applications.
Previous studies report the use of BODIPY dyes as the fluorescent component in
cell-targeted conjugates bearing various targeting moieties for use in in vivo imaging. For
example, Wolfbeis et al. [39] reported fluorescently labeled biomolecules through the use
of copper-free and copper-mediated click chemistry. Wolfbeis et al. designed clickable

Molecules 2021, 26, 593
3 of 14
BODIPY fluorophores for labeling of the azide-modified surface glycans of CHO cells.
Akkaya et al. [40] used click chemistry in the synthesis of a bay region BODIPY dye
attached to a perylenediimide (PDI). Akkaya et al. envisioned the molecule suitable for
light harvesting which allows a large cross-section for the absorption of visible light. These
studies were able to show that the excitation energy was efficiently channeled to the PDI
core. Overkleeft et al. [41] designed three acetylene-functionalized BODIPY dyes to identify
an azido-bearing epoxomicin analogue by fluorescence. Overkleeft et al. performed the
functionalization through the Huisgen 1,3-dipolar cycloaddition, which yielded a group of
fluorescent epoxomicin-derived proteasome probes.
We have previously reported the conjugation of BODIPYs to the EGFR-L1 peptide
via traditional amidation reactions [23], and nucleophilic additions to isothiocyanato-
functionalized fluorophores [21,22]. Herein we report the use of click chemistry for con-
jugation of two EGFR-L1-derived peptides bearing an azide N-terminus (cycloL1.1 and
L1.5) [26] to alkynyl-functionalized BODIPYs. The use of click chemistry allowed the
employment of mild reaction conditions that are compatible with the BODIPY and peptide
scaffolds, increasing the conjugation yield over alternative methodologies, and simpli-
fying conjugate purification. We have previously used click chemistry to conjugate low
molecular weight PEG groups and carbohydrates to BODIPYs, for enhanced aqueous
solubility and cellular permeability [42]. Furthermore, to increase the stability of peptides
in vivo we introduced conformational and configurational constraints, including sequence
cyclization and change in the chirality of amino acids. The structure-activity investigation
of the EGFR-L1 peptide with key modifications in its structure is described in our earlier
report [26]. The modified peptide L1.5 exhibited increased affinity for EGFR compared
to the parent peptide, while cycloL1.1 also exhibited enhanced stability in human serum.
Herein we report the synthesis of two BODIPYs bearing an alkynyl handle for conjugation
via an azide-functionalized lysine side chain to two EGFR-L1 peptide derivatives that have
shown increased EGFR binding affinity and stability relative to EGFR-L1.
2. Results and Discussion
2.1. Synthesis
The alkynyl-functionalized BODIPYs
synthesized as we have previously reported [42]. In brief, p-propargyloxy-benzaldehyde
reacted with 3-ethyl-2,4-dimethylpyrrole in the presence of BF3 OEt2, followed by DDQ
oxidation and boron complexation, to afford BODIPY in 40% yield (see Supporting
Information). Near-IR absorbing and emitting BODIPY was prepared from using
1 and 2 used in the conjugation reactions were
·
1
2
1
indole-3-carbaldehyde in a Knoevenagel condensation [43]. Styryl-derived BODIPY com-
pounds exhibit significant red-shifted fluorescence emissions due to the extension of the
π-conjugation (Scheme 1). Additionally, the incorporation of an indole moiety has been
observed to induce enhanced solubility and cellular permeability, possibly due to its resem-
blance to tryptophan. Tryptophan is a naturally occurring amino acid and an important
component of many enzymes and proteins [44]. Therefore, the attachment of a single
indolyl styryl group to the BODIPYs’ core results in an asymmetric mono-styryl BODIPY
that displays a ca. 90 nm bathochromic shift on its absorption and emission profiles, and
also enhanced solubility, cellular permeability, and overall biological efficacy [42,45].
The Knoevenagel reaction of BODIPY
1 in the presence of indole-3-carbaldehyde,
piperidine, and glacial acetic acid in refluxing toluene, using a Dean-Stark apparatus,
afforded the mono-styryl BODIPY
the distyryl derivative.
2 in 28–36% yields, due to the concomitant formation of
The EGFR-targeting peptide ligand EGFR-L1 (LARLLT) was reported in 2009 by Song
et al. [24] and showed to have high affinity for the extracellular domain of EGFR, both
in vitro and in vivo. In previous work, we reported the conjugation of this peptide to a
porphyrin [19], phthalocyanine [20], and BODIPY dyes [21–23], using either traditional
amidation methodologies or nucleophilic addition to isothiocyanato-functionalized fluo-
rophores. These studies indicated that the mode of conjugation significantly influences

Molecules 2021, 26, 593
4 of 14
the binding affinity of the resulting conjugates to EGFR. In order to conjugate EGFR-L1 to
BODIPYs 1 and 2 using click chemistry, we modified the EGFR-L1 peptide via the introduc-
tion of an azide-functionalized lysine residue, to form L1.5 (K(N3)LARLLT) (Figure 1) [26].
In addition, to improve the stability of the L1.5 peptide in vivo, we performed a cyclization
reaction to afford cycloL1.1 (cyclo(K(N3)larllt). Our previous investigations of peptides
L1.5 and cycloL1.1 revealed that both have high affinity for binding to domain I of the
extracellular domain of EGFR, as well as enhanced stability in human serum [26].
OH
O
Br
K2CO3, acetone
(100%)
N
H
O
O
1. DCM, BF3OEt2
2. DDQ
3. Et3N, BF3OEt2
(40%)
O
O
CHO
N
N
B
N
F
F
H
N
N
B
Piperidine, HOAc
Toluene, reflux
(28-36%)
F
F
N
H
2
1
Scheme 1. Synthetic route to alkynyl-functionalized BODIPYs 1 and 2.
The linear peptide L1.5 was synthesized using solid phase peptide synthesis (SPPS),
as we have previously reported [26]. The cyclic peptide cycloL1.1 was synthesized in a
similar fashion by SPPS, and then cyclized after removal from the resin. The peptides
were purified by HPLC and analyzed by mass spectrometry and NMR. Analytical HPLC
confirmed both peptides were >95% pure.
