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Crown Ether-Metalloporphyrins as Ditopic Receptors
and
Pyropheophorbide-a Conjugates for the Photodynamic Therapy of Tumors
Den Naturwissenschaftlichen Fakultäten
der Friedrich-Alexander-Universität Erlangen-Nürnberg
zur
Erlangung des Doktorgrades
2005
vorgelegt von
Matthias Helmreich
aus Bamberg
Als Dissertation genehmigt von den Naturwissenschaftlichen Fakultäten der
Universität Erlangen-Nürnberg
Tag der mündlichen Prüfung: 14.10.2005
Vorsitzender der Promotionskommission: Prof. Dr. D.-P. Häder
Erstberichterstatter: Prof. Dr. A. Hirsch
Zweitberichterstatter: Prof. Dr. J. Gladysz
Drittberichterstatterin: Prof. Dr. B. Röder
Mein besonderer Dank gilt meinem Doktorvater Prof. Dr. Andreas Hirsch für die
gewährte Unterstützung, sowie das rege Interesse am Fortgang der Arbeit.
Außerdem möchte ich mich herzlichst bei meinem Co-Doktorvater und Betreuer Dr.
Norbert Jux für die Bereitstellung des interessanten Themengebietes, die
Bereitschaft zu fachlichen Diskussionen, sowie die umfassende Betreuung während
der gesamten Promotionszeit bedanken.
Die vorliegende Arbeit entstand in der Zeit vom April 2002 bis Juni 2005 am Institut
für Organische Chemie der Friedrich-Alexander-Universität Erlangen-Nürnberg
Table of Contents
1 Introduction............................................................................1
1.1 Porphyrin Systems and their Applications............................................... 1
1.2 Ditopic Receptors: Crown Ether-Porphyrins............................................ 3
1.3 Photodynamic Therapy ........................................................................... 8
1.3.1 The History of Photodynamic Therapy .................................................... 8
1.3.2 Mechanisms of the Photodynamic Therapy .......................................... 11
1.3.3 Photosensitizers in Photodynamic Therapy .......................................... 14
1.3.4 Photodynamic Therapy as a Therapy for other Diseases than
Cancer .................................................................................................. 17
1.4 Modular Carrier Systems ...................................................................... 20
1.5 Finding a Good Name for Porphyrin- and Chlorophyll-Compounds ...... 22
1.6 C60-Fullerene as a building block .......................................................... 22
2 Aims..................................................................................... 25
3 Results and Discussion ..................................................... 27
3.1 Molecular Recognition – Crown Ether-Porphyrins and their
Coordination Properties ........................................................................ 27
3.1.1 Synthesis of the Parent Crown Ether-Porphyrin 26 and its Metal
Complexes ............................................................................................ 27
3.1.2 Kinetic Experiments – The Stabilizing Effect of the Crown.................... 30
3.1.3 Ditopic Receptors.................................................................................. 33
3.1.4 Synthesis of a Water Soluble System ................................................... 50
3.1.5 A Concept for the Synthesis of Oligomeric Porphyrin Crown Ether
Arrays – Construction of a Library of Building Blocks............................ 52
3.1.6 Rare Earth Metal Porphyrins ................................................................. 56
3.2 The Photodynamic Therapy of Tumors – Construction of Multi-
Pyropheophorbide-a-Fullerene Assemblies .......................................... 59
3.2.1 Isolation of Pyropheophorbide-a 19 ...................................................... 59
3.2.2 Synthesis of Fullerene-Pyropheophorbide-a Conjugates Carrying
two Chromophoric Units........................................................................ 61
3.2.3 Increasing the Number of Chromophores – Introduction of a
Dendritic Unit......................................................................................... 66
3.2.4 Hexa-Substituted C60-Systems as Multiplying Units.............................. 72
3.2.5 Synthesis of a Decapyropheophorbide-a-Antibody-Conjugate.............. 81
3.2.6 Increasing the Solubility in Polar Solvents - Pyropheophorbide-a
Derivatives with Polar Side Chains ....................................................... 88
3.2.7 Photophysical Investigations................................................................. 91
3.2.8 Biological Investigations: In Vitro Experiments with Photosensitizer-
Carrier-Systems; Uptake and Phototoxic Activity on Human
Lymphoid Cells...................................................................................... 97
4 Summary / Zusammenfassung........................................ 104
5 Experimental Part.............................................................. 115
5.1 Chemicals and Instrumentation........................................................... 115
5.2 Synthetic Procedures .......................................................................... 117
6 Crystal Structures..............................................................175
7 Publications........................................................................183
8 References..........................................................................185
Index of Abbreviations
Ac acetyl AFM atomic force microscopy ALA 5-aminolevulinic acid AMD age-related macular degeneration BOC t-butyloxycarbonyl Chl a chlorophyll-a Chl b chlorophyll-b DAPI 4´,6-diamidino-2-phenylindol dihydrochloride DBU 1,8-diazabicyclo[5.4.0]undec-7-ene DCC N,N‘-dicyclohexylcarbodiimide DMA 9,10-dimethylanthracene DMAP 4-dimethylaminopyridine DMF N,N‘-dimethylformamide DMSO dimethylsulphoxide ε extinction coefficient EI-MS electron-impact mass spectrometry eq equivalent ESI electron spray ionisation ET electron-transfer FAB-MS fast atom bombardment mass spectrometry FC flash column chromatography FDA U.S. Food and Drug Administration fs femto seconds GPC gel permeation chromatography HOBT 1-hydroxybenzotriazol Hp hematoporphyrin HpD hematoporphyrin derivative HPLC high performance liquid chromatography IR infra-red ISC Intersystem crossing LAH lithium aluminum hydride LDL low density lipoproteins MAb monoclonal antibody MALDI-TOF matrix assisted laser desorption ionization – time of flight
NMR nuclear magnetic resonance PBS phosphate buffered saline PDT photodynamic therapy Phe a Pheophytin-a PIT photoimmunotherapy ppm parts per million ps pico seconds Pyropheid-a pyropheophytin-a RT room temperature S0 electronic ground state S1 first excited singlet-state SEC size exclusion chromatography T1 first excited triplet-state TB trypan blue TBDMS t-butyl dimethyl silyl tBu t-butyl TCB 1,2,4-trichlorobenzene TFA trifluoroacetic acid THF tetrahydrofuran TLC thin layer chromatography UV/Vis ultraviolet/visible 1O2 first excited singlet-state of dioxygen 3O2 triplet ground-state of dioxygen Φ fl quantum-yield of fluorescence Φ t triplet-state quantum yield Φ Δ singlet-oxygen quantum yield τt triplet-state lifetime
Introduction
1
1 Introduction
1.1 Porphyrin Systems and their Applications
Why does the area of porphyrin chemistry attract so many scientists?
The answer will probably depend on the person you ask. Certainly a large number of
people interviewed will respond that porphyrin systems play a fundamental role in
many biological processes, e. g. photosynthesis (chlorophylls), oxygen transport
(hemoglobine) and oxygen storage (myoglobine), electron-transfer processes
(cytochromes), respiration, and so on.
There is no exact date for the beginning of the history of modern porphyrin research.
It was at the end of the 19th century when several groups started their investigations
on tetrapyrrols, mainly focused on naturally occurring pigments. In 1906, Richard
WILLSTÄTTER published his first work about chlorophyll[1] and was awarded the Nobel
Prize in chemistry in 1915 for his research on plant pigments and especially for his
work on chlorophyll.
The macrocyclic structure of porphyrins was first proposed by KÜSTER in 1912.[2] At
that time, nobody believed him, least of all Hans FISCHER, the father of modern
porphyrin chemistry. Hans FISCHER´S studies on blood and plant pigments, and his
synthesis of hemin[3, 4] were the next milestones in this area which were also awarded
the Nobel Prize in 1929.
After several decades of reduced interest, the next breakthrough was the
determination of the three-dimensional
structure of a bacterial photosynthetic
reaction center by Johann DEISENHOFER,
Robert HUBER, and Hartmut MICHEL (see
Figure 1-1).[5, 6] This was honored with
the Nobel Prize in 1988, and thanks to
their remarkable work, we now have a
more detailed understanding of
photosynthesis, although much still
remains unsolved. Photochemistry,
photophysics, and photobiology joined
the studies of photosynthesis and the Figure 1-1: Photosynthetic reaction center.
Introduction
2
chlorophylls. It has also encouraged researchers to create model systems, which
mimic the structure and photoactivity of natural systems.
A completely different field is the geochemistry of porphyrins in the soil. Traces of
tetrapyrroles in the geosphere, while challenging the sensitivity of current
instrumentation, offer a fascinating way to investigate the fate of biological material
through geological time periods. Probably the most important feature in this area is
the possibility of using this method for geochemical oil prospecting.
Nowadays, it is almost impossible to get a real overview about the enormously wide
field of porphyrin research. Several books have been published to give an overview
about the actual state of research. The latest and also most extensive was given in
the Porphyrin Handbook, which now contains 20 volumes.[7] Other very important and
helpful tools are online databases like SCIFINDER. Nevertheless, performing an online
search by entering the concept porphyrin yields more than 40000 hits. This
enormous number gives a good impression of how intensive and attractive the
research in the field of porphyrin chemistry and related areas is.
The diversity of directions in which the chemistry and science of tetrapyrroles can
lead is quite remarkable. Basic synthesis continues to be an important subject,
combined with new porphyrin-like structures (e.g. porphycenes and texaphyrins[8])
appearing on the scene. Also, the biosynthesis of porphyrins continues to be a major
research area. Associated with this interest is the study of the inborn errors of
porphyrin biosynthesis to be found amongst the porphyrias.[9, 10]
A large new area has emerged in the field of medicinal chemistry. Clinical interest
has developed in photodynamic therapy of cancer and other diseases. In this area
contributions come from across the entire range of disciplines. Porphyrins, chlorins,
and phthalocyanines have proved to be effective photosensitizers with excellent
properties.[11] Additionally, there is an increasing interest in photobactericides and
photoviricides based on tetrapyrroles. The phototherapy of jaundice of the newborn
provides another example of tetrapyrrole photomedicine, this time with the linear
tetrapyrrole bilirubin.[12]
A major direction is emerging in the development and use of porphyrins and
phthalocyanines as electroactive materials. Modern porphyrin chemistry tries to find
solutions for new sources of energy and faster computers. Japanese laboratories are
particularly active here (Solar energy, Molecular wires).[13-15]
Introduction
3
The above-mentioned examples clearly illustrate that porphyrins attract not only
chemists all around the world but also scientists from many other disciplines,
including biochemistry, medicine, geology, chemical engineering, paleobiology,
alternative energy and microelectronics.
Obviously, porphyrins are involved in specialized, highly developed, biological
processes - will we see more and more industrial and commercial applications in the
future? Porphyrins as catalysts, for example? Efficient solar power production? Or
water purification? The world community desperately needs a replacement for the
internal combustion engine and a clean energy source. If scientists continue to learn
more about natural systems and develop new materials based on nature, the
inherent properties of porphyrins and related compounds may play a major role in
satisfying the demands of mankind.
