Identification of the structural determinants for anticancer activity of a ruthenium arene peptide conjugate

Article abstract of DOI:10.1002/chem.201300889

Organometallic Ru(arene)-peptide bioconjugates with potent in vitro anticancer activity are rare. We have prepared a conjugate of a Ru(arene) complex with the neuropeptide [Leu⁵]-enkephalin. [Chlorido(η ⁶-p-cymene)(5-oxo-κO-2-{(4-[(N-tyrosinyl-glycinyl-glycinyl- phenylalanyl-leucinyl-NH₂)propanamido]-1H-1,2,3-triazol-1-yl)methyl}- 4H-pyronato-κO)ruthenium(II)] (8) shows antiproliferative activity in human ovarian carcinoma cells with an IC₅₀ value as low as 13 μM, whereas the peptide or the Ru moiety alone are hardly cytotoxic. The conjugation strategy for linking the Ru(cym) (cym=η⁶-p-cymene) moiety to the peptide involved N-terminal modification of an alkyne-[Leu⁵]- enkephalin with a 2-(azidomethyl)-5-hydroxy-4H-pyran-4-one linker, using Cu ^I-catalyzed alkyne-azide cycloaddition (CuAAC), and subsequent metallation with the Ru(cym) moiety. The ruthenium-bioconjugate was characterized by high resolution top-down electrospray ionization mass spectrometry (ESI-MS) with regard to peptide sequence, linker modification and metallation site. Notably, complete sequence coverage was obtained and the Ru(cym) moiety was confirmed to be coordinated to the pyronato linker. The ruthenium-bioconjugate was analyzed with respect to cytotoxicity-determining constituents, and through the bioconjugate models [{2-(azidomethyl)-5-oxo- κO-4H-pyronato-κO}chloride (η⁶-p-cymene)ruthenium(II) ] (5) and [chlorido(η⁶-p-cymene){5-oxo-κO-2-([(4- (phenoxymethyl)-1H-1,2,3-triazol-1-yl]methyl)-4H-pyronato-κO}ruthenium(II) ] (6) the Ru(cym) fragment with a triazole-carrying pyronato ligand was identified as the minimal unit required to achieve in vitro anticancer activity. Copyright

Full text of DOI:10.1002/chem.201300889

FULL PAPER
DOI: 10.1002/chem.201300889
Identification of the Structural Determinants for Anticancer Activity of a
Ruthenium Arene Peptide Conjugate
Samuel M. Meier,[a, b] Maria Novak,[a] Wolfgang Kandioller,[a, b] Michael A. Jakupec,[a, b]
Vladimir B. Arion,[a] Nils Metzler-Nolte,[c] Bernhard K. Keppler,[a, b] and
Christian G. Hartinger*[a, b, d]
Abstract: Organometallic Ru
(arene)–
of an alkyne-[Leu5]-enkephalin with a
2-(azidomethyl)-5-hydroxy-4H-pyran-4-
one linker, using CuI-catalyzed alkyne–
azide cycloaddition (CuAAC), and sub-
the RuACTHNGUTERNNU(G cym) moiety was confirmed to
peptide bioconjugates with potent in
vitro anticancer activity are rare. We
have prepared a conjugate of a Ru-
be coordinated to the pyronato linker.
The ruthenium-bioconjugate was ana-
lyzed with respect to cytotoxicity-deter-
mining constituents, and through the
bioconjugate models [{2-(azidomethyl)-
5-oxo-kO-4H-pyronato-kO}chloride
R
sequent metallation with the RuACTHNGUTRENNU(G cym)
moiety. The ruthenium-bioconjugate
was characterized by high resolution
top-down electrospray ionization mass
spectrometry (ESI-MS) with regard to
peptide sequence, linker modification
and metallation site. Notably, complete
sequence coverage was obtained and
cymene)(5-oxo-kO-2-{(4-[(N-tyrosinyl-
glycinyl-glycinyl-phenylalanyl-leucinyl-
NH2)propanamido]-1H-1,2,3-triazol-1-
yl)methyl}-4H-pyronato-kO)ruthen-
(h6-p-cymene)ruthen
ACHTNUTRGNEUNGium(II)] (5) and
[chlorido(h6-p-cymene){5-oxo-kO-2-
([(4-(phenoxymethyl)-1H-1,2,3-triazol-
1-yl]methyl)-4H-pyronato-kO}ruthen-
ACHTUNGTRENNUNGium(II)] (8) shows antiproliferative ac-
tivity in human ovarian carcinoma cells
with an IC50 value as low as 13 mm,
whereas the peptide or the Ru moiety
alone are hardly cytotoxic. The conju-
A
ACHTUNGTREN(NUNG cym) fragment
a
ligand was identified as the minimal
unit required to achieve in vitro anti-
cancer activity.
Keywords: antitumor agents · bio-
organometallic chemistry
spectrometry peptide bioconju-
gates · ruthenium complexes
· mass
gation strategy for linking the RuACTHNUTRGNEUG(N cym)
·
(cym=h6-p-cymene) moiety to the pep-
tide involved N-terminal modification
Introduction
dedicated to organometal–peptide conjugation.[7–17] The pep-
tide carrier is typically obtained by solid phase synthesis,
and the metallodrug is linked to the peptide either on solid
support or in solution after peptide cleavage from the resin.
The metal can be conjugated directly via a suitable amino
acid, or a linker with metal-chelating properties can be in-
corporated into the peptide sequence.[14,18] When pursuing
the latter strategy, N-terminal and intrasequence derivatiza-
tions are most commonly used for metal-tagging. Moreover,
most of the reported metal–peptide bioconjugates are syn-
thesized with inert linkers.[14] Based on these strategies, en-
couraging but rare examples of anticancer active metal–pep-
tide bioconjugates were reported with different metal sys-
tems including ferrocenoyl–dipeptides,[19] dicobalt hexacar-
Peptides are useful carriers for the specific accumulation of
drugs in desired tissues and/or cell compartments.[1–5] In di-
agnostic and therapeutic metallodrug research,[6] the ap-
proach of tagging metal fragments to peptides is an emerg-
ing targeting strategy and several recent investigations were
[a] Dr. S. M. Meier, M. Novak, Dr. W. Kandioller, Dr. M. A. Jakupec,
Prof. Dr. V. B. Arion, Prof. Dr. B. K. Keppler,
Prof. Dr. C. G. Hartinger
Institute of Inorganic Chemistry, University of Vienna
Waehringer Strasse 42, 1090 Vienna (Austria)
[b] Dr. S. M. Meier, Dr. W. Kandioller, Dr. M. A. Jakupec,
Prof. Dr. B. K. Keppler, Prof. Dr. C. G. Hartinger
bonyl
[Mn(Cp)(CO)3] tagged to the cell-penetrating peptides sC18
or hCT
(18–32)-k7,[12,15,20] Rh(Cp*)-sandwich,[7] gold(I)[21] or
tagged
to
alkyne-modified
enkephalin,[16]
Research Platform “Translational Cancer Therapy Research”
University of Vienna, Waehringer Strasse 42, 1090 Vienna (Austria)
A
ACHTUNGTRENNUNG
[c] Prof. Dr. N. Metzler-Nolte
platinum(IV) bioconjugates of TAT, pseudoneurotensin or
Inorganic Chemistry I–Bioinorganic Chemistry
Faculty of Chemistry and Biochemistry, Ruhr-University Bochum
Universitꢀtsstrasse 150, 48801 Bochum (Germany)
octreotate.[8,22]
Surprisingly, only little is known about bioconjugates of
anticancer half-sandwich ruthenium(II) and osmium(II) or-
ganometallics with peptide carriers[9,23] or pendant amino
acids.[24,25] This is probably related to synthetic difficulties
[d] Prof. Dr. C. G. Hartinger
School of Chemical Sciences, University of Auckland
Private Bag 92019, Auckland 1142 (New Zealand)
E-mail: c.hartinger@auckland.ac.nz
À
arising from hydrolysis of the metal halido bond in aqueous
solution, which is a crucial activation parameter for these
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201300889.
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9297

classes of anticancer metallodrugs in a biological setting.[26]
The few existing investigations show that loading of the
metalloACHTUNGTRENNUNGdrug on a peptide carrier is associated with a de-
accumulation in a certain (cancer) tissue or even a specific
cell compartment, and they are being studied as promising
vectors in the discovery and design of metal-based anticanc-
er agents with targeting properties.[2,14] Most often, the cyto-
toxic moiety is conjugated to the peptide using an inert
linker with the aim of generating a cytotoxic metal–peptide
bioconjugate. In the case of organometallic Ru- and Os-
crease in antiproliferative activity compared to the non-con-
jugated small metallodrug.[9,23] In particular, conjugation of
an OsACHTUNGTRENNUNG(arene) picolinate moiety to an octaarginine sequence
led to a substantial activity decrease and moderate cytotox-
icity was only obtained in protein depleted fetal calf
serum.[9] Moreover, conjugation of an imidazole-modified
AHCTUNGTREG(NNUN arene) compounds, conjugation to peptides often results in
a significant reduction of the antiproliferative activity of the
metal–peptide bioconjugate compared to the small molecule
metallodrug.[8,9] Therefore, it was aimed to investigate a Ru-
dicarba
analogue
of
octreotide
to
a
Ru-
ACHTUNGTRENNUNG(arene)(triphenylphosphine) moiety also resulted in modest
cytotoxic activity, which was approximately fourfold lower
than that of the analogous small anticancer metallodrug.[23]
The apparent lack of anticancer active organometallic
Ru–peptide conjugates from potent metallodrugs prompted
us to investigate an appropriate model system with the aim
of identifying its cytotoxicity-determining building block(s).
