Water-soluble (phosphane)gold(I) complexes - applications as recyclable catalysts in a three-component coupling reaction and as antimicrobial and anticancer agents

Article abstract of DOI:10.1002/ejic.200900279

Wator-soluble compounds of the typo [AuC-I(PR₃)] with alkylbis(m-sulfonated-phenyl) (mC₆H₄SO₃Na) ₂ and dialkyl-(msulfonated-phenyl) (mC₆H ₄SO₃Na) (R = nBu, Cp) phospha

Full text of DOI:10.1002/ejic.200900279

FULL PAPER
DOI: 10.1002/ejic.200900279
Water-Soluble (Phosphane)gold(I) Complexes – Applications as Recyclable
Catalysts in a Three-Component Coupling Reaction and as Antimicrobial and
Anticancer Agents
Benelita T. Elie,[a] Chaya Levine,[a,b] Iban Ubarretxena-Belandia,[c]
Armando Varela-Ramírez,[d] Renato J. Aguilera,[d] Rafael Ovalle,[b] and María Contel*[a]
Dedicated to Professor Emeritus Martin A. Bennett[‡]
Keywords: Gold / Nanoparticles / Recyclable catalysts / Antimicrobial agents / Cytotoxicity / Apoptosis
Water-soluble compounds of the type [AuCl(PR3)] with alkyl-
bis(m-sulfonated-phenyl) (mC6H4SO3Na)2 and dialkyl-(m-
sulfonated-phenyl) (mC6H4SO3Na) (R = nBu, Cp) phos-
phanes have been prepared. Dialkylphosphane compounds
generate water-soluble nanoparticles of 10–15 nm radius
when dissolved in water. These air-stable complexes have
been evaluated as catalysts in the synthesis of propar-
gylamines by a three-component coupling reaction of alde-
hydes, amines, and alkynes in water. The antimicrobial ac-
tivity of the new complexes against gram-positive and gram-
negative bacteria and yeast has been evaluated. The new
compounds display moderate to high antibacterial activity.
The more lipophilic compounds are also potent against fungi.
Their cytotoxic properties have been analyzed in vitro by uti-
lizing human Jurkat T-cell acute lymphoblastic leukemia
cells. Compounds with dialkyl-(m-sulfonated-phenyl)
(mC6H4SO3Na) phosphanes displayed moderate to high
cytotoxicity on this cell line. The cell death mechanism in-
volves mainly early apoptosis. The catalytic/biological ac-
tivity of the previously described compound with commercial
m-trisulfonated-triphenylphosphane [AuCl(TPPTS)] (6) has
also been evaluated to compare the effects of the higher ba-
sicity and lipophilicity of the alkyl- and dialkyl-(m-sulfon-
ated-phenyl)phosphanes on these new compounds.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2009)
gold(I) compounds with phosphanes display interesting
structural properties, dynamic behavior in solution, or lumi-
nescent properties.[2] Many of these compounds have al-
lowed for the observation of aurophilicity or Au···Au inter-
actions (due to relativistic effects).[3] An isolobal relation-
ship of [Au(PPh3)]+ cations and H+ and Li+ has been pro-
posed on the basis of the behavior of [Au(PPh3)]+ fragments
in many homo- and heterometallic clusters;[4] some of them
having a very unusual coordination number for the central
atom (e.g. C and O).[5] The isolobality can be exploited in
the application of organometallic compounds of gold(I)
[AuR(PPh3)] as a less toxic, air-stable, and room-tempera-
ture alternative to organolithium, organomagnesium, and
organomercury compounds. Thus, these complexes can be
used as transmetallating agents to other metallic centers.[6]
Gold(I) complexes with phosphanes have displayed toxicity
against different tumor cell lines while targeting mito-
chondria.[7] They have also displayed antimalarial proper-
ties,[8] moderate antimicrobial activities,[9] and have found
applications as antirheumatic pharmaceuticals (aurano-
fin).[10] More recently, an increasing number of papers
based on the high catalytic activity of mainly [Au(PR3)]+
cations and other (phosphane)gold(I) derivatives in dif-
ferent chemical processes have been reported.[11]
Introduction
Gold(I) compounds with phosphanes have been known
for many years.[1] Most of gold compounds in the oxidation
state +1 are two-coordinate, linear, and 14-electron species.
Other complexes with different coordination numbers have
also been characterized, although they are not that com-
mon. They are usually three-coordinate trigonal-planar
complexes and four-coordinate gold(I) complexes (includ-
ing those with diphosphane chelating ligands).[2] Several
[a] Department of Chemistry, Brooklyn College and The Graduate
Center, The City University of New York,
2900 Bedford Avenue, Brooklyn, NY 11210, USA
Fax: +1-7189514607
E-mail: MariaContel@brooklyn.cuny.edu
http://userhome.brooklyn.cuny.edu./mariacontel/
[b] Department of Biology, Brooklyn College,
Brooklyn, NY 11210, USA
[c] Department of Structural and Chemical Biology, Mount Sinai
School of Medicine,
1425 Madison Avenue, Box 1677, New York, NY 10029, USA
[d] Department of Biological Sciences, The University of Texas at
El Paso,
El Paso, TX 79968, USA
[‡] In recognition of his outstanding contributions to organometal-
lic and inorganic chemistry.
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejic.200900279.
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M. Contel et al.
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Figure 1. Water-soluble phosphanes used in the preparation of gold(I) compounds.
As green-chemistry principles are incorporated in aca- alkyl-bis(m-sulfonated-phenyl)- and dialkyl-(m-sulfonated-
demic and industrial settings, the development of water- phenyl)phosphanes.[22] The incorporation of other organic
based reactions and water-soluble catalysts has become ex- groups such alkyl groups (cyclopentyl and butyl) in these
tremely important. Because water is the biologically most sulfonated phosphanes can modulate not only the electronic
relevant solvent, the synthesis of metal-based drugs with and steric parameters of these phosphanes (increasing their
enough solubility and stability in water or physiological me- basicity) but their hydrophilicity/lipophilicity properties.
dia is still a matter of increasing interest.
