Intracellular Reactions Promoted by Bis(histidine) Miniproteins Stapled Using Palladium(II) Complexes

Article abstract of DOI:10.1002/anie.202002032

The generation of catalytically active metalloproteins inside living mammalian cells is a major research challenge at the interface between catalysis and cell biology. Herein we demonstrate that basic domains of bZIP transcription factors, mutated to include two histidine residues at i and i+4 positions, react with palladium(II) sources to generate catalytically active, stapled pallado-miniproteins. The resulting constrained peptides are efficiently internalized into living mammalian cells, where they perform palladium-promoted depropargylation reactions without cellular fixation. Control experiments confirm the requirement of the peptide scaffolding and the palladium staple for attaining the intracellular reactivity.

Full text of DOI:10.1002/anie.202002032

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Accepted Article
Title: Intracellular reactions promoted by bis-histidine miniproteins
stapled with Pd(II) complexes
Authors: Jose Luis Mascarenas, Soraya Learte-Aymamí, Cristian Vidal,
and Alejandro Gutiérrez-González
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202002032
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10.1002/anie.202002032
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RESEARCH ARTICLE
Intracellular reactions promoted by bis-histidine miniproteins
stapled with Pd(II) complexes
Soraya Learte-Aymamí,[a] Cristian Vidal,[a] Alejandro Gutiérrez-González,[a] and José L. Mascareñas*[a]
Dedication ((optional))
[a]
S. Learte-Aymamí, Dr. C. Vidal, A. Gutiérrez-González, Prof. J. L. Mascareñas
Centro Singular de Investigación en Química Biolóxica e Materiais Moleculares (CIQUS), and Departamento de Química Orgánica
Universidade de Santiago de Compostela
15782 Santiago de Compostela (Spain)
E-mail: joseluis.mascarenas@usc.es
Supporting Information for this article is given via a link at the end of the document. ((Please delete this text if not appropriate))
Abstract: The generation of catalytically active metalloproteins inside
living mammalian cells is a major research challenge at the interface
between catalysis and cell biology. Herein we demonstrate that basic
domains of bZIP transcription factors, mutated to include two histidine
residues at i, i+4 positions, react with palladium (II) sources to
generate catalytically active, stapled pallado-miniproteins. The
resulting constrained peptides are efficiently internalized into living
mammalian cells, where they can perform palladium-promoted
depropargylation reactions, without cellular fixation. Control
experiments confirm the requirement of the peptide scaffolding and
the palladium staple for attaining the intracellular reactivity.
enzymes.7 Not surprisingly, progress in this matter has been
extremely scarce, and essentially limited to one elegant report in
the internalization of streptavidin-ruthenium constructs in HEK-
293 cells.8 This paucity contrasts with the increasing number of
reports on the development of bioorthogonal and intracellular
reactions promoted by discrete transition metal complexes, 9,10 or
metal nanoparticles.11
Quite recently, we discovered that introducing two histidine
residues in strategic i and i+4 positions of the basic region of a
GCN4 bZIP transcription factor,12 allows to trigger its DNA binding
upon addition of PdCl2(en) (en = ethylenediamine).13 Interestingly,
the palladium additive also sparks an efficient membrane
translocation into mammalian cells. Specific control experiments
confirmed that these properties result from stapling of the two
histidines by the added metal (Figure 1).
Introduction
This discovery raised intriguing questions related to the generality
of the approach, the mechanism of the membrane crossing, and
the stability of the peptide-palladium complex in the cell cytosol,
among others. But more importantly, since the approach can also
be viewed as a peptide-promoted internalization of a palladium
complex, we wondered whether the resulting metallopeptide
could promote designed reactions inside cells. This could
Metalloproteins make up a substantial portion of the human
proteome (between one-third and one-half) and play critical
structural and catalytic role in living cells and whole organisms.1
Catalytic metalloproteins, i.e. metalloenzymes, are involved in
many fundamental metabolic reactions, including, among other,
redox processes, isomerisations, hydrolysis or condensations.2
Interestingly, despite the large number of alternatives offered by
the periodic table, Nature has evolved to use just a few metals as
cofactors to build the palette of metalloenzymes required to
sustain life. Indeed, most metal-based proteins feature either
alkali and alkaline earth elements, or first row transition metals
like Fe, Cu, Mn, Co or Zn.2,3
represent
a significant first step towards the challenging
endeavour of developing intracellular, catalytic metalloproteins.