The neuropeptide [Leu5]-enkephalin was chosen for this
purpose, and an N-terminal alkyne derivative thereof was
further modified by a copper(I)-assisted alkyne–azide cycli-
zation (CuAAC) using 2-(azidomethyl)-5-hydroxy-4H-
pyran-4-one as a linker to the organometallic moiety. Hy-
droxypyrones are well-known metal chelators offering O,O-
bidentate coordination,[27] and recent developments involv-
ing hydroxypyrones as ligands for organometallic Ru- and
ACHTUNGTREN(NUNG arene)-based bioconjugate exhibiting antiproliferative ac-
tivity in vitro and to identify the cytotoxicity-determining
building block by comparing the antiproliferative activity of
the bioconjugate with lower molecular weight fragments of
related structural components.
The linker for the conjugation of the RuACHTNUTRGNEUNG(arene) fragment
to the peptide, that is, 2-(azidomethyl)-5-hydroxy-4H-pyran-
4-one (1), was obtained in two steps starting from kojic acid
by adapting a published procedure.[37] Compound 1 contains
two functional moieties of interest: the pyrone scaffold per-
mits anionic O,O-bidentate metal coordination, and the
azide allows facile and selective modification of the ligand.
Importantly, the azide was modified in a single step by
CuAAC (Scheme 1).[38–40]
OsACHTUNGTRENNUNG(arene) compounds with an-
ticancer properties showed in-
triguing results with respect to
in vitro antitumor activity.[28–34]
For example, dinuclear rutheni-
um bis-pyridonato complexes
display cytotoxic activity in cell
culture assays in dependence of
the spacer length separating the
two Ru-chelating pyridone moi-
eties. In contrast, several mono-
nuclear
organometallic
Ru
compounds based on O,O-bi-
dentate pyrones were found to
exhibit anticancer activity only
in the high micromolar range
whereas a substantial increase
in activity was observed with
S,O-bidentate
thiopyrones,
Scheme 1. Synthetic pathway to neutral pyronato complexes, starting from kojic acid. a) This step features
CuAAC. b) The complexation step involves deprotonation of the hydroxypyrone prior to coordination leading
which have been less investigat-
ed so far.[31,32,34–36] Based on the
mentioned components, that is,
to neutral [Ru(h6-p-cymene)
ACTHNUTRGNE(NUG Pyr)Cl] complexes, where Pyr is the pyronato ligand.
RuACHTUNGTRENNUNG(arene), hydroxypyrone, tria-
zole linker and peptide, we aimed to elucidate the structural
determinants for anticancer activity of this bioconjugate in
human tumor cells.
The cycloaddition involves the reaction of an azide and a
terminal alkyne and allows the introduction of a metal che-
lator in virtually any molecule containing a terminal alkyne.
Copper(I) catalysis yields regioselectively 1,4-substituted tri-
azoles by transient formation of a Cu–acetylide species and
represents a powerful tool for extending SARs of the pyro-
nato class of metallodrugs. In metal-based anticancer re-
search, the 1,2,3-triazole pharmacophore is not yet
well-established in the therapeutic context, although its
Results and Discussion
Synthesis and characterization: Peptide carrier systems are
an attractive means for obtaining specificity with respect to
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Organometal–Peptide Bioconjugates
FULL PAPER
value in diagnostics is well appreciated.[11,41] Copper(I) was
prepared in situ from CuSO4 and sodium ascorbate, and the
cycloaddition reaction products 2 and 3 were isolated in
moderate yields. The molecular structure of 2 was obtained
by single crystal X-ray diffraction analysis confirming the
formation of the 1,4-substituted triazole (Figure S1 in the
Supporting Information). The alkyne-modified [Leu5]-en-
ACHTUNGTRENNUNG
kephalin was prepared adapting a literature procedure.[16]
The cycloaddition reaction with 1 was carried out on solid
support, thus, a suspension of the Rink amide resin was
treated with 2 equiv CuI and an excess of nitrogen base. Di-
ethyldithiocarbamate (Cupral) proved to be suitable for effi-
ciently removing Cu from the suspension after the reaction.
The desired hydroxypyrone-modified [Leu5]-enkephalin 4
was obtained in 58% overall yield after cleavage from the
Rink amide resin.
Complexation of the hydroxypyrones to the RuACTHNUGTRNEUNG(cym)
Figure 1. The molecular structure of an enantiomer of 5 is shown includ-
ing the general numbering scheme. The ellipsoids are drawn at 50%
probability level.
moiety was achieved by deprotonation of the ligand and
subsequent addition of 0.5 equiv bis[dichlorido(h6-p-cy-
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
moved by dissolving the reaction product in DCM, followed
by filtration. In general, subsequent precipitation yielded an-
alytically pure products of 5, 6 and 7 in moderate to high
yields (63–82%). The peptide conjugate 8 was treated simi-
larly, since an attempt of performing the cycloaddition reac-
tion with 5 and the alkyne-modified [Leu5]-enkephalin on
solid support had been unsuccessful, probably due to the
harsh conditions used during work-up and the pyronato or-
ganometallics being known to be acid labile.[34] Complexes
5, 6 and 7 were characterized by 1D/2D NMR spectroscopy,
UHR ESI-TOF MS and elemental analysis. The peptide
conjugate 8 was characterized by 1D/2D NMR spectroscopy,
UHR ESI-TOF MS and HPLC-MS (see Figure S3 in the
Supporting Information for the analytical HPLC-MS experi-
ment).
pyrone moieties are generally in good agreement with those
of the close analogue [chlorido(h6-p-cymene)(2-hydroxy-
methyl-5-oxo-4H-pyronato)ruthenium(II)].[33]
X-ray diffraction analysis: Single crystals of 5 suitable for
X-ray diffraction analysis were obtained by slow diffusion of
n-pentane into a dichloromethane solution of the com-
pound. The result of the X-ray diffraction study is shown in
Figure 1. Details of data collection, structure solution and
refinement, as well as geometrical parameters of 5 are listed
in the Supporting Information (Tables S1 and S2). The com-
plex displays the characteristic half-sandwich “piano-stool”
configuration. The crystal structure confirms anionic O,O-bi-
dentate chelation of the pyronato moiety to the metal
center yielding a neutral monochlorido complex (Figure 1).
Crystal cell parameters and selected bond lengths, angles
and torsion angles are listed in the Supporting Information
(Tables S1 and S2). Interestingly, both enantiomers were ob-
served at a 1:1 ratio in the crystal of 5. The asymmetric unit
of 5 additionally contains two stereo-isoforms, which feature
varying bond lengths in the first coordination sphere (Fig-
ure S2 in the Supporting Information). The isoform 5B
The modification at the azide moiety is directly detectable
1
in the H NMR spectrum (recorded in [D6]DMSO), by com-
paring in particular the H-7 (CH2) signals (see Figure 1 and
Figure S1 in the Supporting Information for the NMR num-
bering scheme). The H-7 signal of 1 displays a singlet with a
chemical shift of 4.42 ppm. Conversion of the azide into a
1,4-substituted triazole is characterized by a down-field shift
of the H-7 singlet (CH2) to 5.61 ppm. The detection of an
additional singlet at approximately 8.3 ppm in 6, 7 and 8 is
indicative of H-8 (HTriaz), the proton in the 1,4-substituted
À
À
shows shorter Ru O3 and Ru Cl bond lengths, but longer
À
À
Ru O2 and Ru centroid bond lengths compared to 5A. In
general, the bond lengths and angles are on the same order
as observed for related Ru–pyronato and –pyridonato com-
plexes.[29,33] It is noteworthy, that the azide does not degrade
during the complexation reaction.
1
triazole ring. Upon complexation, the H NMR signals cor-
responding to H-7 and H-8 did not shift significantly. How-
ever, the H-6 signal underwent an up-field shift by approxi-
mately 0.2 ppm. This indicates that the pyronato moiety but
not the triazole nitrogen is involved in coordination to the
metal, corresponding to a pendant design of triazole com-
plexes, in contrast to the “click-to-chelate” strategy, where
the triazole is directly involved in metal-binding.[11,42]
O,O,N-Chelation is sterically improbable as can be deduced
from Figure 1 and Figure S1 in the Supporting Information.
Finally, the chemical shifts of the coordinated cym and
Top-down ESI-MS characterization of the half-sandwich
ruthenium peptide bioconjugate: In addition to NMR spec-
troscopy, analytical HPLC and UHR ESI-TOF MS analysis,
the peptide bioconjugate 8 was characterized by tandem
mass spectrometric methods in a top-down approach using
both ESI-IT and UHR ESI-TOF MS. In principle, top-down
MS allows the determination of the sequence of amino acids
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9299

C. G. Hartinger et al.
and of the site of pyrone modification and metal coordina-
tion.
