Rational design of gold(III)-dithiocarbamato peptidomimetics for the targeted anticancer chemotherapy

Article abstract of DOI:10.1016/j.jinorgbio.2012.07.001

As a further extension of our research work focusing on the development of gold(III)-dithiocarbamato dtc derivatives of oligopeptides as potential anticancer agents, complexes [Au^IIIX₂(dtc-Sar-l-Ser(t-Bu)- O(t-Bu))] (X = Br (1a)/Cl (

Full text of DOI:10.1016/j.jinorgbio.2012.07.001

Journal of Inorganic Biochemistry 117 (2012) 248–260
Contents lists available at SciVerse ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Rational design of gold(III)-dithiocarbamato peptidomimetics for the targeted
anticancer chemotherapy
a
a
b
c
d
Morelle Negom Kouodom , Giulia Boscutti , Marta Celegato , Marco Crisma , Sergio Sitran ,
b
a
a,
a,
Donatella Aldinucci , Fernando Formaggio , Luca Ronconi ⁎, Dolores Fregona ⁎⁎
Department of Chemical Sciences, University of Padova, Via F. Marzolo 1, Padova 35131, Italy
Division of Experimental Oncology 2, Centro di Riferimento Oncologico (CRO-IRCSS), Via F. Gallini 2, Aviano (PN) 33081, Italy
Institute of Biomolecular Chemistry, CNR, Padova Unit, Via F. Marzolo 1, Padova 35131, Italy
a
b
c
d
Institute of Inorganic and Surfaces Chemistry, CNR Research Area, Corso Stati Uniti 4, Padova 35127, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 1 April 2012
Received in revised form 30 May 2012
Accepted 3 July 2012
Available online 11 July 2012
As a further extension of our research work focusing on the development of gold(III)-dithiocarbamato dtc deriv-
III
atives of oligopeptides as potential anticancer agents, complexes [Au X
2
(dtc-Sar-L-Ser(t-Bu)-O(t-Bu))] (X=Br
III
(
1a)/Cl (1b)), [Au X
AA=D,L-Pro, X=Br (3a)/Cl (3b)), [Au X
dtc-Sar-Aib -Gly-OEt)] (X=Br (5a)/Cl (5b)) (Aib = “alpha”-aminoisobutyric acid, 2-methylalanine) were
2 2
(dtc-AA-Aib -O(t-Bu))] (AA=Sar (sarcosine, N-methylglycine), X=Br (2a)/Cl (2b);
III
III
2
3 2
(dtc-Sar-Aib -O(t-Bu))] (X=Br (4a)/Cl (4b)), and [Au X (-
3
designed to obtain metal-based peptidomimetics that may specifically target two peptide transporters (namely,
PEPT1 and PEPT2) upregulated in several human tumor cells. All the compounds were characterized by means of
Keywords:
Gold
Dithiocarbamates
Peptidomimetics
Anticancer activity
Drug delivery
FT-IR and mono- and multidimensional NMR spectroscopy. According to in vitro cytotoxicity studies, complex
III
[Au Cl
2 2
(dtc-D,L-Pro-Aib -O(t-Bu))] (3b) turned out to be the most effective toward the four human tumor cell
lines evaluated (PC3, 2008, C13, and L540), for which the IC50 values were lower than cisplatin. Remarkably, it
showed no cross-resistance with cisplatin itself and was proved to inhibit tumor cell proliferation by inducing
almost exclusively late apoptosis/necrosis. Biological results are here reported and discussed in terms of the
structure–activity relationship.
Peptide transporters (PEPTs)
©
2012 Elsevier Inc. All rights reserved.
1
. Introduction
So far, a number of strategies have been suggested as drug delivery
systems, including liposomes, nanoparticles and polymers, but very
few have reached the preclinical or clinical trial stage [5–8]. Among
them, drug functionalization with specific peptides may offer higher
flexibility over other approaches [9], and, accordingly, much attention
has been recently given to peptide-based delivery systems targeting
peptide transporters (PEPTs). The proton-coupled peptide transporters
PEPT1 and PEPT2 are integral membrane carrier proteins that mediate
the uptake of most possible physiologically occurring di- and tripeptides.
Notwithstanding the therapeutic efficacy, conventional anticancer
agents suffer from several drawbacks including high toxicity, chemical
instability, unsuitable pharmacokinetic and pharmacodynamic profiles,
no oral bioavailability, and poor specificity [1]. Consequently, much
effort has been recently focusing on shifting from cytotoxic non-specific
chemotherapies to molecularly-targeted rationally designed drugs [2],
because of the potential for minimizing unwanted side-effects as well
as for improving tumor selectivity while retaining the desirable thera-
peutic effectiveness [3].
Tumor cells express many biomarkers and receptors that can be
specifically targeted. Thus, current research has been devoted to the
development of efficient and innovative delivery systems in which
conjugated drugs incorporate a tumor targeting group (carrier) and
a cytotoxic agent (cargo). These systems should ideally reach the tis-
sue of interest and deliver their cargo directly into the cells where it
can exert its therapeutic activity [4].
+
The carriers are symporters that cotransport H and substrates across
cell membranes, the inwardly directed proton gradient providing the
driving force for the accumulation of a substrate against its concentration
gradient [10]. Besides their physiological substrates, PEPTs are also
responsible for membrane transport of many pharmacologically active
peptidomimetic drugs and prodrugs owing to their structural resem-
blance to di- and tripeptides [11,12]. They are expressed in some mam-
malian tissues/organs, including small intestine, kidney, pancreas, bile
duct, liver, mammary glands, lung and choroid plexus, but, remarkably,
seem to be overexpressed in some types of tumors [13]. In fact, cancer
cells require larger amounts of peptide-bound amino acids for growth
and metabolism and, consequently, peptide transporters might be
upregulated [14]. Intriguingly, it was recently shown that PEPTs are
largely overexpressed in several types of tumor cells but not in the
⁎
Corresponding author. Tel.: +39 049 8275214; fax: +39 02 700538450.
⁎
⁎ Corresponding author. Tel.: +39 049 8275159; fax: +39 02 700500560.
E-mail addresses: luca.ronconi@unipd.it (L. Ronconi), dolores.fregona@unipd.it
(D. Fregona).
0162-0134/$ – see front matter © 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.jinorgbio.2012.07.001

M.N. Kouodom et al. / Journal of Inorganic Biochemistry 117 (2012) 248–260
249
healthy counterparts [15,16], thus representing a suitable target for the
site specific delivery of pharmacologically active peptidomimetics.
Among the metal-based anticancer agents, gold complexes have
been gaining increasing attention due to their capability to strongly in-
hibit tumor cell growth by exploiting mechanisms of action different
from clinically established platinum drugs [17–20]. In this context, in
recent years gold(III)-dithiocarbamato derivatives have been carving
Thin layer chromatography was performed on silica gel Merck
Kieselgel 60F254 precoated glass plates. Retention factors (R ) were
f
measured using either chloroform/ethanol 9:1 (Rf1) or 1-butanol/
acetic acid/water 3:1:1 (Rf2) or toluene/ethanol 7:1 (Rf3) as eluents.
Spots were visualized by direct UV irradiation at 254 nm or devel-
oped by exposure to either iodine vapors or NaClO/ninhydrin chro-
matic reaction as appropriate. Flash column chromatography was
performed on MercK Kiesegel 60 silica gel (40–63 μm, 230–400
mesh) as a stationary phase.
out their space in such a research area [21,22]. “First-generation” com-
III
pounds of the type [Au X
2
(dtc)] (X=Cl, Br; dtc=various dithiocarba-
mates) were shown to exert excellent in vivo anticancer activity on
xenografts [23,24] coupled with negligible acute toxicity and an almost
total lack of nephrotoxic side-effects [25].
Optical rotations were measured at 546 nm in spectrophotometric
grade methanol at 20 °C using a Perkin-Elmer 241 polarimeter
equipped with a Haake D thermostat and a cell with an optical path-
way of 10 cm.
CHNS elemental analyses were carried out on either a Fisons
EA1108 or a Thermo Scientific FLASH 2000 CHNS-O microanalyzer.
Positive ion electrospray mass spectra (ESI-MS) were obtained by
means of an ESI-TOF Mariner 5220 mass spectrometer (PerSeptive
Biosystems). The samples were prepared in a 1:1 mixture of water/
methanol containing 0.1% formic acid, to a final concentration of
0.1–1 mM. Samples were injected directly and the ions produced in
an atmospheric pressure ionization (API) ESI ion source. The source
Despite the positive outcomes achieved, alternative routes have been
explored to improve the therapeutic effectiveness of this class of com-
plexes as well as to provide their active delivery into the target tumor
site. Consequently, we have been focusing on “second-generation”
gold(III)-dithiocarbamato derivatives of oligopeptides as improved in-
tracellular drug transfer and delivery systems supported by peptide
transport proteins. The rationale of our research was to design gold(III)
complexes which could preserve both the antitumor properties and the
reduced toxic side-effects of the “first-generation” analogues, together
with an enhanced bioavailability and tumor selectivity owing to the
peptide-mediated cellular internalization provided by PEPTs. In this
regard, we have recently reported on preliminary results related to
2
temperature was 413 K, and the drying gas (N ) flow rate was
−
1
300 L h . A potential of 4.3 kV (capillary voltage) was applied to
the probe tip, and the ions accelerated at 10, 15, 20 or 30 eV. Mass spec-
tra were recorded over the scan range 100–4000 Da at a scan rate of 5 s
and a resolution of 2 m/z. Data acquisition and processing were carried
out using Data Explorer TM version 4.0 (Applied Biosystems).
some novel gold(III)-dipeptidedithiocarbamato derivatives having gen-
III
eral formula [Au X
2
(dtc-Sar-AA-O(t-Bu))] (X=Cl, Br; Sar = sarcosine
(N-methylglycine); AA=Gly, Aib (“alpha”-aminoisobutyric acid, 2-
methylalanine), L-Phe), and in vitro cytotoxicity and subsequent biolog-
ical studies confirmed the goodness of such a designing strategy [26].
Remarkably, to the best of our knowledge, that was the first time that
metal-based peptidomimetic anticancer agents were synthesized on
purpose to target peptide transporters.
In the attempt to improve and tune tumor selectivity, we have been
extending our evaluation to gold(III)-dithiocarbamato derivatives of
additional selected small peptides. Accordingly, we report here on the
synthesis and characterization of a handful of novel oligopeptides
FT-IR spectra were recorded in nujol on a Nicolet Nexus 870 spec-
−
1
trophotometer (1000 scans, resolution 2 cm ) for the range
−
1
50–600 cm , and in solid KBr on either a Nicolet 55XC or a
−
1
Perkin-Elmer 580B spectrophotometer (32 scans, resolution 2 cm
)
−
1
for the range 400–4000 cm . Data processing was carried out using
OMNIC version 5.1 (Nicolet Instrument Corporation).
All NMR spectra were acquired in the appropriate deuterated sol-
vent at 298 K on a Bruker Avance DRX300 spectrometer using a BBI
[ H,X] probe-head equipped with z-field gradients. Data processing
1
(
from di- to pentapeptides, namely H-Sar-L-Ser(t-Bu)-O(t-Bu), H-Sar-
Aib -O(t-Bu), H-D,L-Pro-Aib -O(t-Bu), H-Sar-Aib -O(t-Bu) and H-Sar-
Aib -Gly-OEt) and the corresponding gold(III) compounds of the type
was carried out using MestReNova version 6.2 (Mestrelab Research S.L.).
1
1
2
2
3
Typical acquisition parameters for 1D H NMR spectra ( H:
300.13 MHz): 16 transients, spectral width 7.5 kHz, using 32 k data
points and a delay time of 5.0 s. Spectra were processed using expo-
nential weighting with a resolution of 0.5 Hz and a line-broadening
threshold of 0.1 Hz.
3
III
2
[Au X (pdtc)] (X=Cl, Br; pdtc=oligopeptidedithiocarbamate). In vitro
cytotoxicity and apoptosis induction studies were carried out toward
human prostate cancer (PC3), ovarian adenocarcinoma (2008/C13) and
Hodgkin's lymphoma cell lines (L540), including cisplatin-resistant can-
cer cells (C13) so as to check the possible occurrence of cross-resistance
with platinum drugs. Biological results are compared to those obtained
for the previous dipeptide analogues and discussed.
Typical acquisition parameters for 2D [ H, 13C] HMBC NMR spectra
1
1
13
( H: 300.13/ C: 75.48 MHz): 512 transients of 32 scans/block, spec-
tral width 7.5/18.8 kHz, 2 k/2 k data points and a delay time of 2.0 s.
1
13
1
n
13
1
Sequences were optimized for J( C, H)=145 Hz/ J( C, H)=
5
Hz with no 1H decoupling. Spectra were processed by using sine-
2
. Experimental
square weighting with a resolution of 1.0/3.0 Hz and a line-
broadening threshold of 0.3/1.0 Hz.
1
H and 13C chemical shifts were referenced to TMS at 0.00 ppm via
2
.1. Materials
internal referencing to the residual peak of the deuterated solvent
employed [27].
Sarcosine, NMM, palladium catalyst (10% on activated charcoal),
TEA (Fluka), IBCF (Lancaster), DMSO, DMSO-d
carbon disulfide, HOBt, DMAP, PI, trypan blue (Aldrich), cisplatin
Pharmacia and Upjohn), isobutene (Siad), ZOSu, EDC (Iris Biotech),
Z-Aib-OH, Z-L-Ser(t-Bu)-OH, Z-D,L-Pro-OH, HCl·H-Gly-OEt (Bachem),
potassium tetrachloro- and tetrabromoaurate(III) dehydrate (Alfa
Aesar), RPMI medium, penicillin, streptomycin, L-glutamine, (Cambrex
Bio Science), and FBS (Gibco) were of reagent grade or comparable pu-
rity and were used as supplied. All other reagents and solvents were
used as purchased without any further purification.
6
, acetone-d
6
, CDCl
3
,
Thermogravimetric (TG) and differential scanning calorimetry
(DSC) curves were recorded with a TA Instruments thermobalance
equipped with a DSC 2929 calorimeter. Measurements were carried
out in the range 25–1100 °C in alumina crucibles under air (flux
rate 30 cm min ) at a heating rate of 5 °C min , using alumina
as a reference.
(
3
−1
−1
3
Crystals of HCl·H-Sar-Aib -O(t-Bu) were obtained as trihydrates
from a methanol/diethyl ether mixture. A plate (ca. 0.40×0.40×
3
0.07 mm ) was glued on the tip of a glass fiber. Single crystal diffraction
data were collected at 293(2) K with a Philips PW1100 four-circle
diffractometer in the θ–2θ scan mode using graphite-monochromated
Cu Kα radiation (λ=1.54178 Å). Intensities were corrected for Lorentz
and polarization effects, as well as for absorption, the latter being based
on a psi-scan. The structure was solved by direct methods (SIR2002)
2
.2. Instrumentation
Melting points were determined using a Stuart SMP10 apparatus
and were not corrected.