1.2 Ditopic Receptors: Crown Ether-Porphyrins
Many vitally important biochemical processes rest upon the specific interaction
between proteins and anionic substrates such as carbonates, sulphates, or
phosphates. To achieve the high substrate affinity and selectivity necessary in these
interactions, nature has devised a number of very efficient binding motifs.
In the sulphate binding protein of salmonella typhimurium for example, the affinity
and selectivity is mainly controlled by a defined array of hydrogen bonds between the
anion and NH-groups of the protein backbone.[16] To achieve a similar specificity
using a synthetic receptor is a challenging goal. Reaching it, however, would open up
a large number of interesting applications requiring the selective binding of a
substrate in solution in such fields as medicinal diagnostics, in the analysis of
biological systems, or in environmental monitoring.[17] A receptor that participates
actively in a biological process and predictably changes its outcome might even have
important pharmaceutical use. The design of potent new synthetic receptors is
therefore not only an intellectual challenge, but also provides the possibility of useful
practical applications.[17]
The above-mentioned motifs for research on host-guest systems for ionic species
have played an important role for the development of the field of Supramolecular
Chemistry, the chemistry of non covalent interactions.
A starting point was made by PEDERSEN with his studies on the complexation of alkali
Introduction
4
Figure 1-2: Ditopic receptor.
metal ions by crown ethers in the late 1960s. His work initiated the development of
many other neutral host species for metal ions.[18, 19] Although the first anion receptor
was reported in 1968,[20] the field did not start to develop before 1976, when GRAF
and LEHN reported about the encapsulation of halides by cryptates.[21] Since then,
several other positively charged anion receptors have been synthesized, bearing
protonated nitrogens or metal ions. Most of these host molecules bind their anions by
means of strong electrostatic, coordinative, or hydrogen bonds.[22, 23] In addition to
that, the combination and preorganization of different anion binding groups, like
amides, urea moieties, or Lewis acidic metal centers often leads to receptor
molecules that strongly bind inorganic anions with high selectivity.
The discovery of neutral anion receptors opened the way for neutral ditopic (from
Greek: topos, area) receptors that complex both anions and cations simultaneously.
This ion-pair recognition is an emerging and topical field of coordination chemistry.
Anion binding sites, based on hydrogen bonds or coordination to Lewis acids, have
been combined with cation binding motifs, e. g. crown ethers or calix[4]arene
derivatives.[24-26] The search for new neutral ditopic receptors capable of the
coordination of the ion pair of a target salt is still a subject of great current interest in
the general field of molecular recognition. A second strategy to bind cations and
anions is the use of binary mixtures of cation receptors and anion receptors (dual
receptor strategy).[25]
As already mentioned above, the combination and preorganization of at least two
binding sites is a very important factor which extremely influences the ability of a
neutral receptor to bind guest molecules. At the beginning of
the 1990s, reports on host molecules with the ability for
ditopic binding have been quite rare.[22, 27, 28] The
contributions from SCHMIDTCHEN on ditopic binding of
carboxylates and of REETZ[23, 29] on potassium salts
represent milestones in this field. An illustrating example of a
ditopic receptor is shown in Figure 1-2. System 1 has the
ability to extract solid alkali metal halides into organic
solutions as associated ion pairs. Furthermore, 1 possesses
the capacity to transport alkali metal halides through a liquid
organic membrane.[25, 30]
O
HN
O
NH
OON
ON
O
A
M
1
Introduction
5
Figure 1-3: Porphyrinic ditopic receptor.
Figure 1-4: Ditopic receptors for the recognition of organic molecules.
Another example for the ditopic recognition of salts, also closely related to this work,
was published by KIM et. al. (see Figure 1-3).[31] They synthesized a ditopic receptor 2
which is able to extract sodium cyanide from the solid phase into the organic phase
and bind it strongly. System 2 consists of a
zinc porphyrin as the Lewis acidic binding site
for the anions and an attached crown ether for
the binding of the cations (Lewis base). The
deeply colored porphyrin center reacts to the
coordination of a salt (in this case NaCN) with
a change of color offering the possibility to
monitor the reaction by UV/Vis spectroscopy.
System 2 has the potential to act as a
selective sensor for the recognition of the
highly toxic cyanide ion. Other sodium salts were assumed to bind only in a
monotopic fashion without a change of color.
Other ditopic receptors (see Figure 1-4) are able to bind organic molecules like
pyridines (3), pyrazoles (4 and 5) or even fullerenes.[26, 32-34]
The successful implementation of the molecular complexation properties of anion
receptors into macroscopic applications in membrane separation processes and in
sensors for selective anion detection, reveals the potential behind such systems.
Important industrial applications include the extraction of salts from aqueous and
solid sources.[35] Of particular interest here is the selective extraction of lithium salts,
with potential applications in high technology and medicine.[36]
2
5 4 3
Introduction
6
Oligomeric and Supramolecular Systems
The synthesis and investigations in the field of oligomeric and supramolecular
porphyrin systems which provide a defined structure open up a fast growing field.
The importance of multiporphyrin arrays
firstly comes from nature and its many
assemblies based on sets of several
porphyrins or related molecules arranged
in a well-controlled geometry. These
naturally occurring “devices” often display
a precisely defined electronic structure or
certain catalytic properties. The functions
of such multiporphyrinic structures are
versatile and range from molecular oxygen
transport to electron-transfer and
photosynthesis. In certain cases these
assemblies are set up by covalently bound
monomers, whereas in other cases the assembly is held together only via non-
covalent bonds. Examples are the photosynthetic reaction centers[37] 6 (see Figure
1-5), hemoglobine, and special cytochromes. In
modern Supramolecular chemistry, particular
effort is directed towards studying simple model
systems to mimic important natural processes
like photosynthesis[38] (see Figure 1-6) or other
electron-transfer reactions.[38]
Two major directions are establishing in this
growing field: the first one is the construction of
covalently linked oligomeric systems which have
the advantage of being well characterized and
isolable.[26] Such compounds are used as
receptors (see Figure 1-6), light harvesting
systems, molecular wires and models to study
electron-transfer reactions.
Figure 1-6: Trisporphyrin receptor with guest molecule.
Figure 1-5: Light harvesting system 2. (LH2).
7
6
Introduction
7
OO
OO O
O
OO
OO
OO
OOOO
NN
O
O
NHO
HN
OO
O
O
OOO
H3C
HH
OO
OOO
O
OO
OO
OO
O OOO
NN
O
O
HNO
HN
OO
O
O
OO O
CH3
HH
NH N
HNN
N N
N
N
NN
N
N
N N
NNZn
O
OO
O
OO
OO
OO
OO
O OOO
N
Fe
solar light
holes
electrons
ITO
Scheme 2
The second evolving field is the assembly of oligomeric and supramolecular
structures using the self-organization properties of certain intelligently constructed
monomeric compounds (see Figure 1-7).
The types of interactions include
hydrophobic interaction, hydrogen
bonding and coordinative bonds (metal-
ligand interactions).[32]
These structures include examples for
the utilization of multiporphyrin and
fullerene architectures - yielding artificial
light-harvesting antenna 9 (Figure 1-8)
and reaction center mimics - to tune the
electronic coupling element between
electron donor and electron acceptor and
to affect the total reorganization
energy.[39] Most importantly, with such model systems it is possible to determine the
effects that these
parameters have
on the rate, yield,
and lifetime of the
energetic charge-
separation states.
The supra-
molecular
organization has
also led to nanomaterials for molecular wires (11 and 10, see Figure 1-9),[40]
nonlinear optics materials and other molecular electronics.
Nevertheless, isolation and purification, especially of dynamic oligomeric and
supramolecular systems, remain tough, and the accurate determination of their
molecular weights and structures is successful only in limited cases. The
development of new technologies relevant for supramolecular systems, such as ESI,
MALDI-TOF and AFM, is definitely necessary for further progress in the
characterization of such dynamic systems.[32]
Figure 1-8: Model for light-harvesting antenna.
Figure 1-7: Cyclic porphyrin dodecamer as model for B850 in LH2.
9
8
Introduction
8
Figure 1-9: Linear Porphyrin arrays – Molecular wires.
1.3 Photodynamic Therapy
1.3.1 The History of Photodynamic Therapy
The concept of using light for the treatment of certain diseases is not a new one.
There are reports, that 3000 years ago sunlight, in combination with natural
photosensitizers, was used to treat certain skin diseases. In China, for example,
patients with skin tumors were treated with the excrements of the silkworm and
sunlight whereas the ancient Egyptians used the combination of sunlight and orally
ingested plants to treat vitilago.[41]
Modern photodynamic therapy originated at the end of the nineteenth century when
the medicine student Oscar RAAB discovered that illumination of microbial cultures in
the presence of acridine and related compounds resulted in cell death.[42, 43]
The term photodynamic therapy (PDT) was first introduced in 1904 by TAPPEINER and
JESIONEK.[44] They defined it as a light-induced reaction in biological systems and,
based on the results of Raab, they started their investigations directly with
humans.[45]
In 1912 Friedrich MEYER-BETZ was the first one to show that hematoporphyrin (Hp)
causes photosensitivity in humans by injecting himself with 200 mg of Hp.[46] He
observed severe symptoms of photosensitivity on areas exposed to light (see Figure
1-10).
POLICARD then discovered in 1924 the tendency of porphyrins to accumulate in
tumors when he observed the fluorescence of natural porphyrins in tumors.[47]
In the following decades, several more reports on the use of photosensitizing agents
n = 1, 2, 3, 4, 6, 10, 14, 30, 62, 126 n = 1, 2, 3, 4, 6, 1010 11
Introduction
9
to detect and to treat tumors appeared. However, the first experiments which resulted
in a drug for the treatment of tumors were initiated as late as the 1960s by Richard
LIPSON and Samuel SCHWARTZ.[48]
SCHWARTZ experimented with Hp and isolated a tumor localizing impurity that was
later named hematoporphyrin derivative (HpD). LIPSON started to work with
SCHWARTZ´S HpD first as a tumor detection agent and noticed during his experiments
that it can also be used as a photosensitizer to destroy tumor tissue.[49] SCHWARTZ´S
HpD is a complex mixture of many compounds and after several more years of
isolating and identifying the active fractions of HpD, DIAMOND et al. published the first
results from animal experiments in 1972.[50] The results of the first extensive clinical
trial of the PDT with HpD were given in 1978.[51] However, it took nine more years
until a commercial form of HpD, Photofrin®, became accessible for phase III clinical
trials.[52, 53] Photofrin® was first approved in 1993 in Canada and in the following years
by several other countries including the USA (1995) for the treatment of several types
of cancer like bladder cancers, brain cancers, esophageal cancer, thoracic
malignancies, oral, head and neck cancers.[54, 55]
Unfortunately, these first generation photosensitizers exhibit some unwanted features
like a prolonged and generalized photosensitivity of the skin as their primary side
effect. This is the reason why research activity in the PDT field has expanded
enormously over the last decade. The search for better photosensitizers has created
such a great number of potential photosensitizers for PDT that it is nowadays difficult
to get an overview.[11, 56, 57]
Figure 1-10: Friedrich Meyer-Betz after injection of 200 mg Hp; a) 4 days after injection and with illumination; b) before injection.