H2N
NH
H2N
H2N
NH2
NH
O
H
N
N
H
HN
O
O
O
O
O
O
H
H
H
O
O
HN
O
H
N
N
N
H2N
N
N
N
NH2
O
N
H
H
H
O
O
O
HN
HO
N
H
N3
OH
L1.5
N3
cycloL1.1
Figure 1. Azido peptides used for conjugation to BODIPYs 1 and 2.
BODIPYs
the presence of CuSO4
Scheme 2. To a solution of the BODIPY in THF/water 3:1 were added 1.5 equivalents of
azido-peptide L1.5 dissolved in DMSO. Copper(0) in a catalytic amount was added to the
1
and
2
were conjugated to peptides L1.5 or cycloL1.1 via click chemistry in
5H2O, Cu(0), and L-ascorbic acid in THF/water [46], as shown in
·

Molecules 2021, 26, 593
5 of 14
resulting mixture followed by the addition of 1 equivalent of CuSO4
·
5H2O and L-ascorbic
acid in distilled water. The catalytic amount of copper metal was added as a suitable
reductant for the Cu(II) source, CuSO4 5H2O, in an aqueous solution to generate Cu(I),
·
which is a potent stimulus for the formation of 1,2,3-triazoles. The reaction was allowed to
stir for 24 h, monitored by MALDI-TOF mass spectrometry. Once the desired product was
confirmed, the mixture was quenched with water. Initially, the resulting conjugate was
purified through a molecular weight cut off (MWCO) tube of 1000 Daltons. This method
was used to retain the conjugate in the tube, as the molecular weight for the desired product
conjugates are all above this value, and any starting materials remaining in the mixture
were removed. The resulting conjugates were dried and purified by RP-HPLC, eluted with
a 1% TFA in H2O/0.1% TFA in CH3CN gradient (see Supporting Information) to afford the
corresponding 1,2,3-triazole cycloadducts 3, 4, and 5 in excellent yields (Table 1).
H2N
HN
O
H2N
NH
NH2
NH
O
O
N
N
HN
N
NH
O
O
O
O
HN
NH
O
L1.5
O
Ascorbic Acid
HO
NH2
CuSO4•5H2O, Cu(0)
THF/H2O 3:1
N
N
N
N
B
B
RT, 24 h
(82%)
H2N
F
F
F F
HN
O
H2N
NH
NH2
NH
O
1
3
O
N
N
HN
N
NH
O
O
O
HN
NH
L1.5
O
O
NH2
NH2
Ascorbic Acid
HO
NH2
CuSO4•5H2O, Cu(0)
HN
THF/H2O 3:1
O
O
N
N
H
B
N
RT, 24 h
(70%)
N
H
F
F
NH
O
O
O
O
NH
O
4
H
N
O
N
H
N
N
NH
B
N
H
F
F
N
N
N
HO
2
cycloL1.1
N
H
Ascorbic Acid
O
CuSO4•5H2O, Cu(0)
THF/H2O 3:1
RT, 24 h
(80%)
N
N
B
F
F
5
N
H
Scheme 2. Click conjugation of azido peptides L1.5 and cycloL1.1 to BODIPYs 1 and 2.
Table 1. Molecular weights and click reaction yields of BODIPY-peptide conjugates.
Conjugate
Molecular Weight (g/mol)
Peptide
%Yield
3
4
5
1273.79
1400.89
1383.81
L1.5
L1.5
cycloL1.1
82%
70%
80%
2.2. Spectroscopic Studies
Ultraviolet-Visible (UV/Vis) and fluorescence spectroscopy studies were employed
to determine the spectroscopic properties of the BODIPYs and their conjugates. Exper-
imental results from these studies are shown in Figure S19 (Supplementary Materials).

Molecules 2021, 26, 593
6 of 14
BODIPY
1
was found to have excitation and emission maxima in the visible region of the
electromagnetic spectrum at 525 and 540 nm, respectively. Extension of the π-conjugation
system of the BODIPY through styryl functionalization via Knoevenagel condensation
resulted in bathochromic shifts to 614 nm excitation and 648 nm emission maxima. As
expected, the absorption and emission maxima did not change significantly after click
conjugation with either the linear or cyclic peptides. Multiple shoulder peaks present in
the absorption spectra of
4 and 5 reflect the possibility of electronic transitions between
differing vibrational energy levels.
2.3. Surface Plasmon Resonance (SPR) Studies
We have previously explored the specificity of peptides L1.5 and cycloL1.1 for EGFR
and determined that they both have a fast association with the EGFR protein’s extracellular
domain through SPR studies [26]. To compare the binding affinity of the BODIPY-peptide
conjugates
were performed. All BODIPY-peptide conjugates and precursors were tested at concentra-
tions up to 250 M, and the results obtained are shown in Figures S10–S12 of the Supporting
Information. Conjugates , and were diluted to desired concentrations with a solution
of running buffer HBS-EP+ containing 8% of DMSO.
For conjugate , binding began at around 10 M and further addition of conjugate
did increase the sensorgram in a stepwise fashion, as seen in Figure S12 (Supplementary
Materials). On the other hand, conjugate binding changes in response began around
10 M (Figure S10, Supplementary Materials) and there was a stepwise increase in the
sensorgram with smooth association and dissociation with a total change of 450 RU,
suggesting specific binding of the conjugate to the EGFR protein. At 250 M, conjugate
showed a decrease in RU due to a saturation of binding (Figure S11, Supplementary
3–5 to EGFR in comparison to the peptide sequences alone, SPR [47,48] analyses
µ
3,
4
5
5
µ
3
µ
µ
4
Materials). On the other hand, conjugate
5 exhibited stepwise increase in binding from 25
to 250 µM concentrations (Figure S12, Supplementary Materials). Moreover, conjugate 5
sensorgram exhibited a smooth (and slow) increase in RU from 100 to 180 sec suggesting
specific binding of conjugate to the EGFR extracellular domain, as shown with peptide
cycloL1.1 [26]. Among the three conjugates studied, 3 and 5 exhibited relatively higher and
specific binding to the EGFR protein. This is similar to what we observed for the free cyclic
peptide which was characterized by a stepwise increase in binding with a slow and even
increase in RU, rather than a rapid increase in the response units.