250
M.N. Kouodom et al. / Journal of Inorganic Biochemistry 117 (2012) 248–260
2
[
28], and refined on F with full-matrix least-squares (SHELXL-97) [29]
C, 51.20; H, 8.59; N, 11.94%; found: C, 51.24; H, 8.53; N, 12.08%. FT-IR
(KBr, ˜ν max/cm ): 3421, 3276 (ν, N\H); 2762, 2692 (νa,s, NH ); 1727
2
−
1
+
using all data. Non-hydrogen atoms were refined anisotropically. The hy-
drogen atoms of the peptide were added at calculated positions using a
riding model, whereas those belonging to the three co-crystallized
water molecules were located on a difference Fourier map and their po-
sitions were not refined. Graphical material was prepared with ORTEP-3
for Windows [30]. A summary of data collection and structure refine-
ment for compound P4 is reported in the Supplementary Information
(ν, C_Oester); 1689, 1647 (amide I); 1545, 1518 (amide II); 1258
1
(amide III); 1226 (ν, C\O(t-Bu)); 1150 (ν, O–(t-Bu)). H NMR
2
(DMSO-d
(s, 9H, (CH
CH N Sar); 3.68 (s, 2 H, CH
NH Aib ); 8.95 (s br, 2H, NH
TMS, δ/ppm): 25.0 ((CH C Aib ); 25.2 ((CH
t-Bu ester); 32.9 (CH N Sar); 49.8 (CH N Sar); 55.9 ((CH
C Aib ); 79.6 ((CH C t-Bu ester); 164.9 (C_O Sar);
6
, 300.13 MHz, TMS, δ/ppm): 1.30 (s, 6H, (CH
3
)
2
C Aib ); 1.34
1
3 3 3 2
) C t-Bu ester); 1.39 (s, 6H, (CH ) C Aib ); 2.51 (s, 3H,
2
3
2
N Sar); 7.64 (s, 1 H, NH Aib ); 8.49 (s, 1 H,
1
+
13
2
Sar). C NMR (DMSO-d
6
, 75.48 MHz,
2
1
(
Table S1). X-ray crystallographic data have been deposited at the
)
3 2
3 2
) C Aib ); 27.8 ((CH ) C
3 3
2
Cambridge Crystallographic Data Center and assigned the deposition
number CCDC 871740, and the corresponding CIF file can be obtained
free of charge from http://www.ccdc.cam.ac.uk.
3
2
3 2
) C Aib );
1
56.5 ((CH
)
3 2
3 3
)
1
2
172.8 (C_O Aib ); 173.5 (C_O Aib ). ESI-MS (H
amu): 315.25 for [M–Cl] (calcd. 315.22).
2
O/MeOH 1:1, m/z/
+
2
2
.3. Synthesis of esterified oligopeptide hydrochlorides
1
2
2
.3.3. HCl·H-D,L-Pro-Aib -Aib -O(t-Bu) (P3)
.3.1. HCl·H-Sar-L-Ser(t-Bu)-O(t-Bu) (P1)
A THF solution (15 mL) of Z-Sar-OH (3.10 mmol, see Supplementary
The compound was synthesized as above reported for P1 using Z-D,
1
2
L-Pro-OH (3.92 mmol) and H-Aib -Aib -O(t-Bu) (4.31 mmol, obtained
by previous catalytic hydrogenation in CH Cl of the corresponding
Z-protected derivative, see Supplementary Information) as starting
reagents. Purification of the Z-D,L-Pro-Aib -O(t-Bu) intermediate was
carried out by flash chromatography using petroleum ether/AcOEt 2:3
as eluent.
Name: D,L-prolyl-2-methylalanyl-2-methylalanine 1,1-dimethylethyl
3
ester hydrochloride. Yield: 90%. Mp: 203–205 °C. Rf1 (CHCl /EtOH 9:1):
.20 Rf2 (BuOH/AcOH/H
32ClN O (FW=377.91 g mol ): C, 54.03; H,
3 4
.53; N, 11.12%; found: C, 54.22; H, 8.67; N, 11.01%. FT-IR (KBr,
2
ν max/cm ): 3400, 3284 (ν, N\H); 2761, 2694 (νa,s, NH ); 1723
ν, C_Oester); 1684, 1650 (amide I); 1540, 1505 (amide II); 1250
Information) is treated with NMM (1 equiv), cooled to −15 °C, and then
treated with IBCF (1 equiv) under stirring. After 10 min, a cold suspen-
sion of H-L-Ser(t-Bu)-O(t-Bu) (4.61 mmol, obtained by previous catalytic
hydrogenation in CH
see Supplementary Information) in CHCl
pH adjusted to ca. 8 with NMM. After being stirred overnight at
room temperature, the reaction mixture is concentrated under re-
duced pressure, the residue taken up with AcOEt, washed with 10%
2
2
2
Cl
2 2
of the corresponding Z-protected derivative,
(10 mL) is added and the
3
0
3
O 3:1:1): 0.75; Rf3 (PhMe/EtOH 7:1): 0.05.
4 2 3 2 2 4
KHSO , H O, 5% NaHCO , H O, and dried over Na SO . The organic
−
1
Anal. calcd. for C17
8
H
layer is then concentrated to dryness and the product purified by
flash chromatography using petroleum ether/AcOEt 3:2 as eluent,
to obtain Z-Sar-L-Ser(t-Bu)-O(t-Bu).
Such intermediate is subsequently Z-deprotected by catalytic hydro-
genation in MeOH, and an ether solution of H-Sar-L-Ser(t-Bu)-O(t-Bu) is
dropwise treated with 1 equiv of a dilute ether solution of HCl under
stirring. Eventually, the solvent is evaporated to dryness, the residue
taken up with fresh ether and filtered, and the solvent removed by
evaporation, yielding the corresponding hydrochloride HCl·H-Sar-L-
Ser(t-Bu)-O(t-Bu) as a white powder.
−
1
+
˜
(
(
(
1
amide III); 1225 (ν, C\O(t-Bu)); 1151 (ν, O–(t-Bu)). H NMR
2
DMSO-d
6
, 300.13 MHz, TMS, δ/ppm): 1.29, 1.30 (s, 6H, (CH
.35 (s, 9H, (CH C t-Bu ester); 1.40, 1.42 (s, 6H, (CH
.81–2.29 (m, 4H, CH
3
2
) C Aib );
1
1
1
)
3 3
3
)
2
C Aib );
3
,4
5
2
Pro); 3.15–3.21 (m, 2H, CH Pro); 4.18–4.21
2
2
2
1
(
(
m, 1H, CH Pro); 7.57 (s, 1H, NH Aib ); 8.48 (s, 1H, NH Aib ); 9.30
+
13
s br, 2H, NH
2
Pro). C NMR (DMSO-d
6
, 75.48 MHz, TMS, δ/ppm):
C Aib ); 25.0 ((CH
Pro); 45.3 (CH
4
2
1
2
(
3.3 (CH
2
Pro); 24.8 ((CH
3
)
2
3
)
2
C Aib ); 27.5
Pro); 55.8 ((CH
C Aib ); 58.2 (CH Pro); 79.6 ((CH C t-Bu ester);
Name: N-methylglycyl-O-(1,1-dimethylethyl)-L-serine 1,1-dimethyl-
3
5
(CH C t-Bu ester); 29.6 (CH
)
3 3
2
2
3 2
) C
ethyl ester hydrochloride. Yield: 81%. Mp: 160–162 °C. Rf1 (CHCl
3
/EtOH
O 3:1:1): 0.75; Rf3 (PhMe/EtOH 7:1):
2 4
.10. [α]546 =+4.2° (c=0.5, MeOH). Anal. calcd. for C14 29ClN O
2
1
2
Aib ); 56.3 ((CH
1
)
3 2
3 3
)
9
0
:1): 0.35 Rf2 (BuOH/AcOH/H
3
1
2
66.2 (C_O Pro); 172.5 (C_O Aib ); 173.1 (C_O Aib ). ESI-MS
(H O/MeOH 1:1, m/z/amu): 342.46 for [M–Cl] (calcd. 342.34).
2
20
H
+
−
1
(FW=324.84 g mol ): C, 51.76; H, 9.00; N, 8.62%; found: C, 51.61; H,
−
1
9.18; N, 8.63%. FT-IR (KBr, ˜ν max/cm ): 3300 (ν, N–H); 2781, 2715
+
1
2
3
(ν
a,s, NH
2
); 1738 (ν, C_Oester); 1669 (amide I); 1559 (amide II); 1250
2.3.4. HCl·H-Sar-Aib -Aib -Aib -O(t-Bu) (P4)
(
amide III); 1238 (ν, C\O(t-Bu)); 1167 (ν, O–(t-Bu)); 1110, 907 (νa,s
,
The compound was synthesized as above reported for P1
using Z-Sar-OH (4.52 mmol, see Supplementary Information) and
H-Aib -Aib -Aib -O(t-Bu) (3.77 mmol, obtained by previous catalytic
hydrogenation in MeOH of the corresponding Z-protected derivative,
see Supplementary Information) as starting reagents. Purification of
1
CH
s, 9H, (CH
Sar); 3.56 (m, 2H, CH
Ser); 8.73 (d, 1H, NH Ser); 9.05 (s br, 2H, NH
DMSO-d , 75.48 MHz, TMS, δ/ppm): 26.8 ((CH C Ser); 27.4 ((CH
t-Bu ester); 32.3 (CH N Sar); 48.5 (CH N Sar); 53.0 (CH Ser); 61.4 (CH
Ser); 72.6 ((CH C Ser); 80.7 ((CH C t-Bu ester); 165.1 (C_O Sar);
68.5 (C_O Ser). ESI-MS (H O/MeOH 1:1, m/z/amu): 288.40 for [M–
2
\O\(t-Bu)). H NMR (DMSO-d
C Ser); 1.42 (s, 9H, (CH
Ser); 3.75 (s, 2H, CH
6
, 300.13 MHz, TMS, δ/ppm): 1.13
C t-Bu ester); 2.53 (s, 3H, CH
N Sar); 4.41 (m, 1H, CH
1
2
3
(
3 3
)
3
)
3
3
N
2
2
+
13
2
Sar). C NMR
(
6
3
)
3
3
)
3
C
the Z-Sar-Aib
tography using CH
3
-O(t-Bu) intermediate was carried out by flash chroma-
Cl /MeOH 30:1 as eluent.
3
2
2
2
2
3 3
)
3
)
3
Name: N-methylglycyl-2-methylalanyl-2-methylalanyl-2-methyl-
alanine 1,1-dimethylethyl ester hydrochloride. Yield: 87%. Mp: 146–
1
2
+
Cl] (calcd. 288.20).
147 °C. Rf1 (CHCl
3
/EtOH 9:1): 0.60 Rf2 (BuOH/AcOH/H
37ClN
436.97 g mol ): C, 52.22; H, 8.53; N, 12.82%; found: C, 52.08; H,
3
O 3:1:1): 0.90;
R
f3 (PhMe/EtOH 7:1): 0.40. Anal. calcd. for C19
H
4 5
O
(FW=
1
2
−1
2
.3.2. HCl·H-Sar-Aib -Aib -O(t-Bu) (P2)
The compound was synthesized as above reported for P1 using
−
1
8.60; N, 12.84%. FT-IR (KBr, ˜ν max/cm ): 3406 (ν, N–H); 2764, 2702
1
+
Z-Sar-OH (2.40 mmol, see Supplementary Information) and H-Aib -
Aib -O(t-Bu) (2.64 mmol, obtained by previous catalytic hydrogena-
tion in MeOH of the corresponding Z-protected derivative, see
Supplementary Information) as starting reagents. Purification of the
(νa,s, NH
2
); 1728 (ν, C_Oester); 1681 (amide I); 1520 (amide II);
2
1
1258 (amide III); 1226 (ν, C–O(t-Bu)); 1149 (ν, O–(t-Bu)). H NMR
3
6
(DMSO-d , 300.13 MHz, TMS, δ/ppm): 1.29 (s, 6H, (CH
3
)
2
C Aib ); 1.33
C t-Bu ester); 1.35 (s, 6H,
N Sar); 3.72 (s, 2H, CH N Sar); 7.27
2
(s, 6H, (CH ) C Aib ); 1.34 (s, 9H, (CH )
3 2 3 3
1
Z-Sar-Aib
2
-O(t-Bu) intermediate was carried out by flash chromatogra-
(CH
3
)
2
C Aib ); 2.55 (s, 3H, CH
3
2
3
2
1
phy using petroleum ether/AcOEt 2:3 as eluent.
(s, 1H, NH Aib ); 7.68 (s, 1H, NH Aib ); 8.90 (s, 1H, NH Aib ); 9.12
+
13
Name: N-methylglycyl-2-methylalanyl-2-methylalanine 1,1-dimeth-
(s br, 2H, NH
24.4 ((CH C Aib ); 24.6 ((CH
((CH C t-Bu ester); 32.4 (CH N Sar); 49.0 (CH
2 6
Sar). C NMR (DMSO-d , 75.48 MHz, TMS, δ/ppm):
3
2
1
ylethyl ester hydrochloride. Yield: 87%. Mp: 148–149 °C. Rf1 (CHCl
EtOH 9:1): 0.10 Rf2 (BuOH/AcOH/H O 3:1:1): 0.50; Rf3 (PhMe/EtOH
:1): 0.00. Anal. calcd. for C15 30ClN
3
/
3
)
2
3 2
)
C Aib ); 24.7 ((CH
3 2
) C Aib ); 27.3
3
3
)
3
3
2
N Sar); 55.0 ((CH ) C
3 2
−
1
3
2
1
7
H
3
O
4
(FW=351.87 g mol ):
3 2 3 2 3 3
Aib ); 55.5 ((CH ) C Aib ); 56.2 ((CH ) C Aib ); 78.8 ((CH ) C t-Bu

