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Organometal half-sandwich complexes and their bioconjugates:
Biological activity on cancer cells and potential applications in
biolabelling
Dissertation zur Erlangung des naturwissenschaftlichen Doktorgrades
der
Julius-Maximilians-Universität Würzburg
vorgelegt von
Wanning Hu
aus Liaoning
Würzburg 2012
Eingereicht bei der Fakultät für Chemie und Pharmazie am
Gutachter der schriftlichen Arbeit
1. Gutachter:
2. Gutachter:
Prüfer des öffentlichen Promotionskolloquiums
1. Prüfer:
2. Prüfer:
3. Prüfer:
Datum des öffentlichen Promotionskolloquiums
Doktorurkunde ausgehändigt am
Der größte Liebesdienst, den man einem anderen erweisen kann,
ist, sich selbst weiterzuentwickeln.
-Bruno Bettelheim
Στην αγάπη μου
Danksagung
An dieser Stelle möchte ich mich bei allen Personen bedanken, die mich bei der
Anfertigung dieser Arbeit unterstützt haben:
Mein größter Dank gilt meinem Doktorvater, Herrn Prof. Dr. Ulrich Schatzschneider,
der mich im Hinblick auf meine fachliche, berufliche und persönliche
Weiterentwicklung stets gefördert hat. Speziell bedanken möchte ich mich für seine
Betreuung und Begutachtung meiner Arbeit, für die fachliche Unterstützung, das
Vertrauen und den Freiraum.
Außerdem möchte ich mich ganz herzlich bei Frau Prof. Dr. Ines Neundorf und Frau
Dr. Katrin Splith für die Möglichkeit am Institut für Biochemie der Universität Leipzig
meine zellbiologischen Experimente durchführen zu können, bedanken. Über ihre
Freundschaft, Hilfsbereitschaft und die Einführung in die Zellkultur und Mikroskopie
habe ich mich sehr gefreut. Herrn Jan Hoyer danke ich für die Messungen der
Zytotoxizität. Ich danke auch allen Mitarbeiterinnen und Mitarbeitern von Frau Prof.
Dr. Beck-Sickinger, die mich während meiner Aufenthalte in Leipzig begleitet haben.
Ein großes Dankeschön gilt ebenfalls Herrn Dr. Gregory Smith und seiner
Arbeitsgruppe an der University of Cape Town in Südafrika für die gute
Zusammenarbeit, ihre Gastfreundlichkeit und das schöne Erlebnis während meines
Aufenthalts dort.
Herrn Alexander Damme, Herrn Dr. Klaus Merz und Frau Dr. Vera Vasylyeva danke
ich für die Röntgenstrukturanalysen, Frau Andrea Ewald, Herrn Dr. Lukasz Raszeja
und Frau Regina Reppich-Sacher für das Messen zahlreicher Massenspektren sowie
Herrn Dr. Rüdiger Bertermann, Herrn Prof. Dr. Raphael Stoll und Herrn Martin
Gartmann für die Messungen der NMR-Spektren.
Ich möchte mich bei meinen Kolleginnen und Kollegen in Bochum, Leipzig, Kapstadt
und Würzburg für die gute Kooperation, die Freundschaft und die gemeinsamen
Aktivitäten bedanken.
Ein besonderer Dank gilt meiner Familie und meinen Freunden, die mein Leben
bereichert haben.
I
Table of contents
Abbreviations
1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.1 Metal-based therapeutic agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1
1.2 Platinum anticancer drugs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3
1.3 Non-platinum anticancer drugs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
1.4 Macromolecular carriers for drug delivery. . . . . . . . . . . . . . . . . . . . . . . . . . . 9
1.4.1 Cell-penetrating peptides. . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . 9
1.4.2 Solid-phase peptide synthesis (SPPS) . . . . . . . . . . . . . . . . . . . . . . . . . . . .9
1.4.3 Dendrimers as drug delivery vehicles . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
2 Motivation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
3 Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15
3.1 Biological studies of CpM(CO)3 (M = Mn, Re) carboxylic acids and their
peptide bioconjugates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15
3.1.1 Objective. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15
3.1.2 Synthesis of CpM(CO)3 (M = Mn, Re) carboxylic acids. . . . . . . . . . . . . .17
3.1.3 Conjugation of Cym/Cyr carboxylic acids to the sC18 peptide. . . . . . .29
3.1.4 Fluorescence microscopy of CF-labeled organometal peptides in
MCF-7 human breast cancer cells. . . . . . . . . . . . . . . . . . . . . . . . . . . .34
3.1.5 Cytotoxicity studies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .36
3.2 Biological studies of organometal-dendrimer bioconjugates. . . . . . . . . . . . . .39
3.2.1 Objective. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . .39
3.2.2 Synthesis of organometal and adamantane aldehydes as precursors
for the coupling with dendrimers. . . . . . . . . . . . . . . . . . . . . . . . . . . . .40
3.2.3 Coupling of the aldehyde precursors with DAB dendrimer
G1, G2, G3. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
3.2.4 Synthesis of PAMAM dendrimer G1 and its organometallic
conjugates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
3.2.5 Cytotoxicity studies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
3.3 Metal carbonyl complexes as vibrational peptide labels. . . . . . . . . . .. . . . . . . 57
3.3.1 Objective. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . 57
II
3.3.2 Synthesis of organometal amino acids via Schiff base reactions. . . . . 57
3.3.3 Solid phase peptide synthesis (SPPS) with Mn- and Re-containing
lysines as building blocks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
3.3.4 Synthesis of Fmoc-protected organometal amino acids via amide
bond formation on the solid phase. . . . . . . . . . . . . . . . . . . . . . . . . . . .73
3.3.5 Introduction of organometal carbonyl complexes via an orthogonal
protective group strategy. . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . .87
4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .99
5 Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .103
5.1 General procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .103
5.2 Solid phase synthesis of the sC18 peptide and its bioconjugates. . . . . . . . . .104
5.3 CF-labeling of sC18 on the resin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .104
5.4 Solid phase synthesis of Fmoc-protected organometal amino acids
on a Wang resin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .105
5.5 Solid phase synthesis of peptide H-LKGKFKRG-NH2 and its organometal
conjugates on a Rink amide resin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .105
5.6 RP-HPLC of sC18 peptides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
5.7 RP-HPLC of all other conjugates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .106
5.8 Cell culture and cell viability assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
5.9 Fluorescence microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
5.10 X-ray crystallographic data collection and refinement of 2, 4 and 6. . . . . . 108
5.11 Synthetic procedures. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
6 References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
7 Appendix. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .155
8 Zusammenfassung. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .157
III
Abbreviations
alloc allyloxycarbonyl
ATR attenuated total reflection
Boc tert-butyloxycarbonyl
CAMP cationic antimicrobial peptide
CF (5,6)-carboxyfluorescein
CMIA carbonyl metallo immunoassay
Cp cyclopentadienyl anion
CPP cell-penetrating peptide
CT X-ray computed tomography
Cym cymantrene, cyclopentadienyl manganese tricarbonyl
Cyr cyrhetrene, cyclopentadienyl rhenium tricarbonyl
DAB 1,4-diaminobutane
Dde N-(1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl
DIC N,N-diisopropylcarbodiimide
DIEA N,N-diisopropylethylamine
DMEM Dulbecco's modified Eagle's Medium
DMF N,N-dimethylformamide
DMSO dimethyl sulfoxide
EPR enhanced permeability and retention effect
ER estrogene receptor
ESI electrospray ionization
FBS fetal bovine serum
FDA Food and Drug Administration
Fmoc fluorenylmethyloxycarbonyl
FMT fluorescence tomography
GdNCT gadolinium neutron capture therapy
IV
HATU 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate
HBSS Hanks’ balanced salt solution
HOBt 1-hydroxybenzotriazole
HPLC high performance liquid chromatography
IR infrared
LDH lactate dehydrogenase
MALDI matrix-assisted laser desorption ionization
NMR nuclear magnetic resonanc
MRI magnetic resonance imaging
mtt 4-Methyltrityl
PAMAM polyaminoamine
Pbf 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl
PET positron emission tomography
POPAM poly(propylene amine)
SAR structure–activity relationship
SPECT single-photon emission computed tomography
SPPS solid-phase peptide synthesis
TFA trifluoroacetic acid
THF tetrahydrofuran
TMS tetramethylsilane
Introduction
1
1 Introduction
1.1 Metal-based therapeutic agents
Organisms are made up of about 30 of the more than 90 known naturally occurring elements
while only five of them, carbon, oxygen, hydrogen, nitrogen, and sulfur constitute 99% of
their mass.[1,2] Transition metals only represent a small percentage of them, but play an
indispensable role in biology.[2] In cells, they have important structural, transport and
catalytic functions. For example, iron-containing heme proteins play an important role in
dioxygen activation and transport while iron-sulfur clusters are responsible for electron
transfer. Also, tetrahedral zinc centers are often found in nature as essential catalytic and
structural components. Cobalamines are involved in fermentation processes as isomerases
and in the transfer of methyl groups as in methyltransferases. Manganese is also involved in
many biological processes, such as the detoxification of reactive oxygen species and oxygen
production in photosynthetic organisms.[3] In addition to these natural functions, synthetic
metal complexes are also widely used in diagnostic and therapeutic applications (Figure 1.1).
Figure 1.1: Medicinal applications of metal complexes (modified from ref.[4])
Introduction
2
For early diagnosis of diseases and to understand the curative processes of therapeutic
drugs on a molecular level, different imaging techniques have been developed. They enable
the study of the location of tumors, cellular uptake mechanisms of drugs and targeting
strategies for biomarkers and therapeutics. These techniques can either be grouped by the
energy applied like X-rays, positrons, photons, infrared, near-infrared or sound waves
(Figure 1.2); or by their spatial resolution: macroscopic for clinical applications (CT, MRI or
ultrasound), and mesoscopic or microscopic (PET,SPECT or FMT) for in vitro or in vivo
biological studies.[5]
Figure 1.2: Imaging techniques grouped by the energy utilized (modified from ref.[6])
For example, paramagnetic gadolinium(III), manganese(II), and iron(III) compounds are used
as contrast agents in magnetic resonance imaging (MRI) and some radionuclides like 99mTc
and 188Re are commonly used for imaging and therapy, while 157Ga holds great promise for
applications in gadolinium neutron capture therapy (GdNCT) (Figure 1.3).[4,7,8]
N N
NN
O
O
O
O
O
O
O
O
Gd3+Tc
CC C
C
C
C
N
N
N
N
N
N
R
R
R
R
R
R
Figure 1.3: (left) SPECT radiopharmaceutical Cardiolite® (R = CH2C(CH3)2(OCH3)) and (right) MRIcontrast agent Gd-DOTA Dotarem®
Introduction
3
1.2 Platinum anticancer drugs
For a long time, medicinal applications of metal complexes have primarily been dominated
by the use of cisplatin in anticancer chemotherapy. Its biological activity was discovered by
Rosenberg in 1965, when he investigated the effect of the presence of certain group VIII
transition metals in an electric field on the growth of bacteria. He found that various
transition metal complexes of ruthenium, rhodium and especially platinum at concentrations
of 1-10 ppm inhibited the cell division of E. coli.[9] These compounds were later tested for
antitumor activity and the most active compounds turned out to be cis-[PtCl4(NH3)2] and cis-
[PtCl2(NH3)2].[10] The latter, termed cisplatin, was approved by the FDA in 1978 as a
chemotherapeutic drug.[11] Related platinum compounds also show high efficacy on different
cancer cell lines like ovarian, testicular, head and neck, colon and bladder cancer as well as
melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), lymphomas and
myelomas.[4,11,12]
However, until now, there are only three platinum-based drugs that have been approved
world-wide for clinical use and entered into applications on humans: cisplatin, carboplatin
and oxaliplatin. In addition, nedaplatin, lobaplatin, and heptaplatin are approved in parts of
Asia only (Figure 1.4). The cytotoxicity of platinum compounds is directly associated with
their aquation rate constants. For example, carboplatin was designed with a bis-carboxylate
ligand to overcome the high toxicity of platinum compounds that contain very labile leaving
groups such as nitrate, chloride, or hydroxide groups. As a consequence of its lower
reactivity, carboplatin can be used at much higher doses than cisplatin. Oxaliplatin is the first
platinum compound which is able to overcome cisplatin resistance, due to its capability to
form alternative adducts with DNA.[4,12]
PtH3N
H3N Cl
ClPt
H3N
H3N O
O
O
O
PtNH2
H2N O
O
O
O
cisplatin carboplatin oxaliplatin
PtH3N
H3N O
O O
PtNH2
H2N O
O
O
O
O
O
nedaplatin heptaplatin
Figure 1.4: Examples of platinum-based anticancer drugs approved for clinical use
Introduction
4
The mechanism of transport of platinum compounds into cells is still somewhat unclear. At
first, it was assumed that cisplatin crosses the cell membrane solely by passive diffusion.
Recently, however, there are reports that platinum-based drugs might rather use the
plasma-membrane copper transporter protein Ctr1 as well as organic cation transporters for
the entry into the cytoplasm via an active transport pathway.[13] After cellular internalization
of the platinum drugs, the leaving groups are thought to be replaced by water molecules due
to the lower chloride concentration in the intracellular environment.[4]
The aquated forms of cisplatin target nuclear DNA and bind predominately to the
nucleobases, in particular via the N7 position of guanine, and further with a second
nucleobase, thus forming mostly GpG intrastrand crosslinks. These crosslinks bend the DNA
and cause a distortion which is recognized by proteins that trigger apoptosis of the cells.[4,12]
Due to the high affinity of platinum(II) for soft thiol groups, the binding of platinum drugs to
non-DNA targets can also occur, such as the intracellular redox mediator glutathione
(Scheme 1.1). The glutathione levels of patients can increase after continuous treatment
with platinum drugs and thus lead to the development of drug resistance.[4,12,14]
PtH3N
H3N Cl
Cl
PtH3N
H3N Cl
OH2
PtH3N
H3N Cl
OH
NH
NNH
O
NH2
PtH3N
H3N Cl
N
NH
NNH
O
NH2
PtH3N
H3N OH2
N
NH
NNH
O
NH2
PtH3N
H3N
N
NH
NHN
N
O
NH2
O
HN
NH
O
OH
NH2
O
O
OH
PtH3N
H3N Cl
S
PtH3N
H3N N
SNH
NH2
OO
OO
HO
O
PtN
SHN
H2N
OO
OO
OH
O
N
SNH
NH2
OO
OO
HO
O
Scheme 1.1: (Middle) aquation of cisplatin and its binding to (left) guanine and (right) glutathione
(modified from ref.[12])
Introduction
5
1.3 Non-platinum anticancer drugs
Due to the above mentioned concerns and since cisplatin has only limited activity against
several common malignancies, non-platinum anticancer drugs are also actively developed, to
be able to target cancer cell lines that are resistant to platinum-based drugs, to improve the
selectivity of the drugs, and to reduce the non-specific toxicity of the transition-metal based
drugs and their side effects. Almost all of the transition metals in different coordination
geometries and redox states have been investigated for their in vitro cytotoxicity or in vivo
anti-cancer activity, and some of them have also entered clinical tests.[14-17] Among the non-
platinum metal-based drugs, ruthenium compounds are particularly prominent.[17-19]
There are two main oxidation states of ruthenium in physiological environments, Ru(II) and
Ru(III). The latter is generally believed to be substantially more inert than Ru(II) and thus
could enter biological systems as a prodrug. Due to the rapid growth of tumor tissue
compared to normal microenvironments, a low oxygen level is often found in solid tumors
and thus a reduction of the metal center to a lower oxidation could take place, as proposed
in the “activation-by-reduction” hypothesis.[17,20] After the drug is reduced to the more
reactive Ru(II) oxidation state, the chloride ligands can dissociate and then the drug is able to
bind to its biological target.
RuCl
Cl Cl
Cl
NHN
NNH
KP1019
NHN
H
RuCl
Cl Cl
Cl
NHN
NNH
Na
KP1339
Figure 1.5: Two examples of anti-cancer ruthenium complexes
Ru(III) complexes with imidazole (KP418) or indazole (KP1019, KP1339) ligands (Figure 1.5),
as reported by Keppler et al., show excellent activity against some primary tumors and also
metastases. Indazolium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] (KP1019) has
entered phase I clinical trials in 2004 and the diseases of five out of six patients was
stabilized with no severe side effects observed.[17,21] The cytotoxicity of KP1019 is rather
Introduction
6
moderate compared to that of the platinum-based drugs, probably due to the high binding
affinity of ruthenium to serum proteins (albumin, transferrin) and the selective reductive
activation of Ru(III) in hypoxic tumor microenvironments.[17-19,21] Since KP1019 only has
limited solubility, KP1339 was applied in further investigations because of its higher
bioavailability.[17]
The mechanism and mode of action of these complexes has been extensively studied. They
have a high binding affinity to cellular proteins as well as DNA. The reaction of KP1019 with
plasma proteins was explored and revealed that the major portion of ruthenium is bound to
albumin. This is supposed to be the thermodynamically favored binding partner of KP1019
while transferrin is kinetically favored. Cellular uptake studies showed that addition of
transferrin can support the transfer of the ruthenium complexes into the cell, and due to the
enhanced permeability and retention (EPR) effect, conjugates of transferrin and albumin
with ruthenium could therefore accumulate in tumors.[17,18,21]
RuCl
Cl Cl
Cl
SOH3C
H3C
N
NH
HN
NH
NAMI-A
Figure 1.6: NAMI-A, a ruthenium(II) complex with anti-metastases activity
Imidazolium trans-[tetrachloro(S-DMSO)(1H-imidazole)ruthenate(III)] (NAMI-A) (Figure 1.6),
is another ruthenium complex which has entered clinical trials. The DMSO sulfur atom has a
good π-acceptor property and can stabilize Ru(II), thus facilitating the reduction of Ru(III) to
the bioactive Ru(II) oxidation state.[22] NAMI-A has only negligible in vitro cytotoxicity on
primary tumor cells, but a significant anti-metastatic activity in vivo.[14]
Organometallic ruthenium(II) half-sandwich complexes (Figure 1.7) are the third class of Ru
complexes which has extensively been investigated for their anti-cancer properties. The +II
oxidation state is stabilized by the arene ring with the ruthenium center possessing a
Introduction
7
pseudo-octahedral geometry. Organometallic Ru(II) arene compounds with N,N-, N,O-, or
O,O-chelating ligands were studies and those with an ethylene diamine and chloride ligand
(Figure 1.7 left) exhibited the best anti-cancer activity both in vitro and in vivo. Structure-
activity relationship studies showed an increase of the cytotoxicity with the hydrophobicity
of the arene ligands in the order benzene < p-cymene < biphenyl < dihydroanthracene <
tetrahydroanthracene.[20] The activation of this type of compounds in the body is thought to
be due to rapid chloride dissociation. Inside cells, where the chloride concentration is lower
than in the blood, aquation is favored. DNA is believed to be the main target of these
complexes either via intercalation or hydrogen-bonding.
Ru
ClCl
P
N
NN
Ru
NH2
ClH2N
R R
PF6
Figure 1.7: Bioactive organometallic half-sandwich Ru(II) arene complexes
The RAPTA compounds (Figure 1.7 right) were initially designed to behave in a pH-
dependent manner, since the pH of tumor tissue is often lower (< 6.8) than at normal
physiological conditions (7.4) and protonation of the RAPTA compounds is expected to
improve the cellular uptake of the drug. However, the pKa value of the PTA group is too low
to be protonated. Still, these compounds show anti-cancer properties quite similar to that of
NAMI-A, having in vitro cytotoxicity but significant in vivo anti-metastatic activity.[15]
Iron complexes such as ferrocene with special redox activity also have a strong antitumor
potential in vitro, albeit by a completely different mechanism. For example, tamoxifen is an
organic estrogen receptor (ER) modulator, which acts against hormone-dependent ER(+)
breast cancer cells but is inactive against hormone-independent ER(-) cell lines (Figure 1.8).
The design of ferrocifen is based on the tamoxifen lead structure but the molecule is more
lipophilic than tamoxifen and thus might pass through the cell membrane more easily.
Furthermore, it retains the redox properties of ferrocene.[14] Ferrocifen exhibits a strong
cytotoxicity against ER(+) breast cancer, but is also active on hormone-independent ER(-)
cancer cells.[23] Since the ruthenocen analogue of ferrocifen is inactive on the ER(-) cancer
Introduction
8
cells, the cytotoxicity of ferrocifen towards hormone-independent cells is believed to be
related to redox processes of the ferrocene moiety, leading to quinone methide
formation.[4,24-26]
OH
OH
OH
OH
Fe
tamoxifen ferrocifen
Figure 1.8: Estrogen-receptor modulator therapeutic agents (left) tamoxifen and (right) ferrocifen
with activity against human breast cancer cells
Introduction
9
1.4 Macromolecular carriers for drug delivery
1.4.1 Cell-penetrating peptides
Metal-based drugs often show poor uptake into cells and thus have a limited bioavailability.
Therefore, biomolecules are widely used as carriers to deliver therapeutic cargos into the
cytoplasm and nuclear compartments. Cell-penetrating peptides (CPPs) are 9-35 amino acid
long amphipathic and cationic peptides, which can be conjugated to molecular cargo and
translocate through the cell membrane (Figure 1.9). The development of CPPs is based on
the observation that cationic polyamines such as histones, polylysines, and polyarginines as
well as polycationic proteins can effectively enter cells.[27] CPPs are either derived from
natural proteins or have a synthetic origin. Their delivery mechanisms can be classified into
two major types: a) peptides which are covalently conjugated to the molecular cargo; or b)
peptides which improve the tissue permeability but need not be covalently bond to a
drug.[28,29] The mechanism for the cellular uptake of CPPs is still largely unknown, but the
cationic part of most CPPs is considered to interact with anionic species in the cellular
membrane.[27,29]
M = Mn, Re
MOC CO
CO
N
O
NN
NN
NN
NN
NN
NN
O
O
NH
NHH2N
O
NH2
O
NH
NHH2N
O
O
NH
NHH2N
O
NH2
O
O
NH
NHH2N
O
NH2
O
O
NN
N NH2
NH2
O
O
NH2
O
O OH
O
NH2
organometalmoiety sC18 cell penetrating peptide
Figure 1.9: An example of a cell penetrating peptide (CPP) with conjugated cymantrene carboxylic
acid, here the sC18 sequence is shown
1.4.2 Solid-phase peptide synthesis (SPPS)
For the preparation of polymeric molecules from monomeric building blocks, solid-phase
synthesis methods are more efficient than coupling in solution. Thus, they are widely used in
the preparation of oligonucleotide- and peptide-based biomolecules and conjugates. Metal
complexes are usually attached to the N-terminus of a peptide or to the side-chains on the
resin. The Fmoc strategy is often applied for solid phase peptide synthesis (SPPS)[30]: the first
Introduction
10
amino acid is attached to the resin, and after deprotection of the N-terminal Fmoc
protective group with piperidine or morpholin, additional side-chain protected amino acids
are sequentially coupled to the first amino acid (Scheme 1.2). After the peptide sequence is
assembled, bioactive metal complexes can be attached to the N-terminal amino group of the
peptide or to the amino acid side-chains. Orthogonal side-chain protective group strategies
enable the selective deprotection of different protective groups under special conditions.
Finally, the whole peptide is cleaved from the resin and usually further purified with HPLC.
linkerO
HN
X
O
R
Y
deprotection of the -amino group
linkerOH2N
O
R
Y
resin
resin
coupling with the next protected amino acid
linkerO
HN
O
R
Y
resinNH
X
O
R'
Y'
repetition
of
coupling
cycle
deprotection of the -amino group and side-chains,
followed by cleave from resin
OH
HN
O
R
H2N
O
R'
Scheme 1.2: General principle of solid phase peptide synthesis (SPPS). X: N-terminal amino
protective group, Y: side-chain protective group. (modified from ref.[31])
Introduction
11
1.4.3 Dendrimers as drug delivery vehicles
In addition to cell-penetrating peptides, passive targeting of nanocarriers towards tumour
cells is based on the enhanced permeability and retention (EPR) effect (Figure 1.10).[32] Due
to impaired angiogenesis in tumours, leaky, defective blood vessels are often found in
malignant tissues, leading to a better permeability of the vessel walls for macromolecules.
Typically, the endothelial pore size is increased from < 2 nm in normal to 100-2000 nm in
tumour tissue. In addition, the absent or dysfunctional lymphatic system of tumours leads to
the retention and accumulation of macromolecules with a molecular mass of over 40 kDa in
these tissues.[33,34] The application of macromolecules as carrier systems also improves the
biocompatibility of the drugs, since the retained nanocarriers cannot easily be degraded or
eliminated by the metabolism. In addition, the solubility of drugs is also enhanced by
choosing suitable carrier materials.[35]
Figure 1.10: Enhanced permeability and retention (EPR) effect. Red: blood vessels, blue: cell nuclei,
green: cell cytosol, yellow: lymphatic vessel, yellow circles: nanoparticles
Introduction
12
A large variety of nanoscale carriers like liposomes, polymer-protein conjugates, magnetic
nanoparticles, and quantum dots (QDs) have already found applications in medicine.[32,36,37]
Among them, dendrimers are cascade polymeric molecules with multiple branches, which
are derived from diverse central monoamine or diamine cores (Figure 1.11). The highly
branched structure of dendrimers enables an optimized loading of drugs to the end groups.
Dendrimers can be prepared by convergent pathways, in which the dendrimer molecule is
constructed from the periphery to the core; or divergent strategies, where dendritic
branches are attached stepwise from the core.[38] Some common types of dendrimers are
poly(propyleneimine) (POPAM), polyamidoamine (PAMAM), and polylysine dendrimers,
which are already applied as delivery vehicles for anticancer drugs like cisplatin or ruthenium
arene complexes, but also organic antimalarial drugs, biosensors and imaging agents.[39-44]
NN
NH
NH
NH2
NH2
HN
HN
H2N
H2N
O
O
O
O
H2N NN NH2
NH2
H2N
(a)
(b)N
N
N
N
N
NH2
H2N
NH2
H2N NH2
NH2
N NH2
H2N
(c)
Figure 1.11: Selected examples of dendrimer cores: (a) first generation polyamidoamine (PAMAM)
dendrimer; (b) first generation 1,4-diaminobutane core (DAB) dendrimer; and (c) second generation
DAB dendrimer
Motivation
13
2 Motivation
Due to the development of resistance and many types of cancer currently inaccessible to
chemotherapy, there is an urgent need to prepare and evaluated innovative new medicines.
In contrast to organic drugs, transition metal complexes offer a number of unique properties
that can be exploited for such applications, like redox bistability, tunable ligand exchange
kinetics, and rich photophysics and photochemistry. Furthermore, the wide range of
coordination numbers and geometries accessible for transition metal complexes provides
interesting opportunities for the spatial 3D orientation of ligands. In addition, a large
number of available radioactive isotopes make transition metal complexes good candidates
for development as imaging reporters in medicinal inorganic chemistry.
Organometallic half-sandwich compounds of the type CpM(CO)3 with Cp = cyclopentadienyl
follow the 18-electron rule and show good stability towards water and oxygen and therefore
are interesting building blocks for metal-based drugs. FurtheƌŵŽƌĞƚŚĞĐŚĂƌĂĐƚĞƌŝƐƟĐ൙K
stretching vibrations of the metal carbonyl moiety can be used in analytical techniques like
the carbonyl metallo immuno assay (CMIA) as well as vibrational imaging using IR and
Ramam microscopy.
Thus, the goal of the present work was to synthesize a series of novel organometal half-
sandwich complexes based on the group VI and VII metals. Functional groups suitable for
coupling to carrier molecules were to be introduced into these compounds. Cell-penetrating
peptides as well as different dendrimers were chosen as the carriers to transport the
organometallic cargos into cancer cells for improved activity and targeting. Structure-activity
relationships were to be determined by variation of the metal center, the linker between the
organometal moieties and the carrier peptides, as well as the type, molecular weight, and
number of terminal functional groups of the dendrimer conjugates.
In addition, different metal carbonyl complexes with variable C≡O vibrational band positions
were to be attached to a peptide, to utilize their distinct vibrational signature for
information encoding in biomolecules in a barcoding strategy.
14
Results and discussion
15
3 Results and discussion
3.1 Biological studies of CpM(CO)3 (M = Mn, Re) carboxylic acids and their peptide
bioconjugates
3.1.1 Objective
In previous work,[45-47] cymantrene derivatives with different linkers between the half-
sandwich CpMn(CO)3 moiety and the carboxylate group were conjugated to the cell-
penetrating peptide hCT(18-32)-k7 as well as the antimicrobial peptide sC18 (Figure 3.1.1).
While the organometallic compounds themselves showed no biological activity (Figure
3.1.2a), the cymantrene peptide conjugates were efficiently internalized by cancer cells and
showed moderate cytotoxicity on MCF-7 human breast adenocarcinoma and HT-29 human
colon carcinoma cells (Figure 3.1.2b) compared to cisplatin used as the reference drug.
MnOC CO
CO
O
OH
MnOC CO
CO
O
MnOC CO
CO
O
MnOC CO
CO
O
MnOC CO
CO
O
MnOC CO
CO
O
OH
O
O
OH
O OH O
OH
O
OH
Cym1 Cym2 Cym3
Cym4 Cym5 Cym6
Figure 3.1.1: Functionalized cymantrene carboxylic acids Cym1 - Cym6 prepared for peptide
conjugation
a) b)
Figure 3.1.2: (a) Effect of cymantrene carboxylic acids 1-6 on the cell viability of MCF-7 human breast
cancer cells and (b) IC50 values of their sC-18 peptide conjugates on the same cell line. The cell
viability was determined with the resazurin assay, with cisplatin serving as the positive control
sC18-peptide conjugates IC50 value (µM)
Cym1-sC18 57.4 15.4
Cym2-sC18 62.5 15.5
Cym3-sC18 64.4 5.1
Cym4-sC18 59.5 6.7
Cym5-sC18 50.4 10.4
Cym6-sC18 61.3 12.0
Cisplatin 2.0 0.3
Results and discussion
16
However, the molecular mechanism of the biological activity of these organometal-peptide
conjugates remained elusive in these studies. To obtain further knowledge about the role of
the organometallic CpM(CO)3 moiety and the linker between the cyclopentadienyl ring and
the carboxylate group used for conjugation to the peptide, additional derivatives with
manganese or rhenium as the metal center were prepared and attached to the sC18 peptide
to study the influence of the metal center and the linker on the biological activity of the
peptide conjugates on MCF-7 human breast cancer cells (Figure 3.1.3).
