Bioorthogonal Turn-On BODIPY-Peptide Photosensitizers for Tailored Photodynamic Therapy

Article abstract of DOI:10.1002/chem.202001718

Photodynamic therapy (PDT) leads to cancer remission via the production of cytotoxic species under photosensitizer (PS) irradiation. However, concomitant damage and dark toxicity can both hinder its use. With this in mind, we have implemented a versatile

Full text of DOI:10.1002/chem.202001718

Accepted Article
Accepted Article
Title: Bioorthogonal Turn-On BODIPY-Peptide Photosensitizers
for Tailored Photodynamic Therapy
Authors: Greta Linden and Olalla Vazquez
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To be cited as: Chem. Eur. J. 10.1002/chem.202001718
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01/2020

10.1002/chem.202001718
Chemistry - A European Journal
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Bioorthogonal Turn-On BODIPY-Peptide Photosensitizers for
Tailored Photodynamic Therapy
Greta Linden,[a] and Olalla Vázquez*[a]
Dedicated to Professor Jesús Jiménez Barbero on the occasion of his 60th birthday
[a]
G. Linden, Jun.-Prof. Dr. O. Vázquez
Fachbereich Chemie
Philipps-Universität Marburg
Hans-Meerwein-Straβe 4, 35043 Marburg (Germany)
E-mail: olalla.vazquez@staff.uni-marburg.de
Supporting information for this article is given via a link at the end of the document.
Abstract: Photodynamic therapy (PDT) leads to cancer remission via
the production of cytotoxic species under photosensitizer (PS)
irradiation. However, concomitant damage and dark toxicity can both
hinder its use. With this in mind, we have implemented a versatile
peptide-based platform of bioorthogonally activatable BODIPY-
tetrazine PSs. Confocal microscopy and phototoxicity studies
demonstrated that the incorporation of the PS, as a bifunctional
module, into a peptide enabled spatial and conditional control of
singlet oxygen (1O2) generation. Comparing subcellular distribution,
PS confined in the cytoplasmic membrane achieved the highest
toxicities (IC50 = 0.096 ± 0.003 µM) after activation and without
apparent dark toxicity. Our tunable approach will inspire novel probes
towards smart PDT.
demand Diels–Alder (iEDDA) reaction in presence of a dienophile
such as trans-cyclooctenol (TCO) (Figure 1).
Now, we further expand this concept by exploring a modular
platform, in which the halogenated BODIPY and the quencher
tetrazine are separately integrated into a peptide scaffold via a
cysteine residue (Figure 1, Tz-C(2I-BODIPY)-PEPTIDE, 2-6OFF).
As yet, there are only few examples of BODIPY-peptide conjuga-
Introduction
Recent years have witnessed an immense growth in interest in
the photoregulation of biological events[1] because light offers the
possibility of exerting remote control of cellular functions and,
consequently, cures for a variety of diseases too. A traditional
clinical treatment based on light exposure is photodynamic
therapy (PDT),[2] which is nowadays the standard treatment for
several illnesses,[3] including some cancers.[4] PDT requires only
three primary components to trigger cell death: an efficient
photosensitizer (PS), light, and in situ production of radicals[5]
and/or highly reactive oxygen species (ROS)[6] as cytotoxic
agents. During the last decade, we have moved from
constitutively active PSs to smart compounds,[7] whose toxicity is
triggered by controlled stimuli such as environmental conditions
(pH,[8] glutathione concentration,[9] enzymes,[9b, 10] and cations[11]
)
electrostatic assemblies,[12] oligonucleotide displacement,[13]
aptamer recognition,[14] as well as photochromic switching.[15]
These activatable photosensitizers (aPSs) overcome one of the
biggest limitations of PDT: dark toxicity. Last year, we first
introduced a novel type of aPSs based on halogenated BODIPY-
tetrazine (mTz-2I-BODIPY, 1OFF) probes (Figure 1).[16] We
demonstrated that the direct incorporation of the tetrazine moiety
into the core of the 2I-BODIPY PS results in an efficient FRET
deactivation of the 1O2 production. Importantly, the phototoxic
effect can be restored via the bioorthogonal inverse-electron-
Figure 1. Structures of our precedent work on the unimodular turn-off mTz-2I-
BODIPY (1OFF) photosensitizer[16] (PS) and the turn-on [TCO:mTz]-2I-
BODIPY (1ON) PS, as well as the novel modular peptide-based turn-off Tz-
C(2I-BODIPY)-PEPTIDE (2-6OFF) PSs and turn-on [TCO:Tz]-C(2I-BODIPY)-
PEPTIDE (2-6ON) analogues from this project.
1
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10.1002/chem.202001718
Chemistry - A European Journal
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tes in PDT;[17] they have been mostly used as fluorescent
probes.[18] However, if the new molecular arrangement maintains
the precise control of the FRET process, peptide bioconjugation
may not only improve the properties of our aPS in line with other
hydrophobic PSs,[19] but since peptide signalling sequences are
powerful delivery vehicles,[20] may also localize photodynamic
action to specific subcellular locations. Notably, the efficiency of
cell photodamage is strongly determined by the PS
biodistribution.[21] To investigate our hypothesis, we aimed for the
synthesis and study of five bioorthogonally aPS peptide
conjugates (Fig 1, Tz-C(2I-BODIPY)-PEPTIDE, 2-6OFF) and the
corresponding active PS analogues: [TCO:Tz]-C(2I-BODIPY)-
PEPTIDE, 2-6ON. We selected the conventional polyarginines
(Rn) composed of five (R5) (Tz-C(2I-BODIPY)-R5, 2OFF and
[TCO:Tz]-C(2I-BODIPY)-R5, 2ON) or eight (R8) (Tz-C(2I-
BODIPY)-R8, 3OFF and [TCO:Tz]-C(2I-BODIPY)-R8, 3ON)
consecutive arginine residues capable of delivering cargos to the
cytoplasm and/or nucleus,[22] as well as two mitochondrial-
penetrating peptides (MPPs) from Kelley’s lab (Tz-C(2I-BODIPY)-
MPP1, 4OFF and [TCO:Tz]-C(2I-BODIPY)-MPP1, 4ON; Tz-C(2I-
BODIPY)-MPP2, 5OFF and [TCO:Tz]-C(2I-BODIPY)-MPP2,
5ON), as our targeting vectors. The latter peptides, MPP1[23] and
MPP2,[24] are based on the combination of two unnatural amino
acids: cyclohexylalanine (Fx) and D-arginine (r), the difference
between them is only a single extra D-arginine residue in MPP2.
We also pursued the preparation of a conjugate pair embedded
in the cell membrane. To achieve the appropriate lipophilicity,[25]
we planned to combine the hydrophilic tetra-arginine (R4) moiety
with the palmitic acid (PA) to form the corresponding pair Tz-C(2I-
BODIPY)-K(PA)R4 (6OFF) and [TCO:Tz]-C(2I-BODIPY)-
K(PA)R4 (6ON).