M.N. Kouodom et al. / Journal of Inorganic Biochemistry 117 (2012) 248–260
251
ester); 164.9 (C_O Sar); 171.9 (C_O Aib ); 172.9 (C_O Aib2,3).
1
NH Ser). 13C NMR (acetone-d
Ser); 28.3 ((CH C t-Bu ester); 39.9, 40.8 (CH
4.8, 55.5 (CH N Sar); 63.0 (CH Ser); 73.9 ((CH
, 75.48 MHz, TMS, δ/ppm): 27.7 ((CH
N Sar); 54.8 (CH Ser);
C Ser); 82.2 ((CH
6
3 3
) C
+
ESI-MS (H
2
O/MeOH 1:1, m/z/amu): 401.65 for [M–Cl] (calcd. 401.28).
3
)
3
3
5
2
2
3
)
3
3 3
) C
1
2
3
2
.3.5. HCl·H-Sar-Aib -Aib -Aib -Gly-OEt (P5)
A CH Cl solution (15 mL) of Z-Sar-OH (5.95 mmol, see Supple-
mentary Information) is cooled to −15 °C, treated with HOBt
5.95 mmol) and EDC (5.95 mmol) under stirring. After 10 min, a
cold suspension of H-Aib -Gly-OEt (4.96 mmol, obtained by previous
t-Bu ester); 164.4 (C_O Sar); 169.5 (C_O Ser); 195.9, 199.7 (CSS Sar).
2
2
III
2.4.2. [Au Cl
2
(dtc-Sar-L-Ser(t-Bu)-O(t-Bu))] (1b)
(
Name: Dichlorido[1-(1,1-dimethylethyl) N-(dithiocarboxy-κS,κS')-
N-methylglycyl-O-(1,1-dimethylethyl)-L-serinato]gold(III). Yield: 57%.
3
catalytic hydrogenation in EtOH of the corresponding Z-protected de-
rivative, see Supplementary Information) and NMM (4.96 mmol) is
added. After being stirred at room temperature for 5 days, the reac-
tion mixture is concentrated under reduced pressure, the residue
2 2 4 2
Mp: decomposes at 162 °C. Anal. calcd. for C15H27AuCl N O S (FW=
631.39 g mol ): C, 28.53; H, 4.31; N, 4.44; S, 10.16%; found: C, 28.42;
−
1
H, 4.36; N, 4.34; S, 10.41%. TG (air): calcd. weight loss to Au(0) −68.8%;
−
1
found −69.2%. FT-IR (KBr, ν˜ max/cm ): 3424 (ν, N\H); 1736 (ν,
taken up with AcOEt, washed with 10% KHSO
O, and dried over Na SO . The organic layer is then concentrated
to dryness and the product purified by flash chromatography using
CH Cl /EtOH 95:5 as eluent, to obtain Z-Sar-Aib -Gly-OEt.
The corresponding Z-deprotected hydrochloride HCl·H-Sar-Aib
Gly-OEt was obtained as above reported for P1.
4 2 3
, H O, 5% NaHCO ,
C_Oester); 1685 (amide I); 1561 (ν, N–CSS); 1531 (amide II); 1247
H
2
2
4
(amide III); 1231 (ν, C\O(t-Bu)); 1160 (ν, O–(t-Bu)); 1099, 895 (νa,s,
1
CH
2
\O–(t-Bu)); 1004 (ν
a
, S\C\S). FT-IR (nujol, ν˜ max/cm− ): 549 (ν
a
,
1
2
2
3
S\C\S); 407, 384 (νa,s, S\Au\S); 338, 320 (νa,s, Cl\Au\Cl). H NMR
3
-
(acetone-d
(s, 9H, (CH
CH Ser); 4.56 (m, 1H, CH Ser); 4.78, 4.79 (s, 2H, CH
NH Ser). C NMR (acetone-d
Ser); 28.3 ((CH C t-Bu ester); 40.2, 40.9 (CH
54.9, 55.4 (CH N Sar); 63.0 (CH Ser); 73.8 ((CH
t-Bu ester); 164.4 (C_O Sar); 169.6 (C_O Ser); 194.5, 199.7 (CSS Sar).
6
, 300.13 MHz, TMS, δ/ppm): 1.15 (s, 9H, (CH
Ct-Bu ester); 3.56, 3.57 (s, 3H, CH N Sar); 3.74 (m, 2H,
N Sar); 7.99 (d, 1H,
, 75.48 MHz, TMS, δ/ppm): 27.7 ((CH
N Sar); 54.8 (CH Ser);
C Ser); 82.2 ((CH
3 3
) C Ser); 1.46
3
)
3
3
Name: N-methylglycyl-2-methylalanyl-2-methylalanyl-2-methyl-
alanylglicine ethyl ester hydrochloride. Yield: 73%. Mp: 243–245 °C. Rf1
2
2
1
3
6
3 3
) C
(
CHCl
EtOH 7:1): 0.00. Anal. calcd. for C19
C, 48.97; H, 7.79; N, 14.92%; found: C, 49.02; H, 7.80; N, 14.92%. FT-IR
3
/EtOH 9:1): 0.00 Rf2 (BuOH/AcOH/H
3
O 3:1:1): 0.50; Rf3 (PhMe/
3
)
3
3
−
1
5
H36ClN O
6
(FW=465.97 g mol ):
2
2
3
)
3
3 3
) C
−
1
+
(KBr, ˜ν max/cm ): 3351, 3311 (ν, N\H); 2751, 2712 (νa,s, NH
2
); 1739
III
1
2
(
ν, C_Oester); 1694, 1657 (amide I); 1543, 1537 (amide II); 1248
2
2.4.3. [Au Br (dtc-Sar-Aib -Aib -O(t-Bu))] (2a)
1
(amide III); 1223 (ν, C–OEt); 1037 (ν, O–Et). H NMR (DMSO-d
6
,
Name: Dibromido[1-(1,1-dimethylethyl) N-(dithiocarboxy-κS,κS')-
N-methylglycyl-2-methylalanyl-2-methylalaninato]gold(III). Yield: 67%.
3
00.13 MHz, TMS, δ/ppm): 1.16 (t, 3H, CH Et ester); 1.31 (s, 6H,
3
2
1
3
(
CH
3
)
2
C Aib ); 1.35 (s, 6H, (CH
.57 (s, 3H, CH N Sar); 3.72 (s, 2H, CH
Et ester); 7.26 (s, 1H, NH Aib ); 7.79 (t, 1H, NH
3
)
2
C Aib ); 1.39 (s, 6H, (CH
3
)
2
C Aib );
2 3 4 2
Mp: decomposes at 132 °C. Anal. calcd. for C16H28AuBr N O S (FW=
−
1
2
3
2
N Sar); 3.77 (d, 2H, CH
2
747.32 g mol ): C, 25.71; H, 3.78; N, 5.62; S, 8.58%; found: C, 25.91; H,
3
Gly); 4.04 (q, 2H, CH
Gly); 8.04 (s, 1H, NH Aib ); 8.89 (s, 1H, NH Aib ); 8.96 (s br, 2H, NH
2
3.66; N, 5.64; S, 8.73%. TG (air): calcd. weight loss to Au(0) −73.6%;
2
1
+
−1
2
found −72.9%. FT-IR (KBr, ˜ν max/cm ): 3403 (ν, N\H); 1729 (ν,
Sar). 1 C NMR (DMSO-d
3
, 75.48 MHz, TMS, δ/ppm): 13.6 (CH
Et
C_Oester); 1688 (amide I); 1558 (ν, N–CSS); 1509 (amide II); 1247
6
3
1
2
3
ester); 24.3 ((CH
3 2 3 2 3 2
) C Aib ); 24.4 ((CH ) C Aib ); 24.7 ((CH ) C Aib );
a
(amide III); 1218 (ν, C\O(t-Bu)); 1146 (ν, O–(t-Bu)); 1012 (ν ,
−
1
3
2.4 (CH N Sar); 40.5 (CH
3
2
Gly); 48.9 (CH
2
N Sar); 55.6 ((CH
3
)
2
C
S\C\S). FT-IR (nujol, ˜ν max/cm ): 543 (ν
S\Au\S); 252, 227 (νa,s Br\Au\Br).
300.13 MHz, TMS, δ/ppm): 1.40 (s, 6H, (CH
(CH C t-Bu ester); 1.51, 1.53 (s, 6H, (CH C Aib ); 3.51, 3.55 (s, 3H,
CH N Sar); 4.69, 4.73 (s, 2H, CH N Sar); 7.36 (s, 1H, NH Aib ); 7.83 (s,
H, NH Aib ). C NMR (acetone-d , 75.48 MHz, TMS, δ/ppm): 24.8
C t-Bu ester); 39.4, 40.4 (CH N Sar);
a
, S\C\S); 406, 384 (νa,s
,
3
1
2
1
Aib ); 55.9 ((CH
1
Aib ); 174.7 (C_O Aib ). ESI-MS (H
for [M–Cl] (calcd. 430.27).
3 2
) C Aib ); 56.0 ((CH
3 2
) C Aib ); 59.8 (CH
2
Et ester);
,
H NMR (acetone-d
6
,
2
2
65.2 (C_O Sar); 169.4 (C_O Gly); 173.0 (C_O Aib ); 173.7 (C_O
3 2
) C Aib ); 1.42 (s, 9H,
1
3
1
2
O/MeOH 1:1, m/z/amu): 430.55
3
)
3
3 2
)
+
2
3
2
1
13
,2
1
6
2
.4. Synthesis of gold(III)-oligopeptidedithiocarbamato complexes
((CH
4.9, 55.8 (CH
80.5 ((CH C t-Bu ester); 163.3, 163.5 (C_O Sar); 173.1, 175.6 (C_O
3
)
2
C Aib1 ); 27.8 ((CH
N Sar); 56.4, 57.8 ((CH
3
)
3
3
1
2
5
2
3 2 3 2
) C Aib ); 56.7 ((CH ) C Aib );
As a general synthetic route, an aqueous solution of HCl·H-(AA)
n
-OR
3 3
)
1
2
(
n=2–5) is dropwise treated under stirring with CS (1 equiv) and, sub-
2
Aib ); 173.8 (C_O Aib ); 195.7, 199.3 (CSS Sar).
sequently, with NaOH (1 equiv). After 2–3 h, pH turns from 10 to ca. 6
III
1
2
and the solution is slowly added under stirring to an aqueous solution
2
2.4.4. [Au Cl (dtc-Sar-Aib -Aib -O(t-Bu))] (2b)
III
of K[Au X
4
] (0.5 equiv; X=Cl, Br), leading to the sudden precipitation
Name: Dichlorido[1-(1,1-dimethylethyl) N-(dithiocarboxy-κS,κS')-
N-methylglycyl-2-methylalanyl-2-methylalaninato]gold(III). Yield: 61%.
Mp: decomposes at 143 °C. Anal. calcd. for C16H28AuCl N O S (FW=
2 3 4 2
658.41 g mol ): C, 29.19; H, 4.29; N, 6.38; S, 9.74%; found: C, 29.17; H,
4.48; N, 6.34; S, 9.92%. TG (air): calcd. weight loss to Au(0) −70.1%;
found −69.5%. FT-IR (KBr, ˜ν max/cm ): 3392 (ν, N\H); 1729 (ν, C_
of a solid. The residue is then filtered off, washed with cold water,
purified by recrystallization from ethanol/water, and dried under re-
−
1
duced pressure with P
2
O
5
, yielding the corresponding gold complex
III
[
Au X
2
(dtc-(AA)
n
-OR)], as either a dark orange or a yellow ochre
−
1
powder (for bromo or chloro derivatives, respectively).
O
ester); 1689 (amide I); 1559 (ν, N–CSS); 1512 (amide II); 1248 (amide
III); 1220 (ν, C\O(t-Bu)); 1146 (ν, O–(t-Bu)); 1015 (ν , S\C\S). FT-IR
, S\C\S); 406, 383 (νa,s, S\Au\S); 341,
322 (νa,s, Cl\Au\Cl). H NMR (acetone-d , 300.13 MHz, TMS, δ/ppm):
C t-Bu ester); 1.52, 1.53 (s,
N Sar); 4.73 (s, 2H, CH N Sar); 7.31
C NMR (acetone-d
III
2
.4.1. [Au Br
2
(dtc-Sar-L-Ser(t-Bu)-O(t-Bu))] (1a)
a
−
1
Name: Dibromido[1-(1,1-dimethylethyl) N-(dithiocarboxy-κS,κS')-
N-methylglycyl-O-(1,1-dimethylethyl)-L-serinato]gold(III). Yield: 78%.
Mp: decomposes at 128 °C. Anal. calcd. for C15 27AuBr (FW=
(nujol, ˜ν max/cm ): 543 (ν
a
1
6
2
H
2
N
2
O
4
S
2
3 2 3 3
1.41 (s, 6H, (CH ) C Aib ); 1.42 (s, 9H, (CH )
−
1
1
7
20.29 g mol ): C, 25.01; H, 3.78; N, 3.89; S, 8.90%; found: C, 24.97; H,
6H, (CH
3
2
) C Aib ); 3.55 (s, 3H, CH
3
2
2
1
13
1,2
3
.69; N, 3.83; S, 8.81%. TG (air): calcd. weight loss to Au(0) −72.7%;
(s, 1H, NH Aib ); 7.80 (s, 1H, NH Aib ).
6
,
−
1
found −73.1%. FT-IR (KBr, ν˜ max/cm ): 3428 (ν, N\H); 1736 (ν,
C=Oester); 1675 (amide I); 1564 (ν, N–CSS); 1522 (amide II); 1248
75.48 MHz, TMS, δ/ppm): 26.4 ((CH
ester); 40.6, 41.1 (CH N Sar); 56.1 (CH
Aib ); 57.5 ((CH
3
)
2
C Aib ); 28.5 ((CH
N Sar); 57.1, 58.6 ((CH
C t-Bu ester); 164.0 (C_O Sar);
3 3
) C t-Bu
3
2
3 2
) C
1
2
(
amide III); 1233 (ν, C\O(t-Bu)); 1159 (ν, O–(t-Bu)); 1099, 897 (νa,s
,
3
)
2
C Aib ); 81.2 ((CH
3
)
3
−
1
1
2
CH
S\C\S); 407, 383 (νa,s, S\Au\S); 252, 222 (νa,s, Br\Au\Br). H NMR
acetone-d , 300.13 MHz, TMS, δ/ppm): 1.15 (s, 9H, (CH C Ser); 1.46
s, 9H, (CH C t-Bu ester); 3.53, 3.56 (s, 3H, CH N Sar); 3.73 (m, 2H,
CH Ser); 4.56 (m, 1H, CH Ser); 4.74, 4.78 (s, 2H, CH N Sar); 7.97 (d, 1H,
2 a a
\O–(t-Bu)); 1003 (ν , S\C\S). FT-IR (nujol, ν˜ max/cm ): 547 (ν ,
173.8, 176.3 (C_O Aib ); 174.5 (C_O Aib ); 195.1, 200.1 (CSS Sar).
1
III
1
2
(
(
6
3
)
3
2
2.4.5. [Au Br (dtc-D,L-Pro-Aib -Aib -O(t-Bu))] (3a)
3
)
3
3
Name: Dibromido[1-(1,1-dimethylethyl) N-(dithiocarboxy-κS,κS')-
D,L-prolyl-2-methylalanyl-2-methylalaninato]gold(III). Yield: 94%. Mp:
2
2