M = Mn, Re
MOC CO
CO
N
O
NN
NN
NN
NN
NN
NN
O
O
NH
NHH2N
O
NH2
O
NH
NHH2N
O
O
NH
NHH2N
O
NH2
O
O
NH
NHH2N
O
NH2
O
O
NN
N NH2
NH2
O
O
NH2
O
O OH
O
NH2
organometalmoiety
linker sC18 peptide
Figure 3.1.3: General structure of organometallic peptide bioconjugates prepared in this work
Results and discussion
17
3.1.2 Synthesis of CpM(CO)3 (M = Mn, Re) carboxylic acids
A series of 12 carboxylate-functionalized cyclopentadienyl metal tricarbonyls with different
linkers connecting the carboxylic acid moiety and the cyclopentadiene ring were synthesized
as described below. The linker was varied from aliphatic chains of different length to
phenylene spacers with different connectivity (ortho or para), either with a keto or a
methylene group adjacent to the Cp ring (Figure 3.1.4).
MnOC CO
CO
O
OH
O
MnOC CO
CO
O
OH
O
MnOC CO
CO
OO OH
MnOC CO
COOH
O
MnOC CO
CO
OH
O
MnOC CO
CO
O OH
ReOC CO
CO
O
OH
O
ReOC CO
CO
O
OH
O
ReOC CO
CO
OO OH
ReOC CO
COOH
O
ReOC CO
CO
OH
O
ReOC CO
CO
O OH
3 4
87
1 2
12119
5 6
10
Figure 3.1.4: Functionalized CpM(CO)3 carboxylic acids with M = Mn, Re prepared for peptide
conjugation
The cymantrene keto carboxylic acid 1 as well as its rhenium analogue 3 were obtained by
the Friedel–Crafts acylation of CpM(CO)3 (M = Mn, Re) with succinic anhydride and
aluminium chloride according to a literature procedure.[48,49] The manganese precursor was
more reactive and could be functionalized at room temperature, while heating to reflux was
required for the synthesis of the rhenium analogue (Scheme 3.1.1).
MOC CO
CO
OH
O
O
MOC CO
CO
M = Mn (1), Re (3)
OO O + a) AlCl3, CH2Cl2, RT, 2 d
b) AlCl3, CH2Cl2, under microwave, 1.5 h, 60 °C
Scheme 3.1.1: Synthesis of 1 and 3 by Friedel-Crafts acylation of CpM(CO)3 with M = Mn, Re
Results and discussion
18
The keto group adjacent to the cyclopentadienyl ring was then reduced to a methylene
moiety with a combination of titanium(IV) chloride and triethylsilane in anhydrous
dichlormethane to give 2 and 4 in 65-80% yield.[50] Both steps, the Friedel–Crafts acylation
and the reduction to the methylene group could also be performed under microwave
irradiation to speed up the reaction (Scheme 3.1.2).
M = Mn (2), Re (4)
MOC CO
CO
OH
OM
OC COCO
OH
O
O
a) TiCl4, Et3SiH, CH2Cl2, RT, 2 d
b) TiCl4, Et3SiH, CH2Cl2, under microwave, 1.5 h, 60 °C
Scheme 3.1.2: Synthesis of 2 and 4 by reduction of the keto group with a mixture of titanium (IV)
chloride and triethyl silane
All four manganese and rhenium compounds 1 - 4 with the aliphatic linker prepared this way
were fully characterized by 1H NMR and IR spectroscopy, ESI mass spectrometry, and
elemental analysis. The 1H-NMR spectrum of 1 in acetone-d6 shows three broad peaks with
an intensity ratio of 2:2:4 at 5.75, 5.14, and 3.01 ppm, the latter partially overlapping with
the residual water signal of the solvent (Figure 3.1.5). The expected doublet pattern of the
two aromatic Cp signals at 5.75 and 5.14 ppm is not resolved and the broad signals of the
two methylene groups appear with an identical shift at 3.01 ppm.
Figure 3.1.5: 200 MHz 1H-NMR spectrum of compound 1 in acetone-d6
Results and discussion
19
In the ATR IR spectrum of compound 1, five bands at 2023, 1940, 1915, 1701 and 1678 cm-1
are observed (Figure 3.1.6). The peaks at 2023, 1940 and 1915 cm-1 are due to the
symmetrical and asymmetrical C≡O vibrations of the Mn(CO)3 moiety. There are three
instead of the expected two bands found for this compound. The splitting of the
asymmetrical band is likely due to a lowered local symmetry in the solid state.[51] The bands
at 1678 and 1701 cm-1 are assigned to the keto and ester C=O group of the side-chain,
respectively.
4000 3000 2000 1000
25
50
75
100
Tra
ns
mis
sio
nin
%
Wavenumber in cm-1
2023
19401915
1678
1701
Figure 3.1.6: ATR IR spectrum of compound 1
In the 1H NMR spectrum of methylene compound 2, four peaks are observed at 4.91, 4.86,
2.48 and 1.82 ppm with an integral ratio of 2:2:4:2 in addition to the signals of the solvent
and residual water (Figure 3.1.7). The difference in chemical shift for the two protons on the
cyclopentadiene ring is reduced from 0.61 to 0.05 ppm compared to keto compound 1. In
the aliphatic region, a new peak with an intensity of 2H appears at 1.82 ppm in addition to
the overlapping signals of the two original CH2 groups at 2.48 ppm, which demonstrates the
successful reduction of the keto to a methylene group.
Figure 3.1.7: 200 MHz 1H-NMR spectrum of 2 in acetone-d6
Results and discussion
20
Three intense bands are observed at 2010, 1911, and 1697 cm-1 in the IR for compound 2
(Figure 3.1.8). The latter one is due to the C=O vibration of the carboxylic acid keto group
while the two signals at 2010 and 1911 cm-1 are assigned to the symmetrical and
asymmetrical CO vibrations of the Mn(CO)3 moiety, respectively.
4000 3000 2000 1000
0,6
0,8
1,0
1697
1911
Tra
nsm
issio
nin
%
Wavenumber in cm-1
2010
Figure 3.1.8: ATR IR spectrum of compound 2
In the 1H NMR spectrum of the rhenium compound 3, four peaks at 6.35, 5.73, 3.02 and 2.64
ppm are found with an intensity ratio of 2:2:2:2, which are all split into multiplets (Figure
3.1.9). The protons on the cyclopentadiene ring appear as triplets with 3J = 2.4 Hz at 5.73
and 6.35 ppm, respectively. The aliphatic protons are also split into two triplets, each with 3J
= 6.5 Hz. In the IR spectrum of 3, a pattern similar to compound 1 is observed, with five
bands found at 2022, 1931, 1902, 1699, and 1678 cm-1. All peaks are only slightly shifted
compared to 1 (data now shown).
Figure 3.1.9: 200 MHz 1H-NMR spectrum of 3 in acetone-d6
Results and discussion
21
Compound 4 was isolated as a white solid and characterized by 1H NMR and IR spectroscopy.
The 1H NMR of compound 4 shows a pattern similar to its manganese analog, with two
triplets found at 5.59 and 5.50 ppm with 3J = 2.2 Hz and an integral of 2H, which are due to
the equivalent 1,5- and 3,4-protons of the mono-substituted cyclopentadiene ring. A doublet
of a doublet with 2H is found at 2.55 ppm with 3J = 7.6 Hz and 4J = 0.9 Hz, which is due to the
methylene group in the center of the propylene linker. A triplet found at 2.38 ppm with 3J =
7.4 Hz and an integral of 2H is assigned to the methylene group adjacent to the carboxylic
acid and a triplet with 2H at 1.83 ppm with 3J = 8 Hz results from the methylene group
adjacent to the Cp ring. In the IR spectrum of 4, three peaks at 2010, 1902 and 1706 cm-1 are
observed. The bands at 2010 and 1902 cm-1 result from the Re(CO)3 moiety and the band at
1706 cm-1 is due to the carboxylic acid C=O group. All of them are only slightly shifted
compare to 2.
The compounds were also characterized by electrospray mass spectrometry (ESI) as shown
for 1 and 2 as an example in Figure 3.1.10. The intense peaks at m/z = 302.7 and 288.8,
respectively, are assigned to the CpM(CO)3 carboxylic acids, which are observed in the
negative mode as [M-H]-. The mass difference of m/z = 14 between the two is in line with
the reduction of the keto by a methylene group. The reduced compounds are of somewhat
better purity than the keto analogues. b)
200 300 400 500
m/z = 302.7 [M-H]-
m/z
MnOC CO
CO
OH
O
O
200 300 400 500
m/z = 288.8 [M-H]-
m/z
MnOC CO
CO
OH
O
Figure 3.1.10: Negative mode ESI mass spectra of a) 1 and b) 2 in methanol
Results and discussion
22
Single crystals of 2 and 4 suitable for X-ray structure analysis could be obtained by slow
evaporation of an acetone solution at room temperature. Both compounds crystallized in
the triclinic space group P-1 (Figure 3.1.11). They show the expected coordination of the
manganese or rhenium center by a η5-cyclopentadienyl ring and three carbonyl ligands, with
the substituent on the Cp ring pointing away from the half-sandwich moiety. The C-Mn-C
angles of 2 are all in the range of 92.5°-92.9°, while those of the rhenium compound 4 vary
between 90.3° and 91.1°. For the manganese complex 2, the metal-to-cyclopentadienyl ring
centroid distance is 1.762 Å, whereas for compound 4, it is somewhat longer, at 1.942 Å. The
same trend is also seen in the metal–carbonyl bond lengths. For the manganese compound
M-CO distances vary from 1.778 Å to 1.782 Å while for the rhenium analog they range from
1.890 Å to 1.906 Å. Selected bond lengths and angles are collected in Table 3.1.1.
(a) (b)
Figure 3.1.11: Molecular structures of (a) 2 and (b) 4; Thermal ellipsoids are shown at the
30% probability level
Results and discussion
23
Additional intermolecular interactions exist between the organometal carboxylic acid
monomers. In the crystal lattice, intermolecular hydrogen bonds are observed which lead to
the formation of carboxylate-bridged dimers as the main structural motif (Figure 3.1.12).
Figure 3.1.12: Main intermolecular interaction in 2. Thermal ellipsoids are shown at the 30%
probability level. Dashed lines indicate the intermolecular H-bonding within the dimer
Table 3.1.1: Selected bond lengths [Å] and angles (°) for 2 and 4
2 4
Mn(1)-C(1) 2.144(4)
Mn(1)-C(2) 2.146(4)
Mn(1)-C(3) 2.119(4)
Mn(1)-C(4) 2.123(5)
Mn(1)-C(5) 2.134(4)
Mn(1)-C(6) 1.778(5)
Mn(1)-C(7) 1.788(5)
Mn(1)-C(8) 1.782(5)
C(6)-O(1) 1.155(6)
C(7)-O(2) 1.146(6)
C(8)-O(3) 1.155(5)
C(12)-O(4) 1.220(5)
C(12)-O(5) 1.316(5)
C(6)-Mn(1)-C(7) 92.6(2)
C(6)-Mn(1)-C(8) 92.9(2)
C(7)-Mn(1)-C(8) 92.5(2)
Re(1)-C(1) 2.304(11)
Re(1)-C(2) 2.288(13)
Re(1)-C(3) 2.265(15)
Re(1)-C(4) 2.275(12)
Re(1)-C(5) 2.286(14)
Re(1)-C(6) 1.892(10)
Re(1)-C(7) 1.890(16)
Re(1)-C(8) 1.906(12)
C(6)-O(1) 1.136(8)
C(7)-O(2) 1.136(11)
C(8)-O(3) 1.150(10)
C(12)-O(4) 1.202(10)
C(12)-O(5) 1.307(10)
C(6)-Re(1)-C(7) 90.3(4)
C(6)-Re(1)-C(8) 91.1(5)
C(7)-Re(1)-C(8) 90.8(5)
Results and discussion
24
The cyclopentadienyl tricarbonylmanganese(I) or rhenium(I) carboxylic acids with 1,2-, or
1,4-phenylene linkers were also synthesized via the Friedel-Crafts acylation with anhydrides
or activated acids as shown in Scheme 3.1.3 following reported procedures.[45,47,48,51] In
contrast to the low-temperature Friedel-Crafts acylation of the cymantrene complexes,
CpRe(CO)3 had to be heated with the corresponding reaction partner and aluminium
trichloride in dichloromethane overnight to obtain the desired products due to the lower
reactivity of the rhenium metal center.
a)
b)
O OH
MeOH
SOCl2
MeOH
NaOH
SOCl2
CpM(CO)3
OHO
O OCH3
OCH3
O
O OH
OCH3
O
O Cl
OCH3
O
MeOH
NaOHAlCl3 MOC CO
CO
O
OCH3
O
MOC CO
CO
O
OHO
M = Mn, Re
Scheme 3.1.3: General synthesis of cymantrene and cyrhetrene carboxylic acids with phenylene
linkers. Note the different strategies used to introduce the (a) 1,2-phenylene, and (b) 1,4-phenylene
linkers
The keto group adjacent to the cyclopentadiene ring was reduced with titanium(IV) chloride
and triethylsilane as described above.[50,52]
M = Mn, Re
MOC CO
CO
O
OHO
a) TiCl4, Et3SiH, CH2Cl2, RT, 2 d
b) TiCl4, Et3SiH, CH2Cl2, Microwave, 1.5 h, 60 °CM
OC COCO OH
O
Scheme 3.1.4: Reduction of the keto to a methylene group with titanium(IV) chloride and
triethylsilane
MOC CO
CO
+AlCl3 / CH2Cl2
Friedel-Crafts acylation
OO O
MOC CO
CO
OO OH
Results and discussion
25
All eight compounds prepared were characterized by 1H-NMR and IR spectroscopy as well as
ESI mass spectrometry. For example, in compound 6, six peaks at 7.97, 7.50, 7.38, 4.97, 4.81
and 4.09 ppm, with an intensity ratio of 1:3:2:2:2, as well as the solvent and residual water
signals are found. All peaks are broadened and the expected splitting is not resolved. In the
aromatic region, the peaks of the 1,2-disubstituted phenylene group are found between 7.38
and 7.97 ppm, in which three of the four signals overlap with each other. The Cp protons are
found as two asymmetric singlets between 4.81 and 4.97 ppm, each with an intensity of 2H.
The peak at 4.09 ppm with an integral of 2H is due to the bridging methylene protons and
demonstrates the successful reduction of the keto group.
Figure 3.1.13: 200 MHz 1H-NMR spectrum of compound 6 in acetone-d6
The ATR IR spectrum of compound 6 shows three intense bands at 2014, 1927 and 1666 cm-1.
The peaks at 2014 and 1927 cm-1 result from the symmetrical and asymmetrical C≡O
vibrations of the Mn(CO)3 moiety, while the band at 1666 cm-1 is assigned to the ester C=O
group.
4000 3500 3000 2500 2000 1500 1000
0,80
0,85
0,90
0,95
1,00
Tra
nsm
issio
nin
%
Wavenumber in cm-1
2014
1927
1666
Figure 3.1.14: ATR IR spectrum of compound 6
Results and discussion
26
Yellow single crystals of 6 suitable for X-ray structure analysis were grown from acetone by
slow evaporation at room temperature, and the molecular structure is shown in Figure
3.1.15. The compound crystallized in the triclinic space group P-1. The C-Mn-C angles of the
Mn(CO)3 moiety are all close to 90°. The C-C bonds of the cyclopentadiene and phenylene
groups are all in the expected range for an aromatic system at about 1.40 Å. The metal-to-
cyclopentadiene ring centroid distance is 1.777 Å, which is only a little longer that the
distance of 1.762 Å in complexe 2 with the aliphatic side-chain. The torsion angle between
the cyclopentadienyl and phenyl rings in the reduced compound 6 is 72.0°, a bit smaller than
that of 79.2° in compound 5 with the keto group.[45]
Figure 3.1.15: Molecular structures of 6. Thermal ellipsoids are drawn at the 30%
probability level
Table 3.1.2: Selected bond lengths [Å] and angles (°) for 6
6
Mn(1)-C(1) 2.150(3)
Mn(1)-C(2) 2.163(3)
Mn(1)-C(3) 2.151(3)
Mn(1)-C(4) 2.146(3)
Mn(1)-C(5) 2.144(4)
Mn(1)-C(6) 1.790(4)
Mn(1)-C(7) 1.806(4)
Mn(1)-C(8) 1.786(4)
C(6)-O(1) 1.152(5)
C(7)-O(2) 1.138(4)
C(8)-O(3) 1.158(5)
C(16)-O(4) 1.315(4)
C(16)-O(5) 1.222(5)
C(6)-Mn(1)-C(7) 91.87(16)
C(6)-Mn(1)-C(8) 90.95(17)
C(7)-Mn(1)-C(8) 92.20(17)
Results and discussion
27
The 1H-NMR spectrum of the analogous rhenium compound 8 in acetone-d6 also shows six
peaks at 8.00, 7.54, 7.44, 5.62, 5.47 and 4.22 ppm with an intensity ratio of 1:1:2:2:2:2
(Figure 3.1.16). The multiplets at 8.00, 7.54 and 7.44 ppm are not very well resolved due to
the limited solubility, but can be assigned to the 1,2-disubstituted phenylene linker. The
peaks at 5.62 and 5.47 ppm are due to the Cp protons, and the peak at 4.22 ppm with an
integral of 2H is due to the bridging methylene group.
2.0
0
2.0
02.0
7
2.0
01.0
1
1.0
3
4.2
2
5.4
75.6
2
7.4
47.5
4
8.0
0
Figure 3.1.16: 200 MHz 1H-NMR spectrum of compound 8 in acetone-d6
In the ATR IR of compound 8, three intense bands are observed at 2010, 1888, and 1684 cm-1.
The bands at 2010 and 1888 cm-1 are due to the characteristic pattern of the symmetrical
and asymmetrical C≡O vibrations of the Re(CO)3 moiety. The band at 1684 cm-1 results from
the C=O vibration of the carboxylic acid group (Figure 3.1.17).
4000 3000 2000 1000
0,6
0,8
1,0
Tra
nsm
issio
nin
%
Wavenumber in cm-1
2010
1888
1684
Figure 3.1.17: ATR IR spectrum of compound 8
Results and discussion
28
The ESI mass spectrum of compound 8 shows the characteristic rhenium isotope pattern.
Only one major intense peak at m/z = 468.8 is found and assigned to [M-H]- (Figure 3.1.18).
300 400 500 600
m/z
ReOC CO
CO
O OH
m/z = 468.8 [M-H]-
Figure 3.1.18: Negative mode ESI mass spectrum of compound 8 in methanol
Results and discussion
29
3.1.3 Conjugation of Cym/Cyr carboxylic acids to the sC18 peptide
The carrier peptide sC18 was synthesized by automated solid phase peptide synthesis (SPPS)
on a Rink amide resin following the Fmoc/tBu strategy, with activation by 1-
hydroxybenzotriazole/N,N-diisopropylcarbodiimide (Scheme 3.1.5). The coupling of the
organometallic carboxylic acids 1 (Cym2), 2 (Cym2*), 3 (Cyr2), and 4 (Cyr2*) was done
manually on the resin in the final step with HATU and DIPEA in DMF, after Fmoc-
deprotection with 20% piperidine in DMF.
(Fmoc)HNN
NN
NN
NN
NN
NN
N
O
O
NH
NH(Pbf)HN
O
NH(Boc)
O
NH
NH(Pbf)HN
O
O
NH
NH(Pbf)HN
O
NH(Boc)
O
O
NH
NH(Pbf)HN
O
NH(Boc)
O
O
NN
N NH
NH(Boc)
O
O
NH(Boc)
O
O OtBu
O
NH(Boc)
Rink amideresin
20% piperidine in DMF
H2NN
NN
NN
NN
NN
NN
N
O
O
NH
NH(Pbf)HN
O
NH(Boc)
O
NH
NH(Pbf)HN
O
O
NH
NH(Pbf)HN
O
NH(Boc)
O
O
NH
NH(Pbf)HN
O
NH(Boc)
O
O
NN
N NH
NH(Boc)
O
O
NH(Boc)
O
O OtBu
O
NH(Boc)
Rink amideresin
HN
NN
NN
NN
NN
NN
NN
O
O
NH
NH(Pbf)HN
O
NH(Boc)
O
NH
NH(Pbf)HN
O
O
NH
NH(Pbf)HN
O
NH(Boc)
O
O
NH
NH(Pbf)HN
O
NH(Boc)
O
O
NN
N NH
NH(Boc)
O
O
NH(Boc)
O
O OtBu
O
NH(Boc)
Rink amideresin
HATU, DIPEA in DMF
MnOC CO
CO
OH
O
MnOC CO
COO
5% H2O, 95% TFA
MnOC CO
CO
N
O
NN
NN
NN
NN
NN
NN
O
O
NH
NHH2N
O
NH2
O
NH
NHH2N
O
O
NH
NHH2N
O
NH2
O
O
NH
NHH2N
O
NH2
O
O
NN
N NH2
NH2
O
O
NH2
O
O OH
O
NH2
Scheme 3.1.5: Solid phase peptide synthesis (SPPS) with orthogonal side-chain protective group
strategy for the attachment of organometal carboxylic acids, illustrated here for compound 2. Boc:
tert-butyloxycarbonyl, DIEA: N,N-diisopropylethylamine, Fmoc: fluorenylmethyloxycarbonyl, HATU:
2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, Pbf: 2,2,4,6,7-
pentamethyldihydrobenzofuran-5-sulfonyl, TFA: trifluoroacetic acid
Results and discussion
30
The conjugates were cleaved from the resin with TFA and 5% water as the scavenger. They
were then precipitated by addition of ice-cold diethyl ether, isolated by centrifugation,
washed five times with cold diethyl ether, and then dried for 10 min with a Speed-Vac or IR
Dancer. All conjugates could be successfully cleaved from the resin under these conditions
with no decomposition.
MnOC CO
CO
O
OH
OMn
OC COCO
OH
ORe
OC COCO
O
OH
ORe
OC COCO
OH
O
-GLRKRLRKFRNKIKEK-NH2
MOC CO
CO
linker
-GLRKRLRKFRNKIKEK-NH2
MOC CO
CO
linker
CF
M = Mn, Re
CF =
O OHO
COOH
HOOC
1 2 3 4
Scheme 3.1.6: Functional manganese complexes 1 and 2 as well as the rhenium analogues 3 and 4
conjugated to the sC18 peptide. At the bottom, also the amino acid sequence and position of the
carboxyfluorescein (CF) label is shown
For cellular uptake studies, a carboxyfluorescein (CF) label was attached to the amino group
of the lysine (K) closest to the N-terminus by removing the Dde protective group with 2%
hydrazine in N,N-dimethylformamide, followed by coupling of CF with 1.5 equiv. HATU and
DIEA. The reaction was carried out overnight in the dark, due to the light-sensitivity of the
fluorophore group. The reactive hydroxy groups of CF were protected as the triphenylmethyl
ether before further reaction with the CpMn(CO)3 or CpRe(CO)3 carboxylic acids.
The CpMn(CO)3 and CpRe(CO)3 peptide conjugates were purified by preparative HPLC using
acetonitrile/water (10 to 60%) as the gradient and obtained with more than 98% purity after
preparative HPLC. Analytical HPLC traces showed only a single peak for each conjugate
(Figure 3.1.19). The rhenium peptide conjugate with the keto group (14) has a retention
time of 18.2 min, lower than its methylene analogue (15) with tR = 19.09 min. The peptide
conjugates of the organometal compound with the methylene linker (13 and 15) both have a
retention time of about 19 min, demonstrating that the change of the metal center from Mn
to Re does not have much influence on the retention time.
Results and discussion
31
a)
0 5 10 15 20 25 30
0,0
0,5
1,0
1,5
Absorb
an
ce
Retention Time (min)
tR
= 18.80 min
MnOC CO
CO
sC18
O
b) c)
0 5 10 15 20 25 30
0,0
0,5
1,0
Ab
so
rba
nce
Retention Time (min)
tR
= 18.21 min
ReOC CO
CO
sC18
O
O
0 5 10 15 20 25 30
0,0
0,5
1,0
1,5
Ab
sorb
an
ce
Retention Time (min)
tR
= 19.09 min
ReOC CO
CO
sC18
O
Figure 3.1.19: Analytical HPLC traces of a) Cym2*-sC18 (13) b) Cyr2-sC18 (14) c) Cyr2*-sC18 (15) after
purification by HPLC using a C18 column (acetonitrile/water 10-60%)
Further characterization was done by mass spectrometry. The major peaks for the
organometal peptide conjugates are summarized in Table 3.1.3. For the cymantrene–sC18
conjugate Cym2*-sC18 (13), the most intense peak is observed in the MALDI-MS at m/z =
2203.50 and is assigned to [M+H-Mn(CO)3]+. This is due to photolytic cleavage of the
Mn(CO)3 moiety from the bioconjugate by the laser used in the ion source of the MALDI
mass spectrometer, since its 337 nm wavelength coincides with the main absorption band of
the cymantrene. On the other hand, in the ESI mass spectrum, four peaks at m/z = 781.4,
586.4, 469.4 and 391.3 are found, due to [M+3H]3+, [M+4H]4+, [M+5H]5+ and [M+6H]6+.
Results and discussion
32
Table 3.1.3: Analytical data of the organometal-peptide bioconjugates
peptide amino acid sequence MWcalc. MALDI exp. ESIexp.
Cym2-sC18[46]
Cym2-GLRKRLRKFRNKIKEK-NH2 2355.7 n.d. 471.9 [M+5H]5+
Cym2*-sC18 (13) Cym2*-GLRKRLRKFRNKIKEK-NH2 2341.8 n.d. 469.4 [M+5H]5+
Cyr2-sC18 (14) Cyr2-GLRKRLRKFRNKIKEK-NH2 2487.0 2485.4 [M+H]+
498.5 [M+5H]5+
Cyr2*-sC18 (15) Cyr2*-GLRKRLRKFRNKIKEK-NH2 2473.0 2471.5 [M+H]+
495.6 [M+5H]5+
Cym2*-sC18(CF) (16)Cym2*-GLRK(CF)RLRKFRNKIKEK-
NH2
2700.1 n.d. 541.0 [M+5H]5+
Cyr2-sC18(CF) (17) Cyr2-GLRK(CF)RLRKFRNKIKEK-NH2 2845.3 2843.4 [M+H]+
n.d.
Cyr2*-sC18(CF) (18) Cyr2*-GLRK(CF)RLRKFRNKIKEK-NH2 2831.3 2829.4 [M+H]+
n.d.
Experimental data for Cym2-sC18 adapted from ref.[46] n.d.: not determined.
In contrast, the CpRe(CO)3 conjugates show good stability also under MALDI conditions. In
addition to the main peak resulting from [M+H]+, the [M+2H]2+ peaks could also be observed.
The expected signals were also found in the ESI-MS.
For further proof of the presence of the Mn(CO)3 and Re(CO)3 moieties, IR spectra were
recorded for all conjugates. The IR spectrum of conjugate Cym2*-sC18 (13) shows four peaks
at 2017, 1925, 1651 and 1537 cm-1. For the rhenium conjugate Cyr2-sC18 (14), the peaks at
2020, 1922, and 1653 cm-1 are only slightly shifted, while the last one remains at 1537 cm-1.
The peaks at around 2020 and 1925 cm-1 are due to the symmetrical and asymmetrical
vibrations of the M(CO)3 moieties, and the bands at 1650 and 1540 cm-1 are assigned to the
amide I and II bands of the peptide. Although these dominate the IR spectra because of a
total of 16 amide linkages present in the sC18 peptide conjugates, the two C≡O bands of the
organometallic moiety are still very distinctly visible.
Results and discussion
33
a)
4000 3000 2000 1000
0
25
50
75
100T
ran
sm
issio
nin
%
Wavenumber in cm-1
2017 1925
1651
1537
b)
4000 3000 2000 1000
60
70
80
90
100
Tra
nsm
issio
nin
%
Wavenumber in cm-1
2020
1922
1653
1537
Figure 3.1.20: ATR-IR spectra of a) Cym2*-sC18 (13) and b) Cyr2-sC18 (14) peptide conjugates
Results and discussion
34
3.1.4 Fluorescence microscopy of CF-labeled organometal peptides in MCF-7 human
breast cancer cells
The internalization of the CF-labeled organometal–peptide bioconjugates by MCF-7 human
breast cancer cells was studied using fluorescence microscopy. Living unfixed cells were
incubated for 30 min with 10 or 20 µM solutions of CF-labeled conjugates. The medium was
then exchanged and the cells were quenched with trypan blue solution and incubated with
Hoechst 33342 nuclear stain. Thus, the cell nucleus is visible in blue and the CF-labeled
peptide conjugates in green. As shown in Figure 3.1.21, all peptide conjugates translocated
into the cells after 30 min of incubation as evident from the green fluorescence associated
with the cells. The peptide conjugate sC18(CF) has a vesicular distribution pattern, pointing
to an endosomal uptake mechanism. The Cym4-sC18(CF) conjugate shows an intracellular
distribution similar to sC18(CF), as demonstrated in our previous work.[46,47] No effect of the
exchange of the metal center from manganese to rhenium, or by increasing the
concentration of Cyr2-sC18 from 10µM to 20 µM was observed.
a) b)
c) d)
Figure 3.1.21: Confocal fluorescence microscopy pictures of the sC18 peptide bioconjugates (a) 20
µM sC18(CF) (b) 20 µM Cym4-sC18(CF) (c) 10 µM and (d) 20 µM Cyr2-sC18(CF) (17) applied to MCF-7
breast cancer cells and visualized after 30 min of incubation. Blue: cell nucleus, green: CF-labeled
peptide conjugates. (a) and (b) adapted from ref.[46]
Results and discussion
35
In contrast, the two conjugates Cym2*-sC18(CF) (16) and Cyr2*-sC18(CF) (18) with the
reduced methylene linker showed significant cellular uptake with some degree of nuclear
accumulation already at 10 µM (Figure 3.1.22a+c). Furthermore, the overall uptake seems to
be higher compared to the other substances studied as evident from the brighter
fluorescence. When the applied concentration was increased to 20 µM, the whole cytosol is
filled with the green fluorescence of the CF label (Figure 3.1.22b+d). Also, a pronounced
pattern of intranuclear localization with most intensity associated with a circular structure,
most likely the nucleolus, is observed. Thus, although the influence of the metal center Mn
vs. Re on the intracellular distribution as in Cym2*-sC18(CF) (16) and Cyr2*-sC18(CF) (18) is
negligible, the reduction of the keto linker between the CpM(CO)3 moiety and the
carboxylate group, connecting the complex to the peptide, to a methylene group results in a
significantly different uptake pattern with pronounced nuclear accumulation in both the Mn
and Re peptides.
a) b)
c) d)
Figure 3.1.22: Confocal fluorescence microscopy pictures of the sC18 peptide bioconjugates at: a) 10
µM and b) 20 µM Cym2*-sC18(CF) (16), c) 10 µM and d) 20 µM Cyr2*-sC18(CF) (18) applied to MCF-7
breast cancer cells and visualized after 30 min of incubation. Blue: cell nucleus, green: CF-labeled
peptide conjugates.
