Palladium-mediated intracellular chemistry

Article abstract of DOI:10.1038/nchem.981

Many important intracellular biochemical reactions are modulated by transition metals, typically in the form of metalloproteins. The ability to carry out selective transformations inside a cell would allow researchers to manipulate or interrogate innumera

Full text of DOI:10.1038/nchem.981

ARTICLES
PUBLISHED ONLINE: 6 FEBRUARY 2011 | DOI: 10.1038/NCHEM.981
Palladium-mediated intracellular chemistry
Rahimi M. Yusop , Asier Unciti-Broceta , Emma M. V. Johansson , Rosario M. S a´ nchez-Mart ´ı n
1
,2
1†
1
1
and Mark Bradley1
*
Many important intracellular biochemical reactions are modulated by transition metals, typically in the form of
metalloproteins. The ability to carry out selective transformations inside a cell would allow researchers to manipulate or
interrogate innumerable biological processes. Here, we show that palladium nanoparticles trapped within polystyrene
0
microspheres can enter cells and mediate a variety of Pd -catalysed reactions, such as allylcarbamate cleavage and
Suzuki–Miyaura cross-coupling. The work provides the basis for the customization of heterogeneous unnatural catalysts as
tools to carry out artificial chemistries within cells. Such in cellulo synthesis has potential for a plethora of applications
ranging from cellular labelling to synthesis of modulators or inhibitors of cell function.
ellular biochemistry is governed by a wide range of enzymatic
This innovative catalyst-loaded delivery system was synthesized
entities, with the complex cellular machinery engineered to as shown in Fig. 1. Amino-functionalized polystyrene microspheres
1
,2
C
perform a myriad of diverse chemical reactions . Many of (500 nm) were synthesized by dispersion polymerization from
2
2
these reactions are catalysed by transition metals, typically in the styrene-based monomers as previously described and sub-
form of metalloproteins , which modulate a wide variety of trans- sequently mixed with Pd(OAc) . The electron-rich microsphere
formations, from highly selective oxidations to the efficient network binds Pd by coordination to the free amino groups
formation, lysis or isomerization of multifarious chemical bonds . and aromatic rings, while reduction of Pd to Pd leads to aggrega-
3–5
2
2
þ
1
,2
2þ
0
0
In this context, many groups have taken advantage of the properties tion and the generation of Pd nanoparticles (Fig. 1). Extensive
of these proteins with the generation of bio-inspired devices, based on crosslinking of the available amino groups on the particles means
6
–11
0
coordination complexes aimed at mimicking biological function
.
that the stable Pd nanoparticles are permanently entangled and
0
Bioorganometallic chemistry has therefore evolved as a fascinating trapped within the microsphere. Finally, Pd microspheres were
field, full of possibilities and creative applications, ranging from fluorescently labelled to allow intracellular tracking of the catalysts
highly selective chemistries to the manipulation and in situ interro- (Supplementary Sections S2 and S3). Two different dyes were
. A fluorogenic used for analytical reasons: Cy5.5-labelled Pd microspheres were
chemosensor for the in vivo detection of Pd species has recently used for flow cytometry analyses and Texas Red-labelled Pd micro-
1
2–16
0
gation of innumerable biological processes
2
þ
0
17
0
been described , but the use of Pd catalysts to synthesize exogenous spheres were used for the confocal studies.
materials in cells has never been explored.
Analysis of the resulting microspheres by transmission electron
Of significant relevance is the work of Meggers, who described a microscopy (TEM) showed palladium nanoparticles evenly distrib-
0
water-soluble ruthenium complex that rapidly enters cells and per- uted on the microsphere (Fig. 1b,c), with the presence of the Pd
′
forms an allylcarbamate cleavage from bis-N,N -allyloxycarbonyl confirmed by powder X-ray diffraction (XRD) analysis (Fig. 1d).
rhodamine 110 (1) (ref. 18), and proving to be non-toxic to cells To demonstrate the catalytic activity of the microsphere-captured
0
′
during the short duration of the experiment. We reasoned that Pd nanoparticles, the bis-N,N -allyloxycarbonyl rhodamine 110
1
8
0
the use of a purely heterogeneous catalyst in the form of an ‘artificial (1) used by Meggers was incubated with Pd microspheres
organelle’ would allow the long-term cytoplasmic presence of under different conditions (Supplementary Tables S3–S5). The
0
metals with minimal leakage and toxicity (many transition metals, Pd catalyst mediated the effective deprotection of the allyloxycar-
1
9
20
including palladium , trigger cell death ). This would then allow bonyl group, resulting in the liberation of the strongly fluorescent
0
the exploration of challenging metal-catalysed reactions inside a cell. rhodamine 110 (2). Interestingly, the catalytic activity of the Pd
microspheres was clearly enhanced by the presence of glutathione
Results and discussion
(5 mM), with turnover numbers up to 30 in the presence of cell
Encouraged by the vast possibilities and applications of palladium extract and glutathione.