252
M.N. Kouodom et al. / Journal of Inorganic Biochemistry 117 (2012) 248–260
decomposes at 122 °C. Anal. calcd. for C18
7
H
30AuBr
73.35 g mol ): C, 27.96; H, 3.91; N, 5.43; S, 8.29%; found: C, 28.13;
H, 4.01; N, 5.51; S, 8.11%. TG (air): calcd. weight loss to Au(0) −
4.5%; found −75.1%. FT-IR (KBr, ˜ν max/cm−1): 3410 (ν, N\H); 1728
ν, C_Oester); 1685, 1661 (amide I); 1546 (ν, N−CSS); 1510 (amide
II); 1248 (amide III); 1227 (ν, C\O(t-Bu)); 1147 (ν, O–(t-Bu)); 996
2
N
3
O
4
S
2
(FW=
gold(III). Yield: 84%. Mp: decomposes at 157 °C. Anal. calcd. for
C H35AuCl N O S (FW=743.52 g mol ): C, 32.31; H, 4.74; N,
20 2 4 5 2
−
1
−1
7.54; S, 8.63%; found: C, 32.19; H, 4.87; N, 7.46; S, 8.80%. TG (air): calcd.
−1
7
(
weight loss to Au(0) −73.5%; found −72.9%. FT-IR (KBr, ν˜ max/cm ):
3392 (ν, N\H); 1722 (ν, C_Oester); 1681 (amide I); 1561 (ν, N–CSS);
1520 (amide II); 1252 (amide III); 1218 (ν, C\O(t-Bu)); 1147 (ν,
−
1
−1
(
(
ν
ν
a
, S\C\S). FT-IR (nujol, ˜ν max/cm ): 546 (ν
a
, S\C\S); 406, 380
a a
O–(t-Bu)); 1004 (ν , S\C\S). FT-IR (nujol, ˜ν max/cm ): 551 (ν ,
a,s, S\Au\S); 252, 222 (νa,s, Br\Au\Br). 1H NMR (acetone-d
,
S\C\S); 408, 384 (νa,s, S\Au\S); 340, 327 (νa,s, Cl\Au\Cl). H NMR
1
6
2
3
3
00.13 MHz, TMS, δ/ppm): 1.40, 1.41 (s, 6H, (CH
)
3 2
C Aib ); 1.42
(acetone-d
1.40 (s, 6H, (CH
(s, 6H, (CH
CH
(s, 1H, NH Aib ). C NMR (acetone-d
6
, 300.13 MHz, TMS, δ/ppm): 1.39 (s, 6H, (CH
3
)
2
C Aib );
C t-Bu ester); 1.46, 1.50
N Sar); 4.80, 4.82 (s, 2H,
1
2
(
2
(
s, 9H, (CH C t-Bu ester); 1.51, 1.52 (s, 6H, (CH
3
)
3
3
)
2
C Aib ); 2.23–
Pro); 3.98–4.03
Pro); 5.03–5.06 (m, H, CH Pro); 7.25, 7.29 (s, 1H, NH
3
)
2
C Aib ); 1.42 (s, 9H, (CH
3
)
3
4
3
1
.31 (m, 2H, CH
2
Pro); 2.36-2.66 (m, 2H, CH
2
3 2
)
C Aib ); 3.57, 3.58 (s, 3H, CH
3
5
2
3
2
m, 2H, CH
2
2
N Sar); 7.17, 7.23 (s, 1H, NH Aib ); 7.35, 7.42 (s, 1H, NH Aib ); 8.35
2
1
13
1
13
Aib ); 8.02, 8.09 (s, 1H, NH Aib ). C NMR (acetone-d
TMS, δ/ppm): 23.3 (CH
6
, 75.48 MHz,
6
, 75.48 MHz, TMS, δ/ppm): 25.3,
4
1
1
3
2
2
Pro); 25.2 ((CH
3
)
2
C Aib ); 25.6 ((CH
3
)
2
C
25.7 ((CH
3
)
2
C Aib ); 25.4 ((CH
)
3 2
C Aib ); 26.2 ((CH
3 2
) C Aib ); 28.3
2
3
5
Aib ); 28.1 ((CH
7.1 ((CH C Aib ); 58.2 ((CH
(CH C t-Bu ester); 167.4 (C_O Pro); 173.1 (C_O Aib ); 174.0
C_O Aib ); 190.1 (CSS Pro).
3
)
3
2
C t-Bu ester); 31.1 (CH
2
Pro); 52.3 (CH
2
Pro);
((CH C t-Bu ester); 40.2 (CH
Aib ); 56.9, 58.2 ((CH ) C Aib ); 57.3 ((CH ) C Aib ); 80.0 ((CH )
3 2 3 2 3
3
)
3
3
N Sar); 55.5 (CH
2
N Sar); 56.4 ((CH
3
)
2
C
C
1
2
3
1
2
5
(
(
3
)
2
3
)
2
C Aib ); 65.5 (CH Pro); 80.8
3
1
1
3
)
3
t-Bu ester); 165.1 (C_O Sar); 172.7, 175.7 (C_O Aib ); 173.9 (C_O
2
2
3
Aib ); 174.2 (C_O Aib ); 194.9, 199.8 (CSS Sar).
III
1
2
2
.4.6. [Au Cl
Name: Dichlorido[1-(1,1-dimethylethyl) N-(dithiocarboxy-κS,κS')-
D,L-prolyl-2-methylalanyl-2-methylalaninato]gold(III). Yield: 80%. Mp:
decomposes at 146 °C. Anal. calcd. for C18 30AuCl (FW=
84.45 g mol ): C, 31.59; H, 4.42; N, 6.14; S, 9.37%; found: C, 31.55;
H, 4.56; N, 6.07; S, 9.54%. TG (air): calcd. weight loss to Au(0) −
1.2%; found −69.4%. FT-IR (KBr,˜ν max/cm−1): 3407 (ν, N–H); 1730
ν, C_Oester); 1686, 1663 (amide I); 1543 (ν, N–CSS); 1508 (amide
II); 1249 (amide III); 1228 (ν, C\O(t-Bu)); 1147 (ν, O–(t-Bu)); 997
2
(dtc-D,L-Pro-Aib -Aib -O(t-Bu))] (3b)
III
1
2
3
2
2.4.9. [Au Br (dtc-Sar-Aib -Aib -Aib -Gly-OEt)] (5a)
Name: Dibromido[1-ethyl N-(dithiocarboxy-κS,κS')-N-methylglycyl-
2-methylalanyl-2-methylalanyl-2-methylalanylglycinato]gold(III). Yield:
H
2
N
3
O
4
S
2
−
1
6
2 5 6 2
82%. Mp: decomposes at 179 °C. Anal. calcd. for C20H34AuBr N O S
−
1
(FW=861.42 g mol ): C, 27.89; H, 3.98; N, 8.13; S, 7.44%; found: C,
7
(
28.07; H, 4.02; N, 8.09; S, 7.31%. TG (air): calcd. weight loss to Au(0) −
−
1
77.1%; found −77.0%. FT-IR (KBr, ˜ν max/cm ): 3343 (ν, N\H); 1728
(ν, C=Oester); 1665 (amide I); 1553 (ν, N–CSS); 1528 (amide II); 1274
−
1
(
(
ν
ν
a
, S\C\S). FT-IR (nujol, ˜ν max/cm ): 545 (ν
a
, S\C\S); 406, 380
(amide III); 1216 (ν, C\OEt); 1028 (ν, O–Et); 998 (ν
a
, S\C\S). FT-IR
, S\C\S); 413, 383 (νa,s, S\Au\S); 252,
, 300.13 MHz, TMS, δ/ppm):
a,s, S\Au\S); 341, 321 (νa,s, Cl\Au\Cl). 1H NMR (acetone-d
,
(nujol, ˜ν max/cm ): 560 (ν
−1
6
a
2
1
3
9
(
00.13 MHz, TMS, δ/ppm): 1.40, 1.41 (s, 6H, (CH
H, (CH C t-Bu ester); 1.50, 1.53 (s, 6H, (CH
3
)
2
C Aib ); 1.42 (s,
C Aib ); 2.21–2.37
227 (νa,s, Br\Au\Br). H NMR (acetone-d
6
1
2
3
)
3
3
)
2
1.22 (t, 3H, CH
3
Et ester); 1.38 (s, 6H, (CH
3
)
2
C Aib ); 1.43 (s, 6H,
C Aib ); 3.54, 3.59 (s, 3H, CH N Sar);
Gly); 4.10 (q, 2H, CH Et ester); 4.84, 4.89 (s, 2H, CH
Sar); 7.15, 7.20 (s, 1H, NH Aib ); 7.68 (t, 1H, NH Gly); 7.94, 7.98 (s, 1H,
4
3
5
1
3
m, 2H, CH
2
Pro); 2.38–2.70 (m, 2H, CH
2
Pro); 4.00–4.08 (m, 2H, CH
2
(CH
3
)
2
C Aib ); 1.52 (s, 6H, (CH
3
)
2
3
2
2
Pro); 5.05–5.15 (m, H, CH Pro); 7.25, 7.31 (s, 1H, NH Aib ); 8.08, 8.11
s, 1H, NH Aib ). C NMR (acetone-d , 75.48 MHz, TMS, δ/ppm): 23.6
6
Pro); 25.4 ((CH
t-Bu ester); 31.4 (CH
C Aib ); 66.0 (CH Pro); 80.9 ((CH C t-Bu ester); 167.4
C_O Pro); 173.2 (C_O Aib ); 174.1 (C_O Aib ); 188.6 (CSS Pro).
3.86 (d, 2H, CH
2
2
2
N
1
13
3
(
4
1
2
2
1
13
(CH
2
3
)
2
C Aib ); 26.0 ((CH
Pro); 52.9 (CH
3
)
2
C Aib ); 28.2 ((CH
3
)
3
C
NH Aib ); 8.46, 8.57 (s, 1H, NH Aib ). C NMR (acetone-d
6
, 75.48 MHz,
C Aib ); 26.1 ((CH
N Sar); 42.1, 42.5 (CH Gly);
3
5
2
1
2
2
Pro); 57.1 ((CH
)
3 2
C Aib ); 58.1
TMS, δ/ppm): 15.0 (CH
3
3
Et ester); 25.5 ((CH
C Aib ); 40.4, 41.4 (CH
3
)
2
3 2
) C
1
2
2
(
(CH
3
)
2
)
3 3
Aib ); 26.7 ((CH
3
)
2
3
2
1
2
3
2
(
55.7, 56.7 (CH N Sar); 57.9 ((CH
2
3
)
2
C Aib ); 58.1 ((CH
3 2
) C Aib ); 58.5
1
(
(CH ) C Aib ); 61.5 (CH Et ester); 165.8, 166.2 (C_O Sar); 171.2
3 2 2
III
1
2
3
2
1
3
2
.4.7. [Au Br
2
(dtc-Sar-Aib -Aib -Aib -O(t-Bu))] (4a)
(C_O Gly); 174.4 (C_O Aib ); 175.7 (C_O Aib ); 176.4 (C_O Aib );
197.1, 200.3 (CSS Sar).
Name: Dibromido[1-(1,1-dimethylethyl) N-(dithiocarboxy-κS,κS')-
N-methylglycyl-2-methylalanyl-2-methylalanyl-2-methylalaninato]
gold(III). Yield: 92%. Mp: decomposes at 142 °C. Anal. calcd. for
−
1
III
1
2
3
C
20
H
35AuBr
2
N
4
O
5
S
2
(FW=832.42 g mol ): C, 28.86; H, 4.24; N, 6.73;
2
2.4.10. [Au Cl (dtc-Sar-Aib -Aib -Aib -Gly-OEt)] (5b)
S, 7.70%; found: C, 28.75; H, 4.36; N, 6.60; S, 7.63%. TG (air): calcd. weight
loss to Au(0) −76.3%; found −76.2%. FT-IR (KBr, ν˜ max/cm ): 3400 (ν,
N\H); 1720 (ν, C_Oester); 1682 (amide I); 1560 (ν, N–CSS); 1518
Name: Dichlormido[1-ethyl N-(dithiocarboxy-κS,κS')-N-methylglycyl-
2-methylalanyl-2-methylalanyl-2-methylalanylglycinato]gold(III).
−
1
Yield: 67%. Mp: decomposes at 186 °C. Anal. calcd. for C20
O S (FW=772.52 g mol ): C, 31.10; H, 4.44; N, 9.07; S, 8.30%;
6 2
H
34AuCl
2 5-
N
−
1
(
1
3
(
1
1
(
amide II); 1251 (amide III); 1219 (ν, C\O(t-Bu)); 1147 (ν, O–(t-Bu));
−
1
001 (ν
84 (νa,s
acetone-d
.41 (s, 6H, (CH
.47 (s, 6H, (CH
a
, S\C\S). FT-IR (nujol, ν˜ max/cm ): 550 (ν
S\Au\S); 253, 228 (νa,s Br\Au\Br).
, 300.13 MHz, TMS, δ/ppm): 1.40 (s, 6H, (CH
a
, S\C\S); 410,
found: C, 30.91; H, 4.52; N, 8.94; S, 8.24%. TG (air): calcd. weight loss
1
−1
,
,
H
NMR
to Au(0) −74.5%; found −74.7%. FT-IR (KBr, ˜ν max/cm ): 3340 (ν,
3
6
3
)
2
C Aib );
C t-Bu ester); 1.46,
C Aib ); 3.55, 3.59 (s, 3H, CH N Sar); 4.80, 4.86
N Sar); 7.16, 7.22 (s, 1H, NH Aib ); 7.39, 7.41 (s, 1H, NH
N\H); 1728 (ν, C_Oester); 1667 (amide I); 1557 (ν, N–CSS); 1528
2
3
)
2
C Aib ); 1.42 (s, 9H, (CH
3
)
3
(amide II); 1275 (amide III); 1216 (ν, C–OEt); 1029 (ν, O–Et); 997
1
−1
3
)
2
3
a a
(ν , S\C\S). FT-IR (nujol, ˜ν max/cm ): 562 (ν , S\CS); 413, 382
3
(νa,s, S\Au\S); 338, 322 (νa,s, Cl\Au\Cl). 1H NMR (acetone-d
300.13 MHz, TMS, δ/ppm): 1.22 (t, 3H, CH Et ester); 1.37 (s, 6H,
s, 2H, CH
2
6
,
2
1
13
Aib ); 8.37, 8.48 (s, 1H, NH Aib ).
5.48 MHz, TMS, δ/ppm): 25.4, 25.8 ((CH
C
NMR (acetone-d
6
,
3
1
2
1
3
7
3
)
2
C Aib ); 25.5 ((CH
3
)
2
C
(CH
3.59 (s, 3H, CH
ester); 4.85 (s, 2H, CH
NH Gly); 7.85, 7.89 (s, 1H, NH Aib ); 8.41, 8.47 (s, 1H, NH Aib ).
NMR (acetone-d , 75.48 MHz, TMS, δ/ppm): 14.9 (CH Et ester); 25.5
3
)
2
C Aib ); 1.48 (s, 6H, (CH
N Sar); 3.86 (d, 2H, CH
N Sar); 7.16, 7.22 (s, 1H, NH Aib ); 7.68 (t, 1H,
3
)
2
C Aib ); 1.52 (s, 6H, (CH
3 2
)
C Aib );
3
2
Aib ); 25.9 ((CH
(
(
3
)
2
C Aib ); 28.5 ((CH
3
)
3
C t-Bu ester); 40.0, 41.0
3
2
Gly); 4.11 (q, 2H, CH
2
Et
3
3
CH
(CH
65.3, 165.4 (C_O Sar); 172.9, 175.8 (C_O Aib ); 173.4 (C_O
3
N Sar); 55.3, 56.4 (CH N Sar); 56.6 ((CH
2
3
)
2
C Aib ); 57.2, 58.4
2
1
2
2
1
13
3
)
2
C Aib ); 57.4 ((CH
3
)
2
C Aib ); 80.2 ((CH C t-Bu ester);
3
)
3
C
1
1
6
3
2
3
1
2
3
Aib ); 174.3 (C_O Aib ); 196.6, 199.9 (CSS Sar).
3
((CH )
2
C Aib ); 26.0 ((CH
)
3 2
C Aib ); 26.6 ((CH
N Sar); 57.9 ((CH
((CH ) C Aib ); 58.6 ((CH ) C Aib ); 61.5 (CH Et ester); 165.7 (C_O
3 2 3 2 2
3 2 3
) C Aib ); 40.9 (CH N
3
Sar); 42.3 (CH
2
Gly); 55.9 (CH
2
3
)
2
C Aib ); 58.2
III
1
2
3
2
1
2
.4.8. [Au Cl
Name: Dichlorido[1-(1,1-dimethylethyl) N-(dithiocarboxy-κS,κS')-
N-methylglycyl-2-methylalanyl-2-methylalanyl-2-methylalaninato]
2
(dtc-Sar-Aib -Aib -Aib -O(t-Bu))] (4b)
2
1
Sar); 171.0 (C_O Gly); 174.4 (C_O Aib ); 175.6 (C_O Aib ); 176.4
3
(C_O Aib ); 195.7, 200.4 (CSS Sar).

