Gold(I)-phosphine-N-heterocycles: Biological activity and specific (ligand) interactions on the C-terminal HIVNCp7 zinc finger

Article abstract of DOI:10.1021/ic401535s

The syntheses and the characterization by chemical analysis, ¹H and ³¹P NMR spectroscopy, and mass spectrometry of a series of linear triphenylphosphine gold(I) complexes with substituted N-heterocycle ligands (L), [(PPh₃)Au(I)(L)]⁺, is reported. The reaction of [(PPh₃)Au(L)]⁺ (L = Cl^- or substituted N- heterocyclic pyridine) with the C-terminal (Cys₃His) finger of HIVNCp7 shows evidence by mass spectrometry (ESI-MS) and ³¹P NMR spectroscopy of a long-lived {(PPh₃)Au}-S-peptide species resulting from displacement of the chloride or pyridine ligand by zinc-bound cysteine with concomitant displacement of Zn²⁺. In contrast, reactions with the Cys₂His₂ finger-3 of the Sp1 transcription factor shows significantly reduced intensities of {(PPh₃)Au} adducts. The results suggest the possibility of systematic (electronic, steric) variations of carrier group PR₃ and leaving group L as well as the nature of the zinc finger in modulation of biological activity. The cytotoxicity, cell cycle signaling effects, and cellular accumulation of the series are also reported. All compounds display cytotoxicity in the micromolar range upon 96 h continuous exposure to human tumor cells. The results may have relevance for the reported inhibition of viral load in simian virus by the gold(I) drug auranofin.

Full text of DOI:10.1021/ic401535s

Article
pubs.acs.org/IC
Gold(I)-Phosphine-N-Heterocycles: Biological Activity and Specific
(Ligand) Interactions on the C‑Terminal HIVNCp7 Zinc Finger
Camilla Abbehausen,† Erica J. Peterson,‡ Raphael E. F. de Paiva,† Pedro P. Corbi,† Andre L. B. Formiga,†
́
Yun Qu,‡,§ and Nicholas P. Farrell*,‡,§
†Institute of Chemistry, University of CampinasUNICAMP, P.O. Box 6154, CEP 13083-970, Campinas, Sao Paulo, Brazil
̃
‡Goodwin Laboratory, Massey Cancer Center, Virginia Commonwealth University, 401 College Street, Richmond, Virginia 23298,
United States
§Department of Chemistry, Virginia Commonwealth University, 1001 W. Main Street, Richmond, Virginia 23284-2006, United States
S
* Supporting Information
ABSTRACT: The syntheses and the characterization by chemical
1
analysis, H and 31P NMR spectroscopy, and mass spectrometry of
a series of linear triphenylphosphine gold(I) complexes with
substituted N-heterocycle ligands (L), [(PPh3)Au(I)(L)]+, is
reported. The reaction of [(PPh3)Au(L)]+ (L = Cl− or substituted
N- heterocyclic pyridine) with the C-terminal (Cys3His) finger of HIVNCp7 shows evidence by mass spectrometry (ESI-MS)
and 31P NMR spectroscopy of a long-lived {(PPh3)Au}-S-peptide species resulting from displacement of the chloride or pyridine
ligand by zinc-bound cysteine with concomitant displacement of Zn2+. In contrast, reactions with the Cys2His2 finger-3 of the
Sp1 transcription factor shows significantly reduced intensities of {(PPh3)Au} adducts. The results suggest the possibility of
systematic (electronic, steric) variations of “carrier” group PR3 and “leaving” group L as well as the nature of the zinc finger in
modulation of biological activity. The cytotoxicity, cell cycle signaling effects, and cellular accumulation of the series are also
reported. All compounds display cytotoxicity in the micromolar range upon 96 h continuous exposure to human tumor cells. The
results may have relevance for the reported inhibition of viral load in simian virus by the gold(I) drug auranofin.
INTRODUCTION
■
Gold-based complexes with biological activity represent a wide
variety of chemotypes. The orally active antiarthritic drug
auranofin inhibits tumor growth in vitro, motivating inves-
tigation of the chemical and biological properties of Au(I)-
phosphine compounds.1−3 In blood, the thiosugar of auranofin
is rapidly displaced by serum albumin residues, followed by
phosphine displacement and formation of R3PO.2 The rate
of displacement of the phosphine ligand is determined by the
basicity of the phosphine, but systematic studies of the
influence of the “leaving group” (chloro, organothiols, etc.)
on the activity are relatively sparse for N-donor ligands. In this
Figure 1. Structures of the [(Ph3P)AuL]+ series and the zinc finger
peptides used in this study.
article, we show the synthesis, characterization, cytotoxicity, and
studies on zinc-finger interactions of a new series of
[(PPh3)Au(I)L]+ compounds (where L = Cl− or substituted
N-heterocyclic pyridine) capable of systematic modification on
both ligands, Figure 1.
all ligands attached to the gold center are lost.10−13 An intrinsic
issue in considering zinc fingers as targets is that of specificity.
