Ruthenium-induced allylcarbamate cleavage in living cells

Article abstract of DOI:10.1002/anie.200601752

(Chemical Equation Presented) Easy going: A ruthenium-catalyzed release of amines from their respective allylcarbamates is disclosed and is a step towards the design of catalysts as tools for cellular biology. The reaction tolerates the combination of water, air, and thiols and can be performed inside living mammalian cells. Cp* = pentamethylcyclopentadienyl, cod = 1,5-cyclooctadiene.

Full text of DOI:10.1002/anie.200601752

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tually be used to amplify signals by turning over a substrate
multiple times, catalytically label or deactivate target bio-
molecules, or release prodrugs and all this in a cellular
environment. However, designing catalysts which work under
physiological conditions is a significant challenge owing to the
combined presence of air, water, and a plethora of cellular
components such as millimolar concentrations of thiols, which
are prone to poison organometallic catalysts, especially under
protic and aerobic conditions.[5]
With respect to this new aspect of bioorganometallic
chemistry, we herein disclose a ruthenium-catalyzed release
of amines from their allylcarbamates that tolerates the
combination of water, air, and thiols, and we demonstrate
the utility of this cleavage reaction in living mammalian
cells.[6–10]
In the course of screening several organometallic catalysts
for a variety of reactions, we found that the complex
[Cp*Ru(cod)Cl] (Cp* = pentamethylcyclopentadienyl, cod =
1,5-cyclooctadiene; 1) catalyzes the cleavage of allylcarba-
mates 2 to their respective amines 3 in the presence of an
excess of thiophenol in an open flask experiment, tolerating
water and air (Scheme 1).[11,12] For example, the reaction of p-
Bioorganometallic Chemistry
DOI: 10.1002/anie.200601752
Ruthenium-Induced Allylcarbamate Cleavage in
Living Cells**
Craig Streu and Eric Meggers*
Scheme 1. Reaction of compounds 2 to form the respective amines 3.
[a] Isolated as the Boc-protected amine.
Organometallics are increasingly gaining attention as tools in
chemical biology owing to their distinguished physicochem-
ical properties, reactivities, and three-dimensional struc-
tures.[1] Along these lines, the exceptional ability of organo-
metallic compounds to catalyze a wide variety of chemical
transformations has not yet been sufficiently exploited for
chemical biology, but could yield bioactive molecules with
novel properties.[2–4] For example, such catalysts could even-
methylaniline allylcarbamate 2a (200 mm) with 5 equivalents
of thiophenol in the presence of 10 mol% ruthenium catalyst
1, carried out in MeOH/H2O (95:5) under air and overnight at
room temperature, provides p-methylaniline in 86% yield if
isolated as the tert-butoxycarbonyl (Boc)-protected amine,
and 89% yield according to GC analysis (Table 1, entry 1).
This deprotection is quite general as it works under the same
conditions with high yields also for allylcarbamates of an
electron-acceptor-substituted aniline (2b, 85% yield) and a
primary (2c, 94%, isolated as the Boc-protected amine), as
well as a secondary amine (2d, 87% yield; Scheme 1).[13]
The influence of air, water, and aliphatic thiols on the GC-
determined yields of this ruthenium-catalyzed cleavage is
shown for substrate 2a in Table 1. Accordingly, yields are not
significantly affected by air and water (Table 1, entries 1–3).
In contrast, omitting thiophenol prevents the carbamate
cleavage completely in the presence of water and air (Table 1,
entry 4). Most importantly for cellular applications, the
cleavage reaction can be performed in the presence of
[*] C. Streu, Prof. E. Meggers
Department of Chemistry
University of Pennsylvania
231 South 34th Street, Philadelphia, PA 19104 (USA)
Fax: (+1)215-746-0348
E-mail: meggers@sas.upenn.edu
Homepage: http://www.sas.upenn.edu/~meggers/
[**] We gratefully acknowledge support from the National Institutes of
Health (confocal fluorescence microscope: 1S10RR021113-01) and
the group of Prof. Gary A. Molander (GC measurements).
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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Table 1: Catalytic cleavage of allylcarbamate 2a to p-methylaniline with
[Cp*Ru(cod)Cl].[a]
vent Entry ThiolAStoml .
T
Yield
89%
1
PhSH
MeOH/H2O
(95:5)
MeOH
MeOH/H2O
(95:5)
air
air
RT
2
3
PhSH
PhSH
RT
argon RT
93%
96%
4
5
6
7
no thiols
MeOH/H2O
(95:5)
MeOH/H2O
(95:5)
MeOH/H2O
(95:5)
MeOH/H2O
(95:5)
air
air
air
air
RT
RT
RT
0%
PhSH and
PhCH2CH2SH
PhCH2CH2SH
93%
34%
PhCH2CH2SH
378C 67%
[a] Generalreaction conditions: 2a (200 mm), [Cp*Ru(cod)Cl] (20 mm),
thiol(1 m; each), at room temperature (20–238C) overnight. Yields were
determined by GC analysis.
aliphatic thiols, such as benzeneethanethiol, with virtually no
influence on the yield of the reaction (93%; Table 1, entry 5).
However, if performed at room temperature, the aromatic
thiol is necessary because substitution for an aliphatic thiol
leads to a significant reduction in yield (34%; Table 1,
entry 6). Interestingly, increasing the temperature to 378C
provides the released amine with an improved yield of 67%
by using the aliphatic thiol benzeneethanethiol as the only
nucleophile.
To evaluate the carbamate cleavage under conditions that
more closely resemble a physiological environment, the bis-
allyloxycarbonyl-protected rhodamine 110 (4; Figure 1a) was