Results and discussion
36
3.1.5 Cytotoxicity studies
Only four organometal complexes 1-4 out of the total of twelve CpM(CO)3 carboxylic acids
synthesized were chosen for further biological studies, since bioconjugates with aliphatic or
aromatic linkers showed silimar cytotoxicity according to our previous work.[46] The
cytotoxicity of the four metal complexes as well as their organometal–peptide bioconjugates
on MCF-7 human breast cancer cells was determined with the resazurin assay. Stock
solutions of 1 (Cym2), 2 (Cym2*), 3 (Cyr2), and 4 (Cyr2*) were prepared in acetonitrile/water
(1:2) (5 mM) and diluted with cell medium to concentrations of 0 to 200 µM. Then, all
compounds were incubated with MCF-7 cells at 37 °C for 24 h in the dark. No decrease in cell
viability was observed for any of the four complexes even at the highest tested
concentration of 200 µM (Figure 3.1.23a). Furthermore, the CF-labeled parent peptide sC18
also has no influence on the cell viability (data not shown). Stock solutions of the
organometal–peptide conjugates (1 mM) were prepared in water because of their better
solubility in this medium and were diluted with cell medium before incubation with MCF-7
cells under the same conditions as mentioned above at a range from 10 to 100 µM.
Figure 3.1.23: Change of cell viability of MCF-7 human breast cancer cells upon incubation with (a)
Cym2 (1), Cym2* (2), Cyr2 (3), and Cyr2* (4), (b) Cyr2 (14) and Cyr2*-sC18 (15) determined after 24 h
of incubation with the resazurin assay
Results and discussion
37
Table 3.1.4: IC50 values of the peptide bioconjugates on MCF-7 human breast cancer cells
determined after 24 h with the resazurin assay
Peptide conjugate IC50 [μM]
Cym2-sC18 (ref.[46]) 56.9 ± 2.1
Cyr2-sC18 (14) 59.2 ± 7.3
Cym2*-sC18 (13) 43.8 ± 6.4
Cyr2*-sC18 (15) 40.8 ± 8.9
For all four conjugates, a concentration-dependent decrease of the relative cell viability was
observed as shown in Figure 3.1.23b as an example. The Cym2–sC18 (adapted from ref.[46])
and Cyr2-sC18 (14) bioconjugates show very similar IC50 values of about 60 µM (Table 3.1.4),
which compares well with the biological activity previously observed for other cymantrene–
sC18 bioconjugates.[46] Thus, the substitution of manganese by rhenium in the CpM(CO)3
moiety does not have an effect on the cytotoxicity. On the other hand, reduction of the keto
linker to a methylene group as in Cym2*-sC18 (13) and Cyr2*-sC18 (15) leads to a decrease
of the IC50 value by about 20 µM to around 40 µM as determined for both of these
conjugates (Table 3.1.4). Again, the difference between the corresponding manganese and
rhenium compounds is negligible within the accuracy of the resazurin assay.
Results and discussion
38
To obtain a more detailed picture of the mechanism of cytotoxic activity, the disturbance of
the membrane integrity of the MCF-7 cells by the organometal–peptide conjugates was
studied with the lactate dehydrogenase (LDH) leakage assay. Release of intracellular LDH
into the culture medium is an indicator of irreversible cell death due to membrane damage.
Hence, the activity of LDH in the extracellular medium is measured. The unmodified peptides
without metal complex attached showed no effect on the membrane integrity (results not
shown). In contrast, application of the organometal-peptide conjugates leads to a release of
LDH into the cell culture medium due to damage of the plasma membrane in a
concentration-dependent manner (Figure 3.1.24). Again, conjugates Cym2*-sC18 (13) and
Cyr2*-sC18 (15) with the methylene spacer show a somewhat more pronounced effect on
the LDH release than Cym2–sC18[52] and Cyr2-sC18 (14) with the keto linker.
Figure 3.1.24: Amount of LDH released upon incubation of MCF-7 human breast cancer cells with (a)
Cym2 (1), Cym2* (2), Cyr2 (3), and Cyr2* (4); (b) Cyr2 (14) and Cyr2*-sC18 (15) conjugates for 2 h
Results and discussion
39
3.2 Biological studies of organometal-dendrimer bioconjugates
3.2.1 Objective
Organometal aldehydes instead of carboxylic acids were synthesized to couple them with
the terminal amino groups of PAMAM and DAB dendrimers since no additional activation or
coupling reactants are needed for the Schiff base reaction and the purification of the final
products is thus simplified. Several d-block transition metals were chosen to study the
influence of the metal centers on the biological activity, and adamantane aldehyde was
prepared as a purely organic contrast to the organometal complexes (Figure 3.2.1).
MnOC CO
CO
H
O
N
N
MoCO
CO
CO
CO
OHC
ReOC CO
CO
H
O
CrOC CO
CO
H
OHO
19 22332120
Figure 3.2.1: Aldehydes preperad as precusors for the coupling with dendrimers
The aldehydes were to be coupled to a first generation PAMAM dendrimer as well as
commercial available first-, second- and third-generation DAB dendrimers (Figure 3.2.2). The
amido-amine arms branching from the PAMAM dendrimer core lead to a different structure
compared to the DAB dendrimers, while variation of the dendrimer generation potentially
enables a study of the investigation of the enhanced permeability and retention (EPR) effect,
which is based on a size-dependent selective accumulation in tumor tissue of these
macromolecules.
NN
NH
NH
NH2
NH2
HN
HN
H2N
H2N
O
O
O
O
H2N NN NH2
NH2
H2N
(a)
(b)
Figure 3.2.2: Examples of dendrimers cores used in this work: (a) first-generation PAMAM dendrimer
and (b) first-generation DAB dendrimer
Results and discussion
40
3.2.2 Synthesis of organometal and adamantane aldehydes as precursors for the
coupling with dendrimers
The aldehyde-functionalized half-sandwich complexes were prepared by reaction of the
CpM(CO)3 parent compounds with n-butyllithium and N, N-dimethylformamide at –78 °C
and products were obtained in good yields of over 75% (Scheme 3.2.1). Column
chromatography on silica with ethyl acetate/n-hexane (v/v 2:5) as the eluent allowed the
removal of the non-modified cyclopentadienyl starting materials. The aldehydes could be
further purified by sublimation at 50 °C and 10-2 mbar. The final products were obtained as
crystalline solids.[53,54]
MOC CO
CO
H
O
MOC CO
CO
n-Buli/THF -78°C
DMF
M = Mn (19), Re (20)
Scheme 3.2.1: Reaction of CpM(CO)3 with n-butyllithium and N, N-dimethylformamide
The cymantrene carboxaldehyde 19 was characterized by 1H-NMR in chloroform solution.
Three peaks at 9.61, 5.46 and 4.93 ppm with an intensity ratio of 1:2:2 are found as singlets
(Figure 3.2.3). The signal at 9.61 ppm is due to the aldehyde group, and the peaks at 5.46
and 4.93 ppm are assigned to the 2,5- and 3,4-protons of the mono-substituted
cyclopentadiene ring, for which the expected splitting was not observed.
Figure 3.2.3: 200 MHz 1H-NMR spectrum of 19 in CDCl3
Results and discussion
41
In the IR spectrum of compound 19, three intense bands are observed at 2019, 1903 and
1687 cm-1 (Figure 3.2.4). The two signals at 2019 and 1903 cm-1 are assigned to the
symmetrical and asymmetrical C≡O vibrations of the Mn(CO)3 moiety and the peak at 1687
cm-1 results from the aldehyde C=O group.
4000 3000 2000 1000
0
25
50
75
100
Tra
nsm
issio
nin
%
Wavenumber in cm -1
2019
19031687
Figure 3.2.4: ATR IR spectrum of compound 19
The related rhenium complex 20 was synthesized under the same condition as applied for
cymantrene. The rhenium compound had a better stability during the preparation, since the
reaction solution did not turn dark as observed for the manganese compound. After
purification with column chromatography on silica with ethyl acetate/n-hexane (v/v 2:5) as
the eluent followed by sublimation, the final product was isolated as a white solid in over 85%
yield.
In the 1H-NMR spectrum of 20, three signals at 9.59, 6.01 and 5.47 ppm are observed with
an integral ratio of 1:2:2, which show a similar pattern to the spectrum of the manganese
analog 19 (Figure 3.2.5). The peaks at 6.01 and 5.47 ppm are better resolved than in the
manganese compound and are split into triplets, each with a coupling constant of 3J = 2.2 Hz.
These are assigned to the cyclopentadienyl ring protons while the peak at 9.59 ppm with an
integral of 1H is due to the aldehyde group.
Results and discussion
42
Figure 3.2.5: 200 MHz 1H-NMR spectrum of 20 in CDCl3
The chromium tricarbonyl compound 21 was prepared as shown in Scheme 3.2.2, in a
published three-step procedure starting from benzaldehyde.[55] This compound was purified
by column chromatography on silica with ethyl acetate/n-hexane (v/v 2:5) as the eluent and
isolated as a red solid.
21
CrOC CO
CO
OOO H
O
O
Cr(CO)6
CrOC CO
CO
H
O
HCl/H2O
Scheme 3.2.2: Synthesis of compound 21 from benzaldehyde
Compound 21 was characterized by 1H-NMR at 200 MHz. Besides the solvent signal, four
peaks are observed at 9.46, 5.95, 5.66 and 5.29 ppm, with an integral ratio of 1:2:2:2. The
singlet at 9.46 ppm results from the aldehyde proton. The peak at 5.95 ppm is split into a
doublet with a coupling constant of 3J = 5.8 Hz and is due to the phenyl proton at the para
position to the CHO group, while the signals at 5.66 and 5.29 ppm are split into triplets with
3J = 6.4 Hz and are assigned to the other two aromatic protons positions (Figure 3.2.6).
2.0
0
1.0
1
2.0
0
0.9
9
5.2
65.2
95.3
2
5.6
6
5.9
35.9
6
9.4
6
Figure 3.2.6: 200 MHz 1H-NMR spectrum of 21 in CDCl3
Results and discussion
43
For the DMSO oxidation of 1-hydroxymethyl adamantane to adamantane-1-carboxaldehyde
(22), initially, trifluoroacetic anhydride was tried to activate DMSO, but the desired product
could only be obtained in less than 5% yield. By using an alternative literature procedure
using oxalyl chloride, a better yield of about 50% could be achieved.[56,57]
22
OH HO
trifluoroacetic anhydride/oxalyl chloride
DMSO
Scheme 3.2.3: Synthesis of compound 22
Compound 22 was characterized with 1H-NMR in chloroform. Three main peaks were
observed besides the solvent signal of residual DMSO at 9.31, 2.06 and 1.71 ppm. The signal
at 9.31 ppm is a singlet with an integral of 1H and is due to the aldehyde group. The singlet
at 2.06 ppm results from the three methine protons of the adamantane. The signal at 1.71
ppm with an integral of 12H is due to an overlap of two peaks which cannot be resolved, and
is assigned to the six methylene groups of the adamantane moiety.
Figure 3.2.7: 200 MHz 1H-NMR spectrum of 22 in CDCl3
Results and discussion
44
3.3.3 Coupling of the aldehyde precursors with DAB dendrimer G1, G2, G3
H2N NN NH2
NH2
H2N
Figure 3.2.8: First generation DAB (1,4-diaminobutane core) dendrimer
Cymantrene and cyrhetrene carboxaldehydes 19 and 20 were coupled to the terminal amino
groups of G1, G2 and G3 DAB dendrimers (Figure 3.2.8). The chromium compound 21 also
prepared could not be further investigated due to its limited stability under the coupling
conditions. All reactions were performed in anhydrous ethanol with the addition of 2-3 g of 3
Å molecular sieves to shift the Schiff base condensation reaction to the product side. Two
equivalent of sodium borhydride were then added to reduce the imine to a more stable
amine group.
DAB-dendr N
MnOC CO
COn
DAB-dendr N
ReOC CO
COn
CrOC CO
CO
DAB-dendr N
n
MnOC CO
CO
H
O
ReOC CO
CO
H
O
CrOC CO
CO
H
O
DAB-dendr N
H MnOC CO
COn
DAB-dendr NH Re
OC COCO
n
NaBH4
n = 4, 8, 163Å MS
3Å MS
NaBH4
23, 24, 25
n = 4, 8, 16
26, 27, 28
Scheme 3.2.4: Synthesis of organometal conjugates 23-28 of the G1, G2, and G3 DAB dendrimers
The dendrimer conjugates were purified by preparative HPLC on a C18 column with a
gradient of acetonitrile/water (20-95%) for G1 and G2 and (30-95%) for the G3 DAB
dendrimers conjugates, and characterized with ATR-IR spectroscopy and ESI mass
spectrometry. The rhenium-containing dendrimers could not be detected with ESI or MALDI
spectrometry due to poor ionization. The intense IR bands and MS peaks assignments are
summarized in Table 3.2.1. In addition, the adamantly conjugate 29 was prepared in a
similar way as in Scheme 3.2.4.
Results and discussion
45
Table 3.2.1: ATR-IR and ESI mass spectrometric date of the dendrimer conjugates 23-29
compound end group generation IR (cm-1) ESI (positive mode)
23 CpMn(CO)3 G1 2024, 1921 1081.1 [M+H]+,
591.2 [M+2H]2+
24 CpMn(CO)3 G2 2023, 1927 1251.6 [M+2H] 2+,
835.0 [M+3H]3+,
626.5 [M+4H]4+
25 CpMn(CO)3 G3 2024, 1914 1029.7 [M+5H]5+,
858.4 [M+6H]6+
26 CpRe(CO)3 G1 2027, 1913 n.d.
27 CpRe(CO)3 G2 2024, 1905 n.d.
28 CpRe(CO)3 G3 n.d. n.d.
29 adamantyl G1 n.d. n.d.
n.d. = not determined
The conjugate of cymantrene aldehyde with the G1 DAB dendrimer 23 was purified by
preparative HPLC with a gradient of acetonitrile/water (20-95%). The analytical HPLC of the
purified dendrimer shows only one intense peak at tR = 19.2 min.
0 10 20 30
0
100
200
300
Absorb
an
cein
mA
u
Retention Time in min
tR
= 19.2 min
DAB-dendr N
H MnOC CO
COn
Figure 3.2.9: Analytical HPLC trace of compound 23 with a gradient of ACN/H2O 20-95% over 30 min
Results and discussion
46
The ATR IR of compound 23 shows three intense peaks at 2023, 1921 and 1666 cm-1. The
peaks at 2023 and 1921 cm-1 are due to the symmetrical and asymmetrical vibrations of the
Mn(CO)3 moieties. The peak at 1666 cm-1 appears in all six manganese and rhenium G1, G2,
and G3 dendrimer conjugates after preparative HPLC and is probably due to trifluoroacetic
acid adduts remaining from the aldehyde synthesis.
4000 3000 2000 1000
0
25
50
75
100
Tra
nsm
issi
on
in%
Wavenumber in cm-1
20231921
1666
Figure 3.2.10: ATR-IR of compound 23 after preparative HPLC
Compound 23 was also characterized by 1H-NMR spectroscopy. Six peaks at 5.18, 4.97, 3.90,
3.18, 2.17 and 1.77 ppm are observed with an intensity ratio of 8:8:8:20:8:4. The peaks at
5.18 and 4.97 ppm are split into triplets with 3J = 2.2 Hz and are assigned to the mono-
substituted Cp-ring protons. The singlet at 3.90 ppm is due to the four methylene groups
adjacent to the Cp-ring. The broad signal at 3.18 ppm is attributed to the ten methylene
groups next to the nitrogen atoms, and the peaks at 2.17 and 1.77 ppm with an intensity of
8H and 4H, respectively, result from the methylene protons in the dendrimer branches and
the core which are not adjacent to the nitrogen atoms.
4.0
6
8.0
0
19.8
6
8.0
0
8.0
8
7.8
9
1.7
7
2.1
7
3.1
53.1
8
3.9
0
4.9
7
5.1
8
Figure 3.2.11: 200 MHz 1H-NMR spectrum of 23 in methanol-d4
Results and discussion
47
The 1H-NMR spectrum of the rhenium analogue 26 shows a pattern similar to that of
compound 23. Six peaks are found at 5.83, 5.60, 4.00, 3.15, 2.17 and 1.74 ppm. The triplets
at 5.83 and 5.60 ppm with 3J = 2.2 Hz are due to the mono-substituted Cp ring protons and
are slightly shifted compared to the manganese analogue, while the other peaks are found
with similar chemical shifts. Due to the high similarity of the manganese and rhenium
dendrimer conjugates, only the spectra of the manganese conjugates are shown for the
other compounds.
Figure 3.2.12: 200 MHz 1H-NMR spectrum of compound 26
In the 1H-NMR spectrum of the G2 dendrimer conjugate 24, two triplets at 5.26 and 5.08
ppm with 3J = 2.2 Hz and an integral of 16H are observed, and are due to the mono-
substituted cyclopentadienyl rings. A singlet appears at 3.81 ppm with an intensity of 16H
and is assigned to the methylene groups adjacent to the Cp ring. The broad peak at 2.98 ppm
with an integral of 52H results from the methylene groups in dendrimer branches and the
core next to amino groups, and the peaks at 1.97 and 1.62 ppm with intensities of 20H and
8H, respectively, are due to the dendrimer methylene groups not adjacent to the amines. In
the 1H-NMR of the manganese G3 conjugate 25 and the G2 and G3 rhenium analogues 27
and 28, this pattern is also observed. All protons resulting from dendrimer core are grouped
Results and discussion
48
into three broad peaks, one due to the methylene groups adjacent to the amino groups, and
two signals due to the methylene protons not adjacent to the amino groups.
Figure 3.2.13: 200 MHz 1H-NMR spectrum of compound 24
Results and discussion
49
3.2.4 Synthesis of PAMAM dendrimer G1 and its organometal conjugates
The first generation PAMAM dendrimer was prepared using the procedure of Tomalia et
al.[58,59] In the first step, hexamethylenediamine was heated in methyl acrylate at 87 °C for 4
d, and then the solvent was removed in vacuum.
H2NNH2
OMe
O
NN
OMe
OMe
MeO
MeO
O
O
O
O
+ 4
Scheme 3.2.5: Synthesis of compound 30
The compound was characterized by 1H-NMR. Five peaks at 3.63, 2.73, 2.40, 1.37 and 1.19
ppm are observed. The singlet at 3.63 ppm with an integral of 12H is assigned to the four
ester methyl groups. The signal at 2.73 ppm with an intensity of 8H appear as a triplet with 3J
= 7 Hz and is due to the methylene groups adjacent to the ester -COO moiety. The multiplet
with an integral of 12H at 2.40 ppm results from the six NCH2 groups. Finally, the two broad
signals at 1.37 and 1.19 ppm, each with an integral of 4H, are due to the aliphalic protons in
the 2- to 5-positions of the hexamethylene chain.
Figure 3.2.14: 200 MHz 1H-NMR spectrum of 30 in CDCl3
Results and discussion
50
NN
OMe
OMe
MeO
MeO
O
O
O
O
H2NNH2 N
N
NH
NH
NH2
NH2
HN
HN
H2N
H2N
O
O
O
O
+MeOH
RT
Scheme 3.2.6: Synthesis of compound 31
In the second step, 30 was reacted with 1,2-ethylenediamine in methanol for 3 d at room
temperature. The solvent was then removed in vacuum and the final product isolated as a
yellow gel. Since the reaction solvent is very difficult to be totally removed from the
gelatinous dendrimer formed, the samples for NMR experiments were dissolved in
deuterated water, and the NMR solvent removed in vacuum, with the process repeated
several times. In the 1H-NMR of compound 31, four peaks at 2.66, 2.40, 1.44 and 1.26 ppm
with an integral ratio of 8:12:4:4 are observed and correspond to the signals of the
dendrimer core in compound 30. The two triplets at 2.77 and 3.21 ppm appear each with an
integral of 8H and coupling constants of 3J = 6.0 Hz and 7.0 Hz, respectively. They are
assigned to the ethylene groups in the four “arms” attached to PAMAM core.
Figure 3.2.15: 500 MHz 1H-NMR spectrum of 31 in D2O
Results and discussion
51
N
N
OHC
N
N
SeO2
1,4-dioxane
Scheme 3.2.7: Synthesis of compound 32
Bipyridine ligand 32 was prepared from 4,4’-dimethyl-2,2’-bipyridine using selenium dioxide
as the oxidant (Scheme 3.2.7)[60] and was characterized by 1H-NMR. Two singlets at 2.46 and
10.18 ppm with an integral ratio of 3:1 are observed, which are assigned to the methyl and
the aldehyde group, respectively, and indicate the oxidation of only one methyl group. Six
peaks with an integral of 1H are found between 7 and 9 ppm and are split into multiplets, a
doublet of a doublet at 8.83 ppm with 3J = 1.6 Hz and 4J = 0.8 Hz, a doublet at 8.57 ppm with
3J = 5.0 Hz, a quintet at 8.28 ppm with 4J = 0.8 Hz, a doublet of a doublet at 7.72 ppm with3J =
5.0 Hz and 4J = 1.6 Hz, and a quartet of a doublet at 7.19 with 3J = 5 Hz, 4J = 0.8 Hz,
respectively. They are due to the protons of the bipyridine ring.
Figure 3.2.16: 200 MHz 1H-NMR spectrum of 32 in CDCl3
Results and discussion
52
N
N
OHC
N
N
MoCO
CO
CO
CO
OHC
THF
Mo(CO)6
Scheme 3.2.8: Synthesis of compound 33
Complex 33 was prepared by heating bipyridine ligand 32 with molybdenum hexacarbonyl in
tetrahydrofuran and was then coupled to G1 PAMAM dendrimer 31. However, since the
metal complex had insufficient stability in the coupling solvent upon exposure to daylight
and when stored over longer periods of time, the desired dendrimer conjugate could not be
isolated.
NN
NH
NH
NH2
NH2
HN
HN
H2N
H2N
O
O
O
ON
N
MoCO
CO
CO
CO
OHC
NN
NH
NH
N
N
HN
HN
N
N
O
O
O
O
N
N
MoCO
CO
CO
CO
N
N
MoCO
CO
CO
CO
N
N
MoOC
OC
CO
CO
N
N
MoOC
OC
CO
CO
+
Scheme 3.2.9: Coupling of compelx 33 with G1 PAMAM dendrimer 31
Results and discussion
53
NN
NH
NH
HN
HN
HN
HN
NH
NH
O
O
O
O
MnOC CO
CO MnCOOC
OC
MnOC CO
COMn
COOCOC
NN
NH
NH
NH2
NH2
HN
HN
H2N
H2N
O
O
O
O
MnOC CO
CO
H
O
+a) EtOH, RT
b) NaBH4
31 19
34
Scheme 3.2.10: Coupling of cymantrene aldehyde 19 with G1 PAMAM dendrimer 31 to give 34
Thus, the synthetic target was focused on the half-sandwich compounds only. Different
conditions were examined for the coupling of cymantrene carboxaldehyde 19 with G1
PAMAM dendrimer 31 including toluene, acetonitrile, methanol, and ethanol as the solvent,
using magnesium sulfate or molecular sieves to absorb the water liberated from the
condensation reaction to force the Schiff base reaction to the product side. Sodium
borhydride was added to reduce the imine formed to a more stable amine. The reaction was
monitored by thin layer chromatography with ethyl acetate:n-Hexan (v/v 3:5) as the eluent.
The reaction using ethanol as the solvent and 3 Å molecular sieves to remove the water
proved to be the most suitable conditions. The solvent was then removed and the product
further purified by preparative HPLC using acetonitrile/water (20 to 95%) as the gradient.
Only one major peak at tR = 15.54 min was observed in an analytical run after the
preparative HPLC, which indicates a very good purity.
0 10 20 30
0
1000
2000
3000
Ab
so
rban
ce
inm
Au
Retention Time in min
tR
= 15.54 min
Figure 3.2.17: Analytical HPLC trace of 34 with a 20-95% acetonitrile/water gradient as the eluent
Results and discussion
54
Compound 34 was characterized by ATR-IR spectroscopy. Three intense peaks are observed
at 2013, 1913, and 1643 cm-1. The signals at 2013 and 1913 cm-1 are due to the symmetrical
and asymmetrical vibrations of the Mn(CO)3 carbonyl groups, respectively. The peak at 1643
cm-1 results from the four C=O groups of the amide bond in the dendrimer core.
4000 3000 2000 1000
10
20
30
Tra
nsm
issi
on
in%
Wavenumber in cm-1
2013
1913
1643
Figure 3.2.18: ATR-IR spectrum of compound 34
In the 1H-NMR spectrum of compound 34, two triplets at 5.20 and 4.96 ppm, both with
coupling constants of 3J = 2.2 Hz and an integral of 8H are observed, which are due to the
protons of the mono-substituted cyclopentadiene ring. A singlet at 3.93 ppm with an integral
of 8H results from the methylene group adjacent to the Cp ring. A multiplet with an intensity
of 16H appears at 3.51 ppm, and is from the overlapping signals of the ethylene protons,
which appear in the non-modified PAMAM dendrimer as two triplets. The signals at 3.21,
2.78, 1.79 and 1.46 ppm with an integral ratio of 12:8:4:4 are due to the six NCH2 groups,
COCH2-groups, and -NCH2CH2CH2CH2CH2CH2N- protons of the hexamethylene chain.
Figure 3.2.19: 200 MHz 1H-NMR of compound 34 in methanol-d4
Results and discussion
55
3.2.5 Cytotoxicity studies
Manganese and rhenium G1, G2 and G3 DAB dendrimer conjugates 23-28 as well as
cymantrene PAMAM conjugate 34 and the adamantane-DAB conjugate 29 were incubated
with MCF-7 human breast cancer cells for 24 h and their relative cell viability was
determined with the resazurin assay. Due to the high toxicity of the conjugates, the relative
cell viability at four concentrations in the range of 1 to 25 µM was measured.
a)
b)
Figure 3.2.20: Cell viability of MCF-7 human breast cancer cells after 24 h of incubation with
conjugates 23-29 and 34 determined with the resazurin assay. a) first and b) second set of
experiments.
0
10
20
30
40
50
60
70
80
90
100
1 µM 5 µM 10 µM 25 µM
rela
tive
cell
viab
ility
[%]
23
24
25
34
26
27
28
29
0
20
40
60
80
100
120
140
160
180
1 µM 5 µM 10 µM 25 µM
23
24
25
34
26
27
28
29
rela
tive
cell
viab
ility
[%]
Results and discussion
56
At 1 µM, all conjugates did not influence the cell viability much, with values of around 80%
to 100% (Figure 3.2.20a+b). However, at 5 µM, first differences were observed. The activity
decreased with increasing generation of the dendrimer in both the manganese and the
rhenium series 23-25 and 26-28. The PAMAM dendrimer conjugate 34 showed the least
effect on the cell viability, and the adamantane DAB G1 conjugate 29 was among the most
active ones, with its activity comparable to those of the manganese and rhenium conjugates
23 and 26 of the same G1 generation. Furthermore, slight differences were observed in the
DAB conjugates upon variation of the metal center: the rhenium compounds 26-28 were all
a bit more active than the manganese complexes 23-25. At 10 µM, more significant
differences were observed for the DAB conjugates. Only 20% of the cells remained viable
upon exposure to both manganese and rhenium G1 compounds 23 and 26, while over 60%
of the cells were still alive when incubated with the G3 conjugates 25 and 28. The activity of
the PAMAM G1 conjugate 34 was inbetween those of the manganese and rhenium G3 DAB
conjugates. The adamantane G1 DAB conjugate 29 again showed an activity similar to those
of the corresponding manganese and rhenium G1 compounds. At 25 µM, both DAB G3
conjugates 25 and 28 showed less activity than the other compound at this concentration,
with about 40 and 60% cell viability for the manganese and rhenium conjugates, respectively,
while all other systems led to a reduction of the cell viability to around 20%.
For all conjugates, the relative cell viability decreases with increasing concentration.
However, for the DAB conjugates, a lower activity was observed for higher generation
dendrimers, both in the manganese and rhenium series. The exchange of the metal center
from manganese to rhenium in the DAB dendrimer conjugates lead to a slight increase in the
biological activity. The first generation cymantrene PAMAM dendrimer conjugate 34 was
less active compared to the G1 DAB Mn and Re dendrimers 23 and 26 at 5 and 10 µM. The
adamantane G1 DAB conjugate 29 has an activity similar to those of the organometallic G1
manganese and rhenium DAB conjugates. Thus, the cytotoxicity activity of the dendrimer
conjugates does not seem to directly correlate with the number of organometal or organic
terminal groups, but rather some more subtle effects related to cellular uptake or
intracellular distribution might be operative. The small differences between the Mn/Re
conjugates and the adamantly-substituted analogues also points to a mechanism of activity
in which probably the lipophilicity/hydrophilicity balance of the end groups plays a more
important role than other CpM(CO)3-inherent properties.
Results and discussion
57
3.3 Metal carbonyl complexes as vibrational peptide labels
3.3.1 Objective
In the conventional synthetic procedures to introduce distinct functionalities into peptides,
orthogonal side-chain protective group strategies are used. In this part of the thesis,
different cyclopentadienyl metal carbonyl compounds with distinct IR vibrations of the C≡O
groups are to be conjugated to peptides, to evaluate their potential use as Raman or IR
labels for information encoding. The organometallic moieties were to be attached to the
side chain ε-amino group of L-lysine, thus also blocking the side-chain functionality of the
amino acids (Figure 3.3.1).
36 37
OH
O
HNMnOC CO
CO
NH
OO
OH
O
HNReOC CO
CO
NH
OO
Figure 3.3.1: Organometal amino acids 36 and 37 to be prepared by Schiff base reaction of aldehyde-
functionalized half-sandwich complexes with the ε-side chain amino group of L-lysine followed by
reduction of the imine moiety to an amine
3.3.2 Synthesis of organometal amino acids via Schiff base reaction
Cymantrene and cyrhetrene carboxaldehydes were prepared as described above in section
3.2.2. In L-lysine, there are two amino groups which could undergo a Schiff base reaction.
Since the side-chain ε-amino group of lysine is more reactive, the first attempt was to react
one equivalent of organometal aldehyde with unprotected L-lysine and to further protect
the N-terminal amino group then with Fmoc-OSu (Scheme 3.3.1)[61].