M.N. Kouodom et al. / Journal of Inorganic Biochemistry 117 (2012) 248–260
253
2
.5. Cell lines and culture conditions
Hodgkin's lymphoma (L540) and human androgen-resistant pros-
3. Results and discussion
3.1. Synthesis and spectroscopic characterization
tate cancer (PC3) cell lines were obtained from the German Collection
of Microorganisms and Cell Cultures (Braunschweig, Germany).
Human ovarian adenocarcinoma (2008) cell line was kindly provided
by Prof. G. Marverti (University of Modena, Italy). The parent cisplatin-
resistant subclone (C13) was obtained by weekly treatment of 2008
Five esterified oligopeptides (from di- to pentapeptides) were
prepared as hydrochlorides (namely, HCl·H-Sar-L-Ser(t-Bu)-O(t-Bu)
(P1), HCl·H-Sar-Aib
HCl·H-Sar-Aib -O(t-Bu) (P4), and HCl·H-Sar-Aib
used as starting reagents to synthesize the corresponding gold(III)-
2 2
-O(t-Bu) (P2), HCl·H-D,L-Pro-Aib -O(t-Bu) (P3),
3
3
-Gly-OEt (P5)) and
cells with 1 μM cisplatin [31]. All cells were cultured at 37 °C in 5% CO
2
III
and moisture-enriched atmosphere in RPMI medium supplemented
with 10% heat-inactivated FBS, 0.1% (w/v) L-glutamine and antibiotics
dithiocarbamato derivatives [Au X
2
(dtc-Sar-L-Ser(t-Bu)-O(t-Bu))]
(dtc-AA-Aib -O(t-Bu))] (AA=Sar,
X=Br (2a)/Cl (2b); AA=D,L-Pro, X=Br (3a)/Cl (3b)), [Au X
III
(X=Br (1a)/Cl (1b)), [Au X
2
2
−
1
III
(
0.2 mg mL penicillin and streptomycin).
2
(dtc-
(dtc-Sar-Aib
III
Sar-Aib
3
-O(t-Bu))] (X=Br (4a)/Cl (4b)), and [Au X
2
3
-
2
.6. Samples preparation
Gly-OEt)] (X=Br (5a)/Cl (5b)) (Fig. 1).
Being short peptides, compounds P1–P5 were obtained in good yields
by exploiting and optimizing classical solution synthetic strategies rather
than solid phase synthesis [35–38]. As a general example, the synthetic
Before use, gold(III) complexes 1a–5b and cisplatin were dissolved
in DMSO, aliquoted, and stored at −80 °C. Just before the experiments,
calculated amounts of drug solutions were added to RPMI medium and
filter sterilized, to a final DMSO amount lower than 0.5% (v/v). All the
2
method related to the preparation of the tripeptide HCl·H-Sar-Aib -
α
O(t-Bu) (P2) is reported in Scheme 1. C -tetrasubstituted α-amino
acids are well-known to be slightly reactive “as is” toward the formation
of peptide bonds because of the steric hindrance at the α-carbon atom
1
tested gold(III) compounds were proved by H NMR to be stable in
DMSO over 48 h.
2
[35]. Therefore, the symmetric anhydride (Z-Aib) O was chosen as effec-
2
.7. Cell proliferation assay
tive alternative acylating agent. The first step was the t-butyl esterifica-
tion of the Z-protected amino acid Z-Aib-OH through the acid-catalyzed
reaction with isobutene, followed by the removal of the Z-protecting
group upon catalytic (10% palladium on activated charcoal) hydrogena-
Amounts of 2.5×103 PC3 cells, and 4.0×103 2008 or C13 cells,
were seeded in 96-well flat-bottomed microplates in RPMI medium
100 μL) and incubated at 37 °C in 5% CO atmosphere for 24 h (to
(
2
tion. The dipeptide intermediate H-Aib
condensation of (Z-Aib) O and H-Aib-O(t-Bu) and subsequent catalytic
hydrogenation. The tripeptide H-Sar-Aib -O(t-Bu) was prepared by con-
2
-O(t-Bu) was then obtained by
allow cell adhesion) before drug testing. The medium was then re-
moved and replaced with fresh one containing the compounds to be
tested (previously dissolved in DMSO, see above) at increasing concen-
trations (0.3, 0.6, 1.25, 2.5, 5, 10, 20, 40, 80 μM) at 37 °C for 72 h. Each
treatment was performed in triplicate. Cell proliferation was assayed
using the BrdU Cell Proliferation ELISA kit (Roche Diagnostics,
Germany), according to the manufacturer's protocol [32]. Alternatively,
2
2
densation of such intermediate with Z-Sar-OH in presence of IBCF, again
followed by catalytic Z-deprotection. The resulting purified product was
finally isolated as hydrochloride by slow addition of 1 equiv of HCl in
diethyl ether. This procedure avoided the acid-catalyzed hydrolysis of
the t-butyl ester and, consequently, increased yields as well as reduced
the occurrence of side-products.
5
−1
2
.0×10 L540 cells mL were seeded in 24-well plates and treated as
previously described. After treatment, the viable cells were evaluated
by the trypan blue dye exclusion test [33]. Negative exponential dose–
response curves were obtained by linear regression, and IC50 values
The other oligopeptides were prepared following similar synthetic
pathways, except the pentapeptide P5 for which the segment condensa-
tion through the activation of the C-terminal carboxylic function as
5(4H)-oxazolone was performed (see Supplementary Information) [39].
Since all attempts to isolate the dithiocarbamato derivatives
of oligopeptides P1–P5 without causing their degradation during
the separation/purification process were proved unsuccessful, the
corresponding gold(III) complexes 1a–5b were obtained by a one-pot re-
action. During the first step, the dithiocarbamic acid of an oligopeptide
forms in solution owing to the base-catalyzed nucleophilic attack in
water of the terminal amino group of the starting hydrochloride to carbon
disulfide, the pH consequently decreasing as the reaction proceeds. Once
(i.e. the half maximal inhibitory concentration representing the concen-
tration of a substance required for 50% inhibition in vitro) were calculated
using CalcuSyn version 2.0 (Biosoft).
2
.8. Evaluation of apoptosis
Amounts of 2.5×105 PC3 or 2008 or C13 cells, and 2.0×
5
−1
1
0 L540 cells mL were incubated in six-well Petri dishes with RPMI
medium supplemented with 10% FBS as is (control) or containing
0 μM (30 μM for both cisplatin-sensitive and -resistant human
1
the pH stabilizes at ca. 6, the solution is subsequently reacted with 0.5
III
ovarian adenocarcinoma cells) of either gold complexes 1a–5b or cis-
equiv of K[Au X
4
] (X=Br, Cl) in water, leading to the sudden precipita-
platin at 37 °C for 72 h. Cells were then harvested and resuspended in
tion of the expected gold(III) complex in good yields.
5
2
0 μL of binding buffer (10 mM HEPES/NaOH pH 7.4, 140 mM NaCl,
.5 mM CaCl ), incubated with 5 μL of FITC-conjugated Annexin-V
The choice of the amino acids constituting the peptide chain is not
accidental. We first decided to maintain the sarcosinedithiocarbamato
moiety to resemble our “first-generation” gold(III) analogues [22].
Moreover, the sarcosine-containing dipeptide Gly-Sar is often used as
reference substrate for peptide transport studies because of its good af-
finity toward both PEPT1 and PEPT2 [40]. Only in the case of P3 (and the
associated gold(III) derivatives 3a and 3b) the sarcosine residue was
substituted by a proline unit so as to evaluate possible differences in
the cytotoxic activity upon replacement with another N-substituted
amino acid unit. The prevalent presence of Aib residues in the tri-,
2
(
Pharmigen, USA) in the dark for 15 min, and assayed after the addition
−
1
of 0.3 mL of binding buffer and 10 μL of propidium iodide (10 μg mL
in binding buffer) to each sample. Apoptosis induction was assessed by
flow cytometry detection of Annexin-V-labeled cells according to their
forward and right-angle scattering, electronically gated and analyzed
on a FACScalibur flow cytometer (Becton-Dickinson), using CellQuest
software (Becton-Dickinson), as described elsewhere [34].
2
.9. Statistical analyses
tetra- and pentapeptide derivatives is a consequence of our previous
III
studies from which complex [Au Br
2
(dtc-Sar-Aib-O(t-Bu))] showed
All values are given as means± SD (standard deviation) of measure-
the greater anticancer potency among other gold(III) species containing
different dtc-Sar-AA-O(t-Bu) ligands [26]. The fact that Aib-rich com-
plexes might show higher cytotoxicity is not that surprising. The
α-aminoisobutyric acid is a natural non-coded amino acid and is
ments from three independent experiments. Statistical comparisons
were drawn using Student's t-test. Differences were considered signifi-
cant where pb0.05.