2.4. Cell Studies
To assess the biological efficacy and behavior of the peptide-functionalized BODIPY
conjugates, preliminary in vitro cellular studies, including dark cytotoxicity, phototoxicity
and cellular uptake, were investigated against human squamous cell carcinoma HEp2 cell
line, with high EGFR expression (~75%).
2.4.1. Dark Toxicity
The dark toxicity of all compounds was determined in HEp2 cells at concentrations up
to 200
these studies are shown in Figure 2. BODIPYs
in this study for comparison purposes. Interestingly, BODIPY
devoid of styryl and indole functionalities, were the least toxic compounds in this series.
Conjugates and were found to be more cytotoxic than conjugate , with calculated IC50
values of approximately 100 M. On the other hand, BODIPY bearing an indolyl styryl
group was also found to be more toxic than . These results suggest that the indolyl styryl
µ
M, using a CellTiter Blue (CTB) assay to determine cell viability. The results from
and (lacking peptide) were included
and conjugate , both
1
2
1
3
4
5
3
µ
2
1
moiety lends some cytotoxicity to compounds containing this functional group. This might
be a result of the presence of the indole group, since it is a known pharmacophore and
the basis of many drugs, such as indomethacin [44,49]. Conjugates 4 and 5 containing the
linear and cyclic peptides, respectively, were found to have similar cytotoxicities, with the
compound containing the cyclic peptide having slightly higher cytotoxicity than its linear

Molecules 2021, 26, 593
7 of 14
counterpart at all concentrations investigated. This result might be due to the higher EGFR
binding ability and uptake of the conjugate bearing the cyclic peptide.
Figure 2. Dark toxicity results for BODIPYs 1 (black), 2 (green), 3 (pink), 4 (purple), and 5 (blue) in
human carcinoma HEp2 cells.
2.4.2. Phototoxicity
In addition to dark toxicity, phototoxicity was assessed for the BODIPYs and their con-
jugates, under a low light dose of ~1 J
cm−2 in human carcinoma HEp2 cells using the CTB
·
assay to determine cell viability. Dose-dependent cell survival curves of each compound
over the incubation period of 24 h are shown in Figure S20 (Supporting Information). The
results obtained parallel those obtained for the dark cytotoxicity, with BODIPY
linear peptide conjugate being the least toxic, whereas BODIPY and its cyclic peptide
conjugate were found to be the most toxic, with IC50 values of approximately 100 M. The
phototoxicity of conjugate is similar to that of its precursor BODIPY , both of which were
1 and its
3
2
5
µ
3
1
found to be the least cytotoxic in this series, suggesting that the conjugation of the linear
peptide has little effect on the cytotoxicity of the BODIPY. On the other hand, BODIPY
2
and its conjugates
increase in phototoxicity when compared to
Interestingly, conjugate containing the linear EGFR-targeting peptide showed decrease in
phototoxicity compared with its precursor , although showed increased cellular uptake
in HEp2 cells than . Nevertheless, all conjugates are considered to have low phototoxicity
4
and
5
containing the indolyl styryl moiety, showed a significant
1
and , which lack the indolyl styryl group.
3
4
2
4
2
in HEp2 cells at the low light dose used in this study.
2.4.3. Time-Dependent Cellular Uptake
Cellular uptake studies of all compounds were conducted in a time-dependent manner.
Cellular uptake was determined in human carcinoma HEp2 cells over a 24 h time period
at a non-toxic concentration of 10
assay was employed to determine cell numbers. The results from these studies are shown
in Figure 3. BODIPYs and were also investigated to compare their cellular uptake
with that of the peptide conjugates. After the addition of peptide-containing conjugates 4
and to HEp2 cells, kinetics showed a marked uptake increase and rapid accumulation
in cells in less than 4 h, after which a plateau in uptake is observed for the remaining of
the 24 h time period. On the other hand, conjugate and its parent BODIPY , which are
devoid of the indolyl styryl moiety, showed the lowest uptake in HEp2 cells. BODIPY
showed slightly higher uptake in HEp2 cells compared with as a result of its indolyl styryl
group. However, conjugates and containing the linear and cyclic peptide sequences,
respectively, showed significant higher uptake compared with conjugate in HEp2 cells,
up to 7-fold increase in uptake. Both of these conjugates were readily taken up by HEp2
µ
M for all compounds. A CyQuant® cell proliferation
1
2
5
3
1
2
1
4
5
3

Molecules 2021, 26, 593
8 of 14
cells, with conjugate
5
showing the greatest uptake by far in this series. This result might be
due to the higher affinity of the cyclic peptide for binding to EGFR. We previously reported
that the D-amino acids in cyclo(K(N3)larllt) and its cyclic structure confer a more rigid
conformation relative to the linear peptide that is more suitable for binding to domain I
of EGFR [26]. In addition, the greater stability of the cyclic peptide might confer higher
stability to conjugate
5 compared with 4, thus allowing for a sustained higher uptake
within cells over time. This observed enhanced cellular uptake for
its observed increase in photo- and dark-toxicity.
5 may also contribute to
Figure 3. Cellular uptake results for BODIPYs
1 (black), 2 (green), 3 (pink), 4 (purple), and 5 (blue) in
human carcinoma HEp2 cells.
2.4.4. Competitive Binding Studies
To evaluate the specificity of binding of the most promising conjugate
overexpressing colon cancer cells, competitive binding experiments were carried out with
cyclic peptide (cycloL1.1, without BODIPY) in the presence of conjugate . In these studies,
HT-29 (~78% EGFR expression), DLD-1 (~40% EGFR expression), and LOVO (~6% EGFR
expression) were used [ ]. When constant amount of conjugate (50 M) was added to HT-
5 to EGFR
5
4
5
µ
29 and DLD-1 cells in the presence of variable amount of cyclic peptide, a dose-dependent
decrease in fluorescence intensity was observed as seen in Figure 4, suggesting that the
cyclic peptide was able to replace conjugate
5 competitively, particularly in the high EGFR-
expressing HT-29 cells. The highest inhibition of fluorescence intensity was observed in
the HT-29 cells with the highest EGFR expression. When similar experiments were carried
out in the presence of LOVO cells, there was no dose-dependent decrease in fluorescence
intensity suggesting that both
5 and cyclic peptide bind only non-specifically to the LOVO
cells. These results show that conjugate
cancer cells.