H2NOH
O
NH2
+Mn
OC COCO
H
O
OH
O
NH2MnOC CO
CO
NH
Fmoc-OSu pH 8 - 9
KHSO4, RT
OH
O
HNMnOC CO
CO
NH
OO
Scheme 3.3.1: Proposed sequential introduction of organometal moiety and N-terminal Fmoc
protective group to L-lysine
Results and discussion
58
However, the reaction of cymantrene carboxaldehyde with L-lysine did not occur without
the addition of further reagents. Thus, the reaction was performed using sodium hydroxide
as a base in acetonitrile or methanol for different reaction times, and with the addition of
magnesium sulfate or 4 Å molecular sieves to remove water to facilitate the condensation.
Still, according to thin layer chromatography and 1H-NMR, the starting materials remained
unchanged, under all conditions examined (Scheme 3.3.2).
H2NOH
O
NH2
+OH
O
NH2MnOC CO
CO
NH
+
NaOHacetonitrile
MnOC CO
CO
H
O
orMgSO4
orMS 4 Å
methanolor
Scheme 3.3.2: Attempted Schiff base reaction of cymantrene carboxaldehyde with L-lysine
The free carboxylic acid group was suspected to interfere with this reaction and therefore
was protected as the methyl ester.[62] Thus, L-lysine was dissolved in methanol and cooled to
0 °C. Thionyl chloride was added over 30 min to the mixture which was then heated to reflux
for 4 h. The solvent was removed, methanol was added, and distilled off again to inactivate
remaining thionyl chloride. The methyl ester of L-Lysine was successfully obtained this way
and characterized with 1H-NMR. The further reaction with cymantrene carboxaldehyde was
performed in acetonitrile with addition of sodium carbonate as base (Scheme 3.3.3). This
reaction was monitored with TLC and 1H-NMR but the desired product could also not be
prepared under these conditions.
O
O
NH2MnOC CO
CO
NH
H2NOH
O
NH2
+ CH3OHSOCl2, MeOH
0°C - 90 °C
H2NO
O
NH2
2 HCl
MnOC CO
CO
H
O
Scheme 3.3.3: Protection of carboxyl group of L-lysine as the methyl ester and attempted further
reaction of the ε-amino functionality with cymantrene carboxaldehyde
Results and discussion
59
The same reaction was also examined with commercially available ferrocene carboxaldehyde
instead of cymantrene aldehyde to check if the reactivity or quality of the cymantrene
aldehyde is a crucial factor (Scheme 3.3.4). However, no product formation could be
observed with this aldehyde, either.
Fe
CHO
H2NO
O
NH2
2 HCl +HN
O
O
NH2Fe
Scheme 3.3.4: Attempted reaction of carboxyl-protected L-lysine with ferrocene carboxaldehyde
Next, the procedure of Ivanov et al. was applied for further experiments.[63] Thus, the
reaction of L-lysine with cymantrene carboxaldehyde was carried out in ethanol plus 0.5 wt%
of trifluoroacetic acid. 3 Å molecular sieves were used to absorb the water liberated to
facilitate the Schiff base condensation. The reaction mixture was stirred overnight and 3
equivalents of sodium borhydride were then added for in situ reduction of the imine, and
excess reducing agent quenched by addition of 10% hydrochlorid acid (Scheme 3.3.5). 10 %
hydrochloric acid was added to destroy the exceed sodium borhydride. The light yellow
product formed was extracted with ethyl acetate from the aqueous phase.
19
H2NOH
O
NH2
+Mn
OC COCO
H
O
a) TFA, EtOH, RT
b) NaBH4
OH
O
NH2MnOC CO
CO
NH
Scheme 3.3.5: Reaction of cymantrene carboxaldehyde and L-lysine with trifluoroacetic acid as the
catalyst followed by reduction of the imine intermediate to a more stable secondary amine
The isolated solid was dissolved in ethanol from which pale yellow crystals precipitated upon
slow evaporation. The compound was analyzed with electrospray mass spectrometry (ESI-
MS). Only one intense peak was observed in the negative mode at m/z = 578.9, which
matches with an [M-H]- species 35 in which both amino groups have reacted with
cymantrene aldehyde (Figure 3.3.2).
Since almost equimolar amounts (1.1:1) of cymantrene and L-lysine were used as starting
materials, a possible reason for the unexpected isolation of this bis-functionalized product
Results and discussion
60
could be due to its better solubility compared to the one with only one cymantrene moiety
in the organic phase and therefore the species is isolated. Since the desired mono-
substituted amino acid could not be isolated by recrystallization or column chromatography,
other synthetic strategies were employed in further experiments.
400 500 600 700 800
m/z = 578.9 [M-H]-
m/z
OH
O
HNMnOC CO
CO
NH
MnOC CO
CO
Figure 3.3.2: Negative mode ESI-MS of bis-functionalized compound 35 in methanol
Since the cymantrene carboxaldehyde could not be selectively reacted with the side-chain
ε-amino group of L-Lysine, it was N-terminally protected with Fmoc and employed for the
later introduction of the organometal moiety as an alternative approach (Scheme 3.3.6).
19
OH
O
HN
H2N
OO
MnOC CO
CO
H
O
+
OH
O
HNMnOC CO
CO
NH
OO
36
Scheme 3.3.6: Attempted reaction of cymantrene carboxaldehyde and N-terminal protected L-lysine
followed by imine reduction
Results and discussion
61
The protection of the N-terminal amino group of L-Lysine with Fmoc was performed
according to a literature procedure[61], which involves a dicyclohexylamine amino acid salt
intermediate (Scheme 3.3.8). It is described that side-chain unprotected L-lysine can be
prepared in good yield under these experimental conditions, but the synthesis failed in our
hands. The product of the first step could be isolated but the second one was unsuccessful.
A possible reason could be that this general procedure for the synthesis of Nα-Fmoc amino
acids is based on side-chain protected L-Asp(tBu)-OH, while the side-chain unprotected
lysine has two amino groups with different pKa values. The experimental conditions are
optimized for pH 8-9, which is suitable for the reaction of L-Asp(tBu)-OH with Fmoc-Osu, but
could be inappropriate for L-lysine.
H3NO
O
NH3NH
+ H3NO
O
NH3
H2NAcetone
4 - 5 h, RT
H3NO
O
NH3
H2N N OO
OFmoc
+
OH
O
HN
H2N
OO
pH 8 - 9
KHSO4, RT
Scheme 3.3.8: Synthesis of Fmoc-protected L-lysine according to the literature[61]
A more simple strategy, mimicking the cleavage conditions of solid phase peptide synthesis
(SPPS) was adapted next. 95% trifluoroacetic acid with 5% water as a scavenger was added
to commercially available Fmoc-L-Lys(Boc)-OH and a rapid release of carbon dioxide was
observed. 30 min after the gas release finished, cold diethyl ether was added to the TFA-
amino acid solution and cooled to –20 °C for more than 20 min. The Boc-unprotected amino
acid precipitated as a sticky white solid. It was washed several times with cold diethyl ether
and then dried in vacuum.
OH
O
HN
HN
OO
Boc
TFA 95%/H2O 5%
OH
O
HN
H2N
OO
Scheme 3.3.9: Cleavage of the Boc-protective group from Fmoc-L-Lys(Boc)-OH
with trifluoroacetic acid
Results and discussion
62
The cleavage was monitored with analytical HPLC using acetonitrile/water (20 to 90%) as the
eluent, as shown in Figure 3.3.3. The Boc-protected Fmoc-lysine exhibits a retention time of
tR = 23.4 min. After cleavage, the peak of the Fmoc-L-Lys(Boc)-OH has almost disappeared
and an intense new peak appears at tR = 15.44 min, which indicates a successful removal of
the Boc group and a good purity of the product.
a) b)
0 10 20 30
0
1000
2000
3000
OH
O
HN
HN
OO
Boc
Absorb
ance
Retention Time (min)
tR
= 23.4 min
0 10 20 30
0
1000
2000
3000
OH
O
HN
H2N
OO
tR
= 15.44 min
Ab
sorb
an
ce
Retention Time (min)
Figure 3.3.3: Analytical HPLC of (a) Fmoc-L-Lys(Boc)-OH and (b) Fmoc-L-Lys-OH with a gradient of
acetonitrile/water (20-90%)
Different reaction conditions were then examined for the further condensation of the Fmoc-
lysine with cymantrene or cyrhetrene carboxaldehyde as shown in Scheme 3.3.10, incuding
the addition of different bases (sodium carbonate, DIPEA), and the reaction with Fmoc-lysine
additionally protected with alloc at the carboxylic group, but under none of the conditions,
successful coupling could be observed.
DIPEAOH
O
HN
H2N
O
OMn
OC COCO
H
O
+
OH
O
HNMnOC CO
CO
NH
OO
O
O
HN
H2N
OO
MnOC CO
CO
H
O
+
Acetonitrile, mol sieve
O
O
HNMnOC CO
CO
NH
OO
Na2CO3
or
Scheme 3.3.10: Attempted reactions of cymantrene carboxaldehyde with Fmoc-lysine unter different
conditions
Results and discussion
63
The formation of a TFA adduct with the lysine ε-amino group after cleavage of the Boc-group
could be a possible reason for the unsuccessful Schiff base condensation. Thus, sodium
acetate was added as a base for the removal of the trifluoroacetic acid. The reaction was
carried out in anhydrous ethanol at room temperature overnight and molecular sieves with
a pore size of 3 Å were applied to remove the water formed (Scheme 3.3.11).
OH
O
HN
H2N
OO
MnOC CO
CO
H
O
+
OH
O
HNMnOC CO
CO
NH
OO
NaOAc, EtOH, 3 Å MS
3619
NaBH4
Scheme 3.3.11: Reaction of cymantrene carboxaldehyde with Fmoc-lysine using sodium acetate as
the base to give 36
The reaction was monitored with analytical HPLC. The peaks at 15.65 min and 17.74 min are
assigned to the Fmoc-lysine and cymantrene starting materials, respectively. A new peak
appeared at 21.48 min (Figure 3.3.4). It was later isolated with preparative HPLC and
identified with IR, 1H-NMR and ESI-MS as the desired coupling product 36.
0 10 20 30
0
500
1000
1500
Ab
sorb
an
ce
Retention Time (min)
OH
O
HN
H2N
OO
MnOC CO
CO
H
O
OH
O
HNMnOC CO
CO
NH
O
O
Figure 3.3.4: Analytical HPLC of the reaction between cymantrene carboxaldehyde and Fmoc-lysine
using sodium acetate as base
Results and discussion
64
Five different concentrations of sodium acetate ranging from 0.25 to 3 equivalents were
used for the reaction to identify optimum condition. Each reaction was monitored with
analytical HPLC as shown in Figure 3.3.5. The best yield of product 36 was obtained when
two equivalents of sodium acetate were applied, and thus these conditions were used for
further synthesis.
0 5 10 15 20 25 30
0
440
880
1320
1760
0.25 eq.
0.5 eq.
1 eq.
2 eq.
Retention Time (min)
Abso
rba
nce
NaOAc
3 eq.
OH
O
HN
H2N
OO
OH
O
HNMnOC CO
CO
NH
O
O
Figure 3.3.5: HPLC traces of the reaction of cymantrene carboxaldehyde with Fmoc-lysine at different
concentrations of sodium acetate base added
Compound 36 was further purified by preparative HPLC. Figure 3.3.6 shows the analytical
HPLC trace of the compound after purification, using acetonitrile/water (20 to 95%) as the
gradient. Only one major peak at tR = 21.79 min indicates a good purity of the product and
its stability under HPLC conditions.
0 10 20 30
0
1000
2000
3000
tR
= 21.79 min
Abso
rban
ce
Retention Time (min)
OH
O
HNMnOC CO
CO
NH
OO
Figure 3.3.6: Analytical HPLC of 36 after preparative HPLC purification with acetonitrile/H2O 20-90%
Results and discussion
65
In the ATR IR spectrum of compound 36, five intense peaks are observed at 2023, 1927,
1660, 1196 and 1138 cm-1 (Figure 3.3.7). The two signals at 2023 and 1927 cm-1 are the
characteristic symmetrical and asymmetrical vibrational bands of the Mn(CO)3 moiety. The
broad signal at 1660 cm-1 is due to the overlapping C=O vibration of the carboxylic acid and
the keto function in the Fmoc urethane group. The peaks at 1196 and 1138 cm-1 are typical
for the aromatic ring vibration of the fluorenyl group.
4000 3000 2000 1000
0
20
40
60
80
100
120
Tra
nsm
issio
nin
%
Wavenumber in cm-1
20231927
1660 11961138
Figure 3.3.7: ATR IR spectrum of compound 36
The 500 MHz 1H-NMR of 36 was measured in methanol-d4 (Figure 3.3.8). The four peaks at
7.80, 7.66, 7.40 and 7.31 ppm all exhibit an integral of 2H. They belong to the fluorenyl ring
of the Fmoc-protective group. The peak at 7.80 ppm appears as a doublet with a coupling
constant of 3J = 7.5 Hz, and the one at 7.66 ppm is a doublet with 3J = 7.5 Hz. The peak at
7.40 ppm is a triplet with 3J = 7.5 Hz and the one at 7.31 ppm is a doublet of a triplet with 3J
= 7.5 Hz and 4J = 1.0 Hz, respectively. The signals at 5.15 and 4.95 ppm are split but are not
very well resolved and a determination of the coupling constants is thus not possible.
However, the chemical shifts of around 5 ppm and the integral ratio of 2:2 are indications
that they are due to the protons of the monosubstituted cyclopentadiene ring. The singlet at
3.85 ppm with an integral of 2H can be assigned to the methylene group adjacent to the Cp
ring. In addition to the water and methanol solvent signals at 3.31 and 4.90 ppm, there are
four peaks at 4.41, 4.32, 4.23 and 4.17 ppm, all with an integral of 1H and another peak at
3.85 ppm with an integral of 2H. The ones at 4.41 and 4.32 ppm both appear as a doublet of
a doublet with 3J = 5.0 and 6.0 Hz, respectively, and the one at 4.23 ppm is a triplet with 3J =
7.0 Hz, while the peak at 4.17 ppm appears as a singlet. Together with the other not very
well resolved multiplets in the aliphatic range at 3.01, 1.89, 1.71 and 1.49 ppm, all non-
Results and discussion
66
aromatic protons of the lysine and the Fmoc group could be identified. Thus, all 1H NMR
signals expected for 36 could be assigned.
Figure 3.3.8: 500 MHz 1H-NMR spectrum of 36 in methanol-d4
The compound was also characterized by ESI mass spectrometry. There is only one intense
peak for 36 at m/z = 585.14 in the negative mode which can be assigned to the [M-H]- of the
desired product (Figure 3.3.9).
400 500 600 700 800
m/z
OH
O
HNMnOC CO
CO
NH
OO
m/z = 585.14 [M-H]-
Figure 3.3.9: Negative mode ESI-MS of compound 36 in methanol
Results and discussion
67
The rhenium analog 37 of compound 36 was also prepared under the same reaction
conditions as applied for the cymantrene carboxaldehyde but with longer reaction times,
due to the lower reactivity of rhenium compare to manganese (Scheme 3.3.12). Two
equivalents of sodium acetate were applied as base and the reaction mixture was stirred at
room temperature for 2 d. Sodium borhydride was then added to reduce the imine group to
the more stable secondary amine.
NaOAc, EtOH, 3 Å MS
37
OH
O
HN
H2N
OORe
OC COCO
H
O
+
OH
O
HNReOC CO
CO
NH
OONaBH4
Scheme 3.3.12: Reaction of cyrhetrene carboxaldehyde with Fmoc-lysine using sodium acetate as
base
After isolation of the product 37, it was purified by preparative HPLC under the same
condition as applied for 36. The IR spectrum of compound 37 shows the similar pattern as
observed for its manganese analogue, five intense bands are found at 2023, 1913, 1666,
1182 and 1140 cm-1, only slightly shifted compared to 36.
4000 3000 2000 1000
20
40
60
80
100
Tra
nsm
issi
on
in%
Wavenumber in cm-1
20231913
16661140
1182
Figure 3.3.10: ATR IR spectrum of compound 37
Results and discussion
68
In the negative mode of ESI-MS, there is only one peak at m/z = 717.16 which is assigned to
[M-H]-. It shows the typical isotope pattern of the naturally occurring 185Re and 187Re
isotopes as depicted in Figure 3.3.10.
500 600 700 800 900 1000
700 710 720 730 740
m/z
m/z = 717.16 [M-H]-
m/z
OH
O
HNReOC CO
CO
NH
OO
Figure 3.3.11: Negative mode ESI-MS of 37 in methanol and (inset)
showing the enlarged Re isotope pattern
Results and discussion
69
3.3.3 Solid phase peptide synthesis (SPPS) with Mn- and Re-containing lysines as
building blocks
3.3.3.1 Introduction of the Mn- and Re-containing lysines to the peptide H-TRKKRKRG-NH2
The peptide with the sequence H-TRKKRKRG-NH2 was prepared manually on a Rink amide
AM resin. Manual coupling of each commercially available amino acid as well as cymantrene-
and cyrhetrene-containing lysines 36 and 37 was achieved by activation with 1.5 equiv HATU
and N,N-diisopropylethylamine following our reported procedure[45,46,52]. Peptides were
cleaved from the resin with TFA under addition of 5% water as a scavenger for 3 h and were
precipitated as white solids by addition of ice-cold diethyl ether (Figure 3.3.12).
H2N
HN
NH
HN
NH
HN
NH
HN
H3C
O
O
O
NH2
O
O
NH2
O
NH
NHH2N
O
NH2
OOH
NH
NHH2N
NHMn
OC COCO
NH
NHH2N
H2N
HN
NH
HN
NH
HN
NH
HN
H3C
O
O
O
NH2
O
O
NH2
O
NH
NHH2N
O
NH2
OOH
NH
NHH2N
NHRe
OC COCO
NH
NHH2N
38
39
Figure 3.3.12: Mn- and Re-lysine containing peptides prepared from organometal amino acid building
blocks 36 or 37
Results and discussion
70
However, signals of the desired products 38 and 39 could not be detected in the ESI mass
spectra after cleavage from the solid phase. Still, in the ATR-IR spectrum of 38, two
vibrational bands at 2020 and 1932 cm-1 can be observed together with other strong bands
at 1655 and 1167 cm-1 (Figure 3.3.12). The intense signals at 1655 and 1167 cm-1 are the
amide I and II bands which dominate in the octapeptide. The peaks at 2020 and 1932 cm-1
are the characteristic metal carbonyl vibrations of M(CO)3 and correspond well with the C≡O
vibrations at 2023 and 1927 cm-1 for the cymantrene amino acid 36. This indicates a possible
introduction of the organometal amino acid to the peptide but potentially incomplete
cleavage from the resin or other difficulties in the isolation and characterization.
4000 3000 2000 1000
40
60
80
100
Tra
nsm
issio
nin
%
Wavenumber in cm-1
20201932
1655 1167
Figure 3.3.13: ATR IR of the peptide 38 after cleavage from the resin
Results and discussion
71
3.3.3.2 Introduction of Mn- and Re-containing lysines in the peptide H-LKGKFKRG-NH2
Therefore, a new peptide with the sequence H-LKGKFKRG-NH2 was prepared, due to the
problems with cleavage encountered with the peptide with the H-TRKKRKRG-NH2 sequence.
Phenylalanine (F) was introduced to facilitate the detection of the peptide with the UV
detector of the HPLC due to the strong and characteristic absorption of the aromatic ring at
around 260 nm. In addition, lysines were not placed next to each other again to avoid
possible problems caused in the cleavage. The rhenium-containing building block was to be
introduced before the other organometal amino acids in the peptide sequence due to its
better stability compared to the manganese analogue.
H2N
HN
NH
HN
NH
HN
NH
HN
O
NH2
O
O
NH
O
O
O
NH
NHH2N
O
MnOC CO
CO
NH2
O
NH
ReOC CO
CO
Figure 3.3.14: Peptide 40 containing Mn- and Re-lysine building blocks
During the peptide synthesis, the resin was repeatedly examined with IR spectroscopy to
determine if the metal carbonyl moieties were successfully coupled to the solid phase.
During this process, problems due to the insufficient stability of the organometal building
blocks 36 and 37 were observed. DMF solutions containing the resin and coupling reagents,
as well as a 30% piperidine in DMF solution used in the Fmoc cleavage turned dark. Also, the
resin lost its mechanical stability and could not be swollen again after the introduction of the
Mn- and Re-compounds.
Still, cleavage from the resin was performed with 5% water in TFA and the product which
precipitated upon addition of diethyl ether and purified with preparative HPLC. In the IR
spectrum of this product 40 (Figure 3.3.15), two vibrational bands at 2020 and 1932 cm-1 can
Results and discussion
72
be observed, which indicates that species with an organometal moiety are still present after
the preparative HPLC purification, but the desired product could not be detected with ESI
mass spectrometry.
4000 3000 2000 1000
40
60
80
100
Tra
nsm
issio
nin
%
Wavenumber in cm-1
20201932
1662
1203 1126
Figure 3.3.15: ATR IR of peptide 40 after cleavage from the resin
Thus, the stability and purity of the organometal building blocks and the sequence of the
peptide seem to be crucial factors for their successful introduction in a peptide. The
secondary amine linkage of CpMn(CO)3 or CpRe(CO)3 to the ε-amino side chain group of
L-lysine is a possible reason for the poor stability of the peptide conjugates under SPPS
conditions.
Results and discussion
73
3.3.4 Synthesis of Fmoc-protected organometal amino acids via amide bond formation
on the solid phase
3.3.4.1 Synthesis of the half-sandwich precursors for solid phase synthesis
Finally, the amino acids 45-47 shown in Scheme 3.3.13 were prepared on a Wang resin, in
which the introduction of an amide linker between the organometal moiety and the side-
chain ε-amino group of lysine is expected to improve the stability of the building blocks
under solid phase peptide synthesis conditions. The W(CO)3CH3 moiety was additionally
selected to obtain vibrational bands more distinct from the closely spaced ones of the
CpMn(CO)3 and CpRe(CO)3 moieties. These compounds were prepared on the resin to
simplify the purification, since the activating reagents, side products, and solvents can easily
be washed away from the solid phase.
b) cleavage from wang resin with 50% TFA in DCM ReOC CO
CO
O
OH
O
HNO
O
NH
OCOC
WCH3
CO
O
OH
O
HNO
O
NH
MnOC CO
CO
O
OH
O
HNO
O
NH
Wang ResinO
O
O
NH
NH2
O
O
a) CpM(CO)3X, HATU, DIPEA
45
46
47
Scheme 3.3.13: Solid phase synthesis of organometal amino acids 45-47 with the half-sandwich
moiety connected to the ε-amino group of L-lysine
Results and discussion
74
The synthesis of the cymantrene carboxylic acid 41 was performed by addition of n-
butyllithium to cymantrene in tetrahydrofuran followed by crushed solid carbon dioxide at
low temperature (Scheme 3.3.14).[64] The final product was separated and isolated from the
organic phase by addition of 10% hydrochloric acid to the reaction mixture.
MOC CO
CO
n-Buli, CO2
THF MOC CO
CO
OH
O
M = Mn, Re
Scheme 3.3.14: Synthesis of compounds 41 and 42 from cymantrene or cyrethrene with n-
butyllithium and solid carbon dioxide
The 1H NMR spectrum of 41 in methanol-d4 shows two peaks at 5.54 and 4.98 ppm besides
the solvent and residual water signals. Each integrates as 2H and is split into a triplet with 3J
= 2.3 Hz, demonstrating the successful mono-substitution of the cyclopentadiene ring
(Figure 3.3.16).
Figure 3.3.16: 200 MHz 1H-NMR spectrum of 41 in methanol-d4
Results and discussion
75
In the IR spectrum of compound 41, three intense bands at 2020, 1926 and 1673 cm-1 are
observed. The two signals at 2020 and 1926 cm-1 are assigned to the symmetrical and
asymmetrical C≡O vibrations of the Mn(CO)3 moiety. The band at 1673 cm-1 results from the
C=O vibration of the carboxylic acid group (Figure 3.3.17).
4000 3000 2000 1000
20
40
60
80
100
Tra
nsm
issi
on
in%
Wavenumber in cm-1
2020
1926
1673
Figure 3.3.17: ATR IR spectrum of compound 41
The rhenium analog of compound 42 was also synthesized from CpRe(CO)3, n-butyllithium
and crushed solid carbon dioxide under the same condition as applied for the manganese
compound. The compound was isolated as a pale yellow solid and characterized by 1H NMR
and IR spectroscopy. The 1H NMR of compound 42 shows a pattern similar to its manganese
analog, with two triplets found at 6.13 and 5.19 ppm with 3J = 2.3 Hz, which are due to the
mono-substituted cyclopentadiene ring. In the IR spectrum of 42, three peaks at 2027, 1900
and 1680 cm-1 are observed, the bands at 2027 and 1900 cm-1 resulting from the Re(CO)3
moiety and the band at 1680 cm-1 due to the carboxylic acid, all of which are only slightly
shifted compare to 41.
To prepare cyclopentadienyltricarbonylmethyltungsten(I) 44, two routes using either lithium
cyclopentadienide or sodium cyclopentadienide were evaluated (Scheme 3.3.15).[65] During
the reaction with lithium cyclopentadienide, the mixture with tungsten hexacarbonyl turned
from colorless to red. The color change could be an evidence for a conversion taking place,
but after the addition of one equivalent of methyl iodide, a mixture of some white and a bit
of pale yellow solid was obtained, which could be separated by column chromatography on
Results and discussion
76
silica using n-hexane/ethyl acetate (3:2) as the eluent. However, only a small amount of the
desired product could be isolated as a yellow solid, in less than 5% of the theoretical yield.
43
LiW(CO)6 +CH3I
THF
OCOC
WCH3CONaW(CO)6 +
CH3I
THF
Scheme 3.3.15: Synthesis of compound 43 using lithium and sodium cyclopentadienide
When sodium cyclopentadienide was applied instead, rapid gas evolution could be observed
while heating. The reaction mixture was maintained at reflux for about 30 h, after which gas
release ceased. The cooled mixture was then quenched with methyl iodide. Anhydrous
ethanol was added to destroy remaining traces of sodium cyclopentadienide. After
purification with column chromatography on silica using n-hexane/ethyl acetate (v/v 3:2) as
the eluent, a yellow solid was obtained in about 90% yield.
In the 1H NMR spectrum of 43, two intense peaks at 5.42 and 0.40 ppm are observed in an
intensity ratio of 5:3. Both signals appear as singlets, which corresponds well with the
expected equivalent five cyclopentadienyl and three methyl protons.
Figure 3.3.18: 200 MHz 1H-NMR spectrum of 43 in dichloromethane-d2
Results and discussion
77
The IR spectrum of 43 (Figure 3.3.19) shows two intense bands at 1997 and 1867 cm-1, which
are due to the symmetrical and asymmetrical C≡O vibrations of the W(CO)3CH3 moiety.
4000 3000 2000 1000
0
25
50
75
100
1867
Tra
nsm
issi
on
in%
Wavenumber in cm-1
1997
Figure 3.3.19: ATR IR spectrum of compound 43
The tungsten methyl tricarbonyl carboxylic acid 44 was prepared following the same
procedure as applied for the manganese and rhenium carboxylic acid by reaction of 43 with
n-butyllithium and solid carbon dioxide. The isolated product was not further purified, since
the following coupling step was to be performed on a solid phase and impurities from the
previous step could easily be washed away during this procedure.
43
OCOC
WCH3
CO
n-Buli, CO2
THF OCOC
WCH3
CO
O
OH
44
Scheme 3.3.16: Synthesis of 44 from 43 with n-butyllithium and solid carbon dioxide
The crude tungsten compound 44 was characterized with 1H-NMR and IR spectroscopy. In
the 1H NMR spectrum of 44, three main peaks are observed at 5.88, 5.66 and 0.44 ppm
besides the residual solvent and water signals (Figure 3.3.20). The peaks at 5.88 and 5.66
ppm, each with an integral of 2H, are split into triplets with 3J = 2.4 Hz, and belong to the
Results and discussion
78
mono-substituted cyclopentadienyl ring, while the singlet at 0.44 ppm has an integral of 3H
and indicates the presence of the metal-bound methyl group.
Figure 3.3.20: 200 MHz 1H NMR spectrum of 44 in methanol-d4
The ATR IR spectrum of 44 shows four intense peaks at 2015, 1928, 1900 and 1678 cm-1
(Figure 3.3.21). The band at 2015, 1928, and 1900 cm-1 are due to the symmetrical and
asymmetrical C≡O vibrations of the W(CO)3CH3 moiety. The asymmetrical vibrational band at
around 1900 cm-1 is split into two peaks in the solid state. The band at 1678 cm-1 is assigned
to the carboxylic acid C=O group.
4000 3000 2000 1000
20
40
60
80
100
Tra
nsm
issio
nin
%
Wavenumber in cm-1
2015
19001928
1678
Figure 3.3.21: ATR IR spectrum of compound 44
Results and discussion
79
3.3.4.2 Synthesis of Fmoc-protected organometallic amino acids on the solid phase
The Fmoc-protected organometallic amino acids 45-47 of the previously prepared Mn, Re
and W carboxylic acids 41, 42 and 44 were synthesized on the solid phase after removal of
the side-chain Mtt-protective group of the L-lysine with 1% trifluoroacetic acid in
dichloromethane. In the first step, Fmoc-L-Lys(Mtt)-OH was loaded on Wang resin with three
equivalents of HOBt and HBTU as well as 10 equivalents of DIPEA used. The ester formation
on the Wang resin is less effective compare to the amide bond formation during the peptide
synthesis. Since the loading of the first amino acid on the Wang resin is crucial, a reaction
time of 2–3 d is necessary. After washing with N,N-dimethylformamide, dichloromethane
and diethyl ether, the Mtt protective group was removed by repeated cleavage using 1%
trifluoroacetic acid in dichloromethane with 4% triisopropylsilane or phenol added as the
scavengers. The colorless cleavage solution immediately turned yellow when added to the
Wang resin, and the syringe with the reaction mixture was shaken for 2 min, the solvent
removed, the resin washed with dichloromethane until the washing solution was colorless.
This process was repeated until no color change of the resin could be observed anymore
after addition of a 1% TFA solution (Scheme 3.3.17).
removal of mtt with1% TFA in DCM
Wang ResinO
O
O
NH
NH2
OO
Wang ResinO
OH
O
NH
NH
OO
CH3
HO
+HOBt, HBTU, DIPEA
Wang ResinO
O
O
NH
NH
OO
CH3
Scheme 3.3.17: Loading of Fmoc-L-Lys(Mtt)-OH on the Wang resin
Results and discussion
80
The coupling of the manganese, rhenium and tungsten tricarbonyl carboxylic acids 41, 42,
and 44 were then performed using 1.5 equiv. of HATU and organometal compound, and 10
equiv. of DIPEA in DMF with a reaction time of 3-5 h. The cleavage from the Wang resin was
achieved using 50% TFA in dichloromethane. The resin was washed with TFA and
dichloromethane, all solutions were collected and the solvents were removed in vacuum
using an extra cold trap for safe removal of TFA, and the products obtained as brown solids
(see Scheme 3.3.13). The purity of the compounds was checked with analytical HPLC. A
gradient of 20-95% acetonitrile/water over 30 min was applied. For example, only one main
peak at a retention time of 26.6 min was found for compound 45 (Figure 3.3.22).