Introduction
10
Many of these so-called second or third generation photosensitizers are already in
phase I, II or III clinical trials or, like 5-aminolevulinic acid (ALA) and benzoporphyrin
derivative (BPD-Ma), approved for the PDT.[57]
Table 1-1 gives a chronological overview of the major experimental results leading to
the development of PDT.
1899 RAAB First report on using light and eosin
1904 TAPPEINER Introduction of the term Photodynamic
1907 HAUSMANN Chlorophylls and light cause erythrocyte hemolysis
1909 HASSELBACH O2 is required for erythrocyte photohemolysis
1911 HAUSMANN Extensive experimentation with photosensitization of
mice using Hp
1912 FISCHER and MEYER-
BETZ
First structure-activity study of porphyrins using mice
1913 MEYER-BETZ Hp causes photosensitizing in man
1916 FISCHER Structure-activity study of porphyrins using mice
1924 POLICARD Fluorescing natural porphyrins are observed in
tumors
1942 AULER and BANZER Hp accumulates in animal tumors causing
photonecrosis
1948 FIGGE et al. Hp and its Zn-complex accumulate in mouse tumors
1960 LIPSON and BALDES HpD is first synthesized
1961 LIPSON et al. HpD accumulates in tumors and fluoresces
1972 DIAMOND et al. First clear description of HpD PDT treatment in the
rat
1975 DOUGHERTY et al. Successful treatment of tumors in mice and rats using
HpD PDT
1975 KELLY et al. Treatment of human tumors transplanted in mice
using HpD PDT
1976 KELLY and SNELL First clear description of HpD PDT in clinical use
1978 DOUGHERTY et al. Extensive clinical trial of HpD PDT
1993 Canada Photofrin approved against bladder cancer
Table 1-1: Experimental results leading to the development of PDT.[48]
Introduction
11
1.3.2 Mechanisms of the Photodynamic Therapy
Photodynamic therapy is a promising new treatment for several diseases, most
notably cancer.[11, 57, 58] PDT is based on the photodynamic effect, when special
drugs (photosensitizers) become cytotoxic after illumination due to the generation of
singlet oxygen. Basically, it is the combination of the following three factors:
HO O
N
NH N
HN
O
O O
O O
O O
O O
O O
O O
O OO O
laserlaser lightlightoxygenoxygenphotosensitizerphotosensitizer
Figure 1-11: Three components of the PDT.
A typical PDT-session consists of three steps.
Step 1
A solution of a photosensitizer (drug) with negligible dark toxicity is injected and
accumulates in the targeted tissue, preferentially in rapidly dividing cells during
6-96 h, depending on the photosensitizer used. For skin tumors a local application is
also possible.[59]
Step 2
After the accumulation period, when the drug reaches an appropriate ratio in
diseased versus healthy tissue, the activation of the photosensitizer in the targeted
tissue occurs by illumination with light of a suitable wavelength. In the presence of
oxygen, this results in the formation of reactive oxygen species like singlet oxygen.
These reactive species damage vital structures and functions of cells as well as of
the tumor itself which in the end results in tissue destruction.[60, 61]
Step 3
Following the illumination of the tissue, a massive cell death occurs by apoptosis or
necrosis. In addition to direct cell damage the degradation of the vascular system
plays an important role in the destruction of the tumor.[55, 62, 63] In the ideal case this
causes a total dissolving of the tumor over a period of 4-6 weeks.[58]
Introduction
12
1.3.2.1 Action of Light
To explain the formation of singlet oxygen in cells (or anywhere else) it is very helpful
to look at a simplified JABLONSKI diagram (see Figure 1-12).[11, 64, 65]
3P*
1O2
3O2
1P*
0P
ISC
E
ICAb Fl
Ph
Type I -Photoprocess
Type II -Photoprocess
Figure 1-12: Modified Jablonski diagram. Photophysical processes: Ab (absorbtion); Fl (fluorescence); IC (internal conversion); ISC (intersystem crossing); Ph (phosphorescence).
Irradiating a photosensitizer in the ground state (0P) with light of a suitable
wavelength causes its excitation to the first excited singlet state (1P*). The singlet
excited photosensitizer can relax back to the ground state (0P) by emitting the
absorbed energy in the form of fluorescence (Fl) - enabling the identification of tumor
tissue - or by internal conversion (IC). If the singlet state lifetime of the
photosensitizer is long enough (and this is true for many porphyrins), it is possible for
the 1P* photosensitizer to convert to the first excited triplet state (3P*) by intersystem
crossing (ISC). In the first-order approximation this transition is spin-forbidden, but
nevertheless a good photosensitizer has a high triplet-state yield. From triplet exited
states the photosensitizer can relax back to the ground state by emitting a
phosphorescent photon (Ph) or transferring energy to another molecule via a
radiationless transition. Often the lifetime of the 3P*-state is long enough for taking
part in chemical reactions and therefore the photodynamic action is mostly mediated
by the 3P*-state.
There are two types of photodynamic reactions: The so called Type-I photoreactions
are electron or hydrogen-transfer reactions between the 3P*-photosensitizer and
other organic substrates. These processes create reactive intermediates like
superoxide, hydroperoxyl, and hydroperoxyl-radicals as well as hydrogen peroxide
(Figure 1-13).[57]
Introduction
13
In oxygenated environments the Type-II photoprocess which is an electron spin
exchange between the 3P*-photosensitizer and 3O2, prevails. It produces the
cytotoxic singlet oxygen (1O2) by inverting the spin of one of the π*-electrons. 1O2 is
regarded as the main mediator of the phototoxicity in PDT.[57] The energy required for
the triplet to singlet transition in oxygen is only 22.5 kcal mol-1, which corresponds to
a wavelength of 1274 nm.[57]
hν+ 1P
3P*
3P*
3P*
P-
1O2
3O21O2
O2 O2-
O2* O2
-
S
S
1P
P
S+
S(O)
P+
P-
3P*
Type-I photoreactions
Type-II photoreactions
Figure 1-13: Type-I and Type-II photoreactions.
Singlet oxygen is a powerful oxidant and reacts with many kinds of biomolecules like
amino acids, nucleic acid bases, phospholipids and cholesterol.[11, 64, 66, 67] The
lifetime of the 1O2 in a cellular environment is very short and it reacts at its site of
formation. Therefore the PDT-induced photodamage is highly localized to regions not
larger in diameter than a cell membrane.[11] The area where the photodamage occurs
depends on the photosensitizer used, as different photosensitizers accumulate in
different cellular compartments.[63] Additionally, the cell membranes are important
targets of photodynamic damage. Nevertheless, accumulation inside the cells is
preferable versus the accumulation on the cell membrane because apoptosis will
happen easier in the first case whereas necrosis is preferred in the second case.
Type I and type II reactions both induce oxidation processes (oxidative stress) of
biomolecules and as a result, the cells die after a certain while via necrosis or
apoptosis. The tumor response after illumination is not necessarily a result of the
total destruction of each tumor cell. From animal studies it is known that after the
PDT treatment still viable tumor cells reside in the targeted tissue.[68] The complete
death of the tumor is also, at least in part, due to the damaging effect of the PDT on
the vasculature.[69]
Introduction
14
1.3.3 Photosensitizers in Photodynamic Therapy
In order to develop new and improved photosensitizers, the characteristics of an
ideal photosensitizer must be known. Beside certain demands every drug should
possess, a good photosensitizer should additionally comprise the following
features:[11, 57]
● strong absorption, preferably between 600 and 800 nm, because with increasing
the wavelength, light penetrates deeper into the tissue
● low dark toxicity
● have a pharmacokinetic profile where it is rapidly eliminated from the body to
avoid generalized skin photosensitization
● high singlet-oxygen yield (long-lived exited states)
● no self-aggregation in the body because this reduces the 1O2 quantum yield
In addition to those above-mentioned characteristics, there are others that could
prove to be useful in PDT.
The properties of chlorins often satisfy the demands given for a good photosensitizer;
therefore they are good candidates for better photosensitizers in the future.
1.3.3.1 First-Generation Photosensitizers
The first photosensitizer tested in the clinic was hematoporphyrin derivative (HpD). It
is easily synthesized by treating hematoporphyrin (Hp) with 5% sulphuric acid in
acetic acid. Subsequent hydrolysis with base yields a crude material that is
commonly referred to as HpD.[70, 71] It is a complex mixture of monomeric and
oligomeric porphyrins. In detailed experiments it was shown that the ability to act as a
photosensitizer is mainly due to the oligomeric components.[70, 72-74]
Commercial forms of HpD (Photofrin®[75], Photosan®, Haematodrex®,
Photocarcinorin®) are obtained by removing the low-molecular weight components of
this mixture. Even the purified form is still a mixture of hematoporphyrin monomers,
dimers and oligomers with up to nine porphyrin units as well as their dehydration
products. The ratio of monomers, dimers and oligomers has been estimated in HpD
to be 22:23:55 and in Photofrin® to be 14:19:67.[48] The porphyrin units are connected
either via ether or ester linkages.
Introduction
15
N
NH N
HN
CO2Na CO2Na
O
O
nn=1-9Photofrin
N
NH N
HN
CO2H CO2H
HOOH
Hematoporphyrin
Figure 1-14: Hp 12 and HpD 13.
Even though HpD was the first photosensitizer approved and which turned out to be
very successful for the treatment of certain types of tumors, it is far from being an
ideal photosensitizer because of the severe limitations. Photofrin®´s longest
wavelength absorption maximum is a relatively weak Q-band at 630 nm. The light
penetration into the tissue at 630 nm (~5 mm)[11] is not optimal due to the weak
absorption (3500 M-1cm-1). Therefore, only small tumors can be treated with
Photofrin®. Other limitations are:
● it is a complex mixture of about 25 components
● the precise composition of the mixture is not known and varies from batch to
batch
● the components of HpD are subject to changes in tissues
● due to the chemical heterogenity dose-response studies are difficult to interpret
● the photodynamic characteristics and distribution in tissue vary from one
preparation to another.
Another major side effect is the accumulation of Photofrin® in the skin, urging every
patient to avoid sunlight and high intensity light for approximately six weeks after
treatment.[56, 57]
1.3.3.2 Second-Generation Photosensitizers
The problems encountered with Photofrin® have led to the development of new
molecules, so called second-generation photosensitizers. Although these new
photosensitizers still do not match all the requirements a perfect photosensitizer
should have, there are a lot of improvements compared to the HpD-type
photosensitizers.