Results and Discussion
For the assembly of the hybrids, we followed a convergent
strategy that involves the synthesis of the 2-bromoacetamide
BODIPY 9 and the corresponding cysteine-containing peptides
(11-14) (Scheme 1). The tetrazine could successfully be
introduced in solution via standard N-hydroxysuccinimide (NHS)
conjugation[26] to yield the expected turn-off BODIPY-peptide PSs
(Tz-C(2I-BODIPY)-PEPTIDE, 2-6OFF). These could be activated,
in the presence of TCO, to afford the pyridazine analogues
([TCO:Tz]-C(2I-BODIPY)-PEPTIDE, 2-6ON) with the intention of
generating the 1O2 upon irradiation.
The synthetic route to these bioconjugates is shown in
Scheme 1. Thus, the azide-functionalized BODIPY 7, which had
previously been described as an accessible scaffold for the
generation of various BODIPY dyads,[27] was first iodinated with
N-iodosuccinimide (NIS) in DCM to maximize the intersystem
1
crossing process, and hence O2 production.[28] The resulting 2I-
BODIPY compound 8 was then converted to bromoacetamide 9
via a sequence of a 1,3-dipolar cycloaddition followed by
substitution. For the modular attachment of our PS/quencher pair,
all peptides included an N-terminal cysteine. The synthesis of
these cysteinyl fragments (11-14) was performed using the
classical Fmoc solid-phase methodology. After cleavage from the
solid support and reversed-phase (RP) HPLC purification, the
single cysteine residue enabled the efficient alkylation with the
thiol-reactive 2I-BODIPY 9. Taking advantage of the preference
of the NHS active esters for primary amines, the peptides were
tetrazine derivatized at the N-terminus in the final step. The
structures of the activatable BODIPY-peptide PSs (Tz-C(2I-
BODIPY)-PEPTIDE, 2-5OFF) were verified by high-resolution
mass spectrometry. For the Tz-C(2I-BODIPY)-K(PA)R4 (6OFF),
the palmitic acid (PA) was on-resin coupled to the lysine Nε-
amino group, which was previously protected as allyloxycarbonyl.
Scheme 1. Convergent synthetic approach for the preparation of the turn-off Tz-C(2I-BODIPY)-PEPTIDE conjugates (2-6OFF), and the corresponding turn-on
[TCO:Tz]-C(2I-BODIPY)-PEPTIDE analogues (2-6ON).
2
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The remainder of the synthesis was then analogous to those
described above. Lastly, all of the obtained Tz-C(2I-BODIPY)-
PEPTIDE conjugates (2-6OFF) reacted successfully with the
strained dienophile TCO to form the desired active peptide-based
PSs ([TCO:Tz]-C(2I-BODIPY)-PEPTIDE, 2-6ON).
Next, we explored the photodynamic activity and subcellular
localization of all BODIPY peptide-based conjugates. First, we
1
analysed their ability to produce O2 under irradiation. We used
1,3-diphenylisobenzofuran (DPBF) as a trap compound.[29] In the
presence of 1O2, DPBF decomposes in a 1:1 stoichiometry via a
[4+2] cycloaddition to give 1,2-dibenzoylbenzene, resulting in
reduced absorbance at 415 nm, and thereby, allowing the
measurement of 1O2 production. Our kinetics (Fig 2, Fig S43-
Figure 2. Consumption of DPBF in MeCN (200 μM) over time due to 1O2
generation in the presence of 0.1 μM of: 3OFF (solid dark blue circle); 3ON
(white circle with dark blue border); 5OFF (solid dark green triangle); 5ON (white
triangle with dark green border); 6OFF (solid yellow hexagon); 6ON (white
hexagon with yellow border); 8 (pink star) under irradiation at 525 nm with a
custom-made 96-well plate LED array (69.4 ± 0.6 W m-2). Control: solution of
DPBF in MeCN (200 μM, white squares). Mean data-points and standard
deviations are derived from at least two independent experiments, each
concentration in triplicate. For better comprehension only the turn-off/turn-on
photosensitizer (PS) pairs with the highest difference between off and on states
for each vector type are shown. The remaining pairs: 2OFF/ON and 4OFF/ON
are analysed in Figure S44.
1
Fig S45 and Table 1) together with the determined O2 quantum
yields (Table 1) showed that bioconjugation, i.e. conjugation to
the peptide sequences, does not have any impact on the in vitro
production of 1O2. The PS capacity of the turn-on [TCO:Tz]-C(2I-
BODIPY)-PEPTIDE conjugates (2-6ON) is similar (ΦΔ ∼ 0.60) to
the one of standard PSs.[30] Under 525 nm irradiation, all
pyridazine
BODIPY
[TCO:Tz]-C(2I-BODIPY)-PEPTIDE,
analogues (2-6ON) retained the same PS capacity as the
halogenated azide-functionalized BODIPY 8 (k ∼ 4.52 s-1 10-3
Afterwards, we evaluated the cellular uptake and subcellular
localization of these derivatives in HeLa cells by flow cytometry
and live-cell confocal microscopy. For this purpose, we prepared
the fluorescent non-halogenated H2N-C(BODIPY)-PEPTIDE
analogues (2-6FL) lacking the tetrazine group. (Fig 3 A). Flow
cytometry enables cellular-uptake quantification by measuring
fluorescence intensity. Thus, HeLa cells were incubated with the
BODIPY-labelled peptides (H2N-C(BODIPY)-PEPTIDEs
versus k = 4.97 s-1 10-3; ΦΔ
∼
0.59 versus ΦΔ = 0.61,
respectively) while, gratifyingly, there was a slower decrease in
1
the O2 generation of all Tz-C(2I-BODIPY)-PEPTIDE conjugates
1
(2-6OFF) (k ∼ 2.61 s-1 10-3; ΦΔ ∼ 0.55). Despite the efficient O2
quenching observed, as expected from a FRET-quenching
mechanism,[16, 31] this effect is higher when the tetrazine is directly
conjugated with the 2I-BODIPY core, as in PSs 1[16] than in the
case of the Tz-C(2I-BODIPY)-PEPTIDE compounds (2-6OFF).
Fig 3. Evaluation of cellular uptake via flow cytometry. A) Structure of the fluorescent H2N-C(BODIPY)-PEPTIDE conjugates (2-6FL). B) Representative dot plots
of the flow cytometry analysis of conjugate-untreated and -treated HeLa cells with the conjugates 2-6FL, including quantification at 2 μM (top) and 0.02 μM (bottom).
C) Overlayed histogram profiles of representative flow cytometry of conjugate-untreated (grey) and 2FL (light blue), 3FL (dark blue), 4FL (light green), 5FL (dark
green) and 6FL (yellow) treated cells showing the shift of flow cytometry fluorescence intensity signal at 2 µM conjugate concentration. D) Quantified fluorescent
signatures of the HeLa cells treated with the conjugates 2-6FL at concentrations: 2 μM (grid pattern), 0.2 μM (solid pattern) and 0.02 μM (diagonal line pattern).
Data are normalized to the vehicle-treated (ultrapure H2O, 1.25% MeCN) treated cells, which are assigned a value of 1.