In the context of metallated peptides, capital letters are
used to denote metallated peptide fragments.[46] The occur-
rence of a complete set of B-fragments indicates that the
charged metal must be near the N terminus, where [Leu5]-
Investigations were performed using collision-induced dis-
sociation (CID) and electron transfer dissociation (ETD,
only for IT), which often deliver complementary informa-
tion due to underlying differences in the fragmentation
mechanisms.[43,44] In general, ESI-IT- and ESI-TOF-MS of 8
yield singly and doubly charged species corresponding to
enkephalin was modified with the pyrone. Again, the radical
+
C
b-fragment corresponding to [Ru
(cym)
G
(m/z
360.0292, mex =360.0298, 2 ppm) underlines that the metal–
peptide conjugate is formed via metal coordination to the
pyronato moiety. Additional secondary fragmentation prod-
ucts (A-fragments) were observed, which were assigned to
aldimine ions.
8
hydr, [8hydr +H]2+ and [8hydr +Na]2+ (all experimental and
theoretical signals are listed in Table S3 in the Supporting
Information). The notation 8hydr describes the mass signal
corresponding to [8ÀCl]+.
ETD relies on the transfer of electrons from a radical
anion to the analyte and leads to specific fragmentations of
the peptide backbone.[44] ETD of the doubly charged ion
Stability in aqueous solution: Aqueous stability of metallo-
drugs destined for clinical applications is a crucial prerequi-
site for drug development. In addition, when considering
the class of (thio)pyr(id)onato metallodrugs the ability to
resist ligand cleavage from the metal was determined as a
second key parameter for obtaining cytotoxic com-
pounds.[31,47] Therefore, the stability of the organometallic
complexes 5, 6 and the bioconjugate 8 was investigated in
aqueous solution by ESI-IT mass spectrometry over a
period of 48 h. The stability of 7 was not determined be-
cause the solubility in aqueous solution is insufficient for
performing cytotoxicity assays. Compounds 5 and 8 proved
to be stable over the entire incubation period. For each
compound the characteristic Mhydr mass signal was detected
corresponding to [MÀCl]+, where M=5, 6, 8. An additional
signal at m/z 373.94Æ0.02 (mex =374.03, <5%) was detect-
ed in the mass spectrum of 5, which can be assigned to a
species formed through loss of N2 from the azide during the
[8hydr +H]2+ yields primarily three species; a charge-reduced
+
species [8hydr +H]+, a [(cym)Ru
N
C
and the most abundant mass signal corresponding to
A
À
ACHTUNGTRENNUNG
leads primarily to arene cleavage, probably induced by a
one-electron reduction of the metal during the electron
transfer process (Figure S4 in the Supporting Information).
This result was underlined by additional CID investigation
of the isolated charge-reduced species (CRCID)[45] corre-
sponding to [8hydr
MS mass spectrum corresponds to [8hydr
This species was found at m/z 512.02 suggesting a ruthen-
ium(I) species, since ruthenium(II) would have led to the
detection of a signal at m/z 511.02. The mass signal of [Ru-
À
ACHTUNGTRENNUNG
(cym)]+. The most abundant signal in the
À
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
+
C
N
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
ronato moiety to the metal
center, however, no information
on the peptide sequence was
obtained.
On the other hand, CID of
the 8hydr parent signal with
75 eV yielded a more detailed
fragmentation
picture
(Figure 2). The IT and TOF in-
struments yielded similar frag-
mentation patterns in the CID
spectra, but a higher fragmenta-
tion efficiency was observed on
the IT instrument. In fact, com-
plete sequence coverage was
obtained confirming the amino
acid sequence of the modified
[Leu5]-enkephalin. CID leads to
strand breaks of the peptide
À
backbone at the amide CO N
bond, which generates predomi-
nately positively charged acyli-
um ions corresponding to
b-fragments and the comple-
Figure 2. CID tandem mass spectrum of 8hydr measured on an ESI-IT MS using a collision energy of 75 eV.
The identified fragments were labeled according to classical peptide fragment nomenclature; b denotes the
mentary amine y-fragments.[43] radical cation, whereas capital letters denote metallated fragments.
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Organometal–Peptide Bioconjugates
FULL PAPER
Figure 3. The investigated metabolic pathways of pyronato complexes are illustrated. Organometallic Ru pyronato complexes have to be considered as
anticancer prodrugs, which are activated by hydrolysis. For details regarding the stability of the [ub+Ru
AHCTUNGTRENNUNG
and other biological nucleophiles, see ref. [47].
Table 1. IC50 (50% inhibitory concentration) values in three human
cancer cell lines after 96 h.
spraying process. Compound 6 turned out to be somewhat
less stable in aqueous solution. After 24 h, ligand cleavage
led to the formation of the dinuclear species
Compound
IC50 [mm]
ACHTUNGTRENNUNG[Ru2ACHTUNGTRENNUNG(cym)2ACHTUNGTRENNUNG
(m-OCH3)3]+ (6%), which increased to 15% rela-
CH1
SW480
A549
n.d.[b]
>640
>640
>640
Ru-kojic acid[a]
234Æ21
264Æ2
429Æ10
>640
>640
tive to the 6hydr mass signal after 48 h (Figure 3 and Fig-
ure S5 in the Supporting Information). The m-methoxide
probably stems from the sample preparation, which involved
dilution with H2O/MeOH (1:1). Aliquots of the same incu-
bation solutions were additionally measured on the MaXis
UHR ESI-TOF, and the identities of the Mhydr complexes
were confirmed with an accuracy of ꢀ5 ppm as listed in the
Experimental Section.
1
2
4
5
6
8
194Æ11
>640
>640
168Æ35
7.6Æ2.7
13Æ5
224Æ24
170Æ32
>320
520Æ46
159Æ52
>320
[a] Taken from ref. [33]; [b] not determined.
In vitro anticancer activity: Organometallic Ru
(arene) anti-
range. The selective activity in the CH1 cell line parallels
the findings with previously reported pyronato com-
plexes.[31–33] It must be noted that pyronato metallodrugs dis-
play modest anticancer activity in vitro, whereas the cyto-
toxicity of the pyronato-based bioconjugate 8 is of the same
order of magnitude as that of S,O-bidentate thiopyronato
complexes.[31,33] Roughly comparable to the cytotoxicity of 8
in CH1 cells is that of the bioconjugate model 6, displaying
cancer agents containing bidentate (thio)pyronato ligands
exhibit an intriguing anticancer activity profile. The in vitro
cytotoxicity of these metallodrugs as expressed by the IC50
value covers a wide range from inactive to active represen-
tatives. The anticancer activity of these compounds depends
specifically on the ligand choice, and notably hydroxypyrone
derivatives are generally noncytotoxic.[32,33,36] Altering the
inner coordination sphere form O,O- to S,O-chelates leads
to a dramatic activity increase and IC50 values in the low mm
range.[33] Furthermore, the stability of the complexes in the
presence of biomolecules appears to be the second parame-
ter determining anticancer activity.[31,47] When peptide carri-
er systems are employed, organometallic Ru and also Os
conjugation often results in a decrease of the antiprolifera-
tive activity compared to the structurally related small mole-
cule metallodrugs.[8,9] In the present study, the antiprolifera-
tive activity was evaluated in ovarian (CH1), colon (SW480)
and non-small lung (A549) cancer cell lines by means of the
colorimetric MTT assay.
an IC50 value of 7.6
N
5 featuring the free azide is hardly active in vitro, while 7
was found to be too poorly soluble in aqueous media for
performing cytotoxicity assays. In our approach, triazole for-
mation in a pendant design seems to be an important pa-
rameter with respect to antiproliferative activity of the Ru
bioconjugate based on noncytotoxic pyrones. In fact, the an-
tiproliferative activity seems to be independent of the pep-
tide carrier, as 6 and 8 show similar anticancer activities in
vitro. Consequently, it is suggested that RuACTHNURTGNEU(GN cym) in combi-
nation with the triazolyl-pyronato linker represents the anti-
cancer-determining building block, which is also supported
by the fact that 4 and 5 are inactive in vitro. In principle,
this might offer a promising bioconjugation strategy to other
cell-penetrating peptides with potential retention of the bio-
logical activity.
The bioconjugate precursor 4 is anticancer inactive in
vitro, as are the ligands 1 and 2 and the hydrolysis product
(m-OH)3]+.[31,34] In
of the RuACHTUNGTRENNUNG(cym) moiety, that is, [Ru2ACHTUNRGTEG(NNNU cym)2ACHTNUGTRENNUGN
contrast, the bioconjugate 8 displayed high antiproliferative
activity and an IC50 value of 13(Æ5) mm was determined in
chemosensitive CH1 cells (Table 1). This represents, to the
best of our knowledge, the first example of a half-sandwich
Ru bioconjugate with antiproliferative activity in the low mm
Reactivity toward biomolecules: Encouraged by the promis-
ing results obtained in the in vitro assay, the interaction of
the bioconjugate 8 and the model complexes 5 and 6 with
Chem. Eur. J. 2013, 19, 9297 – 9307
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9301

C. G. Hartinger et al.
small biomolecules and proteins was studied. Since metallo-
drugs would usually be administered intravenously, they
may react with a broad range of biomolecules in the blood
stream, in particular plasma proteins, before reaching the
drug target. In order to estimate the reactivity of 5, 6 and 8
toward biological nucleophiles, they were incubated with the
amino acids glycine (Gly), l-cysteine (Cys) and l-histidine
(His) and the DNA model 9-ethylguanine (EtG) as well as
the proteins ubiquitin (ub) and cytochrome c (cyt). Mass
spectrometry has emerged as a powerful technique for the
analysis of the interactions between metallodrugs and bio-
molecules, both with respect to the nature of binding as well
as location of binding sites.[46–52] Herein, the experiments
were performed with ESI-ion trap (IT) and UHR ESI-time-
of-flight (TOF) mass spectrometry. ESI-IT MS was em-
ployed for analyzing small molecules, whereas high resolu-
tion ESI-TOF MS was used for protein interaction studies.