21
0
chemistry , the preparation of a bio-friendly internalizable Pd -
Although the microspheres have shown remarkable cellular com-
based heterogeneous catalyst was undertaken. This was achieved patibility and have been widely used as a highly efficient cellular
through the application of two technologies. First, a microsphere delivery vehicle for a variety of materials22–26, the presence of Pd
0
(
500 nm) mediated delivery system was used that had previously on the beads required an investigation of their biological compatibility.
0
been applied in both cellular labelling and intracellular delivery of Pd microspheres were therefore incubated with HeLa cells and
biomaterials, showing remarkable cellular biocompatibility and exo- uptake and toxicity evaluated. After 24 h, the intracellular presence
nuclear localization2 . The second technology involved the appli- of fluorescently labelled Pd microspheres was determined by flow
2–26
0
cation of palladium nanoparticles entrapped within crosslinked cytometry, demonstrating that more than 75% of cells had taken
0
resin beads, which have been shown to operate catalytically in a up one or more of the Pd microspheres, uptake that was further
27
truly heterogeneous manner .
corroborated by confocal imaging (Supplementary Figs S6 and
1
2
School of Chemistry, University of Edinburgh, West Mains Road, Edinburgh EH9 3JJ, UK, School of Chemical Sciences and Food Technology, Faculty of
†
Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM, Bangi, Selangor Darul Ehsan, Malaysia, Present address: Edinburgh Cancer Research
UK Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Crewe Road, Edinburgh EH4 2XR, UK. *e-mail: mark.bradley@ed.ac.uk
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NATURE CHEMISTRY DOI: 10.1038/NCHEM.981
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a
O
O
O
Cl
Cl
H
NH2
NH2
NH2
Pd(OAc)2
N
Pd(OAc)22
NHFmoc
Et3N, dry DMF
Pd(OAc)2
Toluene, 80°C
NH2
N
H
O
NHFmoc
10% NH2NH2
in MeOH
O
O
O
H
N
H
N
i) 20% piperidine
in DMF
Pd0
Pd0
NH2
ii) Preactivated DYE
Pr2EtN, dry DMF
N
N
DYE
N
NHFmoc
H
H
H
O
NH2
b
c
d
2,000
1
1
,500
,000
(
(
1)
2)
3)
500
(
0
35 40 45 50 55 60 65 70 75 80
50nm
5nm
2θ (deg)
0
0
Figure 1 | Synthesis and characterization of Pd microspheres. a, Synthesis of fluorescently labelled Pd microspheres. Amino-functionalized polystyrene
microspheres22 were treated with Pd(OAc) for 3 h to ensure uptake and interaction with the beads. The coordinated Pd was subsequently trapped by
2þ
2
2þ
0
extensive crosslinking with the bis acid chloride of racemic Fmoc-glutamic acid (generated in situ). Hydrazine in methanol was used to reduce Pd to Pd .
0
Labelling of the Pd microspheres was carried out by deprotection of the Fmoc group and subsequent treatment with an activated dye under basic
0
0
conditions. b,c, TEM of Pd nanoparticles within microspheres at two different magnifications: image of Pd nanoparticles physically captured and entrapped
0
on the microspheres (b); image of a single microsphere-captured Pd nanoparticle (arrow, c). Average diameter of nanoparticles: 5+2.5 nm. d, Powder XRD
patterns of (1) Pd microspheres, (2) commercial Pd powder, and (3) naked microspheres.