3
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10.1002/chem.202001718
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2-6FL) at different concentrations for two hours, followed by a
washing protocol including
a trypsin treatment before the
cytometry trials. At 2 μM concentration, all peptides displayed a
pronounced fluorescence shift, which is clearly different to that of
the untreated cells. This signal corresponds to quantitative
labelling (Fig
3 B-D). However, if the concentration were
decreased 100 times (0.02 μM), cells were hardy labelled, except
when using lipopeptide H2N-C(BODIPY)-K(PA)R4 (6FL) (28%
labelled cells). Interestingly, this effect was more dramatic for the
oligoarginine conjugates, where low labelling rates (∼10%) were
already detected at 0.2 μM (Fig S67 and S68). The fluorescence
intensity was directly proportional to the concentration and
dependent on the type of peptide (Fig 3 D). Lipopeptide H2N-
C(BODIPY)-K(PA)R4 (6FL) displayed at least an ~ three-fold
signal increase in comparison to 2FL, 3FL, 4FL and 5FL at all
concentrations (Table S4), with a maximum of 15-fold signal
intensity increase for the 2 µM concentration of H2N-C(BODIPY)-
K(PA)R4 (6FL) compared to H2N-C(BODIPY)-R5 (2FL) .
Since standard flow cytometry cannot discriminate between
fluorescence from internalized fluorophores and that resulting
from membrane-bound ones, we employed live-cell confocal
microscopy (Fig 4). Images recording across consecutive Z-
stacks and the use of commercial fluorescent markers specific for
lysosomes (LysoTracker® Red DND-99),[32] mitochondria
(tetramethylrhodamine ethyl ester perchlorate: TMRE )[33] and
cytoplasmic membranes (1,1’-dioctadecyl-3,3,3’-tetramethyl-
indocarbocyanine perchlorate: DiI)[34] allowed us to decipher the
intracellular distribution of our fluorescent conjugates. For
comparison, we used the same parameters for all BODIPY-
peptide derivatives (H2N-C(BODIPY)-PEPTIDEs, 2-6FL), i.e.
2 µM concentration and two hours of incubation. The analysis of
the results revealed that the lipopeptide H2N-C(BODIPY)-
K(PA)R4 (6FL) was unable to penetrate the cells, despite having
an oligoargenine sequence. Indeed, the incapacity of other R4
peptide constructs to efficiently translocate the cell membrane
has previously been reported.[25, 35] In this case, we observed a
very intense fluorescence signal around the cytoplasmatic
membrane.
Co-localization experiments with H2N-C(BODIPY)-K(PA)R4
conjugate (6FL) and the membrane stain DiI demonstrated a
close-to-perfect overlay with a Pearson’s correlation coefficient
(Rr) of 0.907. However, the conjugates containing either at least
five arginine residues (R5 and R8) or either the MPP sequences
were efficiently taken up by the HeLa cells under the same
conditions (Fig 4). For polyarginine vectors, in general, it is
assumed that a minimum of six arginine residues (R6) are needed
to induce intracellular uptake.[22a, 36] However, we observed that
five consecutive arginines (R5) in our BODIPY-peptides H2N-
C(BODIPY)-R5, (2FL) are enough to achieve almost the same
cell-penetration as the R8 derivatives (H2N-C(BODIPY)-R8, 3FL).
Regarding the subcellular localization, significant differences
between the Rn and MPP hybrids were observed. Thus,
LysoTracker® colocalization experiments verified that the
punctate BODIPY-Rn distribution — hybrids H2N-C(BODIPY)-R5
(2FL) and H2N-C(BODIPY)-R8, (3FL) — is predominantly within
Figure 4. Localization of the fluorescent BODIPY-peptides 2-6FL in live HeLa
cells. Images displayed in green correspond to these peptides at 488 nm
excitation/491-535 nm emission. Before confocal imagining, cells were
incubated for 2 h (in FluoroBrite media, 2.5% FBS, 1.25% MeCN, 8.75%
ultrapure H2O). Concentrations of 2 μM BODIPY conjugates were used. The
conditions were: A) BODIPY-oligoarginine (Rn) peptides 2FL or 3FL: following
a DPBS washing step, 50 nM of LysoTracker® Red DND-99 in FluoroBrite
media (2.5% FBS, 0.5% DMSO) was added and left 2 h; cells were DPBS
washed again before image acquisition; images displayed in red correspond to
LysoTracker® Red DND-99 fluorescence (633nm excitation/635-700 nm
emission); or B) BODIPY-mitochondria penetrating peptides (MPPs) 4FL or
5FL: following a DPBS washing step, 25 nM of TMRE in FluoroBrite media
(2.5% FBS, 0.5% DMSO) was added and left for 30 min; cells were DPBS
washed again before image acquisition; images displayed in orange correspond
to TMRE fluorescence (543 nm excitation/545-589 nm emission); or C)
BODIPY-lipopeptide 6FL: following a DPBS washing step, 10 μM of DiI in
FluoroBrite media (2.5% FBS, 0.5% DMSO) was added and left for 15 min; cells
were DPBS washed again before image acquisition; images displayed in orange
correspond to DiI fluorescence parameters (543 nm excitation/545-589 nm
emission). All images collected were analysed by Zeiss ZEN; ImageJ software
was used for the calculation of the Rr to determine the extent of overlay.[37]
Values reported were calculated by linear regression,[38] using more than 10
cells analysed in two independent experiments. Scale bar: 20 µm; TMRE:
tetramethylrhodamine ethyl ester perchlorate; DiI: 1,1’-dioctadecyl-3,3,3’-
tetramethyl-indocarbocyanine perchlorate.
that the MPP derivatives were located within the lysosomes (Rr ~
0.6 and Rr ~ 0.65 for 2 hour and 30 minute incubation,
respectively).
Long-distance diffusion of 1O2 is impossible due to its brief
lifetime.[39] Consequently, PS distribution may determine the effi-
ciency of photoinduced cell death.[40] Once we had achieved
different subcellular localization by using peptides capable of
targeting specific cellular compartments, we next studied this
effect on both phototoxicity and in situ intracellular turn-on
activation of our bioorthogonally activatable PS. To this end, the
different OFF/ON BODIPY-peptide PSs were incubated at
different concentrations, and irradiated at 525 nm with a custom-
made 96-well plate LED array (69.4 ± 0.6 W m-2 for 160 s) after
the lysosomes (Rr
∼ 0.7). Unexpectedly, the compounds
designed specifically to target mitochondria (H2N-C(BODIPY)-
MPP1, 4FL and H2N-C(BODIPY)-MPP2, 5FL) showed poor
colocalization with TMRE (Rr ∼ 0.34) even when the incubation
time was reduced to 30 min (Rr ∼ 0.42, Fig S65). Further
colocalization studies with LysoTracker® (Fig S64) suggested
4
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10.1002/chem.202001718
Chemistry - A European Journal
FULL PAPER
two hours of incubation. Before the viability assays, the total
incubation time was 20 hours. The obtained IC50 values (Table 1;
Fig S47-S49) displayed a clear difference between the tetrazine
Tz-C(2I-BODIPY)-PEPTIDE derivatives (2-6OFF) and the
corresponding pyridazine [TCO:Tz]-C(2I-BODIPY)-PEPTIDE
ones (2-6ON) in the intracellular environment. Importantly,
without the PS motif, the peptidic vectors for cellular-compartment
targeting displayed a total absence of toxicity at the studied
concentration range, up to 4 μM (Fig S51).