254
M.N. Kouodom et al. / Journal of Inorganic Biochemistry 117 (2012) 248–260
Fig. 1. Oligopeptides and the corresponding gold(III)-dithiocarbamato complexes studied in this work.
abundant in a class of peptide antibiotics (namely, antibiotic peptaibols)
exhibiting antiviral and anticancer activity [41,42]. It was recently
suggested that Aib, owing to his rigidity, plays a crucial role in the
biological activity of peptaibols because it forces the peptide backbone
to fold into helical arrangements, providing a relevant capability to
cross and/or perturb cell membranes [43]. Therefore, the presence of
Aib residues might increase the bioavailability and, consequently, the
biological activity.
Among all, the only dipeptide here reported (P1), together with
the corresponding gold(III) complexes 1a and 1b, represent an excep-
tion since they contain an L-serine (instead of Aib) residue as terminal
amino acid. L-Ser side-chain was t-butylated to avoid the possible
Scheme 1. Synthetic scheme for the preparation of the tripeptide HCl·H-Sar-Aib
SO (cat.), CH Cl /H O, −60 °C/r.t., 7 d; (d) 10% Pd-C, H , CH Cl ; (e) NMM, THF/H
h) HCl, Et O.
2
-O(t-Bu) (P2): (a) ZOSu, TEA, CH
3
CN/H
2
O, r.t., 4 d; (b) EDC, CH
2
Cl
2
/H
2
O, r.t., 4 h; (c) isobutene,
/H O, −15 °C/r.t., o.n.;
H
(
2
4
2
2
2
2
2
2
2
O, r.t., 2 d; (f) 10% Pd-C, H
2
, MeOH; (g) NMM, IBCF, THF/CHCl
3
2
2

M.N. Kouodom et al. / Journal of Inorganic Biochemistry 117 (2012) 248–260
255
formation of by-products upon reaction of the free hydroxo group and
carbon disulfide during the synthesis of the corresponding dithiocarba-
mate (that is, the possible formation of a xanthate OCSS moiety). On
the other hand, this approach allowed us to evaluate the influence of
two hydrophobic groups (i.e. two t-Bu residues) on the cytotoxic activity.
Finally, esterification of the C-terminus was proved necessary
because it was previously shown that analogues containing a free
carboxylic functional group are not active [44]. Additionally, although
conferring less water solubility, the bulky t-butyl group is unlikely to
undergo easy hydrolysis (occurring only in strongly acidic conditions),
thus providing stability to the final compounds and, consequently,
avoiding intramolecular cyclization of the peptide chain.
In the pentapeptide P5, the terminal Gly residue and the ethyl
ester were chosen on purpose to reduce the hydrophobicity of the
molecule while maintaining the structural properties provided by
longer Aib-containing oligopeptides.
The starting oligopeptides and the corresponding gold(III)-
dithiocarbamato derivatives were characterized by means of several
techniques in order to assess the structure as well as to investigate
their stability in solution.
As concerns the other main frequencies arising from the oligopeptide
chains, the vibrational modes strictly related to the peptide bonds –C(O)
N(H)– seem to be slightly affected by the formation of the dithiocarba-
mate and the subsequent metal coordination. Bands referred to as
amide I and amide II show relatively strong infrared absorptions and
are very sensitive to the formation of hydrogen bonds [49]. According
to literature data [50,51], both the peptide amide and carbonyl groups
of complexes 1a–4b do not seem to form any hydrogen bonds whereas
in the starting oligopeptides P1–P3 (but not P4) some of the NH groups
are involved in hydrogen bonds in the solid state (and some of the pep-
tide CO groups as well, for the tripeptides P2 and P3). On the contrary, all
the NH groups belonging to the free pentapeptide P5 form weak to
strong hydrogen bonds (and also part of the CO units), and this behavior
seems to be maintained in the corresponding gold(III)-dithiocarbamato
complexes 5a and 5b (except for the carbonyls).
1
Further characterization was carried out by means of mono- ( H)
1
13
and multidimensional ([ H, C] HMBC) NMR spectroscopy. With ref-
erence to the spectral data collected in Table S5 (see Supplementary
Information), compared with the starting oligopeptide ester hydro-
chlorides P1–P5, a general downfield shift of the proton signals is ob-
served for the peptide backbone upon formation of the corresponding
gold(III)-dithiocarbamato derivatives, the larger variation (ca. 1 ppm)
involving the groups directly bound to the ditiocarbamato nitrogen
atom. On moving away from the NCSS moiety, the magnitude of the
chemical shift change decreases along the peptide chain until being
negligible for the further ester group. Only for the amide proton(s)
peaks lower chemical shift values (ca. 0.5–1 ppm) were recorded for
the complexes compared to the free oligopeptides. This overall trend,
in agreement with data previously reported [26,46], is also maintained
The proposed stoichiometry of the gold complexes was confirmed
by both elemental and thermogravimetric analyses, the latter indicat-
ing a good correlation between calculated and found weight loss
values to metallic gold (Table S4 in the Supplementary Information).
IR spectroscopy was proved useful to identify the synthesized com-
pounds. With reference to Table 1, by comparison with the spectra
recorded for the starting oligopeptide ester hydrochlorides P1–P5, the
−
1
+
disappearance of the broad bands at 2780–2690 cm
45] together with the detection of two new bands attributed to the
ν(N–CSS) and ν (S\C–S) vibrational modes in the spectra of the
2
(νa,s(NH ))
[
13
a
for C signals (Table S6 in the Supplementary Information). In this re-
gard, the most significant peaks were recorded at around 198 ppm and
attributed to the dithiocarbamic carbon atoms [44,52]. For high oxida-
corresponding derivatives 1a–5b, are consistent with the formation of
the gold complexes depicted in Fig. 1. In fact, the strong signal at
−
1
13
1
560–1540 cm
(the so-called “thioureide” band) is in accordance
tion state transition metal derivatives such as gold(III), δ(N CSS) are
with a chelating dithiocarbamato ligand coordinated to a gold(III) cen-
found at 195–201 ppm. Therefore, the presence of such peaks, together
−
1
+
ter [46]. Additionally, the detection of a single band at ca. 1000 cm in
all the complexes (commonly attributed to the ν (S\C\S)) is a clear
with the disappearance of the NH
2
proton signals, is consistent with
a
the formation of the gold(III)-oligopeptidedithiocarbamato complexes
depicted in Fig. 1.
indication for the symmetrical bidentate coordination of the NCSS
moiety to the metal ion, as based on the Bonati–Ugo criterion [47].
New bands, absent in the IR spectra of the free oligopeptides, were
The solution chemistry of complexes 1a–5b was investigated
in-depth in order to evaluate their stability. As example, the 1
H
–1
observed in the range 420–380 cm and assigned to the gold–sulfur
6
NMR spectrum of complex 2a in acetone-d (Fig. 2) is discussed.
stretching vibrations, in agreement with data previously reported
First of all, the proton signals associated to the amide protons are
not detectable anymore within 24 h. Such experimental evidence is
in agreement with that previously observed for shorter dipeptide ana-
logues [26], and is likely to be due to the possible hydrogen/deuterium
[
46]. Finally, other informative bands were recorded at lower wave-
−
1
numbers (340–220 cm ) and attributed to metal-halide stretching
modes for terminal cis-chlorides/bromides [46,48].
Table 1
Selected IR frequencies of the starting oligopeptides P1–P5 and the corresponding gold(III)-dithiocarbamato derivatives 1a–5b.
Compound
Vibrational mode [cm−1]
ν
NH
ν
NH
a/s
ν
C_O
Amide I
ν
Amide II
ν
SCS
a/s
ν
SAuS
a/s
ν
a/s
XAuX
+
2
N−CSS
P1
3300
3428
3424
3421/3276
3403
3392
3400/3284
3410
3407
3406
2781/2715
–
1738
1736
1736
1727
1729
1729
1723
1728
1730
1728
1720
1722
1739
1728
1728
1669
1675
1685
1689/1647
1688
–
1559
1522
1531
1545/1518
1509
1512
1540/1505
1510
1508
1520
1518
1520
1543/1537
1528
1528
–
–
–
a
1a
1564
1561
–
1003/547
1004/549
–
407/383
407/384
–
252/222
1b
–
338/320b
–
P2
2762/2692
–
2a
1558
1559
–
1012/543
1015/543
–
406/384
406/383
–
252/227a
341/322b
–
2b
–
1689
P3
2761/2694
–
1684/1650
1685/1661
1686/1663
1681
1682
1681
1694/1657
1665
1667
a
3a
1546
1543
–
996/546
997/545
–
406/380
406/380
–
252/222
3b
–
341/321b
P4
2764/2702
–
–
a
4a
3400
1560
1561
–
1001/550
1004/551
–
410/384
408/384
–
253/228
4b
33792
3351/3311
3343
–
340/327b
P5
2751/2712
–
–
a
5a
1553
1557
998/560
997/562
413/383
413/382
252/227
5b
3340
–
338/322b
a
X = Br.
X = Cl.
b