We therefore examined the interactions of the [(PPh3)Au(L)]+
series with prototypical zinc finger peptides differing in the zinc
coordination sphere: the Cys2His2 finger-3 of the Sp1
transcription factor and the Cys3His C-terminal finger of
HIVNCp7 nucleocapsid protein. The latter produces a long-
lived [{(PPh3)Au}-peptide(S-donor)] species upon reaction,
the first observation of a Au(ligand)-zinc finger species. The
The inherently “soft” nature of Au(I) has long suggested
thiol interactions as possible modes of action of these drugs.1,2
Notably, thioredoxin has been reviewed as a target1,4 as have
cysteine proteases,5 and most recently even glutathione
peroxidase and trypanothione reductase.6,7 Early studies on
antiarthritic gold complexes suggested a plausible mode of
action as inhibition of zinc fingers through metal ion
displacement.8,9 Biophysical studies of a variety of Au(I) and
Au(III) compounds on zinc fingers do indeed show rapid
displacement of Zn(II) with formation of “gold fingers” where
Received: June 17, 2013
Published: September 24, 2013
© 2013 American Chemical Society
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a
1
Table 1. H, 13C, and 31P NMR; ESI-MS Spectrometry m/z Ions; and λmax for the Studied Compounds
b
c
b
1H δ(ppm) multiplicity, integration
31P δ(ppm)
13C δ(ppm)
m/z
λmax (nm)
Compound I
7.42−7.58 (0.16) m, 15H (Ph)
30.8 (38.7)
127.91 (−0.78)
128.81 (0.01)
128.97 (0.02)
131.64 (−5.76)
133.70 (−0.14)
133.88 (−0.22)
721 [(Ph3P)Au(PPh3)]+
204 (199.5)
476 [(Ph3PO)Au]+
+
952 [Au(Ph3PO)]2
Compound II
8.69 (0.24), d, 2H
2.47 (0.14), s, 3H
27.1 (−3.7)
28.2 (−2.6)
27.5 (−3.3)
21.83 (0.61)
127.51 (2.68)
150.73 (0.97)
153.73 (6.51)
721 [(Ph3P)Au(PPh3)]+
552 [(Ph3P)Au(C6H7N)]+
459 [(Ph3P)Au]+
201 (204)
200 (199)
476 [Au(Ph3PO)+H]+
500 [Au(PPh3)(CH3CN)]+
Compound III
6.84 (0.52), s, 1H
6.51 (0.05), br, 1H
6.42 (2.19), d, 1H
2.40 (0.07), s, 3H
21.70 (0,48)
113.68 (4.54)
115.24 (0.55)
153.05 (5.06)
158.59 (0.00)
567 [(Ph3P)Au(C6H8N2)]+
721 [(Ph3P)Au(PPh3)]+
Compound IV
8.33 (−0.10), d 2H
6.49 (0.30), d, 2H
3.11 (0.64), s, 6H
39.22 (0.01)
107.44 (0.73)
148.78 (1.28)
155.28 (0.90)
581 [(Ph3P)Au(C7H10N2)]+
721 [(Ph3P)Au(PPh)3]+
200 (203, 256)
a
b
c
Numbers in parentheses indicate the difference between the gold(I) complex and the free ligand (L or PPh3). Referenced to TMS. Referenced to
CH3PO4.
instrument operated in positive ion mode. Solutions were injected in
3:1 acetonitrile (ACN)/acetic acid mixture at a flow rate of 3 μL
min−1,. using a source voltage of 2.34 kV and a cone voltage of 30 V.
The source temperature was maintained at 85 °C throughout.
Collision gas was introduced into the hexapole to aid in ion cooling.
Elemental analysis was performed by Quantitative Technologies Inc.,
Whitehouse, NJ. UV−vis spectra were recorded with a JascoV550
UV−vis spectrophotometer (Easton, MD) equipped with a Jasco ETC
505T temperature controller and a VWR thermostat using a low
headspace microcuvette with 10 mm path and 150 μL volume.
Fluorescence experiments were conducted with a Cary Eclipse
fluorescence spectrophotometer (Agilent Technologies, Santa Clara,
CA) using a cuvette with 10 mm path and 3 mL volume. The
temperature adjustment was made by a Peltier module and magnetic
stirring. Inductive coupled plasma−mass spectrometry measurements
were performed with a Varian ICP-MS. Potentiometric titrations were
performed using the Metrohm Titrino Plus 848 automatic titrator,
with a dosing buret of 10 000 steps and a glass electrode. The
measurements were performed in the dynamic mode. Samples were
prepared dissolving compounds I, II, and IV, 1 mmol L−1, in CH3CN/
H2O 5:1, with KCl 0.05 mol L−1. KOH 3 mmol L−1 in CH3CN/H2O
5:1, with KCl 0.05 mol L−1, was used as titrant. Potentiometric
titration curves were analyzed with CurTiPot 3.6.1, to obtain the pKa
values.14
Synthesis of Chlorotriphenylphosphinegold(I) (I). [(Ph3P)-
AuCl] I was synthesized as previously described.15 In brief,
tetrachloroauric acid trihydrate H[AuCl4]·3H2O (1 mmol, 0.3938 g)
was dissolved in a 1:1 ethanol/acetone (3 mL) mixture.
Triphenylphosphine, PPh3 (2 mmol, 0.5245 g) in chloroform (6
mL), was added to the solution of chloroauric acid with stirring. The
yellow color of the solution disappeared and a white precipitate
immediately formed. The solid was recovered by filtration, washed
with ethanol/acetone and ethyl ether and dried in the desiccator.
Yield: 78% (0.3859 g). Anal. Calcd (%) for C18H15ClPAu: C, 43.7; H,
3.06. Found (%): C, 43.9; H, 3.17.
reaction is zinc-finger specific and is not observed under similar
conditions for the Cys2His2-based Sp1 transcription factor.
EXPERIMENTAL METHODS
■
Materials. All reagents used in this study were reagent grade and
used without further purification. Aurochloric acid trihydrate,
H[AuCl4]·3H2O, was purchased from StremChem (Newburyport,
MA). Triphenylphosphine (PPh3) was purchased from Sigma Aldrich
in 99% purity grade. 4-Picoline (L2, pic), 2-amine-4-picoline (L3),
dimethylaminopyridine (L4, DMAP), and N-acetyl-L-cysteine (N-
AcCys) were purchased from Sigma Aldrich. Apo-peptides were
purchased by GenScript Corporation and have the following
sequences: NCp7-F2 (KGCWKCGKEGHQMKDCTER), monoiso-
t o p i c m a s s 2 2 2 4 . 4 7 D a ; S p 1 - F 3 ( K K F A C P E C P -
KRFMSDHLSKHIKTHQNKK), monoisotopic mass 3366.95 Da.