12 13
Introduction
16
One of the main drawbacks of Photofrin® is the absorption at 630 nm combined with
an unsatisfactory light penetration into the tissue. Therefore one of the most
important goals was to develop new photosensitizers absorbing light of longer
wavelengths (see Table 1-2). The photosensitizers that are currently used in clinical
trials or that are already approved belong to the groups of porphyrins,
phthalocyanines, texaphyrins, chlorins or bacteriochlorins.
λmax (nm) ε (M-1 cm-1)
Porphyrins 620-640 10000
Phthalocyanines 700 200000
Naphthalocyanines 780 350000
Porphycenes 610-650 50000
Texaphyrinato-Lu(III) 732 42000
Chlorins 680 40000
Bacteriochlorins 780 150000
Table 1-2: Some groups of second-generation photosensitizers with their longest absorbtion wavelenght and extinction coefficient.[76]
Other improvements are a higher 1O2 quantum yield, higher purity, better
accumulation in the target tissue and better pharmacokinetics (lower side effects).
Figure 1-15 shows the structures of some second-generation photosensitizers and
their long wavelength absorption.
An interesting strategy is the use of the endogenous photosensitizer protoporphyrin
IX. This photosensitizer is an intermediate product in the protoheme biosynthesis and
can be over-expressed by applying the prodrug 5-aminolevulinic acid 20 (ALA). The
conversion of ALA to protoporphyrin IX is much faster than the conversion of the
latter to protoheme. This results in an accumulation of the photosensitizer
protophorphyrin IX in the cells which makes a photodynamic effect induced by
illumination possible.
All compounds shown in Figure 1-15 share certain characteristics that make them
suitable for PDT. They all have a high singlet oxygen yield, absorb light of long
wavelength and have no dark toxicity. Good overviews are given by PANDEY[77],
DETTY[78] and HYNNINEN.[57]
Introduction
17
RO O
N
NH N
HN
O
N
N N
NSn
CO2C2H5
Tin Etiopurpurin (SnEt2 660 nm)
N
NH N
HNHO
HO
OH
OH
m-Tetrahydroxyphenylchlorin(m-THPC) Foscan (650 nm)
N
NH N
HN
CO2HHN CO2H
Mono-aspartyl-chlorin-e6(650 nm)
NN
N
NN
OO (C2H4O)3CH3H3C(OC2H4)3
HOC3H6 C3H6OH
Lu
Lutetium-texaphyrin (732 nm)
HO2C NH2
O
5-Aminolevulinic acid (630-635 nm)
N
NH N
HN
CO2R CO2R
H3CO2C
H3CO2C
Benzoporphyrin derivative(BPD 690 nm)
Pyropheophorpbide-aand its derivatives
(660 nm)
HH
H H
OCO2HHO2C
Figure 1-15: Some second generation photosensitizers with their longest absorption wavelength.
1.3.3.3 Third-Generation Photosensitizers
The third generation photosensitizers which are currently under development
combine the improved properties of the second-generation sensitizers with methods
for the selective accumulation of the sensitizers in the tumor-tissue. Especially
conjugates with monoclonal antibodies and other targeting vehicles like liposomes,
nanoparticles and proteins show promising results (see also chapter 1.4).[79-81]
1.3.4 Photodynamic Therapy as a Therapy for other Diseases than Cancer
Apart from treating cancer, PDT has shown the potential to treat several other types
of diseases. In addition to psoriasis, arthritis, atherosclerosis and purifying blood
infected with viruses, including HIV, the treatment of age related macular
14 15 16
17 18 19
20
Introduction
18
degeneration (AMD) and microbial infections are already in clinical use or current
areas of research.[77]
1.3.4.1 PDT for Age-Related Macular Degeneration (AMD)
Age-related macular degeneration (AMD) is the leading cause for blindness of people
aged over 50 in the western world. With AMD, patients experience the loss of central
vision in varying degrees while retaining their side or peripheral vision.
AMD is classified into 2 types: the “dry” (atrophic) form, marked by the appearance of
small yellowish deposits known as “drusen” within the retina, and the more severe
“wet” (neovascular) form.
Dry AMD accounts for about 90% of AMD cases. In dry AMD, drusen accumulate in
the retinal pigment epithelium, causing the macula to thin and dry out. Although this
form of the disease usually only produces mild vision loss, patients may progress to
wet AMD and, therefore, must be monitored continually.
Among patients with any sign of AMD, estimates indicate that only about 10% to 20%
have the wet form of the disease. Nonetheless, wet AMD is responsible for 90% of
the severe vision loss associated with this condition. Each year approximately
200,000 new cases of wet AMD occur worldwide.
In wet AMD, choroidal neovascularization (CNV) occurs, in which abnormal choroidal
blood vessels break through Bruch’s membrane into the subretinal space and retinal
pigment epithelium. These weak and underdeveloped vessels leak blood and fluid
into tissue behind the retina, causing damage to the macula, which destroys central
vision in as little as 2 months to 3 years.[82]
The photosensitizer VISUDYNE® (16 in Figure 1-15) was the first approved drug for the
light-activated therapy indicated for the treatment of patients with wet AMD.
It is intended to support the preservation of visual acuity and to slow down or stop the
advancement of AMD. VISUDYNE® utilizes a photosensitizer, known as verteporfin, to
occlude abnormal blood vessels found in the eye while sparing overlying retinal
tissue. PDT with VISUDYNE® has been shown to significantly reduce the risk of severe
vision loss and slow the progression of AMD.[83]
In Figure 1-16 are the typical steps of a PDT-treatment with VISUDYNE® shown.
Introduction
19
Figure 1-16: VISUDYNE® therapy.
1. Photodynamic drug is applied into
the blood stream through an injection.
2. Low Density Lipoproteins (LDL) form
complex protein molecules that carry
fatty material in blood, and thus form a
complex with verteporfin to take the
drug to all parts of the body.
3. Verteporfin structure (LEVY and other
scientists at QLT).[83]
4. The drug accumulates in the
abnormal blood vessels of the diseased
macula, part of the retina at the back of
the eye, where new blood vessels are
growing improperly causing the
disease. The abnormal vessels attract
and absorb the LDL-VISUDYNE complex.
5. Because new blood vessel cells grow faster than normal cells, they invade one of
the membranes of the retina and start leaking. This is the cause of one form of
macular degeneration disease. Their faster growth rate also makes them take up
verteporfin about ten times quicker than normal cells.
6. About 10 - 15 minutes after the injection, doctors shine cool red laser diode light
into the eye for about 90 s. The light has a wavelength of 690 nm which activates the
photosensitizer producing singlet oxygen. The singlet oxygen reacts with the
abnormal blood vessel cells and effectively “burns” them up.
7. The abnormal vessels are destroyed.[84]
1.3.4.2 Bactericidal Photodynamic Therapy
Even though bactericidal photodynamic effects have been known for a long time,
only recently has there been increased interest in practical use.[85-89]
Particularly, the emergence of antibiotic resistance among pathogenic bacteria has
led to efforts to find alternative antimicrobial therapeutics to which bacteria will not
easily develop resistance to.[90] It seems that PDT has this potential and so far it has
been used to kill pathogenic microorganisms in vitro. Its use to treat infections in
animal models or patients has not been much developed so far.
Introduction
20
Nevertheless, the application of PDT to treat infections in selected animal models
and some clinical trials, mainly for viral lesions[91], but also for acne, gastric infection
by Helicobacter pylori and brain abscesses was reported. Possible future clinical
applications include infections in wounds and burns, rapidly spreading and intractable
soft-tissue infections and abscesses, infections in body cavities such as the mouth,
ear, nasal sinus, bladder and stomach, and surface infections of the cornea and
skin.[92]
1.4 Modular Carrier Systems
Beside the major improvements related to the development of the second-generation
sensitizers there are some unsolved problems left. Probably the main goal is still the
delivery of a sufficient amount of the photosensitizer to the tumor cells combined with
a high selectivity. The lack of selectivity can result in severe normal tissue damage
after PDT. Furthermore, PDT can result in skin phototoxicity with the consequence
that patients must stay out of bright sunlight for several weeks after the treatment.[93]
Therefore, it is still one major goal to develop suitable delivery systems to obtain a
high accumulation of the sensitizers in the target tissue. As an answer to that
problem, the concept of modular drug delivery systems was proposed.[94] This
concept comprises the following three parts (see Figure 1-17):
Drug (green), Multiplier (brown), and Addressing Unit (red)
MULTIPLIER
HO O
N
NH N
HN
O
HOO
NNH
NHN
O
HOO
N HN
NNHO
OHO
NHN
NNH
OOHO
NHN
NNH
O
OHO
N
HNN
NH
O
HOO
NNH
NHN
O
Y
Y
YY
YTumorTumor ADDRESSINGUNIT
Figure 1-17: Modular-Drug-Delivery-System.
Introduction
21
It is a known fact that monoclonal antibodies (MAb) or antibody fragments can be
used very efficiently as addressing units directed to tumor-associated antigens. The
combination with attached photosensitizers is called photoimmunotherapy (PIT)
which can be used for the selective delivery of the drug to the tumors. Unfortunately,
the direct usage of these biomolecules as selective carriers for photosensitizers is
limited due to the decrease of the immune activity of the dye-antibody complexes
with an increasing number of covalently linked dye molecules.[95] Also, the
hydrophobicity of the coupled photosensitizer, as well as the type, number, and
arrangement of charged groups, can strongly influence the physicochemical
properties of the MAb, resulting in alteration of pharmacokinetics, biodistribution,
specific and non-specific binding and internalization.[93] Another, non-negligible
problem is that the antigen expression on the tumor cells is neither uniform nor
static.[93]
The above-mentioned problems have to be considered when thinking about the
synthesis of such photosensitizer immuno-conjugates. Possible solutions are the
introduction of multiplying units carrying many dye molecules as well as spacer units
to create a distance between the hydrophobic sensitizer and the MAb. These
multiplying units and spacers might also be useful for increasing the solubility of the
multi-sensitizer unit and therefore for increasing the sensitizer-to-MAb ratio of the
conjugate. Dendrimers in combination with long spacer units for example, with their
well-defined structure and their large number of active end groups, would be ideal for
serving as multiplying units.[96, 97] C60-hexakisadducts with their octahedral addition
pattern can be regarded as dendritic systems and be used as multiplying units.
Indeed, it is possible to use C60 as a versatile building block for the construction of
globular dendritic systems.[98-101] Further multipliers, including polyglutamic acid, poly-
L-lysine, dextran, and polyvinyl alcohols are also reported.[93]
Beside the antibody-based carrier systems, other transporters include
photosensitizers conjugated with lipoproteins (active targeting) or with polymer-
conjugates, oil-based dispersions, nanoparticles and liposomes (passive
targeting).[79-81]
The above-mentioned as well as other targeting systems are now being examined
with a view toward enhanced PDT efficacy. In order to justify the additional expense
of these highly selective delivery systems, it will be necessary to show a clearly
Introduction
22
improved PDT efficacy or other clear advantages before the widespread acceptance
in clinical use is likely.[80]
Nevertheless, current reports by the groups of HASAN[102], CARCENAC[103] and
VROUENRAETS[104, 105] show very encouraging results in the field of PIT and are
promising for further development.