5 binds specifically to EGFR over-expressing colon

Molecules 2021, 26, 593
9 of 14
Figure 4. Evaluation of binding of conjugate
5
to EGFR on different cell lines that express differential
amount of EGFR. Cyclic peptide cyclo(K(N3)larllt) (without BODIPY) was used with conjugate
5
and BODIPY fluorescence was monitored to observe the competitive binding. Binding of
) HT-29 (high EGFR expression), ( ) DLD-1 (moderate EGFR expression) and (
5
to
(A
B
C
) LOVO (low
EGFR expression). Zero concentration fluorescence refers to fluorescence without the addition of
cyclo(K(N3)larllt). Concentrations on the X-axis are for various concentrations of cyclic peptide
added with constant concentration of conjugate 5 (50 µM).
3. Materials and Methods
3.1. Synthesis
All reagents are commercially available and were purchased from Sigma-Aldrich
(St. Louis, MO, USA) and VWR International (Radnor, PA, USA). All solvents and reagents
used in the synthesis of conjugates were performed with reagents purchased from Sigma
Aldrich peptide synthesis grade. Required amino acids were purchased from either
Anaspec (Fremont, CA, USA), AAPPTec (Louisville, KY, USA) or Applied Biosystems
(Foster City, CA, USA). Analytical thin-layer-chromatography (TLC) was performed on
polyester backed TLC plates 254 (pre-coated, 200 µm, Sorbent Technologies, Norcross, GA,

Molecules 2021, 26, 593
10 of 14
USA). Column chromatography was performed on silica gel (Sorbent Technologies, 60 Å,
1
40–63
µ
m). H-NMR and HSQC spectra were recorded using a Bruker AVIII-500 spectrom-
eter (Birrica, MA, USA) (operating at 500 MHz for 1H-NMR and HSQC) in DMSO-d6. All
chemical shifts are given in parts per millions (ppm) relative to tetramethylsilane (TMS,
0 ppm). All spectra were recorded at 308 K. High resolution mass spectra were obtained
at the LSU Department of Chemistry Mass Spectrometry Facility using Bruker Omniflex
MALDI Time-of-Flight Mass Spectrometer. The absorption measurements were carried
out on a Varian 212 Cary 50 UV/Vis spectrophotometer (Palo Alto, CA, USA) and the
steady-state fluorescence spectroscopic studies were performed on a PTI Quantum Mas-
ter4/2006SE spectrofluorometer (Northampton, UK). All spectra were recorded at 298 K
using non-degassed samples, spectroscopic grade solvents, and a 10-mm quartz cuvette.
RP-HPLC was used to purify BODIPY-peptide conjugates, as shown in our previous work.
The RP-HPLC system is comprised of a 2489 UV/Vis detector, a 2545 quaternary gradient
module pump, and a FlexInject sample injector (Waters, Milford, MA, USA) to a purity of
≥
95% (unless otherwise noted). To achieve full separation, X-Bridge BEH300 Prep C18
column (5 m, 10 250 mm) armed with an X-Bridge BEH300 Prep guard column (300 Å,
m, 10 10 mm) operating at a gradient of 50% A for 5 min, 50% A to 10% A over 1 min,
µ
×
5
µ
×
10% A to 0% A over 13 min, 0% A to 50% A over 2 min, then hold at 50% A for 5 min at a
flow rate of 4 mL/min (unless otherwise noted). Conjugates UV detection was recorded for
peptides at 220 nm, and for BODIPY at 540 and 640 nm. Fractions of HPLC purity (>95%)
with the anticipated mass were combined and lyophilized. The chromatographs were
analyzed using Empower 2 software. BODIPYs
1 and 2 were synthesized as previously
described in the literature [42] (see Supplementary Materials). Peptides L1.5 and cycloL1.1
were synthesized as previously reported [26].
General Procedure for 1,3-cycloaddition: Alkynyl-functionalized BODIPY and azido-peptide
(1.5 equiv.) were dissolved in a mixture of THF/H2O (1.8 mL, 3:1), followed by addition
of Cu(0) (1.0 equiv.). A solution of CuSO4 5H2O (1 equiv.) and L-ascorbic acid (1 equiv.)
·
in H2O was added to the reaction mixture. The solution was allowed to stir at room
temperature for 24 h. Once the reaction was complete, the mixture was quenched with
water and lyophilized. The fluffy product was then purified by RP-HPLC under varying
gradients of water (with 0.1% TFA) and acetonitrile (with 0.1% TFA).
Conjugate 3 (1,3,5,7-tetramethyl-2,6-diethyl-8-(4-phenoxymethyltriazole K(N3)LARLLT)-BODIPY):
This conjugate was obtained as a reddish orange solid (4.8 mg, 82%). MALDI-TOF m/z
1273.82; calc. for C63H100BF2N16O9+: 1273.79. For 1H-NMR and HSQC see Supporting
Information.
Conjugate 4 (1,3,7-trimethyl-5-indolylstyryl-2,6-diethyl-8-(4-phenoxymethyltriazole K(N3)LARLLT)-
BODIPY): This conjugate was obtained as a dark blue solid (3.49 mg, 70%). MALDI-TOF
m/z 1400.87; calc. for C73H106BF2N16O9+: 1400.89. For 1H-NMR and HSQC see Supporting
Information.
Conjugate 5 (1,3,7-trimethyl-5-indolylstyryl-2,6-diethyl-8-(4-phenoxymethyltriazole cycloK(N3)larllt)-
BODIPY): This conjugate was obtained as a deep blue solid (6.1 mg, 80%). MALDI-TOF
m/z 1383.91; calc. for C72H102BF2N16O9+: 1383.81. For 1H-NMR and HSQC see Supporting
Information.
3.2. Spectroscopy Studies
UV-Visible and emission spectroscopy studies were measured using a Varian Cary
spectrophotometer (Palo Alto, CA, USA) and a Perkin Elmer LS55 spectrophotometer
(Waltham, MA, USA) at room temperature. Quartz cuvettes (1 cm path length) were used
for each study. To analyze the data, integrated absorbance against the corresponding
solution concentrations were plotted to conclude the extinction coefficients (
ε) at the
maximum absorption.