The zinc finger peptides were prepared according to our published
procedures (refs 12 and 33) and characterized with known samples by
CD (Figure 5A,B), 1H NMR and mass spectrometry. The other
reagents used in this study are common.
Physical Measurements. For complex characterization, H, 13C,
1
and 31P NMR spectra were recorded on a Varian Mercury series 300
MHz NMR spectrometer using a 5 mm probe. 1H and 13C were
referenced to trimethylsilane (TMS), and 31P was referenced to
trimethylphosphate (CH3)3PO4. Samples were dissolved in deuterated
chloroform (CDCl3) with 99% deuterium content and 0.1% (v/v)
TMS. Trimethylphosphate was added in 0.1% (v/v) amount for 31P
NMR acquisition. For gold compound−peptide studies, the stock
solutions of the peptide, zinc acetate, and gold compounds were
prepared in the same conditions described above using deuterated
solvents D2O and CD3CN. pH adjustments were made using NaOD.
The molar ratio was 1:1.3 (peptide/Zn2+) and 1:1.3 (ZF/Au+). 31P
NMR spectra were recorded on a Bruker Avance series 600 MHz
NMR spectrometer using a 5 mm probe. 31P was referenced to
trimethylphosphate (CH3)3PO4.
Electrospray ionization time-of-flight mass spectrometry (ESI-TOF-
MS) evaluations were conducted on a Waters/Micromass QTOF-2
Synthesis of [(Ph3P)AuL]NO3 (II−IV). A 0.3 mmol portion of I
was dissolved in chloroform (3 mL) under stirring. A 0.3 mmol
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portion of AgNO3 dissolved in 2 mL of a 1:1 mixture of ethanol/
acetone was added to the solution. AgCl precipitated immediately and
was removed by filtration. Portions (0.3 mmol) of L2−4 were
dissolved in 1 mL of chloroform and added to the activated gold-
triphenylphosphine solution. The mixture was kept under stirring for
24 h followed by removal of the solvent under vacuum. The yellowish
film was dissolved in acetone, and drops of ether were added
precipitating the white solid. Solution was removed, and the solid was
dried under vacuum. Principal characterizing data shown; full details in
Table 1.
[(Ph3P)Au(C6H7N)]NO3 (II). Characterization data follow. White
powder. Yield: 43% (80 mg). NMR δ (1H) 7.42−7.58 (m, 17H); 8.69
(d, 2H); 2.47 (s, 3H). δ (31P) 27.1 ppm; m/z 552 [(Ph3P)Au-
(C6H7N)]+. Anal. Calcd (%) for C24H22N2O3PAu: C, 46.9; H, 3.32; N,
4.31. Found (%): C, 46.9; H, 3.61; N, 4.56.
Interaction with N-Acetyl-tryptophan. Solutions of gold(I)
complexes I−IV were prepared in acetonitrile to a final concentration
of 7.5 mmol L−1. A 10 mmol L−1 stock solution of N-acetyltryptophan
was prepared in tris buffer 20 mmol L−1 pH 7.4. In a fluorescence
cuvette, 3 mL of 5 μmol L−1 solution of N-acetyltryptophan was
titrated with volumes of 20 μL of the stock solution of the gold(I)
compound, and the fluorescence emission was measured in a range
300−450 nm, with an excitation wavelength of 280 nm and a detector
voltage of 750 V. The temperature was kept at 25 °C during the
experiment. The Ka values were determined from Eadie Hofstee plots
and were determined from an average of 3 trials using the following
equation: ΔF = (Ka)−1ΔF/[quencher] + ΔFc.
Interaction with Zinc Fingers. Zinc fingers (ZFs) were prepared
for circular dichroism (CD) and ESI-MS analysis according to the
following procedure: 0.5 mg of the apo-peptide was dissolved in 0.5
mL of degassed water, and the pH was then adjusted to 7.4 with
NH4OH. The concentration based on the isotopic mass was 0.3 mmol
L−1. A 10 mmol L−1 stock solution of zinc acetate in degassed water
was prepared, and 19.3 μL of this solution was added to the peptide
for a final molar ratio of 1:1.3 (peptide/Zn2+). The solution was then
incubated at 37 °C for 2 h and could be kept in freezer as a stock for
72 h. Stock solutions of the gold(I) compounds were prepared in
acetonitrile for a final concentration of 10 mmol L−1. For general ESI-
MS or CD evaluations the molar ratio 1:1.3 (ZF/Au) was used.
Titrations for CD analysis were made adding different volumes of this
stock solution of compounds to a stock solution of the ZF and the
final molar ratios reported. For NMR purposes the stock solutions of
the peptide, zinc acetate, and gold compounds were prepared in the
same conditions described above using deuterated solvents D2O and
CD3CN. pH adjustments were made using NaOD. The molar ratio
was 1:1.3 (peptide/Zn2+) and 1:1.3 (ZF/Au+).
Cytotoxicity Assays. HCT116, A2780, and MCF7s cell lines were
used in this study and maintained according to published
procedures.19,20 The IC50 values were calculated similar to that
described previously.21 In brief, cells were cultured in RPMI (Gibco,
Carlsbad, CA), 5% FBS, 5% BCS, 0.25% penicillin/streptomycin. Cells
were routinely passaged at 80−90% confluence. For the MTT assay,
cells were plated at a density of 5000 cells per well in a 96-well plate
and incubated overnight at 37 °C in a 5% CO2 atmosphere. Cells were
treated with a varying drug concentration for 96 h. After drug removal,
cells were incubated with MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-
diphenyl-2H-tetrazolium bromide) (0.5 mg/mL in RPMI) for 3 h. The
excess MTT was then removed. The dye was dissolved in 100 μL of
DMSO (Sigma; St Louis, MO), and absorbance was read at 540 nm.
