Hollow nanoreactors for Pd-catalyzed Suzuki-Miyaura coupling and O-propargyl cleavage reactions in bio-relevant aqueous media

Article abstract of DOI:10.1039/C8SC04390F

We describe the fabrication of hollow microspheres consisting of mesoporous silica nanoshells decorated with an inner layer of palladium nanoparticles and their use as Pd-nanoreactors in aqueous media. These palladium-equipped capsules can be used to promote the uncaging of propargyl-protected phenols, as well as Suzuki-Miyaura cross-coupling, in water and at physiologically compatible temperatures. Importantly, the depropargylation reaction can be accomplished in a bioorthogonal manner in the presence of relatively high concentrations of biomolecular components and even in the presence of mammalian cells.

Full text of DOI:10.1039/C8SC04390F

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DOI: 10.1039/C8SC04390F
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Hollow Nanoreactors for Pd-catalyzed Suzuki- Miyaura
Couplings and O-Propargyl Cleavage Reactions in Bio-relevant
Aqueous Media
Paolo Destito,a,§ Ana Sousa,b,§ José R. Couceiro,a Fernando López,*a,c Miguel A. Correa-Duarte,*,c José L.
Mascareñas*,a
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
We describe the fabrication of hollow microspheres consisting of mesoporous silica nanoshells decorated with an inner layer
of palladium nanoparticles, and their use as Pd-nanoreactors in aqueous media. These palladium-equipped capsules can be
used to promote the uncaging of propargyl-protected phenols, as well as Suzuki-Miyaura cross-couplings, in water, and
under physiologically compatible temperatures. Importantly, the depropargylation reaction can be accomplished in a
bioorthogonal manner in presence of relatively high concentrations of biomolecular components, and even in the presence of
mammalian cells.
explored, palladium occupies a prominent position owing to its
well-known transformative potential in organic solvents.18-27
Despite palladium complexes can be deactivated in aqueous
Introduction
solvents, Davis and Lin have independently demonstrated that
in presence of specific additives it is possible to achieve Pd-
mediated protein Suzuki-Miyaura or Sonogashira cross-
couplings in aqueous milieu.28-32 Chen has reported the use of
Pd salts, including [Pd(allyl)Cl]2, to promote depropargylation
and deallenylation reactions in proteins containing caged
lysines and tyrosines.33-35 However, recent studies suggest that
achieving these deallylation or depropargylation reactions in
complex aqueous media, such as standard tissue cultures
(HBSS and MEM), requires the use of more elaborated,
specifically designed phosphine-palladium complexes.36-38
An alternative to small-sized, well-defined palladium reagents
consists of the use of Pd nanoparticles (Pd-NPs). Indeed, a
number of Pd-NP-promoted processes, including Suzuki-
Miyaura cross couplings, have been achieved in aqueous media.
However, most of them require the addition of bases and
temperatures higher than 50 °C, which represent significant
limitations in terms of translating this type of reactivity to
biological media.39-45 Moreover, and importantly, catalytic Pd-
NPs tend to aggregate, and/or can be readily passivated when
used in aqueous mixtures containing biological components.
Chen and coworkers have shown that Pd-NPs can promote the
uncaging of propargyloxy protected amines in cell-surface
glycans,46 however they indicate that the nanostructures need to
be immediately used after their generation (within 30 s). Unciti-
Broceta and Bradley have devised an elegant method to protect
the NPs from the media, by embedding them within polystyrene
microspheres, which allowed their use in different biological
set-ups.47-51 While these constructs have found interesting
applications, the difficulties in controlling the architecture of
the resulting structures, especially with regard to the
During the last decade, bioorthogonal reactions have emerged
as prominent tools in chemical biology, and have facilitated the
study and rational modification of different biological
processes.1-3 Among these reactions, the Cu-catalyzed azide-
alkyne cycloaddition (CuAAC) remains as a main reference in
the field.4-7 However, the coordination and oxidation lability of
Cu(I) complexes, and the toxicity associated to this metal, have
prompted the development of alternative, metal-free reactions
(e.g. strain promoted azide-alkyne cycloaddition, inverse
electron demand Diels-Alder reactions or photoclick
cycloadditions, among others).8-17
While these transformations represent important tools in
chemical biology, bioorthogonal reactions catalyzed by metals
are especially attractive owing to the additional control
possibilities offered by the presence of the catalyst. However,
achieving such metal-mediated processes in aqueous biological
environments is far from obvious. Indeed, only recently several
bioorthogonal metal-promoted reactions that take place in
aqueous media, and even in cellular settings, have been
developed.18-24 Among the different metals that have been
a. Centro Singular de Investigación en Química Biolóxica e Materiais Moleculares
(CiQUS) and Departamento de Química Orgánica. Universidad de Santiago de
Compostela, 15782, Santiago de Compostela, Spain
b. Department of Physical Chemistry, Center for Biomedical Research (CINBIO),
Southern Galicia Institute of Health Research (IISGS), and Biomedical Research
Networking Center for Mental Health (CIBERSAM), Universidade de Vigo, 36310
Vigo, Spain
c. Instituto de Química Orgánica General CSIC, Juan de la Cierva 3, 28006, Madrid,
Spain
† Footnotes relating to the title and/or authors should appear here.
