opus. · PDF fileDanksagung An dieser Stelle möchte ich mich bei allen Personen bedanken, die mich bei der Anfertigung dieser Arbeit unterstützt haben: Mein größter

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


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


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


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


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


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


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


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


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


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)


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


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.


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



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)


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


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



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


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


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.


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


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


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+.


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.


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


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]


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.


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


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.


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


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


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


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


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.


42


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



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.



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.


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


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


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


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


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


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.



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


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


51

N

N

OHC

N

N

SeO2

1,4-dioxane


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.



52

N

N

OHC

N

N

MoCO

CO

CO

CO

OHC

THF

Mo(CO)6


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


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


tR

= 15.54 min

Figure 3.2.17: Analytical HPLC trace of 34 with a 20-95% acetonitrile/water gradient as the eluent


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


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

[%]


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.


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


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


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


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


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


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


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


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


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


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


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.


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


OH

O

HNMnOC CO

CO

NH

OO

Figure 3.3.6: Analytical HPLC of 36 after preparative HPLC purification with acetonitrile/H2O 20-90%


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


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-


66

aromatic protons of the lysine and the Fmoc group could be identified. Thus, all 1H NMR

signals expected for 36 could be assigned.


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


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



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


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


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


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


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.


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


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).



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


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


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


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


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


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



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


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


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



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.


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.


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


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


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


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.


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


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


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)


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.


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


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




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




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


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.


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


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


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


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


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


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).


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


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


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.


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).


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.


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.


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.


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.


112

Cyclopentadien-1-yl-(3-carboxylatopropyl) rhenium tricarbonyl (4)[52]

USC-WH067

ReOC CO

CO

OH

ORe

OC COCO

OH

O

O


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.


113

Cyclopentadien-1-yl-(2-carboxylatophenoxyl) manganese tricarbonyl (5)

USC-WH030

+AlCl3 / CH2Cl2


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.


114

Cyclopentadien-1-yl-(2-carboxylatophenyl) manganese tricarbonyl (6)

USC-WH065


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





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.


115

Cyclopentadien-1-yl-(2-carboxylatophenoxyl) rhenium tricarbonyl (7)

USC-WH033

C8H4O3

148.1 g/mol

ReOC CO

CO

+AlCl3 / CH2Cl2/reflux


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;




116

Cyclopentadien-1-yl-(2-carboxylatophenyl) rhenium tricarbonyl (8)

USC-WH070

ReOC CO

CO

O OH


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)









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




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


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);


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.


119

Cyclopentadien-1-yl-(4-carboxylatophenyl) manganese tricarbonyl (10)

USC-WH068

MnOC CO

CO

O

OH

O

9

C16H9MnO6

352.18 g/mol


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)









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




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.




121

Cyclopentadien-1-yl-(4-carboxylatophenyl) rhenium tricarbonyl (12)

USC-WH071

ReOC CO

CO

O

OH

O

11


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)







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,




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






MALDI-MS (positive): 2485.4 [M+H]+

IR (ATR, cm-1): 2020, 1922, 1653, 1537.


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






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


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.




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





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





MALDI-MS (positive): m/z = 2829.4 [M+H]+


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;




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



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;




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.


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;




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);




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


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




the solvent removed under evaporation to obtain the product as a yellow oil.

Yield: 209 mg, 74.8%.


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


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






Yield: 165.9 mg, 88%


was obtained.


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


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






the solvent removed under evaporation to obtain the product as a colorless oil.

Yield: (148.2 mg, 96.5%)


was obtained.


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


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







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,


1.66 (m, 4H, NCH2CH2CH2CH2Ncore)


IR (ATR, cm-1): 2024, 1905


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







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,


1.40 (m, 4H, NCH2CH2CH2CH2Ncore)


IR (ATR, cm-1): not done


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%)


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))


IR (ATR, cm-1): not done.


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-)




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-)




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);




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






Yield: 282 mg, 0.20 mmol, 86.5%;


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-);


IR (ATR, cm-1): 2013, 1913, 1643


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%;


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-.


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%;


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.


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



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


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




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



h. Crushed dry ice (20 g) was added and the cold bath was removed after one hour, and the




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;




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;




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




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;




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)


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



IR (ATR, cm-1): 2025, 1913;

ESI-MS (negative): m/z = 729.12 [M-H]-.


148

USC-WH167 (47)

OCOC

WCH3

CO

O

OH

O

HN

OO

NH

47


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



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]-


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


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;


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


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.



IR (ATR, cm-1): 2025, 1948, 1938.


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





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





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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References

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.

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