Cell Uptake. HCT116 cells were plated in 10 cm dishes at 5 × 105
cells per dish and allowed to attach overnight. The cells were then
treated with IC90 concentrations of each compound. After 3 and 6 h
the medium was collected in 15 mL falcon tubes. The cells were
removed from the dish with trypsin and then centrifuged for 3 min at
1000g at 4 °C. The medium was removed, the cell pellet was washed
three times with cold PBS, and the cells were counted. The samples
were digested by addition of 1 mL of fuming hydrochloric acid. The
acid content was diluted by the addiction of 1 mL of deionized water.
Analysis of the gold content was performed on a Varian ICP-MS using
H[AuCl4] for the analytical curve. Standards and blank were prepared
similarly. Plates were prepared in duplicate. The results shown are a
combination of three different experiments.
[(Ph3P)Au(C6H8N2)]NO3. (III). Characterization data follow. White
powder. Yield: 47% (89 mg). NMR δ (1H) 7.42−7.58 (m, 15H); 6.84
(s, 1H); 6.51 (br, 1H); 6.42 (d, 1H). δ 31P 28.2 ppm; m/z 567
[(Ph3P)Au(C6H8N2)]+. Anal. Calcd (%) for C24H23N3O3PAu: C, 45.8;
H, 3.68; N, 6.68. Found (%): C, 45.6; H, 3.55; N, 6.56.
[(Ph3P)Au(C7H10N2)] NO3 (IV). Characterization data follow. White
powder. Yield: 41% (79 mg). NMR δ (1H) 8.33 (d, 2H); 7.42−7.58
(m, 15H); 6.49 (d, 2H); 3.11 (s, 6H). δ (31P) 27.5 ppm; m/z 581
[(Ph3P)Au(C7H10N2)]+. Anal. Calcd (%) for C25H25N3O3PAu: C,
46.7; H, 3.92; N, 6.53. Found (%): C, 46.3; H, 3.80; N, 6.21.
DNA Melting Point Determination in the Presence and
Absence of Gold Compounds I−IV. Calf thymus DNA (CT-DNA)
solution (expressed as nucleotides) was prepared in 10 mmol L−1
NaClO4 and dialyzed for 48 h. The 1 mmol L−1 stock solutions of the
gold compounds were prepared in DMSO. A 100 μmol L−1 DNA
solution was incubated with the gold(I) compounds (I−IV) at various
ratios (ri = 0−0.10; ri being the ratio of compound per nucleotide) at
37 °C for 24 h. The maximum concentration of DMSO was 1% (v/v),
and this amount was added to the ri = 0 and incubated for 24 h. To
avoid the formation of air bubbles during the experiment, the samples
were degassed by stirring under vacuum for 5 min. The resulting
melting curves were normalized, and the melting temperature was
determined by sigmoidal curve fitting using Origin 8.0 software.
Experiments were performed in triplicate.
Ethidium Bromide Interaction with DNA in the Presence
and Absence of Gold Compounds I−IV. CT-DNA was prepared in
buffer solutions containing 10 mmol L−1 PO43‑ and 50 mmol L−1 NaCl
(pH 7.4). The concentrations of nucleic acids (expressed as
nucleotides) and ethidium bromide were determined spectrophoto-
metrically using the following extinction coefficients: ε260 = 6000 M−1
cm−1 for CT-DNA16 and ε338 = 5680 M−1 cm−1 for ethidium
bromide.17,18 Stock solutions of the CT-DNA incubated with the
gold(I) compounds (I−IV) at an ri of 0.1 (ri being the ratio of
compound per nucleotide) were prepared and incubated at 37 °C for
24 h. Varying amounts of ethidium bromide in the range 0−0.25
ethidium bromide per nucleotide were added to CT-DNA and CT-
DNA treated with I−IV. The final concentration of CT-DNA in
samples was 5 μmol L−1. Fluorescence emission spectra were recorded
on a Cary Eclipse (Varian) fluorimeter at 25 °C. Fluorescence was
excited at λex = 525 nm and registered at λem = 600 nm. Spectra were
recorded thrice and averaged. Because the absorbance of all samples at
excitation wavelength was less than 0.05, the inner filter was
disregarded.
Cell Cycle Arrest. HCT 116 cells were plated in Petri dishes and
allowed to settle in the incubator overnight. The next day, the gold
compounds were added from a 10 mmol L−1 stock solution in DMSO
to the final IC90 concentration. To the untreated control, the
maximum volume of DMSO added was 1% v/v the final amount.
The cells were incubated for 24−48 h. Cells were detached using
trypsin (Gibco), washed twice with PBS, and suspended in propidium
iodide staining solution (50 μg mL−1 propidium iodide, 0.1% Triton-X
in PBS) containing RNase A (200 U mL−1). Cell cycle analysis was
done after 15 min incubation on a FACSort flow cytometer (Becton
Dickinson).
Interaction with Model Amino Acids (N-Acetyl-L-cysteine).
Gold(I) complexes II−IV were dissolved in 750 μL of CD3CN to a
final concentration of 20 mmol L−1. An equivalent amount of N-acetyl-
L-cysteine was dissolved separately in 750 μL of CD3CN. The
interaction of the compounds with each amino acid was evaluated by
1H and 31P NMR spectroscopy by mixing one solution of gold(I)
compound and the amino acid at the time of the spectral acquisition.
The formation of solid precipitate was observed prior to the transfer of
the solution to the NMR tube. Centrifugation was used to separate
solid particles when necessary. The NMR tubes were kept at 37 °C
bath for spectral acquisition after 24, 48, and 72 h.
