Intracellular Deprotection Reactions Mediated by Palladium Complexes Equipped with Designed Phosphine Ligands

Article abstract of DOI:10.1021/acscatal.8b01606

Discrete palladium(II) complexes featuring purposely designed phosphine ligands can promote depropargylation and deallylation reactions in cell lysates. These complexes perform better than other palladium sources, which apparently are rapidly deactivated

Full text of DOI:10.1021/acscatal.8b01606

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Letter
Intracellular deprotection reactions mediated by palladium
complexes equipped with designed phosphine ligands
Miguel Martínez-Calvo, Jose R. Couceiro, Paolo Destito,
Jessica Rodriguez, Jesús Mosquera, and José L. Mascareñas
ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.8b01606 • Publication Date (Web): 01 Jun 2018
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Intracellular deprotection reactions mediated by palladium complexes
equipped with designed phosphine ligands
Miguel Martínez-Calvo,‡ José R. Couceiro, Paolo Destito,‡ Jéssica Rodríguez,† Jesús Mosquera,§ José
L. Mascareñas*
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Centro Singular de Investigación en Química Biolóxica e Materiais Moleculares (CIQUS) and Departamento de Química
Orgánica Universidade de Santiago de Compostela Rúa Jenaro de la Fuente s/n, 15782, Santiago de Compostela, Spain
ABSTRACT: Discrete palladium (II) complexes featuring purposely designed phosphine ligands can promote depropargyla-
tion and deallylation reactions in cell lysates. These complexes perform better than other palladium sources, which appar-
ently are rapidly deactivated in such hostile complex media. This good balance between reactivity and stability allows the
use of these discrete phosphine palladium complexes in living mammalian cells, whereby they can mediate similar trans-
formations. The presence of a phosphine ligand in the coordination sphere of palladium also provides for the introduction
of targeting groups, such as hydrophobic phosphonium moieties, which facilitate the accumulation of the complexes in
mitochondria.
KEYWORDS: Palladium • discrete complexes • bioorthogonal • intracellular catalysis • mitochondrial confinement.
Bradley, Unciti 2014
“Pd”= Nanospheres; removal of poc
H
N
H
N
The functioning of the cell depends on the regulated ac-
tion of thousands of enzymes, many of which require metal
cofactors for their activity. In most of the cases, the metal
works either as a Lewis acid or as an electron-transfer cen-
tre.1 In recent years, there have been impressive advances
towards the development of artificial metalloenzymes ca-
pable of achieving transformations that do not occur in
nature,2–4 with a special emphasis on those promoted by
late transition metals.5,6 However, translating these metal-
loprotein catalysts to living settings is far from obvious, and
has only been proved in isolated cases, and in bacterial
periplasms.7,8
O
O
O
Chen, 2014
O
O
P
P
“Pd”= Pd(dba)2 or [Pd(allyl)Cl]2; removal of poc
O
Weissleder, 2017
“Pd”= PdCl2(TFP)2 encapsulated in PGLA-PEG
removal of alloc
O
(alloc)
(poc)
P
=
This work
"Pd"
"Living mammalian cells"
"cellular settings"
Ar
Ar
O
+
NH2
OH
H2N
O
Cl
Pd
Ph
P
O-
O
Ph
R
O
Rho
Rho
N
H
O
NH2
More success has been attained with discrete, small tran-
sition metal complexes which are capable of crossing cell
membranes, and thereby able to promote intracellular
transformations.9–13 This is the case for several rutheni-
um(II) complexes that can mediate the uncaging of allyl-
carbamate (alloc) protected amines in living mammalian
cells.14–17
Scheme 1. Previous examples of in cellulo uncaging of poc
and alloc probes mediated by Pd catalysts and reactions de-
scribed in this work.
Discrete palladium complexes have also been used, but
only sporadically, and with somewhat differing results.