256
M.N. Kouodom et al. / Journal of Inorganic Biochemistry 117 (2012) 248–260
NCSS carbon atom in the [ H, 13C] HMBC spectrum (Fig. 3), each giving
1
rise to long-range couplings with one set of N-methyl and N-methylene
sarcosine groups. Two forms have been observed also in DMSO-d but,
6
remarkably, there is no interconversion of one into the other over
time (data not shown), confirming the crucial role played by the solvent
in the solution behavior of the object complex. The major final species in
solution is likely to be the expected gold(III)-dithiocarbamato deriva-
tive 2a depicted in Fig. 1, whereas the identification of the second
species is still uncertain, although this evidence has been previously
reported and discussed for some analogues [46].
The same results were obtained for all the other complexes, in-
cluding 3a and 3b in which the sarcosine residue is replaced by a pro-
line, no matter what the oligopeptide chain or the halide type (data
not shown).
3.2. Conformational studies
The Aib-containing tri-, tetra- and pentapeptides are long enough
to adopt secondary structures. Therefore, specific IR studies were car-
ried out, aimed at elucidating their conformation in solution. The ex-
istence of hydrogen bonds between a donor (the amide NH) and an
acceptor (the amide CO) influences the vibrational modes associated
with the functional groups themselves. The stretching of “free” NH
groups (that is, not involved in hydrogen bonding) is generally
detected at wavenumbers higher than 3400 cm , whereas a shift to-
ward lower frequencies is consistent with the presence of hydrogen
bonds [56,57]. The discrimination between solvated (“free”) and
hydrogen-bonded N\H groups is possible only in low polarity sol-
Fig. 2. Detail of the 1H NMR spectra of complex 2a in acetone-d
over 24 h.
6
exchange with the solvent. Peptide amides are well-known to undergo
hydrogen exchange reactions with protic deuterated solvents (e.g. D
and methanol-d ) [53], whereas, to the best of our knowledge, such a
behavior is hardly observed for aprotic analogues. In addition, this ex-
change reaction occurs for complex 2a in acetone-d but does not take
place in DMSO-d (data not shown), thus supporting the hypothesis
2
O
4
−
1
6
6
of a solvent-dependent process. As far as we are aware of, apart from
this class of gold(III)-oligopeptidedithiocarbamato derivatives, a similar
observation has been reported only once for a zinc(II) complex bound
to an amide-like-containing ligand [54].
3
vents (such as CDCl ) which, contrary to polar ones, do not signifi-
cantly alter the position of the bands arising from solvent exposed
N\H groups.
It is also worth noting that two sets of signals are detected for both
N-methyl and N-methylene functions belonging to the sarcosine res-
In the present study, since the oligopeptide ester hydrochlorides
P2, P4 and P5 are not soluble in CDCl , the corresponding
3
idue. With reference to Fig. 2, soon after dissolution of complex 2a in
Z-protected analogues were investigated. The superimposed IR spec-
1
−3
acetone-d
served for NCH
:2.3 ratio. Intriguingly, the downfield peaks interconvert into their
highfield counterparts over time, the final ratio after 24 h being ca.
:0.5. The coexistence of two different forms of compound 2a, in
agreement with data previously reported for analogous compounds
6
two H signals at 3.51/3.55 and 4.69/4.73 ppm are ob-
tra in CDCl
3
(1.0×10
2 3
M) of Z-Sar-Aib -O(t-Bu), Z-Sar-Aib -O(t-Bu)
3
and NCH
2
groups, respectively, at approximately
and Z-Sar-Aib -Gly-OEt in the ν(N\H) region are shown in Fig. 4.
3
1
Among the bands observed, for all three oligopeptides only one signal
attributable to N–H groups involved in hydrogen-bonding in solution
−
1
1
was detected (at 3362, 3368 and 3352 cm
Z-Sar-Aib -O(t-Bu) and Z-Sar-Aib -Gly-OEt, respectively). According
to the absorbance values, the Z-pentapeptide shows the greater
2
for Z-Sar-Aib -O(t-Bu),
3
3
[26,46,55], is also confirmed by the presence of two signals for the
Fig. 3. Detail of the [ H, 13C] HMBC spectrum of complex 2a in acetone-d
1
6
.

M.N. Kouodom et al. / Journal of Inorganic Biochemistry 117 (2012) 248–260
257
A: 3456, 3426, 3402, 3362 cm-
B: 3458, 3428, 3402, 3368 cm-
C: 3458, 3428, 3402, 3352 cm-
1
1
1
C
8.0
7.5
7.0
6.5
6.0
NH Aib1
NH Gly
NH Aib3
NH Aib2
B
A
3500
3400
Wavenumber [cm ]
3300
3200
-
1
Fig. 4. Superimposed IR spectra of (A) Z-Sar-Aib
(
2
-O(t-Bu), (B) Z-Sar-Aib
3
-O(t-Bu), and
3 3
C) Z-Sar-Aib (ν(N\H) region).
-Gly-OEt 1.0×10− M in CDCl
3
0
1
2
3
4
5
6
7
8
DMSO-d in CDCl [%]
6
3
involvement in the formation of hydrogen bonds through some of the
amide NH groups.
Intramolecular hydrogen bonds are insensitive to dilution where-
as intermolecular ones are favored at higher concentrations. There-
fore, to ascertain whether the observed hydrogen bonds are due to
1
2
3
Fig. 6. Variation of the chemical shifts of the amide protons of Z-Sar-Aib -Aib -Aib -
Gly-OEt (1.0×10− M in CDCl
3
) upon addition of DMSO-d .
3 6
the corresponding 1H signals, those already involved in hydrogen
bonds are negligibly affected. With reference to Fig. 6, upon addition
inter- or intramolecular interactions, IR investigations in CDCl
3
were
performed for the Z-protected pentapeptide at different concentra-
6 3
of calculated amounts of DMSO-d to a solution of Z-Sar-Aib -Gly-OEt
−
2
−3
−4
−3
tions (1.0×10 , 1.0×10 , 1.0×10
M). According to results
in CDCl
3
(1.0×10
M), only the signal assigned to the NH group of
1
reported in Fig. 5, it is clear that dilution has negligible effects on
Aib is significantly shifted toward higher chemical shift values
−
1
2
3
the intensity of the strong band at 3352 cm , confirming the occur-
rence of intramolecular hydrogen bonds. Consequently, we can spec-
ulate about the conformation of the pentapeptide in solution since
such a strong band is recurrent for peptides assuming a helical struc-
ture. In fact, the presence of three Aib residues supports the hypoth-
esis of a 310-helix stabilized by three intramolecular hydrogen bonds.
Additional NMR experiments provided further insights about the
whereas the other three amide protons belonging to Aib , Aib and
Gly are almost unaffected, consistent with their actual involvement
in intramolecular hydrogen bonds (in agreement with results previ-
ously obtained by IR studies in solution).
1
1
Subsequent [ H, H] ROESY experiments (Fig. S1 in the Supplementa-
ry Information) allowed the detection of four NH(i)→NH(i+1) consec-
1
α
utive cross-peaks (from Aib to Gly) and of the C H(i)→NH(i+1) cross-
peak of Sar, all of which are diagnostic for the assumption of a folded
conformation [59]. Although the sarcosine unit does not have an amide
proton, a cross-peak between the N-methyl group and the amide protons
3
conformation of the pentapeptide Z-Sar-Aib -Gly-OEt in solution.
Useful information related to the involvement of the peptide NH
groups in hydrogen bonds (and, thus, on the peptide conformation)
can be obtained by recording successive 1H NMR spectra in CDCl
of both Aib and Aib are consistent with an helical structure, most likely
of the 310-type [60].
1
3
3
upon addition of increasing amounts of a suitable substance capable
to form hydrogen bonds with the peptide itself (for instance,
DMSO) [58]. “Free” amide protons exposed to the solvent may form
hydrogen bonds with the DMSO, thus causing a downfield shift of
3.3. X-ray crystal structure determination
1
2
3
The structure of HCl·H-Sar-Aib -Aib -Aib -O(t-Bu) (P4) was de-
termined as trihydrate by single-crystal X-ray diffraction (Fig. S2 in
the Supplementary Information). The corresponding crystallographic
data as well as a list of relevant backbone torsion angles [61] and
intra- and intermolecular hydrogen bond parameters are reported
in the Supplementary Information (Tables S1, S2 and S3, respective-
ly). Since the structure is centrosymmetric, molecules of both hand-
edness occur in the unit cell, and the following description refers to
the molecule in the left-handed screw sense, chosen as asymmetric
unit.
-
2
1
.0x10
.0x10
.0x10
M
-3
1
M
-4
1
M
The peptide backbone is folded into a β-turn [62] stabilized by an
3
intramolecular hydrogen bond between the NH group of Aib and the
carbonyl oxygen of Sar. The sets of ϕ and ψ torsion angles adopted by
1
2
Aib and Aib , which occupy the i+1 and i+2 corner positions, are
close to those of an ideal type-III′ β-turn (ϕi+1=ϕi+2=60°; ψi+1
i+2=30°), their values being ϕ =56.4(4)°/ψ =29.1(4)° and ϕ
0.3(4)°/ψ =41.8(4)°. At the N-terminus, the ψ
=
=
ψ
5
2
2
3
3
1
torsion angle of
3
500
3400
Wavenumber [cm ]
3300
3200
-
1
the Sar residue is extended (156.6(3)°) and the value of the
C1N-N1-C1A-C1 torsion angle, which describes the orientation of the
C1N methyl group relative to the peptide backbone, is −66.5(5)°. The
-Gly-OEt at 1.0×10−2, 1.0×10−3 and
Fig. 5. Superimposed IR spectra of Z-Sar-Aib
3
−
4
3
1.0×10
M in CDCl
3
(ν(N\H) region).
Aib residue adopts an helical conformation with a screw sense opposite