phototoxicity is higher than that of the other constructs (2-5ON)
yet DPBF behaviour (Fig 2) is comparable. The superior
phototoxicity of 6ON was verified in other cell types too (Fig S52-
S54, Table S3). Importantly, in the highly metastatic PC-3 cells,
the conjugates 6 displayed similarly the highest toxicity as well as
the best OFF/ON ratio (3.3-fold; IC50 = 0.392 ± 0.028 µM for 6OFF
versus IC50 = 0.119 ± 0.012 µM for 6ON) among the pairs. In
addition, we demonstrated that the acidic pH of the lysosomes
does not affect the 1O2 generation by our PSs (Figure S44), unlike
in other lysosome targeted BODIPYs.[41] Consequently, the
highest phototoxicity of [TCO:Tz]-C(2I-BODIPY)-K(PA)R4 (6ON)
must be due to the specific localisation within the cell. These
factors jointly reveal the importance of photoinduced membrane
damage. This observation is in agreement with the general theory
that cell membranes are essential sites for the photosensitized
cell damage, [42] as reported for other PSs like rose bengal,[43] or
KillerRed.[44] Of note, no dark toxicity was detected for any
compound up to a maximum of 4 µM concentration (Fig S50).
Similarly, conjugates of the well-validated PS tetraphenyl-
porphyrin (TPP) with our peptides 12, 14 and 15 displayed an
increased phototoxicity for the H2N-C(TPP)-K(PA)R4 conjugate
(31, IC50 = 2.310 ± 0.301 µM) compared to the H2N-C(TPP)-R8
Table 1. Photophysical properties and photodynamic activity of the 2I-BODIPY-
peptide conjugates in HeLa cells.
[b]
Compound
2OFF
3OFF
4OFF
5OFF
6OFF
2ON
k [s-1 10-3][a]
2.62
ΦΔ
IC50 [μM][c]
0.55
0.55
0.56
0.56
0.55
0.59
0.60
0.59
0.59
0.59
0.617 ± 0.024
0.716 ± 0.078
0.599 ± 0.043
0.598 ± 0.037
0.369 ± 0.030
0.201 ± 0.012
0.196 ± 0.017
0.228 ± 0.008
0.225 ± 0.011
0.096 ± 0.003
2.55
2.82
2.70
2.40
(29, IC50 = 3.894 ± 0.453 µM) and H2N-C(TPP)-MPP2 (30, IC50
=
4.50
3.923 ± 0.469 µM) conjugates (Fig S49). These results suggest a
general behaviour of PA-R4 conjugates, which is independent of
the cargo, and therefore, applicable to other PSs.
3ON
4.96
Finally, we investigated whether we could achieve activation
of our turn-off BODIPY-peptide PSs via an in-situ bioorthogonal
iEDDA, and whether there is an influence of PS localization. For
this purpose, HeLa cells were treated with the corresponding turn-
off BODIPY-peptide PSs (2-6OFF) in varied concentrations from
0.13 µM to 1 µM for 90 minutes, followed by a washing step to
remove any traces of extracellular derivatives. Afterwards, the
dienophile TCO was added in excess (12.5 eq). After 30 min, the
plates were irradiated with 69.4 ± 0.6 W m-2 for 160 s and
incubated for another 18 hours, before determining cell viability
via a resazurin-based assay.[42] We observed that the addition of
TCO to the turn-off Tz-C(2I-BODIPY)-PEPTIDE probes (2-6OFF)
caused more cellular death than either compound alone, across
the entire range of concentrations studied (Fig S56-S58).
Importantly, such toxicity perfectly aligns to that measured for the
synthesized turn-on [TCO:Tz]-C(2I-BODIPY)-PEPTIDE PSs, (2-
6ON) (Fig S47-S49). Altogether, these results demonstrate that
4ON
4.14
5ON
4.32
6ON
4.68
[a] Rate constant of the DPBF consumption in MeCN; [b] 1O2 quantum yields
calculated relative to: rose bengal (ΦΔ = 0.76) and erythrosine B (ΦΔ = 0.60) in
MeOH;[30] [c] half maximal inhibitory concentration obtained from resazurin cell
viability assays (irradiation dose: 69.4 ± 0.6 W m-2 for 160 s). Mean values
derived from two independent experiments with errors ± 6 % for k, ± 7 % for ΦΔ;
In concordance with the in vitro 1O2 quenching experiments
(Fig 2), the phototoxicity window increases when the tetrazine is
directly conjugated with the 2I-BODIPY core, for example PS 1
(IC50 = 1.92 ± 0.27 μM for the tetrazine derivative 1OFF versus
IC50 = 0.354 ± 0.017 μM for its pyridazine analogue 1ON after
iEDDA with TCO).[16] However, divergence remains evident in the
new bioorthogonally activatable BODIPY-peptide conjugates.
Furthermore, all the turn-on [TCO:Tz]-C(2I-BODIPY)-PEPTIDE
compounds (2-6ON) showed higher cytotoxic potency than the
pyridazine analogue of the unimodular PS [TCO:mTz]-2I-BODIPY
(1ON), which must be due to the advantages of peptide
conjugation. It is known than peptides increase aqueous solubility
and reduce the general aggregation tendency observed in
hydrophobic photosensitizers such as BODIPYs.[19] As expected,
there was almost no difference in the intracellular phototoxicity of
the turn-off/turn-on pairs bearing oligoarginines (Rn) or
mitochondria penetrating peptides (MPPs), which were almost
identical in both vitro reactivity (Fig 2 and Table 1) and cellular
localization (Fig 4). It is noteworthy to highlight the BODIPY-
lipopeptide conjugates 6 because, among our BODIPY-peptide
PSs, the membrane-tagging turn-on [TCO:Tz]-C(2I-BODIPY)-
K(PA)R4 (6ON) was the most cytotoxic, and, simultaneously, this
construct displayed the highest phototoxicity variation between
turn-off Tz-C(2I-BODIPY)-K(PA)R4 (6OFF) /turn-on [TCO:Tz]-
Figure 5. Intracellular activation and phototoxicity effects on HeLa cells of the
turn-off BODIPY-peptides 2-6OFF at concentrations A) 0.5 μM of 2 (light blue),
3 (dark blue), 4 (light green) or 5 (dark green), and B) 0.25 μM for 6 (yellow) in
the presence (grid pattern) and absence (solid pattern) of the dienophile trans-
cyclooctenol (TCO), and the corresponding turn-on BODIPY-peptides 2-6ON as
controls (diagonal line pattern; same colours). After standing for 30 min, cells
were irradiated (69.4 ± 0.6 W m-2 for 160 s) at 525 nm with a custom made 96-
well plate LED array, followed by 18 h incubation in the dark. Mean data-points
and standard deviations are derived from two independent experiments.
C(2I-BODIPY)-K(PA)R4,
(6ON)
(3.8-fold).