Therefore, Chen and coworkers have shown that commer-
cial Pd precursors like Pd(dba)2 and Pd2(allyl)2Cl2 can
cleave propargyloxy groups (poc) in PBS at 37 °C, and even
in HeLa cells.23 However, a recent study by the Weissleder
group reveals that these commercial Pd sources perform
poorly when using standard tissue culture (HBSS and MEM)
as reaction milieu. Remarkably, these authors were able to
obtain good yields in alloc-removing reactions by using
bis[tris(2-furyl)phosphine]palladium(II) dichloride as cata-
lyst; although stability and solubility issues precluded a
direct use of the complex in living settings. This problem
was solved by encapsulating the metal complex within a
biocompatible polymer (Scheme 1).24
Particularly exciting is the possibility of using Pd-
catalysts in biological contexts, owing to the well-known
transformative power of palladium in organometallic chem-
istry.18,19 However, the use of palladium complexes in the
complex environment of living cells is seriously compro-
mised by a number of issues including solubility, stability,
biorthogonal reactivity or toxicity. Therefore, most applica-
tions so far described involve the use Pd nano- or
mesostructures, rather than discrete complexes. Hence,
Bradley, Unciti-Broceta and coworkers have claimed that
small Pd(0) microspheres can induce depropargylation
reactions or even Suzuki-Miyaura cross-coupling in biologi-
cal settings;20,21 and Chen reported that freshly made palla-
dium nanoparticles can promote similar reactions.22
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a)
b)
Cl
c)
Pd
10 mol%
I
[Pd(allyl)Cl]2
Pd(OAc)2
Pd2(dba)3
I
N
N
L
Pd1
Pd2
Pd3
O
OH
24h, 37ºC
200 µM
1
2
O
OH
Ph
P
Ph
Ph
Ph
HO
O
O
O
75
60
45
30
N
P
L=
H2O
PBS 1x
HeLa Lysate
N
H
S
Ph
Pd4
Pd7
Pd6
Pd5
PF6
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Ph
Ph
Ph
P
Ph
H
N
H
N
TFA
P
Ph
Ph
P
P
Ph
Ph
Ph
O
O
Pd8
Pd9
15
12
8
4
0
Pd1
Pd2
Pd3
Pd4
Pd5
Catalysts
Pd6
Pd7
Pd8
Pd9
Figure 1. a) Representation of the uncaging reaction of 1, b) bars diagram representation of the yields obtained for each catalyst
under biological compatible conditions in water, PBS buffer and cell lysates. Reaction conditions: 1 (0.2 mmol, 1 eq.), Pd (0.02
mmol, 0.1 eq.) in 1 mL, 37 °C, 24 h (mean ± s.e.m, n=2); c) Pd catalysts used in this work for the study of their catalytic perfor-
mance in biological compatible media and in living cells. Yields were calculated by RP-HPLC-MS using coumarin as internal stand-
ard.
The above results raise doubts on whether discrete palla-
dium complexes can indeed be used as catalysts in complex
biological media and in living cells. Such small-sized palla-
dium complexes are very attractive because of their well-
defined nature, and the possibility of playing with the lig-
ands to tune solubility, stability and reactivity properties,
and eventually favour cell transport and intracellular tar-
geting.
and PBS; however, in cell lysates the conversion was ex-
tremely low. Likely, these palladium complexes do not sur-
vive to the complex mixture of components present in a cell
lysate, and decompose to form inactive Pd species.26 The
palladium complexes with phosphine ligands Pd6-Pd9 gave
lower conversions in water, with yields ranging from ca.
40% for Pd6 to ca. 15 % for Pd7-9, and performed very
poorly in PBS, likely because of the low solubility of the
complexes in high ionic strength media.
Herein, we demonstrate that discrete palladium complex-
es with designed phosphine ligands can promote propargyl
and alloc cleavage reactions in cell lysates, and even in liv-
ing mammalian cells. Importantly, appropriate engineering
of the ligand allows the mitochondria accumulation of an
active palladium catalyst.
However, complexes Pd6, Pd7 and Pd9 were able to
promote the depropargylation reaction in cell lysates. Alt-
hough the yields were low, their performance is better than
that of the other palladium sources (Pd1-Pd5). Possibly,
albeit a phosphine ligand is not the best one from a reactivi-
ty point of view, the corresponding palladium complexes
are more stable, and can better resist hostile media like that
present in cell lysates.
Given that previous studies did not establish clearly the in
vitro reactivity of different palladium sources to cleave
propargyloxy groups, we investigated the catalytic perfor-
mance of several complexes in PBS and cell lysates.
We also investigated the ability of these latter phosphine
palladium reagents to remove alloc protecting groups, a
process that likely involves Pd(0) reagents. The uncaging
reaction was evaluated in substrate 3, in absence or pres-
ence of added reducing agents (Fig. 2a and SI pag. S9-13).