0 10 20 30
0
1000
2000
3000
Ab
sorb
ance
Retention Time (min)
MnOC CO
CO
O
OH
O
HNO
O
NH
tR=26.6 min
Figure 3.3.22: Analytical HPLC trace of 45 with a gradient of 20-95% acetonitrile/water over 30 min
In the ATR IR spectrum of compound 45, two intense bands at 2023 and 1928 cm-1 are
ŽďƐĞƌǀ ĞĚ dŚĞLJĂƌĞĚƵĞƚŽƚŚĞƐLJŵŵĞƚƌŝĐĂůĂŶĚĂƐLJŵŵĞƚƌŝĐĂů൙Kǀ ŝďƌĂƟŽŶƐŽĨƚŚĞD Ŷ;KͿ3
moiety.
4000 3000 2000 1000
20
40
60
80
100
Tra
nsm
issio
nin
%
Wavenumber in cm-1
20231928
Figure 3.3.23: ATR IR spectrum of compound 45
Results and discussion
81
Compound 45 was also characterized with 1H-NMR in methanol-d4. In the aromatic region,
three peaks at 7.80, 7.66 and 7.35 ppm are observed with an intensity ratio of 2:2:4 as
multiplets, belonging to the fluorenyl moiety of the Fmoc group. A determination of the
coupling constants was not possible due to the low concentration achievable. Two additional
peaks are found at 5.56 and 4.93 ppm with an integral of 2H, with the latter overlapping
with the residual water signal. These are assigned to the protons of the monosubstituted
cyclopentadienyl ring. The broad peak at 1.53 ppm with an integral of 6H is due to a part of
the aliphatic side chain of the lysine. There are also three singlets at 3.02, 2.95 and 2.85 ppm,
with a total integral of 2H, which can be assigned to the ε-protons of the lysine. The other
protons are found between 4.33 and 4.12 ppm with a total intensity of 4H. The poor quality
of the spectrum results from the low solubility of the compound.
Figure 3.3.24: 200 MHz 1H-NMR spectrum of 45 in methanol-d4
Compound 45 was also characterized by ESI mass spectroscopy in the negative mode. Two
intense peaks are observed at m/z = 597.11 and 1195.21, which are due to [M-H]- and [2M-
2H+Na]- of the desired target compound.
Results and discussion
82
(a) (b)
600 900 1200 1500
m/z = 1195.21 [2M-2H+Na]-
m/z
m/z = 597.11 [M-H]-
MnOC CO
CO
O
OH
O
HNO
O
NH
580 590 600 610 620
m/z
m/z = 597.11 [M-H]-
Figure 3.3.25: (a) negative mode ESI mass spectra of 45, and (b) an enlarged view showing the
isotope pattern of the major peak
The rhenium compound 46 was also characterized by ESI mass spectrometry. One intense
peak at m/z = 729.12 is observed, which is due to [M-H]-. In ATR IR spectrum of 46, two
intense peaks at 2025 and 1913 cm-1 were found, which are due to the symmetrical and
ĂƐLJŵŵĞƚƌŝĐĂůǀ ŝďƌĂƟŽŶƐŽĨƚŚĞ൙KŐƌŽƵƉƐ dŚŝƐĐŽŵƉŽƵŶĚǁ ĂƐƚŚƵƐŶŽƚĨƵƌƚŚĞƌƵƟůŝnjĞĚĨŽƌ
ŝŶĐŽƌƉŽƌĂƟŽŶŝŶƚŽƚŚĞƉĞƉƟĚĞƐŝŶĐĞŝƚƐƐLJŵŵĞƚƌŝĐĂů൙Kǀ ŝďƌĂƟŽŶďĂŶĚŝƐĂƚĂƚŽŽƐŝŵŝůĂƌ
position to that of the manganese compound 45 to be applied in the labeling. Tungsten
compound 47 was prepared the same way as applied for the manganese analog 45. Due to
its low stability, a preparative HPLC purification was carried out immediately after the
cleavage of the amino acid from the Wang resin. Two main peaks were observed before the
preparative purification and the latter one at tR = 27.11 min was collected. The final product
was isolated as a red solid after removal of the solvent.
(a) (b)
0 10 20 30
0
500
1000
1500
2000
2500
tR=24.6 min
tR=27.11 min
Abso
rba
nce
inm
Au
Retention Time in min
OCOC
WCH3
CO
O
OH
O
HNO
O
NH
0 10 20 30
0
1000
2000
3000tR=28.17 min
OCOC
WCH3
CO
O
OH
O
HNO
O
NH
Ab
sorb
an
cein
mA
u
Retention Time in min
Figure 3.3.26: Analytical HPLC traces of 47 with a gradient of 20-95% ACN/water over 30 min (a)
before and (b) after preparative HPLC purification
Results and discussion
83
In the 1H-NMR spectrum of 47 in methanol-d4, three signals at 7.80, 7.64 and 7.35 ppm can
be observed with an intensity ratio of 2:2:4, which are assigned to the protons of the Fmoc
protective group. Two peaks at 6.38 and 6.06 ppm, both with an integral of 2H, are
attributed to the protons of the mono-substituted Cp ring. The peak at 4.34 ppm is due to
the methylene group next to the fluorenyl ring, while the peak at 4.17 ppm and the broad
signal at about 1.52 ppm result from the lysine side chain. However, the signal of the methyl
ligand directly bound to the tungsten center expected to show up at around 0.4 ppm could
not be observed.
Figure 3.3.27: 200 MHz 1H-NMR spectrum of 47 in methanol-d4
47
OCOC
WCH3
CO
O
OH
O
HNO
O
NH
OCOC
W
CO
O
OH
O
HNO
O
NH
Scheme 3.3.18: 1H-NMR spectroscopy indicates the loss of the metal-coordinated
methyl ligand from 47
Results and discussion
84
The loss of the methyl ligand from the tungsten coordination sphere was also confirmed by
ESI mass spectrometry. When the measurement is performed in the negative mode with
methanol as the solvent, three intense peaks at m/z = 811.11, 839.11 and 1679.21 are found,
which can be assigned to the species [M-CH3-CO+CF3COO-H]-, [M-CH3+CF3COO-H]-, and [2M-
2CH3+2CF3COO-H]-. The cleavage of 47 from Wang resin with 50% trifluoroacetic acid in
dichloromethane thus might be the cause for the loss of the methyl group and the formation
of a trifluoroacetic acid adduct.
(a)
600 900 1200 1500 1800
m/z = 1679.21
[2M-2CH3+2CF
3COO-H]-
m/z = 839.11
[M-CH3+CF
3COO-H]-
m/z = 811.11
[M-CH3-CO+CF
3COO-H]-
m/z
OCOC
WCH3
CO
O
OH
O
HNO
O
NH
OCOC
WOOCCF3
CO
O
OH
O
HN
OO
NH
(b)
800 820 840 860
m/z = 839.11
[M-CH3+CF
3COO-H]-
m/z = 811.11
[M-CH3-CO+CF
3COO-H]-
m/z
Figure 3.3.28: Negative mode ESI-MS of (a) compound 47 and (b) enlarged scan of the range of m/z =
800-860, showing the typical tungsten isotope distribution of these peaks
Results and discussion
85
3.3.4.3 Solid phase peptide synthesis (SPPS) with CpMn(CO)3- and CpW(CO)3CH3-
containing L-lysines as the building blocks
Since the analytical HPLC traces of the manganese compound 45 showed good purity, it was
applied in further solid phase peptide synthesis without purification, while the less stable
tungsten-containing compound 47 was used after preparative HPLC purification. The
peptides 48 and 49 were prepared manually following the procedure described before. After
the cleavage from the Rink amide resin and precipitation with cold diethyl ether, the
peptides were isolated as pale yellow solids. The IR spectrum of peptide 48 shows only the IR
vibrational signature of the cymantrene moiety above 1900 cm-1, with two bands at 2027
and 1936 cm-1 ǁ ŚŝĐŚĂƌĞĚƵĞƚŽƚŚĞƐLJŵŵĞƚƌŝĐĂůĂŶĚĂƐLJŵŵĞƚƌŝĐĂůǀ ŝďƌĂƟŽŶƐŽĨƚŚĞ൙K
group in the Mn(CO)3 moiety (Figure 3.3.30). However, in ESI mass spectrometry, species
corresponding to the desired product could not be detected. Peptide 49 could also not be
identified in the ESI-MS and the typical metal tricarbonyl vibrational pattern was not found
in the IR spectrum.
H2N
HN
NH
HN
NH
HN
NH
HN
O
NH
O
O
NH
O
O
NH2
O
NH
NHH2N
O
O
OCOC
WCH3
CO
O
MnOC CO
CO
NH2
O
H2N
HN
NH
HN
NH
HN
NH
HN
O
NH
O
O
NH2
O
O
NH2
O
NH
NHH2N
O
O
OCOC
WCH3
CO
NH2
O
Figure 3.3.29: Mn- and W-lysine containing peptides 48 (left) and 49 (right)
Results and discussion
86
4000 3000 2000 1000
70
80
90
100
Tra
nsm
issio
nin
%
Wavenumber in cm-1
20271936
1658
1535
Figure 3.3.30: ATR IR spectrum of peptide 48
The unsuccessful preparation of the peptides could be due to the loss of the methyl ligand of
the tungsten complex 47 and the trifluoroacetic acid adduct formed, which might interfere
with the amine bond formation upon activation with HATU. In the synthesis of peptide 48,
the application of the manganese compound 45 without preparative HPLC purification might
also be a factor leading to the unsuccessful coupling of the organometal amino acid building
blocks.
Results and discussion
87
3.3.5 Introduction of organometal carbonyl complexes via an orthogonal protective
group strategy
Thus, the organometal carbonyl complexes were attached to the peptide on a Rink amide
resin using the orthogonal methyltrityl (Mtt) protective group on the ε-amino group of
L-lysine. The general procedure is shown below for a peptide containing both CpMn(CO)3-
and CpW(CO)3CH3 groups. The peptide with the sequence H-LKGKFKRG-NH2 was synthesized
sequentially until the second glycine had been attached, with the N-terminus still protected
with Fmoc (Scheme 3.3.19). Then, the mtt-protective group was removed with 1% TFA in
dichloromethane, and the cymantrene carboxylic acid 41 was coupled to the free side chain
ε-amino group of the L-lysine. The completeness of this coupling step could easily be
monitored with the Kaiser test. Then, the N-terminal Fmoc group was removed with 20%
piperidine in N,N-dimethylformamide for the further extension of the polypeptide chain.
HN
NH
HN
NH
HN
NH(Mtt)
O
O
NH(Boc)
O
NH
NH(Pbf)HN
O
NH
O
Rink amideresin
(Fmoc)HN
O
1% TFA in DCM
HN
NH
HN
NH
HN
NH2
O
O
NH(Boc)
O
NH
NH(Pbf)HN
O
NH
O
Rink amideresin
(Fmoc)HN
O
MnOC CO
CO
OH
O
+
HATU, DIPEA in DMF
HN
NH
HN
NH
HN
NH
O
O
NH(Boc)
O
NH
NH(Pbf)HN
O
NH
O
Rink amideresin
(Fmoc)HN
O
O
MnOC CO
COFmoc removal
Scheme 3.3.19: Peptide synthesis with an orthogonal side chain protective group strategy for the
attachment of two different metal-carbonyl markers
Results and discussion
88
Then, the N-terminal amino acids, Fmoc-L-Lys(Mtt)-OH and Fmoc-L-leucine were
sequentially coupled. The Mtt group of the lysine was removed under the same conditions
as before and the second organometal moiety, tungsten carboxylic acid 44, was attached to
the free side chain ε-amino group (Scheme 3.3.20).
HN
NH
HN
NH
HN
NH
O
O
NH(Boc)
O
NH
NH(Pbf)HN
O
NH
O
Rink amideresin
H2N
O
O
MnOC CO
CO
coupling with Fmoc-K(Mtt)-OHand Fmoc-L-OH
HN
NH
HN
NH
HN
NH
O
O
NH(Boc)
O
NH
NH(Pbf)HN
O
NH
O
Rink amideresin
NH
O
O
MnOC CO
CO
O
NH(Mtt)
(Fmoc)HN
HN
O
1% TFA in DCM
HN
NH
HN
NH
HN
NH
O
O
NH(Boc)
O
NH
NH(Pbf)HN
O
NH
O
Rink amideresin
NH
O
O
MnOC CO
CO
O
NH2
(Fmoc)HN
HN
O
OCOC
WCH3CO
O
OHHATU, DIPEA
in DMF
HN
NH
HN
NH
HN
NH
O
O
NH(Boc)
O
NH
NH(Pbf)HN
O
NH
O
Rink amideresin
NH
O
O
MnOC CO
CO
O
NH
(Fmoc)HN
HN
O
O
OCOC
WCH3
CO
Scheme 3.3.20: Peptide synthesis with an orthogonal side chain protective group strategy for the
attachment of two different metal-carbonyl markers
Results and discussion
89
In the last two steps, the Fmoc group of the N-terminal leucine was first removed with 20%
piperidine solution in N,N-dimethylformamide, followed by the final cleavage of all side
chain protective groups and the cleavage of the peptide from the resin with 95% TFA using
5% water as the scavenger (Scheme 3.3.21). The peptide was then precipitated with cold
diethyl ether and isolated by centrifugation.
HN
NH
HN
NH
HN
NH
O
O
NH(Boc)
O
NH
NH(Pbf)HN
O
NH
O
Rink amideresin
NH
O
O
MnOC CO
CO
O
NH
(Fmoc)HN
HN
O
O
OCOC
WCH3
CO
HN
NH
HN
NH
HN
NH
O
O
NH(Boc)
O
NH
NH(Pbf)HN
O
NH
O
Rink amideresin
NH
O
O
MnOC CO
CO
O
NH
H2N
HN
O
O
OCOC
WCH3
CO
H2N
HN
NH
HN
NH
HN
NH
HN
O
NH
O
O
NH
O
O
NH2
O
NH
NHH2N
O
O
OCOC
WCH3
CO
O
MnOC CO
CO
NH2
O
20% piperidine in DMF
5% H2O, 95% TFA
Scheme 3.3.21: Peptide synthesis with an orthogonal side chain protective group strategy for the
attachment of two different metal-carbonyl markers
Results and discussion
90
In total, four peptides 50-53 with either no, one CpMn(CO)3- or CpW(CO)3CH3 group,
respectively, or both a manganese and a tungsten organometal moiety were synthesized
using Fmoc-L-Lys(Mtt)-OH for the lysine positions to be modified with the organometal
carboxylic acids on the side chain. The three peptides with organometal moieties were
prepared to obtain distinct IR vibrational patterns, and the metal-free peptide to serve as a
negative control. The peptides were purified by preparative HPLC with a gradient of 30 to
90% of an acetonitrile/water mixture and were characterized with ESI mass spectrometry
and IR spectroscopy.
H2N
HN
NH
HN
NH
HN
NH
HN
O
NH
O
O
NH
O
O
NH2
O
NH
NHH2N
O
O
OCOC
WCH3
CO
O
MnOC CO
CO
NH2
O
H2N
HN
NH
HN
NH
HN
NH
HN
O
NH
O
O
NH2
O
O
NH2
O
NH
NHH2N
O
O
OCOC
WCH3
CO
NH2
O
H2N
HN
NH
HN
NH
HN
NH
NH2
O
NH2
O
O
NH2
O
O
NH2
O
NH
NHH2N
O
H2N
HN
NH
HN
NH
HN
NH
HN
O
NH2
O
O
NH
O
O
NH2
O
NH
NHH2N
O
O
MnOC CO
CO
NH2
O
50 51
5352
Figure 3.3.31: Peptides 50-53 with or without organometall moieties
Results and discussion
91
Table 3.3.1: Mass spectrometric data of the organometal peptides
Peptide MWcalc. in g/mol ESI in Da
50 931.61 932.6 466.82
[M+H]+ [M+2H]2+
51 1161.62 1162.69 581.84
[M+H]+ [M+2H]2+
52 1305.59 631.78 1404.55
[M-CO-CH3+H]2+ [M-CH3+CF3COO+H]+
53 1535.53 1606.5 1634.50
[M-CO-CH3+CF3COO+H]+ [M-CH3+CF3COO+H]+
1747.6 [M-CH3+2CF3COO+H]+
In the positive mode of ESI-MS, peptide 50 with the free lysine side chain amino groups gave
two signals at m/z = 932.6 and 466.8 for [M+H]+ and [M+2H]2+, respectively (data not
shown). Peptide 51, which is only functionalized with the cyclopentadienyl manganese
tricarbonyl moiety, gave a strong peak at m/z = 1162.6 for [M+H]+ (Figure 3.3.32a) and at
m/z = 581.8 for [M+2H]2+. Tungsten carbonyl-modified peptide 52 gave signals at m/z =
1404.5 for [M-CH3+CF3COO+H]+ and m/z = 631.8 for [M-CO-CH3+H]2+ (Figure 3.3.32b). Bis-
functionalized peptide 53 shows three signals at m/z = 1747.6, 1634.5 and 1606.5 for [M-
CH3+2CF3COO+H]+, [M-CH3+CF3COO+H]+, and [M-CO-CH3+CF3COO+H]+, respectively (Figure
3.3.33). Both peptides with the tungsten moiety show the characteristic isotope pattern of
this metal and were detected at as the TFA adducts after loss of the metal-coordinated
methyl ligand.
Results and discussion
92
(a)
1050 1100 1150 1200 1250
m/z
m/z = 1162.6 [M+H]+
H2N
HN
NH
HN
NH
HN
NH
HN
O
NH2
O
O
NH
O
O
NH2
O
NH
NHH2N
O
O
MnOC CO
CO
NH2
O
51
(b)
1200 1300 1400 1500
m/z = 1404.5
[M-CH3+CF
3COO+H]+
m/z
H2N
HN
NH
HN
NH
HN
NH
HN
O
NH
O
O
NH2
O
O
NH2
O
NH
NHH2N
O
O
OCOC
WCH3
CO
NH2
O
52
Figure 3.3.32: Positive mode ESI-MS of the peptides (a) 51 and (b) 52
Results and discussion
93
1550 1600 1650 1700
1620 1630 1640 1650
m/z
m/z = 1634.5
[M-CH3+CF
3COO+H]+
m/z
m/z = 1634.5
[M-CH3+CF
3COO+H]+
H2N
HN
NH
HN
NH
HN
NH
HN
O
NH
O
O
NH
O
O
NH2
O
NH
NHH2N
O
O
OCOC
WCH3
CO
O
MnOC CO
CO
O
53
Figure 3.3.33: Positive mode ESI-MS of peptide 53 with the inset showing the typical tungsten
isotope pattern of the main peak
Results and discussion
94
All peptides were also characterized with ATR IR spectroscopy. The spectrum of peptide 50
without any organometal moieties is shown in Figure 3.3.34. Several peaks below 2000 cm-1
can be observed at 1666, 1631 and 1529 cm-1, which are mostly due to the amide linkages
and the aromatic side chain groups on the peptide.
4000 3000 2000 1000
40
60
80
100
Tra
nsm
issio
nin
%
Wavenumber in cm-1
16661631
1529
Figure 3.3.34: ATR-IR spectrum of peptides 50
Results and discussion
95
The spectrum of peptide 51 with a cymantrene unit coupled to one of the L-lysine ε-amino
groups is shown in Figure 3.3.35. In addition to the signals of the peptide backbone and the
side chains, three peaks are observed at 2025, 1948, and 1938 cm-1 which are due to the
symmetrical and asymmetrical C≡O vibrations of the Mn(CO)3 moiety.
4000 3000 2000 1000
40
60
80
100
Tra
nsm
issio
nin
%
Wavenumber in cm-1
2025
19381948
Figure 3.3.35: ATR-IR spectrum of peptide 51
Results and discussion
96
In the ATR IR spectrum of the CpW(CO)3CH3-modified peptide 52, three intense signals are
found at 2056, 1971 and 1971 cm-1, which can be assigned to the C≡O vibrations of the
W(CO)3 moiety, in addition to the usual peptide signals.
4000 3000 2000 1000
60
70
80
90
100
Tra
nsm
issio
nin
%
Wavenumber in cm -1
2056
19711963
Figure 3.3.36: ATR-IR spectrum of peptide 52
Results and discussion
97
The IR spectrum of bis-functionalized peptide 53 incorporating both the manganese and the
tungsten tricarbonyl groups is shown in Figure 3.3.37. Four intense peaks plus one shoulder
can be observed at 2056, 2025, 1971, 1948, and 1938 cm-1. The two peaks at 2056 and 2025
cm-1 are due to the symmetrical vibrational bands of the Mn(CO)3 and W(CO)3 moieties,
respectively. The signals at 1948 and 1938 cm-1 result from the asymmetrical vibration of the
tricarbonyl moiety of cymantrene, which overlaps with the asymmetrical vibration of the
W(CO)3 moiety for which only a small shoulder is observed at 1971 cm-1.
4000 3000 2000 1000
40
60
80
100
Tra
nsm
issi
on
in%
Wavenumber in cm-1
2056
2025
19381948
1971
Figure 3.3.37: ATR-IR spectrum of bis-functionalized peptide 53
TŚĞƉŽƐŝƟŽŶƐŽĨƚŚĞƐLJŵŵĞƚƌŝĐĂůĂŶĚĂƐLJŵŵĞƚƌŝĐĂů൙Kǀ ŝďƌĂƟŽŶĂůďĂŶĚƐŽĨƉĞƉƟĚĞƐ50-53
are summarized in Table 3.3.2. Figure 3.3.35 shows an overlay of the IR spectra of the four
peptides in the 1400 to 2400 cm-1 region. The asymmetrical bands of all organometal
peptides are difficult to discuss since these bands are relatively broad. However, the
ƐLJŵŵĞƚƌŝĐĂůďĂŶĚƐƐŚŽǁ ĚŝƐƟŶĐƚ൙Kǀ ŝďƌĂƟŽŶĂůƐŝŐŶĂƚƵƌĞƐĨŽƌƚŚĞMn(CO)3 and W(CO)3
moieties which can clearly be distinguished although the difference in peak maxima is only
31 cm-1 (2025 vs. 2056 cm-1).
Results and discussion
98
2400 2200 2000 1800 1600 1400
40
60
80
100
Tra
nsm
issi
on
in%
Wavenumber in cm-1
50515253
Figure 3.3.38: Overlay of the ATR IR spectra of 50, 51, 52, 53
Table 3.3.2: IR vibrations of peptides 50, 51, 52 and 53
Peptide IR bands assigned to Mn(CO)3 (cm-1) IR bands assigned to W(CO)3 (cm-1)
symmetrical asymmetrical symmetrical asymmetrical
50 -- -- -- --
51 2025 1948, 1938 -- --
52 2056 1963, 1971
53 2025 1948, 1938 2056 1971
Conclusion
99
Conclusion
In the first part of this work, a series of twelve carboxylic acid-functionalized
cyclopentadienyl manganese and rhenium tricarbonyl complexes CpM(CO)3 with different
linkers between the half-sandwich moiety and the carboxylate group were prepared,
characterized by IR, 1H-NMR spectroscopy, ESI mass spectrometry, and elemental analysis as
well as X-ray structure determination of some selected examples, and then conjugated to
the cell-penetrating peptide sC18 in a solid-phase protocol. The intracellular distribution of
the CpM(CO)3-sC18 conjugates was studied with fluorescence microscopy of their CF-labeled
derivatives. IC50 values were determined with the resazurin assay and the LDH release
leakage assay was also applied to study the cytotoxicity. The organometal compounds
themselves and the unmodified sC18 peptide showed no cytotoxicity on MCF-7 human
breast cancer cells at up to 200 µM. However, the organometal-peptide conjugates were
efficiently internalized by the cells and showed some cytotoxic activity. An exchange of the
metal center from manganese to rhenium had no significant effect on the biological
properties of the conjugates. However, the reduction of the keto group in the linker to a
methylene moiety led to a more pronounced nuclear accumulation associated with higher
cytotoxicity, with IC50 values reduced from 60 µM for the C=O to 40 µM for the CH2 linker.
Thus, even a small variation of the linker between the cyclopentadienyl and carboxylic acid
moieties significantly influences the intracellular localization and cytotoxic activity.
In the second part, a series of organometal half-sandwich complexes were prepared,
functionalized with an aldehyde group, and coupled with the terminal amino groups of first
generation PAMAM and first, second, and third generation DAB dendrimers. The
organometal-dendrimer conjugates as well as a purely organic adamantane-dendrimer
conjugate synthesized for comparison were purified by preparative HPLC and characterized
with ATR-IR and 1H-NMR spectroscopy as well as ESI mass spectrometry. The cytotoxicity of
the organometal-dendrimer conjugates as well as the adamantane conjugate was
determined on MCF-7 breast cancer cells. Four concentrations in the 1 to 25 µM range were
chosen to study the correlation of the cytotoxicity with dendrimer structure and generation
as well as the effect of variation of the metal center. For all conjugates, higher
concentrations led to more pronounced cytotoxicity. However, the replacement of
manganese by rhenium in the G1, G2 and G3 DAB dendrimer conjugates did not have any
Conclusion
100
significant effect on the biological activity. Surprisingly, the activity decreased with
increasing generation of the dendrimer, in both the manganese and the rhenium cases. The
adamantane G1 DAB conjugate showed an activity similar to those of the corresponding
organometal G1 manganese or rhenium DAB conjugates. Thus, the cytotoxicity of the
dendrimer conjugates does not seem to directly correlate with the type or number of
terminal functional groups, and the small differences between the organometal conjugates
and their adamantane analogue point to a mechanism of cytotoxicity which is different from
that observed for the peptide conjugates, where a small modification of the conjugated
organometal moiety led to a significant modulation of the biological activity of these systems.
In the third part of the present thesis, organometal carbonyl complexes with different
ǀ ŝďƌĂƟŽŶĂů൙KďĂŶĚƉŽƐŝƟŽŶƐǁ ĞƌĞĐŽŶũƵŐĂƚĞĚƚŽĂŵŽĚĞůƉĞƉƟĚĞƚŽĞdžƉůŽƌĞthe use of the
distinct vibrational signature of the M(CO)n moiety to encode information in biomolecules.
Initially, Fmoc-protected organometal amino acids were prepared as building blocks for solid
phase peptide synthesis. Thus, the CpM(CO)3 groups were attached to the side-chain ε-
amino group of L-lysine via a Schiff base reaction. Different strategies were evaluated and
finally, the coupling of cymantrene or cyrhetrene carboxaldehydes to L-lysine N-terminally
protected with Fmoc using sodium acetate as a base was successful. However, these building
blocks showed insufficient stability during the solid-phase peptide synthesis. Therefore,
organometal amino acids were attached via an amide bond instead of the amine linkage to
improve their stability. These conjugates were prepared by a solid-phase strategy on a Wang
resin. However, for the CpW(CO)3CH3-modified L-lysine, trifluoroacetic acid adducts were
detected by ESI mass spectrometry, which replaced the metal-bound methyl group. This
again caused significant problems during peptide synthesis and therefore, yet another
strategy had to be explored. Thus, the introduction of the organometal groups to the
peptide was finally achieved via an orthogonal protective group strategy on a rink amide
resin, in which a side-chain Mtt protective group was selectively removed from an N-
terminal L-lysine and then the organometal complex was coupled to the side-chain on the
solid phase before N-terminal Fmoc deprotection and further chain elongation. Four
peptides with either no, one CpMn(CO)3- or CpW(CO)3CH3 group, respectively, or both
organometallic moieties were synthesized using Fmoc-L-Lys(Mtt)-OH at the L-lysine positions
to be modified with the organometal complexes. These peptides conjugates showed the
expected distinct IR vibrational patterns. In the metal-free peptide, only bands due to the
Conclusion
101
amide linkages and the aromatic side chain groups can be observed at higher wavenumbers.
In contrast, all three organometal modified peptides showed the characteristic symmetrical
and asymmetrical vibrations of the CpM(CO)3 moieties in the 2056 to 1938 cm-1 range. The
broad asymmetrical bands show significant overlap, and thus cannot be evaluated
ƵŶĞƋƵŝǀ ŽĐĂůůLJ, Žǁ Ğǀ ĞƌƚŚĞƐLJŵŵĞƚƌŝĐĂů൙Kǀ ŝďƌĂƟŽŶŝƐĨŽƵŶĚŝŶƚŚĞƉĞƉƟĚĞǁ ŝƚŚ
CpMn(CO)3-group at 2025 cm-1 and in the CpW(CO)3CH3-modified peptide at 2056 cm-1. Thus
in the bis-functionalized peptide incorporating both the manganese and the tungsten
tricarbonyl groups both bands at 2025 and 2056 cm-1 could clearly be observed with near
base-line resolution, thus demonstrating the general applicability of this encoding strategy.
In summary, structure-activity relationships in peptide and dendrimer carriers modified with
different organometal complexes were studied on a human breast cancer cell line. Variation
of the organometal cargo and carrier can significantly influence their biological properties
and might open the way to new approaches in chemotherapy. Furthermore, the
incorporation of complexes with different C≡O vibrational signatures in a model peptide was
explored to examine information encoding in biomolecules in a barcoding strategy for
potential imaging applications. In particular for the latter, additional stable metal-carbonyl
markers need to be prepared in future work to expand the pool of vibrational labels
available.
Conclusion
102
Materials and methods
103
5 Materials and methods
5.1 General procedures
All reactions were carried out in oven-dried Schlenk glassware under an atmosphere of pure
dinitrogen or argon when necessary. Solvents were dried over molecular sieves and
degassed prior to use. 1,4-Diaminobutane poly(propylenimine) octaamine dendrimers (DAB-
(NH2)4-G1) (DAB-(NH2)8-G2) and (DAB-(NH2)16-G3) were purchased from SyMOChem. Fmoc
amino acid derivatives were purchased from Iris Biotech, CBL, and Novabiochem. 1-
Hydroxybenzotriazole (HOBt) and 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxy-
methyl-linked polystyrene (Rink amide) were obtained from Novabiochem. Piperidine was
purchased from Fluka. Terephthalic acid monomethyl ester chloride was prepared from
terephthalic acid according to literature procedure.[66] Dichloromethane (DCM) and N,N-
dimethylformamide (DMF) were Biosolve products. N,N-diisopropylcarbodiimide (DIC) was
obtained from Iris Biotech, anisole and trifluoroacetic acid (TFA) from Sigma-Aldrich. All
other chemicals were obtained from commercial sources and used without further
purification. NMR spectra were recorded on Bruker Avance 200, DPX 200, DPX 250, DRX 300,
DRX 400, and Avance 500 spectrometers (1H at 200.13, 250.13, 400.13 and 500.13 MHz,
respectively). Chemical shifts δ in ppm indicate a downfield shift relative to
tetramethylsilane (TMS) and were referenced relative to the residual 1H signal of the
solvent.[67] Individual peaks are marked as singlet (s), doublet (d), triplet (t), or multiplet (m).