The recognition that introducing other transition metals like Ni, Ru,
Rh, Pd or Ir at designed sites of proteins might provide for non-
natural transformations with enzymatic characteristics has led to
very significant advances in the field of “artificial
metalloenzymes”.4 Therefore, a number of transition metal
protein hybrids capable of promoting otherwise difficult reactions
have been recently reported.5 Most successful results involve the
use of prosthetic groups as metal coordinating units, albeit some
examples in which the artificial metalloproteins result from direct
metal coordination to amino acid side chains have also been
described.6
Figure 1. a) Representation of the bis-Histidine modified basic region protein,
and conformational constrain resulting from the Pd(II) stapling. The clipped
miniprotein is internalized into mammalian cells. b) Some questions
approached in this work. Note: Amino acid numbering according to the natural
bZIP protein.
Whereas these efforts are ascribed to the realm of synthetic
chemistry, a challenging, remaining goal in the field consists of
the introduction or generation of artificial metalloenzymes in living
mammalian cells, which is the native environment of many natural
1
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10.1002/anie.202002032
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RESEARCH ARTICLE
Herein we demonstrate the viability of the approach by describing
the first metal-equipped, synthetic miniproteins made by natural
amino acids that are capable of promoting abiotic reactions inside
living mammalian cells. Our results indicate that the bis-histidine
containing peptide and the palladium complex are both required
for the cellular internalization and for the intracellular reactivity,
which in this work is demonstrated for depropargylation reactions.
Specifically, we synthesized the bipyridine-anthracene palladium
derivative Pd1 (Figure 2), and found that it also reacts efficiently
with the bis-histidine peptide to give the expected complex, as
corroborated by LC-MS (see Figure S21). HeLa cells were then
incubated for 1 h with the complex resulting from mixing
equivalent amounts of TMR-brHis2 and Pd1 (30 min). After
washing (PBS, 2X), cells were observed by microscopy at
different times. Figures 2a and 2b show the intracellular
emissions corresponding to both fluorophores after 1 h, while
Figure 2c is a colocalization image. Observation after 4 h did not
show perceptible changes in the colocalization of the
fluorophores. A control experiment using only the metal complex
(Pd1), at the same concentration, revealed a very poor
internalization (Figure 2d), confirming the role of the peptide as
palladium transporter.
With a method to internalize a relatively stable Pd(II) complex, we
wondered whether this species could be engaged in catalytic
reactions. As testing reaction, we explored the depropargylation
of the probe HBTPQ (1, Figure 3a),10i which is not fluorescent but
upon uncaging generates a product 2 that emits light at 635 nm
when excited in the far-ultraviolet (far-UV) region (λexc = 330 nm).
Importantly, previous work from our and other groups had shown
that typical palladium sources like PdCl(allyl)2 or Pd(OAc)2 fail to
promote this reaction inside HeLa cells,10i,11d likely because of a
poor cell penetration of the palladium species, as well as a rapid
deactivation in the presence of cellular components.
Results and Discussion
Before testing catalytic reactivities, we considered fundamental
to obtain more information on the internalization requirements of
the miniprotein. Our previous studies had demonstrated that
mixing the peptide brHis2 with PdCl2(en) produces relatively
stable peptide palladium complexes (detected by ESI-MS), and
that these metallopeptides go inside HeLa cells at concentrations
as low as 5 μM.13
We have now found that keeping the six native arginines of the
basic region fragment is key for the internalization, because
replacing any of them by alanines leads to a significant decrease
in the intracellular fluorescence of tetramethylrhodamine (TMR)-
labelled derivatives (see the SI, Figure S32). This is consistent
with previous studies with cell penetrating peptides, where
arginines play a critical role to favour the cell uptake.14 Not
surprisingly, when the internalization experiments were carried
out at 4ºC instead of at 37ºC, the intracellular fluorescence, as
measured by flow cytometry, was considerably lower (Figure
S35). This result suggests that the cell penetration is energy
dependent, and therefore very likely occurring by endocytosis,
with the resulting peptide accumulated in vesicular structures.