The reactions were performed using 200 µM solutions of 3
in water, 10 mol% of the palladium complexes, at 37 °C, in
absence or presence of 2 mM of NaAsc or 0.4 mM of GSH,
and monitored by HPLC-MS at 1, 3, 6 and 24 hours. In the
absence of the additives, the yields were very poor, howev-
er in presence of NaAsc, we obtained yields of over 50 and
30% using Pd7 and Pd9 respectively (24 hours). With GSH
the reaction with Pd7 was more efficient, and hence, we
observed 60% yield after 1h, and 94% after 24h. When the
experiments were performed in presence of cell lysates,
Pd7 and Pd9 led to 21% and 33% yields of the product
after 24 hours.
In addition to standard commercial species [Pd(allyl)Cl]2
(Pd1), Pd(OAc)2 (Pd2) and Pd2(dba)3 (Pd3),23–25 we pur-
posely made a series of Pd(II) complexes (Pd4-9) exhibiting
designed pyridine, thioether or phosphine ligands (Fig. 1
and SI pg. S3-8). In the case of the phosphine containing
derivatives, we built complexes with neutral (Pd6), anionic
(Pd7) or positively charged phosphonium tethers (Pd8 and
Pd9).
Instead of the typical assay consisting of the cleavage of a
propargylic carbamate, we studied the depropargylation of
phenol derivative 1. We chose this probe because of its
good aqueous solubility, ease of synthesis and good separa-
tion by HPLC-MS. Furthermore, in contrast to standard
Rhodamine probes, it presents only one propargylic ap-
pendage, therefore simplifying the analysis.
The reactions were carried out using a 200 µM solution of
substrate 1 and 10 mol% of the Pd sources in either water,
PBS (phosphate buffered saline, pH 7.2) or HeLa cell lysates
for 24 hours at 37 °C (Fig. 1 and SI pg. S9-S13). Pd1 and
Pd2, and the complexes bearing pyridine and methionine
ligands (Pd4-Pd5) provided good yields of 2, in both, water
With further addition of GSH (0.4 mM) to the lysate the
yields were not very different (24% and 22% respectively,
Fig. 2).
The above results confirm that phosphine palladium(II)
complexes can promote depropargylation and deallylation
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reactions in water with good yields. While in cell lysates the
emission (Fig. S15), with species Pd6-Pd8, equipped with
phosphine ligands, we detected strong red emission due to
the release of the product 6 inside Vero cells (Fig. 3b). The
overlap between the absorption of the pyrene unit in Pd9
and that of product 6, precluded the analysis of the reaction
with this complex. Overall, these results, are consistent with
the in vitro studies, and confirm that the phosphine ligands
are beneficial for observing “in cell” reactivity, likely be-
cause the complexes present a good balance between reac-
tivity and stability in the cellular milieu.
efficiency is considerably lower, we were intrigued to know
whether the reactions could be achieved inside living cells,
as the intracellular environment does not necessarily
Cl
Pd
10 mol%
I
I
N
N
L
a)
NH
O
NH2
24h, 37ºC
O
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3
200
H2O
NaAsc (2 mM)
GSH (0.4 mM)
µM
4
100
90
80
70
60
50
40
30
20
10
0
50
H2O
b)
b)
i)
ii)
NaAsc (2 mM)
GSH (0.4 mM)
40
30
20
10
0
Considering the amount of palladium that was present in
cells, as measured by ICP-MS of cell extracts after extensive
washing steps and associating the normalized fluorescence
intensity with the amount of product, using calibration
curves (See SI, page S18), it is possible to estimate whether
there is some turnover. Indeed, for Pd7 we calculated an
average TON of 10, while for Pd8 it is over 5, suggesting
that catalytic cycles can operate in cellular settings.
0
1
2
3
4
5
6
15 20 25
0
1
2
3
4
5
6
15 20 25
Time / h
Time / h
45
40
35
30
25
20
15
10
5
HeLa Lysate
HeLa Lysate + GSH (0.4mM)
c)
Ph
P
HO
L =
O
Ph
The cellular reactions can also be carried out in mamma-
lian cells other than Vero, such as HeLa (Fig S17). We also
checked an inverse protocol in which cells are first treated
with the palladium reagents, and the probe is added after
30 min, after the corresponding washing steps. But in these
cases, we did not observe fluorescence, likely because the
palladium complexes, upon time, are converted to species
that are not enough active in the depropargylation process.
Pd7
Ph
Ph
Ph
H
N
TFA
P
P
Ph
O
Pd9
0
Pd7
Pd9
Catalysts
Figure 2. a) Uncaging of 3, b) plot of the yields of reaction
obtained with catalysts i) Pd7 and ii) Pd9 at 1, 3, 6 and 24 h in
only water, or with additives: NaAsc (2 mM) or GSH (0.4 mM).