Mass spectra of small molecules were measured on a Bruker Esquire 6000 or on a Bruker
microTOF ESI instrument. Only characteristic fragments are given for the most abundant
isotope peak. IR spectra were recorded on pure solid samples with a Bruker Tensor 27 FT-IR
spectrometer equipped with a Pike MIRacle Micro ATR accessory or a Nicolet 380 FT-IR
spectrometer with a smart iTR setup. The elemental composition of the compounds was
determined with a VarioEL analyzer from Elementar Analysensysteme GmbH.
Materials and methodes
104
5.2 Solid phase synthesis of the sC18 peptide and its bioconjugates
The sC18 peptide with the sequence H-GLRKRLRKFRNKIKEK-NH2 was synthesized by
automated solid-phase peptide synthesis (SPPS) on a Rink amide resin (30 mg, resin loading
0.45 mmol g-1) using the Fmoc/tBu-strategy on a multiple synthesizer (Syro II, MultiSyntech,
Witten, Germany). Manual coupling of cymantrene acids 1 and 2 as well as rhenium
compounds 3 and 4 to the N-terminal amino group was achieved by activation with 1.5 eq.
2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU)/
N,N-diisopropylethylamine (DIEA), following our reported procedure.[68] Peptides were
cleaved from the resin with trifluoroacetic acid (TFA) under addition of 5% water as a
scavenger for 3 h, precipitated by addition of ice-cold diethyl ether and isolated by
centrifugation. The compounds were analyzed by reversed-phase (RP) HPLC, matrix-assisted
laser desorption ionization time of flight (MALDI-ToF) mass spectrometry (MS), and
electrospray ionization (ESI) MS. ESI ion trap measurements were performed on a Bruker
HCT mass spectrometer. MALDI-ToF mass spectrometry was carried out on a Bruker
Daltonics Ultraflex III instrument in reflection mode.
5.3 CF-labeling of sC18 on the resin
(5,6)-Carboxyfluorescein (CF) was manually coupled to the ε-amino group of the lysine
residue closest to the N-terminus (H-GLRKRLRKFRNKIKEK-NH2). For this purpose, Fmoc-
Lys(Dde)-OH was introduced at this position. After complete assembly of the peptide, the
Dde group was cleaved off with a freshly prepared solution of 2% hydrazine in N,N-
dimethylformamide (DMF), which leaves the other protective groups unmodified. Then, 3
equiv. of (5,6)-Carboxyfluorescein (CF) and 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-
tetramethyluronium hexafluorophosphate (HATU) in N,N-dimethylformamide (DMF)
solution were added to the peptide on the resin. The reaction was allowed to proceed
overnight in the dark, due to the light-sensitivity of the (5,6)-Carboxyfluorescein (CF) group.
The reactive hydroxy groups of CF had to be protected as the triphenylmethyl ether before
further reaction with the organometallic compounds. Thus, 4 equiv. of tritylchloride/ N,N-
diisopropylethylamine (DIEA) were added to the CF-labeled peptide on the resin and reacted
for 16 h in the dark.[69] The coupling of the metal complexes 1, 2, 3, and 4 was then carried
out as described above. Conjugates were cleaved from the resin using trifluoroacetic acid
including 5% water as scavenger, leading to concomitant removal of all other protective
Materials and methods
105
groups. The peptide conjugates were precipitated by addition of cold diethyl ether, the solid
was isolated by centrifugation, and washed five times with cold ether and dried for 10 min.
The CF-labeled manganese and rhenium peptide conjugates were purified with preparative
HPLC and identified with ESI-MS or MALDI-ToF MS, respectively.
5.4 Solid phase synthesis of Fmoc-protected organometal amino acids on a Wang
resin
Fmoc-L-Lys(Mtt)-OH was loaded on Wang resin with three equivalents of HOBt and HBTU as
well as 10 equivalents of DIPEA used. The reaction mixture was shaken at room temperature
for 2d. After washing with N,N-dimethylformamide, dichloromethane and diethyl ether, the
Mtt protective group was removed by repeated cleavage using 1% trifluoroacetic acid in
dichloromethane with 4% triisopropylsilane or phenol added as the scavengers. The
colorless cleavage solution immediately turned yellow when added to the Wang resin, and
the syringe with the reaction mixture was shaken for 2 min, the solvent removed, the resin
washed with dichloromethane until the washing solution was colorless. This process was
repeated until no color change of the resin could be observed anymore after addition of a
1% TFA solution The coupling of the manganese, rhenium and tungsten tricarbonyl
carboxylic acids 41, 42, and 44 were then performed using 1.5 equiv. of HATU and
organometal compound, and 10 equiv. of DIPEA in DMF with a reaction time of 3-5 h. The
cleavage from the Wang resin was achieved using 50% TFA in dichloromethane. The resin
was washed with TFA and dichloromethane, all solutions were collected and the solvents
were removed in vacuum using an extra cold trap for safe removal of TFA, and the products
obtained as brown solids
5.5 Solid phase synthesis of peptide H-LKGKFKRG-NH2 and its organometal conjugates
on a Rink amide resin
Metal complex 41 and 44 were manually coupled to the ε-amino group of the two lysine
residues closest to the N-terminus H-LKGKFKRG-NH2. Fmoc-L-Lys(Mtt)-OH was introduced at
these positions. For the metalfree peptide or the peptide with only one organometal group,
Fmoc-L-Lys(Boc)-OH was applied for the lysine positions not to be modified. The peptide
with the sequence H-LKGKFKRG-NH2 was synthesized sequentially on a Rink amide resin until
the second glycine had been attached, with the N-terminus still protected with Fmoc. Then,
the Mtt-protective group was removed with 1% TFA in dichloromethane, and metal complex
Materials and methodes
106
41 was coupled to the free ε-side chain amino group of the lysine. Then, the N-terminal
Fmoc group was removed with 20% piperidine in N,N-dimethylformamide for the further
extension of the polypeptide chain. The terminal amino acids, Fmoc-L-Lys(Mtt)-OH and
Fmoc-L-leucine were sequentially coupled. The Mtt group of the N-terminal lysine was
removed under the same conditions as before and the second organometal moiety,
tungsten carboxylic acid 44, was attached to the free ε-side chain amino group. The Fmoc
group of the N-terminal leucine was first removed with 20% piperidine solution in N,N-
dimethylformamide, followed by the final cleavage of all side chain protective groups and
the cleavage of the peptide from the resin with 95% TFA using 5% water as the scavenger.
The peptide was then precipitated with cold diethyl ether and isolated by centrifugation.
5.6 RP-HPLC of sC18 peptides
Analytical RP-HPLC was done on a Merck-Hitachi system with a Grace Vydac 218TP54
column (4.6 x 250 mm; 5 µm; 300 Å) using a linear gradient of 10 to 60% of
acetonitrile/0.08% TFA and water/0.1% TFA over 30 min with a flow rate of 0.6 ml/min
(System A). The functionalized peptides were purified by preparative RP-HPLC on a Shimadzu
Chromatopac system using the same binary elution system as used in the analytical HPLC.
Preparative HPLC was carried out utilizing a Vydac 218TP1022 C18 column (250 x 20 mm; 10
µm; 300 Å) and the same linear gradients as described above. Fractions containing peptide
conjugates were collected and analyzed by analytical HPLC and MALDI-ToF MS. Pure
fractions were combined and frozen at -80 °C followed by subsequent lyophilization.
5.7 RP-HPLC of all other conjugates
For the other molecules, analytical measurements and preparative purifications were
performed on a Dionex Ultimate 3000 HPLC system using a ReproSil 100 column (C18, 5 µm,
4.6 or 10 mm diameter for analytical and preparative separations, respectively, 250 mm
length) and a linear gradient of 20-90% acetonitrile/water (system B) or 30-90%
acetonitrile/water (system C) over 30 min at a flow rate of 0.6 ml/min for analytical and 3.0
ml/min over 40 min for preparative chromatography respectively.
Materials and methods
107
5.8 Cell culture and cell viability assays
MCF-7 human breast adenocarcinoma cells were used for cytotoxicity studies. Cells were
grown to confluency at 37 °C and 5% CO2 in a humidified atmosphere in 75 cm2 cell culture
flasks. DMEM/Ham’s F12 medium supplemented with 10% heat-inactivated fetal bovine
serum (FBS) (v/v) and 2 mM L-glutamine (Q) was used. The effect of the organometallic
peptide conjugates on the cell viability was examined using a resazurin-based in vitro
toxicology assay. MCF-7 cells were seeded in 96-well plates at 40000 cells per well. Having
reached 80% confluency, the medium was removed and cells were incubated for 24 h at
37 °C with the test substance at different concentrations. Negative controls were incubated
with cell culture medium only. After 24 h, the cells were washed and incubated for 2 h with
10% resazurin in cell medium without fetal bovine serum (v/v) at 37 °C. As a positive control,
cells treated with 70% ethanol were used since this is known to be highly toxic to cells. The
fluorescence was measured using a Spectrafluor plus multiwell reader (Tecan) at 595 nm
with an excitation wavelength of 550 nm.
LDH release into the cell culture supernatant and consequently lysis of the cell membrane
was quantified using a Promega CytoTox-ONE™ assay kit, following the instructions of the
manufacturer. Therefore, the cells were grown and pre-treated like it was described before
for the cell viability assay. Without washing steps, the CytoTOX-ONE (100 µL) reagent was
added after 2 h incubation time. After 10 min incubation at room temperature the stop
solution (50 µL) was added. The positive control had previously been treated with lysis
solution (2 µL). The fluorescence was measured using a Spectrafluor plus multiwell reader
(Tecan) at 595 nm at an excitation wavelength of 550 nm.
5.9 Fluorescence microscopy
To investigate the cellular uptake of the organometal-sC18-conjugates by fluorescence
microscopy, unfixed MCF-7 cells were used. After growing to subconfluency, cells were
incubated with 10 or 20 µM of the CF-labeled conjugate in OptiMEM at 37 °C for 30 min. The
cell nuclei were stained with Hoechst benzimide H33342 for 10 min prior to the end of
peptide incubation. After the incubation, the conjugate solution was removed, cells were
treated for 1 min with trypan blue (6.5 mM in sodium acetate buffer, pH 4.5) to quench
external CF fluorescence and washed twice with HBSS (Hanks’ balanced salt solution).
Materials and methodes
108
Visualization was done with a Zeiss Axiovert 200 inverted fluorescence microscope with
ApoTome.
5.10 X-ray crystallographic data collection and refinement of 2, 4 and 6
X-ray crystal structures of 2, 4 and 6 were solved by Dr. Klaus Merz at the Lehlstuhl für
Anorganische Chemie, Ruhr-Universität Bochum. A single crystal of each compound was
coated with perfluoropolyether, picked up with a glass fiber, and immediately mounted in
the nitrogen cold stream of the diffractometer. Intensity data were collected at 223(2) or
173(2) K using graphite monochromated MoKα radiation (λ = 0.71073 Å). Final cell constants
were obtained from a least squares fit of a subset of a few thousand strong reflections. Data
collection was performed by hemisphere runs taking frames at 0.3° in ω on a Bruker AXS
CCD 1000 diffractometer. The program SADABS was used to account for absorption.[70] The
SHELXL-97 software package was used for solution, refinement, and artwork of the
structures.[71] The structures were readily solved by Patterson methods and difference
Fourier techniques. All nonhydrogen atoms were refined anisotropically and hydrogen
atoms were placed at calculated positions and refined as riding atoms with isotropic
displacement parameters. Crystallographic parameters for 2, 4 and 6 are collected in
Appendix A and relevant bond lengths and angles are given in Table 3.1.1 and Table 3.1.2.
Materials and methods
109
5.11 Synthetic procedures
Cyclopentadien-1-yl-(3-carboxylato-1-oxopropyl) manganese tricarbonyl (1)[51]
USC-WH064
MnOC CO
CO
OH
O
O
MnOC CO
CO
OO O +AlCl3, CH2Cl2,
RT, 2 d
C8H5MnO3
204.06 g/mol
C4H4O3
100.07 g/mol
C12H9MnO6
304.13 g/mol
1
Cymantrene (500 mg, 2.45 mmol) was dissolved in anhydrous dichloromethane (20 ml) and
the solution was cooled to 0-5°C. Then, succinic anhydride (245 mg, 2.45 mmol) was added
to the mixture followed by anhydrous aluminium chloride (667 mg, 5 mmol). Stirring was
continued overnight at room temperature. Then, a 10% hydrochloric acid in ice-water
mixture (100 ml) was added to the solution and the aqueous phase was extracted with
dichloromethane (3 x 50 ml). The combined dichloromethane extracts were first washed
with water and then with saturated sodium carbonate solution until the disappearance of
the yellow color in the organic phase. The sodium carbonate solution was acidified with 20%
hydrochloric acid, extracted with diethyl ether, and the ether phase dried over magnesium
sulfate. After the removal of the solvent in vacuum, the product was isolated as yellow
crystals.
Yield: 600 mg, 2.0 mmol (80%);
1H-NMR (200 MHz, acetone-d6, δppm): 5.75 (s, 2H, Cp), 5.14 (s, 2H, Cp), 3.01 (m, 4H,
CH2CH2COOH);
ESI-MS: m/z = 302.75 [M-H]-;
IR (ATR, cm-1): 2023, 1940, 1915, 1701, 1678;
Elemental analysis (%): calc. for C12H9O6Mn: C 47.37, H 2.96, found: C 47.33, H 3.06.
Materials and methodes
110
Cyclopentadien-1-yl-(3-carboxylatopropyl) manganese tricarbonyl (2)[52]
USC-WH063
MnOC CO
CO
OH
OMn
OC COCO
OH
O
O
TiCl4, Et3SiH, CH2Cl2,
RT, 2d
C12H11MnO5
290.15 g/mol
C12H9MnO6
304.13 g/mol
1 2
Cyclopentadien-1-yl-(3-carboxylato-1-oxopropyl) manganese tricarbonyl (1) (200 mg, 0.66
mmol) was dissolved in anhydrous dichloromethane (30 ml). Then, titanium tetrachloride
(71.8 µL, 124 mg, 0.65 mmol) in anhydrous dichlormethane (10 ml) was added to the
solution in small portion, followed by triethylsilane (420 µL, 2.64 mmol). The mixture was
stirred at room temperature for 2 d. Then, a 5% aqueous sodium carbonate solution (10 ml)
was added. The layers were separated and the organic phase was washed with sodium
carbonate solution (2 x 25 ml). The aqueous phases were combined and 20% hydrochlorid
acid was added until a yellow solid precipitated. After extraction with ethyl acetate and
drying over magnesium sulfate, the final product was isolated as yellow crystals after
removal of the solvent.
Yield: 150 mg, 0.52 mmol (79%);
1H-NMR (200 MHz, acetone-d6, δppm): 4.91 (m, 2H, Cp), 4.86 (m, 2H, Cp), 2.48 (m, 4H,
CH2CH2COOH), 1.82 (m, 2H, CpCH2);
ESI-MS: m/z = 288.70 [M-H]-;
IR (ATR, cm-1): 2010, 1911, 1691;
Elemental analysis (%): calc. for C12H11O5Mn: C 49.63, H 3.79, found: C 50.03, H 4.12.
Materials and methods
111
Cyclopentadien-1-yl-(3-carboxylato-1-oxopropyl) rhenium tricarbonyl (3)[51]
USC-WH066
ReOC CO
CO
OH
O
O
ReOC CO
CO
OO O +AlCl3, CH2Cl2,
reflux, overnight
C8H5ReO3
335.33 g/mol
C4H4O3
100.07 g/mol
C12H9O6Re
435.40 g/mol
3
Cyrhetrene (200 mg, 0.60 mmol) was dissolved in anhydrous dichloromethane (20 ml).
Then, succinic anhydride (60 mg, 0.60 mmol) was added to the mixture followed by
anhydrous aluminium chloride (160 mg, 1.20 mmol). The solution was refluxed
overnight. Then, a 10% hydrochloric acid in ice-water mixture (100 ml) was added to the
solution and the aqueous phase was extracted with dichloromethane (3 x 50 ml). The
combined dichloromethane extracts were first washed with water and then with
saturated sodium carbonate solution (100 ml). The sodium carbonate solution was
acidified with 20% hydrochloric acid, extracted with diethyl ether, and the ether phase
dried over magnesium sulfate. After the removal of the solvent in vacuum, the product
was obtained as a dark brown powder and could be purified by washing with
dichloromethane or chloroform.
Yield: 180 mg, 0.41 mmol, 68%;
1H-NMR (200 MHz, acetone-d6, δppm): 6.35 (t, 3J = 2.4 Hz, 2H, Cp), 5.73 (t, 3J = 2.4 Hz, 2H, Cp),
3.02 (t, 3J = 6.5 Hz, 2H, CH2), 2.64 (t, 3J = 6.5 Hz, 2H, CH2);
ESI-MS: m/z = 434.76 [M-H]-;
IR (ATR, cm-1): 2022, 1931, 1902, 1699, 1678;
Elemental analysis (%): calc. for C12H9O6Re: C 33.02, H 2.06, found: C 33.12, H 1.94.
Materials and methodes
112
Cyclopentadien-1-yl-(3-carboxylatopropyl) rhenium tricarbonyl (4)[52]
USC-WH067
ReOC CO
CO
OH
ORe
OC COCO
OH
O
O
TiCl4, Et3SiH, CH2Cl2,
RT, 2 d
C12H11O5Re
421.42 g/mol
C12H9O6Re
435.40 g/mol
3 4
Cyclopentadien-1-yl-(3-carboxylato-1-oxopropyl) rhenium tricarbonyl (3) (85 mg, 0.19 mmol)
was dissolved in anhydrous dichloromethane (30 ml). Then, titanium tetrachloride (21.4 µL,
36 mg, 0.19 mmol) in anhydrous dichlormethane (10 ml) was added to the solution in small
portion, followed by triethylsilane (125 µL, 0.77 mmol). The mixture was stirred at room
temperature for 2 d. Then, a 5% aqueous sodium carbonate solution (10 ml) was added. The
layers were separated and the organic phase was washed with sodium carbonate solution (2
x 25 ml). The aqueous phases were combined and 20% hydrochloric acid was added until a
white solid precipitated. After extraction with ethyl acetate and drying over magnesium
sulfate, the final product was isolated as a dark brown powder after removal of the solvent
and could be purified by washing with dichloromethane or chloroform.
Yield: 54 mg, 0.13 mmol, 67%;
1H-NMR (200 MHz, acetone-d6, δppm): 5.59 (t, 3J = 2.2 Hz, Cp), 5.50 (t, 3J = 2.2 Hz, 2H, Cp), 2.53
(dd, 3J = 7.6 Hz, 4J = 0.9 Hz, 2H, CH2CH2CH2), 2.38 (t, 3J = 7.4 Hz, 2H, CH2CH2CH2), 1.83 (t, 3J = 8
Hz, 2H, CpCH2);
ESI-MS: m/z = 420.64 [M-H]-;
IR (ATR, cm-1): 2010, 1902, 1706;
Elemental analysis (%): calc. for C12H11O5Re: C 34.12, H 2.6, found: C 34.55, H 2.37.
Materials and methods
113
Cyclopentadien-1-yl-(2-carboxylatophenoxyl) manganese tricarbonyl (5)
USC-WH030
+AlCl3 / CH2Cl2
Friedel-Crafts acylation
OO O
MnOC CO
CO
OO OH
5
C16H9MnO6
352.18 g/mol
MnOC CO
CO
C8H5MnO3
204.06 g/mol
C8H4O3
148.1 g/mol
Cymantrene (700 mg, 3.39 mmol) was dissolved in anhydrous dichloromethane (20 ml) and
the solution was cooled to 0-5°C. Then, phthalic anhydride (500 mg, 3.39 mmol) was added
to the mixture followed by anhydrous aluminium chloride (920 mg, 10.20 mmol). Stirring
was continued overnight at room temperature. Then, a 10% hydrochloric acid in ice-water
mixture (100 ml) was added to the solution and the aqueous phase was extracted with
dichloromethane (4 x 50 ml). The combined dichloromethane extracts were first washed
with water and then with saturated sodium carbonate solution until the disappearance of
the yellow color in the organic phase. The sodium carbonate solution was acidified with 20%
hydrochloric acid, extracted with diethyl ether, and the ether phase dried over magnesium
sulfate. After the removal of the solvent in vacuum, the product was isolated as yellow
crystals.
Yield: 550 mg, 1.56 mmol, 46%;
1H-NMR (200 MHz, acetone-d6, δppm): 8.09 (m, 1H, HAr), 7.74 (m, 2H, HAr), 7.50 (m, 1H, HAr),
5.36 (s, 2H, Cp), 5.09 (s, 2H, Cp);
ESI-MS: m/z = 350.9 [M-H]-;
IR (ATR, cm-1): 2014, 1921, 1693, 1664;
An elemental analysis for 5 was not performed since the other analytical data were in full
accordance with published results.
Materials and methodes
114
Cyclopentadien-1-yl-(2-carboxylatophenyl) manganese tricarbonyl (6)
USC-WH065
TiCl4, Et3SiH, CH2Cl2,
RT, 2dMn
OC COCO
OO OH
5
C16H9MnO6
352.18 g/mol
MnOC CO
CO
O OH
6
C16H9MnO6
338.19 g/mol
Cyclopentadien-1-yl-(2-carboxylatophenoxyl) manganese tricarbonyl (5) (171 mg, 0.48 mmol)
was dissolved in anhydrous dichloromethane (30 ml). Then, titanium tetrachloride (53 µL, 92
mg, 0.48 mmol) in anhydrous dichlormethane (10 ml) was added to the solution in small
portion, followed by triethylsilane (310 µL, 1.95 mmol). The mixture was stirred at room
temperature for 2 d. Then, a 5% aqueous sodium carbonate solution (10 ml) was added. The
layers were separated and the organic phase was washed with sodium carbonate solution (2
x 25 ml). The aqueous phases were combined and 20% hydrochloric acid was added until a
yellow solid precipitated. After extraction with ethyl acetate and drying over magnesium
sulfate, the final product was isolated as yellow crystals after removal of the solvent.
Yield: 55 mg, 0.16 mmol, 33%;
1H-NMR (200 MHz, acetone-d6, δppm): 7.97 (m, 1H, HAr), 7.50(m, 2H, HAr), 7.38(m, 1H, HAr),
4.97 (m, 2H, Cp), 4.81 (m, 2H, Cp), 4.09 (m, 2H, CH2);
ESI-MS: m/z = 336.8 [M-H]-;
IR (ATR, cm-1): 2021, 1927, 1666
An elemental analysis of 6 could not be performed due to the limited amount of product
obtained, which was completely used for subsequent reactions.
Materials and methods
115
Cyclopentadien-1-yl-(2-carboxylatophenoxyl) rhenium tricarbonyl (7)
USC-WH033
C8H4O3
148.1 g/mol
ReOC CO
CO
+AlCl3 / CH2Cl2/reflux
Friedel-Crafts acylation
OO O
ReOC CO
CO
OO OH
7
C16H9ReO6
483.45 g/mol
C8H5ReO3
335.33 g/mol
Cyrhetrene (235 mg, 0.70 mmol) was dissolved in anhydrous dichloromethane (20 ml).
Then, phthalic anhydride (105 mg, 0.70 mmol) was added to the mixture followed by
anhydrous aluminium chloride (187 mg, 1.40 mmol). The solution was refluxed
overnight. Then, a 10% hydrochloric acid in ice-water mixture (100 ml) was added to the
solution and the aqueous phase was extracted with dichloromethane (3 x 50 ml). The
combined dichloromethane extracts were first washed with water and then with
saturated sodium carbonate solution (100 ml). The sodium carbonate solution was
acidified with 20% hydrochloric acid, extracted with diethyl ether, and the ether phase
dried over magnesium sulfate. After the removal of the solvent in vacuum, the product
was obtained as a dark brown powder and could be purified by washing with
dichloromethane or chloroform.
Yield: 100 mg, 0.21 mmol, 30%;
1H-NMR (250 MHz, (CD3)2CO, δppm): 8.08 (d, H, 3J = 7.5 Hz, HAr), 7.69 (m, 2H, HAr); 7.41 (d, H,
3J = 7.5 Hz, HAr), 5.95 (m, 2H, HCp), 5.68 (m, 2H, HCp);
ESI-MS (negative): m/z = 482.9 [M-H]-;
IR (ATR, cm-1): 2024, 1916, 1656, 1573;
An elemental analysis of 7 could not be performed due to the limited amount of product
obtained, which was completely used for subsequent reactions.
Materials and methodes
116
Cyclopentadien-1-yl-(2-carboxylatophenyl) rhenium tricarbonyl (8)
USC-WH070
ReOC CO
CO
O OH
TiCl4, Et3SiH, CH2Cl2,
RT, 2d
C16H11O5Re
469.46 g/mol
8
ReOC CO
CO
OO OH
7
C16H9O6Re
483.45 g/mol
Cyclopentadien-1-yl-(2-carboxylatophenoxyl) rhenium tricarbonyl (7) (105 mg, 0.22 mmol)
was dissolved in anhydrous dichloromethane (30 ml). Then, titanium tetrachloride (24 µL, 42
mg, 0.22 mmol) in anhydrous dichlormethane (10 ml) was added to the solution in small
portion, followed by triethylsilane (142 µL, 0.89 mmol). The mixture was stirred at room
temperature for 2 d. Then, a 5% aqueous sodium carbonate solution (10 ml) was added. The
layers were separated and the organic phase was washed with sodium carbonate solution (2
x 25 ml). The aqueous phases were combined and 20% hydrochloric acid was added until a
yellow solid precipitated. After extraction with ethyl acetate and drying over magnesium
sulfate, the final product was isolated as yellow crystals after removal of the solvent.
Yield: 57 mg, 0.12 mmol, 55%.
1H-NMR (200 MHz, acetone-d6, δppm): 8.04-8.00 (m, 1H, HAr), 7.60-7.55 (m, 1H, HAr), 7.47-
7.36 (m, 2H, HAr), 5.62 (m, 2H, Cp), 5.47 (m, 2H, Cp), 4.22 (m, 2H, CH2);
ESI-MS: m/z = 468.8 [M-H]-;
IR (ATR, cm-1): 2010, 1888, 1684
An elemental analysis of 8 could not be performed due to the limited amount of product
obtained, which was completely used for subsequent reactions.
Materials and methods
117
Cyclopentadien-1-yl-(4-carboxylatophenoxyl) manganese tricarbonyl (9)[45]
USC-WH034
O
OO
Cl
CH3
MnOC CO
CO
O
O
O
CH3
MeOH
NaOHMn
OC COCO
O
OH
O
9
C16H9MnO6
352.18 g/mol
MnOC CO
CO
C8H5MnO3
204.06 g/mol
C9H7ClO3
198.60 g/mol
+
C17H11MnO6
352.18 g/mol
AlCl3 / CH2Cl2
Friedel-Crafts acylation
Aluminium chloride (333.8 mg, 2.5 mmol) was added in anhydrous dichlormethane (20 ml)
and cooled to 0-5 °C with ice while stirring. Then, Terephthalic acid monomethyl ester
chloride (243.3 mg, 1.23 mmol) was added in small portions followed by cymantrene (250
mg, 1.23 mmol) while the temperature was maintained at 0-5 °C. After 1 h, the reaction
mixture was allowed to warm to room temperature and stirring continued overnight. The
reaction mixture was then poured into a mixture of ice/water (100 ml) and concentrated
hydrochloric acid (10 ml). The layers were separated and the aqueous phase extracted with
dichlormethane (3× 50 ml). The combined organic phases were then washed with water and
saturated aqueous sodium carbonate solution. The solvent was removed from the organic
phase and the residue purified by column chromatography on silica using a mixture of n-
hexane/ethyl acetate 5:2 (v/v) as the eluent (Rf = 0.45) to give the product as a yellow solid.
The solid was added to an aqueous solution of sodium hydroxide (1 M, 10 ml) (200 mg, 0.55
mmol) in methanol (50 ml) and stirred at room temperature. The reaction was monitored
with TLC (silica, n-hexane/ethyl acetate 5:2 v/v) until the disappearance of the starting
material was completed. Then, the solvent was removed and water (200 ml) added to the
residue. The solution was washed with dichlormethane (3× 50 ml), and acidified with
concentrated hydrochloric acid to pH 1. The precipitate was taken up in ethyl acetate and
dried over magnesium sulfate. After removal of the solvent, compound 9 was obtained as a
yellow solid.
Yield: 370 mg, 1.05 mmol, 85%.
1H-NMR (200 MHz, (CD3)2CO, δppm): 8.19 (s, 2H, HAr), 7.94 (s, 2H, HAr), 5.76 (s, 2H, Cp), 5.25 (s,
2H, Cp);
Materials and methodes
118
13C-NMR (62.9 MHz, (CD3)2SO, δppm): 223.44 (C=O), 191.21 (C=O), 166.21 (C=O), 140.47 (CAr),
133.96 (CHAr), 90.94 (Cp), 89.20 (Cp), 85.81 (Cp) 85.13 (Cp);
ESI-MS(negative): m/z = 350.9 [M-H]-;
IR (ATR, cm-1): 2543, 2028, 1928, 1683, 1632,;
Elemental analysis (%): calc. for C16H9MnO6: C 54.57, H 2.58, found: C 54.69, H 3.02.
Materials and methods
119
Cyclopentadien-1-yl-(4-carboxylatophenyl) manganese tricarbonyl (10)
USC-WH068
MnOC CO
CO
O
OH
O
9
C16H9MnO6
352.18 g/mol
TiCl4, Et3SiH, CH2Cl2,
RT, 2d
10
C16H9MnO6
338.19 g/mol
MnOC CO
COOH
O
Cyclopentadien-1-yl-(4-carboxylatophenoxyl) manganese tricarbonyl (9) (100 mg, 0.28 mmol)
was dissolved in anhydrous dichloromethane (30 ml). Then, titanium tetrachloride (31 µL, 54
mg, 0.28 mmol) in anhydrous dichlormethane (10 ml) was added to the solution in small
portion, followed by triethylsilane (182 µL, 1.14 mmol). The mixture was stirred at room
temperature for 2 d. Then, a 5% aqueous sodium carbonate solution (10 ml) was added. The
layers were separated and the organic phase was washed with sodium carbonate solution (2
x 25 ml). The aqueous phases were combined and 20% hydrochloric acid was added until a
yellow solid precipitated. After extraction with ethyl acetate and drying over magnesium
sulfate, the final product was isolated as yellow crystals after removal of the solvent.