Its
cellular
5
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10.1002/chem.202001718
Chemistry - A European Journal
FULL PAPER
MedChemExpress (USA); LysoTracker® Red DND-99 from Thermo
Fisher (USA); bromoacetic anhydride, palmitic acid, trypsin-EDTA and
penicillin-streptomycin from Sigma Aldrich (USA); Oxyma, Fmoc-
Lys(Alloc)-OH and Fmoc-D-Arg(Pbf)-OH from Carbolution (Germany);
Fmoc-Fx-OH from Fluorochem; DMF (peptide grade), Fmoc-protected
amino acids and TentaGel S RAM resin from Iris Biotech (Germany);
DPBS, DMEM and FluoroBrite DMEM from Gibco Thermo Fisher Scientific
(USA); FBS from PAN Biotech (Germany); MeCN (HPLC grade) from
VWR (France). TEA, THF and DCM were dried using standard
procedures.[45] Ultrapure H2O of type 1 was obtained with a MicroPure
Water Purification System (TKA, Germany).
regardless of the cellular localization of the PSs, all conjugates
are assembled inside the cells, and upon irradiation, the newly
synthesized [TCO:Tz]-C(2I-BODIPY)-PEPTIDE cycloadducts (2-
6ON) display enhanced phototoxicity. The Figure 5 compares the
in-situ activation processes for the different peptide-based PSs at
concentrations, where the cytotoxic difference between the turn-
off and in-situ activated analogues is the highest. Despite similar,
satisfactory tendencies across conjugates in the in-situ activation
process, the membrane-embedded conjugates 6OFF/ON again
surpassed the rest of the conjugates. Indeed, its effectiveness in
triggering cellular mortality due to its specific membrane
localization enabled reduction of the concentration of the turn-off
Tz-C(2I-BODIPY)-K(PA)R4 PS (6OFF) by half, which minimized
background toxicity and improved its applicability as an aPS.
Characterization
NMR spectra were recorded at 300 K on Bruker AV III HD 300 and 600
MHz spectrometers, whereas HRMS (ESI) results were acquired with a
LTQ-FT Finnigan Ultra mass spectrometer (Thermo Fischer Scientific), the
resolution was set to 100,000. Peptide and conjugate characterization was
performed via RP-HPLC-MS on an Agilent 1260 Infinity II HPLC-System
(Agilent Technologies) with an UV detector (220 nm). The analytical
column was an Agilent eclipse XDB-C18 column (5 μm, 4.6 × 150 mm) or
Macherey Nagel Nucleodur 100-C18 ec column (3 µm, 2 × 125 mm). The
following two eluents were used: A) ultrapure H2O with addition of 0.05%
TFA, and B) MeCN with addition of 0.03% TFA.
Conclusion
Most small-molecules fail to target cellular compartments
specifically: instead, they distribute either between several areas
simultaneously, or at random without reaching the desired
localization. In this study, we successfully provided access to a
modular peptide-based platform, in which the halogenated
BODIPY photosensitizer (PS) and the quencher tetrazine are
Singlet oxygen measurements
Singlet oxygen production: This was determined in black µclear 96 well
plates (Greiner Bio-One, Item: 655096) by recording endpoint absorbance
at 415 nm with a Tecan Spark 20M plate reader at 25°C. Compound
concentrations were determined from stocks using the molar extinction
coefficients (Table S1). 50 µL of 800 µM DPBF solution (200 µM as final
concentration per well) and 20 µL of 1 µM PS (100 nM as final
concentration per well) were added in MeCN (200 µL as final volume per
well). As controls the background (MeCN), and a sample containing
200 µM DPBF were used. Irradiation was performed in 20 s intervals with
an irradiation of 69.4 ± 0.6 W m-2 until 320 s by a custom-made LED-array
bearing 96 LEDs (Broadcom Limited, 525 nm, 16000-27000 mcd, viewing
angle: 23°, AVAGO HLMP-CM2B-120DD). For all measurements, before
the first irradiation cycle, an initial measurement was performed as time
zero without irradiation. Results were given as mean of the individual
values obtained, in which the background was previously subtracted,
displayed as percentage of DPBF present in each sample in triplicate.
Measurements were performed at least in two independent experiments,
i.e. from two different stocks.
separated. Peptide conjugation not only represents
a
straightforward approach to improve the photochemical
properties of PSs but also enables spatial activation due to the
accumulation of the PS in specific organelles. We reported a
range of dual-labelled peptides that only differ in their peptidic
vectors, sharing the same PS/quencher pair. This type of
bioorthogonally activatable PSs achieved control of the confined
photodynamic effect. Thus, the bioorthogonal activation in
specific subcellular organelles was successful. Importantly, PS
distribution determined the cellular response to photodamage in
multiple cell lines. Among our conjugates, the example capable of
being activated specifically in the cytoplasmic membrane ,Tz-
C(2I-BODIPY)-K(PA)R4, (6OFF) via an bioorthogonal iEDDA
surpassed the rest. Once active, the in-situ formed, [TCO:Tz]-
C(2I-BODIPY)-K(PA)R4 PS, (6ON) attained superior PDT
performance, i.e. enhancement of the photodynamic effect at
lower concentrations without dark toxicity. Importantly, since our
Singlet oxygen quantum yields (ΦΔ): They were determined as we
described before.[16] The procedure was alike the determination of 1O2
described above, but using 1 µM as final PS concentration. Erythrosine B
(EB) and rose bengal (RB) in MeOH were used as reference (ΦΔ = 0.76
for RB and 0.6 for EB).[30] Irradiation was performed in 10 s intervals with
irradiation of 69.4 ± 0.6 W m-2 for 160 s.
approach used
a
cysteine residue as workhorse, this
methodology is compatible with the common bioconjugation
strategies, making it highly versatile and tunable for the
incorporation of other PS as well as other biopolymers. Thus, we
believe that our modular approach will assist the exploration of
further subcellular compartments and bioconjugation possibilities,
as well as additional peptidic functionalities such as tumour-
targeting peptides and antibodies to tackle the selectivity issues
in PDT. Therefore, this modular peptide-based platform will
further advance the applicability of the bioorthogonally activatable
PSs.
Peptide synthesis
Peptides 11-14 were synthesized in a 20 µmol scale according to the
standard SPPS methodology, using Fmoc-amino acids and Oxyma/DIC as
coupling agent. Obtained peptides were purified by semipreparative RP-
HPLC on a Varian (USA) ProStar Preparative HPLC system with a semi-
preparative Phenomenex Juptier 10 u C18 300 Å column. The eluents
were ultrapure H2O (A) and MeCN (B) with addition of 0.1% TFA. Analytical
RP-HPLC chromatograms and used gradients are in Fig S1-S4.
Experimental Section
H2N-CRRRRR-CONH2 (11): White solid (13.3 mg, 8.4 µmol, 42%).
tR = 13.16 min (Fig S1). Chemical formula: C33H68N22O6S. HRMS-ESI+
(m/z): [M+2H]2+calcd: 451.2779; found: 451.2778.
Materials
All commercially available compounds were used without further
purification as delivered from the corresponding companies: 3-
diphenylisobenzofuran from Alfa Aesar (USA); propagylamine from Acros
Organics (Belgium); TMRE from Cayman Chemical (USA); DiI from
H2N-CRRRRRRRR-CONH2 (12): White solid (15.4 mg, 6.4 µmol, 32%),
tR =16.48 min (Fig S2). Chemical formula: C51H104N34O9S. HRMS-ESI+
(m/z): [M+3H]3+ calcd: 457.2888; found: 457.2887.