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Figure 2. ESI-MS full scan (positive mode) of reaction between IV [(PPh3)Au(DMAP)]+ and ZF NCp7 immediately upon incubation. The species
at m/z 721.29 is assigned to [Au(PPh3)2]+. Other species are as follows: (b) [apoNCp7]3+m/z 741.8, (c) [AuF]3+ m/z 807.15, (d) [Au2F]3+ m/z
873.65, (e) [apoNCp7-{Au(PPh3)}]3+ m/z 894.52, (f) [apoNCp7]2+, (g) [AuF]2+ m/z 1210.18, (h) [apoNCp7-{Au(PPh3)}]2+m/z 1341.29.
apoNCp7 (or F) refers to the ZF peptide with loss of Zn2+. (apoNCp7 represents the apopeptide of C-terminal HIV NCp7 zinc finger; [AuF],
[Au2F] represent Au complexed to apoNCp7.)
producing the [Au(PPh3)2]+ ion, Table 1. The intensity of the
RESULTS AND DISCUSSION
■
[Au(PPh3)2]+ ion varies with L: the least intense occurs in the
The N-heterocycles are good σ-donor and π-acceptor groups
spectrum of strongest base, 4-Me2N-pyr (IV), which also has
and can modulate the properties of the trans phosphine. The
the sharpest 31P NMR spectrum. In the case of II, L = 4-
compounds were characterized by elemental analysis; H, 13C,
picoline, mass spectra show peaks at m/z of 459 for
and 31P NMR spectroscopy (Supporting Information Figure
[Au(PPh)3]+ and at m/z = 500 corresponding to [Au(PPh3)-
S1); mass spectrometry; and electronic spectroscopy, Table 1.
(CH3CN)]+ (from solvent) indicating substitution lability of
The 31P NMR signals of all compounds were shifted 3−4
this ligand, Supporting Information Figure S2. It is of interest
ppm upfield compared to the [(PPh3)AuCl] starting material
that this compound also shows the broadest 31PNMR spectrum
(30.8 ppm), consistent with a shielding of this atom,
suggesting susceptibility to rearrangement, depending on donor
Supporting Information Figure S1. The peaks are broad
strength of ligand.
1
which could be due to some line broadening or possible
Potentiometric studies of II and IV in H2O/CH3CN (1:5)
give pKa values for pyridine protonation of 4.31 and 8.42,
exchange between discrete [(PPh3)AuL] complexes and a
statistical mixture of [Au(PPh3)2]+ and [AuL2]+ . The former
respectively, Supporting Information Figure S3. These are
1
ion but not the latter is seen in the mass spectra. In the H
lower than literature values in H2O of 5.99 and 9.70,
NMR spectrum, for II, the ortho-(2,6) protons of the pic ligand
respectively. Interestingly, the kinetics of L (N-heterocycle)
are shifted downfield by 0.24 ppm, and the CH3 group is
substitution by Cl− in AuCl3L + Cl− → [AuCl4]− + L showed a
likewise shifted downfield by 0.14 ppm compared to free ligand.
The para-protons are obscured by the PPh3 resonance. In
contrast, the ortho-protons of IV are only moderately affected
(0.1 ppm upfield), but the NMe2 ligands undergo a significant
deshielding of 0.64 ppm consistent with donation of electron
density to the Au−P bond. In this case the meta-(3,5) protons
are also shifted downfield by 0.30 ppm.
The exocyclic amine and the pyridine nitrogen represent two
possible binding sites for ligand L3. The 31P NMR shift is
consistent with binding through the pyridine nitrogen. The
singlet of proton 3 is shifted downfield by 0.52 ppm while the
pair of doublets at 6.46 and 7.94 ppm in the 1H NMR spectrum
of the L3 corresponding to the protons 5 and 6 are shifted
upon coordination to 7.50 ppm (obscured within Ph3P
multiplet) and 6.51 ppm, respectively. The C(2)−NH2 protons
undergo a downfield shift of 2.09 ppm, suggesting the amine
nitrogen is uncoordinated. This was further confirmed by
disappearance of the signal upon addition of D2O. In the 13C
NMR C(2)−NH2 does not shift whereas the C6 signal is
shifted downfield by 5.06 ppm. Overall, the pyridine N
represents the most likely binding site.
linear relationship between the basicity of the leaving group and
the reactivity (expressed as the logarithm of the second order
rate constant) with respect to chloride substitution, where the
entering group is acting essentially as a σ-bonding nucleo-
phile.22 These trends may also be reflected in the differential
susceptibility of II−IV to rearrangement to the [Au(PPh3)2]+
ion and the electron donation to the Au(I) center as evidenced
by NMR parameters. A second inflection point in the
potentiometric titration at approximately 3.4−3.7 is also
present in I and is assigned to the protonation of the
phosphine ligand. These values are broadly consistent with
those reported in the literature for tertiary phosphines and
diphosphines, although the solvent conditions vary greatly.23,24
Reactivity with Biomolecules. Melting points of calf
thymus DNA (CT-DNA) after incubation with the complexes
in the molar ratios (ri) 0, 0.01, 0.03, 0.05, 0.075, and 0.1 for 24
h showed little variation, Supporting Information Table S1.
Competition assays of the DNA−ethidium bromide intercalator
interaction also showed little difference in fluorescence
quenching confirming that the DNA is unlikely to be the
principal target for these compounds, Supporting Information
Figure S4.25,26
In the ESI-MS of the compounds, the molecular ion of I is
not observed, and the main peak corresponds to that of the
disproportionation product [Au(PPh3)2]+. In contrast, for
[(PPh3)AuL]+ all species show the molecular ion, and also
the loss of the N-heterocycle ligand (L) and rearrangements
As stated, protein thiols and selenols are indicated as
biological targets of Au(I). The reaction of II and IV with N-
acetylcysteine (N-AcCys, 1:1 stoichiometry) showed immediate
changes in the 31P NMR spectrum with significant shifts from
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30 to 38 ppm followed by an immediate white precipitate,
consistent with displacement of L followed by coordination of a
sulfur donor ligand, giving a P−Au−S coordination unit,
Supporting Information Figure S5.27 Early studies on
antiarthritic gold complexes suggested a plausible mode of
action as inhibition of zinc fingers through metal ion
displacement.8,9 A number of Au(I) and Au(III) compounds
have been studied for their interactions with zinc fingers.