Reaction conditions: 3 (0.2 mmol, 1 eq.), Pd complex (0.02 mM,
0.1 eq.) in 1 mL, 37 °C, 24 h, and c) Uncaging reaction in cell
lysates (green column) and in cell lysate + 0.4 mM of GSH (red
column) promoted by catalysts Pd7 and Pd9. Reaction condi-
tions: 10 (0.2 mmol, 1 eq.), Pd (0.02 mM, 0.1 eq.) in 1 mL, 37 °C,
24 h. Inset: Pd7 and Pd9. Yields were calculated by RP-HPLC-
MS using coumarine as internal standard (mean ± s.e.m., n=2).
parallel that of a cell lysate. A key element to take into ac-
count before moving to intracellular reactions is uptake of
the potential catalysts. ICP-MS analysis of cellular extracts
(Vero cells) obtained after extensive washing and lytic
treatment, revealed a higher amount of palladium after the
addition of complexes Pd6-9 than of species Pd1-5 (Fig S12
and S13). Importantly, cell viability tests (MTT in Vero cells,
24 h) demonstrated that the phosphine-containing com-
plexes are essentially non toxic below 50 µM, and some of
them can be even used at higher concentrations (Figure
S14). While substrate 1 was good for in vitro studies, the
change in fluorescence after the reactions is not strong
enough for an appropriate monitoring in cellular settings.
Therefore, we changed to the propargylated probe 5, which
after uncaging generates a product 6 which emits at 635 nm
when excited in the far UV region (Fig. 3a).27
Figure 3. a) Schematic representation of the uncaging reaction
of the propargylated probe 5, and b) imaging of the reaction in
Vero cells. First row – red channel; second row – brightfield
and red channels merged (i: mock, ii: Pd6; iii: Pd7 and iv: Pd8).
Reaction conditions: 5 (50 µM) was incubated in DMEM with
5% Fetal Bovine Serum for 30 min. Culture medium was re-
moved and cells were washed twice before addition of the Pd
catalyst (50 µM). Cells were then incubated for another 30 min,
washed twice and observed under the microscope (Scale bar =
20 µm).
The experiments were carried out by incubation of Vero
cells at a final concentration of 50 μM of the probe 5 in cul-
ture medium containing 5% fetal bovine serum (FBS-
DMEM) for 30 min at 37º C. The milieu was removed, and
cells were washed twice with FBS-DMEM to ensure removal
of extracellular probe. Pd reagents (50 μM) were then add-
ed in fresh media (0.1 % DMSO), and after 30 min, cells
were imaged by widefield fluorescence microscopy.
We next explored the removal of alloc groups inside liv-
ing HeLa cells, in this case using the caged Rhodamine 110
derivative 7, which is essentially non-fluorescent, but emits
green light after removing the alloc protecting groups. Ini-
tial experiments were carried out by mixing HeLa cellswith
7 (50 µM in FBS-DMEM) for 30 minutes, twofold washing
with FBS-DMEM, and addition of the Pd complexes. After 30
While in the experiments with Pd sources Pd1-Pd3 and
complexes Pd4-Pd5 we observed almost negligible red
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min at 37ºC, cells were visualized under the microscope. As
in the case of the depropargylation reaction, the complexes
Pd1-Pd5 raised only a very low intracellular fluorescence,
however, with species Pd6-8 we could observe strong in-
7
tracellular
green
fluorescence
(Fig.
S18).
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Figure 4. a) Representation of the uncaging reaction. b) Co-localization of Pd9 with TMRE in HeLa cells, observed 2 h after incuba-
tion with the palladium complex: i) blue light emission of Pd9; ii) red light emission from the mitochondrial dye TMRE, iii) merged
image of i) and ii). c) Reaction promoted by Pd9: i) blue light emission of Pd9; ii) green light emission from released 8, iii) merged
image of i) and ii). d) Reaction promoted by Pd7: i) blue channel; ii) green light emission from released 8, iii) merged image of i)
and ii). e) Graphical representation of the ICP-MS results from mitochondrial isolation of fractioned Vero cells using complexes
Pd6-9 (see supporting information for details). Reaction conditions: Pd complexes (50 µM) were incubated with HeLa cells in
DMEM with 5% Fetal Bovine Serum for 2h at 37 °C before substrate 7 (50 µM) was added; the cells were incubated for another 30
min and observed under the microscope (Scale bar = 20 µm).