This makes the new compounds good candidates to study
Gold(I) compounds with water-soluble phosphanes (di-, their application as homogeneous catalysts in water and to
tri-, or even four-coordinate) have been described.[12] Some explore some biological properties oriented to their use as
of the phosphanes used are depicted in Figure 1. Sulfonated pharmaceuticals. We have chosen a three-component cou-
arylphosphanes (abbreviated TPPMS, TPPDS, TPPTS),[13] pling reaction of aldehydes, amines, and alkynes in water to
mono-, or diphosphanes containing pyridyl groups such as synthesize propargylamines, which are extremely versatile
DnPYPE, DnPYPP,[14] or maleic acid (DPMAA),[15] building blocks for organic synthesis.[23,24] We have also
phosphanes like 1,3,5-triaza-7-phosphaadamantane (TPA) evaluated the antimicrobial properties of these compounds
or 3,7-diacetyl-1,3,7-triaza-5-phosphabicyclo[3.3.1]nonane against bacteria (gram-negative and gram-positive) and
(DAPTA)[16] have been employed in the preparation of gold yeast. The cytotoxic and apoptotic properties of these com-
compounds with simple halide ligands (Cl, Br) or other co- plexes have been evaluated in vitro by utilizing human Jur-
ordination/organometallic complexes with thiolato, thion- kat T-cell acute lymphoblastic leukemia cells. We have used
ato, or alkynyl ligands. More recently, phosphanes contain- the previously described [AuCl(TPPTS)][19a] compound 6
ing hydroxy groups[17] and water-soluble cyclodiphosphaz- with commercially available phosphane [P(mC6H4SO3Na)3]
anes[18] have been employed in the preparation of gold(I) (TPPTS) (1) in order to compare the behavior of alkyl-sub-
compounds. Some of the compounds have been structurally stituted and unsubstituted sulfonated phosphanes in the
characterized (affording linear coordinated or polymeric catalytic/biological properties of the water-soluble gold
structures depending on the phosphane) in the solid state. compounds.
Some of the complexes with these phosphanes display inter-
esting photoluminescence[13,15] and cytotoxic properties
against different tumor cell lines.[14,15,16j,17,18]
Results and Discussion
[AuCl(TPPTS)] is a good co-catalyst in the biphasic pal-
ladium-catalyzed alkynylation reaction,[19a] whereas
[AuR(TPPTS)] compounds (R = alkynyl) are efficient and
recyclable catalysts in the hydration of alkynes.[20] Surpris-
ingly, studies on the microbial activity of gold compounds
with water-soluble phosphanes have been neglected.
Synthesis of Gold(I) Compounds with Alkyl-bis(m-
sulfonated-phenyl)- and Dialkyl-(m-sulfonated-phenyl)-
phosphanes
The preparation of the new complexes and compound
[AuCl(TPPTS)] (6)[19a] is achieved by displacing the labile
The increasing interest on homogeneous catalysis in ligand tht (tetrahydrothiophene) from the starting gold(I)
water as alternative reaction medium with the recovery of material [AuCl(tht)] by the water-soluble phosphanes
the catalyst as well as the development of gold catalysis (a (Scheme 1).[19] The complexes are obtained as white solids
current scientific hotspot)[21] warrants the study of new that are stable in air. They are all soluble in water (with
water-soluble gold compounds. The study of the biological solubilities in the range of 125 to 75 g/L, see Experimental
properties of these compounds becomes relevant in the se- Section), but they have different stabilities in solution. The
arch of new metallopharmaceuticals.
most stable compounds (in water solution and at room
We describe here the synthesis of new water-soluble temp.) are derivatives with three sulfonated-aryl groups in
gold(I) compounds that contain the recently described the phosphane 6, or one alkyl group and two sulfonated-
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Water-Soluble (Phosphane)gold(I) Complexes
Scheme 1. Preparation of the new water-soluble compounds and complex 6.[19]
aryl groups (7, 8). Complexes with two alkyl groups and case of 10 the extra signal at δ = 65.61 ppm becomes a
one sulfonated-aryl group (9, 10) display a different behav- major signal after 48 h (see Supporting Information). While
ior. The new compounds have been characterized by dif- we do not have information about the nature of these de-
1
ferent spectroscopic (31P{1H}, 13C{1H}, H, NMR), spec- composition products, complexes 9 and 10 are stable
trometric (ESI-MS), and analytical techniques (C, H, S mi- enough at room temp. for 24 h to allow us to perform the
croanalysis). 31P{1H} NMR spectroscopy is quite useful in biological tests described here.
the characterization and study of the stability of these com-
plexes over time. In all cases the chemical shifts correspond
to usual (phosphane)gold(I) chemical shifts for linear com-
pounds. The 31P{1H} NMR spectroscopic data for the free
phosphane ligands, for 6 and new gold(I) compounds 7–10
are collected in Table 1 (see also Supporting Information).
Table 1. 31P{1H} NMR chemical shifts (ppm) in D2O (25 °C) for
the water-soluble phosphane ligands 1, 2–5[22] and for the new
gold(I)-phosphane complexes 6–10.
Free
ligands
1
2
3
4
5
δ31P NMR –5.88 (s) –15.22 (s) –3.11 (s) –23.56 (s) 3.00 (s)
[AuClL] 10
6
7
8
9
δ31P NMR 33.40 (s) 30.40 (s) 46.06 (s) 29.68 (s) 56.77 (s)
Compounds 1–3 are stable in aqueous solution at room
temp. for 48 h as shown by 31P{1H} NMR spectroscopy.
After this time, a little decomposition to metallic gold can
be observed, but the signals in the 31P{1H} NMR spectra
remain the same for weeks. Compounds with the more ba-
sic dialkyl-(m-sulfonated-phenyl) (mC6H4SO3Na) phospha-
nes 9 and 10 have a different behavior. They can be ob-
tained as analytically pure white compounds (see Experi-
mental Section), but when dissolved in water, after a few
minutes they give a solution of intense purple color indica-
tive of water-soluble colloids. The 31P{1H} NMR spectra
do not change much over time, and only small signals (sing-
lets) accompanying the main singlet at δ = 51.94 (9) and
Figure 2. Top: TEM field-view image at 10000ϫ of nano-9. The
inset shows a detail at 50000ϫ. Bottom: Histogram of particle
65.61 (10) ppm can be seen at room temp. after 24 h. In the count as a function of particle radius (nm) in the image.
Eur. J. Inorg. Chem. 2009, 3421–3430
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M. Contel et al.
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Unfortunately, attempts to obtain crystals of high reactions were run at 100 °C and with 5 mol-% of catalysts
enough quality for X-ray diffraction studies (6–10) were un- (Entries 2, 7, and 9). Yields improve by addition of NaPF6
successful.