1.5 Finding a Good Name for Porphyrin- and Chlorophyll-Compounds
Though the IUPAC has established a nomenclature system for porphyrins and
chlorophylls, trivial nomenclature is still widely used, especially for naturally occurring
chlorophylls. The crown ether-tetraphenyl porphyrin derivatives in the first part of this
thesis will be named after the more systematic IUPAC nomenclature as far as
possible. In those cases where this would lead to long and complicated names, a
less confusing, non-systematic nomenclature will be used.
HO O
N
NH N
HN
O
221
331
32
771
881
82
12112
13
131132
17171
172173
1818114
16
19
HO O
N
NH N
HN
O
11a2
2a2b
33a
44a
4b
5a5
6910
77a7b7c
88a
11
12 13
1415
1617
18
1
4 5 6
910
11
15
20
α
βγ
δ
Figure 1-18: IUPAC and Fischer numering of pyropheophorbide-a 19
The Chlorophyll-type compounds in the second part of this thesis will be named by
using the Fischer and trivial nomenclature because this nomenclature is still
commonly used in the literature. Especially the assignment of the NMR spectra was
done by using the Fischer system. The more systematic IUPAC nomenclature would
result in long and complex names and will therefore not be used.
1.6 C60-Fullerene as a building block
The C60 fullerene 21 with its highly symmetrical core is particularly suitable for the
construction of three dimensional structures. Various well-established methods for
19 19
Introduction
23
the controlled covalent exohedral functionalization of C60 21 make polyfunctional
macromolecules accessible. Template and tether techniques lead to
stereochemically well-defined multiple adducts with up to six addends. The
octahedral topology obtained by a six-fold addition to the C60-core is a unique
structural motif which offers interesting possibilities to synthesize molecules with new
properties.
The most important nucleophilic addition reaction for the synthesis of exohedral
functionalized fullerenes is the nucleophilic cyclopropanation with malonic esters
introduced by BINGEL in 1993 (see Scheme 1-1).[106, 107]
OOR
ORO
OOR
ORO
DBU, CBr4toluene
ORO
ROO
Br-
-Br
Scheme 1-1: Cyclopropanation by modified Bingel reaction.
The template-mediated method using 9,10-dimethylanthracene 23 (DMA) makes a
six-fold cyclopropanation possible and gives rise to a Th-symmetrical addition
pattern.[108, 109] This one-pot method greatly improves the yield of hexakisadducts.
(regioisomers)
(regioisomers)
C60(DMA)n
O OO
O
O
OOORRO
RO ORRO
OR
ORO
ORO
OR
O OR
ORO
ROO
O ORO OR
CBr4, DBU
Scheme 1-2: DMA mediated template method for the synthesis of hexakisadducts. 24
22 21
23
Introduction
24
It is possible to synthesize hexakisadducts with six similar addends 24 starting from
C60 21 as well as mixed hexakisadducts starting from the corresponding fullerene
derivatives.
Compared to the conjugated π-electron system of C60, which is extended over the
entire molecule, the hexakisadducts exhibit an enhanced aromatic character. As a
result of the octahedral addition pattern in hexakisadducts the π-electron system is
reduced to a cubic cyclophane-type structure.
The remaining π-system consists of eight isolated benzoic rings with only marginal
bond length alterations. These circumstances lead to major changes in the
spectroscopic properties of the hexakisadducts, compared to the less symmetrical
adducts. The hexakisadducts, are only light yellow, compared to the intensively
colored lower addition products. As a consequence, even small impurities of
pentakisadducts can be easily recognized by a change of color from orange to red.
The high symmetry of the hexakisadducts is also visible in the 13C NMR spectra in
the sp2-region of the fullerene resonances. For the Th-symmetrical hexakisadducts
only two resonances around 140 ppm are found.
Aims
25
2 Aims The work presented in this thesis is divided into two independent projects.
The aim of the first project is the expansion of the work on crown ether-porphyrin
conjugates starting with motifs obtained in my diploma thesis. Primarily it is
necessary to optimize the synthesis of the parent crown ether-porphyrin. As soon as
larger quantities of the crown ether porphyrin are available, several different metal
complexes should be synthesized.
The stabilizing effect of the crown ether moiety onto metal complexes with large
central metals should be evaluated. This should be done by kinetic investigations
where the crown ether-porphyrin is compared with the crown ether free porphyrin as
the reference. Exemplarily, the metal exchange reaction of the cadmium center by a
zinc ion can serve for this purpose.
A major goal is the examination of the ditopic binding properties of the zinc and
cobalt complexes. Especially, potassium salts shall be used for the investigations. To
monitor such ditopic behavior UV/Vis spectroscopy, NMR and most importantly X-ray
crystallography can serve as good indicators.
A forth goal is the expansion of the crown ether-porphyrin motive towards larger
arrays. By the introduction of bisfunctional porphyrins and diazacrown ethers it
should be possible to synthesize a library of building blocks for the construction of
oligomeric porphyrin-crown ether systems. These building blocks of different
symmetry make the selective construction of oligomeric porphyrin-crown ether
structures possible.
The last goal in this field is the synthesis of rare earth metal monoporphyrin
complexes.
The second project is aimed at the development of a strategy for the construction of
multi-pyropheophorbide-a-fullerene conjugates as selective photosensitizer carrier
systems for the photodynamic therapy of tumors. Such systems should be
synthesized based on the concept of modular drug delivery systems. These
conjugates should comprise a highly functionalized fullerene as the carrier molecule,
a large number of photosensitizer molecules (pyropheophorbide-a) attached to the
multiplying unit (fullerene, dendrimer) and a highly selective addressing unit
Aims
26
(monoclonal antibody) covalently linked to the multiplying unit via a spacer molecule.
The first goal was the evaluation of different ways for the attachment of several
photosensitizer molecules to the fullerene core. After establishing a protocol to
achieve that, a molecule should be synthesized bearing an additional selective
coupling site for the tumor selective antibody. The final goal should be the coupling of
such a multipyropheophorbide-a-fullerene system with a tumor-selective antibody.
Additionally the photophysical and photobiological properties of the
pyropheophorbide-a conjugates should be investigated in collaboration with groups
from the Humboldt University of Berlin. The main emphasis here should be the
estimation of the singlet oxygen quantum yield as well as the ability of the
compounds to kill tumor cells under illumination with light.
Results
27
3 Results and Discussion
3.1 Molecular Recognition – Crown Ether-Porphyrins and their Coordination Properties
3.1.1 Synthesis of the Parent Crown Ether-Porphyrin 26 and its Metal Complexes
Starting from the bromomethylated tetraphenyl porphyrin 25[110] the crown ether-
porphyrin 26 is easily accessible via a nucleophilic substitution reaction with 1-aza-
18-crown 6-ether (Scheme 3-1).
HN
NNH
N
OO
OO
N
O
HN
NNH
N
Br
OO
OO
NH
O
toluene refluxNaHCO3
Scheme 3-1: Synthesis of crown ether porphyrin 26.
The corresponding metal complexes of the free base porphyrin 26 with various
central metal ions like zinc, cobalt, iron, nickel or rare earth metals were obtained,
using standard procedures known from literature (Scheme 3-2).[111, 112]
MHN
NNH
N
OO
OO
N
O
N
NN
N
OO
OO
N
O
M = Zn2+, Co2+/3+, Fe3+, Cd2+, Ni2+, Gd3+, Eu3+
M(ac)2 orM(acac)3
MeOH or DMFor THF
Scheme 3-2: Synthesis of metalloporphyrins.
The yields for the metallation reactions are usually high and typically range between
85 % and 95 %, depending on the inserted central metal and the reaction conditions
25 26
Results
28
applied. Because the metallation of the free base porphyrin 26 is always associated
with a more or less intensive change of color, the UV/Vis spectroscopy provides an
excellent tool for monitoring the progress of the reaction (Figure 3-1).
350 400 450 500 550 600 650 7000
100000
200000
300000
400000
500000
ε [l
mol
-1 c
m-1
]
λ nm
Figure 3-1: UV/Vis spectra (CH2Cl2) of some synthesized metalloporphyrins.
Table 3-1 shows the Soret band absorptions (λmax) of the synthesized
metalloporphyrins.
Central metal λmax Compound number
2H 421 nm 26
Zn2+ 430 nm 30
Cd2+ 439 nm 29
Co2+ 414 nm 37
Fe3+ 419 nm 27
Ni2+ 418 nm 28
Gd3+ 430 nm 59
Eu3+ 425 nm 60
Table 3-1: λmax of some synthesized metalloporphyrins.
Beside the observed shifts of the bands in the UV/Vis spectra, the metallation of the
free base porphyrin causes also distinct changes in the NMR spectra. The most
prominent changes exhibit the crown ether resonances. Figure 3-2 shows
exemplarily the NMR spectrum of the zinc porphyrin 30. The resonances of the crown
26
26 + ZnII
26 + FeIII
26 + CoII
26 + CdII
Results
29
ether moiety (red resonances) are shifted to higher field for about 1-1.5 ppm,
indicating the close proximity of the ether protons to the porphyrin´s anisotropy cone.
Such behavior is known for similar systems.[113, 114] It can also be assumed that one
of the oxygen atoms of the crown ether binds to the central zinc ion which would
enhance the shielding effect. The red shifts of the UV/Vis absorptions (~7 nm) also
support this conclusion.
8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5ppm
HN
NNH
N
OO
OO
N
O
ZnN
NN
N
OO
OO
N
O
A B
A
B
Figure 3-2: 1H-NMR spectra (CDCl3) of zinc porphyrin 30 and free base porphyrin 26.
All central metals which do not favor a square planar coordination in porphyrin
complexes are coordinatively unsaturated. These metals are often able to form
square pyramidal, octahedral or cubic complexes (Zn, Cd, Co, Fe, Eu, Gd) in which
the crown ether´s O-donor atoms may partcipate as ligands. From the complexes
synthesized in this thesis, only the nickel complex 28 favors a strictly square planar
coordination geometry and therefore no shifts of the crown ether resonances can be
observed in its 1H NMR spectrum.
The paramagnetic Fe3+, Co2+, Eu3+and Gd3+ complexes are of course much more
difficult to characterize by NMR spectra. Nevertheless, it can be assumed that they
show a similar behavior because they also prefer a non square planar coordination.
Results
30
3.1.2 Kinetic Experiments – The Stabilizing Effect of the Crown
From the analysis of the NMR spectra we deduced that the crown ether moiety
interacts with the central metal atom of most metalloporphyrins. Compared to the
crown ether-porphyrin free base 26, all crown ether protons are shifted to higher field
due to the ring current effect of the porphyrin core. As already mentioned above, one
explanation for this effect would be that an oxygen atom of the crown ether acts as
an intramolecular electron-pair donor and forms a coordinative bond to a free
coordination site of the central metal. In analogy to a clam the metal ion in this case
is enclosed between the porphyrin core and the crown ether.