6
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10.1002/chem.202001718
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FULL PAPER
H2N-CFxrFxrFxr-CONH2 (13): White solid (18.8 mg, 12.5 µmol, 62%).
tR =22.15 min (Fig S3). Chemical formula: C48H89N17O7S. HRMS-ESI+
(m/z): [M+2H]2+ calcd: 524.8499; found: 524.8516.
260 nm. The 2I-BODIPY-peptides were characterized as explained before
(characterization section above). Analytical RP-HPLC chromatograms and
used gradients are in Figures S23-S26. Afterwards, to a 0.07 M solution of
the corresponding 2I-BODIPY-peptide (1.00 eq) dissolved in DMF, it was
added first DIPEA (2.00 eq), and subsequently the NHS-active ester 10
(2.00 eq, 0.20 M in DMF). The reaction was mixed at room temperature on
an Eppendorf Thermoshaker, and followed by analytical RP-HPLC-MS
until complete conversion was observed. 2-5A conjugates were purified by
semi-preparative RP-HPLC. A linear gradient over 30 minutes from 5% to
75% of solvent B was applied for compounds 2-5A, Detection of the
signals was achieved with a dual wavelength UV detector at 220 nm and
260 nm. Analytical RP-HPLC chromatograms and used gradients are in
Figures S28-31.
H2N-CrFxrFxrFxr-CONH2 (14): White solid (16.4 mg, 9.2 µmol, 46%).
tR =25.05 min (Fig S4). Chemical formula: C54H101N21O8S. HRMS-ESI+
(m/z): [M+2H]2+ calcd: 602.9004; found: 602.9022.
Organic synthesis
NMR spectra for compounds 19 and 9 are in Fig S12-S17.
Compound 9: This was obtained in a two-step synthesis from 8. 8 (165 mg,
250 µmol, 1.00 eq) and CuI (7.6 mg, 40.0 µmol, 0.16 eq) were dissolved
in dry THF (3.00 mL) under inert atmosphere. Propargylamine (240 µL,
3.75 µmol, 15.0 eq) was added last, and the solution was stirred for 3 h.
The reaction mixture was concentrated under reduced pressure and
purified by column flash chromatography (DCM to DCM/MeOH/TEA;
94:5:1, v/v/v). The intermediate 19 was obtained as red solid (82.9 mg,
116 µmol, 46%). TLC: Rf = 0.30 (DCM/MeOH/TEA, 94:5:1, v/v/v). 1H-NMR
(300 MHz, 300 K, CDCl3): δ = 8.68 (brs, 3H, NH3), 8.09 (s, 1H,Htriazole),
7.13 (d, 3J = 8.5 Hz, 2H, 2×Harom), 7.01 (d, 3J = 8.6 Hz, 2H, 2×Harom), 4.81
(t, 3J = 4.6 Hz, 2H, CH2), 4.44-4.41 (m, 4H, 2×CH2), 2.61 (s, 6H, 2×CH3),
1.38 (s, 6H, 2×CH3) ppm. 13C-NMR (75 MHz, 300K, CDCl3): δ = 158.8 (1C,
Cq.), 157.0 (2C, 2×Cq.), 145.3 (2C, 2×Cq.), 141.0 (1C, Cq.), 140.3 (1C, Cq),
131.7 (2C, 2×Cq.), 129.5 (2C, 2×Carom), 128.1 (1C, Cq.triazole) 125.5 (1C,
HCtrialzole), 115.6 (2C, 2×Carom), 85.9 (2C, 2×CI), 66.2 (1C, CH2), 50.1 (1C,
CH2), 34.9 (1C, CH2-NH3), 17.4 (2C, 2×CH3), 16.2 (2C, 2×CH3) ppm. 19F-
NMR (470 MHz, 300K, CDCl3): δ = -146.2 (q, 2F, 1J = 32.4 Hz, BF2) ppm.
11B-NMR (160 MHz, 300 K, CDCl3): δ = 0.28 (t, 1B, 1J = 32.4 Hz, BF2) ppm.
HRMS-ESI+ (m/z): calcd for C24H26BF2I2N6O [M+Na]+: 740.0216; found:
740.0212. The intermediate 19 (50.0 mg, 69.8 µmol, 1.00 eq), and pyridine
(5.6 µL, 69.8 µmol, 1.00 eq) were dissolved in degassed DMF (2.50 mL)
under inert atmosphere and placed in an ice bath. Bromoacetic anhydride
(54.4 mg, 209 µmol, 3.00 eq) dissolved in degassed DMF (1.50 mL) was
added dropwise. After 15 minutes the ice bath was removed, and the red
solution was stirred for three hours at room temperature. The mixture was
then concentrated under reduced pressure, diluted with EtOAc (10 mL),
washed with distilled H2O (2× 25 mL) and brine (1× 25 mL). The organic
phase was dried over anhydrous MgSO4, filtered, concentrated under
reduced pressure and purified by column flash chromatography (EtOAc)
to obtain 9 as dark red solid (37.5 mg, 44.8 µmol, 64%). TLC: Rf = 0.36
(EtOAc). 1H-NMR (300 MHz, 300 K, CDCl3): δ = 7.79 (s, 1H, CHtriazol), 7.15
(d, 3J = 8.7 Hz, 2H, 2×Harom), 7.01 (d, 3J = 8.7 Hz, 2H, 2×Harom), 4.81 (t, 3J
= 5.1 Hz, 2H, CH2), 4.59 (d, 3J = 5.9 Hz, 2H, CH2), 4.44 (t, 3J = 5.1 Hz, 2H,
CH2), 3.88 (s, 2H, CH2), 2.63 (s, 6H, 2× CH3), 1.41 (s, 6H, 2× CH3) ppm.
13C-NMR (75 MHz, 300K, CDCl3): δ = 165.9 (1C, CO), 158.9 (1C, Cq.),
157.0 (2C, 2×Cq.), 145.4 (2C, 2×Cq.), 144.2 (1C, Cq), 141.0 (1C, Cq.), 131.8
(2C, 2×Cq.), 129.6 (2C, 2×Carom), 128.1 (1C, Cq.triazole), 123.8 (1C, HCtriazole),
115.6 (2C, 2×Carom), 85.8 (2C, 2×CI), 66.5 (1C, CH2), 49.9 (1C, CH2), 35.7
(1C, CH2-NH), 28.9 (1C, CH2-Br), 17.4 (2C, 2×CH3), 16.2 (2C, 2×CH3) ppm.
19F-NMR (470 MHz, 300 K, CDCl3): δ = -145.8 (q, 2F, 1J = 32.4 Hz, BF2)
ppm. 11B-NMR (160 MHz, 300 K, CDCl3): δ = 0.27 (t, 1B, 1J = 32.4 Hz,
BF2) ppm. HRMS-ESI+ (m/z): calcd for C26H26BBrF2I2N6O2 [M+Na]+:
858.9348; found: 858.9346.