Importantly, the palladium complex used as stapling agent can
feature other bidentate ligands than ethylenediamine (en), e.g. a
bipyridine (bpy). We therefore envisioned the use of a bipyridine
ligand equipped with a fluorophore other that TMR (already
present in the peptide), to simultaneously track the localization of
the peptide and the metal complex.
We first assessed the reactivities “in vitro”, using PBS as solvent.
Pd(OAc)2 and the palladium chloride sources used in the
preparation of the hybrid bis-histidine metal complexes,
PdCl2(en) and PdCl2(bpy), were able to promote the reaction
(conditions: 200 μM solution of 1 and 10 molꢀ% of the catalyst
Figure 3b).
Figure 2. Upper: Structure of Pd1. Bottom: Fluorescence micrographies of
HeLa cells. The palladopeptide complex was made by mixing TMR-brHis2 (10
μM) with Pd1 (1:1 ratio) in water for 30 min. Cells were incubated with the
mixture for 1 h at 37 ºC, washed twice with PBS, replenished with fresh DMEM,
and observed under the microscope. a) Red emission channel corresponding
to the TAMRA fluorophore; b) Blue emission channel corresponding to the
anthracene; c) Colocalization image processed with Image J; d) Blue emission
channel after incubation with Pd1 alone. Incubations were made in Dulbecco´s
modified Eagle medium (DMEM) completed with 5% of fetal bovine serum
(FBS-DMEM). Red channel: λexc = 550 nm, λem = 590-650 nm. Blue channel:
λexc = 385 nm, λem = 450-510 nm. Scale bar: 5 µm, the image shows a single
cell.
Figure 3. a) Uncaging of HBTPQ (1). b) Bar diagram representation of the
yields obtained for each catalyst. Reaction conditions: HBPTQ (200 μM), Pd
source (20 μM, 10 mol%) in 1 mL of PBS at 37 ºC, for 24 h. Yields were
calculated by RP-HPLC-MS using internal standards (see the SI). Data are
mean +/- SEM for experimental performed in triplicates and repeated in three
independent times.
2
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10.1002/anie.202002032
Angewandte Chemie International Edition
RESEARCH ARTICLE
Noticeably, the palladopeptide resulting from mixing
stoichiometric amounts of peptide and PdCl2(en) in water was
much less effective (Figure 3b).15 Despite this result could be
discouraging, it is not so surprising, as in this complex the
ethylendiamine ligand is not labile enough to allow an efficient
coordination of the substrate. Indeed, a similar metallopeptide
made from PdCl2(COD) (COD = 1,8-cyclooctadiene),17 gave the
product in 65% yield after 24 h of reaction (Figure 3b). This result
confirms the need of using Pd(II) reagents with suitable labile
ligands to obtain good reactivities. As a control reagent we also
consequence of the lack of reactivity, because the
palladopeptides are inside the cells, as can be deduced from the
TMR fluorescence (Figures 5b-c, bottom row). Control
experiments with a peptide lacking the TMR label gave similar
reactivity results, confirming that the fluorophore label doesn’t
affect the process. As expected, using the peptide TMR-brHis2
alone, we didn’t detect any fluorescence (see Figure S37).
explored the reactivity of
a purposely made bis-histidine
dichloride complex, PdCl2(Boc-His)2 (see the Supporting
Information) which gave the product albeit with lower yields (30%)
than the metallopeptide, suggesting a positive effect of the
peptide scaffold in the reactivity.