Similar to 5 and 6, the reactions of the bioconjugate 8
with amino acids are characterized by ligand cleavage from
formation seems to depend again on the steric demand of
the ligand, with 5 forming 36% of adducts after 48 h and
the bioconjugate 8 forming only 6% adducts with EtG. The
following trend for the reactivity toward EtG was observed:
5>6>8. The stability of the [6hydr +EtG]+ adduct in the
presence of His and Gly was additionally investigated (Fig-
ure S7 in the Supporting Information). For this purpose, 6
was incubated with EtG for 5 days prior to the addition of
2 equiv of either Gly or His. Addition of His led to quantita-
tive conversion of 6hydr and [6hydr +EtG]+ to the [Ru
ACHTUNGTRENNUNG(cym)-
AHCTUNGTRENNUNG
(His)]+ adduct within 3 h. Addition of Gly did not entirely
deplete the [6hydr +EtG]+ adduct within 24 h. However, Gly
seems to react with 6hydr forming a mixed ligand adduct with
(Gly)]+, which
free EtG corresponding to [RuACHTNUGTREN(NGUN cym)ACHNUTRTGEG(NNUN EtG)ACHTUNGTRENNUGN
becomes the most abundant species after 24 h. The absence
of an analogous adduct upon addition of His indicates a pos-
sible tridentate binding mode of His to the metal.
The subtle differences in reactivity of the bioconjugate 8
(and its cytotoxic model 6) on the one hand and 5 on the
other hand with small biomolecules suggest that slower ki-
netics of adduct formation and ligand cleavage from the
metal are associated with increased in vitro cytotoxic activi-
ty, which is also in accordance with an earlier study on pyro-
nato metallodrugs.[47]
Moreover, it seems that the compounds in this study inter-
act preferentially with amino acids rather than with nucleo-
bases, suggesting that Ru metallodrugs containing the pyro-
nato scaffold probably have a cellular target different from
DNA and more likely related to proteins (Figure 3). Conse-
quently, 5, 6 and 8 were additionally analyzed with respect
to their reactivity toward the proteins ubiquitin (ub) and cy-
tochrome c (cyt) by using UHR ESI-TOF mass spectrome-
try.
the metal and formation of [Ru(aa)ACHTUNGTRNEUNG
(cym)]+ (aa=amino
acid) adducts similarly to related Ru-pyr(id)onato metallo-
drugs (Figure 3).[29,47] Such a behavior was observed in the
presence of Cys and His, but also to some extent in the pres-
ence of Gly (Figure S6 and Table S3 in the Supporting Infor-
mation). Cys turned out to be the most potent amino acid
and induced quantitative ligand cleavage, which may be re-
lated to the trans effect of the thiol group. Initially, several
Cys adducts were detected in the mass spectra, which con-
vert after 48 h, however, to the thermodynamically most
(Cys)]+. His with
stable adduct corresponding to [RuACTHNUTRGENNUG(cym)ACHTUNGTRENNUGN
its imidazole side chain was also able to form metal adducts
through ligand cleavage, although the kinetics of this reac-
tion were slightly slower than for Cys. His leads to quantita-
tive depletion of the signals assignable to 5hydr, 6hydr and 8hydr
in the mass spectra within 6 h. The conjugate model 6hydr
was slightly more resistant to ligand cleavage, which was re-
Binding to ub was found to be accompanied by ligand
cleavage from the metal, giving rise to the characteristic
[ub+RuACHTUNGTRNEUNG
(cym)]+ adducts when incubating ub with the bio-
flected in a lower percentage of [Ru
G
N
conjugate 8 or its models 5 or 6 (Table S3 in the Supporting
Information). Ubiquitin features two probable binding part-
ners for a Ru ion, namely the N-terminal methionine
(Met1) and histidine at position 68 (His68). It was suggested
that Met1 may act as a bidentate binding partner to a
formation (68%) compared to 5hydr and 8hydr (>85%) after
1 h. In contrast to previous reports, the reaction with Gly
was also characterized by adduct formation and ligand
cleavage from the metal, albeit to a low extent. Interestingly,
mono- and bis-adducts were formed during the reaction cor-
metalloACHTUNGTRNEUNG
drug,[50,53] which may increase reaction kinetics and
responding to [Ru
(cym)
E
(cym)
N
also adduct stability. Mono-adducts are most probably
formed by metallation of Met1, as recently determined in a
occurrence of bis-adducts is probably related to the reduced
steric demand of the amino acid. However, Gly was not
able to completely consume the free complexes over an in-
cubation period of 48 h (Figure S6 in the Supporting Infor-
mation). Binding of Gly was least pronounced for the bio-
conjugate 8 possibly due to steric reasons.
Furthermore, the reaction with EtG, used as a DNA
model, resulted in the formation of [Mhydr +EtG]+ adducts
(M=5, 6 and 8) possibly via N7 of guanine and was charac-
terized by ligand retention, which can be explained by the
monodentate character of the interaction. Despite the in-
creased molar ratio (2:1 EtG/Ru), EtG adducts did not
exceed a relative abundance of 36% (relative to Mhydr) and
only monoadducts were observed within 48 h. EtG adduct
top-down ESI-MS analysis of the [ub+RuACHTUNGTRENNUNG
(cym)]+
adduct.[54] The ESI-TOF MS experiments yielded detailed
information on the adduct types, that is, bis-adducts corre-
sponding to [ub+2RuACTHNUTRGNEUNG
(cym)]+ were also detected in small
amounts indicating metallation of His68 to a minor degree.
Interestingly, bis-adducts with ub were detected only at 1
and 6% relative abundance for 8 and 6, respectively, where-
as they were present at 12% relative abundance for 5.
The incubation with cyt at a molar ratio of 3:1 was char-
acterized by two types of mono-adducts. Similar to the ex-
periments with ub, the most abundant adduct corresponds
to [cyt+Ru
of the metallodrug, bis-adducts were not observed. Addi-
(cym)]+. Despite the addition of a molar excess
ACHTUNGTRENNUNG
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Chem. Eur. J. 2013, 19, 9297 – 9307

Organometal–Peptide Bioconjugates
FULL PAPER
MeOH, 0.2% formic acid) in the broadband ESI-TOF mass
spectrum, most of the charge states for free cyt are found
between +12 and +19 referring to entirely denatured pro-
+
tein (labeled with *, Figure 4a).[55] The adduct [cyt+5hydr
]
displays roughly the same charge distribution, indicating
that mono-dentate binding of the organometallic fragment
to the protein does not influence its tertiary structure (la-
beled with D, Figure 4a). Interestingly, the [cyt+RuACHTUNGTRENNUNG
(cym)]+
adduct shows two charge state populations, with the first
population showing charge states of +13 to +17 indicative
of a completely unfolded protein (Figure 4a, grey) similar to
free cyt. In contrast, the second population with charge
states from +8 to +13 refers to a partially folded protein
and suggests a cross-linking of the protein backbone by the
Ru
Ru(cym) binding to His26 and/or His33, which does not
affect the protein tertiary structure. On the other hand, the
second population may correspond to Ru(cym) binding to
ACHTUNGTREN(NGNU cym) fragment. The first population may correspond to
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
His33 and Met65, leading to the observed cross-linking
effect in MS experiments. This behavior was observed exclu-
sively for 5, 6 and 8 incubated with cyt but not with ub. This
is an intriguing aspect, since the function of proteins highly
depends on their tertiary structure and consequently, protein
cross-linking might play a role in the mode of action of the
investigated metallodrugs.
Figure 4. a) Charge state distributions observed in the broadband ESI-
TOF mass spectrum of the incubation mixture containing 5 and cyt at a
3:1 metal-to-protein ratio. Free cyt is detected at high charge states (*)
+
Conclusion
indicating complete loss of the tertiary structure, similar to [cyt+5hydr
]
(D), which can only bind in a monodentate coordination mode. On the
other hand, the lower charge state distribution of the [cyt+Ru
(cym)]+
adduct (grey) indicates the cross-linking ability of the Ru(cym) moiety.
The spectrum was recorded under denaturing conditions. b) The decon-
voluted UHR ESI-TOF mass spectrum of the same experiment is shown.
A cyt adduct with intact 5hydr was detected at 12760.25 Da.
AHCTUNGTRENNUNG
Attaching a cytotoxic moiety to peptide carrier systems has
attracted much interest, for example, because this opens up
new possibilities for drug targeting with cell-penetrating
peptides. However, conjugation of organometallic Ru and
also Os anticancer agents to a peptide carrier usually result-
ed in a reduction of the antiproliferative activity compared
to the small metallodrug alone. Here, a metal bioconjuga-
tion strategy is pursued in which the opposite effect was ob-
served, and to the best of our knowledge, the first organo-
metallic half-sandwich Ru bioconjugate displaying antiproli-
ferative activity in the low micromolar range (in CH1 ovari-
an cancer cells) is reported, whereas the non-metallated
peptide 4 is completely inactive. The comparison of the Ru
bioconjugate 8 with the smaller bioconjugate models 5 and
6, which can be considered building blocks of 8, revealed
that the anticancer potency seems to be independent of the
peptide carrier, whereas the triazole moiety is essential, sug-
AHCTUNGTRENNUNG
+
tional [cyt+Mhydr
]
adducts were observed in all three cases
and were characterized by ligand retention, for example,
+
[cyt+5hydr
]
(13%, Figure 4b). After 48 h, the relative
abundance of the combined mono-adducts formed with the
bioconjugate 8 and the model 6 corresponds to 41(Æ4)%,
compared to 64% for the inactive model 5.