Molecules 2021, 26, 593
11 of 14
3.3. Surface Plasmon Resonance (SPR) Studies
Surface plasmon resonance analysis was conducted using the extracellular domain of
EGFR protein (Leinco Technologies, St. Louis, MO, USA) immobilized on a CM5 sensor
chip via standard amine coupling with a Biacore X100 (GE Healthcare Life Sciences, Marl-
borough, MA, USA) as described in our previous publications [22
used was 8% DMSO in 1X HBS-EP+ (0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.005%
Tween, pH 7.5) (GE Healthcare Biosciences, Chicago, IL, USA), at a flow rate of 5 L/min.
,26]. The running buffer
µ
DMSO was added to the running buffer and peptide solutions to enhance the solubility
of the peptides studied. The solutions were prepared in running buffer and filtered using
a 0.45 µm filter. All SPR sensorgrams relay the rates of association and dissociation of
analytes at concentrations of 0 to 250 µM performed at room temperature.
3.4. Cell Studies
For cellular studies, commercially available reagents and culture media were pur-
chased from Life Technologies (Carlsbad, CA, USA) and human carcinoma HEp2 (ATCC
CCL-23) cells were purchased from the American Type Culture Collection (ATCC) (Manas-
sas, Virginia, USA). HEp2 cells were cultured and maintained at 37 ◦C under 5% CO2 and
95% humidity in minimum essential media (MEM) supplemented with 10% fetal bovine
serum (FBS) and 1% penicillin-streptomycin (P/S).
3.4.1. Dark Cytotoxicity
Stock solutions (32 mM) for compounds
DMSO as the solvent. Working solutions were then prepared at concentrations of 0, 6.25,
12.5, 25, 50, 100, and 200 M by diluting stock solutions with culture medium. HEp2
cells were exposed to the working solutions up to 200 M with 24 h incubation (5% CO2,
1, 2, 3, 4, and 5 were prepared using 100%
µ
µ
95% humidity, 37 ◦C). After 24 h treatment, the loading medium was removed, and cells
were washed thrive with phosphate buffered saline (PBS) solution to remove residual
compound. Then, medium containing 20% CellTiter Blue (CTB) (Promega, Madison, WI,
USA) was added to the cells and allowed to incubate it for another 4 h. Following the
incubation period, cell viability was assessed by determining fluorescence intensity at
570/615 nm using a BMG FLUOstar Optima microplate reader (BMG Labtech, Cary, NC,
USA). The fluorescence intensity was normalized to 100% for untreated cells and the results
are expressed as a percentage of viable cells.
3.4.2. Phototoxicity
HEp2 cells were exposed to working solutions of each compound up to 100
3.125, 6.25, 12.5, 25, 50, and 100
µ
M (0,
◦
µ
M) and incubated for 24 h (5% CO2, 95% humidity, 37 C).
The loading medium was removed and the cells were washed with PBS solution. Fresh
culture medium was introduced to the cells, followed by light exposure using a halogen
lamp (600 W) light source that included a beam turning mirror (200 nm to 30
range, Newport) and a water filter (transmits radiation 250–950 nm), for 20 min. The cells
received a total light dose of approximately 1 J
cm−2. After 20 min light exposure, the cells
µm spectral
·
were returned to the incubator for another 24 h. The culture medium was replaced with
media containing 20% CellTiter Blue and the cells incubated for 4 h. Cell viability was
assessed as described above.
3.4.3. Time-Dependent Cellular Uptake
Cellular uptake was investigated in HEp2 cells exposed to 10
µM solutions of com-
pounds 4, and at incremental time intervals (0, 1, 2, 4, 8, and 24 h). At the end
1,
2,
3,
5
of the allotted time, the loading medium was removed, and the cells washed with PBS
solution to remove residual compound not taken up by the cells. Cells were solubilized
using 0.25% Triton X-100 in PBS. Compound standard curves were created by diluting the
32 mM stock solutions with 0.25% Triton X-100 in PBS to final concentrations of 10, 5, 2.5,
1.25, 0.625, and 0.3125 µM. A cell standard curve was created by plating 10,000, 20,000,

Molecules 2021, 26, 593
12 of 14
40,000, 60,000, 80,000, and 100,000 cells per well (five replicates for each concentration).
To quantify cells, CyQuant Cell Proliferation Assay (Invitrogen, Carlsbad, CA, USA) was
used. The concentration of the compounds was determined using the standard curves and
a BMG FLUOstar Optima microplate reader at 485/520 nm. Cellular uptake results are
expressed in nM of compound per cell.
3.4.4. Competitive Binding Studies
Cells HT-29 (~78% EGFR) DLD-1 (~37% EGFR) and LOVO (~6% EGFR) with high,
moderate, and low EGFR expressions, respectively, were used in these studies. About
ten thousand cells of each cell line were coated in a 96 well clear bottom tissue culture
black plate and incubated overnight at 37 ◦C with 5% CO2. After 24 h, the medium was
removed, and different concentrations (0.25 to 100
concentration (50 M) of conjugate , was added to the wells in triplicate. PBS was used
for dilution of compounds. The plate was then covered in aluminum foil and incubated at
37 ◦C with 5% CO2. After 45 min, each well was washed twice with 100
L of PBS, and
µM) of cyclo(K(N3)larllt) with a constant
µ
5
µ
fluorescence was determined with a microplate reader using an excitation wavelength of
614 nm and an emission wavelength of 648 nm. Autofluorescence values were subtracted
from each well. A plot of concentrations vs. relative fluorescence was plotted. Results in
Figure 4 are expressed as ±S.E.