After these in vitro results, the stage was set for the intracellular
studies. In consonance with the results observed with PdCl2(en),
the internalization of TMR-brHis2 can also be triggered by
pre-treatment with the complex PdCl2(COD) (Figure 4a)
Interestingly, circular dichroism spectra of the peptide (TMR-
brHis2) after adding increasing amounts of the Pd(II) complex, at
4ºC, confirmed the propensity of the peptide to acquire an alpha-
helical structure (Figure 4b).18
Figure 5. Intracellular depropargylation of
1 monitored by fluorescence
microscopy in HeLa cells. Cells were treated with 50 μM of probe 1 for 30 min
at 37 ºC, and washed twice to remove excess of the probe. Peptides (10 μM),
or solutions of peptide-palladium complexes obtained by mixing equivalent
amounts of the peptide and the palladium source for 10 min, were added in
fresh media, and the cells were incubated for 1 h. a) Incubation with the
palladopeptide obtained from PdCl2(COD); b) Incubation with the
palladopeptide obtained from PdCl2(en); c) Incubation with the palladopeptide
obtained from PdCl2(bpy); (d) Incubation with PdCl2(COD), in absence of
peptide. The cells were washed with PBS before being observed under the
microscope. All the incubations were made in FBS-DMEM. Bright fields images
are superimposed to the red or orange emission channel (the orange colour is
arbitrarily chosen to differentiate the emission with the red channel). Upper row:
Orange channel: λexc = 385 nm, λem = 515-700 nm; Bottom row: Red channel:
λexc = 550 nm, λem = 590-650 nm; Scale bar: 5 µm.
We observed a similar reactivity using lower amounts of the
reactive metallopeptide (10 μM), which demonstrates the
effectivity of the system (see Figure S38).
Given that the palladopeptide is preformed just before the cellular
incubations, it could not be discarded that the intracellular
reaction was promoted by discrete palladium entities derived from
the palladium source. However, in the absence of the peptide, the
complex PdCl2(COD) is incapable of generating any intracellular
fluorescence (Figure 5d and S38). Other palladium sources like
Pd(OAc)2 or PdCl2(allyl) also failed to generate intracellular
fluorescence under the above conditions (Figure S39).
ICP-MS analysis of cellular extracts revealed that the palladium
content is substantially higher in cells treated with the preformed
palladoprotein hybrid than with equivalent amounts of
PdCl2(COD) (Figure S42). Nonetheless, this doesn’t account for
the lack of reactivity observed with these palladium sources,
which suggests that the stapled peptide structure also plays a key
role to favour the intracellular reaction.
Additional insights were obtained using as reagents the peptides
brHis (His 230 mutated to Leu), or br (native GCN4-basic region,
without histidines), instead of brHis2, which were not able to
promote any intracellular reaction when premixed with
PdCl2(COD) (Figure S40). Using a mutated peptide that lack key
arginines for cellular uptake (R249A, R245A), also led to similar
fails. These results confirm the requirement of a bis-histidine
Figure 4. a) Fluorescence micrographies of HeLa cells (confocal). Upper:
TMR-brHis2 (5 μM) was pre-mixed with PdCl2(COD) (1:1 ratio) in water for 10
min and incubated with cells for 30 min at 37 ºC. Bottom: Incubation with TMR-
brHis2 alone. The cells were washed with PBS before being observed under
the microscope. All the incubations were made in FBS-DMEM. Red channel:
λexc = 561 nm, λem = 620/60 nm. Scale bar: 10 µm. b) Circular Dichroism titration
of a 5 μM solution of brHis2 (narrow solid line) and increasing concentrations
of PdCl2(COD): 1 equiv. (dashed line), 3 equiv. (dotted line) or 10 equivalents
(thick solid line). The experiments were carried out at 4 °C, in 10 mM phosphate
buffer pH 7.5 and 100 mM of NaCl. Mean residue molar ellipticity (MRE) was
calculated considering a 24-mer.