Yield: 40 mg, 0.12 mmol, 42%;
1H-NMR (200 MHz, acetone-d6, δppm): 7.98 (m, 2H, HAr), 7.49 (m, 2H, HAr), 4.98 (m, 2H, Cp),
4.88 (m, 2H, Cp), 3.76 (m, 2H, CH2),;
ESI-MS: m/z = 336.7 [M-H]-;
IR (ATR, cm-1): 2006, 1921, 1680
An elemental analysis of 10 could not be performed due to the limited amount of product
obtained, which was completely used for subsequent reactions.
Materials and methodes
120
Cyclopentadien-1-yl-(4-carboxylatophenoxyl) rhenium tricarbonyl (11)[45]
USC-WH069
O
OO
Cl
CH3
ReOC CO
CO
O
O
O
CH3
MeOH
NaOHRe
OC COCO
O
OH
O
11
ReOC CO
CO
C9H7ClO3
198.60 g/mol
+AlCl3 / CH2Cl2
reflux overnight
C16H9O6Re
483.45 g/mol
C8H5O3Re
335.33 g/mol
A mixture of aluminium chloride (54 mg, 0.40 mmol), terephthalic acid monomethyl ester
chloride (40 mg, 0.20 mmol) and cyrhetrene (68 mg, 0.20 mmol) in anhydrous
dichlormethane (20 ml) was refluxed overnight. The reaction mixture was then poured into a
mixture of ice/water (100 ml) and concentrated hydrochloric acid (10 ml). The layers were
separated and the aqueous phase extracted with dichlormethane (3× 50 ml). The combined
organic phases were then washed with water and saturated aqueous sodium carbonate
solution. The solvent was removed from the organic phase and the residue purified by
column chromatography on silica using a mixture of n-hexane/ethyl acetate 5:2 (v/v) as the
eluent (Rf = 0.45) to give the product as a white solid. The solid was added to an aqueous
solution of sodium hydroxide (1 M, 10 ml) (200 mg, 0.55 mmol) in methanol (50 ml) and
stirred at room temperature. The reaction was monitored with TLC (silica, n-hexane/ethyl
acetate 5:2 v/v) until the disappearance of the starting material was completed. Then, the
solvent was removed and water (200 ml) added to the residue. The solution was washed
with dichlormethane (3× 50 ml), and acidified with concentrated hydrochloric acid to pH 1.
The precipitate was taken up in ethyl acetate and dried over magnesium sulfate. After
removal of the solvent, compound 11 was obtained as a white solid.
Yield: 67.2 mg, 0.14 mmol, 69%;
1H-NMR (200 MHz, acetone-d6, δppm): 8.16 (m, 2H, HAr), 7.95 (m, 2H, HAr), 6.35 (m, 2H, Cp),
5.85 (m, 2H, Cp);
ESI-MS: m/z = 482.7 [M-H]-;
IR (ATR, cm-1): 2039, 1919, 1684, 1636.
An elemental analysis of 11 could not be performed due to the limited amount of product
obtained, which was completely used for subsequent reactions.
Materials and methods
121
Cyclopentadien-1-yl-(4-carboxylatophenyl) rhenium tricarbonyl (12)
USC-WH071
ReOC CO
CO
O
OH
O
11
TiCl4, Et3SiH, CH2Cl2,
RT, 2d
C16H9O6Re
483.45 g/mol
ReOC CO
COOH
O
C16H11O5Re
469.46 g/mol
12
Cyclopentadien-1-yl-(4-carboxylatophenoxyl) rhenium tricarbonyl (11) (60 mg, 0.12 mmol)
was dissolved in anhydrous dichloromethane (30 ml). Then, titanium tetrachloride (13 µL, 23
mg, 0.12 mmol) in anhydrous dichlormethane (10 ml) was added to the solution in small
portion, followed by triethylsilane (77 µL, 0.49 mmol). The mixture was stirred at room
temperature for 2 d. Then, a 5% aqueous sodium carbonate solution (10 ml) was added. The
layers were separated and the organic phase was washed with sodium carbonate solution (2
x 25 ml). The aqueous phases were combined and 20% hydrochloric acid was added until a
white solid precipitated. After extraction with ethyl acetate and drying over magnesium
sulfate, the final product was isolated as white crystals after removal of the solvent.
Yield: 37.6 mg, 0.08 mmol, 69%.
1H-NMR (250 MHz, (CD3)2CO, δppm): 8.01 (d, 2H, 3J = 8.2 Hz, HAr), 7.49 (d, 2H, 3J = 8.2 Hz, HAr);
5.65 (m, 2H, HCp), 5.52 (m, 2H, HCp), 3.89 (s, 2H, CH2);
ESI-MS (negative): m/z = 468.91 [M-H]-;
IR (ATR, cm-1): 2933, 2011, 1897, 1683,
An elemental analysis of 12 could not be performed due to the limited amount of product
obtained, which was completely used for subsequent reactions.
Materials and methodes
122
Cym2*-sC18 (13)
MnOC CO
CO
N
O
NN
NN
NN
NN
NN
NN
O
O
NH
NHH2N
O
NH2
O
NH
NHH2N
O
O
NH
NHH2N
O
NH2
O
O
NH
NHH2N
O
NH2
O
O
NN
N NH2
NH2
O
O
NH2
O
O OH
O
NH2
Peptide 13 was prepared on a Rink amide resin using the amino acids Fmoc-Gly-OH, Fmoc-
Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, Fmoc-Asn(Trt)-OH and Fmoc-
Ile-OH applying the procedure described in section 5.2.
RP-HPLC: system A, tR = 18.80 min
ESI-MS (positive): m/z = 469.4 [M+5H]5+
IR (ATR, cm-1): 2017, 1925, 1651, 1537.
Cyr2-sC18 (14)
ReOC CO
CO
N
O
NN
NN
NN
NN
NN
NN
O
O
NH
NHH2N
O
NH2
O
NH
NHH2N
O
O
NH
NHH2N
O
NH2
O
O
NH
NHH2N
O
NH2
O
O
NN
N NH2
NH2
O
O
NH2
O
O OH
O
NH2
O
Peptide 14 was prepared on a Rink amide resin using the amino acids Fmoc-Gly-OH, Fmoc-
Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, Fmoc-Asn(Trt)-OH and Fmoc-
Ile-OH applying the procedure described in section 5.2.
RP-HPLC: system A, tR = 18.21 min
ESI-MS (positive): m/z = 498.5 [M+5H]5+
MALDI-MS (positive): 2485.4 [M+H]+
IR (ATR, cm-1): 2020, 1922, 1653, 1537.
Materials and methods
123
Cyr2*-sC18 (15)
ReOC CO
CO
N
O
NN
NN
NN
NN
NN
NN
O
O
NH
NHH2N
O
NH2
O
NH
NHH2N
O
O
NH
NHH2N
O
NH2
O
O
NH
NHH2N
O
NH2
O
O
NN
N NH2
NH2
O
O
NH2
O
O OH
O
NH2
Peptide 15 was prepared on a Rink amide resin using the amino acids Fmoc-Gly-OH, Fmoc-
Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, Fmoc-Asn(Trt)-OH and Fmoc-
Ile-OH applying the procedure described in section 5.2.
RP-HPLC: system A, tR = 19.09 min
ESI-MS (positive): m/z = 495.6 [M+5H]5+
MALDI-MS (positive): 2471.5 [M+H]+
IR (ATR, cm-1): 2031, 1933, 1653, 1539.
Cym2*-sC18(CF) (16)
NH(CF)
MnOC CO
CO
N
O
NN
NN
NN
NN
NN
NN
O
O
NH
NHH2N
O
O
NH
NHH2N
O
O
NH
NHH2N
O
NH2
O
O
NH
NHH2N
O
NH2
O
O
NN
N NH2
NH2
O
O
NH2
O
O OH
O
NH2
Peptide 16 was prepared on a Rink amide resin using the amino acids Fmoc-Gly-OH, Fmoc-
Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Dde)-OH, Fmoc-Phe-OH, Fmoc-
Asn(Trt)-OH and Fmoc-Ile-OH applying the procedure described in section 5.2 and 5.3.
RP-HPLC: system A, tR = 20.29 min
ESI-MS (positive): m/z = 541.0 [M+5H]5+
Materials and methodes
124
Cyr2-sC18(CF) (17)
NH(CF)
ReOC CO
CO
N
O
NN
NN
NN
NN
NN
NN
O
O
NH
NHH2N
O
O
NH
NHH2N
O
O
NH
NHH2N
O
NH2
O
O
NH
NHH2N
O
NH2
O
O
NN
N NH2
NH2
O
O
NH2
O
O OH
O
NH2
O
Peptide 17 was prepared on a Rink amide resin using the amino acids Fmoc-Gly-OH, Fmoc-
Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Dde)-OH, Fmoc-Phe-OH, Fmoc-
Asn(Trt)-OH and Fmoc-Ile-OH applying the procedure described in section 5.2 and 5.3.
RP-HPLC: system A, tR = 20.69 min
MALDI-MS (positive): m/z = 2843.4 [M+H]+
Cyr2*-sC18(CF) (18)
NH(CF)
ReOC CO
CO
N
O
NN
NN
NN
NN
NN
NN
O
O
NH
NHH2N
O
O
NH
NHH2N
O
O
NH
NHH2N
O
NH2
O
O
NH
NHH2N
O
NH2
O
O
NN
N NH2
NH2
O
O
NH2
O
O OH
O
NH2
Peptide 18 was prepared on a Rink amide resin using the amino acids Fmoc-Gly-OH, Fmoc-
Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Dde)-OH, Fmoc-Phe-OH, Fmoc-
Asn(Trt)-OH and Fmoc-Ile-OH applying the procedure described in section 5.2 and 5.3.
RP-HPLC: system A, tR = 21.20 min
MALDI-MS (positive): m/z = 2829.4 [M+H]+
Materials and methods
125
(Formylcyclopentadienyl)manganese tricarbonyl (19)
USC-WH086
MnOC CO
CO
H
O
n-Buli/THF -78°C
DMF
19
C9H9MnO4
232.07 g/mol
MnOC CO
CO
C8H5MnO3
204.06 g/mol
Cymantrene (500 mg, 2.45 mmol) was dissolved in anhydrous tetrahydrofuran (30 ml) and
cooled to -78 °C with an aceton/dry ice bath. Then, n-butyllithium (1.6 M solution in hexane,
3.13 ml, 4.9 mmol) was added dropwise and the reaction mixture was stirred at -78 °C for 1
h. N,N-Dimethylformamide (0.8 ml, 9.8 mmol) was added and stirring continued for another
1 h. Then the reaction was allowed to warm up to room temperature. After the addition of a
10% solution of hydrochloric acid (50 ml) and dichloromethane (100 ml), the phases was
separated, the organic phase washed with water, and dried over magnesium sulfate. After
filtration and the removal of the solvent, a yellow-brown solid was obtained. The product
was further purified by column chromatography on silica using ethyl acetate/n-hexane 2:5 as
the eluent (Rf = 0.5), or by sublimation at 50 °C and 10-2 mbar and the final product was
isolated as yellow crystals.
Yields: 445 mg, 78%;
1H-NMR (200 MHz, CDCl3, δppm): 9.61 (s, 1H, HCHO), 5.46 (m, 2H, HCp); 4.93 (m, 2H, HCp);
IR (ATR, cm-1): 2019, 1903, 1687;
An elemental analysis for 19 was not performed since the other analytical data were in full
accordance with published results.
Materials and methodes
126
(Formylcyclopentadienyl)rhenium tricarbonyl (20)
USC-WH087
ReOC CO
CO
H
O
ReOC CO
CO
n-Buli/THF -78°C
DMF
20
C9H5O4Re
363.34 g/mol
C8H5O3Re
335.33 g/mol
Cyrhetrene (500mg, 1.49 mmol) was dissolved in anhydrous tetrahydrofuran (30 ml) and
cooled to -78 °C with an aceton/dry ice bath. Then, n-butyllithium (1.6 M solution in hexane,
1.9 ml, 2.98 mmol) was added dropwise and the reaction mixture was stirred at -78 °C for 1
h. N,N-Dimethylformamide (0.48 ml, 5.96 mmol) was added and stirring continued for
another 1 h. Then the reaction was allowed to warm up to room temperature. After the
addition of a 10% solution of hydrochloric acid (50 ml) and dichloromethane (100 ml), the
phases was separated, the organic phase washed with water, and dried over magnesium
sulfate. After filtration and the removal of the solvent, a white solid was obtained. The
product was further purified by column chromatography on silica using ethyl acetate/n-
hexane 2:5 as the eluent (Rf = 0.5), or by sublimation at 50 °C and 10-2 mbar and the final
product was isolated as a white solid.
Yield: (486 mg, 89.7%);
1H-NMR (200 MHz, CDCl3, δppm): 9.59 (s, 1H, HCHO), 6.01 (m, 2H, HCp); 5.47 (m, 2H, HCp);
IR (ATR, cm-1): 2021, 1882, 1687;
An elemental analysis for 20 was not performed since the other analytical data were in full
accordance with published results.
Materials and methods
127
Benzaldehyde diethyl acetal[55]
USC-WH097
C11H16O2
180.24 g/mol
OOO Hethyl orthoformate
H2SO4, RT, 24 h
C7H6O
106.12 g/mol
To a mixture of benzaldehyde (10 g, 0.094 mol) and ethyl orthoformate (14.82 g, 0.1 mol),
two drops of sulphuric acid were added. The reaction mixture was then stirred at room
temperature for 24 h and the product was isolated by distillation. At 10-1 mbar, it has a
boiling point of 95 °C. The final product was obtained as a colorless liquid.
1H-NMR (200 MHz, CDCl3, δppm): 7.51-7.29 (m, 5H, HAr), 5.52 (s, 1H, HCH), 3.73-3.46 (m, 4H,
CH2CH3), 5.52 (t, 3J = 7 Hz, 6H, CH2CH3)
An elemental analysis for benzaldehyde diethyl acetal was not performed since the other
analytical data were in full accordance with published results.
Materials and methodes
128
Benzaldehyde chromium tricarbonyl (21)[55]
USC-WH104
C14H16CrO5
316.27 g/mol
21
CrOC CO
CO
OO
O
O
Cr(CO)6
CrOC CO
CO
H
O
HCl/H2O
C11H16O2
180.24 g/mol
C10H6CrO4
242.15 g/mol
Benzaldehyde diethylacetal (2 ml, 39.2 mmol), and chromium hexacarbonyl (0.4 g, 1.8 mmol)
were heated in dry 1,4-dioxane (20 ml) to 100 °C overnight. The yellow-green solution was
filtered through Celite and the solvent was evaporated under vacuum. 0.5 M hydrochlorid
acid (30 ml) was added to the yellow residue and stirring continued at room temperature.
The color turned from yellow to orange. Then, diethyl ether (100 ml) was added and the
phases were separated. The aqueous phase was extracted with diethyl ether (2×100 ml). The
organic phases were combined, dried over magnesium sulfate and the solvent then removed
in vacuum. The product was further purified by column chromatography on silica using ethyl
acetate/n-hexane 2:5 as the eluent (Rf = 0.3)
1H-NMR (200 MHz, CDCl3, δppm): 9.46 (s, 1H, HCHO), 5.94 (d, 3J = 6.0 Hz, 2H, HAr), 5.69 (t, 3J =
6.4 Hz, 1H, HAr), 5.29 (t, 3J = 6.4 Hz, 2H, HAr);
IR (ATR, cm-1): 1954, 1855, 1687;
An elemental analysis for 21 was not performed since the other analytical data were in full
accordance with published results.
Materials and methods
129
1-Adamantane carboxaldehyde (22)[56,57]
USC-WH138
OH HO
trifluoroacetic anhydride/oxalyl chloride
DMSO
C11H18O
166.26 g/mol
C11H16O
164.24 g/mol
A mixture of dichloromethane (30 ml) and dimethyl sulfoxide (30 ml) was cooled to -78 °C.
Oxalylchloride (1.7 ml, 2.49 g, 20 mmol) was added to the solution and further stirred for 20
min. 1-Adamantanyl methanol (2.5 g, 15.04 mmol) was dissolved in dichloromethane (25 ml)
and added dropwise to the mixture and stirred at -78 °C for 1 h. Then, triethylamine (5 ml)
was added slowly and stirring continued for another 30 min. Then, the solution was allowed
to warm up to room temperature. 20% sodium dihydrogen phosphate solution (12.5 ml) and
ice-water mixture (50 ml) was added, stirred for 15 min and extracted with diethyl ether (3 ×
50 ml). The organic phase was separated and washed with 20% sodium dihydrogen
phosphate solution (200 ml) as well as saturated sodium chloride (2 × 50 ml) and dried over
magnesium sulfate. After removal of the solvent, the product was isolated as a white solid.
Yield: 1.53 g, 9.32 mmol (46%);
1H-NMR (200 MHz, CDCl3, δppm): 9.31 (s, 1H, HCHO), 2.06 (s, 3H, CH), 1.71 (s, 12H, CH2);
An elemental analysis for 22 was not performed since the other analytical data were in full
accordance with published results.
Materials and methodes
130
USC-WH120 (23)
C9H5MnO4
232.07 g/mol
MnOC CO
CO
H
O
H2N NN NH2
NH2
H2N
+ 43Å MS, ethanol
NaBH4
NN
MnOC CO
CO
HN
NH
HN
HN
MnCOOC
OC
MnOC CO
CO
MnCOOC
OC
b)
a)
C16H40N6
316.53 g/mol
C52H60MnN6O12
1180.82 g/mol
2319
(Formylcyclopentadienyl)manganese tricarbonyl 19 (200 mg, 0.86 mmol) and DAB-(NH2)4-G1
dendrimer (65 mg, 0.21 mmol) were added to anhydrous ethanol (30 ml). Molecular sieves
(3 Å, 3-5 g) were added and the mixture stirred overnight. On the next day, sodium
borhydride (65.22 mg, 1.72 mmol) was added. After 1 h, water (10 ml) was added to the
solution and the molecular sieves were filtered off. The product was extracted with
dichloromethane (3 x 100 ml), organic phase was separated, dried over sodium sulfate, and
the solvent removed to obtain the product as a yellow oil.
Yield: 210, 79%.
A part of it was purified with preparative HPLC (System B, tR = 19.20 min) and a white solid
was obtained.
1H-NMR (200 MHz, CD3OD, δppm): 5.19 (m, 8H, Cp), 4.97 (m, 8H, Cp), 3.90 (s, 8H, NCH2Cp),
3.16 (m, 20H, NCH2CH2CH2N, NCH2CH2CH2CH2Ncore), 2.18 (m, 8H, NCH2CH2CH2N), 1.77 (m, 4H,
NCH2CH2CH2CH2Ncore)
ESI-MS (positive): m/z = 1081.1 [M+H]+, 591.2 [M+2H]2+;
IR (ATR, cm-1): 2024, 1928
Materials and methods
131
USC-WH124 (24)
C9H5MnO4
232.07 g/mol
DAB-dendr N
MnOC CO
CO8
MnOC CO
CO
H
O
DAB-dendr NH Mn
OC COCO
8
NaBH4
19
C112H136Mn8N14O24
2500.87 g/mol
24
(Formylcyclopentadienyl)manganese tricarbonyl 19 (200 mg, 0.86 mmol) and DAB-(NH2)8-G2
dendrimer (79.36 mg, 0.102 mmol) were added to anhydrous ethanol (30 ml). Molecular
sieves (3 Å, 3-5 g) were added and the mixture stirred overnight. On the next day, sodium
borhydride (65.22 mg, 1.72 mmol) was added. After 1 h, water (10 ml) was added to the
solution and the molecular sieves were filtered off. The product was extracted with
dichloromethane (3 x 100 ml), organic phase was separated, dried over sodium sulfate, and
the solvent removed under evaporation to obtain the product as a yellow oil.
Yield: 209 mg, 74.8%.
A part of it was purified with preparative HPLC (System B, tR = 23.24 min) and a white solid
was obtained.
1H-NMR (200 MHz, (CD3)2SO, δppm): 5.25 (m, 16H, Cp), 5.08 (m, 16H, Cp), 3.81 (m, 16H,
NCH2Cp), 2.98 (m, 52H, NCH2CH2CH2N, NCH2CH2CH2CH2Ncore), 1.97 (m, 20H, NCH2CH2CH2N),
1.61 (m, 8H, NCH2CH2CH2CH2Ncore, NCH2CH2CH2N)
ESI-MS (positive): m/z = 1251.6 [M+2H] 2+, 835.0 [M+3H]3+, 626.5 [M+4H]4+;
IR (ATR, cm-1): 2023, 1914
Materials and methodes
132
USC-WH127 (25)
C9H5MnO4
232.07 g/mol
DAB-dendr N
MnOC CO
CO16
MnOC CO
CO
H
O
DAB-dendr NH Mn
OC COCO
16
NaBH4
19
C232H288Mn16N30O48
5143.96 g/mol
25
(Formylcyclopentadienyl)manganese tricarbonyl 19 (150 mg, 0.65 mmol) and DAB-(NH2)16-
G3 dendrimer (64.92 mg, 0.039 mmol) were added to anhydrous ethanol (30 ml). Molecular
sieves (3 Å, 3-5 g) were added and the mixture stirred overnight. On the next day, sodium
borhydride (65.22 mg, 1.72 mmol) was added. After 1 h, water (10 ml) was added to the
solution and the molecular sieves were filtered off. The product was extracted with
dichloromethane (3 x 100 ml), organic phase was separated, dried over sodium sulfate, and
the solvent removed under evaporation to obtain the product as a yellow oil.
Yield: 165.9 mg, 88%
A part of it was purified with preparative HPLC (System B, tR = 27.21 min) and a white solid
was obtained.
1H-NMR (200 MHz, CD3OD, δppm): 5.18 (m, 32H, Cp), 4.95 (m, 32H, Cp), 3.89 (s, 32H, NCH2Cp),
3.15 (m, 116H, NCH2CH2CH2N, NCH2CH2CH2CH2Ncore), 2.17 (m, 56H, NCH2CH2CH2N), 1.84 (m,
4H, NCH2CH2CH2CH2Ncore)
ESI-MS (positive): m/z = 1029.7 [M+5H]5+, 858.4 [M+6H]6+;
IR (ATR, cm-1): 2024, 1914
Materials and methods
133
USC-WH122 (26)
ReOC CO
CO
H
O
H2N NN NH2
NH2
H2N
+ 43Å MS, ethanol
NaBH4
NN
ReOC CO
CO
HN
NH
HN
HN
ReCOOC
OC
ReOC CO
CO
ReCOOC
OC
b)
a)
C16H40N6
316.53 g/mol
C52H60N6O12Re4
1147.27 g/mol
2620
C9H5O4Re
363.34 g/mol
(Formylcyclopentadienyl)rhenium tricarbonyl 20 (150 mg, 0.412 mmol) and DAB-(NH2)4-G1
dendrimer (31.06 mg, 0.098 mmol) were added to anhydrous ethanol (30 ml). Molecular
sieves (3 Å, 3-5 g) were added and the mixture stirred overnight. On the next day, sodium
borhydride (65.22 mg, 1.72 mmol) was added. After 1 h, water (10 ml) was added to the
solution and the molecular sieves were filtered off. The product was extracted with
dichloromethane (3 x 100 ml), organic phase was separated, dried over sodium sulfate, and
the solvent removed under evaporation to obtain the product as a colorless oil.
Yield: (148.2 mg, 96.5%)
A part of it was purified with preparative HPLC (System B, tR = 20.04 min) and a white solid
was obtained.
1H-NMR (200 MHz, CD3OD, δppm): 5.83 (m, 8H, Cp), 5.61 (m, 8H, Cp), 4.01 (s, 8H, NCH2Cp),
3.15 (m, 20H, NCH2CH2CH2N, NCH2CH2CH2CH2Ncore), 2.14 (m, 8H, NCH2CH2CH2N), 1.76 (m, 4H,
NCH2CH2CH2CH2Ncore)
Cannot be characterized by mass spectrometry due to poor ionization under ESI conditions;
IR (ATR, cm-1): 2026, 1913
Materials and methodes
134
USC-WH125 (27)
C9H5O4Re
363.34 g/mol
DAB-dendr N
ReOC CO
CO8
ReOC CO
CO
H
O
DAB-dendr NH Re
OC COCO
8
NaBH4
20
C112H136N14O24Re8
3552.02 g/mol
27
(Formylcyclopentadienyl)rhenium tricarbonyl 20 (150 mg, 0.412 mmol) and DAB-(NH2)8-G2
dendrimer (37.94 mg, 0.049 mmol) were added to anhydrous ethanol (30 ml). Molecular
sieves (3 Å, 3-5 g) were added and the mixture stirred overnight. On the next day, sodium
borhydride (65.22 mg, 1.72 mmol) was added. After 1 h, water (10 ml) was added to the
solution and the molecular sieves were filtered off. The product was extracted with
dichloromethane (3 x 100 ml), organic phase was separated, dried over sodium sulfate, and
the solvent removed under evaporation to obtain the product as a colorless oil.
Yield: colorless oil, (176 mg, 93.6%).
Part of the oil was further purified with preparative HPLC (System B, tR = 24.02 min) and a
white solid was obtained.
1H-NMR (200 MHz, (CD3)2SO, δppm): 5.89 (m, 16H, Cp), 5.71 (m, 16H, Cp), 3.92 (s, 16H,
NCH2Cp), 2.99 (m, 52H, NCH2CH2CH2N, NCH2CH2CH2CH2Ncore), 1.99 (m, 24H, NCH2CH2CH2N),
1.66 (m, 4H, NCH2CH2CH2CH2Ncore)
Cannot be characterized by mass spectrometry due to poor ionization under ESI conditions;
IR (ATR, cm-1): 2024, 1905
Materials and methods
135
USC-WH128 (28)
C9H5O4Re
363.34 g/mol
DAB-dendr N
ReOC CO
CO16
ReOC CO
CO
H
O
DAB-dendr NH Re
OC COCO
16
NaBH4
20
C232H288N30O48Re16
7244.26 g/mol
28
(Formylcyclopentadienyl) rhenium tricarbonyl 20 (200mg, 0.549 mmol) and DAB-(NH2)16-G3
dendrimer (55.17 mg, 0.033 mmol) were added to anhydrous ethanol (30 ml). Molecular
sieves (3 Å, 3-5 g) were added and the mixture stirred overnight. On the next day, sodium
borhydride (41.57 mg, 1.099 mmol) was added. After 1 h, water (10 ml) was added to the
solution and the molecular sieves were filtered off. The product was extracted with
dichloromethane (3 x 100 ml), organic phase was separated, dried over sodium sulfate, and
the solvent removed under evaporation to obtain the product as a colorless oil.
Yield: colorless oil, (228.7 mg, 89.7%).
Part of the oil was further purified with preparative HPLC (System C, tR = 28.74 min) and a
white solid was obtained.
1H-NMR (200 MHz, CD3OD, δppm): 5.40 (m, 32H, Cp), 5.28 (m, 32H, Cp), 3.53 (s, 32H,
NCH2Cp), 2.68 (m, 116H, NCH2CH2CH2N, NCH2CH2CH2CH2Ncore), 1.62 (m, 56H, NCH2CH2CH2N),
1.40 (m, 4H, NCH2CH2CH2CH2Ncore)
Cannot be characterized by mass spectrometry due to poor ionization under ESI conditions;
IR (ATR, cm-1): not done
Materials and methodes
136
USC-WH139 (29)
HO
H2N NN NH2
NH2
H2N
NN
HN
NH
HN
HN
+ molecular sieve 3 Å, ethanol
NaBH4
C16H40N6
316.53 g/mol
C11H16O
164.24 g/mol
22
C60H48N6
853.06 g/mol
29
1-Adamantane carboxaldehyde 22 (213 mg, 1.30 mmol) and DAB-(NH2)4-G1 dendrimer (89
mg, 0.28 mmol) were added to anhydrous ethanol (30 ml). Molecular sieves (3 Å, 3-5 g) were
added and the mixture stirred overnight. On the next day, sodium borhydride (97.98 mg,
37.83 mmol) was added. After 1 h, water (10 ml) was added to the solution and the
molecular sieves were filtered off. The product was extracted with dichloromethane (3 x 100
ml), organic phase was separated, dried over sodium sulfate, and the solvent removed under
evaporation to obtain the product as a colorless oil.
Yield: (206 mg, 86.5%)
A part of it was purified with preparative HPLC (System B, tR = 22.24 min) and a white solid
was obtained.
2.06 (s, 3H, CH), 1.71 (s, 12H, CH2);
1H-NMR (200 MHz, CD3OD, δppm): 2.75 (s, 8H, NCH2Cp), 2.04 (m, 20H, NCH2CH2CH2N,
NCH2CH2CH2CH2Ncore), 1.84-1.73 (m, 24H, NCH2CH2CH2N, NCH2CH2CH2CH2Ncore,
CH(adamantane)) 1.64 (s, 48H, CH2(adamantane))
Cannot be characterized by mass spectrometry due to poor ionization under ESI conditions;
IR (ATR, cm-1): not done.
Materials and methods
137
USC-WH088 (30)[58,59]
H2NNH2
OMe
O
NN
OMe
OMe
MeO
MeO
O
O
O
O
+ 4
C6H16N2
164.24 g/mol
C4H6O2
164.24 g/mol
C22H40N2O8
460.28 g/mol
30
Hexamethylenediamine (1 g, 8.6 mmol, 1.124 ml) was heated in methyl acrylate (40.56 g,
258 mmol, 43.47 ml) at 87 °C for 4 d, then the solvents were removed in vacuum and the
product was isolated as a yellow gel.
Yield: about 4 g, 8.6 mmol, 100%.
1H-NMR (200 MHz, CD3OD, δppm): 3.63 (s, 12H, -CH3), 2.73 (t, 3J = 7 Hz, 8H, COCH2-), 2.40 (m,
12H, NCH2-), 1.37 (m, 4H, -NCH2CH2CH2CH2CH2CH2N-), 1.19 (m, 4H, -
NCH2CH2CH2CH2CH2CH2N-)
An elemental analysis for 30 was not performed since the other analytical data were in full
accordance with published results.