4. Conclusions
Two terminal alkyne-functionalized BODIPY dyes and two EGFR azido-peptide lig-
ands, one linear and one cyclic, were conjugated via click chemistry in 70–82% yields. Of all
the conjugates,
and cytotoxicity compared with
presence of the cyclic peptide in conjugate
4
and
5
bearing an indolyl styryl group showed increased cellular uptake
, which bears no indolyl styryl group. However, the
induced the highest accumulation in HEp2
3
5
cells that overexpress EGFR of all compounds in this series. This might be a result of the
more rigid conformation of the cyclic peptide, which is more suitable for binding to EGFR,
compared with the linear peptide. SPR results suggest that all conjugates bind to EGFR, in
particular, conjugate 5. Competitive binding studies using three cell lines with different
EGFR expression showed that conjugate 5 specifically binds to colon cancer cells overex-
pressing EGFR (HT-29), while non-specific binding was also observed in cells with low
EGFR expression (LOVO). The click conjugation of fluorophores to azide-functionalized
peptides is anticipated to provide a resourceful methodology for the preparation of vast
arrays of functionalized BODIPYs possessing potential utility in in vivo imaging.
Supplementary Materials: The following are available online, Figure S1: HPLC for 3, Figure S2:
HPLC for 4, Figure S3: HPLC for 5, Figure S4: MALDI-TOF for 3, Figure S5: MALDI-TOF-TOF for
3, Figure S6: MALDI-TOF for 4, Figure S7: MALDI-TOF-TOF for 4, Figure S8: MALDI-TOF for 5,
Figure S9: MALDI-TOF-TOF for 5, Figure S10: SPR sensorgram for 3, Figure S11: SPR sensorgram
for 4, Figure S12: SPR sensorgram for 5, Figure S13: 1H-NMR spectrum for 3, Figure S14: HSQC
1
NMR spectrum for 3, Figure S15: H-NMR spectrum for 4, Figure S16: HSQC NMR spectrum for 4,
1
Figure S17: H-NMR spectrum for 5, Figure S18: HSQC NMR spectrum for 5, Figure S19: Normalized
UV-Vis and fluorescence spectra, Figure S20: Phototoxicity results in human HEp2 cells.
Author Contributions: Conceptualization, M.d.G.H.V. and S.D.J.; methodology, T.M.W., N.E.M.K.,
Z.Z., and S.S.S.; software, M.d.G.H.V. and S.D.J.; validation, T.M.W., N.E.M.K., Z.Z., and S.S.S.;
formal analysis, T.M.W., N.E.M.K., Z.Z.; investigation, T.M.W., N.E.M.K., Z.Z., and S.S.S.; resources,
M.d.G.H.V. and S.D.J.; data curation, T.M.W., N.E.M.K., Z.Z., and S.S.S.; writing—original draft
preparation, T.M.W., N.E.M.K. and M.d.G.H.V.; writing—review and editing, M.d.G.H.V. and S.D.J.;
visualization, T.M.W., N.E.M.K., Z.Z., and S.S.S.; supervision, M.d.G.H.V. and S.D.J.; project adminis-
tration, M.d.G.H.V.; funding acquisition, M.d.G.H.V. and S.D.J. All authors have read and agreed to
the published version of the manuscript.
Funding: This research was supported by the National Institutes of Health, grant number R01
CA179902, and the National Science Foundation, grant number 1800126.

Molecules 2021, 26, 593
13 of 14
Data Availability Statement: The data presented in this study are available in Supplementary
Materials.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds are available from the authors upon request.
References
1.
2.
3.
Kar, G.; Gursoy, A.; Keskin, O. Human cancer protein-protein interaction network: A structural perspective. PLoS Comput. Biol.
2009, 5, e1000601. [CrossRef] [PubMed]
Kann, M.G. Protein interactions and disease: Computational approaches to uncover the etiology of diseases. Brief. Bioinform.
2007, 8, 333–346. [CrossRef] [PubMed]
Kennedy, S.A.; Jarboui, M.-A.; Srihari, S.; Raso, C.; Bryan, K.; Dernayka, L.; Charitou, T.; Bernal-Llinares, M.; Herrera-Montavez,
C.; Krstic, A.; et al. Extensive rewiring of the EGFR network in colorectal cancer cells expressing transforming levels of KRAS
G13D. Nat. Commun. 2020, 11, 1–14. [CrossRef] [PubMed]
4.
5.
6.
7.
Yang, J.L.; Qu, X.J.; Russell, P.J.; Goldstein, D. Regulation of Epidermal Growth Factor Receptor in Human Colon Cancer Cell
Lines by Interferon α. Gut 2004, 53, 123–129. [CrossRef]
Yewale, C.; Baradia, D.; Vhora, I.; Patil, S.; Misra, A. Epidermal growth factor receptor targeting in cancer: A review of trends and
strategies. Biomaterials 2013, 34, 8690–8707. [CrossRef]
Hynes, N.E.; Lane, H.A. ERBB receptors and cancer: The complexity of targeted inhibitors. Nat. Rev. Cancer 2005, 5, 341–354.
[CrossRef]
Burgess, A.W.; Cho, H.-S.; Eigenbrot, C.; Ferguson, K.M.; Garrett, T.P.J.; Leahy, D.J.; Lemmon, M.A.; Sliwkowski, M.X.; Ward,
C.W.; Yokoyama, S. An Open-and-Shut Case? Recent Insights into the Activation of EGF/ErbB Receptors. Mol. Cell 2003, 12,
541–552. [CrossRef]
8.
9.
Patra, C.R.; Bhattacharya, R.; Wang, E.; Katarya, A.; Lau, J.S.; Dutta, S.; Muders, M.; Wang, S.; Buhrow, S.A.; Safgren, S.L.; et al.
Targeted delivery of gemcitabine to pancreatic adenocarcinoma using cetuximab as a targeting agent. Cancer Res. 2008, 68,
1970–1978. [CrossRef]
Saki, M.; Toulany, M.; Sihver, W.; Zenker, M.; Heldt, J.-M.; Mosch, B.; Pietzsch, H.-J.; Baumann, M.; Steinbach, J.; Rodemann, H.P.
Cellular and molecular properties of 90Y-labeled cetuximab in combination with radiotherapy on human tumor cells in vitro
.
Strahlenther. Onkol. 2012, 188, 823–832. [CrossRef]
10. Yang, L.; Mao, H.; Wang, Y.A.; Cao, Z.; Peng, X.; Wang, X.; Duan, H.; Ni, C.; Yuan, Q.; Adams, G.; et al. Single chain epidermal
growth factor receptor antibody conjugated nanoparticles for in vivo tumor targeting and imaging. Small 2009, 5, 235–243.