The cellular reactions were carried out by incubation of HeLa cells
with 50 μM of probe 1 for 30 min at 37 °C. The medium was
removed, and the cells washed twice with FBS-DMEM to ensure
removal of extracellular probe. The peptide-palladium reagents
(50 μM) were then added in fresh media and, after 1 h and two
washes with PBS, the cells were imaged by wide-field
fluorescence microscopy. Gratifyingly, when cells were treated
with the palladopeptide resulting from mixing TMR-brHis2 and
PdCl2(COD), we could observe a very intense intracellular
fluorescence arising from the expected product 2 (Figure 5a). Not
so surprisingly, with the metallopeptides made from PdCl2(en)
and PdCl2(bpy), we didn’t observe fluorescence, which must be a
3
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10.1002/anie.202002032
Angewandte Chemie International Edition
RESEARCH ARTICLE
stapling and an appropriate set of arginines for cellular uptake
and intracellular reactivity.
Also importantly, the approach can be used in other human cells
such as A549 and mammalian cells like Vero (see Figure S41),
which supports the generality of the approach.
The chemistry can be extended to other challenging substrates,
like the bis-propargyl derivative 3, which upon deprotection
produces a far-red emitting product. As in the case of the phenol
derivative 1, only the palladominiprotein was able to perform the
reaction inside mammalian cells (Figure 6).
Figure 7. 1) Bis-Histidine RGD derivative and stapling produced by treatment
with the palladium complex. 2) Intracellular depropargylation using the peptide
TMR-HRGDH or control derivatives. HeLa cells were treated with 50 μM of
probe 1 for 30 min at 37 ºC and washed twice. Peptides (10 μM) or solutions of
peptide-palladium complexes obtained by mixing equivalent amounts for 10 min,
were added in fresh media, and the cells were incubated for 1 h. a) Pallado-
miniprotein resulting from PdCl2(COD) and brHis2; b) Mixture of TMR-HRGDH
and PdCl2(COD); c) Mixture of TMR-HRGDA and PdCl2(COD); d)
PdCl2(Boc-His)2. The cells were washed twice with PBS before being observed
under the microscope. All incubations were in FBS-DMEM. λexc = 385 nm, λem
= 515-700 nm. Scale bar: 10 µm. 3) CTFC measurements after the intracellular
reaction. Data are mean +/- SEM for experimental performed in triplicates and
repeated in three independent times.
Figure 6. Fluorescence micrographies of HeLa cells (confocal). Standard
conditions as in the previous cases, with 75 μM of probe 3 and 10 μM of the
reagents. Bright field images are superimposed to far-red emission channel. a)
Incubation with the metallopeptide made from brHis2 and PdCl2(COD); b)
Incubation with PdCl2(COD) alone; c) Incubation with substrate 3 alone. λexc
=
561 nm, λem = 620/60 nm. Scale bar: 10 µm.
It is important to note that cell viability tests (MTT in HeLa Cells,
24 h) demonstrated that the none of palladopeptide hybrids or
palladium complexes were toxic below 50 μM (more than 90%
viability after 24 h, Figure S43).
Finally, given that the cellular uptake of the palladium chelated
complex of HRGDH in cells might be partially associated to the
presence of integrin receptors, we anticipated that its cell
penetration would depend on the type of cells and the presence
of these receptors. Indeed, we were glad to observe that while in
A549 or HeLa cells there is intracellular catalysis, in the MCF-7
breast cancer cell line, which expresses integrin at low levels, we
barely detect any fluorescence. This preliminary result prompts
well for the development of target selective intracellular metal
catalysis (Figure 8).
Altogether, the above results confirm that engineering a bis-
histidine handle in a basic miniprotein can provide for cellular
internalization and cytosolic palladium catalysis owing to its ability
to form palladium stapled derivatives. An ensuing question is
whether the palladium stapling strategy is also effective for other,
shorter peptides.18 Considering the well-known integrin targeting
motif RGD (Arg-Gly-Asp), we wondered whether it could be also
converted into a reactive metallopeptide by adding two histidines
at the edges (Figure 7, panel 1). A TMR-labelled derivative
revealed that while the peptide HRGDH presents a relatively poor
cellular uptake in HeLa cells, the internalization is significantly
improved when pre-treated with PdCl2(COD), albeit the uptake is
not as efficient as with the above basic miniprotein (Figures S33
and S34).