Continuing our systematic studies on the chemistry of
coordination compounds with zinc fingers,28 we studied the
reactions of the series with two representative zinc finger
peptides: the C-terminal Cys3His finger of HIV Nucleocapsid 7
(ZFNCp7) and finger 3 of the Cys2His2-based Sp1 tran-
scription factor.
The ESI-MS (positive mode) of the reaction between
[(Ph3P)Au(DMAP)]+, IV, and ZF NCp7 immediately upon
incubation shows a number of new peaks. The most prominent
are those at 894.52 (3+) and 1341.29 m/z (2+) corresponding
to the [apoNCp7-{Au(PPh3)}]n+ ion. Other notable species
besides unreacted zinc finger itself correspond to [AuapoNCp7,
AuF]3+ and [Au2apoNCp7, Au2F]3+ species caused by zinc
ejection and free peptide itself (apoNCp7, 15% intensity),
Figure 2. The [apoNCp7-{Au(PPh3)}] species persists even up
to 78 h of reaction time (Supporting Information Figure S6).
The MS/MS dissociation of the 894.52 (3+) species gave new
peaks corresponding to free phosphine [PPh3 + H+]+ and the
previously observed “gold finger” [AuF]3+ species (data not
shown). The results are consistent with, first, displacement of
the DMAP ligand and formation of an Au(PPh3) adduct with
Zn displacement, followed by loss of PPh3 to give [AuF].
Similar spectra are observed for the chloride derivative and also
for the 4-picoline (II) and 2-amino-4-picoline analogues (III)
(Supporting Information Figure S7). The MS−MS of the
[Au2F]3+ peak at m/z 873.65 is remarkably resistant to
fragmentation, suggesting by definition a very stable structure
(Supporting Information Figure S8).
auranofin [Au(PEt3)(S-thioglucose)] and the reaction products
of [AuCl(PEt3)] with cysteine and protein thiolate residues in
serum albumin.27,29,30 The presence of two peaks implies at
least two species, which is reasonable given the presence of
three thiolate ligands (Cys) and one histidine residue (His) in
the peptide sequence. Further, the broadness of the peaks first
suggests different microenvironments and perhaps some
conformational flexibility.
Zinc finger cysteinates vary in chemical reactivity, and
nucleophilicity is very dependent on the nature of the Zn-
binding core. The Zn-Cys49 thiolate of NCp7 is one of the
residues most susceptible to electrophilic attack.31 To examine
this point further we compared the reactivity of ZFNCp7 with
that of zinc finger 3 of the Sp1 transcription factor (Sp1F3).
Previous studies with aurothiomalate and Sp1F3 showed rapid
formation of [AuapoSp1F3, (or AuF3)] with indications of an
ordered structure.10(apoSp1F3 is the apopeptide of Finger 3 of
transcription factor Sp1; [AuF3], [Au2F3] represent Au
complexed to apoSp1.)
Incubation of the Sp1F3 with IV again showed a number of
species but with significantly reduced intensity for any putative
{Au(Ligand)}-apoSp1 species. The ejection of zinc is quick,
and the dominant species in all cases (I−IV) shows a set of
peaks at 891.74 (4+) and 1188.32 (3+) corresponding to
[AuapoSp1F3]n+. The presence of [Au2Sp1F3, Au2F3]n+ (3+
and 4+) is also seen, Figure 4 and Supporting Information
Figure S9. No apoSp1F3-{Au(PPh3)} was observed for any of
these compounds. However, for compound IV only, peaks at
1006 and 1341 m/z (charge 4+ and 3+, respectively)
correspond to a mass of [apoSp1F3-{(PPh3)Au2}] implying
the presence of one coordinated Au(PPh3) unit and one Au
devoid of starting ligands (Figure 4). The reaction with the Sp1
finger is thus more ligand-specific since no evidence for any
equivalent Au(PPh3) species is seen for compounds I−II
containing as ligands L1 = Cl− or L2 = 4-picoline (Supporting
Information Figure S9). The prototypical [Au(III)Cl(dien)]2+
reacts readily with the C-terminal zinc finger of the HIV
nucleocapsid Cys3His zinc finger (NCp7) producing Au2F and
Au4F species, as monitored by ESI-MS.12 Thus, the nature of
these reactions depends both on the compound and leaving
group (Cl− or substituted pyridine) as well as the coordination
sphere of the zinc finger itself (Cys3His or Cys2His2).
Conformational changes upon binding were further analyzed
by circular dichroism spectroscopy (CD) to confirm these
differences. The interactions of I and IV with ZFNCp7 and
Sp1F3 are shown in Figure 5. No significant differences were
found for compounds II and III (data not shown). The
apoNCp7 and apoSp1F3 present typical random coil spectra
with a negative ellipticity centered at 195−200 nm.12,32 Zn2+
coordination to the peptide in a tetrahedral environment
decreases the intensity with a slight red shift at 200−210 nm
and a positive signal at 190 nm indicating a more α-helical
conformation, Figure 5A,B.
The 31P NMR spectrum of [(PPh3)Au(DMAP)]+, IV,
incubated with ZF NCp7 gave two new peaks at 32.92 and
34.35 ppm representing shifts of 6.32 and 7.56 ppm,
respectively, from the complex, Figure 3. The species is stable
over time [water pH 7.4 containing 10% (v/v) acetonitrile]
with no signals detected assignable to PPh3O or free PPh3. The
chemical shifts are entirely consistent with literature values of
For ZFNCp7 reaction with I and IV, unusual and shapeless
CD spectra were produced. In the case of the reaction of
[AuCl(dien)]2+ with ZFNCp7, the negative ellipticity is
decreased in the range 190−200 nm and the minimum is
shifted in comparison to apoNCp7.12 CD spectra do represent
the sum of conformations present, but it is expected that the
conformation of the peptide will change upon coordination of
gold(I) in a linear geometry rather than the tetrahedral
geometry of Zn2+. The presence of different species in solution,
apoNCp7-{Au(PPh3)}, AuF, and AuF2, with different con-
Figure 3. 31P NMR spectra of compound IV and ZFNCp7: (A)
compound IV, (B) IV + ZFNCp7 immediately after mixing, and (C)
IV + ZFNCp7 at time 7 days.