[CrossRef]
11. Bell, A.; Wang, Z.J.; Arbabi-Ghahroudi, M.; Chang, T.A.; Durocher, Y.; Trojahn, U.; Baardsnes, J.; Jaramillo, M.L.; Li, S.; Baral, T.N.;
et al. Differential tumor-targeting abilities of three single-domain antibody formats. Cancer Lett. 2010, 289, 81–90. [CrossRef]
[PubMed]
12. Zarschler, K.; Prapainop, K.; Mahon, E.; Rocks, L.; Bramini, M.; Kelly, P.M.; Stephan, H.; Dawson, K.A. Diagnostic nanoparticle
targeting of the EGF-receptor in complex biological conditions using single-domain antibodies. Nanoscale 2014, 6, 6046–6056.
[CrossRef] [PubMed]
13. Sexton, K.; Tichauer, K.; Samkoe, K.S.; Gunn, J.; Hoopes, P.J.; Pogue, B.W. Fluorescent affibody peptide penetration in glioma
margin is superior to full antibody. PLoS ONE 2013, 8, e60390.
14. Nordberg, E.; Friedman, M.; Göstring, L.; Adams, G.P.; Brismar, H.; Nilsson, F.Y.; Ståhl, S.; Glimelius, B.; Carlsson, J. Cellular
studies of binding, internalization and retention of a radiolabeled EGFR-binding affibody molecule. Nucl. Med. Biol. 2007, 34,
609–618. [CrossRef] [PubMed]
15. Hamzeh-Mivehroud, M.; Mahmoudpour, A.; Dastmalchi, S. Identification of new peptide ligands for epidermal growth factor
receptor using phage display and computationally modeling their mode of binding. Chem. Biol. Drug Des. 2012, 79, 246–259.
[CrossRef]
16. Franek, J.; Cappelleri, J.C.; Larkin-Kaiser, K.A.; Wilner, K.D.; Sandin, R. Systematic review and network meta-analysis of first-line
therapy for advanced EGFR-positive non-small-cell lung cancer. Future Oncol. 2019, 15, 2857–2871. [CrossRef]
17. Zhang, X.; Gureasko, J.; Shen, K.; Cole, P.A.; Kuriyan, J. An allosteric mechanism for activation of the kinase domain of epidermal
growth factor receptor. Cell 2006, 125, 1137–1149. [CrossRef]
18. Bhirde, A.A.; Patel, V.; Gavard, J.; Zhang, G.; Sousa, A.A.; Masedunskas, A.; Leapman, R.D.; Weigert, R.; Gutkind, J.S.; Rusling,
J.F. Targeted killing of cancer cells in vivo and in vitro with EGF-directed carbon nanotube-based drug delivery. ACS Nano 2009
3, 307–316. [CrossRef]
,
19. Fontenot, K.R.; Ongarora, B.G.; LeBlanc, L.E.; Zhou, Z.; Jois, S.D.; Vicente, M.G.H. Targeting of the epidermal growth factor
receptor with mesoporphyrin IX-peptide conjugates. J. Porphyr. Phthalocyanines 2016, 20, 352–366. [CrossRef]
20. Ongarora, B.G.; Fontenot, K.R.; Hu, X.; Sehgal, I.; Satyanarayana-Jois, S.D.; Vicente, M.G.H. Phthalocyanine–peptide conjugates
for epidermal growth factor receptor targeting. J. Med. Chem. 2012, 55, 3725–3738. [CrossRef]
21. Zhao, N.; Williams, T.M.; Zhou, Z.; Fronczek, F.R.; Sibrian-Vazquez, M.; Jois, S.D.; Vicente, M.G.H. Synthesis of BODIPY-peptide
conjugates for fluorescence labeling of EGFR overexpressing cells. Bioconjugate Chem. 2017, 28, 1566–1579. [CrossRef] [PubMed]

Molecules 2021, 26, 593
14 of 14
22. Kaufman, N.E.; Meng, Q.; Griffin, K.E.; Singh, S.S.; Dahal, A.; Zhou, Z.; Fronczek, F.R.; Mathis, J.M.; Jois, S.D.; Vicente, M.G.H.
Synthesis, characterization, and evaluation of near-IR boron dipyrromethene bioconjugates for labeling of adenocarcinomas by
selectively targeting the epidermal growth factor receptor. J. Med. Chem. 2019, 62, 3323–3335. [CrossRef] [PubMed]
23. Williams, T.M.; Zhou, Z.; Singh, S.S.; Sibrian-Vazquez, M.; Jois, S.D.; Vicente, M.G.H. Targeting EGFR overexpression at the
surface of colorectal cancer cells by exploiting amidated BODIPY-peptide conjugates. Photochem. Photobiol. 2020, 96, 581–595.
[CrossRef] [PubMed]
24. Song, S.; Liu, D.; Peng, J.; Deng, H.; Guo, Y.; Xu, L.X.; Miller, A.D.; Xu, Y. Novel peptide ligand directs liposomes toward EGFR
high-expressing cancer cells in vitro and in vivo. FASEB J. 2009, 23, 1396–1404. [CrossRef]
25. Li, Z.; Zhao, R.; Wu, X.; Sun, Y.; Yao, M.; Li, J.; Xu, Y.; Gu, J. Identification and characterization of a novel peptide ligand of
epidermal growth factor receptor for targeted delivery of therapeutics. FASEB J. 2005, 19, 1978–1985. [CrossRef]
26. Williams, T.M.; Sable, R.; Singh, S.; Vicente, M.G.H.; Jois, S.D. Peptide ligands for targeting the extracellular domain of EGFR:
Comparison between linear and cyclic peptides. Chem. Biol. Drug Design 2018, 91, 605–619. [CrossRef]
27. Loudet, A.; Burgess, K. BODIPY dyes and their derivatives: syntheses and spectroscopic properties. Chem. Rev. 2007, 107,
4891–4932. [CrossRef]
28. Ulrich, G.; Ziessel, R.; Harriman, A. The chemistry of fluorescent BODIPY dyes: Versatility unsurpassed. Angew. Chem. Int. Ed.