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
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distribution of Pd-NPs, might represent a limitation in terms of solution in ethanol (5%). The use of a polystyrene core
View Article Online
obtaining improved designs.
facilitated its removal under soft condiDtiOonI:s10(.130:319/TC8HSFC/0H432O90)F,
Therefore, the development of alternative palladium-based avoiding alternative calcination processes that could sinter and
nanocatalytic constructions that work in biological media reduce the catalytic potential of the Pd-NPs (see the Supporting
represents a major current challenge. In this context, we Information for details, Fig. S2).
envisioned a new strategy to achieve bioorthogonal palladium
heterogeneous catalysis based on the use of hollow
nanoreactors. Specifically, we reasoned that porous hollow
microspheres equipped with palladium nanoparticles at the
internal surface might work as a biocompatible, smart reactive
chambers.52-54 The thickness and porosity of the chamber shell
could be rationally tuned to allow an effective flow of low
molecular weight reactants and products, while avoiding both
metal leakage and the entrance of large biomolecules that could
Scheme 1. Strategy for the synthesis of hollow spheres equipped with Pd-NPs at their
passivate the catalytic surface (Fig. 1).
inner cavity.
While there are some precedents on the use of yolk@shell
nanoparticles to carry out Pd-promoted processes (Suzuki-
Miyaura couplings), the reactions have been performed in
alcoholic, rather than aqueous solvents, under non-
physiological temperatures, and required high concentrations of
the reactants.55-59
Herein we demonstrate that hollow microspheres consisting of
mesoporous silica nanoshells equipped with Pd-NPs at their
inner side, behave as efficient Pd-nanoreactors for O-
depropargylation reactions and Suzuki-Miyaura cross-couplings
in aqueous buffer solutions (pH = 7.2). Importantly, while
discrete palladium complexes like [Pd(allyl)Cl]2, failed to
promote the propargyl-uncaging process when proteins like
BSA are added to the reaction mixture (vide infra), our hollow
nanoreactors remain active. Indeed, the capsules also work in
cell culture medium and even in the presence of living cells.
Fig. 2 shows typical transmission electron microscopy (TEM)
images of the resulting nanocapsules exemplified for Pd-Cap1
with an homogeneous average size of 500 nm and a
mesoporous silica shell thickness of 25 nm.60-62 The Pd-NPs are
homogeneously distributed through the inner surface of this
mesoporous shell as was confirmed by STEM and EDS
analysis (Fig. 2 b,d and Fig. S3). The content of Pd in these
capsules, measured by ICP-MS, turned out to be of 0.61g/mg.
Fig. 2. Characterization of Pd-Cap1. (a) Representative TEM image (500 nm diameter);
(b) STEM Image; (c) Zoom-in detail showing the vertically oriented mesoporous in the
silica shell with a thickness of 25 nm; (d) XEDS mapping showing the elemental
distribution of Pd and Si.
Prior to the reactivity tests, we checked the stability of the Pd
nanocapsules (Pd-Cap1) in water at 37 °C. Gratifyingly, the
structural features of the spheres and the size of the internalized
Pd-NPs remained unaffected even after 30 days, confirming the
protective role of the mesoporous shells on the characteristics
of the Pd-NPs. The catalytic activity was preliminary tested in
the decaging of propargyl phenol derivative 1, a pro-
fluorogenic substrate which upon deprotection provides a
fluorescent benzothiazole (2, Scheme 2, eq 1). The green
fluorescence emission of this product (exc = 460 nm, em = 535
nm), which arises from an excited state intramolecular proton
transfer process (ESIPT), enables an efficient quantification of
reaction yields at low M concentrations.63 Treatment of 1 (50
M) with Pd-Cap1 (5 mol% Pd content) in a 2:8 mixture of
DMSO:water provided, after 3h at 37 °C, the desired product
Fig. 1. Prototype reactive chambers for metal-catalyzed biocompatible processes.