Tz-C(2I-BODIPY)-RRRRR-CONH2 (2OFF): obtained from peptide 24 and
compound 10; reaction time: 3 h; red solid; yield: 46 %; tR= 20.81 min (Fig
S28). Chemical formula: C69H99BF2I2N32O9S. HRMS-ESI+ (m/z):
[M+2H]2+calcd: 928.3150; found:928.3153.
Tz-C(2I-BODIPY)-RRRRRRRR-CONH2 (3OFF): obtained from peptide 25
and compound 10; reaction time: 3 h; red solid; yield: 61 %; tR= 23.00 min
(Fig S29). Chemical formula: C87H135BF2I2N44O12S. HRMS-ESI+ (m/z):
[M+2H]2+ calcd: 1162.9677; found: 1162.9682.
Tz-C(2I-BODIPY)-FxrFxrFxr-CONH2 (4OFF): obtained from peptide 26
and compound 10; reaction time: 3 h; pink solid; yield: 53%; tR= 26.32 min
(Fig S30). Chemical formula: C84H120BF2I2N27O10S. HRMS-ESI+ (m/z):
[M+2H]2+ calcd: 1001.8871; found: 1001.8899.
Tz-C(2I-BODIPY)-rFxrFxrFxr-CONH2 (5OFF): obtained from peptide 27
and compound 10; reaction time: 3 h; pink solid; yield: 57%; tR= 23.92 min
(Fig S31). Chemical formula: C90H132BF2I2N31O11S. HRMS-ESI+ (m/z):
[M+2H]2+ calcd: 1079.9375; found: 1079.9415.
Compound 6OFF: Following the explained SPPS methodology, l-
RRRRK(Alloc)C-Fmoc was synthesised. The allyloxycarbonyl group was
removed on-resin. Thus, under nitrogen, the resin was washed with DCM,
and then Pd(OAc)2 (0.05 eq) and triphenylphosphine (1.50 eq) were added
to the reactor together with a solution of phenylsilane (20.0 eq) and NMM
(20.0 eq) in 1 mL DCM (0.02 M). The resin was shaken for 90 min, and
afterwards, washed with sodium diethyldithiocarbamate (5 mg/mL in DMF,
5× 1.5 mL), DCM (5× 1.5 mL) and DMF (5× 1.5 mL). Then, palmitic acid
(4.00 eq), Oxyma (4.00 eq) and DIC (4.00 eq) were dissolved in 400 μL of
DMF and pre-activated for 3 min. The mixture was added to the resin. After
90 min, the coupling was repeated to ensure quantitative conversion. Test
cleavage and MS (Fig S5) confirmed the identity of the Fmoc-
CK(PA)RRRR-CONH2 (15) intermediate. Afterwards, the N-terminus was
deprotected under standard SPPS conditions, this lipopeptide was cleaved
from the solid support. The obtained compound underwent the same
bioconjugation conditions as 24-27 but using DMSO as solvent to avoid
the micelle formation of the starting material lipopeptide, which was
observed in the Tris buffer/MeCN solvent mixture. The reaction mixture
was further diluted with ultrapure H2O/MeCN (7:3) to have a DMSO
concentration ≤5% before semipreparative RP-HPLC purification at a
linear gradient over 30 minutes from 15% to 95% of solvent B. This
procedure yielded the H2N-C(2I-BODIPY)-K(PA)RRRR-CONH2 (28) (Fig
S27). Finally the tetrazine conjugation proceed as for 2-5OFF using a
purification gradient from 15% to 95% of solvent B, which afforded the
compound 6OFF. Analytical RP-HPLC chromatogram and used gradients
are in Figure S32.
Conjugate synthesis
Compounds 2-5A: They were obtained in a two-step synthesis. Initially, in
a microcentrifuge tube DIPEA (2.20 eq) was added to a 0.12 M solution of
the corresponding peptide (1.20 eq) in degassed Tris buffer (0.10 M,
pH= 8.5). Afterwards, the 2-bromoacyl photosensitizer 9 (1.00 eq, 0.10 M
in MeCN) was added. The reaction was mixed at room temperature on an
Eppendorf Thermoshaker, and followed by analytical RP-HPLC-MS until
the complete conversion of the starting material 9 was observed, and the
desired 2I-BODIPY-peptides 24-27 were observed (Fig S23-26). These
compounds were purified by semipreparative RP-HPLC using a linear
gradient over 30 minutes from 5% to 75% of solvent B. Detection of the
signals was achieved with a dual wavelength UV detector at 220 nm and
Tz-C(2I-BODIPY)-K(PA)RRRR-CONH2 (6OFF): obtained from peptide 28
and compound 10; reaction time: 4 h; pink solid; yield: 42%; tR= 31.16 min
(Fig S32). Chemical formula: C85H129BF2I2N30O10S. HRMS-ESI+ (m/z):
[M+2H]2+ calcd: 1033.4269; found: 1033,4309.
Compounds 2-6ON: In a microcentrifuge tube, TCO (10.0 eq) was added
to the corresponding 2I-BODIPY peptide OFF (1.00 eq, 50 mM) dissolved
in MeCN. The reaction was mixed at room temperature on an Eppendorf
Thermoshaker for 30 minutes. The crudes were purified by
semipreparative RP-HPLC purification to obtain the compounds 2-6ON. A
linear gradient over 30 minutes from 5% to 75% of solvent B was applied
7
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10.1002/chem.202001718
Chemistry - A European Journal
FULL PAPER
for compounds 2-5ON, and from 15% to 95% of solvent B for compound
6ON. Detection of the signals was achieved with a dual wavelength UV
detector at 220 nm and 260 nm. Analytical RP-HPLC chromatograms and
used gradients are in Figures S33-37.
conditions were assessed in triplicates per sample/control. The results
derived from, at least, two independent experiments.
Flow cytometry measurements
[TCO:Tz]-C(2I-BODIPY)-RRRRR-CONH2 (2ON): Pink solid; yield: 54%;
(Fig S33). Chemical formula: C77H111BF2I2N30O10S. HRMS-ESI+ (m/z):
[M+2H]2+ calcd: 976.3564; found: 976.3571.
HeLa cells (1×106) were seeded in 3.0 mL DMEM (2.5% FBS) into 6-well
plates (Sartetedt, Item: 83.3920), and incubated for 18 h. The cells were
treated with three different concentrations: 2.00 µM, 0.20 µM and 0.02 µM
in 1.25% MeCN in ultrapure H2O of the fluorescent peptides 2-6FL for 2 h.
Afterwards they were washed DPBS (2× 2.0 mL), and treated with a
trypsin-EDTA solution (500 µL) for 3 min at 37°C. After that time, DMEM
(1000 µL, 2.5% FBS) was added, and the cell suspensions were
transferred to microcentrifuge tubes and centrifuged (6000 rpm, 1 min) in
a Biofuge pico (Heraeus, Germany), the supernatant was discarded. This
was repeated one more time using DPBS. Finally, the cells were
suspended in DPBS containing 0.5% FBS for measurement. Fluorescence
analysis was performed immediately with a LSR Fortessa cell analyser
(BD Biosciences, USA). A minimum of 50,000 events per sample were
analysed. The experiment was performed in two independent experiments.