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Figure 4. ESI-MS full scan (positive mode) of the reaction between [(PPh3)Au(DMAP)]+ and Sp1F3 immediately upon incubation. Species (a)
[DMAP + H+]+, (b) [Au(PPh3)2]+, (c)[Au-apoSp1F3, AuF3]4+, (d) [Au2-apoSp1F3, Au2F3]4+, (e) [apoSp1F3-{(PPh3)Au2}]4+, (f) [AuF3]3+, (g)
[Au2F3]3+, (h) [apoSp1F3-{PPh3)Au2}]3+. apoSp1F3 or F3 refers to the zinc finger peptide with loss of Zn2+.
Figure 5. CD spectra of apo-peptides, zinc fingers, and zinc finger + Au(I) drugs (I and IV): (A) NCp7, (B) Sp1F3, (C) Sp1F3 + IV titration.
formations, obviates against observation of well-defined signals
especially as the Au(PPh3) adduct is the dominant species.
However it is clear that the peptide conformation changed and
no free apoNCp7 is detected as the main species in solution,
Figure 5A.
reactivity of the zinc fingers with Au compounds through
molecular recognition and targeting with π-stacking ligands.32,33
“Noncovalent” association with tryptophan and ZFNCp7 has
been confirmed for [Pt(dien)(nucleobase)]2+ (nucleobase = 9-
EtGua, 1-MeCyt).33,34 Pyridine ligands may also of course
engage in π stacking interactions. Assessment of stacking with
ZFNCp7 was not feasible due to the rapid ejection of Zn2+, but
use of N-acetyl-tryptophan indicated possible π stacking with
IV, enhanced over free DMAP ligand, L4, alone (Table 2). The
Try-RNA(DNA) interaction is a critical feature of NCp7
function.35−37
In contrast, the Sp1F3 showed a concentration-dependent
alteration where the structure is clearly ordered but distinct
from the starting Sp1F3 or free peptide (apoSp1F3), Figure 5B.
The chloride compound, I, presented a slight decrease in the
negative and positive ellipticity, being consistent with
previously reported results.11,12 Compound IV showed a red
shift, an increase in the positive signal in the range 190−195
nm and a significant increase in the positive signal in the range
210−220 nm. Interestingly, the changes for IV are concen-
tration-dependent, and the presence of a putative isodichroic
point at ∼200 nm suggests that the same products or a mixture
of products is produced. The results for the Sp1 finger are
generally consistent with those previously reported for Au(I)
compounds and zinc finger model peptides. Binding of
[AuCl(PEt3)] to consensus sequences for Cys2His2, Cys3His,
and Cys4 models showed a stoichiometry of 1Au:2Cys binding
for all peptides, and the gold fingers produced were more
ordered than free peptide but again conformationally distinct
from the canonical Zn finger structure.11 As stated, CD
spectroscopy showed differences in conformation upon treat-
ment of zinc fingers with aurothiomalate, with zinc ejection and
formation of Au peptides, resulting in inhibition of the DNA
binding of the two model Cys2His2 transcription factors TFIIIA
and Sp1.10
Table 2. Association Constants for Au Compounds with N-
a
AcTryptophan by Fluorescence Quenching
quencher
Ka (×103 M‑1)
SD (×103)
I
b
II
2.55
b
0.01
L2
IV
L4
25.0
3.19
0.20
0.01
a
The Ka was determined from Eadie Hofstee plots and determined
from an average of 3 trials using the equation ΔF = (Ka)−1ΔF/
[quencher] + ΔFc. No quenching observed
b
Cellular Effects. All complexes show strong antiproliferative
effects in vitro against human colon carcinoma (HCT116),
human ovarian carcinoma (A2780), and human breast
carcinoma (MCF7), Table 3 and Supporting Information
Figure S10. All compounds displayed greater cytotoxicity than
cisplatin control in the human colon and ovarian tumor lines
The presence of the aromatic residue tryptophan (Trp) in
the C-terminal NCp7 is a potential source of differentiation for
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and other P−Au−N compounds have been reported in
isolation.41,42
Table 3. IC50 Values for Gold(I) Compounds in HCT116,
A2780, and MCF7 Tumorigenic Cell Lines
As stated, the inherently “soft” nature of Au(I) has long
suggested thiol interactions as possible modes of action of these
drugs. The studies reviewed here confirm the suggestions that
zinc fingers are also suitable receptors. In this study we show
for the first time that the interactions may be modulated by
both the nature of the ligand and the zinc finger coordination
motif. This study represents the first observation of gold-
(ligand) interactions on these biologically important zinc finger
proteins: all other examples have had Au stripped of all ligands.
(PR3)Au-protein species have been confirmed in other proteins
such as HAS2,27,29,30 and Ubiquitin,41 but these generally are
not as thiol-rich as the zinc fingers. Some organometallic
Au(III) compounds also show Au(ligand)-protein adducts by
mass spectrometry.43 The apoNCp7-Au(PPh3) species is
remarkably long-lived as assessed by both MS and NMR
studies, and it is further noteworthy that the Au2apoNCp7
species (Au2F, see Figure 2) produced is also remarkably stable:
it does not fragment under MS/MS conditions, Supporting
Information Figure S11. The chemistry is best described in
Figure 7. Given evidence of multiple (PPh3)Au−S(cysteine)
a
IC50/μmol L‑1
compd
A2780
HCT116
1.478
MCF7
I
0.303
0.324
0.319
0.238
2.31
0.068
0.061
0.085
0.038
0.06
0.430
0.248
0.075
0.170
1.70
2.467
2.245
2.265
2.271
4.8
0.051
0.140
0.065
0.078
0.80
II
1.269
1.226
0.914
7.6
III
IV
b
cisplatin
a
Values are an average of three different experiments. Free ligands
show no cytotoxicity at <25 μmol L−1 under identical conditions.
b
Values for cisplatin performed under identical conditions.
and equivalent cytotoxicity in the breast cancer cell line. No
significant differences are observed among I−IV themselves
with only slight differences in values in any individual cell line.