(which should facilitate the removal of chloride from the
We characterized these purple solutions to test for the gold compounds). Moreover, with compound 6 we found
presence of gold colloids. Indeed, TEM analysis confirmed good conversions at room temperature if reactions were run
the formation of homogeneous nanoparticles with a 10– for 42 h (Entry 5). When reactions were run at room temp.
15 nm radius (Figure 2, see Experimental Section for de- or 40 °C, we did not observe decomposition to metallic gold
tails). Smaller dimensions (2–3 nm) have been previously (we observed this decomposition at 100 °C), and we decided
observed for fluorous-soluble gold nanoparticles that were to recycle the water layers from these runs in order to study
generated from gold(I)-fluorous phosphane derivatives.[25]
the recyclability of the system.
Table 2. Three-component coupling of benzaldehyde, piperidine,
and phenylacetylene catalyzed by water-soluble gold compounds
6–8.
Catalytic Activity in the Synthesis of Propargylamines by a
Three-Component Coupling Reaction of Aldehydes, Amines,
and Alkynes
Run
Catalyst
Co-catalyst Temp.
Time Yield Conv.
[°C]
[h]
(%)
(%)[b]
We used the water-soluble compounds in a coupling re-
action with formation of C–C and C–N bonds. The reac-
tion is a one-pot reaction with three components (alde-
hydes, alkynes, and amines) by C–H activation. Gold salts
like AuCl or AuBr3 had been shown to be very good cata-
lysts for this reaction (in amounts of 5 or 1 mol-%).[24a]
However, these salts are hygroscopic, highly acidic, and can
not be handled in air. The reactions were generally run at
100 °C, and water was obtained as the only byproduct of
the reaction (Scheme 2).
1
2
3
4
6 (1 mol-%)
6 (5 mol-%)
6 (5 mol-%)
6 (5 mol-%)
–
–
–
100
100
40
12
17
42
12
17
62
71
78
54
44
100
56
NaPF6
(5 mol-%)
–
100
5
6
6 (5 mol-%)
6 (2.5 mol-%) NaPF6
(5 mol-%)
r.t.
100
42
12
81
78
98
56
TPPTS
(5 mol-%)
–
–
7
8
9
7 (5 mol-%)
7 (7 mol-%)
8 (5 mol-%)
100
r.t.
100
17
42
12
99
12
94
85
70
93
NaPF6
(5 mol-%)
10
11
8 (5 mol-%)
8 (5 mol-%)
NaPF6
(5 mol-%)
NaPF6
(5 mol-%)
50
40
24
21
48
25
91
88
Scheme 2. Three-component coupling of benzaldehyde, piperidine,
and phenylacetylene catalyzed by gold.[23,24]
12
13
8 (7 mol-%)
6 (7 mol-%)
–
r.t.
r.t.
42
42
39
75
97
96
NaPF6
(7 mol-%)
More recently, other gold catalysts have been employed
in this reaction;[23,24b–24g] (salen)gold(III) complexes af-
forded excellent yields at 40 °C, and when chiral prolinol
derivatives were used as the amine component, excellent
14
15
16
17
18
19
20
RP[a] (run 13)
RP[a] (run 14)
RP[a] (run 15)
6 (7 mol-%)
–
–
–
–
–
–
–
r.t.
r.t.
r.t.
r.t.
r.t.
r.t.
r.t.
42
42
42
93
42
42
42
65
53
38
91
88
83
77
83
77
85
87
85
80
74
diastereoselectivities (up to 99:1) were obtained.[24c]
A
RP[a] (run 17)
RP[a] (run 18)
RP[a] (run 19)
gold(III) C,O metallacycle (with a phosphinamide ligand)
has also proved to be highly efficient for this three-compo-
nent coupling.[24g] The synthesis of quinoline derivatives
can be achieved by a sequential catalytic process by AuCl3/
CuBr (5 mol-%/30 mol-%).[24e] The recyclability of the sys-
tem was achieved for 4 runs with gold nanoparticles
(10 mol-%) at 75–80 °C.[24d] Supported gold catalysts (lay-
[a] RP: recycled water phase containing the gold catalyst. [b] Deter-
mined by 1H NMR spectroscopic analysis of the crude reaction
mixture on the basis of benzaldehyde conversion.
The study of recycling catalyst 6 is collected in En-
ered double-supported gold[24b] and gold nanoparticles sup- tries 13–16. With 7 mol-% of 1 and 7 mol-% of NaPF6 the
ported on nanocrystalline ZrO2 and CeO2, 2.8 mol-%[24f]
)
catalyst can be separated and re-used 4 times. However, the
are highly active, selective (high diastereoselectivities for yields of product decrease steadily from run to run from
chiral amines), and recyclable in this A3 coupling in water 75 to 38 (4th run). Addition of NaPF6 was, however, not
at 100 °C.[24f] However, apart from the report on AuCl by necessary, and we could separate catalyst 6 and recycle it
Liu, there are no reports on the catalytic activity of gold(I) with higher conversion and less decrease in the yield/con-
compounds in these reactions.
version from run to run (Entries 17–19) of product. It seems
We tested the most stable of our compounds (6–8) under that at room temp. the addition of NaPF6 is not as benefi-
different reaction conditions in a biphasic system consisting cial as at higher temperatures.
of 1 mL of H2O (where we dissolved the gold compounds)
While we were preparing this manuscript, a study on the
and the organic reagents. After the reaction had taken recyclability of an organogold(III) complex was re-
place, the separation of the gold catalyst was performed by ported.[23] The compound of type [Au(C–N)Cl2] (N–CH =
simple decantation of the two layers. The results of these 2-phenylpyridine) was an efficient and selective catalyst
studies are collected in Table 2. We found good yields when (1 mol-%) for this three-component coupling reaction in
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Water-Soluble (Phosphane)gold(I) Complexes
water at 40 °C. Notably this complex could be repeatedly isms, clearance zones were 1.0–1.4 cm in diameter regard-
used for 10 cycles, leading to an overall turnover number of less of the quantity of each compound.
812.[23] While our system is not so efficient, it has allowed
The behavior for Bacillus cereus and Staphyloccocus au-
for the synthesis of propargylamines at room temp. These reus is nearly identical, and compounds 9 and 10 (the more
compounds are stable in water under mild conditions (up lipophilic) turned out to be the more toxic to these organ-
to 40 °C) for several hours, and this may allow their use as isms. For both compounds the zones of growth suppression
catalysts in some other processes in the future in which a exceeded 2.0 cm in diameter at 100 µg/disk. The relative dif-
higher basicity of the phosphanes coordinated to the gold ference in toxicity between 9 and 10 could be due to the
centers are preferred.
fact that the phosphane in compound 9 contains a bulkier
Cp group (because both phosphanes should have similar
lipophilicity).