0
0
0
S7). Cytotoxicity associated with the Pd microspheres, determined mediated catalysis inside a cell for the first time. As shown in
using both (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Fig. 2f, confocal microscopy verified the simultaneous presence of
0
bromide (MTT) and propidium iodide assays, indicated low levels Texas Red-labelled Pd microspheres and intracellular compound 2.
of necrosis (,4%) and good cell viability (.91%) after 48 h of
contact with the Pd microspheres (Supplementary Figs S8 and liminary cytotoxicity study based on an allylcarbamate-protected
To expand the scope of the in cellulo deprotection method, a pre-
0
S9, Tables S1 and S2).
derivative of amsacrine (a commercially available antineoplastic
2
8
agent ) was performed (Supplementary Fig. S22). Alloc protection
led to a measurable reduction in the cytotoxic properties of this
chemically modified derivative relative to free amsacrine, a fact that
was used to investigate if amsacrine could be released in situ by the
0
Pd -mediated allylcarbamate cleavage within cells. The catalytic
0
activity of the Pd microspheres was investigated within living
cells, using HeLa cells as a model system. Cells were loaded with
0
fluorescently labelled Pd microspheres, washed to eliminate
0
catalytic activity of cell-containing Pd microspheres. Cells incubated
0
extracellular Pd microspheres, then fresh media containing
with alloc-protected amsacrine showed up to a sevenfold increment of
′
3
0 mM bis-N,N -allyloxycarbonyl rhodamine 110 (1) was added.
0
cytotoxicity in cell cultures preloaded with Pd microspheres.
The lipophilic nature of non-fluorescent compound 1 allows its
cellular internalization, whereas upon allylcarbamate cleavage Making C–C bonds within cells. The Suzuki–Miyaura cross-
most of the resulting fluorescent compound 2 is retained within coupling of arylboronates or esters with aryl halides (or triflates) in
0
the cell (Fig. 2a). As observed in Fig. 2, flow cytometry analysis of the presence of Pd permits ready access to a spectacular range of
2
9,30
an untreated cell control (Fig. 2b), a control treated with only the biaryls . To explore the intracellular formation of a carbon–carbon
0
Cy5.5-labelled Pd microspheres (Fig. 2c) or incubated just with cross-coupled product, an unambiguous fluorescence-detectable cell-
reagent 1 (Fig. 2d) showed no fluorescence under the FITC based experiment was designed, based on the palladium-mediated
0
31
emission filter (530/35 nm). In contrast, Cy5.5-labelled Pd
synthesis of a fluorescent dye (anthofluorescein ) via an aryl–aryl
microsphere-loaded HeLa cells incubated with reagent 1 showed cross-coupling reaction (Fig. 3a). The lipophilic non-fluorescent
fluorescence emission under both Cy5.5 (indicating the presence mono-triflate 3, containing a fluoran-based pro-fluorophore, and the
0
of the Pd microspheres) and FITC-like bandpass filters alkylaminophenylboronate 4 were therefore synthesized. Cross-
0
(
associated with the deprotection of dye 2) (Fig. 2e), showing Pd - coupling of these two components would restore the p-electron
240
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NATURE CHEMISTRY DOI: 10.1038/NCHEM.981
ARTICLES
a
O
O
H
N
+
Pd0
H2N
O
NH2
O
O
O
O
O
O
O
O
N
DYE
N
H
H
HN
O
NH
HN
O
NH
–
COO
O
O
O
O
2
x/ = 498/521nm
m
1
Cytoplasm
Non-fluorescent
b
c
1
1
1
1
1
04
104
103
102
101
100
f
03
02
01
00
1
00 101 102 103
FITC
104
100 101 102 103
FITC
104
e
d
1
1
1
1
1
04
03
02
01
00
104
103
102
101
100
1
00 101 102 103
FITC
104
100 101 102 103
FITC
104
0
0
Figure 2 | Pd -mediated allylcarbamate cleavage within HeLa cells. a, Pd -catalysed intracellular deprotection of reagent 1 generates fluorescent compound
0
2
. b–e, Flow cytometry analysis of HeLa cells showing intracellular Pd catalysis of allylcarbamate cleavage. The y-axis represents Cy5.5 fluorescence intensity
0
due to the Cy5.5-labelled Pd microspheres (bandpass emission filter, 780/60 nm) and the x-axis the FITC-like intensity of the cell due to compound 2
0
(
bandpass emission filter, 530/30 nm). b, Untreated cell control (no fluorescence emission). c, Cells after 24 h incubation with Cy5.5-labelled Pd
microspheres (positive fluorescence emission from Cy5.5 channel). d, Cells after 24 h incubation with reagent 1 (no fluorescence emission). e, Cy5.5-labelled
0
0
Pd microsphere-loaded cells after 24 h incubation with reagent 1, showing fluorescence emission under both Cy5.5 (indicating the presence of the Pd
microspheres) and FITC-like bandpass filters (confirming the synthesis of deprotected dye 2). f, Merged confocal image of a single HeLa cell (fixed with
0
paraformaldehyde) showing a Hoechst 33342-stained nucleus (blue) with Texas Red-labelled Pd microspheres (red) and deprotected compound 2 (green).