Results and discussion
The fabrication of the hollow-structured mesoporous systems
equipped with palladium nanostructures at their inner cavity
was achieved according to a protocol which ensures the tuning
60-62,56
of the silica shell (Scheme 1).
Namely, homogeneous
polystyrene beads used as sacrificial templates are coated with
polyallylamine hydrochloride (PAH). Then, freshly prepared Pd
nanoparticles (3.8 ±0.5 nm, Fig. S1) are deposited onto these
polystyrene particles and subsequently coated with
homogeneous mesoporous silica layer using a tetraethoxysilane
a
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(2) in 32% yield (the remaining starting material (1) accounts strong blue fluorescence (exc = 330 nm, em = 43V4iewnmArt,ic1le0OnliMne
for the rest of the mass balance). Control experiments in EtOH:H2O 1:1),65 facilitating the reaction monitoring.
DOI: 10.1039/C8SC04390F
confirmed that the reaction does not proceed in the absence of Gratifyingly, when a water suspension of Pd-Cap1 (10 mol%
Pd-Cap1.64 The yield could be improved to 53% by doubling Pd content), 5 (150 M), 6 (100 M) and K2CO3 (1 mM) was
the concentration of Pd-Cap1 (10 mol% of Pd content) and mixed at 37 °C for 3 h, the desired product 7 was obtained in
running the reaction for 24 h. Importantly, control experiments 55% yield. Moreover, by running the reaction for 24 h, the
with
non-encapsulated
Pd-NPs,
stabilized
with yield of 7 could be further increased up to 78%. Not
polyvinylpyrrolidone (PVP, Pd-NP1), under otherwise surprisingly, in the absence of base the reaction did not occur.
identical reaction conditions, provided yields below 10% after However, we were pleased to find that the reaction could be
24 h, confirming that confinement of the Pd-NPs in the carried out using PBS 10X (pH 7.2) as solvent, without any
nanoreactor has a pivotal influence in their catalytic activity.64
additional base. In this case, a 65% yield of 7 was obtained
Remarkably, the reaction promoted by Pd-Cap1 could also be after 3h at 37 °C, while after 24 h the yield was 86%. Notably,
carried out in PBS, instead of water, with similar results (48% this reaction represents the first application of hollow
yield). To avoid the use of DMSO as co-solvent, we checked palladium nanocapsules in Suzuki-Miyaura cross couplings in
the reactivity of the styryl indolinium probe 3 (Scheme 2, eq. an aqueous solvent, and at physiological temperatures.
2), which presents excellent water solubility in the M range. Although it was not the main goal of this research, it is worth to
Interestingly, the reaction outcome can be easily judged by note that these nanoreactors can be easily recovered by
naked-eye detection of the colorimetric change, and yields are centrifugation after reaction completion, and preliminary
easily quantified by HPLC-MS.64 Treatment of a water solution experiments demonstrate that they can be reused, at least three
of 3 with Pd-Cap1 (10 mol% of Pd) provided, after 24 h at 37 times, without an important loss of catalytic activity (Fig.
°C, the depropargylated product 4 in 47 % yield, while a S16).64 On the other hand, leaching was insignificant, as
significantly better value of 73% could be obtained if the determined by ICP-MS analysis of the supernatants.
reaction was carried out for 48h (Scheme 2, eq. 2). A similar Accordingly, mother liquids obtained by centrifugation after a
yield of 4 after 24 h was obtained when the reaction was carried reaction did not show catalytic activity.64
out in PBS (Scheme 2, eq. 2). Overall, and despite yields and
S
Pd-Cap1
(10 mol% Pd)
O
O
S
B(OH)2
reaction rates are moderate, these results validate the potential
of the palladium nanoreactors in water, at low temperatures and
under high dilution conditions.
I
Solvent, time
37 ºC
5
6
7
Solvent
Additive
7, % yield (time)
Water
Water
K2CO3 (10 eq)
-
55 (3h); 78 (24h)
0 (24h)
HO
O
O
O
Pd-Cap1
(X mol% Pd)
PBS
-
65 (3h); 86 (24h)
S
N
S
(1)
Solvent, t (h)
37 ºC
N
Scheme 3. Suzuki-Miyaura cross-coupling of 5 and 6.