A similar general trend (more toxic for gram-positive
bacteria than gram-negative ones or fungi) has been noted
previously with auranofin[9a] and some other (phosphane)-
gold(I) derivatives.[9b–9d,9f,9g] However, caution has to be ap-
plied when generalizing the results, because many factors
are involved in the antimicrobial activities. Thus, the nature
of other ligands coordinated to the gold centers (besides
the phosphanes) plays a decisive role. For instance, a more
complicated pattern is found for thiol-phosphanegold deriv-
atives, and the antimicrobial potency can be higher for
gram-negative ones or fungi depending on the combination
thiol-phosphane.[9a]
An explanation for the behavior displayed by the new
complexes 7–10 could be as follows: if these compounds
interfere with respiration (a mitochondrial function), then
the fungi should be most resistant, because electron trans-
port is shielded inside internal organelles, whereas in bacte-
ria electron transport occurs at the plasma membrane.
Gram-negative bacteria have a protective outer second
membrane; therefore, the compounds do not have ready ac-
cess to the electron-transport chain. Finally, the most lipo-
philic (hydrophobic) molecules are the most effective for
gram-positive ones. This is explained by the ability of 9 and
10 to partition into or through the membrane, whereas 6,
the most highly charged compound, is less likely to pass
through the plasma membrane.
Biological Activity: Antimicrobial Activity and Cytotoxic
Properties in vitro
Antimicrobial Activity
The antimicrobial activity of the new compounds as well
as that of compound 6 were evaluated against yeast (Sac-
charomyces cerevisiae) gram-negative (Escherichia coli and
Salmonella tiphymurium) and gram-positive (Bacillus cereus
and Staphyloccocus aureus) bacteria. The results of the ex-
periments are collected in Table 3. They all have moderate
to high antibacterial activity. The compounds with the
highest lipophilicity (9 and 10) are also potent against
fungi.
Table 3. Toxicity assesment of compounds 6–10 against microbial
organisms.[a]
Compd. S. cerevisiae E. coli S. tiphymurium B. cereus
S. aureus
6
7
8
9
–
–
–
100
100
10
100
100
10
100
10
10
100
100
1
100
100
100
1
100
100
100
100
10
10
10
[a] Minimum number of µg required to create a zone of clearing of
0.5 cm diameter on a lawn of fungal or bacterial cells: 100, 10, 1,
or – no toxicity.
Saccharomyces cerevisiae appears resistant to this class of
compounds. The plasma membrane appears to be an effec-
tive barrier to the more hydrophilic compounds 6–8,
whereas the more lipophilic compounds with dialkyl-(m-
sulfonated-phenyl) (mC6H4SO3Na) phosphanes 9, 10 are
Cytotoxic Properties and Apoptosis Studies
The cytotoxic properties of gold(I) compounds 6–10
able to penetrate the plasma membrane to suppress growth. were analyzed in vitro according to the procedures de-
Compounds 9 and 10 suppress yeast growth in a zone of scribed by Montoya et al.[26] and our laboratories[27] by uti-
about 2.5 cm diameter. Further testing with other fungal lizing Jurkat T-cell acute lymphoblastic leukemia cells
species is required to see if this pattern of resistance is uni- (Table 4). Before use, all tested compounds were dissolved
form across the fungi.
in physiological saline solution (NaCl 0.9%), and dilutions
Among the bacteria we found that gram-positive ones of each compound were then added to the cells in normal
are 100 times more sensitive to [AuCl{PR2- growth medium. All the tested complexes have proven, by
(mC6H4SO3Na)}] than gram-negative ones, whether R is 31P NMR spectroscopic studies, to be stable in D2O over
cyclopentyl (9) or n-butyl (10). Gram-negative bacteria are 24 h or more. Compounds 9 and 10 produce stable water-
moderately resistant, showing growth suppression at only at soluble nanoparticles when dissolved in water.
100 µg/disk; however, individual species may show selective
Cisplatin was used as a positive control as previously re-
sensitivity to a particular molecule. Thus, Escherichia coli ported by our laboratories with organogold(III) complexes
was more sensitive to [AuCl{PmC6H4SO3Na}3] (6), whereas containing iminophosphorane ligands.[27]
the cyclopentyl group in [AuCl{PCp(mC6H4SO3Na)}] (8)
Compounds
with
dialkyl-(m-sulfonated-phenyl)
and [AuCl{PCp2(mC6H4SO3Na)}] (10) increases toxicity to (mC6H4SO3Na) phosphanes that produced nanoparticles
Salmonella typhimurium. For both gram-negative organ- when dissolved in water displayed moderate to high cytoto-
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M. Contel et al.
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Table 4. IC50 values [µ][a] of compounds 6–10 compared to cispla- mental Section for details). As cells may undergo pro-
tin.
grammed cell death (apoptosis) or necrosis, the mode of
cisplatin[b]
31
6
7
8
9
10
death mediated by our compounds was investigated. In
early stages of apoptosis, one of the significant biochemical
features is loss of plasma membrane phospholipid asym-
metry, due to translocation of phosphatidylserine (PS) from
the cytoplasmic to the extracellular side. This characteristic
allows detection of externalized PS by the specific binding
of Annexin V (FITC-conjugatated). Initiation of cell death
will eventually result in the permeabilization of the cell
membrane, allowing PI to stain DNA within the nucleus.
The results of these studies are summarized in Figure 3.
All five compounds tested demonstrated that the in-
flicted cell damage was preferentially due to apoptosis in-
stead of necrosis. The preferential apoptosis induced is
markedly much higher than that for the gold(I) phosphane
compounds described by Bandoli and co-workers.[28] Ap-
parently, compound 10 may be slightly more toxic than the
other four compounds tested, since it induces higher levels
of late apoptosis in 16 h of incubation time. As described
in the Experimental Section, three controls were included:
untreated cells, cells treated with H2O2, and samples treated
with physiological saline solution. There was no significant
difference between untreated cells and cells treated with so-
dium chloride. Auranofin is known to also induce apoptosis
in HL-60 cells,[29] ovarian cancer cells,[30] and cardiac tis-
sue.[31] Other (phosphane or phosphite)gold(I) complexes
such as water-soluble compounds with the D2PYPP di-
phosphane ligand,[32] cyclodiphosphazanes,[17] or com-
pounds with bis(phosphite) ligands containing mesocyclic
thioether moieties[33] have induced apoptosis in HeLa,
breast cancer, and/or HCT-116 cell lines by activation of
p53 protein.