Scale bar, 10 mm.
conjugation of the fluoran polycycle 3 by opening of the lactone, mitochondria labelled with a far-red dye (MitoTracker Deep Red,
resulting in molecular fluorescence while at the same time localizing E /E ≈ 640/662 nm). Fixed cells were labelled with Hoechst
x
m
the product 5 to the mitochondria due to the presence of a 33342 (nuclei stain), imaged by confocal microscopy and the
triphenylphosphonium
accumulation of several hundredfold within mitochondria ).
moiety
(lipophilic
cations
direct images processed for three-dimensional analysis. As shown in
3
2
0
Fig. 3b, confocal images showed co-localization of both Pd -syn-
Suzuki–Miyaura dye in cellulo synthesis was initiated by the thesized dye and MitoTracker, demonstrating the unambiguous
0
incubation of fluorescently labelled Pd microspheres with HeLa identification of the Suzuki–Miyaura product 5, with in cellulo syn-
cells for 24 h, followed by intensive washing of the cells to eliminate thesis confirmed by flow cytometry analysis (Supplementary
0
extracellular Pd microspheres before the addition of the two Fig. S15). Controls were carried out, both in solution and in cell
reagents 3 (20 mM) and 4 (20 mM). After 48 h incubation, cellular experiments, to rule out the possibility of any fluorescence being
fluorescence was investigated by confocal microscopy with the result of hydrolysed mono-triflate 3 (Supplementary Figs S15,
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NATURE CHEMISTRY DOI: 10.1038/NCHEM.981
ARTICLES
a
CF3
CF3
O
S
O
O
S
O
+
O
O
O
O
O
O
Ph3P
O
O
O
H
+
N
NH
O
O
Pd0
O
O
3
N
N
DYE
H
H
O
Non-fluorescent
–
COO
5
Lipophilic
cation
+
x/ = 488/540nm
m
P
+
P
HN
HN
B
4
O
O
Non-fluorescent
B
O
O
Mitochondrion
Cytoplasm
c
b
0
0
Figure 3 | Pd -mediated Suzuki–Miyaura cross-coupling within HeLa cells. a, Pd -catalysed intracellular cross-coupling of reagents 3 and 4 generates the
0
0
mitochondria-localized fluorescent compound 5. b,c, HeLa cells were loaded with Pd microspheres, washed to eliminate extracellular Pd microspheres, and
subsequently incubated for 48 h with reagents 3 and 4. Cells were incubated for 30 min with MitoTracker Deep Red (mitochondrial stain), fixed with
paraformaldehyde, incubated with Hoechst 33342 (nuclei stain) and imaged by confocal microscopy. Deconvolved confocal images (b) of a single cell
showing co-localization of MitoTracker-labelled mitochondria and Suzuki–Miyaura product 5. Left panel: cell nucleus (blue) and mitochondria (red). Centre
panel: cell nucleus (blue) and in cellulo synthesized compound 5 (green). Right panel: merged image (orange indicates co-localization). Merged image (c) of
0
the same cell observed from a different angle. White arrow indicates the presence of a Texas Red-labelled Pd microsphere (not localized within the
mitochondria), which was imaged using a 550/20 nm emission filter together with compound 5.
S19 and S20, Table S5). Indeed, mono-triflate was remarkably before achieved within cells to be performed, such as the Suzuki–
0
robust, surviving unchanged in the presence of Pd for over 48 h. Miyaura cross-coupling reaction. This investigation provides the
Compound 5 was also extracted from the cells and its identity con- basis for the customization of heterogeneous unnatural catalysts as
firmed by high-performance liquid chromatography (HPLC) and tools for creative applications in chemical biology (such as in situ
mass spectrometry (MS) studies, confirming intracellular synthesis. labelling of cellular structures), pharmacology (for example,
Owing to the broad excitation/emission spectra of Texas Red, both in cellulo pro-drug activation of hydrophilic molecules with low cell
0
the Suzuki–Miyaura product 5 and the Texas Red-labelled Pd
penetrability for functional screening in cellular disease models)
microspheres were imaged under the same fluorescent channel. and, potentially, in medicine (forexample, the systemic administration
0
The Pd microspheres could be identified, because they did not of a pro-drug with local activation via implant-captured catalysts).