2
1 (50 M)
t (h)
Solvent
X mol% Pd
% yield
We next analyzed the influence of the structural properties of
the nanostructures in the reactivity. Thus, two additional Pd-
nanocapsules of different size and shell thickness were
prepared.64 Capsules Pd-Cap2, which are significantly smaller
(280 nm) but of equal shell thickness (25 nm), were prepared
from polystyrene beads of 230 nm (see Scheme 4 for TEM
image). On the other hand, Pd-Cap3, comprising a thicker
mesoporous shell (50 nm) and an overall diameter of 500 nm
were obtained following a slightly modified procedure.62 As
can be deduced from the results of Scheme 4, Pd-Cap2
performed very similar in the cross-coupling between 5 and 6,
providing a 85% yield of 7 after 24 h at 37 °C. On the other
hand, the efficiency of Pd-Cap3, which exhibits a 50 nm shell
width, turned out to be slightly lower (7, 71 % yield), indicating
that increasing shell thickness disrupts the catalytic activity,
likely because of a less efficient flow of reactants and products
throughout the shell. These results confirm that tuning the
thickness of the surface allows an extra control over the
reaction kinetics.
DMSO-H2O (2:8)
DMSO-H2O (2:8)
DMSO-H2O (2:8)
5
32
41
53
3
3
10
10
24
DMSO-PBS (1:9)
10
48
24
Pd-Cap1
(10 mol% Pd)
N
N
I
I
(2)
OH
Solvent
37 ºC, 24h
O
4
3 (100 M)
% yield
Solvent
47 (73)a
49
H2O
PBS
a Yield after 48 h
Scheme 2. Pd-Cap1-promoted uncaging of the 1 and 3. Conditions: A suspension of Pd-
Cap1 (0.2 mL, 8.33 mg/ml) in EtOH was centrifuged (8000 rpm) for 10 min. The
supernatant was removed and a mixture of H2O:DMSO (4:1) was added, followed by
addition of 1 or 3. The mixture was stirred at 37 °C, under air, for the indicated time,
and centrifuged (8000 rpm) prior to quantification by fluorescence analysis (for 2) or by
HPLC (for 4).
To check whether these Pd-nanoreactors could be applied to
more challenging bimolecular processes, we tested their
effectivity in Suzuki-Miyaura couplings between 2-thiophenyl
boronic acid (5) and p-iodo benzaldehyde (6) (Scheme 3). The
expected product, 4-(thiophen-2-yl)benzaldehyde (7) shows a
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Pd-Cap
(10 mol% Pd)
O
O
S
S
View Article Online
B(OH)2
I
N
I
N
I
Pd-Cap1
(10 mol%)
DOI: 10.1039/C8SC04390F
PBS, 24h, 37 ºC
5
6
7
Additive (10 mol%)
H2O, 37 ºC, 24h
O
OH
Pd-Cap
7, % yield
3 (100 M)
4
b) 70
60
Pd-Cap1
Pd-Cap2
Pd-Cap3
86
85
71
60
50
40
30
20
10
0
a)
Pd-Cap1
[PdCl(allyl)]2
63
55
50
49
47
44
40
30
20
10
44
36
34
24
18
Scheme 4. Reactivity of Pd-capsules with other diameter and thickness. a) TEM image
of Pd-Cap3; b) TEM image of Pd-Cap2.
6
0.04
0.02
0.00
0
0
0
0
100
300
600
e
e
Valin
BSA concentration / M
Glycine
Glutamic acid
Glucose
Tyrosine
At this point, we explored the viability of exporting the above
reactivities to aqueous media containing additional
biomolecules, in order to test the bioorthogonality of the
approach. Gratifyingly, the Suzuki-Miyaura coupling between 5
and 6, promoted by Pd-Cap1, proceeds with similar yields in
the presence of one equivalent (with respect to the Pd content)
of carbohydrates such as glucose, or different amino acids like
glycine, tyrosine or valine (Fig. 3). We later found that it also
tolerates larger excesses of the additives (Fig. S11). However,
the reaction was inhibited by biorelevant thiols such as
glutathione, and did not proceed in cell lysates or in the
presence of Vero cells. This inactivation is likely associated to
the mechanistic complexity of the bimolecular process and the
lateral reactivity of transient palladium intermediates with some
of the components of the mixture (specially with thiols).
Glutathion
Fig. 4. Pd-Cap1-promoted depropargylation of 3 (mean ± s.e.m., n = 2) in presence of
different additives, 1.0 equiv with respect to Pd (a), and of increasing concentrations of
BSA (b). The reaction is also effective in presence of higher amounts of the additives
indicated in (a), see Fig. S12.