Materials and methodes
138
USC-WH091 (31)[58,59]
C22H40N2O8
460.28 g/mol
30
NN
OMe
OMe
MeO
MeO
O
O
O
O
H2NNH2 N
N
NH
NH
NH2
NH2
HN
HN
H2N
H2N
O
O
O
O
+MeOH
RT
C2H8N2
60.07 g/mol
C26H56N10O4
572.45 g/mol
31
30 was reacted with 1,2-ethylenediamine in methanol for 3 d at room temperature. The
solvent was then removed in vacuum and the final product isolated as a yellow gel.
Yield: about 4 g, 8.6 mmol, 100%.
1H-NMR (200 MHz, CD3OD, δppm): 3.21 (s, 3J = 6.0 Hz, 8H, NH2CH2CH2NH-), 2.77 (s, 3J = 7.0 Hz,
8H, NH2CH2CH2NH-), 2.66 (t, 3J = 7 Hz, 8H, COCH2-), 2.40 (m, 12H, NCH2-), 1.44 (m, 4H, -
NCH2CH2CH2CH2CH2CH2N-), 1.26 (m, 4H, -NCH2CH2CH2CH2CH2CH2N-)
An elemental analysis for 31 was not performed since the other analytical data were in full
accordance with published results.
Materials and methods
139
4’-methyl-2,2’-bipyridine-4-carboxaldehyde (32) )[60]
USC-WH096
32
N
N
OHC
N
N
SeO2
1,4-dioxane
C12H12N2
184.24 g/mol
C12H10N2O
198.22 g/mol
A suspension of 4,4’-dimethyl-2,2’-bipyridine (2 g, 10.9 mmol) and selenium dioxide (1.33 g,
11.9 mmol) was heated to reflux in 1,4-dioxane for 24 h.[60] The solution was filtered hot and
the filtrate cooled to room temperature and filtered again. The residue was partially
dissolved in ethyl acetate and was filtered again. The ethyl acetate solution was first washed
with 10% sodium carbonate (2 × 100 ml), and then with 10% sodium metabisulfite (3 × 100
ml). Solid sodium carbonate was added to the sodium metabisulfite phase until pH 10 and
the solution extracted with dichloromethane (5 × 100 ml), dried over magnesium sulfate,
and the product was isolated as a white solid.
Yield: 700 mg, 3.53 mmol (32.4%);
1H-NMR (200 MHz, CDCl3, δppm): 10.18 (s, 1H, HCHO), 8.89 (d, 3J = 5.4 Hz, 1H, Hbipy), 8.83 (dd, 3J
= 1.6 Hz, 4J = 0.8 Hz, 1H, Hbipy), 8.57 (d, 3J = 5 Hz, 1H, Hbipy), 8.28 (quintet, 4J = 0.8 Hz, 1H,
Hbipy), 7.72 (dd, 3J = 5 Hz, 4J = 1.6 Hz, 1H, Hbipy), 7.19 (qd, 3J = 5 Hz, 4J = 0.8 Hz, 1H, Hbipy), 2.46
(s, 3H, -CH3);
An elemental analysis for 32 was not performed since the other analytical data were in full
accordance with published results.
Materials and methodes
140
USC-WH134 (34)
C9H5MnO4
232.07 g/mol
C26H56N10O4
572.45 g/mol
NN
NH
NH
HN
HN
HN
HN
NH
NH
O
O
O
O
MnOC CO
CO MnCOOC
OC
MnOC CO
COMn
COOCOC
NN
NH
NH
NH2
NH2
HN
HN
H2N
H2N
O
O
O
O
MnOC CO
CO
H
O
+EtOH, RT
NaBH4
1931
C62H76Mn4N10O16
1437.08 g/mol
34
(Formylcyclopentadienyl) manganese tricarbonyl 19 (220 mg, 0.948 mmol) and PAMAM G1
dendrimer (130 mg, 0.227 mmol) were added to anhydrous ethanol (30 ml). Molecular
sieves (3 Å, 3-5 g) were added and the mixture stirred overnight. On the next day, sodium
borhydride (97.98 mg, 2.59 mmol) was added. After 1 h, water (10 ml) was added to the
solution and the molecular sieves were filtered off. The product was extracted with
dichloromethane (3 x 100 ml), organic phase was separated, dried over sodium sulfate, and
the solvent removed under evaporation to obtain the product as a yellow oil.
Yield: 282 mg, 0.20 mmol, 86.5%;
A part of it was purified with preparative HPLC (System B, tR = 15.54 min) and a white solid
was obtained.
1H-NMR (200 MHz, CD3OD, δppm): 5.20 (t, 3J = 2.2 Hz, 8H, Cp), 4.96 (t, 3J = 2.2 Hz, 8H, Cp),
3.93 (s, 8H, NCH2Cp), 3.51 (m, 16H, NH2CH2CH2NH-), 3.21 (m, 12H, COCH2-), 2.78 (m, 8H,
NCH2-), 1.79 (m, 4H, -NCH2CH2CH2CH2CH2CH2N-), 1.46 (m, 4H, -NCH2CH2CH2CH2CH2CH2N-);
Cannot be characterized by mass spectrometry due to poor ionization under ESI conditions;
IR (ATR, cm-1): 2013, 1913, 1643
Materials and methods
141
USC-WH146 (36)
C9H5MnO4
232.07 g/mol
19
C21H24N2O4
368.17 g/mol
OH
O
HN
H2N
OO
MnOC CO
CO
H
O
+
OH
O
HNMnOC CO
CO
NH
OO
NaOAc, EtOH, 3Å MS
C30H30MnN2O7
586.14 g/mol
36
95% Trifluoroacetic acid with 5% water as a scavenger was added to commercially available
Fmoc-L-Lys(Boc)-OH and a rapid release of carbon dioxide was observed. 30 min after the
gas release finished, cold diethyl ether was added to the TFA-amino acid solution and cooled
to –20 °C for more than 20 min. The Boc-unprotected amino acid precipitated as a sticky
white solid. It was washed several times with cold diethyl ether and then dried in vacuum. A
mixture of the Boc-unprotected lysine (193 mg, 0.52 mmol), (Formylcyclopentadienyl)
manganese tricarbonyl 19 (104 mg, 0.448 mmol), molecular sieves (3 Å, 3-5 g) and sodium
acetate (73.54 mg, 0.90 mmol) in anhydrous ethanol (30 ml) were stirred overnight at room
temperature On the next day, sodium borhydride (50.87 mg, 1.35 mmol) was added. After 1
h, water (10 ml) was added to the solution and the molecular sieves were filtered off. The
product was extracted with ethyl acetate (3 x 100 ml), organic phase was separated, dried
over magnesium sulfate, and the solvent removed under evaporation to obtain the product
as a white solid.
Yield: 180 mg, 0.30 mmol, 68.8%;
A part of it was purified with preparative HPLC (System B, tR = 21.79 min) and a white solid
was obtained.
1H-NMR (200 MHz, CD3OD, δppm): 7.80 (d, 3J = 7.5 Hz, 2H, fluorenyl), 7.66 (d, 3J = 7.5 Hz, 2H,
fluorenyl), 7.40 (t, 3J = 7.5 Hz, 2H, fluorenyl), 7.31 (dt, 3J = 7.5 Hz, 4J = 1.0 Hz, 2H, fluorenyl),
5.15 (m, 2H, Cp), 4.95 (m, 2H, Cp), 4.41 (d, 3J = 5 Hz, 1H), 4.32 (d, 3J = 6 Hz, 1H), 4.23 (t, 3J =
7.0 Hz, 1H), 4.17 (s, 1H), 3.85 (s, 2H, NCH2Cp), 3.01 (m, 2H), 1.89 (m, 1H), 1.71 (m, 3H), 1.49
(m, 2H);
IR (ATR, cm-1): 2023, 1927, 1660, 1450, 1196, 1136
ESI-MS (negative): m/z = 585.14 [M-H]1-.
Materials and methodes
142
USC-WH150 (37)
20
C9H5O4Re
363.34 g/mol
C21H24N2O4
368.17 g/mol
OH
O
HN
H2N
OO
ReOC CO
CO
H
O
+
OH
O
HNReOC CO
CO
NH
OO
NaOAc, EtOH, 3Å MS
C30H28N2O7Re
716.78 g/mol
37
95% Trifluoroacetic acid with 5% water as a scavenger was added to commercially available
Fmoc-L-Lys(Boc)-OH and a rapid release of carbon dioxide was observed. 30 min after the
gas release finished, cold diethyl ether was added to the TFA-amino acid solution and cooled
to –20 °C for more than 20 min. The Boc-unprotected amino acid precipitated as a sticky
white solid. It was washed several times with cold diethyl ether and then dried in vacuum. A
mixture of the Boc-unprotected lysine (220 mg, 0.60 mmol), (Formylcyclopentadienyl)
rhenium tricarbonyl 20 (200 mg, 0.55 mmol), molecular sieves (3 Å, 3-5 g) and sodium
acetate (90.31 mg, 1.10 mmol) in anhydrous ethanol (30 ml) were stirred overnight at room
temperature On the next day, sodium borhydride (80 mg, 2.2 mmol) was added. After 1 h,
water (10 ml) was added to the solution and the molecular sieves were filtered off. The
product was extracted with ethyl acetate (3 x 100 ml), organic phase was separated, dried
over magnesium sulfate, and the solvent removed under evaporation to obtain the product
as a white solid.
Yield: 210 mg, 0.29 mmol, 48.8%;
A part of it was purified with preparative HPLC (System B, tR = 21.90 min) and a white solid
was obtained.
IR (ATR, cm-1): 2023, 1913, 1666, 1182, 1140
ESI-MS (negative): m/z = 717.16 [M-H]1-.
An elemental analysis or NMR of 37 could not be performed due to the limited amount of
product obtained, which was completely used for subsequent reactions.
Materials and methods
143
Cyclopentadien-1-yl manganese tricarbonyl carboxylic acid (41)[64]
USC-WH159
MnOC CO
CO
n-Buli, CO2
THF MnOC CO
CO
OH
O
C9H5MnO5
248.07 g/mol
C8H5MnO3
204.06 g/mol
41
Cymantrene (500 mg, 2.45 mmol) was dissolved in anhydrous tetrahydrofuran (30 ml) and
cooled to -50 °C with an aceton/dry ice bath. Then, n-butyllithium (1.6 M solution in hexane,
3.13 ml, 4.9 mmol) was added dropwise and the reaction mixture was stirred at -50 °C for 1
h. Crushed dry ice (20 g) was added and the cold bath was removed after one hour, and the
reaction mixture was allowed to warm up to room temperature. After the addition of a 10%
solution of hydrochloric acid (50 ml) and dichloromethane (100 ml), the phases was
separated, the organic phase washed with water, and dried over magnesium sulfate. After
filtration and the removal of the solvent, a yellow solid was obtained.
Yield: 550 mg, 2.22 mmol, 90.5%;
1H-NMR (200 MHz, CD3OD, δppm): 5.54 (t, 3J = 2.3 Hz, 2H, Cp), 4.98 (t, 3J = 2.3 Hz, 2H, Cp);
IR (ATR, cm-1): 2020, 1926, 1673
An elemental analysis for 41 was not performed since the other analytical data were in full
accordance with published results.
Materials and methodes
144
Cyclopentadien-1-yl rhenium tricarbonyl carboxylic acid (42)[64]
USC-WH164
C9H5O5Re
379.034 g/mol
42
ReOC CO
CO
n-Buli, CO2
THF ReOC CO
CO
OH
O
C8H5O3Re
335.33 g/mol
Cyrhetrene (300 mg, 0.89 mmol) was dissolved in anhydrous tetrahydrofuran (30 ml) and
cooled to -50 °C with an aceton/dry ice bath. Then, n-butyllithium (1.6 M solution in hexane,
1.12 ml, 1.75 mmol) was added dropwise and the reaction mixture was stirred at -50 °C for 1
h. Crushed dry ice (20 g) was added and the cold bath was removed after one hour, and the
reaction mixture was allowed to warm up to room temperature. After the addition of a 10%
solution of hydrochloric acid (50 ml) and dichloromethane (100 ml), the phases was
separated, the organic phase washed with water, and dried over magnesium sulfate. After
filtration and the removal of the solvent, a white solid was obtained.
Yield: 240 mg, 0.63 mmol, 71.2%;
1H-NMR (200 MHz, CD3OD, δppm): 6.13 (t, 3J = 2.3 Hz, 2H, Cp), 5.19 (t, 3J = 2.3 Hz, 2H, Cp);
IR (ATR, cm-1): 2027, 1900, 1680;
An elemental analysis for 42 was not performed since the other analytical data were in full
accordance with published results.
Materials and methods
145
Cyclopentadien-1-yl methyl tricarbonyl tungsten (43)[65]
USC-WH161
43
C9H8O3W
348.00 g/mol
OCOC
WCH3
CO
NaW(CO)6 +CH3I
THF
C5H5Na
88.08 g/mol
C6O6W
351.9 g/mol
A mixture of sodium cyclopentadienide (870 mg, 9.88 mmol), and tungsten hexacarbonyl
(3.48 g, 9.98 mmol) in anhydrous tetrahydrofuran (20 ml) were heated to reflex for 30 h.
Then the reaction mixture was allowed to cool to room temperature and later to 0 °C with
an ice bath. Methyl iodide (0.6 ml, 10 mmol) was added followed by dichloromethane (50
ml). The mixture was washed with 5% sodium hydrogen carbonate solution (100 ml), and the
aqueous phase extracted with dichloromethane (2 × 100 ml). The organic phase was dried
with magnesium sulfate. After removal of the solvent, a yellow solid was obtained.
Yield: 1.764 g, 5.07 mmol, 51.3%;
1H-NMR (200 MHz, CD2Cl2, δppm): 5.42 (s, 5H, Cp), 0.40 (s, 3H, CH3-);
IR (ATR, cm-1): 1997, 1867;
An elemental analysis for 43 was not performed since the other analytical data were in full
accordance with published results.
Materials and methodes
146
Cyclopentadien-1-yl methyl tricarbonyl tungsten carboxylic acid (44)[64]
USC-WH163
43
C9H8O3W
348.00 g/mol
OCOC
WCH3
CO
n-Buli, CO2
THF OCOC
WCH3
CO
O
OH
44
C10H8O5W
392.01 g/mol
43 (610 mg, 1.75 mmol) was dissolved in anhydrous tetrahydrofuran (30 ml) and cooled
to -50 °C with an aceton/dry ice bath. Then, n-butyllithium (1.6 M solution in hexane, 2.19 ml,
3.42 mmol) was added dropwise and the reaction mixture was stirred at -50 °C for 1 h.
Crushed dry ice (20 g) was added and the cold bath was removed after one hour, and the
reaction mixture was allowed to warm up to room temperature. After the addition of a 10%
solution of hydrochloric acid (50 ml) and dichloromethane (100 ml), the phases was
separated, the organic phase washed with water, and dried over magnesium sulfate. After
filtration and the removal of the solvent, a white solid was obtained.
Yield: 224 mg, 0.57 mmol, 32.7%;
1H-NMR (200 MHz, CD3OD, δppm): 5.88 (t, 3J = 2.4 Hz, 2H, Cp), 5.66 (t, 3J = 2.4 Hz, 2H, Cp); 0.44
(s, 3H, CH3-);
IR (ATR, cm-1): 2015, 1928, 1900, 1678;
An elemental analysis for 44 was not performed since the other analytical data were in full
accordance with published results.
Materials and methods
147
USC-WH160 (45)
b) cleavage from wang resin with 50% TFA in DCM
MnOC CO
CO
O
OH
O
HNO
O
NHWang Resin
OO
O
NH
NH2
OO
a) HATU, DIPEA
45
MnOC CO
CO
OH
O
+
C9H5MnO5
248.07 g/mol
41
C30H27MnN2O8
598.48 g/mol
45 was prepared applying the procedure described in section 5.4 on a 0.22 mmol scale on a
Wang resin (200 mg, 1.1 mmol/g).
1H-NMR (200 MHz, CD3OD, δppm): 7.80 (m, 2H, fluorenyl), 7.66 (m, 2H, fluorenyl) 7.35 (m, 4H,
fluorenyl), 5.56 (m, 2H, Cp), 4.93 (m, 2H, Cp), 1.53 (m, 6H), 3.02-2.85 (m, 2H), 4.33-4.12 (m,
4H);
IR (ATR, cm-1): 2023, 1928;
ESI-MS (negative): m/z = 597.11 [M-H]-, 1195.21 [2M-2H+Na]-
USC-WH166 (46)
b) cleavage from wang resin with 50% TFA in DCM
Wang ResinO
O
O
NH
NH2
OO
a) HATU, DIPEA
+
C30H27N2O8Re
729.75 g/mol
ReOC CO
CO
O
OH
O
HNO
O
NH
46C9H5O5Re
379.034 g/mol
42
ReOC CO
CO
OH
O
46 was prepared applying the procedure described in section 5.4 on a 0.22 mmol scale on a
Wang resin (200 mg, 1.1 mmol/g).
IR (ATR, cm-1): 2025, 1913;
ESI-MS (negative): m/z = 729.12 [M-H]-.
Materials and methodes
148
USC-WH167 (47)
OCOC
WCH3
CO
O
OH
O
HN
OO
NH
47
b) cleavage from wang resin with 50% TFA in DCM
Wang ResinO
O
O
NH
NH2
O
O
a) HATU, DIPEA+
OCOC
WCH3
CO
O
OH
44
C10H8O5W
392.01 g/mol
C31H30N2O8Re
742.42 g/mol
47 was prepared applying the procedure described in section 5.4 on a 0.22 mmol scale on a
Wang resin (200 mg, 1.1 mmol/g).
1H-NMR (200 MHz, CD3OD, δppm): 7.80 (m, 2H, fluorenyl), 7.64 (m, 2H, fluorenyl) 7.35 (m, 4H,
fluorenyl), 6.38 (m, 2H, Cp), 6.06 (m, 2H, Cp), 4.34 (m, 2H), 4.17 (m, 2H), 1.81-1.52 (m, 6H);
IR (ATR, cm-1): 2023, 1928;
ESI-MS (negative): m/z = 811.11 [M-CH3-CO+CF3COO-H]-, 839.11 [M-CH3+CF3COO-H]-,
1679.21 [2M-2CH3+2CF3COO-H]-
Materials and methods
149
USC-WH157 (50)
H2N
HN
NH
HN
NH
HN
NH
NH2
O
NH2
O
O
NH2
O
O
NH2
O
NH
NHH2N
O
50
Peptide 50 was prepared on a Rink amide resin using the amino acids Fmoc-Gly-OH, Fmoc-
Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH, and Fmoc-Phe-OH applying the procedure
described in section 5.5.
RP-HPLC: system B, tR = 10.06 min;
ESI-MS (positive): m/z = 932.6 [M+H]+, 466.82 [M+2H]2+;
IR (ATR, cm-1): 1666, 1631, 1529.
USC-WH172 (51)
H2N
HN
NH
HN
NH
HN
NH
HN
O
NH2
O
O
NH
O
O
NH2
O
NH
NHH2N
O
O
MnOC CO
CO
NH2
O
51
Peptide 51 was prepared on a Rink amide resin using the amino acids Fmoc-Gly-OH, Fmoc-
Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Mtt)-OH and Fmoc-Phe-OH
applying the procedure described in section 5.5.
RP-HPLC: system B, tR = 19.90 min;
ESI-MS (positive): m/z = 1162.69 [M+H]+, 581.84 [M+2H]2+;
IR (ATR, cm-1): 2025, 1948, 1938.
Materials and methodes
150
USC-WH171 (52)
H2N
HN
NH
HN
NH
HN
NH
HN
O
NH
O
O
NH2
O
O
NH2
O
NH
NHH2N
O
O
OCOC
WCH3
CO
NH2
O
52
Peptide 52 was prepared on a Rink amide resin using the amino acids Fmoc-Gly-OH, Fmoc-
Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Mtt)-OH and Fmoc-Phe-OH
applying the procedure described in section 5.5.
RP-HPLC: system B, tR = 16.70 min;
ESI-MS (positive): m/z = 631.78 [M-CO-CH3+H]2+, 1404.55 [M-CH3+CF3COO+H]+;
IR (ATR, cm-1): 2056, 1971 and 1971.
USC-WH170 (53)
H2N
HN
NH
HN
NH
HN
NH
HN
O
NH
O
O
NH
O
O
NH2
O
NH
NHH2N
O
O
OCOC
WCH3
CO
O
MnOC CO
CO
NH2
O
53
Peptide 53 was prepared on a Rink amide resin using the amino acids Fmoc-Gly-OH, Fmoc-
Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Mtt)-OH and Fmoc-Phe-OH
applying the procedure described in section 5.5.
RP-HPLC: system B, tR = 22.90 min;
ESI-MS (positive): m/z = 1606.5 [M-CO-CH3+CF3COO+H]+, 1634.50 [M-CH3+CF3COO+H]+,
1747.6 [M-CH3+2CF3COO+H]+
IR (ATR, cm-1): 2056, 2025, 1971, 1948, and 1938
References
151
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154
Appendix
155
Compound 2 4 6
Empirical formula C12H11MnO5 C12H11ReO5 C16H11MnO5
Formula weight 290.15 421.41 338.19
dimensions (mm) 0.10 x 0.10 x 0.05 0.13 x 0.12 x 0.10 0.15 x 0.15 x 0.10
Space group P-1 P-1 P-1
a (Å) 7.484(4) 7.49(6) 8.275(4)
b (Å) 8.845(4) 9.03(5) 9.784(4)
c (Å) 10.481(5) 10.51(6) 10.077(5)
(°) 114.257(9) 65.50(15) 71.667(9)
(°) 95.124(10) 85.09(19) 83.770(9)
(°) 91.489(10) 90.1(2) 69.667(9)
V (Å3) 628.5(5) 644(7) 726.2(6)
2 2 2
calc (g cm-3) 1.533 2.173 1.547
T (K) 223(2) 173(2) 223(2)
(mm-1) 1.059 9.444 0.929
(Å) (Mo K) 0.71073 0.71075 0.71073
2max (°) 24.98 27.49 25.69
Reflections measured 3341 6684 5671
Unique refl. / [I >2(I)] 2109/1639 2900 / 2462 2666/2206
Variables 168 163 203
R (I > 2(I)) 0.0728 0.0470 0.0644
wR [I > 2(I)] 0.1582 0.0696 0.1430
Appendix
156
Zusammenfassung
157
Zusammenfassung
Im ersten Teil dieser Arbeit wurde eine Serie von zwölf Carbonsäure-funktionalisierten
Cyclopentadienylmangan- und Rheniumtricarbonyl-Komplexen des Typs CpM(CO)3 mit
verschiedenen Linkern zwischen der Halbsandwich-Einheit und der Carboxyl-Gruppe
synthetisiert und mit IR- und NMR-Spektroskopie sowie ESI-Massenspektrometrie
charakterisiert. Ausgewählte Verbindungen konnten auch mittels Röntgenstrukturanalyse
weiter untersucht werden. Die Verbindungen wurden dann mittels Festphasensynthese an
das cell penetrating peptide sC18 angeknüpft. Die intrazelluläre Verteilung von
Carboxyfluorescein-markierten Derivaten wurde in humanen MCF-7-Brustkrebszellen mittels
Fluoreszenz-Mikroskopie untersucht und die IC50-Werte der Konjugate mit dem Resazurin-
Assay ermittelt. Sowohl die unkonjugierten Organometall-Verbindungen wie auch das nicht-
modifizierte Peptid zeigen bei Konzentrationen von bis zu 200 µM keinerlei biologische
Aktivität. Dagegen werden die Organometall-Peptid-Konjugate effizient in Zellen
aufgenommen und zeigen eine Dosis-abhängige zytotoxische Wirkung. Der Tausch des
Metallzentrums von Mangan gegen Rhenium hatte jedoch keinen messbaren Effekt auf die
biologischen Eigenschaften der Konjugate. Dagegen führt der Austausch der Keto-Gruppe im
Linker gegen eine Methylen-Funktion zu einer effizienteren Anreicherung in Zellkern, was
mit einer höheren Zytotoxizität einhergeht, wobei sich die IC50-Werte von 60 µM für die
Keto-Gruppe auf 40 µM für den Methylen-Linker reduzieren. Somit können bereits kleine
Änderungen im Linker zwischen den Cyclopentadienyl- und Carbonsäure-Gruppen die
intrazelluläre Verteilung und Zytotoxizität der Peptid-Biokonjugate wesentlich beeinflussen.
Im zweiten Teil wurde eine Serie von organometallischen Halbsandwich-Komplexen
hergestellt, mit einer Aldehyd-Gruppe funktionalisiert, und mit den terminalen
Aminogruppen eines G1-PAMAM-Dendrimers und der G1-, G2- und G3-DAB-Dendrimere
gekuppelt. Sowohl die organometallischen Dendrimer-Konjugate als auch ein rein
organisches Adamantan-Dendrimer-Konjugat als Vergleich wurden mittels präparativer HPLC
gereinigt und mit IR- und NMR-Spektroskopie sowie ESI-Massenspektrometrie
charakterisiert. Die Zytotoxizität der Organometall- und Adamantan-Dendrimer-Konjugate
wurde auf humanen MCF-7-Brustkrebszellen getestet. Vier Konzentrationen zwischen 1 und
25 µM wurden verwendet um eine Korrelation der zytotoxischen Aktivität mit der Struktur
des Dendrimer-Kerns, deren Generation sowie der Variation des Metallzentrums zu
Zusammenfassung
158
untersuchen. Für alle Konjugate führten höhere Konzentrationen zu deutlich höherer
Zytotoxizität. Der Ersatz von Mangan gegen Rhenium in den G1-, G2- und G3-DAB-
Dendrimer-Konjugaten hat dagegen wiederum keinen signifikanten Einfluss auf deren
biologischen Aktivität. Überraschenderweise nahm die Aktivität mit zunehmender
Generation der Dendrimere ab, sowohl für die Mangan- wie auch die Rhenium-
Verbindungen. Das Adamantan-G1-DAB-Konjugat hatte eine ähnliche Aktivität wie die
analogen Organometall-G1-Mangan- und Rhenium-DAB-Konjugate. Somit besteht keine
direkte Korrelation zwischen der Art und der Anzahl der terminalen funktionellen Gruppen.
Die beobachteten geringen Unterschiede zwischen den Organometall-Konjugaten und dem
Adamantan-Analog weisen auf einen Wirkmechanismus hin, der von dem der Peptid-
Konjugate verschieden ist, wo bereits eine kleine Modifikation der konjugierten
Organometall-Einheit zu einer signifikanten Änderung der biologischen Aktivität dieser
Systeme führt.
In dem dritten Teil der vorliegenden Arbeit wurden Organometall-Carbonylkomplexe mit
unterschiedlichen C≡O-Streckschwingungsbanden an ein Modellpeptid angeknüpft, um
deren Verwendung für ein Barcoding von Biomolekülen zu untersuchen. Zunächst wurden
Fmoc-geschützte Aminosäuren als Bausteine für die Festphasenpeptidsynthese hergestellt.
Dafür wurden CpM(CO)3-Bausteine an die ε-Aminogruppe des L-Lysins über eine Schiff-Base
Reaktion angeknüpft. Dabei erwies sich die Kopplung von Cymantren- oder
Cyrhetrencarboxylaldehyd mit N-terminal Fmoc-geschütztem L-Lysin und Natriumacetat als
Base als erfolgreich. Leider wiesen diese Bausteine jedoch eine ungenügende Stabilität unter
den Bedingungen der Festphasenpeptidsynthese auf. Daher wurden Organometall-
Aminosäuren-Konjugate über eine Amid-Bindung anstelle der Amin-Verknüpfung hergestellt,
um deren Stabilität zu verbessern. Diese Konjugate wurden mittels Festphasensynthese an
einem Wang-Harz hergestellt. Allerdings bildete das CpW(CO)3CH3-modifizierte L-Lysin unter
Verlust der Metall-gebundenen Methyl-Gruppe Addukte mit Trifluoressigsäure. Dies führte
wiederum zu erheblichen Problemen während der Peptidsynthese. Daher wurde schließlich
auf die Einführung der Organometall-Gruppen in das Peptid über eine orthogonale
Schutzgruppe-Strategie an einem Rink-Amid-Harz zurückgegriffen, wobei jeweils eine Mtt-
Schutzgruppe selektiv von der ε-Aminogruppe eines N-terminalen Fmoc-L-Lysins
abgespalten wurde. Danach wurden die Organometall-Komplexe an der Festphase an die
Lysin-Seitenkette gekuppelt bevor der N-Terminus entschützt und die Kette weiter
Zusammenfassung
159
verlängert wurde. Vier Peptide wurden so synthetisiert, die entweder keine, jeweils nur eine
CpMn(CO)3- oder CpW(CO)3CH3 Gruppe, oder aber beiden Organometall-Einheiten
enthielten. Diese Peptidkonjugate zeigen die erwarteten IR-Spektren, wobei im metallfreien
Peptid nur die Banden der Amid-Bindung und der aromatischen Seitenketten bei niedrigeren
Wellenzahlen beobachtet werden konnten. Im Vergleich dazu zeigten alle drei
Organometall-Peptide jeweils die charakteristischen symmetrischen und asymmetrischen
C≡O-Streckschwingungen der CpM(CO)3-Gruppen im Bereich von 1940 bis 2050 cm-1. Die
breiten asymmetrischen Banden der verschiedenen Organometallgruppen überlagern sich
und konnten nicht aufgelöst werden. Dagegen beobachtet man die symmetrischen C≡O-
Schwingungen der an das Peptid konjugierten CpMn(CO)3-Gruppe wohl separiert bei 2025
cm-1 und das der CpW(CO)3CH3-Gruppe bei 2056 cm-1. Im bis-funktionalisierten Peptid, das
sowohl den Mangan- wie den Wolframtricarbonyl-Baustein enthält, werden zwei Banden bei
2025 und 2056 cm-1 mit nahezu Basislinie-Trennung beobachtet, was die generelle
Anwendbarkeit dieser Markierungsstrategie belegt.
In der vorliegenden Arbeit konnten also Struktur-Wirkungs-Beziehungen für Organometall-
Peptid- und Dendrimer-Konjugate mit unterschiedlichen funktionellen Gruppen an einer
humanen Brustkrebs-Zelllinie untersucht werden. Die Variation der Organometall-Gruppen
und des Trägermoleküls führen zu signifikanten Unterschieden in ihrer biologischen Aktivität.
Zusätzlich wurden Modell-Peptide mit verschiedenen Metallcarbonyl-basierten IR-Markern
versehen um diese in einer Barcoding-Strategie zu labeln. In Zukunft soll das Spektrum der
verfügbaren, nicht-überlappenden Schwingungsmarker noch deutlich erweitert werden.