258
M.N. Kouodom et al. / Journal of Inorganic Biochemistry 117 (2012) 248–260
to that of the preceding residues, as usually observed in peptide esters
containing a C-terminal Aib residue [63,64].
3.5. Induction of apoptosis
Within the same asymmetric unit, the protonated secondary
amino group of Sar is hydrogen-bonded to both the chlorine ion
and the O1W water molecule, whereas the N2H and N3H groups
form hydrogen bonds with the O2W and O3W water molecules,
respectively. In the packing mode, additional hydrogen bonds are ob-
served, involving the three co-crystallized water molecules as donors,
and symmetry equivalents of the chlorine ion and of the O2 and O3
peptide carbonyl oxygen atoms as the acceptors.
Although the mechanism of action of this class of gold(III)
peptidomimetics is still under investigation, the capability of complexes
1a–5b to induce apoptosis was tested by incubating the various cancer
cell lines with a single cytotoxic dose (10 μM for PC3/L540 cells, 30 μM
for 2008/C13 cells) using the Annexin-V/PI double staining assay over
72 h. One of the earliest features of apoptosis is the translocation of
the plasma membrane phospholipid phosphatidylserine (PS) from the
inner to the outer leaflet of the membrane itself, thereby exposing PS
to the external environment. The phospholipid binding protein
Annexin-V specifically binds to PS exposed on the cell surface and,
once detected by flow cytometry, it allows the identification of early
stage apoptotic cells. Remarkably, the relative integrity of the mem-
brane of cells undergoing early apoptosis prevents internalization of
PI. On the other hand, because of extensive membrane damage, necrotic
cells are quickly stained by both Annexin-V and PI, thus allowing the
differentiation between early apoptotic and necrotic cells. A major
drawback of this assay is that as apoptosis progresses and the plasma
membrane becomes compromised (i.e. late apoptotic cells) PI, is also in-
ternalized. Therefore, for longer exposure times there is no clear-cut
parameter to distinguish by flow cytometry between cells undergoing
either necrosis or late apoptosis.
With reference to Fig. 7, treatment of both cisplatin-sensitive (2008)
and -resistant (C13) ovarian adenocarcinoma cells with complexes 1a–
3b induced a massive amount of FITC-Annexin-V/PI stained cells owing
to the large majority of cells undergoing late apoptosis/necrosis. Con-
versely, cisplatin induced both apoptosis and late apoptosis/necrosis
in 2008 cells whereas, as expected, had negligible effect against the
C13 resistant subclone. A similar trend was observed for Hodgkin's lym-
phoma L540 cell line since cells underwent almost exclusively late
apoptosis/necrosis upon incubation with either the gold(III) complexes
or cisplatin.
3
.4. In vitro cytotoxicity
Complexes 1a–5b were tested for in vitro cytotoxic activity toward
the human androgen receptor-negative prostate cancer PC3 cells, ovar-
ian adenocarcinoma 2008 cells and the parent cisplatin-resistant C13
subclone, and Hodgkin's lymphoma L540 cells (Table 2). The choice of
such cell lines is not accidental; in fact, all of them were shown to
overexpress both peptide transporters PEPT1 and PEPT2 to a good ex-
tent [26], whereas they have not been localized in either the normal
genitourinary apparatus or healthy lymphocytes [13], thus making
them suitable for preliminary anticancer screening of the object
gold(III)-based peptidomimetics. The inhibition of cell proliferation
was evaluated over 72 h by means of the trypan blue dye exclusion
test for the floating lymphoma cells, and the BrdU ELISA assay (allowing
cell proliferation to be assessed by quantifying DNA synthesis during
cells activation and proliferation) for adherent prostate and ovarian
cancer cell lines. Results are expressed in terms of IC50 values and
are compared to the reference drug cisplatin as well as to the model
III
gold(III)-dipeptidedithiocarbamato analogue [Au Br
2
(dtc-Sar-Aib-O
(t-Bu))] previously reported [26], tested under the same experimental
conditions.
Exposure of L540 cells to increasing concentrations of complexes
1
a–5b resulted in a significant inhibition of cell growth, with IC50
Finally, prostate cancer PC3 cells appeared to be less sensitive to
the treatment. Nevertheless, substantially equivalent amounts of
FITC-Annexin-V (accounting for PS exposure) and FITC-Annexin-V/
PI stained cells were detected following the treatment with com-
plexes 1a–3b, thus confirming that both apoptosis and late apoptosis/
necrosis are involved in cell growth inhibition. Remarkably, results
were widely comparable with those recorded for cisplatin.
Overall, in agreement with IC50 values reported in Table 2, the
gold(III)-dithiocarbamato derivatives 4a–5b showed modest or even
no capability to induce either apoptosis or late apoptosis/necrosis. Con-
versely, such a biological evaluation confirmed complex 3b as the most
effective among the gold-based peptidomimetics here investigated.
values (1.4–5.4 μM) comparable to that recorded for cisplatin
2.5 μM). The gold(III) compounds 1a–3b exhibited cytotoxicity
(
levels comparable or even lower than cisplatin on both prostate can-
cer and ovarian adenocarcinoma cells, whereas the tetra- and penta-
peptide derivatives 4a–5b resulted to be less effective. When tested
toward the cisplatin-resistant C13 subclone, the gold(III) complexes
were proved much more active than the reference drug, including
4
a–5b.
Overall, the tripeptide derivative [Au Cl
3b) turned out to be the best performer on all the investigated tumor
cell lines, showing IC50 values comparable to those previously reported
III
2 2
(dtc-D,L-Pro-Aib -O(t-Bu))]
(
III
2
for the model complex [Au Br (dtc-Sar-Aib-O(t-Bu))].
4
. Conclusions
Table 2
In vitro growth inhibition of human prostate cancer (PC3), ovarian adenocarcinoma
cisplatin-sensitive (2008) and -resistant (C13), and Hodgkin's lymphoma (L540) cells
Ehrlich's “magic bullet” concept [65] may be summarized in a
over 72 h by gold(III)-oligopeptidedithiocarbamato derivatives 1a–5b, and comparison
word: targeting. Tumor site specific delivery and targeting bio-
markers overexpressed in cancer cells are two aspects of drug design
that have been receiving much attention in recent years. Conjugated
drugs incorporating a tumor targeting group and a cytotoxic “smart
bomb” are emerging as promising therapies for cancer treatment.
In particular, this approach has been successfully used for peptide-
mediated delivery of metal complexes into cells [66].
In this context, the proton-coupled peptide transporters PEPT1
and PEPT2 are currently under intense investigation. Owing to their
capability to promote the cellular uptake of physiologically occurring
di- and tripeptides, they can internalize into the cells suitable
peptidomimetic substrates with therapeutic properties or precursors
of pharmacologically active agents [10–13]. Together with their actual
localization in some tissues/organs (e.g. small intestine, kidney, lung,
etc.), intriguingly, they were proved to be ubiquitously overexpressed
in various types of tumors but not in the normal counterparts [15,16].
III
2
with the reference drug cisplatin and the model complex [Au Br (dtc-Sar-Aib-O(t-Bu)].
Compound
IC50 [μM]
PC3
2008
C13
L540
Cisplatin
3.3± 0.3 19.4± 1.2 117.2± 9.1 2.5± 0.1
1
1
2
2
3
3
4
4
5
5
a
b
a
b
a
b
a
b
a
b
5.2± 0.5 17.5± 1.6
6.5± 0.7 15.2± 1.5
5.8± 0.6 12.0± 1.1
5.8± 0.5 14.2± 1.5
6.3± 0.7 16.3± 1.4
17.0± 1.6 3.4± 0.2
16.1± 1.3 1.4± 0.1
15.0± 1.3 3.8± 0.4
15.4± 1.4 4.1± 0.5
11.5± 1.1 2.2± 0.1
7.8± 0.5 2.2± 0.2
17.5± 1.6 3.9± 0.4
11.5± 1.2 3.5± 0.2
22.9± 1.9 5.4± 0.5
35.5± 3.9 5.0± 0.8
3.7± 0.3 1.5± 0.2
3.0± 0.2
8.2± 0.7
16.0± 1.7 42.8± 4.1
16.1± 0.6 43.5± 1.2
15.0± 1.4 29.2± 3.0
20.0± 1.9 27.8± 2.3
[
AuIIIBr
2
(dtc-Sar-Aib-O(t-Bu))]a
0.8± 0.1
4.5± 0.2
a
Ref. [26].

M.N. Kouodom et al. / Journal of Inorganic Biochemistry 117 (2012) 248–260
259
affecting healthy tissues, thus acting as “Trojan horses” and strongly im-
proving the cellular uptake and minimizing unwanted side-effects.
According to the objectives outlined above, we designed five novel
oligopeptides (from di- to pentapeptides, P1–P5) and successfully syn-
thesized the corresponding gold(III)-oligopeptidedithiocarbamato de-
rivatives 1a–5b (Fig. 1).
Results related to the in vitro cytotoxic activity of complexes 1a–5b
toward human prostate (PC3), ovarian adenocarcinoma cisplatin-
sensitive (2008) and -resistant (C13), and Hodgkin's lymphoma (L540)
cells (Table 2), need to be evaluated in comparison to those previously
III
reported for the model dipeptide analogue [Au Br
2
(dtc-Sar-Aib-
O(t-Bu))] [26]. In this regard, the dipeptide derivatives 1a and 1b showed
IC50 values higher than those observed for the model complex,
suggesting that such an increase of hydrophobicity might be detrimental
for the anticancer activity.
III
As concern the tripeptide derivatives 2a–3b, complex [Au Cl
2
(dtc-D,
2
L-Pro-Aib -O(t-Bu))] (3b) turned out to be the overall best performer
on all the tested tumor cell lines, showing IC50 values lower than the ref-
erence drug cisplatin, although slightly higher than those recorded for
III
the model complex [Au Br
2
(dtc-Sar-Aib-O(t-Bu))] tested under the
same experimental conditions. Remarkably, when cisplatin-resistant
ovarian adenocarcinoma C13 cells were used, activity levels (15-fold
lower than cisplatin) comparable to those observed on the parent
cisplatin-sensitive 2008 cells were detected, thus ruling out the occur-
rence of cross-resistance with cisplatin itself. Additionally, complex 3b
seems to exert its cytotoxic effect on all the tumor cell lines mainly by
inducing almost exclusively late apoptosis/necrosis over 72 h. Surpris-
ingly, it resulted to be more effective than the sarcosine-containing
counterparts 2a and 2b, thus confirming the crucial role played by the
N-substituted amino acid residue directly bound to the dithiocarbamato
moiety.
Finally, for the tetra- and pentapeptide-containing complexes 4a–5b
modest cytotoxicity levels were observed, in agreement with the fact
that, notwithstanding the exceptionally broad substrate specificity,
PEPTs are unable to transport peptides longer than tripeptides [14].
Therefore, such experimental evidence might support the involve-
ment of PEPTs in the biological activity of the shorter gold(III)-
dithiocarbamato peptidomimetics.
As far as we are aware of, this appears to be the first time that
metal-based peptidomimetic anticancer agents have been designed
specifically to target peptide transporters. Eventually, this innovative
strategy may lead to the development of novel and more selective
chemotherapeutic agents that could provide a valuable alternative
to the platinum-based anticancer drugs in clinical use.
III
Together with [Au Br
2
(dtc-Sar-Aib-O(t-Bu))], complex 3b is cur-
rently under investigation for its in vivo anticancer activity as well
as for the elucidation of the mechanism of action. Should it really re-
semble the behavior of its previous analogues, we are expecting the
proteasome to be a major biological target, and preliminary outcomes
seem to confirm this hypothesis.
These results represent a solid starting point for the recognition of
this class of gold(III) peptidomimetics as suitable candidates for fur-
ther pharmacological testing and, remarkably, allowed us to file an
international patent for their use in cancer chemotherapy (just ex-
tended in several countries worldwide) [67].
Fig. 7. FACS analysis of cells after 72 h incubation at 37 °C with either complexes 1a–5b
or cisplatin (10 μM for PC3/ L540 cells, 30 μM for 2008/C13 cells) and double stained
with FITC-Annexin-V and PI.
Abbreviations
As a further extension of our previous research on gold(III)-
dithiocarbamato anticancer agents, we have been developing “second
generation” gold(III) compounds that may be able to maintain both
the promising antitumor activity and the favorable toxicological profile
of their previous analogues, together with an improved selectivity to-
ward tumor cells by targeting PEPT1 and/or PEPT2 via the oligopeptide
chain. The basic idea relies on the potential recognition of the whole
metal complex that, once recognized and transported by PEPTs and
delivered inside the tumor cell, could exert its anticancer activity without
AA
Aib
BrdU
DMAP
DSC
amino acid
α-aminoisobutyric acid (2-methylalanine)
bromodeoxyuridine
4-dimethylaminopyridine
differential scanning calorimetry
dithiocarbamate
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
enzyme-linked immunoadsorbent assay
fluorescence-activated cell sorting
Dtc
EDC
ELISA,
FACS

260
M.N. Kouodom et al. / Journal of Inorganic Biochemistry 117 (2012) 248–260
[
15] K. Mitsuoka, S. Miyoshi, Y. Kato, Y. Murakami, R. Utsumi, Y. Kubo, A. Noda, Y.
FBS
fetal bovine serum
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19] S. Nobili, E. Mini, I. Landini, C. Gabbiani, A. Casini, L. Messori, Med. Chem. Res. 30
(2010) 550–580.
FITC
HEPES
HMBC
HOBt
IBCF
NMM
oxl
pdtc
PEPT
PI
fluorescein isothiocyanate
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
heteronuclear multiple bond correlation
1-hydroxybenzotriazole
isobutyl chloroformiate
N-methylmorpholine
5(4H)-oxazolone
peptidedithiocarbamate
peptide transporter
[
[
[
[
[20] S.J. Berners-Price, A. Filipovska, Metallomics 3 (2011) 863–873.
[
[
21] L. Ronconi, D. Fregona, Dalton Trans. (2009) 10670–10680.
22] L. Ronconi, D. Aldinucci, Q.P. Dou, D. Fregona, Anti-Cancer Agents Med. Chem. 10
(2010) 283–292.
[23] V. Milacic, D. Chen, L. Ronconi, K.R. Landis-Piwowar, D. Fregona, Q.P. Dou, Cancer
Res. 66 (2006) 10478–10486.
24] L. Cattaruzza, D. Fregona, M. Mongiat, L. Ronconi, A. Fassina, A. Colombatti, D.
propidium iodide
phosphatidylserine
[
PS
Aldinucci, Int. J. Cancer 128 (2011) 206–215.
RPMI
Sar
TEA
TFA
Roswell Park Memorial Institute (culture medium)
sarcosine (N-methylglycine)
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