Cell line
Jurkat-GFP
97
113
76
50
23
[a] IC50 is defined as the concentration of drug required to disrupt
the plasma membrane of 50% of the cell population, as compared
to untreated cells, after 22 h of incubation. Cells with compromised
plasma membranes were monitored with Propidium iodide (PI)
and flow cytometry. Cisplatin was used as reference compound. [b]
Ref.[27]
xicity on this cell line. It appears that an increase in the
lipophilic properties of the phosphanes increases the cyto-
toxicity of this type of compounds. The cytotoxicity of 9
was not much higher than that displayed by cisplatin. Com-
pound 10 was the most cytotoxic compound of all tested,
even more than cisplatin for this cell line. The IC50 values
for 9 and 10 are similar to those found for some organogol-
d(III) complexes with iminophosphorane C,N skeletons
and chloride and/or water-soluble phosphanes recently de-
scribed by us,[27] but not as high as those with the combina-
tion of iminophosphorane and dithiocarbamate ligands (1–
2 µ).[27] Very recently, Bindoli and co-workers have re-
ported that well-known (phosphane)gold(I) complexes such
S-[(triethylphosphane)gold(I)]-2,3,4,6-tetra-O-acetyl-1-thio-
B--glucopyranoside (auranofin) and (triethylphosphane)-
gold(I) chloride induced apoptosis in Jurkat T cells.[28] Al-
though IC50 values were not provided, different experiments
were run with 1 µ of gold compounds for which cell mor-
tality was between 20 and 27%. This apoptosis appeared to
be triggered by inhibition exerted by the gold(I) compounds
on the cytosolic and mitochondrial isoforms of thioredoxin
reductase, which resulted in an increase in H2O2. However,
no significant lipid peroxidation or nitric oxide formation
We also performed a study of the cytotoxicity of com-
were observed after incubation with gold(I) complexes, in- pound 10 after it was dissolved in the medium and kept in
dicating that the cells had not been subjected to extensive the absence of light and at –20 °C for 30 d. In Figure 4, the
oxidative stress. Polymeric gold(I) compound aurothiomal- cytotoxicity of a freshly prepared solution of 10 is shown
ate was poorly effective, both in inhibiting thioredoxine re- as 1. The cytotoxicity of this complex after 30 d at –20 °C
ductase and in inducing apoptosis.[28]
is shown as 2. We also measured the cytotoxicity of solu-
In order to gain some insight of the type of cell death tions that were kept in the dark and at –20 °C for 28 d and
that complexes 6–10 induce in this cancer cell line, we per- then kept at room temp. for 2 d extra either in the dark
formed some apoptosis assays with Jurkat cells (see Experi- (4) or in the presence of artificial light (3). Basically, the
Figure 3. Preferential induction of early apoptosis in Jurkat cells after treatment with the gold(I) complexes. The IC50 concentration
shown in Table 4 was used for each gold compound.
3426
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Water-Soluble (Phosphane)gold(I) Complexes
Figure 4. Testing of cytotoxic activity against Jurkat cells of compound 10 after exposure to artificial light at room temperature and time
after dissolution. (1) Freshly prepared sample of 10 (25 µ in saline solution); (2) solution of 10 kept at –20 °C for 30 d in the dark; (3)
solution of 10 kept at –20 °C for 28 d in the dark and additionally at room temp. in the presence of light for 2 d; (4) solution of 10 kept
at –20 °C in the dark for 28 d and additionally at room temp. in the dark for 2 d; (5) untreated cells; (6) cells treated with 1 µL of NaCl
(0.9%); (7) cells treated with 500 µ of H2O2 (positive control). Incubation time: 22 h.
cytotoxicity of these solutions decreases to approximately pounds have shown to be not as efficient catalysts as 6 (the
half of the value of that for a freshly prepared solution of most hydrophilic complex) in a biphasic three-component
compound 10.
coupling reaction in the synthesis of propargylamines, they
Compounds 6–10 yield [AuPR3]+ species in solution, have displayed interesting biological activities (antimicro-
which may enable them to pass more easily through the bial and anticancer). They all have moderate to high antimi-
lipid bilayer of membranes and, possibly, mitochondria, as crobial activity. The compounds with the highest lipophil-
it happens for some delocalized lipophilic cations (DLCs) icity (9 and 10) are the most potent for gram-positive
of gold(I).[33] The degree of lipophilicity of the ligands has bacteria and fungi. Also the higher lipophilicity of,
been recently connected to the in vitro cytotoxicity. A especially, compounds with dialkyl-(m-sulfonated-phenyl)
higher lipophilicity of the gold(I) drugs with phosphanes (mC6H4SO3Na) phosphanes (9 and 10) makes them the
results in a higher in vitro antitumor and hepatotoxic po- most cytotoxic of the group (6–10) in vitro against human
tency.[34] We believe that further modification of the gold(I) Jurkat T-cell acute lymphoblastic leukemia cells. Com-
compounds described here by exchange of chloride by other pound 10 is even more cytotoxic than cisplatin for this cell
organic or inorganic ligands may render promising stable, line. The cell-death mechanism involves mainly early
water-soluble cytotoxic compounds with an optimized lipo- apoptosis for all the compounds studied (6–10) as for some
philicity value. Interestingly, gold nanoparticles have been other (phosphane)gold(I) complexes (although compounds
found to enhance the anti-leukemia action of a 6-mercapto- 6–10 are more apoptotic than those described previously).
purine chemotherapeutic agent.[35] The most cytotoxic com-
The higher basicity of the new gold complexes can be
pounds described here (9 and 10) generate water-soluble further exploited for some other homogeneous biphasic
nanoparticles when dissolved in water. Further biochemical catalytic processes. Subsequent modification of these com-
assays will be performed in order to determine the mecha- plexes by exchange of chloride for other ligands may afford
nism that triggers apoptosis for these complexes as well as complexes with an optimized lipophilicity value with poten-
their toxicity against normal human cells.
tial applications as more efficient antimicrobial and anti-
cancer agents.