Not surprisingly, the peptide-palladium complex is also capable
of performing the intracellular depropargylation of substrate 1
(Figure 7, panel 2b), however the reactivity was lower compared
with the pallado-miniprotein (Figure 7, panel 3). A similar peptide
with only one histidine (HRGDA) presents some capacity to
internalize (the same with or without adding PdCl2(COD), see
Figure S34), but failed to act as a catalyst (Figure 7, panel 2c).
Interestingly, the non-cyclic bis-histidine palladium complex
PdCl2(Boc-His)2 also failed to generate the intracellular product
(Figure 7, panel 2d).
Figure 8. Fluorescence micrographies of A549 (a) and MCF-7 cells (b), using
standard incubation conditions with the peptide-palladium complex resulting
from TMR-HRDGH and PdCl2(COD) (1:1 ratio). λexc = 385 nm, λem = 515-700
nm. Scale bar: 10 µm.
4
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10.1002/anie.202002032
Angewandte Chemie International Edition
RESEARCH ARTICLE
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Conclusion
[5]
[6]
In summary, arginine-rich miniproteins made of natural amino
acids, and equipped with i, i+4 bis-histidine residues, can
efficiently react with different Pd(II) salts to form stapled
derivatives that show enhanced cell internalization properties.
More importantly, the pallado-miniproteins obtained when using
PdCl2(COD) as palladium source, work as effective
metalloreactors to promote depropargylation reactions inside
living mammalian cells; transformations that cannot be performed
using just the palladium sources. The effectivity of approach is
likely associated to a synergistic beneficial effect of the
conformational constrain introduced by the metal bridge, and a
protective role of the peptide scaffolding, which avoids a rapid
deactivation of the metal.
Our results represent a first step towards the development of a
“bottom-up” strategy for the generation of artificial catalytic
metalloproteins capable of working in the native living
environment of enzymes. Additionally, the well-known
transformative potential of palladium catalysis prompts well for
further applications of the strategy in other type of reactions.
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Acknowledgements
This work has received financial support from Spanish grants
(SAF2016-76689-R, ORFEO-CINQA network CTQ2016-81797-
REDC) the Consellería de Cultura, Educación e Ordenación
Universitaria (2015-CP082, ED431C-2017/19 and Centro
Singular de Investigación de Galicia Accreditation 2019-2022,
ED431G 2019/03), the European Union (European Regional
Development Fund-ERDF corresponding to the multiannual
financial framework 2014-2020), and the European Research
Council (Advanced Grant No. 340055). S. L.-A and A. G.-G
thanks the Ministerio de Educación, Cultura y Deporte for the FPI
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fellowship
(BES-2017-080555)
and
FPU
fellowship
(FPU17/00711). C. V. thank the Ministerio de Economía y
Competitividad for the Juan de la Cierva-Formación (FJCI-2017-
33168). The authors thank R. Menaya-Vargas for technical
assistance and M. E. Vázquez for the illustration support.
Keywords: Metalloproteins • Intracellular catalysis • Palladium
promoted uncaging • Stapling • bZIP proteins
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5
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10.1002/anie.202002032
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RESEARCH ARTICLE
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[17] Not surprising, while in the MS of the palladapeptide resulting from
mixing brhis2 and PdCl2(en) the ethylenediamine ligand is conserved,
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depropargylation of rhodamine derivatives, using fixed cells: E. Indrigo,
J. Clavadetscher, S. V. Chankeshwara, A. Megia-Fernandez, A.
Lilienkampf, M. Bradley, Chem. Commun. 2017, 53, 6712-6715.
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RESEARCH ARTICLE
Entry for the Table of Contents
Stapling bis-histidine equipped basic miniproteins with Pd(II) produces cell-permeable derivatives that enable depropargylation
reactions in the interior of living mammalian cells. Both the metal bridge and the peptide scaffolding are essential for the
internalization, and for observing the intracellular transformations.
Institute and/or researcher Twitter usernames:
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