Due to a chelating effect of the crown ether and for entropic reasons, those
metalloporphyrin complexes should gain more stability compared to the non-crown
ether-metalloporphyrins. The largest effect should be observable for metals with large
ionic radii like Cd2+, Pb2+, Eu3+ and Gd3+. Those complexes are often labile because
the metal ion is too large to fit properly into the central porphyrin cavity. Therefore
these complexes are often sensitive to acids or have the tendency to form double-
decker complexes.
To investigate and verify the expected stabilizing effect of the crown ether moiety on
the metalloporphyrin complexes, kinetic investigations were performed.
In the literature, the stabilizing effect of free 18-crown 6-ether for zinc, cadmium, and
lead tetrakis (sulfonatophenyl) porphyrins in water is already described.[115] However,
a large excess of 18-crown 6-ether (≈ 1000 equivalents) is necessary to see any
stabilizing effect. Therefore we chose this well-established metal-metal metathesis
and determined the rate constant of the cadmium-zinc exchange of our system 29
(Scheme 3-3). To quantify the effect, we also prepared the corresponding cadmium
tetraphenylporphyrin 31 without the attached crown ether and used it as a reference
system (Scheme 3-3).
Processing of the Experiments
The metal exchange reaction of cadmium by zinc (Figure 3-3) was monitored via
UV/Vis spectroscopy (Figure 3-3). Firstly, the kobs values were determined for
different zinc concentrations by observing the time-dependent change of the
absorbance at the Soret band region. The data obtained was analyzed by using the
program OLIS. Plotting the kobs values against the zinc concentrations (Figure 3-4)
Results
31
furnished the rate constants for both systems.
CdN
NN
N
OO
OO
N
O
ZnN
NN
N
OO
OO
N
O
Zn(ac)2 / DMF
CdN
NN
NZn
N
NN
N
Zn(ac)2 / DMF
A
B
Scheme 3-3: Investigated metal exchange reactions.
Figure 3-3 shows the blue shift of the Soret band from 436 nm to 428 nm as a
function of time caused by the replacement of cadmium as central metal by zinc.
CdZn
Zn+Zn2+ Cdfast slow
-Cd2+
Scheme 3-4: Proposed mechanism of Cd-Zn exchange.
The addition of the colorless zinc solution to the green cadmium porphyrin solution
instantly causes a shift of the Soret band from 441 nm to 436 nm (fast pre-
equilibrium). The real exchange
is much slower and can be
observed by the shift from
436 nm to 428 nm (Zn-
porphyrin). The typical time
frame for these measurements
was between 16 h (50
equivalents of zinc) and 45 min
(500 equivalents of zinc). The
exchange reaction proceeds
nicely through an isosbestic
point which shows that there is
400 410 420 430 440 450 4600,0
0,2
0,4
0,6
0,8
1,0
1,2
1,4
1,6
1,8
2,0
abso
rptio
n rel
λ in nm
Figure 3-3: UV/Vis spectrum (DMF) of the Cd-Zn-exchange.
29 30
31 32
Results
32
only the cadmium complex (educt) and the zinc complex (product) involved in that
reaction. These measurements were performed for both cadmium systems 29 and 31
with varying zinc concentrations from 50 equivalents to 500 equivalents in DMF as
the solvent.
Figure 3-4 shows the plot of the kobs values against the concentration of zinc for both
systems. The linear fit gives the rate constants.
y = 0,7765x - 9E-06
y = 0,2179x - 2E-05
0
0.0002
0.0004
0.0006
0.0008
0.001
0.0012
0.0014
0.0016
0 0.25 0.5 0.75 1 1.25 1.5 1.75 2c [Zn2+] mmol/l
k [ob
s]
CdN
NN
N
OO
OO
N
O
ZnN
NN
N
OO
OO
N
O
Zn(ac)2 / DMF
CdN
NN
NZn
N
NN
N
Zn(ac)2 / DMF
Figure 3-4: Time constants of cadmium-zinc exchange.
The data clearly shows that the exchange rate of the reference system without the
internal crown ether moiety is 3.6 times faster compared to our crown ether-porphyrin
system. This fact emphasizes that the introduction of the crown ether moiety into our
system has a distinct stabilizing effect on the cadmium center. It can also be
assumed that this fact is true for other metalloporphyrins with large central metals. In
contrast to the literature known stabilizing effect of external 18-crown 6-ether, system
31 did not show this effect at all.
Results
33
3.1.3 Ditopic Receptors
The development of novel chemosensors/receptors has received major interest over
the last few years (see also chapter 1.2). In particular, systems that simultaneously
bind cations and anions constitute a growing field.[116] Whereas in such molecules
crown ethers frequently act as recognition sites for ammonium and alkali metal ions,
the anion is typically (but not necessarily[117, 118]) bound by metal centers with free
coordination sites, for example boronic acid esters[119, 120] and uranyl cations.[121-123]
The combination of porphyrins and crown ethers has also led to receptors for
diamines[124], pyridinium salts[125], alkali metal salts[31], and peptide-binding
systems[126, 127] which offer one of the most promising strategies for length- and
sequence-selective recognition of natural peptides in aqueous media. NMR studies
on a zinc porphyrin system with a benzo 15-crown 5-ether addend were performed in
the presence of sodium cyanide showing a strong ditopic binding.[31] Our novel
porphyrin-crown ether conjugates 30 and 37 bind potassium cyanide and other salts
in a ditopic fashion. The variation of the attached crown ether offers the possibility to
construct analogous systems for the selective binding of other cations like sodium or
cesium.
3.1.3.1 Investigation of the Zinc-Crown Ether-Porphyrin System
Due to the strong UV/Vis absorption of zinc porphyrins and the occurring distinct
color changes upon the coordination of different axial ligands,[128] these systems may
be used as sensors for anions. The zinc porphyrin 30 is obtained by stirring a
methanolic solution of the free base porphyrin 26 together with an excess of zinc
acetate for 4 h at room temperature. Successive column chromatography on silica
yields the pink metalloporphyrin in high yields.
Figure 3-5 shows the Soret band of the zinc-crown ether porphyrin 30 in the
presence of different potassium salts. All investigations were performed in DMF-
solutions with the salts added in solid form. The strongest shifts can be observed for
the coordination of the hard ligands CN-, O2- and OCN-. These changes of color are
so intensive that they can be recognized even by eye. One reason for the observed
large shifts (~20 nm) is that the central zinc atom is pulled further out of the porphyrin
plane. The result is a distortion of the plane porphyrin core which induces altered
Results
34
electronic properties. In the case of weak ligands like halides, no shifts can be
observed in DMF.
400 410 420 430 440 450 4600
100000
200000
300000
400000
500000
600000
ε [l m
ol-1
cm
-1]
λ in nm
Figure 3-5: UV/Vis spectra (DMF) of 30 coordinated with different potassium salts.
30 428 nm
KCN 439 nm
KHCO3 439 nm
KOH 439 nm
KO2 438 nm
KOCN 435 nm
KSCN 433 nm
K-formiat 433 nm
KOAc 432 nm
KNO2 431 nm
KCl 429 nm
KBr 428 nm
KJ 428 nm
KNO3 428 nm
Table 3-2 shows the observed
wavelength of the Soret band
maximum absorption (λmax) of the
zinc porphyrin 30 with several
coordinated potassium salts in DMF.
30
30 + KOCN
30 + KCN
30 + KSCN
30 + KBr
30 + KNO2
30 + KO2
Table 3-2:Soret band λmax.
Results
35
Characterization of the Zinc Porphyrin Potassium Cyanide Complex 33
Solutions of 30 do not only take up solid KCN but also from methanolic or aqueous
solutions with the expected change of color [128] from purple to an intense green and
form a stable complex (Scheme 3-5). Such a solution can be evaporated to dryness
without destroying this complex. Afterwards the dry complex 33 can be redissolved in
nearly all organic solvents.
CN
ZnN
NN
N
OO
OO
N
O
ZnN
NN
N
OO
OO
N
O
CH2Cl2
K
K+ CN-
Scheme 3-5: Uptake of solid KCN by zinc porphyrin 30.
Whereas the main UV/Vis absorptions of 30 in CH2Cl2 can be found at 429, 559, and
603 nm, the corresponding bands for 33 are shifted to 438, 576, and 620 nm
respectively.
Crystals suitable for X-ray analysis grew (Figure 3-6) when water was carefully
layered on a THF/CHCl3 solution of 33. The structure proves unambiguously that one
molecule of KCN is bound within 30. Several structural details are noteworthy: first,
the cyanide is clearly bound to the zinc atom because the latter is pulled out of the
N4-plane of the porphyrin (displacement 0.5 Å); second, all bond lengths within the
coordination sphere of the zinc ion are quite normal (average 2.081 Å); third, the
bond lengths within the coordination sphere of the potassium ion are also in the
expected range of values for such a system (average 2.858 Å); fourth, the length of
the C-N bond (1.137 Å) of the cyanide anion clearly indicates a triple bond. Also, CN-
sits nearly perpendicular on the Zn-atom with regard to the N4-plane of the porphyrin
(deviation from the normal axis of the plane 5.35°); fifth, the crown ether moiety with
the coordinated potassium sits above the porphyrin core and K+ is clearly attached to
the CN- ion (2.704 Å) with an angle of 145°.
The complex 33 co-crystallizes with three THF molecules in the unit cell.
33 30
Results
36
CNZnKO
Figure 3-6: Structure of 33 in the crystal (THF omitted for clarity).
Due to the size of the molecule, a differentiation between the C- and the N-atom of
the cyanide anion can not be made by X-ray analysis (see also later in this chapter).
High-field shifts of all crown ether proton resonances are observed in the 1H NMR
spectra of 30, indicating the close proximity to the porphyrins anisotropy cone – a
behaviour known from similar systems. One of the oxygen atoms of the crown ether
may bind to the zinc ion which would enhance the shielding effect. Clearly, 30 is pre-
organized in an oyster-like fashion. Contrary to that the proton signals of the crown
ether moiety of 33 experience an interesting shift behavior (Figure 3-7). All these
resonances are shifted to downfield for about 1.5-2.0 ppm and are closer to the
resonances known for free 1-aza-18-crown 6-ether. Because the proton resonances
of azacrown ethers do not shift strongly upon complexation with K+,[129] this
observation suggests that the crown ether moiety is moved away from the immediate
vicinity of the porphyrin.
Results
37
9.0 8.5 8.0 7.5 3.5 3.0 2.5 2.0 1.5 ppm
CN
ZnN
NN
N
OO
OO
N
OK
ZnN
NN
N
OO
OO
N
O
B A
B
A
CHCl3
Figure 3-7: 1H NMR spectra (CDCl3) of compounds 30 (B) and 33 (A).