co-localize with the mitochondria tracker dye or the nucleus stain,
Methods
thus corroborating their cytoplasmatic location (Fig. 3c;
0
Pd microsphere synthesis. Aminomethyl polystyrene microspheres were prepared
Supplementary Figs S16 and S17). This experiment represents the
first non-enzymatic aryl–aryl bond formation ever achieved
22
as previously described . Microspheres (1 ml, aminomethyl polystyrene
21
microsphere, 0.5 mm, 0.08 mmol g , 4% solid content (SC)) were placed in a 1 ml
within living cells.
eppendorf tube and the water removed after centrifugation at 13,000 r.p.m. for
0
In conclusion, the first Pd -based heterogeneous catalyst with the 5 min. The beads were subsequently washed with toluene (2 × 1 ml). A volume of
21
0
.2 ml of a 0.4 mg ml palladium acetate solution in toluene was added and heated
ability to cross cell membranes, stay harmlessly within the cyto-
plasm for days, and carry out artificial chemistry has been described.
This engineered pseudo-organelle demonstrated intracellular cataly-
to 80 8C and sonicated every 2 min (5×). Subsequently, the reaction mixture was
shaken at room temperature for 2 h to give a light-brown coloured mixture. The
bead mixture was washed with toluene (3 × 1 ml) to ensure excess of palladium
tic activity towards exogenous materials, allowing chemistry never acetate was removed. Cross-coupling reagent (10 mg, Fmoc-Glu(Cl)-Cl) was freshly
242
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NATURE CHEMISTRY DOI: 10.1038/NCHEM.981
ARTICLES
prepared and dissolved in 1 ml of dry dimethylformamide (DMF) together with
triethylamine (11 ml, 0.08 mmol). Subsequently, 0.5 ml of this coupling solution was
added to the beads and shaken for 1 h at room temperature to afford 100%
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0
0
.5 ml) and water (3 × 0.5 ml). The Pd microspheres were stored in water as a light-
grey suspension. The Fmoc group was deprotected using 20% piperidine in DMF,
0
and the Pd microspheres were washed with DMF (3 × 1 ml), then treated with pre-
2
activated dye (5 equiv. Cy5.5 or Texas Red, 0.5 mg ml in DMF) and
1
diisopropylethylamine (DIPEA) (10 equiv.). The resulting mixture was shaken for
0
2
4 h at room temperature. The fluorescently labelled Pd microspheres were washed
with MeOH (3 × 1 ml) and water (3 × 1 ml) and stored in water in the dark. Cy5.5-
0
labelled Pd microspheres were used for the flow cytometry analysis, and Texas Red-
0
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1
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1
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serum and antibiotics (RPMI Complete Media (RPMI-CM)) in a 12-well plate with a
density of 40,000 cells per well and the cells were grown for 24 h. Thereafter, 1.0 ml of
0
Pd microspheres per ml (Cy5.5 labelled for flow cytometry analysis and Texas Red
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0
Pd microspheres were removed by washing with phosphate buffered saline (PBS)
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incubation with a 10 mg ml solution of Hoeschst 33342 in media for 5 min at
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0
10
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0
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2
0 mM and incubated at 37 8C and in 5% CO for 48 h. Subsequently, mitochondria
were stained by 30 min incubation with 50 nM of MitoTracker Deep Red in RPMI-
CM at 37 8C. HeLa cells were then fixed with 4% formaldehyde in PBS (30 min at
room temperature), and nuclei were stained by the addition of a 10 mg ml solution
of Hoechst 33342 (5 min). Microscope settings: excitation laser lines at 488 nm,
95 nm and 633 nm, with emission filters of 385–470 nm for Hoechst 33342,
40–560 nm for compound 5 and 650–670 nm for MitoTracker Deep Red.
2
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Received 23 September 2010; accepted 16 December 2010;
published online 6 February 2011
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This work was supported financially by the Government of Malaysia (R.M.Y.), the Swiss
National Science Foundation (E.M.V.J.) and the Engineering and Physical Sciences
Research Council. R.M.S.M. thanks the Royal Society for a Dorothy Hodgkin Fellowship.
The authors thank D. Kelly for his help with the confocal microscopy studies.
(
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