[TCO:Tz]-C(2I-BODIPY)-RRRRRRRR-CONH2 (3ON): Pink solid; yield:
47%; (Fig S34). Chemical formula: C95H147BF2I2N42O13S. HRMS-ESI+
(m/z): [M+2H]2+ calcd: 1211.0092; found: 1211.0106.
[TCO:Tz]-C(2I-BODIPY)-FxrFxrFxr-CONH2 (4ON): Pink solid; yield: 61%;
(Fig S35). Chemical formula: C92H132BF2I2N25O11S. HRMS-ESI+ (m/z):
[M+2H]2+ calcd: 1049.9283; found: 1049.9305.
[TCO:Tz]-C(2I-BODIPY)-rFxrFxrFxr-CONH2 (5ON): Pink solid; yield: 57%;
(Fig S36). Chemical formula: C98H144BF2I2N29O12S. HRMS-ESI+ (m/z):
[M+2H]2+ calcd: 1127.9788; found: 1127.9813.
[TCO:Tz]-C(2I-BODIPY)-K(PA)RRRR-CONH2 (6ON): Pink solid; yield:
52%; (Fig S37). Chemical formula: C93H141BF2I2N28O11S. HRMS-ESI+
(m/z): [M+2H]2+ calcd: 1081.4683; found: 1081.4720.
Live-cell microscopy measurements
HeLa cells (2.5×104) were seeded in 8-well on cover glass cell culture
chambers (Sarstedt, Item: 94.6190.802) in 500 µL FluoroBrite DMEM
(2.5% FBS) and incubated for 18 h. Then, fluorescent peptides 2-6FL were
added from freshly prepared stocks (20 µM in 12.5% MeCN ultrapure H2O)
resulting into a final 2 µM concentration and incubated for 2 h. After that,
cells were rinsed with DPBS (3× 500 µL). Cells were then treated with
commercial dyes for labelling cell compartments. For lysosome labelling:
cells were treated with 100 nM LysoTracker® Red DND-99 in FluoroBrite
media (2.5% FBS, 0.5% DMSO) for 2 h. For mitochondria labelling, cells
were treated with 25 nM TMRE in FluoroBrite media (2.5% FBS, 0.5%
DMSO) for 30 min. For cytoplasmic membrane labelling, cells were treated
with 10 µM DiI in FluoroBrite media (2.5% FBS, 0.5% DMSO) for 15 min.
Afterwards, cells were rinsed with DPBS (2× 500 µL), then, 500 µL
FluoroBrite DMEM with 2.5% FBS was added to each well. Images were
acquired on a LSM880 Laser Scanning Confocal Microscope (Zeiss,
Germany) with a 63 × 1.4 oil immersion objective at 37°C. Z-stack images
(1 µm steps) corresponding to images in Figure 4 are depicted in Figures
S59-S63.
Cell-bassed assays
Cell culture: Most of the studies were performed in HeLa cells (details on
PC-3 cells see cell culture in SI). HeLa cells were grown in flat-bottomed
culture flasks (T75, Sarstedt, Germany) in a fully humidified cell-culture
incubator (Galaxy CO-170 S incubator, New Brunswick Scientific, USA) at
37°C under CO2 (g) (5 % v/v). Growth medium was DMEM supplemented
with FBS (10 % v/v), penicillin (100 units/mL), and streptomycin
(100 μg/mL). DMEM and DPBS were stored at 6°C, additives and trypsin
at -20°C, solutions were tempered to 37°C prior usage by a water bath.
Cells were grown to confluency and passaged every 2 to 4 days using
trypsin-EDTA solution till passage 17. Cells were counted using a
Neubauer improved cell counter (Laboroptik Ltd, Germany) for the
determination of seeding densities. All cell-based assays were performed
under reduced media conditions (2.5% FBS). A detailed step-wise protocol
of the cell-bassed assays is in the supporting information (Cell Culture).
IC50 determinations: HeLa cells (2×104) were seeded into black µclear 96-
well microtiter plates (Greiner Bio-One, Item: 655-090) in DMEM (200 µL,
2.5% FBS). After 21 h, 110 µL media were removed from each well, and
cells were treated with 10 µL of the conjugates 2-6OFF, 2-6ON and 29-31
in ultrapure H2O with 12.5% MeCN at different concentrations (4.00-0.03
μM as final conjugate concentrations), each concentration in triplicate.
Compounds were incubated for 2 h. Then, the plate was irradiated 69.4 ±
0.6 W m-2 for 160 s at 525 nm and further incubated for 18 h. Resazurin
fluorescence-based cell viability assay was performed to evaluate cell
viability. IC50 values were determined using GraphPad Prism version 6
(GraphPad Software, USA) applying the log(inhibitor) vs. response –
variable slope (four parameters) fit. Untreated cells and cells treated only
with the vehicle solvent (12.5% MeCN in ultrapure H2O), were considered
as negative controls. All conditions were assessed in triplicates per
sample/control. The IC50 derived from, at least, two independent
experiments.
Acknowledgements
We gratefully thank Prof. Dr. E. Meggers (Philipps-Universität
Marburg) for accessibility to the S1 laboratory; Dr. G. Malengo
(Max Plack Institute for Terrestrial Microbiology) for microscopy
image acquisition; Nina Büttner (Max Plack Institute for Terrestrial
Microbiology) for flow cytometry acquisition; Jun.-Prof. Dr. L.
Schulte for initial assistance in flow cytometry analysis. Prof. Dr.
U. Bakowsky (Philipps-Universität Marburg) for providing
LysoTracker® Red DND-99; N. Frommknecht (Philipps-
Universität Marburg) for design assistance and construction of
custom-made 96-well plate LED arrays, Dr. K. Hoogewijs (Ghent
University) for his insight into mitochondria-tagging and T. Roider
(Philipps-Universität Marburg) for LED irradiance measurements.
Intracellular iEDDA cell viability assays: HeLa cells were seeded as for the
IC50 determination experiments. 21 h after seeding, 110 µL media per well
were removed, and cells were treated with 10 µL of the conjugates 2-6ON
in ultrapure H2O with 12.5% MeCN at different concentrations (1.00-
0.13 µM as final conjugate concentration), each concentration in triplicate.
Compounds were incubated for 90 min. Then, the medium was carefully
aspirated, and the cells were washed twice with DPBS, and once with
media (DMEM, 2.5% FBS). Afterwards, fresh reduced serum culture media
(100 µL DMEM, 2.5% FBS) was added to the wells as well as 12.5 eq
(according to the peptide concentration) of TCO. After 30 min of incubation,
the plate was irradiated 69.4 ± 0.6 W m-2 for 160 s at 525 nm and
incubated for 18 h. Untreated cells, cells treated only with the vehicle
solvent (12.5% MeCN in ultrapure H2O) and only TCO were controls. All
Keywords: BODIPY • peptides • photodynamic therapy
•
tetrazine • bioorthogonal reactions
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Entry for the Table of Contents
Modular peptide-based platform for organelle-confinement of the photodynamic effect. We report a general approach capable
of directing the bioorthogonal activation of tetrazine-BODIPY photosensitizers to specific subcellular organelles.
11
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