The kinetics of adduct formation are significantly lower
for cyt than for ub, which is attributed to the presence of
different binding partners in these proteins. Cytochrome c
contains three accessible metal binding sites, His26, His33
and Met65, that are mono-dentate binding partners similar
to His68 in ub. It is, therefore, assumed that the ability of bi-
dentate Met1 binding in ub is responsible for the kinetic dif-
ferences of adduct formation observed for ub and cyt. Fur-
thermore, the lack of potential bidentate binding partners in
cyt may be responsible for the observation of stable
gesting the RuACTHUNTGRNEUNG(cym) species with the triazolyl-pyronato
linker as the minimal structural requirement for activity in
vitro. The identification of this cytotoxic moiety may open
an approach to a wider range of anticancer active Ru bio-
conjugates. High resolution top-down ESI-MS was per-
formed confirming the expected amino acid sequence, linker
modification and metallation site in 8 and providing a useful
approach for characterizing organometal–peptide bioconju-
gates. Finally, ESI-MS studies provided insights into their
molecular reactivity toward biomolecules and proteins, and
a delayed reactivity of the bioconjugate toward the biomole-
M
hydr–cyt adducts. Again, the slightly slower kinetics of
adduct formation with proteins for 6 and 8 compared to 5
are reflected in their increased antiproliferative activity in
vitro.
When comparing the charge state distributions of free cyt
and the mono-adduct under denaturing conditions (50%
Chem. Eur. J. 2013, 19, 9297 – 9307
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9303

C. G. Hartinger et al.
and tables were used: structure solution SHELXS-97,[59] refinement
SHELXL-97,[59] molecular diagrams Mercury CSD 3.0.[60]
cules seems to be reflected in an increased in vitro cytotox-
icity.
Interaction with biomolecules: Samples for ESI-MS were analyzed by
direct infusion at a flow rate of 3 mLminÀ1 and at typical concentrations
of 1–5 mm. Protein samples were analyzed under denaturing conditions by
diluting them with water/methanol/formic acid (50:50:0.2), whereas the
reference complexes and small molecule samples were diluted with
water/methanol (50:50). Stock solutions of compound 5 and 8 were pre-
pared in H2O, whereas 6 was dissolved in aqueous solution containing
1% DSMO. The following molar ratios were used for interaction studies
at 378C and pH 5.5: ub/M (1:2), cyt/M (1:3), amino acid/M (1:1) and
EtG/M (2:1), where M is the respective Ru metallodrug. Mass spectra
were recorded after 1, 3, 6, 24 and 48 h. In general, relative intensities
correspond to percentages of the area under peaks of the sum of all as-
signable metal signals in the spectrum.
Experimental Section
Materials and methods: All reactions were carried out in dry solvents
and under inert atmosphere. Chemicals obtained from commercial sup-
pliers were used as received and were of analytical grade. Methanol and
dichloromethane were dried using standard procedures. RuCl3·3H2O
(40.4%) was purchased from Johnson Matthey; ubiquitin (bovine eryth-
rocytes), horse heart cytochrome c, dimethyl sulfoxide (DMSO) and
9-ethylguanine from Sigma; N,N-dimethylformamide (extra dry), 2-prop-
anol, propargylbromide (80%, stabilized in toluene), a-terpinene and tet-
rahydrofuran from Acros; copper(II) sulfate pentahydrate, phenol and
3,4,5-trimethylphenol from Fisher; l-histidine and potassium carbonate
from Merck; l-methionine, sodium ascorbate, sodium azide, sodium
methoxide, thionyl chloride and triphenylphosphite from Sigma–Aldrich
and kojic acid from TCI Europe. Methanol (VWR Int., HiPerSolv
CHROMANORM), formic acid (Fluka) and MilliQ H2O (18.2 MW, Ad-
vantage A10, 185 UV Ultrapure Water System, Millipore, France) were
used in ESI-MS studies. The dimer bis[dichlorido(h6-p-cymene)ruthen-
Cell lines and culture conditions: CH1 cells (ovarian adenocarcinoma,
human) were provided by Lloyd R. Kelland (CRC Centre for Cancer
Therapeutics, Institute of Cancer Research, Sutton, UK), SW480 (adeno-
carcinoma of the colon, human) and A549 (non-small cell lung cancer,
human) cells were provided by Brigitte Marian (Institute of Cancer Re-
search, Department of Medicine I, Medical University of Vienna, Aus-
tria). All cell culture reagents were purchased from Sigma–Aldrich. Cells
were grown in 75 cm2 culture flasks (Starlab) as adherent monolayer cul-
tures in Eagleꢂs minimal essential medium (MEM) supplemented with
heat-inactivated fetal calf serum (10%), sodium pyruvate (1 mm), l-gluta-
mine (4 mm) and non-essential amino acids (1% v/v; from 100ꢃ ready-
to-use stock). Cultures were maintained at 378C in a humidified atmo-
ACHTUNGTRENNUNG
ium(II)][56,57] and the ligand 2-(azidomethyl)-5-hydroxy-4H-pyran-4-one[37]
were synthesized as previously described.
NMR spectra were recorded at 258C on a Bruker FT NMR spectrometer
Avance IIITM 500 MHz at 500.10 (1H), 202.63 MHz (31P{1H}) and
125.75 MHz (13C{1H}) and 2D NMR data were collected in a gradient-en-
hanced mode. Hydrogen and carbon atoms were numbered according to
crystal structure numbering. Elemental analysis was carried out on a
Perkin–Elmer 2400 CHN elemental analyzer by the Laboratory for Ele-
mental Analysis, Faculty of Chemistry, University of Vienna. An analyti-
cal HPLC system (TM100, Dionex) was equipped with a reversed-phased
column (Zorbax Eclipse Plus C18, Agilent, 5 mm pore size, 4.6 mm inner
diameter and 250 mm column length), that was thermostated at 258C,
and a UV-detector (UVD 170U, Dionex). The flow rate was 1 mLminÀ1
using a H2O/MeOH (95:5) eluent ratio for the first 3 min. Afterwards, a
linear gradient was employed starting with H2O/MeOH (95:5) and
ending with H2O/MeOH (5:95) for 15 min, which was then kept for
5 min. TFA (0.1%) was added to all eluents. The HPLC was coupled to
an AmaZon ESI-ion trap mass spectrometer (Bruker Daltonics GmbH,
Bremen, Germany) and controlled by Chromeleon 6.8 software (Thermo
Scientific, Bremen, Germany). Electrospray ionization mass spectra were
recorded on a Bruker AmaZon SL, a Bruker AmaZon Speed ETD ion
trap (IT) and on a UHR MaXis time-of-flight (TOF) mass spectrometer
(Bruker Daltonics GmbH, Bremen, Germany). Collision-induced dissoci-
ation (CID) was performed using 50–75 eV collision energy, whereas
100–300 ms reaction times were employed for electron transfer dissocia-
tion (ETD) experiments on the AmaZon Speed ETD. Data were ac-
quired and processed using Compass 1.3 and Data Analysis 4.0 (Bruker
Daltonics GmbH, Bremen, Germany). Deconvolution was obtained by
applying the maximum entropy algorithm with a 0.1 m/z mass step and
0.5 m/z instrument peak width for the IT and automatic data point spac-
ing and 30000 instrument resolving power for the TOF. X-ray diffraction
AHCTUNGERTGsNNUN phere containing 95% air and 5% CO2.
Cytotoxicity in cancer cell lines: Cytotoxicity was determined by the col-
orimetric MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
bromide; Sigma–Aldrich) microculture assay. For this purpose, cells were
harvested from culture flasks by trypsinization and seeded in aliquots
(100 mL) of complete MEM (see above) into 96-well microculture plates
(Starlab). Cell densities of 1.0ꢃ103 cells per well (CH1), 2.0ꢃ103 cells per
well (SW480) and 3.0ꢃ103 cells per well (A549) were chosen in order to
ensure exponential growth of untreated controls throughout the experi-
ment. Cells were allowed to settle and resume exponential growth for
24 h. Compound 1 was dissolved directly in complete MEM, whereas all
other test compounds were dissolved in DMSO first and then serially di-
luted in complete MEM such that the effective DMSO content did not
exceed 0.5% (v/v). Dilutions were added in aliquots (100 mL) to the mi-
crocultures, and cells were exposed to the test compounds for 96 h. At
the end of the exposure period, all media were replaced with 100 mL per
well of a 6:1 mixture of RPMI1640 medium (supplemented with 10%
heat-inactivated fetal calf serum and 2 mm l-glutamine) and MTT solu-
tion (5 mgmLÀ1 phosphate-buffered saline). After incubation for 4 h, the
supernatants were removed, and the formazan product formed by viable
cells was dissolved in 150 mL DMSO per well. Optical densities at 550 nm
were measured with a microplate reader (BioTek ELx808), by using a
reference wavelength of 690 nm to correct for unspecific absorption. The
quantity of viable cells was expressed in terms of T/C values by compari-
son to untreated control microcultures, and 50% inhibitory concentra-
tions (IC50) were calculated from concentration-effect curves by interpo-
lation. Evaluation is based on means from at least three independent ex-
periments, each comprising three replicates per concentration level.
measurements of single crystals were performed on
a Bruker X8
Synthesis
APEX II CCD diffractometer at 100 K. CCDC-902337 (2), and 902338
(5) contain the supplementary crystallographic data for this paper. These
data can be obtained free of charge from The Cambridge Crystallograph-
ic Data Centre via www.ccdc.cam.ac.uk/data_request/cif. The single crys-
tals of 2 and 5 were positioned at 35 mm from the detector. A total of
1141 frames for 60 s over 18 were measured for 2 and 2039 frames for
20 s over 18 for 5. The data were processed using the SAINT Plus soft-
ware package.[58] Crystal data, data collection parameters, and structure
refinement details are given in Table S1 in the Supporting Information.