Conclusions
Experimental Section
We have prepared gold(I) compounds with water-soluble
phosphanes with higher basicity and lipophilicity than pre-
viously reported {[AuCl{P(mC6H4SO3Na)3}] (6)}[19a] by in-
troduction of one or two alkyl groups {R = nBu or Cp)
in the (sulfonated-aryl)phosphane ([PR(mC6H4SO3Na)2] or
[PR2(mC6H4SO3Na)]}. The complexes with two alkyl
groups on the phosphanes (9 and 10) produce nanoparticles
when dissolved in water as opposed to 6 or compounds
1. Sythesis and Characterization of the Water-Soluble Gold(I) Com-
plexes: Solvents were purified by use of a PureSolv purification unit
from Innovative Technology, Inc.; all other chemicals were used as
received. Elemental analyses were carried out by Atlantic Microlab,
Inc. (US). The 1H, 13C{1H} and 31P{1H} NMR spectra were re-
corded in D2O solutions at 25 °C with a Bruker 400 spectrometer
(δ, ppm; J, Hz); 1H and 13C{1H} NMR data were referenced by
using the solvent signal as internal standard, whereas 31P{1H}
with just one alkyl group (7 and 8). Whereas these com- NMR data were externally referenced to H3PO4 (85%). Mass spec-
Eur. J. Inorg. Chem. 2009, 3421–3430
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3427

M. Contel et al.
FULL PAPER
tra (ESI) were recorded from acetone or water solutions by the
were removed under vacuum. By addition of 5 mL of acetone/
mass spectrometry facility of the University of California Riverside 10 mL of diethyl ether, compound 9 was obtained as a white solid.
(US). The preparation[19] of compound 6 is slightly different from
that previously reported.[19a] Phosphanes 2–5 [PR(mC6H4SO3Na)2]
and [PR2(mC6H4SO3Na)] (R = nBu, Cp) were prepared as recently
described.[22] [AuCl(tht)] was prepared according to the procedure
of Uson et al.[36] The rest of chemicals and solvents were purchased
from Sigma–Aldrich.
Yield: 0.22 g, 71%. Solubility in H2O: 75 g/L. C14H22AuClNaO3PS
(556.77): calcd. C 30.20, H 3.98, S 5.76; found C 30.11, H 4.20, S
6.01. MS (ESI-): m/z = 533.03.94 [M – Na]–. 31P{1H} NMR (D2O):
1
δ = 29.68 (s) ppm. H NMR (D2O): δ = 0.61 (br., 6 H, CH3, Bu),
1.27 (br. m, 8 H, CH2CH2CH2P), 2.07 (br., 4 H, CH2P), 7.43 (br.,
1 H, H5, mC6H4SO3Na), 7.5–8.05 (v. br., 3 H, H6, H4, H2,
mC6H4SO3Na) ppm. 13C{1H} NMR (D2O): δ = 12.99 (s, CH3, Bu),
[AuCl{P(mC6H4SO3Na)3}] (6):[19a] A solution of [P(mC6H4SO3-
Na)3] (0.341 g, 0.6 mmol) in 5 mL of acetone and 9 drops of water
was added dropwise to a solution of [AuCl(tht)] (0.192 g, 0.6 mmol)
in 15 mL of dichloromethane. The reaction mixture was stirred at
room temp. for 30 min, and all the solvents were removed under
vacuum. By addition of 5 mL of acetone/10 mL of diethyl ether,
compound 6 was obtained as a white solid that was filtered off and
dried under vacuum. Yield: 0.480 g, 99%. Solubility in H2O: 125 g/
L. 31P{1H} NMR (D2O): δ = 33.4 (s) ppm.
23.21 (d, 3JP,C = 16.4 Hz, CH2CH2P, Bu), 27.05 (s, CH3CH2), 40.10
1
(s, CH2P), 129.26 (br., C2, mC6H4SO3Na), 129.72 (d, JP,C
=
12.68 Hz, C1, mC6H4SO3Na), 130.16 (br., C4, mC6H4SO3Na),
130.29 (br., C5, mC6H4SO3Na), 136.14 (d, JP,C = 12.98 Hz, C6,
2
mC6H4SO3Na), 143.83 (br., C3, mC6H4SO3Na) ppm.
[AuCl{PCp2(mC6H4SO3Na)}]
(10):
A
solution
of
[PCp2(mC6H4SO3Na)] (0.1021 g/0.3 mmol) in 1.5 mL of deoxy-
genated of water was added dropwise to a solution of [AuCl(tht)]
(0.0961 g/0.3 mmol) in 6 mL of dry acetonitrile. The reaction mix-
ture was stirred under argon at room temp. for 30 min, and all
the solvents were removed under vacuum. By addition of 5 mL of
acetone/10 mL of diethyl ether, compound 10 was obtained as a
white solid. Yield: 0.0468 g, 92%. Solubility in H2O: 75 g/L.
C16H20AuClNaO3PS (578.78): calcd. C 33.20, H 3.48, S 5.54; found
C 33.19, H 4.43, S 5.60. MS (ESI–): m/z = 557.03 [M – Na]–.
31P{1H} NMR (D2O): δ = 56.77 (s) ppm. 1H NMR (D2O): δ =
0.85–1.99 (v br. m, 10 H, Cp), 7.46 (m, 1 H, H5, mC6H4SO3Na),
[AuCl{PnBu(mC6H4SO3Na)2}] (7): A solution of [PnBu(mC6H4-
SO3Na)2] (0.134 g/0.3 mmol) in 5 mL of acetone and 9 drops of
water was added dropwise to a solution of [AuCl(tht)] (0.0962 g/
0.3 mmol) in 15 mL of dichloromethane. The reaction mixture was
stirred at room temp. for 30 min, and all the solvents were removed
under vacuum. By addition of 5 mL of acetone/10 mL of diethyl
ether, compound 7 was obtained as a white solid. Yield: 0.195 g,
96%. Solubility in H2O: 125 g/L. C12H15AuClNa2O6PS2 (628.74):
calcd. C 28.31, H 2.52, S 9.44; found C 27.75, H 3.15, S 9.33. MS
(ESI–): m/z = 654.94 [M – Na]–. 31P{1H} NMR (D2O): δ = 30.40
3
7.84 (m, 2 H, H6, H4, mC6H4SO3Na), 7.82 (d, JH,H = 16 Hz, 2 H,
3
H4, mC6H4SO3Na), 8.04 (d, JP-H
=
10.8 Hz,
1
H, H2,
1
(s) ppm. H NMR (D2O): δ = 0.71 (br., 3 H, CH3 Bu), 1.33 (br.