The 13C NMR data of 33 is more or less identical with that of 30 itself, but the carbon
resonance of the cyanide ion was only assignable after the preparation of a sample
with K13CN. The cyanide resonance appears at 144.8 ppm in the 13C NMR spectrum.
Unfortunately, no 13C NMR data for cyanide-complexed zinc porphyrins is available in
the literature which could help to determine the orientation of the cyanide ion. The 13C resonance for K2[Zn(CN)4] in aqueous solution is reported with a value of 147.0
ppm,[130] whereas uncomplexed CN- absorbs at 166.2 ppm in solution.[131] The high-
field shift of the cyanide carbon resonance in K2[Zn(CN)4] when compared to that of
free cyanide was attributed to the increased polarization of the triple bond of CN- due
to an inductive withdrawal through the metal-carbon σ-bond. The π-accepting
properties of the cyanide anion seemed to be of lesser importance in this complex.
Results
38
150 140 130 120 110 100 90 80 70 60 50 40 30 20 ppm
13CN
*
Figure 3-8: 13C NMR spectrum (CDCl3) of 33 with 13C labeled KCN.
The comparison of the results for K2[Zn(CN)4] and 33 gives a first indication that the
C-atom and not the N-atom of the cyanide anion binds to the central zinc atom.
Surprisingly, only a very weak absorption for the CN stretching vibration was found in
the infrared spectrum of 33. The CN-absorption of 33 was found at 2130 cm-1
whereas for the labelled compound 33-K13CN it appears at 2082 cm-1 (see Figure
3-9). At this point a theoretical analysis of the vibrational modes and their intensities
seemed the appropriate method to determine the orientation of the cyanide ion, i.e. a
Zn-C-N-K versus a Zn-N-C-K arrangement. The calculations were performed in the
group of Prof. Dr. Marcus REIHER from the Friedrich-Schiller University of Jena.
The “missing” CN stretching vibration was the starting point of the analysis (details
see[132]). The mode-tracking protocol[133] which is particularly suited for tracking
structure-characteristic vibrations in large molecules[134, 135] was applied in order to
calculate the CN stretching frequency for the two possible isomers. As a prerequisite
both isomers of the CN complex, 33-a (Zn-C ≡ N-K) and 33-b (Zn-N ≡ C-K) (see
Figure 3-10) were optimized.
Results
39
Wavenumber [cm-1]4000 3500 3000 2500 2000 1500 1000 500
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
208213CN
213012CN
Tran
smis
sion
Figure 3-9: IR-spectra of 13CN-33 and 12CN-33.
Table 3-3 shows some structural data of BP86/RI/TZVP optimized 33-a and 33-b
(distances d in pm and angles a in degrees)
Isomer 3-KCN-a 3-KCN-b
d(ZnN) - 206.5
d(ZnC) 208.7 -
d(CN) 117.3 117.5
d(NK) 277.9 -
d(CK) - 291.9
a(ZnNC) - 175.9
a(ZnCN) 176.6 -
a(NCK) - 130.8
a(CNK) 128.8 -
Table 3-3: BP86/RI/TZVP optimized data for 33-a and 33-b.
12CN-33 13CN-33
Results
40
Note that the substitution pattern of the porphyrin was not simplified. The most
relevant structural data obtained from the optimized structures is given in Figure
3-10. It is clear that the structural differences are very small. However, the CN isomer
33-a is more stable than the NC isomer 33-b: the two isomers are energetically
separated by 24.2 kJ/mol. Based on the relative energy of both isomers, the
conclusion may be drawn that the CN isomer 33-a is the one which has been
obtained in experiment. Interestingly, the inherent binding energy of CN- in 33-a
amounts to -478.6 kJ/mol, and is diminished to -409.0 kJ/mol after structural
relaxation of the CN--free metal fragment.
The subsequent mode-tracking calculations converged fast within only two iterations
(starting from a pure CN bond elongation as a guess for the stretching mode) to the
harmonic wavenumbers. For 33-a and 33-b, 2152.8 cm-1 and 2133.1 cm-1 were
obtained respectively. The difference of about 20 cm-1 is not significantly large in
order to distinguish both isomers from each other within the quantum chemical
methodology employed as they depend on the harmonic approximation as well as on
the density functional and basis set chosen.
The experimental IR spectrum shows a very weak peak at 2131 cm-1 and it is thus
tempting to assume that this originates from the NC isomer 33-b. However, one
should keep in mind that the calculated frequencies were obtained within the
harmonic approximation and should thus deviate from experiment. Nevertheless it is
possible to use the additional information obtained in the experimental vibrational
spectrum, namely the infrared intensities, as a starting point for further investigations.
3-KCN-a 3-KCN-b
Figure 3-10: BP86/RI/TZVP optimized structures of the two possible isomers 33-a and 33-b.
Results
41
The vanishing peak in the experimental IR spectrum is rather unusual for CN-
coordination to a metalloporphyrin. However, it corresponds well to the small intensity
calculated for isomer 33-a. But the intensity calculation for 33-b also yields a less
intense peak though its intensity is almost two times larger than in the case of isomer
33-a. Despite the factor of two, both intensities are small and the energy criterion
should be considered decisively. However, the vibrational analyses of the K+-crown-
ether-free analogues of 33-a and 33-b show a vanishing IR intensity for the Zn-CN derivative, while the corresponding Zn-NC system possesses a rather strong IR
absorption for the NC stretching vibration.
These results seem to contradict the expectation that the hard ligand field of the
porphyrin should favor the attachment of the hard N-atom of the cyanide anion to the
central zinc atom. This would disregard the fact that K+ coordinated by the crown
ether is certainly the harder ion and should therefore prefer the coordination of the N-
atom. It seems reasonable to assume that the orientation of the cyanide anion is
controlled by the potassium ion and not by the zinc ion.
Characterization of the Zinc Porphyrin Potassium Superoxide Complex
Another very interesting potassium salt which is coordinated by 30 is potassium
superoxide (KO2). Solutions of 30 in anhydrous aprotic solvents also take up solid
KO2 with a color change[128] from purple to an intense green forming a stable complex
(Scheme 3-6).
O2
ZnN
NN
N
OO
OO
N
O
ZnN
NN
N
OO
OO
N
O
CH2Cl2
K
K+ O2-
Scheme 3-6: Formation the potassium superoxide complex 34.
The uptake and coordination of KO2 in DMF occurs very fast (in minutes) and the
main UV/Vis absorptions of 34 are shifted to 438, 578, and 621 nm. For 30, the corresponding bands can be found at 429, 559, and 603 nm.
34 30
Results
42
Solutions of 34 can be evaporated to dryness without destroying this complex,
although even traces of water have to be avoided carefully.
The coordination of KO2 offers a new, interesting application for our crown ether
system. Porphyrin 30 can incorporate the cheap oxidant potassium superoxide from
the solid phase and transfer it into the organic phase where it can be used for the
oxidation of organic substrates. This process can be seen as a kind of two phase
reaction. In order to determine the phase transfer catalysator capabilities of the zinc
system 30, we investigated the oxidation reaction of benzylalkohol to benzaldehyde
by potassium superoxide (see Scheme 3-7). It could be shown that the complex 34
has the ability to oxidize benzylalkohol 35 to benzaldehyde 36 in cyclohexane
solutions.
OH Ocyclohexane / KO2
Scheme 3-7: Oxidation of benzylalkohol to benzaldehyde by KO2 and 30.
The reaction was performed by adding an excess of solid KO2 (5 eq.) to a solution of
35 (1 eq.) and zinc complex 30 (0.1 eq.) in dry cyclohexane. Monitoring the reaction
by GC revealed that 36 was formed almost quantitatively after 5 h, whereas only
traces of benzoic acid were formed.
The 1H NMR spectrum of compound 34 in dry benzene-d6 (Figure 3-11) shows some
interesting changes compared to the potassium cyanide complex 33. While the
aromatic resonances appear well-resolved in the expected region, the signals of the
crown ether moiety are not so much shifted to higher field. They appear as several
not well-resolved multiplets with low intensities between 1.8 and 3.0 ppm.
Interestingly, the spin density of the O2--ion seems to be strongly localized and
influences the crown ether proton resonances only slightly. The signals of the t-butyl-
groups appear as three singlets with an intensity of 1:2:1 at 1.40, 1.45 and 1.58 ppm
which clearly reveals the Cs-symmetry of the system.
The 13C NMR spectrum of 34 is more or less identical with those of 33 or 30 itself.
30
35 36
Results
43
9.0 8.5 8.0 7.5 3.5 3.0 2.5 2.0 1.5 ppm
A
B
A B
O2
ZnN
NN
N
OO
OO
N
OK
CN
ZnN
NN
N
OO
OO
N
OK
CHCl3
C6H6
Figure 3-11: 1H NMR spectra of KO2-complex 34 (A, C6D6) and KCN-complex 33 (B, CDCl3).
It was not possible to obtain crystals suitable for X-ray crystallography up to now.
Certainly one reason is that the successfully used solvent mixture (THF/water)
cannot be applied here due to the sensitivity of superoxide anions to water.
3.1.3.2 The Cobalt Crown Ether-Porphyrin 37 - A Selective Clamp for Molecules with Two Atoms?
When the zinc ion in the center of the porphyrin is replaced by a cobalt ion, the
situation becomes more complicated. This is not only due to the possible axial
coordination of external ligands but also to redox processes being accessible in this
system.
The metallation is performed by heating an excess of cobalt(II) acetate in THF with
the free base porphyrin 26 to reflux for 12 h. After chromatography on silica the
paramagnetic orange cobalt(II) porphyrin 37 is obtained. The major differences to the
zinc system 30 are the ability of cobalt porphyrins to form octahedral complexes in
contrast to the square pyramidal complexes of 30 and the above-mentioned
possibility of an oxidation reaction by dioxygen leading from cobalt(II) to cobalt(III).
Results
44
The oxidation usually takes place readily as soon as a strong ligand like cyanide is
present. Depending on the used ligand an equilibrium between both oxidation states
is often reached.
Due to their strong UV/Vis absorptions cobalt porphyrins may also be used as
sensors for anions but, in contrast to the zinc system 30, the possible oxidation step
has to be taken into account.
300 350 400 450 500 550 600 650 7000
30000
60000
90000
120000
150000
180000
ε [l
mol
-1 c
m-1]
λ nm
Figure 3-12: UV/Vis spectra (DMF) of Co-porphyrin 37 with different potassium salts.
Figure 3-12 shows the UV/Vis spectra of the cobalt crown ether porphyrin 37
coordinated with different potassium salts. The strongest shifts can be observed for
the coordination of potassium cyanide and potassium thiocyanate. A direct
consequence of the coordination of these ligands is an one-electron oxidation
reaction yielding the corresponding cobalt(III) porphyrins. Both complexes can be
obtained by stirring a solution of 37 in DMF or CH2Cl2 together with solid KCN or
KSCN.