The structure was solved by direct methods and refined by full-matrix
least-squares techniques. Non-hydrogen atoms were refined with aniso-
tropic displacement parameters. H atoms were inserted at calculated po-
sitions and refined with a riding model. The following software programs
(Prop-2-yn-1-yloxy)benzene: Phenol (3.00 g, 32 mmol) and propargyl bro-
mide (3.56 mL, 32 mmol) were added in a 100 mL round flask containing
dry DMF (30 mL) and potassium carbonate (6.62 g, 48 mmol). The reac-
tion mixture was stirred for 18 h under argon atmosphere at room tem-
perature. The product was extracted with dichloromethane (3ꢃ50 mL),
the combined extracts were washed with H2O (50 mL) and dried over an-
hydrous Na2SO4. The solution was concentrated yielding the crude prod-
uct as a yellow oil, which was purified by flash-column chromatography
using n-hexane/dichloromethane as eluent (1:1.1). Yield: 2.20 g (52%);
1H NMR (500.10 MHz, [D6]DMSO): d=7.31 (dd, 3J
G
3J
4J
ACHTUNGTRENNUNG
ACHUTNGRENNUG CAHTUNGTRENNUNG
(H,H)=2 Hz, 2H, CH2), 3.55 ppm (t, 4J
À
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Organometal–Peptide Bioconjugates
FULL PAPER
13C{1H} NMR (125.75 MHz, [D6]DMSO): d=157.1 (CAr1’), 129.4 (CAr2’,6’),
1.8 mmol) in deoxygenated, argon-flushed H2O/THF (7.5 mL, 1:2). The
reaction mixture was stirred for 24 h at room temperature after which
the solvent was evaporated. The residue was dissolved in ethyl acetate,
dried over anhydrous Na2SO4 and filtered. After removal of the solvent
and recrystallization from iPrOH, the yellowish product was obtained
after filtration and dried in vacuo. Yield: 260 mg (64%); 1H NMR
(500.10 MHz, [D6]DMSO): d=9.28 (s, 1H, ÀOH), 8.29 (s, 1H, H-8), 8.05
(s, 1H, H-6), 6.67 (s, 2H, HAr2’,6’), 6.38 (s, 1H, H-3), 5.60 (s, 2H, H-7),
À ꢁ
À
121.09 (CAr4’), 114.7 (CAr3’,5’), 78.0 ( C CH), 55.2 ppm ( CH2).
1,2,3-Trimethyl-5-(prop-2-yn-1-yloxy)benzene: In a 100 mL round flask,
3,4,5-trimethylphenol (1.36 g, 10 mmol) was dissolved in acetone
(50 mL). Propargyl bromide (0.90 mL, 11 mmol) and potassium carbo-
nate (1.52 g, 11 mmol) were added. The reaction mixture was refluxed at
658C for 24 h. Water (20 mL) was added and the pH adjusted to 12 with
NaOH. The product was quickly extracted with dichloromethane (3ꢃ
20 mL). The combined organic phase was dried over anhydrous Na2SO4
and subsequently filtered. The solvent was removed yielding the product
À
À
5.08 (s, 2H, H-10), 2.19 (s, 6H, CAr3’,5’ CH3), 2.03 ppm (s, 3H, CAr4’
CH3); 13C{1H} NMR (125.75 MHz, [D6]DMSO): d=173.5 (C-4), 160.5 (C-
2), 155.2 (CAr1’), 145.9 (C-5), 143.4 (C-9), 139.9 (C-6), 136.9 (CAr3’,5’), 126.7
(CAr4’), 125.1 (C-8), 113.7 (CAr2’,6’), 112.9 (C-3), 60.7 (C-10), 49.8 (C-7),
1
as a yellow oil. Yield: 1.61 g (89%); H NMR (500.10 MHz, [D6]DMSO):
d=6.62 (s, 2H, HAr), 4.69 (d, 4J
G
À
20.3 (CAr4’ CH3), 14.2 ppm (CAr3’5’ CH3); MS (ESI+): m/z 342.10
À
À
À ꢁ
G
AHCTUNGTRENNUNG
[M+H]+ (mex =342.15).
CAr4’ CH3); 13C{1H} NMR (125.75 MHz, [D6]DMSO): d=154.3 (CAr1’),
À ꢁ
136.9 (CAr3’,5’), 126.7 (CAr4’), 113.8 (CAr2’,6’), 77.7 ( C CH), 55.1 ( CH2),
5-Hydroxy-2-{((4-(N-tyrosinyl-glycinyl-glycinyl-phenylallanyl-leucinyl-
NH2)propanamido)-1H-1,2,3-triazol-1-yl)methyl}-4H-pyran-4-one
À
20.4 (CAr3’,5’ CH3), 14.2 ppm (CAr4’ CH3); MS (ESI ): m/z 175.13
[M+H]+ (mex =175.11).
(4):
The Fmoc-protected [Leu5]-enkephalin was manually prepared on Rink
amide resins (500 mg, 0.36 mmol) according to standard solid-phase syn-
thesis procedures[16, 61] using a fourfold excess of the Fmoc-protected
amino acid. Each coupling was performed in the presence of TBTU
(433 mg, 1.35 mmol), HOBT (192 mg, 1.35 mmol) and DIPEA (618 mL,
0.36 mmol) and 4-pentynoic acid was coupled in the same manner. The
solid-phase CuAAC was performed adapting a procedure by Tornoe
et al.[39] and Koester et al.[62] Compound 1 (119 mg, 0.71 mmol) was dis-
solved in ACN/DCM (2:1, 6 mL) and purged with N2. CuI (135 mg,
0.71 mmol) was dissolved in ACN/DCM (2:1, 8 mL) and both compounds
were transferred into the syringe containing the resin (500 mg,
0.36 mmol). Finally, DIPEA (2.98 mL, 18 mmol, 50 equiv) was taken up
in the syringe and the mixture was shaken at room temperature in the
absence of light for 18 h under N2. After washing with DMF (5ꢃ4 mL,
2 min), Cu impurities were removed with a 0.14m solution of cupral
(0.71 mmol) in DMF and again washed with DMF (5ꢃ4 mL, 2 min) and
DCM (5ꢃ4 mL, 2 min). The modified peptide was cleaved from the resin
by treatment with 7 mL of TFA/TIS/H2O (95:2.5:2.5) for 2 h and then
precipitated by addition of cold diethyl ether. After centrifugation, the
solution was decanted and the product was washed twice with cold dieth-
yl ether and finally dried in vacuo. After purification by preparative
HPLC and lyophilization, the product was obtained as a colourless solid.
Yield: 166 mg (58%); 1H NMR (500.10 MHz, [D6]DMSO): d=9.18 (brs,
ACHTUNGTRENNUNG
2-(Azidomethyl)-5-hydroxy-4H-pyran-4-one (1): The procedure of Atkin-
son et al. was used with minor modifications.[37] Step 1: thionyl chloride
(19.5 mL, 271 mmol) was added to kojic acid (7.00 g, 50 mmol) under vig-
orous stirring at 08C. After complete addition, the reaction was stirred
for 4 h and the remaining thionyl chloride was removed under reduced
pressure using a cooling trap. The dry yellowish reaction product was sus-
pended in n-hexane and filtered. Recrystallization from iPrOH yielded
colorless crystals of 2-(chloromethyl)-5-hydroxy-pyran-4H-one, which
were dried in vacuo. Yield: 6.60 g (84%); 1H NMR (500.10 MHz,
À
[D6]DMSO): d=9.29 (s, 1H, OH), 8.12 (s, 1H, H-6), 6.56 (s, 1H, H-3),
4.66 ppm (s, 2H, H-7). Step 2: in a round-bottom flask, sodium azide
(1.22 g, 19 mmol) was suspended in dry DMF (12.3 mL) under argon at-
mosphere at 08C. Then 2-(chloromethyl)-5-hydroxy-pyran-4H-one
(3.00 g, 19 mmol) was slowly added and the reaction mixture became
turbid after several minutes. The suspension was allowed to warm to
room temperature and was stirred in the absence of light for 24 h, before
it was slowly poured into H2O (7.5 mL, 08C). A colorless solid precipitat-
ed, which was separated by filtration and dried in vacuo. Yield: 2.71 g
1
À
(87%); H NMR (500.10 MHz, [D6]DMSO): d=9.24 (s, 1H, OH), 8.11
(s, 1H, H-6), 6.45 (s, 1H, H-3), 4.42 ppm (s, 2H, H-7); 13C{1H} NMR
(125.75 MHz, [D6]DMSO, 258C): d=174.1 (C-4), 162.3 (C-2), 146.4
ACHTUNGTRENNUNG(C-5), 140.5 (C-6), 113.0 (C-3), 50.6 ppm (C-7); elemental analysis calcd
1H, OHTyr), 8.26 (t, 3J
(H,H)=6 Hz, 1H, NHGly), 8.14 (d, 3J
(H,H)=
À
À
for C6H5N3O3: C 43.12, H 3.02, N 25.14; found: C 43.11, H 2.76, N 24.97;
3
MS (ESI+): m/z 168.11 [M+H]+ (mex =168.04).