mC6H4SO3Na) ppm. 13C{1H} NMR (D2O): δ = 25.63 (d, JP,C
=
2
m, 4 H, CH2CH2CH2P), 2.53 (br., 2 H, CH2P), 7.53 (br., 2 H, H5,
mC6H4SO3Na), 7.67 (br., 2 H, H6, mC6H4SO3Na), 7.87 (br., 2 H,
H4, mC6H4SO3Na), 7.97 (d, 3JP-H = 12 Hz, 2 H, H2, mC6H4SO3Na)
ppm. 13C{1H} NMR (D2O): δ = 12.99 (s, CH3, Bu), 23.21 (d, 3JP,C
= 16.4 Hz, CH2CH2P, Bu), 27.05 (s, CH3CH2), 40.10 (s, CH2P),
2
7.02 Hz, Cb, Cp), 26.54 (d, JP,C = 6.172 Hz, Cb, CpЈ), 30.51 (br.,
Cc, Cp), 30.90 (br., Cc, CpЈ), 36.05 (br., Ca, Cp), 36.42 (br., Ca,
CpЈ), 129.29 (br., C2, mC6H4SO3Na), 129.60 (br., C1, C4,
mC6H4SO3Na), 130.14 (br., C5, mC6H4SO3Na), 130.10 (br., C5,
mC6H4SO3Na), 136.78 (br., C6, mC6H4SO3Na), 144.58 (d, JP,C
=
3
1
129.26 (br., C2, mC6H4SO3Na), 129.72 (d, JP,C = 12.68 Hz, C1,
8.78 Hz, C3, mC6H4SO3Na) ppm.
mC6H4SO3Na), 130.16 (br., C4, mC6H4SO3Na), 130.29 (br., C5,
2
mC6H4SO3Na), 136.14 (d, JP,C = 12.98 Hz, C6, mC6H4SO3Na),
2. General Procedure for the Gold(I)-Catalyzed A3 Coupling of
Benzaldehyde, Piperidine, and Phenylacetylene: In a Schlenk or
Kontex (for reactions at temperature higher than room tempera-
ture) tube, under N2 (g), the appropriate amount of the catalyst was
dissolved in 1 mL of deoxygenated water. Then, phenylacetylene
(150 µL, 1.5 mmol), benzaldehyde (100 µL, 1.0 mmol), and piper-
idine (105 µL, 1.1 mmol) were added. The reaction mixtures were
stirred for the time and temperature specified in Table 1. Once the
reaction had taken place, 2–3 mL of Et2O was added, and the two
layers (water/organic) were separated. Another 20 mL of Et2O was
added to the organic layer, and it was washed with 10 mL of a
brine solution and 2ϫ10 mL of H2O. After drying with anhydrous
MgSO4, the organic solvents were completely removed, and the
residue was weighed (yield) and analyzed by NMR spectroscopy
(conversion). When recycling the catalyst, we used the aqueous
layer and added an extra amount (0.5 mL) of deoxygenated water.
143.83 (br., C3, m-C6H4-SO3Na) ppm.
[AuCl{PCp(mC6H4SO3Na)2}]
(8):
A
solution
of
[PCp(mC6H4SO3Na)2] (0.136 g/0.3 mmol) in 5 mL of acetone and
9 drops of water was added dropwise to a solution of [AuCl(tht)]
(0.0962 g/0.3 mmol) in 15 mL of dichloromethane. The reaction
mixture was stirred at room temp. for 30 min, and all the solvents
were removed under vacuum. By addition of 5 mL of acetone/
10 mL of diethyl ether, compound 8 was obtained as a white solid.
Yield: 0.188 g, 92%. Solubility in H2O: 125 g/L. C17H16AuClNa2-
O6PS2 (700.33): calcd. C 29.15, H 2.30, S 9.15; found C 29.11, H
3.22, S 10.1. MS (ESI-): m/z = 666.94 [M – Na]–. 31P{1H} NMR
1
(D2O): δ = 45.06 (s) ppm. H NMR (D2O): δ = 1.4–1.6 (br. m, 9
3
H, Cp), 7.51 (t, JH,H = 8 Hz, 2 H, H5, m-C6H4-SO3Na), 7.76 (q,
3
2 H, H6, m-C6H4-SO3Na), 7.82 (d, JH,H = 16 Hz, 2 H, H4, m-
3
C6H4-SO3Na), 8.11 (d, JP-H = 12 Hz, 2 H, H2, m-C6H4-SO3Na)
2
ppm. 13C{1H} NMR (D2O): δ = 26.5 (d, JP,C = 9.33 Hz, Cb, Cp),
3. Antimicrobial Assays: The gold compounds were tested for mi-
crobial toxicity in a Kirby–Bauer disk diffusion assay. Compounds
6–10 were solvated in methanol to a concentration of 10 mg/mL
and serially diluted in methanol by factors of 10 to create solutions
ranging down to 0.1 mg/mL. 10 µL aliquots of the solution were
transferred onto filter paper disks (5 mm diameterϫ1 mm thick-
3
1
30.31 (d, JP,C = 5.82 Hz, Cc, Cp), 34.51 (d, JP,C = 38.47 Hz, Ca,
Cp), 129.24 (br., C2, mC6H4SO3Na), 129.87 (d, JP,C = 75.68 Hz,
1
C1, mC6H4SO3Na), 129.93 (br., C4, mC6H4SO3Na), 130.10 (br., C5,
mC6H4SO3Na), 136.44 (d, JP,C = 12.98 Hz, C6, mC6H4SO3Na),
2
143.83 (d, JP,C = 10.72 Hz, C3, mC6H4SO3Na) ppm.
3
[AuCl{PnBu2(m-C6H4SO3Na)}] (9): A solution of [PnBu2(mC6H4- ness), which were then vacuum-dried and stored at –20 °C prior to
SO3Na)] (0.181 g, 0.556 mmol) in 1.5 mL of deoxygenated of water
was added dropwise to solution of [AuCl(tht)] (0.178 g,
0.556 mmol) in 6 mL of dry acetonitrile. The reaction mixture was
stirred under argon at room temp. for 30 min, and all the solvents
the experiment. For the assay, 100 µL of a cell suspension contain-
ing 3ϫ107 cells/mL were spread uniformly on a Mueller–Hinton
(MH for bacteria) or Yeast Extract (YPD for fungi) agar plate
(100 mmϫ15 mm). After spreading, four paper disks impregnated
a
3428
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Eur. J. Inorg. Chem. 2009, 3421–3430

Water-Soluble (Phosphane)gold(I) Complexes
with either 100, 10, 1, or 0.1 µg of a compound were placed on the
agar surface with a solvent control in the center of the plate. The
plates were incubated at either 30 °C (fungi) or 37 °C (bacteria) for
48 h, and the resulting zones of growth suppression were measured.
flow cytometer was set up and calibrated by utilizing unstained,
single- (PI or Annexin V-FITC) and double- (Annexin V-FITC plus
PI) stained cells. The same three control groups (as described in
the previous section) were used: untreated cells, cells treated with
250 µ H2O2, and cells treated with 4 µL of physiological saline
solution.