Extensive surveys of cytotoxicity and biological activity of
Au(I)-phosphine complexes have emphasized variation of the
phosphine ligand.15,38
Intracellular gold accumulation studies showed the highest
accumulation for the neutral I compared to the cationic
complexes, Figure 6. This similarity may reflect rapid
Figure 7. Stepwise coordination of gold(I) drugs and zinc ejection.
species no specific binding sites should be assigned, but the
AuF species would be expected to be linear S−Au−S while the
Au2F species can be easily envisaged as a Au2Cys3 system
arising from monodentate and bridging cysteinates.
The chemistry described here suggests [(PR3)AuL]+ may be
complementary to agents such as iodoacetate and N-ethyl-
maleimide as probes of zinc finger structure and function.44
The susceptibility to oxidative attack on the cysteine ligands
also suggests approaches to the design of electrophilic agents
for inactivation of zinc finger medicinal targets.28 The full 2-
finger HIV NCp7 is an attractive drug target for inhibition of
HIV infectivity.45−47 Electrophilic agents target NCp7 causing
Zn2+ ejection, resulting in loss of native protein structure and
nucleic acid binding capacity, effectively causing disruption of
virus replication. A consideration for any anti-HIV drug
potential is selectivity among zinc fingers. Notable examples
are DIBA-1, dithiane, and ADA which show some selectivity of
attack on NCp7 without significantly affecting other cellular
proteins such as poly(ADP-ribose) polymerase (PARP) and the
Sp1 and GATA-1 transcription factors.48 The results presented
here confirm the value of coordination chemistry for systematic
modulation of protein chemical behavior. The series presented
here allows systematic (electronic, steric, lipophilicity)
variations of “carrier” group PR3 and “leaving” group L in
modulation of biological parameters of cellular accumulation
and cytotoxicity, similar to the mechanistic concept for cisplatin
anticancer agents. It is of considerable interest that auranofin
itself has been shown to inhibit viral load in simian virus.49 The
results presented here may well be relevant to these
observations. Systematic study of the potential of gold
compounds against this HIV target is warranted.50
Figure 6. Bar graphs showing the total gold accumulation for HCT116
cells treated with compounds I, II, III, and IV.
displacement of either Cl− or L by intracellular thiol. However,
the fact that accumulation levels for II−IV are similar at 3 and 6
h implies a more rapid uptake than I and a possible carrier-
mediated uptake if saturation is achieved at these early time
points. Cellular accumulation of Au(I)-diphosphine and N-
heterocycle carbene complexes may be modulated through
lipohilicity of the ligand, leading to development of Au(I)
compounds as mitochondrial poisons.1,39 Cell cycle effects on
HCT116 cells by flow cytometry staining showed a significant
increase in the percentage of the cells in the G1 phase
compared to the control set, suggesting induction of cell
growth inhibition by elongation of the G1 phase in the cell
cycle, Supporting Information Figure S11.
CONCLUSIONS
■
The set of [(PR3)Au(I)(L)]+ (L = N-heterocyclic) compounds
represents a new series of Au(I) compounds with interesting
biological activity. Their interaction with N-acetyl-cysteine in
acetonitrile showed displacement of the planar amine with
precipitation of insoluble compounds. Gold phosphine-imido
complexes have been reported for their antiarthritic activity,40
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Article
(21) Hegmans, A.; Qu, Y.; Kelland, L. R.; Roberts, J. D.; Farrell, N.
Inorg. Chem. 2001, 40, 6108−6114.
ASSOCIATED CONTENT
■
S
* Supporting Information
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1H and 31P NMR spectra, ESI-MS, pH titrations, and species
distribution of compounds I−IV, ethidium bromide fluores-
cence assay and table of DNA melting temperatures, 31P NMR
spectra of II and IV and their interaction with N-AcCysteine,
ESI-MS spectra of reactions of I−IV with zinc finger proteins,
cytotoxicity curves, and cell cycle effects of I−IV. This material
is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
(28) Quintal, S. M.; dePaula, Q. A.; Farrell, N. P. Metallomics 2011, 3,
121−139.
■
Corresponding Author
*E-mail: npfarrell@vcu.edu.
Author Contributions
All authors have given approval to the final version of the
(29) Roberts, J.; Xiao, J.; Schleisman, B.; Parsons, D.; Shaw, C. Inorg.
Chem. 1996, 35, 424−433.
(30) Malik, N. A.; Sadler, P. J. Biochem. Soc. Trans. 1979, 7, 731−732.
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manuscript.
(32) Quintal, S.; Viegas, A.; Erhardt, S.; Cabrita, E. J.; Farrell, N. P.
Biochemistry 2012, 51, 1752−1761.
Notes
The authors declare no competing financial interest.
(33) Anzellotti, A. I.; Liu, Q.; Bloemink, M. J.; Scarsdale, J. N.;
Farrell, N. Chem. Biol. 2006, 13, 539−548.
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483−485.
ACKNOWLEDGMENTS
■
This work was financially supported by NSF CHE 1058726.
The authors acknowledge the award of a scholarship to C.A. by
Coordination for the Improvement of High-Level Personnel
(CAPES) Process 2276-11-9. We thank K. Nelson and K.
Knitter for mass spectra.
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