2008, 47, 1184–1201. [CrossRef]
29. Lu, H.; Mack, J.; Yang, Y.; Shen, Z. Structural modification strategies for the rational design of red/NIR region BODIPYs. Chem.
Soc. Rev. 2014, 43, 4778–4823. [CrossRef]
30. Kolb, H.C.; Finn, M.G.; Sharpless, K.B. Click chemistry: Diverse chemical Function from a few good reactions. Angew. Chem. Int.
Ed. 2001, 40, 2004–2021. [CrossRef]
31. Rostovtsev, V.V.; Green, L.G.; Fokin, V.V.; Sharpless, K.B. A stepwise huisgen cycloaddition process: Copper(I)-catalyzed
regioselective “ligation” of azides and terminal alkynes. Angew. Chem. Int. Ed. 2002, 41, 2596–2599. [CrossRef]
32. Mukosera, G.T.; Adams, T.P.; Rothbarth, R.F.; Langat, H.; Akanda, S.; Barkley, R.G.; Dolewski, R.D.; Ruppel, J.V.; Snyder,
N.L. Synthesis of glycosylated zinc (II) 5,15-diphenylporphyrin and zinc (II) 5,10,15,20-tetraphenylporphyrin analogs using
Cu-catalyzed azide-alkyne 1, 3-dipolar cycloaddition reactions. Tetrahedron Lett. 2015, 56, 73–77. [CrossRef]
33. Parthiban, V.; Yen, P.Y.M.; Uruma, Y.; Lai, P.S. Designing synthetic glycosylated photosensitizers for photodynamic therapy.
Bull. Chem. Soc. Jpn. 2020, 93, 978–984. [CrossRef]
34. Dumoulin, F.; Ahsen, V. Click chemistry: The emerging role of the azide-alkyne Huisgen dipolar addition in the preparation of
substituted tetrapyrrolic derivatives. J. Porphyr. Phthalocyanines 2011, 15, 481–504. [CrossRef]
35. Ranyuk, E.; Cauchon, N.; Klarskov, K.; Guérin, B.; Van Lier, J.E. Phthalocyanine–peptide conjugates: Receptor-targeting
bifunctional agents for imaging and photodynamic therapy. J. Med. Chem. 2013, 56, 1520–1534. [CrossRef]
36. Best, M.D. Click chemistry and bioorthogonal reactions: Unprecedented selectivity in the labeling of biological molecules.
Biochemistry 2009, 48, 6571–6584. [CrossRef]
37. Hoyle, C.E.; Bowman, C.N. Thiol–ene click chemistry. Angew. Chem. Int. Ed. 2010, 49, 1540–1573. [CrossRef]
38. Tron, G.C.; Pirali, T.; Billington, R.A.; Canonico, P.L.; Sorba, G.; Genazzani, A.A. Click chemistry reactions in medicinal chemistry:
Applications of the 1, 3-dipolar cycloaddition between azides and alkynes. Med. Res. Rev. 2008, 28, 278–308. [CrossRef]
39. Kele, P.; Li, X.; Link, M.; Nagy, K.; Herner, A.; Lo˝rincz, K.; Béni, S.; Wolfbeis, O.S. Clickable fluorophores for biological
labeling—with or without copper. Org. Biomol. Chem. 2009, 7, 3486–3490. [CrossRef]
40. Yilmaz, M.D.; Bozdemir, O.A.; Akkaya, E.U. Light harvesting and efficient energy transfer in a boron-dipyrrin (BODIPY)
functionalized perylenediimide derivative. Organic Lett. 2006, 8, 2871–2873. [CrossRef]
41. Verdoes, M.; Hillaert, U.; Florea, B.I.; Sae-Heng, M.; Risseeuw, M.D.P.; Filippov, D.V.; van der Marel, G.A.; Overkleeft, H.S.
Acetylene functionalized BODIPY dyes and their application in the synthesis of activity based proteasome probes. Bioorg. Med.
Chem. Lett. 2007, 17, 6169–6171. [CrossRef] [PubMed]
42. Uppal, T.; Bhupathiraju, N.V.S.D.K.; Vicente, M.G.H. Synthesis and cellular properties of near-IR BODIPY–PEG and carbohydrate
conjugates. Tetrahedron 2013, 69, 4687–4693. [CrossRef]
43. Deniz, E.; Isbasar, G.C.; Bozdemir, O.A.; Yildirim, L.T.; Siemiarczuk, A.; Akkaya, E.U. Bidirectional switching of near IR emitting
boradiazaindacene fluorophores. Organ. Lett. 2008, 10, 3401–3403. [CrossRef] [PubMed]
44. Sharma, V.; Kumar, P.; Pathak, D. Biological importance of the indole nucleus in recent years: A compreensive review. J. Heterocyclic
Chem. 2010, 47, 491–502. [CrossRef]
45. Gibbs, J.H.; Zhou, Z.; Kessel, D.; Fronczek, F.R.; Pakhomova, S.; Vicente, M.G.H. Synthesis, spectroscopic, and in vitro investiga-
tions of 2,6-diiodo-BODIPYs with PDT and multi-mode imaging applications. J. Photochem. Photobiol. B Biol. 2015, 145, 35–47.
[CrossRef] [PubMed]
46. Wang, Q.; Chan, T.R.; Hilgraf, R.; Fokin, V.V.; Sharpless, K.B.; Finn, M. Bioconjugation by copper (I)-catalyzed azide-alkyne [3+ 2]
cycloaddition. J. Am. Chem. Soc. 2003, 125, 3192–3193. [CrossRef]
47. Komolov, K.E.; Koch, K.W. Application of surface plasmon resonance spectroscopy to study G-protein coupled receptor signaling.
In Surface Plasmon Resonance; Humana Press: Totova, NJ, USA, 2010; pp. 249–260.
48. Wilson, W.D. Analyzing biomolecular interactions. Science 2002, 295, 2103–2105. [CrossRef]
49. Chimerel, C.; Field, C.M.; Piñero-Fernandez, S.; Keyser, U.F.; Summers, D.K. Indole prevents Escherichia coli cell division by
modulating membrane potential. Biochem. Biophy. Acta 2012, 1818, 1590–1594. [CrossRef]