4. Cytotoxicity Assay: Jurkat cells, non-adherent human T leuke-
mia lymphoblast-like cell line (American Type Culture Collection),
were seeded at 100000 cells/well by using RPMI media (HyClone,
Logan, UT) supplemented with antibiotics and 10% heat-inacti-
vated newborn calf serum (also referred to as complete media).
After overnight incubation, cells were exposed for 22 h to several
concentrations of chemical compounds. Cells from each individual
well were collected in an ice-cold tube, placed on ice, and washed
with cold complete media, and then, with cold PBS. After centrifu-
gation at 1400 rpm at 4 °C for 5 min, the supernatant was removed,
and the cell pellets were resuspended in 500 µL of staining solution,
containing 2 µg/mL propidium iodide dissolved in FACS buffer
(PBS, 0.5 m EDTA, 2% heat-inactivated fetal bovine serum, and
0.1% sodium azide). The cells were then incubated in the dark at
room temperature for 15 min and analyzed by flow cytometry,
using Cytomic FC 500 (Beckman-Coulter, Miami, FL). The data
were acquired and analyzed by using CXP software (Beckman-
Coulter). To calculate the IC50, the average of PI-positive cells
(dead cells) obtained from triplicates of untreated cells was sub-
tracted from each experimental point to obtain normalized values,
and also to eliminate culture-inherent effects. In these studies, only
untreated cells that were at 90% viability or higher were utilized.
The average cytotoxicity (annotated as a percentage) over three in-
dependent experiments was plotted against chemical compound
concentration in an xy (scatter) chart function (Microsoft Excel).
The IC50 values were then calculated from the triplicate values by
using the linear regression plot with its respective R2 value between
data points, to calculate the concentration of compounds dis-
rupting 50% of the plasma membranes of the test cells. For each
experimental set, three controls were prepared in triplicate: (1) un-
treated cells, (2) cells treated with hydrogen peroxide, and (3) cells
treated with physiological saline solution. Hydrogen peroxide,
which is well known as inducer of apoptosis and necrosis,[37] was
added to a final concentration of 500 µ as a positive control of cell
death and utilized to ensure that the flow cytometer was calibrated
properly. Physiological saline solution (NaCl 0.9%), the diluent of
chemical compounds, was tested at the same concentration as con-
tained in the experimental samples, as a control for nonspecific
diluent effects. The average of triplicate values from untreated cells
was used as 100% of viability. No significant differences were ob-
served in cells treated with physiological saline solution when com-
pared with untreated cells.
6. Electron Microscopy and Image Analysis: A 2 mL aliquot of com-
pound 9 in aqueous solution was applied onto a glow discharged
carbon coated copper grid and blotted. The specimen was imaged
with a JEOL-2100 transmission electron microscope, and images
were recorded with a Tietz 2kx2k CCD camera at a nominal mag-
nification of 50000x. At this magnification, the effective pixel size
at the specimen was 3.06 Å. Particle selection and measurement
were performed with the program ImageJ.[38]
Supporting Information (see footnote on the first page of this arti-
cle): Selected 31P{1H} NMR spectra of compounds 1–10.
Acknowledgments
We thank Brooklyn College (start-up budget M. C.), the Profes-
sional Staff Congress – City University of New York (PSC-CUNY)
research award PSCOOC-38-78 (M. C.), National Institutes of
Health (NIH) – National Institute of General Medical Sciences
(NIGMS) – Support of Competitive Research (SCORE) – Min-
ority Biomedical Research Support (MBRS) (Grant number
3S06GM076168-02S1) (R. O.), National Science Foundation
(NSF) (Grant number MCB-0546087) (I. U.-B.) for financial sup-
port and the Arnold and Ruth T. Kaufman Chemistry Fund at
Brooklyn College for a summer research undergraduate award
(C. L.). We thank the staff of the Cell Culture and High Through-
put Screening (HTS) Core Facility for services and facilities pro-
vided; this core facility is supported by grant 5G12RR008124 to
the Border Biomedical Research Center (BBRC), granted to the
University of Texas at El Paso from the National Center for Re-
search Resources (NCRR) of the NIH. We also thank the New
York Structural Biology Center for access to their electron micro-
scope. We are indebted to Prof. István T. Horváth (Eötvös Univer-
sity, Budapest, Hungary) for the generous donation of the water-
soluble phosphanes used in this work. We also thank undergradu-
ate student Tasmin Chowdhury and high school student Syeda Ta-
bassum (recipient of the American Chemical Society – Summer
Educational Experience for the Disadvantaged ACS-SEED pro-
gram 2007-08) for their assistance with some of the experiments.
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5. Apoptosis Assay: Jurkat cells were seeded and cultured as de-
scribed above and exposed to IC50 concentrations of the chemical
compounds as determined by the cytotoxicity assays for 16 h. Cells
from each individual well were collected and washed as described
above. After centrifugation at 1400 rpm at 4 °C for 5 min, the me-
dia and PBS were removed. The staining procedure was performed
by resuspending the cell pellets in 100 µL of binding buffer (0.1 
HEPES, pH = 7.4; 140 m NaCl; 2.55 m CaCl2) containing 1 µL
of 25 µg/mL Annexin V-FITC (Beckman Coulter, Miami, FL) and
5 µL of 250 µg/mL PI. After incubation for 15 min on ice in the
dark, ice-cold binding buffer (400 µL) was added to the cell suspen-
sions, gently homogenized, and immediately analyzed by flow cyto-
metry. For each sample, 10000 individual events were acquired and
analyzed by using CXP software (Beckman Coulter, Miami, FL).
After exposure of cells to the chemical compounds, all subsequent
procedures were carried out on ice or at 4 °C to arrest or slow
down progression of cell damage. Prior to data acquisition, the
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Eur. J. Inorg. Chem. 2009, 3421–3430
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Received: March 24, 2009
Published Online: June 30, 2009
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