À
À
8 Hz, 1H, NHLeu), 8.06 (d, J
G
8), 7.99 (t, 3J
(H,H)=6 Hz, 1H, NHPhe), 7.96 (d, 3J
E
À
5-Hydroxy-2-{(4-(phenoxymethyl)-1H-1,2,3-triazol-1-yl)methyl}-4H-
pyran-4-one (2): Copper sulfate pentahydrate (30 mg, 10 mol%) and
sodium ascorbate (95.1 mg, 40 mol%) were suspended in deoxygenated
H2O (2.5 mL) and stirred until the reaction mixture turned yellow. This
reaction mixture was added to a suspension of 1 (200 mg, 1.2 mmol) and
(prop-2-yn-1-yloxy)benzene (237 mg, 1.8 mmol) in deoxygenated argon-
flushed H2O/THF (7.5 mL, 1:2). The reaction mixture was stirred for 24 h
at room temperature under argon atmosphere and then the solvent was
removed. The residue was dissolved in ethyl acetate, dried over anhy-
drous Na2SO4 and filtered. After removal of the solvent and recrystalliza-
tion from iPrOH, the yellowish product was obtained after filtration and
dried in vacuo. Yield: 165 mg (46%); 1H NMR (500.10 MHz,
NHGly), 7.81 (s, 1H, H-6), 7.24 (d, 3J
(H,H)=5 Hz, 4H, HAr,Phe), 7.17–
À
7.14 (m, 1H, HAr,Phe), 7.09 (s, 1H, NH2), 7.00 (d, 3J
G
À
À
H
Ar,Tyr), 6.97 (s, 1H, NH2), 6.62 (d, 3J
(H,H)=9 Hz, 2, HAr,Tyr), 6.36 (s,
1H, H-3), 5.49 (s, 2H, H-7), 4.53–4.48 (m, 1H, Ha,Phe), 4.42–4.39 (m, 1H,
H
a,Tyr), 4.22–4.17 (m, 1H, Ha,Leu), 3.72–3.69 (m, 4H, Ha,Gly), 3.03 (dd, 2J-
(H,H)=14 Hz, 3J(H,H)=5 Hz, 2H, Hb,Tyr), 2.92 (dd, 2J(H,H)=14 Hz, 3J-
(H,H)=5 Hz, 2H, Hb,Phe), 2.76 (t, J
(H,H)=9 Hz, 2H, Hb,pent), 1.60–1.52 (m, 1H, Hg,Leu), 1.48–1.45 (m, 2H,
b,Leu), 0.84 ppm (dd, 2J(H,H)=26 Hz, 3J
(H,H)=7 Hz, 6H, Hd,Leu); MS
(ESI+): m/z 802.23 [M+H]+ (mex =802.35), MS (ESIÀ): m/z 914.44
[MÀH+TFA]À (mex =914.33); anal. HPLC: 11.26 min.
General procedure for the synthesis of [chlorido(h6-p-cymene)-
(pyronato)ruthenium(II)] complexes: The pyrone ligand (1–1.1 equiv) and
A
R
ACHTUNGTRENNUNG
3
3
E
ACHTUNGRTNEGNUN(H,H)=9 Hz, 2H, Ha,pent), 2.64 (t, J-
AHCTUNGTRENNUNG
H
N
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
À
[D6]DMSO): d=9.28 (s, 1H, OH), 8.33 (s, 1H, H-8), 8.06 (s, 1H, H-6),
3
AHCTUNGTRENNUNG
7.30 (t, J
G
R
6.95 (t, 3J
ACHTUNGTRENNUNG(H,H)=8 Hz, 1H, HAr4’), 6.40 (s, 1H, H-3), 5.61 (s, 2H, H-7),
sodium methoxide (1–1.1 equiv) were suspended in dry methanol under
argon atmosphere at room temperature and stirred for 15 min. Then bis-
5.16 ppm (s, 2H, H-10); 13C{1H} NMR (125.75 MHz, [D6]DMSO): d=
173.5 (C-4), 160.5 (C-2), 157.9 (CAr1’), 145.9 (C-5), 143.1 (C-9), 139.9 (C-
6), 129.4 (CAr3’,5’), 125.3 (C-8), 120.8 (CAr4’), 114.6 (CAr2’,6’), 113.0 (C-3),
60.8 (C-10), 49.9 ppm (C-7); elemental analysis calcd for
C15H13N3O4·0.4H2O: C 58.78, H 4.54, N 13.71; found: C 59.05, H 4.14,
N 13.32; MS (ESIÀ): m/z 298.15 [MÀH]À (mex =298.08).
AHCTUNGTRENNUNG
[dichlorido(h6-p-cymene)ruthenium(II)] (0.5 equiv) was added and the
clear, orange-red colored solution was stirred for further 6–18 h. The sol-
vent was removed under reduced pressure, the residue was dissolved in
dichloromethane, filtered and the filtrate was concentrated to a final
volume of about 2–3 mL. The product was precipitated by addition of
n-hexane, filtered and dried in vacuo.
5-Hydroxy-2-{(4-[(3,4,5-trimethylphenoxy)methyl]-1H-1,2,3-triazol-1-yl)-
methyl}-4H-pyran-4-one (3): Copper sulfate pentahydrate (30 mg,
10 mol%) and sodium ascorbate (95 mg, 40 mol%) were suspended in
deoxygenated H2O (2.5 mL) and stirred until the mixture turned yellow.
This reaction mixture was then added to the suspension of 1 (200 mg,
1.2 mmol) and 1,2,3-trimethyl-5-(prop-2-yn-1-yloxy)benzene (314 mg,
[{2-(Azidomethyl)-5-oxo-kO-4H-pyronato-kO}chlorido(h6-p-cymene)ru-
thenium(II)] (5): The reaction was performed according to the general
procedure using pyrone 1 (40 mg, 0.23 mmol), sodium methoxide (13 mg,
0.23 mmol),
bis[dichlorido(h6-p-cymene)ruthenium(II)]
(70 mg,
0.16 mmol) and methanol (8 mL). The product was isolated as brownish
Chem. Eur. J. 2013, 19, 9297 – 9307
ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
9305

C. G. Hartinger et al.
crystals. Reaction time: 18 h. Yield: 65 mg (65%); 1H NMR
(500.10 MHz, [D6]DMSO): d=7.93 (s, 1H, H-6), 6.68 (s, 1H, H-3), 5.70–
5.68 (m, 2H, HCym3’,5’), 5.42–5.40 (m, 2H, HCym2’,6’), 4.43 (s, 2H, H-7), 2.76
3J
14 Hz, 3J
2H, Hb,Phe), 2.78–2.74 (m, 2H, Hb,Pent), 2.66–2.61 (m, 2H, Ha,Pent), 2.16 (s,
3H, HCyma H-1’), 1.60–1.52 (m, 1H, Hg,Leu), 1.46 (t, 3J
(H,H)=7 Hz, 2H,
b,Leu), 1.27 (d, 3J(H,H)=7 Hz, 6H, HCymc), 0.85 ppm (dd, 3J
(H,H)=
N
ACHTUNGTRENNUNG(H,H)=
R
ACHTUNGTRENNUNG
(sept, 3J
ACHTUNGTRENNUNG
(H,H)=7 Hz, 1H, HCymb), 2.14 (s, 3H, HCyma), 1.25 ppm (dd, 3J-
AHCTUNGTRENNUNG
(H,H)=7 Hz, 4J(H,H)=2 Hz, 6H, HCymc); 13C{1H} NMR (125.75 MHz,
N
ACHTUNGTRENNUNG
N
[D6]DMSO): d=185.2 (C-4), 161.5 (C-2), 159.9 (C-5), 140.8 (C-6), 108.8
(C-3), 97.9 (CCym1’), 94.9 (CCym4’), 79.9 (CCym3’,5’), 76.9 (CCym2’,6’), 49.8 (C-7),
30.5 (CCymb), 22.0 (CCymc), 17.9 ppm (CCyma); elemental analysis calcd for
H]2+ (mex =518.6794, 4 ppm); anal. HPLC: 12.2 min.
C16H18ClN3O3Ru·1= H2O: C 43.39, H 4.25, N 9.48; found: C 43.79, H 3.97,
3
N 9.09; MS (ESI+): m/z 402.0377 [MÀCl]+ (mex =402.0391, 4 ppm),
374.0311 [MÀClÀN2]+ (mex =374.0329, 5 ppm).
A
Acknowledgements
zol-1-yl]methyl)-4H-pyronato-kO}ruthenium(II)] (6): The reaction was
performed according to the general procedure using pyrone 2 (100 mg,
0.33 mmol), sodium methoxide (18 mg, 0.34 mmol), bis[dichlorido(h6-p-
cymene)ruthenium(II)] (99 mg, 0.16 mmol) and methanol (10 mL). The
product was isolated as yellow microcrystals. Reaction time: 18 h. Yield:
115 mg (63%); 1H NMR (500.10 MHz, [D6]DMSO): d=8.30 (s, 1H, H-
We would like to thank COST D39 and CM1105 and the Johanna
Mahlke nꢄe Obermann Foundation for financial support, and the Mass
Spectrometry Center (Faculty of Chemistry, University of Vienna) for
access to the UHR ESI-TOF mass spectrometer. We gratefully acknowl-
edge Alexander Roller for collecting the X-ray diffraction data. S.M.M.
thanks in particular S. David Kçster, H. Bauke Albada and Kathrin
Klein for their support at RUB, and Elisabeth Jirkovsky for several ESI-
MS measurements.