synthesized.[14] This caged fluorophore is virtually nonfluor-
escent (Figure 1b, entry 1) and stable in the presence of E.
coli cell extract (Figure 1b, entry 2).[15] However, upon
allylcarbamate deprotection, strongly green-fluorescent rhod-
amine 110 (5) is released (Figure 1a). This allows for mon-
itoring of the ruthenium-catalyzed reaction by fluorescence
analysis. For example, the reaction of 4 with 1 (20 mol%) in
the presence of cell extract and an additional glutathione
(GSH; 3.5 mm)[16] leads to the release of the fluorophore with
a yield for 5 of 6% at 378C (Figure 1b, entry 3). Under the
same conditions, but with additional thiophenol, the yield
increases to 80% as measured after 2.5 h (Figure 1b, entry 4),
resulting in a turnover number of 8. These results demon-
strate that [Cp*Ru(cod)Cl] is able to catalyze the cleavage of
allylcarbamates under biological conditions consisting of
millimolar concentrations of GSH as well as a plethora of
cellular components from the whole-cell extract.
Figure 1. Influence of cell extract on the ruthenium-catalyzed allylcarba-
mate cleavage. a) Ruthenium-catalyzed deprotection of the caged
fluorophore 4 to rhodamine 110 (5). b) Fluorescence development with
4. Reaction conditions: Entry 1: 4 (0.5 mm) in DMSO/H2O (1:1).
Entry 2: 4 (0.5 mm), in DMSO/cell extract (1:1), and GSH (3.5 mm),
yielding an overall pH of 7.0. Entry 3: Same as entry 2, but with
additional[Cp*Ru(cod)C]l at 100 mm. Entry 4: Same as entry 2, but
with additional[Cp*Ru(cod)C]l at 100 mm and 3.5 mm PhSH. Reaction
mixtures were shaken at 120 rpm for 2.5 h at 378C. Yields were then
determined by comparing the fluorescence of diluted samples with a
standard curve of different concentrations of rhodamine 110. For the
shown vials, the solutions were diluted 20-fold with H2O/MeOH (1:1)
and excited with a long-wavelength UV lamp.
changes in fluorescence by live-cell imaging with a confocal
fluorescence microscope demonstrates that green fluores-
cence increases in intensity by around 10-fold over a time
period of 15 minutes within the cytoplasm of the cells
(Figure 2a and b). For comparison, in the absence of
thiophenol, the fluorescence increases more modestly by
around 3.5-fold, indicating that thiophenol is beneficial,
although not absolutely required, for the induction of
fluorescence. This is consistent with our model experiments
in cell extract (see Figure 1).
Additional cell-staining experiments with the red fluo-
rescent membrane carbocyanine dye DiIC18(5) illustrate that
the fluorescence only develops in the interior of the cell
(Figure 2c–f). In control experiments, we verified that no
fluorescence evolves in the absence of ruthenium complex 1
or the absence of the caged fluorophore 4 (data not shown).
Together, these experiments demonstrate that ruthenium
complex 1 is capable of passing through the cell membrane
and inducing the cleavage of allylcarbamates within living
cells. Notably, cytotoxicity experiments with the 3-(4,5-
Finally, the caged fluorophore 4 was used as a tool to
investigate the ruthenium-induced allylcarbamate cleavage in
living mammalian cells. For this, cultured HeLa cells were
loaded with caged fluorophore 4 by incubating the HeLa cells
with 4 (100 mm) for half an hour, followed by washing the cells
with phosphate-buffered saline (PBS) buffer solution, and the
subsequent addition of fresh media. Thereafter, catalyst 1 was
added to the medium in final concentrations as low as 20 mm.
Fluorescence should now only develop inside the cell where
the caged fluorophore 4 is located. Indeed, observing the
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide
(MTT) method verify that treatment with the organoruthe-
nium compound 1, thiophenol, and the caged fluorophore 4
does not influence the viability of the cells (see the Supporting
Information).
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Figure 2. Fluorescence microscopy imaging of ruthenium-induced uncaging of allylcarbamate-protected rhodamine 110 (4) inside HeLa cells.
Shown are superimposed phase-contrast and fluorescence images. Images a) and b): Cells were preincubated with 4 (100 mm) for 30 min, washed
with PBS buffer solution, and then treated with [Cp*Ru(cod)Cl] (20 mm) and thiophenol(500 mm). The shown images are a) right after this
addition and b) after 15 min. Images c)–f): Cells were preincubated with 4 (100 mm) for 30 min and at the same time with the membrane
carbocyanine dye DiIC18(5) (Molecular Probes). After washing with PBS buffer solution, cells were treated with [Cp*Ru(cod)Cl] (40 mm) and
thiophenol(100 mm). The shown images are c) right after this addition and d)–f) after the indicated times.
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In conclusion, [Cp*Ru(cod)Cl] has been shown to be
capable of inducing the uncaging of amines from their
respective allylcarbamates under physiological conditions
such as in living mammalian cells. The described reaction is
an encouraging starting point towards the goal of designing
catalytic organometallics as tools in chemical biology.
Received: May 3, 2006
Published online: July 20, 2006
Keywords: bioorganometallic chemistry · cleavage reactions ·
.
homogeneous catalysis · mammalian cells · ruthenium
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[5] Testing of many organometallic compounds in our laboratory for
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À
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