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Inverting the order of addition, first the palladium com-
plexes, and after 30 min, washing and addition of the
probe 7, we detected intracellular fluorescence, but only
with complexes Pd8 (Fig. S19) and Pd9 (Fig. 4). Similar
results were obtained in Vero cells (Fig S20).
Our results confirm that while many Pd(0) and Pd(II)
sources can effect depropargylation and deallylation
reactions in water and PBS, they are much less effective in
complex cellular media or living cells. However, palladium
complexes featuring designed phosphine ligands can
promote the transformations in cell lysates with variable
efficiencies, which depend on the characteristics of the
ligands. Despite this modest activity, these palladium
phosphine complexes can be translated into living
mammalian cells and promote both depropagylation and
alloc cleavage reactions.
In the case of Pd9, the presence of a pyrene group in the
ligand structure allowed a direct monitoring of the species
in living cells. As shown in Figure 4, the blue emission of
living cells after addition of this complex shows a very
good colocalization with the red color of a mitotracker
(Fig. 4b, i-iii). This was further confirmed by ICP-MS analy-
sis of cells lysed under conditions that allowed the isola-
tion of the mitochondria from the rest of cytoplasmic mass.
As indicated in Figure 4e, Pd9 presents a substantial high-
er mitochondrial accumulation than the other complexes.
Likely, the presence of the pyrene and phosphonium
groups in the ligand allows the right combination of charge
and hydrophobicity to favour a significant mitochondrial
accumulation of the complex.
We have also demonstrated that using a phosphine
ligand tethered to phosphonium and hydrophobic pyrene
groups, the palladium complex presents a preferencial
accumulation in mithochondria wherein it remains active.
This type of subcellular targeting might open new
opportunites for promoting enhanced and selective
biological effects in prodrug activation processes. Our
results confirm the viability of engineering well-defined,
discrete palladium complexes to promote relevant
reactions in cellular milieu. The small-size and ligand
tunability of these complexes offer advantages with
respect to other alternatives based on metalloenzymes or
nanoconstructs in terms of simplicity and synthetic access,
as well as of controlling properties such as reactivity,
targetability and cellular uptake.
Importantly, in cells treated with Pd9 we observed an
efficient transformation of 7 into 8, as deduced from the
quite strong build-up of green fluorescence, that also ac-
cumulates in the mitochondria surroundings (Fig. 4c, i-iii).
Other phosphine-containing catalyst, such as Pd7 or Pd8
were less active (Fig. 4d, i-III and Fig. S21).
Given the positive results with the rhodamine alloc
probe (7) and Pd8 and Pd9 using the inverse protocol
(palladium complexes added first), we also tested this
protocol with the rhodamine derivative having poc instead
of alloc protecting groups (Rho-poc).23 The results,
detailed in the supporting information, indicated that
while Pd8 failed to raise a significant fluorescence, Pd9
was able to promote the depropargylation. This might be
associated to a protective effect of the mitochondria
sourroundings on the stability of the palladium complex
(Fig. S22).
AUTHOR INFORMATION
Corresponding Author
* José Luis Mascareñas (joseluis.mascarenas@usc.es)
Present Addresses
†
Laboratoire Hétérochimie Fondamentale et Appliquée, Uni-
versité Paul Sabatier/CNRS UMR 5069, 118 Route de Nar-
bonne, 31062 Toulouse Cedex 09, France.
§
CIC biomaGUNE and Ciber-BBN, Paseo de Miramón 182,
20014 Donostia-San Sebastián, Spain.
Conclusions
Author Contributions
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Supporting Information. The Supporting Information is
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ACKNOWLEDGMENT
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We are thankful for the financial support from the Xunta de
Galicia (Centro Singular de Investigación de Galicia
Acreditación 2016–2019, ED431G/09) and the European
Union (European Regional Development Fund – ERDF). We
also thank support given by the Spanish grant SAF2016-
76689-R, the Xunta de Galicia (grants 2015-CP082, ED431C
2017/19), and the European Research Council (Advanced
Grant No. 340055). MMC thanks the Ministerio de Economía y
Competitividad for the Postdoctoral fellowship (IJCI-2014-
19326). JR and JM thank to Xunta de Galicia and Ministerio of
Educacion for predoctoral fellowships, respectively. The au-
thors thank R. Menaya-Vargas for technical assistance.
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