8), 7.87 (s, 1H, H-6), 7.30 (t, 3J
(H,H)=8 Hz, 2H, HAr2’,6’), 6.95 (t, 3J
1H, H-3), 5.69–5.67 (m, 2H, HCym3’,5’), 5.62 (s, 2H, H-7), 5.41–5.39 (m,
2H, HCym2’,6’), 5.14 (s, 2H, H-10), 2.76 (sept, 3J
(H,H)=7 Hz, 1H, HCymb),
2.13 (s, 3H, HCyma), 1.25 ppm (d, 3J(H,H)=7 Hz, 6H, HCymc); 13C{1H}
ACHTUNGTRENNUNG
A
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
NMR (125.75 MHz, [D6]DMSO, 258C): d=185.2 (C-4), 160.1 (C-2),
160.0 (C-5), 157.9 (CAr1’), 143.2 (C-9), 140.8 (C-6), 129.4 (CAr3’,5’), 126.0
(C-8), 120.8 (CAr4’), 114.6 (CAr2’,6’), 109.1 (C-3), 97.9 (CCym1’), 94.9 (CCym4’),
79.9 (CCym3’,5’), 77.0 (CCym2’,6’), 60.7 (C-10), 49.6 (C-7), 30.6 (CCymb), 22.0
[1] N. Yamamoto, N. S. Bryce, N. Metzler-Nolte, T. W. Hambley, Bio-
conjugate Chem. 2012, 23, 1110–1118.
[2] F. Milletti, Drug Discovery Today 2012, 17, 850–860.
[3] C. G. Hartinger, N. Metzler-Nolte, P. J. Dyson, Organometallics
2012, 31, 5677–5685.
(CCymc),
17.9 ppm
(CCyma);
elemental
analysis
calcd
for
[4] Q. Geng, X. Sun, T. Gong, Z. R. Zhang, Bioconjugate Chem. 2012,
23, 1200–1210.
[5] K. L. Horton, K. M. Stewart, S. B. Fonseca, Q. Guo, S. O. Kelley,
Chem. Biol. 2008, 15, 375–382.
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91.
[7] H. B. Albada, F. Wieberneit, I. Dijkgraaf, J. H. Harvey, J. L. Whis-
tler, R. Stoll, N. Metzler-Nolte, R. H. Fish, J. Am. Chem. Soc. 2012,
134, 10321–10324.
[8] S. Abramkin, S. M. Valiahdi, M. A. Jakupec, M. Galanski, N. Met-
zler-Nolte, B. K. Keppler, Dalton Trans. 2012, 41, 3001–3005.
[9] S. H. van Rijt, H. Kostrhunova, V. Brabec, P. J. Sadler, Bioconjugate
Chem. 2011, 22, 218–226.
[10] A. Gross, M. Neukamm, N. Metzler-Nolte, Dalton Trans. 2011, 40,
1382–1386.
[11] H. Struthers, T. L. Mindt, R. Schibli, Dalton Trans. 2010, 39, 675–
696.
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Onambele, A. Prokop, I. Neundorf, Bioconjugate Chem. 2010, 21,
1288–1296.
[13] A. Pinto, U. Hoffmanns, M. Ott, G. Fricker, N. Metzler-Nolte,
ChemBioChem 2009, 10, 1852–1860.
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(Eds: G. Jaouen, N. Metzler-Nolte), Springer, Heidelberg, 2009,
pp. 195–217.
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Schatzschneider, Chem. Commun. 2008, 5604–5606.
[16] M. A. Neukamm, A. Pinto, N. Metzler-Nolte, Chem. Commun. 2008,
232–234.
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2007, 18, 88–100.
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Chem. 2009, 694, 823–827.
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Rubbiani, I. Ott, P. Riesterer, A. Prokop, K. Merz, N. Metzler-
Nolte, Chem. Sci. 2012, 3, 2062–2072.
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2012, 4, 260–266.
C25H26ClN3O4Ru·0.5H2O: C 51.95, H 4.71, N 7.27; found: C 52.14, H
4.36, N 7.30; MS (ESI+): m/z 534.0947 [MÀCl]+ (mex =534.0968, 4 ppm).
ACHTUNGTRENNUNG
[Chlorido(h6-p-cymene){5-oxo-kO-2-[(4-((3,4,5-trimethylphenoxy)meth-
yl)-1H-1,2,3-triazol-1-yl)methyl]-4H-pyronato-kO}ruthenium(II)] (7): The
reaction was performed according to the general procedure using pyrone
3 (112 mg, 0.33 mmol), sodium methoxide (18 mg, 0.34 mmol), bis[di-
chlorido(h6-p-cymene)ruthenium(II)] (99 mg, 0.16 mmol) and methanol
(10 mL). The product was isolated as yellow solid. Reaction time: 18 h.
Yield: 160 mg (82%); 1H NMR (500.10 MHz, [D6]DMSO): d=8.26 (s,
1H, H-8), 7.88 (s, 1H, H-6), 6.67 (s, 2H, HAr2’,6’), 6.59 (s, 1H, H-3), 5.69–
5.67 (m, 2H, HCym3’,5’), 5.61 (s, 2H, H-7), 5.41–5.39 (m, 2H, HCym2’,6’), 5.06
(s, 2H, H-10), 2.76 (sept, 3J
ACTHUNGRTENGNU(H,H)=7 Hz, 1H, HCymb), 2.19 (s, 6H, CAr3’,5’
ÀCH3), 2.13 (s, 3H, HCyma), 2.03 (s, 3H, CAr4’ CH3), 1.25 ppm (d, 3J-
À
ACHTUNGTRENNUNG
(H,H)=7 Hz, 6H, HCymc); 13C{1H} NMR (125.75 MHz, [D6]DMSO): d=
185.2 (C-4), 160.2 (C-2), 160.0 (C-5), 155.2 (CAr1’), 143.4 (C-9), 140.8 (C-
6), 136.9 (CAr3’,5’), 126.7 (CAr4’), 126.0 (C-8), 113.6 (CAr2’,6’), 109.0 (C-3),
97.9 (CCym1’), 94.9 (CCym4’), 79.9 (CCym3’,5’), 77.0 (CCym2’,6’), 60.7 (C-10), 49.6
À
(C-7), 30.6 (CCymb), 22.0 (CCymc), 20.3 (CAr3’,5’ CH3), 17.9 (CCyma),
À
CH3);
14.2 ppm
(CAr4’
elemental
analysis
calcd
for
C28H32ClN3O4Ru·0.5H2O: C 54.23, H 5.36, N 6.78; found: C 53.98, H
4.97, N 6.81; MS (ESI+): m/z 576.1406 [MÀCl]+ (mex =576.1433, 5 ppm).
ACHTUNGTRENNUNG
[Chlorido(h6-p-cymene)(5-oxo-kO-2-{(4-[(N-tyrosinyl-glycinyl-glycinyl-
phenylallanyl-leucinyl-NH2)propanamido]-1H-1,2,3-triazol-1-yl)methyl}-
4H-pyronato-kO)ruthenium(II)] (8): The reaction was performed accord-
ing to the general procedure using pyrone
4 (18.3 mg, 0.02 mmol),
sodium methoxide (2.2 mg, 0.04 mmol), bis[dichlorido(h6-p-cymene)ru-
thenium(II)] (6.4 mg, 0.01 mmol) and methanol (4 mL). The reaction was
stirred for 6 h in the absence of light at room temperature and under an
inert atmosphere. A yellow solution was obtained, which was concentrat-
ed and the residue was dried in vacuo. Preparative HPLC and subse-
quent lyophilization yielded the desired product as a yellow powder.
Yield: 11.7 mg (51%); 1H NMR (500.10 MHz, [D6]DMSO): d=9.15 (s,
1H, OHTyr), 8.24 (t, 3J
G
À
À
8.12 (d, 3J
A
3
NHTyr), 7.99 (d, JACHTUNGTRENNUNG(H,H)=6 Hz, 1H, NHPhe), 7.95 (t, JACHTGNUTRENNUNG
NHGly), 7.80 (s, 1H, H-6), 7.24 (d, 3J
À
7.15 (m, 1H, HAr,Phe), 7.09 (brs, 1H, NH2), 7.00 (d, 3J
Ar,Tyr), 6.96 (brs, 1H, NH2), 6.62 (d, 3J
(H,H)=9 Hz, 2H, HAr,Tyr), 6.35
À
H
(s, 1H, H-3), 5.69–5.66 (m, 2H, HCym3’,5’), 5.59 (s, 2H, H-7), 5.41–5.39 (m,
2H, HCym2’,6’), 4.51–4.49 (m, 1H, Ha,Phe), 4.44–4.40 (m, 1H, Ha,Tyr), 4.20 (q,
9306
www.chemeurj.org
ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2013, 19, 9297 – 9307

Organometal–Peptide Bioconjugates
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Received: March 7, 2013
Published online: May 27, 2013
Chem. Eur. J. 2013, 19, 9297 – 9307
ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
9307