Finally, a preliminary analysis of the toxicity in Vero cells
revealed a superior biocompatibility of the nanoreactor Pd-
Cap1, compared to that of [Pd(allyl)Cl]2. Indeed, while at low
concentrations (2.5 M), none of the Pd-systems probed to be
significantly toxic, at a concentration of 20 M an important
toxicity of [Pd(allyl)Cl]2 was observed observed (85% of cells
were dead after 24h), whereas the Pd-nanocapsules still
provided a good cell viability > 75% (Fig. S14).64
Given their low toxicity, we studied the catalytic activity in
cellular environments. With this purpose, we performed the
depropargylation of the probe HBTPQ (8)66 in the presence of
Vero mammalian cells.
Pd-Cap1
(10 mol%)
O
O
S
S
B(OH)2
I
Additive (10 mol%)
PBS (10X, pH 7.2)
37 ºC, 3 h
7
6
5
150 M
100 M
70
60
50
40
30
20
10
0
64
55
51
I
I
S
OH
N
S
O
N
45
Pd-Cap1
(10 mol%)
N
N
PBS / HeLa cells
HBTP (9)
HBTPQ (8)
50 M
< 0.1
e
e
Valin
Glycine
Glucose
Tyrosine
Glutathion
Fig. 3. Pd-Cap1-promoted Suzuki-Miyaura reaction of 5 and 6 (top, mean ± s.e.m., n =
2), in presence of additives.
However, to our delight, the depropargylation of probe 3
proceeded in the presence of added glucose, different types of
amino acids and even of in the presence of glutathione (Fig. 4a
and Fig S12). Interestingly, the reaction can also be performed
at acidic pH's of 5.3 and 4.5 (45% and 46% yield, respectively).
Moreover, the reaction is also effective in presence of
significant amounts of proteins like BSA (up to 600 M, Fig.
4b). Importantly, under these conditions, discrete Pd complexes
that had been previously used in lysine uncaging reactions, like
[Pd(allyl)Cl]2,33-35 turned out to be completely ineffective, even
using BSA concentrations as low as 100 M. These results
confirm the superiority of the Pd-nanocapsules, which prevents
an otherwise rapid passivation.
Fig. 5. Micrographs of Vero cells incubated for 30 min in DMEM containing Pd-Cap1 (
5 M Pd) and 50 M HBTPQ (8), washed and analyzed.
This probe does not emit red fluorescence when irradiated with
UV light but the cleavage of its propargyl moiety generates the
red fluorescent compound HBTP (9, exc= 330 nm, em= 635
nm). In this experiment, the extracellular medium was removed
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4
H. C. Kolb, M. G. Finn and K. B. Sharpless, Angew. Chem.
View Article Online
and substituted by a solution of 50 µM HBTPQ (8) in DMEM
or in PBS. Pd-Cap1 was added to reach a final 5 M
concentration of Pd, and the resulting cellular fluorescence was
monitored by confocal microscopy. Gratifyingly, red
fluorescence spots arising from the intracellular presence of the
product were observed in the cells incubated with the probe 8
(Fig. 5).
Int. Ed., 2001, 40, 2004.
5
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To sum up, we have demonstrated that hollow microspheres
consisting of mesoporous silica nanoshells with Pd-NPs at their
inner face, behave as efficient nanoreactors for Pd-catalyzed
depropargylations and Suzuki-Miyaura intermolecular cross-
couplings under physiologically compatible aqueous
conditions. The aqueous stability, monodispersity, well-defined
architecture and fabrication reproducibility of the capsules
represent important advantages with respect to other polymer-
based structures. Moreover, the low toxicities to cells owing to
an inert external surface, and the bioorthogonality of the
depropargylation, augurs well for further developments with
hollow nanoreactors that present well-defined metal catalysts
inside the capsule. The development of this type of
biocompatible reactors might allow the implementation of new
therapies based on prodrug activations,67 and/or contribute to
the establishment of biocompatible, non-natural metabolic
networks. Work in this direction is underway.
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Conflicts of interest
There are no conflicts to declare.
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Acknowledgements
This work has received financial support from Spanish grants
(SAF2016-76689-R, CTQ2017-84767-P, CTM2014-58481-R,
CTM2017-84050-R and Orfeo-Cinqa network CTQ2016-
81797-REDC), the Consellería de Cultura, Educación e
Ordenación Universitaria (2015-CP082, ED431C/2017119 and
Centro Singular de Investigación de Galicia accreditation 2016-
2019, ED431G/09), 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).
Notes and references
‡ Footnotes relating to the main text should appear here. These
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