As a result of the coordination and associated oxidation, the color changes from
orange to an intense green in the case of KCN or brown in the case of KSCN. Both
complexes are stable, and the solutions can be taken down to dryness without
destroying the complexes. Afterwards the dry complexes 38 and 39 can be
redissolved in nearly all organic solvents. One difference between both
37
37 + KCN
37 + KNO2
37 + KSCN
37 +KOH
Results
45
complexations was that the oxidation reaction from cobalt(II) to cobalt(III) took place
in a few minutes in the case of KCN whereas the same reaction needed several
hours in the case of KSCN. This is certainly due to the different electronic properties
of both ligands.
L
CoII
N
NN
N
OO
OO
N
O
CoIII
N
NN
N
OO
OO
N
O
CH2Cl2 / O2
K
K+ L-
LL = CN-
L = SCN-
Scheme 3-8: Cobalt porphyrin 37 with potassium salts.
The reactions with other salts such as potassium hydroxide or potassium nitrite give
rise to equilibria between both cobalt species.
Cobalt Porphyrin Potassium Cyanide Complex 38
As mentioned above, the oxidation and coordination is very fast in the case of KCN
and can be observed through a change of the color from orange to green. The main
absorption of the Soret band is shifted bathochromically from 414 nm for 37 to
454 nm for 38.
In contrast to the paramagnetic cobalt(II) porphyrin 37, the NMR spectra of the
diamagnetic cobalt(III) species are clearly resolved and can be fully assigned.
The proton resonances of the crown ether moiety of 38 appear all between 2.2 and
3.1 ppm and are close to the resonances known for the free base porphyrin 26
(Figure 3-16). This behavior was already observed for the zinc porphyrin 33 which
again strongly suggests that the crown ether moiety is moved away from the
immediate vicinity of the porphyrin. Like in the case of the superoxide system 34, the
resonances appear as broad, not well-resolved signals which indicates the dynamic
behavior of the crown ether moiety.
The 13C NMR data of 38 is also more or less identical with those of the zinc porphyrin
33. Again, the carbon resonances of the two cyanide ions were only assignable after
the preparation of a sample with K13CN. Both carbon atoms of the cyanide ions
couple with each other through the cobalt center, and their resonances appear as
37 38
39
Results
46
dublets at 130.7 ppm and 124.4 ppm with a coupling constant of 54.9 Hz (Figure
3-13:). This 2J coupling clearly reveals that the cyanides are bound to the cobalt
center via their carbon atom.
150 140 130 120 110 100 90 80 70 60 50 40 30 20 ppm
13CN
Figure 3-13: 13C NMR spectrum (CDCl3) of K13CN coordinated 38.
The IR spectrum of 38 shows the stretching vibration of the CN- ion at 2079 cm-1
which is in contrast to the IR spectrum of the zinc potassium cyanide system 33. Due
to the fact that there are two cyanide ions present, each in a different environment,
we would expect to see two independent CN vibrations. A possible explanation for
the missing vibration would be that the CN ion enclosed between the cobalt and the
potassium center again has a very low IR vibration intensity.
Crystals suitable for X-ray analysis grew when water was carefully layered on a
THF/CHCl3 solution of 38 (see Figure 3-14). Due to the moderate quality of the
available crystals the accuracy of the geometrical parameters is limited. The
cobalt(III)-ion is situated in an octahedral environment with two coordinated cyanide
ions. By the complexation of a potassium ion in the crown ether the neutrality of the
complex is retained. The strong distortion of the porphyrin macrocycle 38 towards a
saddle-shaped conformation is also described for other six-coordinated cobalt(III)
porphyrin complexes.[136] This is a direct result the very small CoIII-ion in the center of
the porphyrin. The distances between the C-atom and N-atom in the cyanide ions
(1.151 Å and 1.144 Å respectively) are indicative of triple bonds. Both cyanides sit
Results
47
nearly perpendicular on the cobalt atom with regard to the N4-plane of the porphyrin.
The angle between the potassium atom and the CN ion is 151.7°. In contrast to the
zinc system 33, the cobalt atom is almost precisely centered in the porphyrin plane
with Co-NP distances (1.926-1.951 Å) comparable to the reported values for similar
systems.[136] The Co-CCN distances (1.91 Å and 1.94 Å) are in a normal range for
ligands without a steric hindrance.
CNCoKO
Figure 3-14: Structure of 38 in the crystal (protons obmitted).
Cobalt Potassium Thiocyanate Complex
In the case of potassium thiocyanate the oxidation and coordination reaction in
CH2Cl2 takes more than 24 h and can be observed through a change of color from
orange to brown. The main absorption of the Soret band is shifted bathochromically
from 414 nm for 37 to 442 nm for 39.
Crystals suitable for X-ray analysis could be obtained, when water was carefully
layered on a THF solution of 39 (Figure 3-15).
Again, several structural details are noteworthy: first, in contrast to the structure of
Results
48
38, the crown ether moiety with the coordinated potassium sits no longer above the
porphyrin core (distance S-K: 7.759 Å). The crown ether is turned away and points in
the direction of one thiocyanate of a neighboring molecule. Therefore, the distance of
the potassium ion to one thiocyanate of an adjacent molecule is clearly shorter
4.19 Å) than to the thiocyanate in the same molecule.
S
CNCoKO
Figure 3-15: Structure of 39 in the crystal; THF and water have been omitted for clarity.
The main reason for that behavior is probably that the thiocyanate with its three
atoms is just too large to fit well between the cobalt center and the potassium in the
crown ether. The porphyrin macrocycle is again distorted towards a saddle-shaped
conformation which was already described for other six-coordinated cobalt(III)
porphyrin complexes.[136] Both thiocyanates sit again nearly perpendicular on the Co
atom with regard to the N4-plane of the porphyrin. The cobalt atom is almost precisely
centered in the porphyrin plane with Co-NP distances (1.95 Å). The Co-NNCS
distances (1.925 Å and 1.921 Å) are again in a normal range for ligands without a
Results
49
steric hindrance. Four THF molecules and one water molecule are also found in the
unit cell.
Figure 3-16 shows a comparison between the 1H NMR spectra of the free base
porphyrin 26 (C) and the cobalt porphyrins 38 (B) and 39 (A).
9 8 7 6 5 4 3 2 1 ppm
L
CoIII
N
NN
N
OO
OO
N
OK
LA : L = SCN-
B : L = CN-
HN
NNH
N
OO
OO
N
O
C
C
B
A
CH2Cl2
CHCl3
Figure 3-16: 1H NMR spectra (CDCl3) of the porphyrin 26 and cobalt porphyrins 38 and 39.
In the 1H NMR spectra of 39 the proton signals of the crown ether moiety appear at
lower field, shifted for about 1.5-2.0 ppm, and are close to the resonances known for
free monoaza[18]crown-6. However, in this case the signals are better resolved in
comparison to the cobalt porphyrin with coordinated potassium cyanide 38. This
observation is also an important evidence for the crown ether moiety being moved
away from the immediate vicinity of the porphyrin.
The 13C NMR shows only minor changes compared to the cobalt porphyrin 38. It was
not possible to assign the carbon resonances of the thiocyanates.
Looking at the IR-spectrum of compound 39, the C=N stretching vibration of the
thiocyanate groups appears at 2078 cm-1 which is in good accordance with other
thiocyanate coordinated cobalt systems described in the literature.[137, 138] The
frequency of the C=N stretching vibration of solid potassium thiocyanate appears at
2053 cm-1.[139]
Results
50
3.1.4 Synthesis of a Water Soluble System
The crown ether porphyrin 26 and its metal complexes are well-soluble in almost all
organic solvents but not in water. However, most inorganic salts are only soluble in
water or very polar organic solvents. Therefore, it seemed reasonable to investigate
the coordination behaviour of the crown ether systems also in the biologically
relevant solvent water. To achieve water solubility, additional polar addends had to
be introduced into the system.
Starting from the readily available tetrabromoporphyrin 40,[140] the mono crown ether-
porphyrin 41 was easily accessible via a nucleophilic substitution.
ZnN
NN
N
OO
OO
N
O
Br
BrBr
ZnN
NN
N
BrBr
BrBr
ZnN
NN
N
OO
OO
N
O
OO
OO
NH
O
OHO
HOO
OH
OHO
O
ZnN
NN
N
OO
OO
N
OOEtO
EtOO
EtO
OEtO
O
OEt
OEtO
O
OHO
HO O
OEtO
OOEt
/ KH
NaOH
Scheme 3-9: Synthesis of the crown ether-porphyrin 43.
To obtain the crown ether compound 41, a slight excess of the tetrabromo compound
40 was reacted with 1-aza-18-crown 6-ether in toluene yielding 28 % of product
besides unreacted porphyrin 40 which can be recovered (yield based on recovered
material: 76%). 41 still comprises three reactive benzylic bromides where a further
functionalization is possible. In the following step 41 was reacted with a large excess
of diethyl malonate anions in DMF. After chromatography on silica, compound 42
was obtained pure and in good yields (45 %).
40 41
42 43
Results
51
1,60 1,55 1,50
9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm
CH2Cl2
CHCl3 ZnN
NN
N
OO
OO
N
OOEtO
OEtO
EtO
OEtO
O
OEt
OEtO
O
Figure 3-17: 1H-NMR spectrum (CDCl3) of zinc crown ether malonate porphyrin 42.
Figure 3-17 shows the 1H NMR spectrum of 42. It clearly reveals the Cs-symmetry of
the compound, and all expected signals can be assigned. The resonances of the
ethyl ester protons appear as a broad multiplet at 3.7 ppm (CH2) and as three
independent triplets (CH3) around 0.8 ppm (green signals). The crown ether signals
appear as broad, not very well resolved signals between 1.6 and 2.9 ppm (red
signals) whereas the pyrrolic β-protons appear at 8.9 and 8.7 ppm (blue signals).
Even though compound 42 exhibits a substantially higher polarity because of its six
ester groups, it is still not soluble in water.
Nevertheless, water solubility can be achieved by the cleavage of the six ethyl ester
groups with an ethanolic NaOH-solution. The obtained porphyrin 43 possesses six
carboxylic acid groups and it is, as expected, soluble in water, depending on the pH-
value. At high pH-values, 43 is very well-soluble in water because the carboxylic acid
groups are deprotonated and a salt is formed. By lowering the pH-value, the solubility
of 43 drops and at low values insolubility is reached.
Contrary to 30, no shifts in the UV/Vis spectrum can be observed by adding
potassium cyanide to a solution of 43 in water.
Results
52
3.1.5 A Concept for the Synthesis of Oligomeric Porphyrin Crown Ether Arrays – Construction of a Library of Building Blocks
Using the well-established bromomethylated tetraphenylporphyrins[140] with different
symmetries and numbers of substituents as starting material, a large variety of
oligomeric and polymeric systems is imaginable. The nucleophilic substitution
reaction of the different tetraphenylporphyrin precursors with the bisfunctional crown
ether 1,4,10,13-tetraoxa-7,16-diaza-cyclooctadecane 46 was performed to generate
several novel porphyrin-crown ether conjugates. These conjugates bear additional
amino groups in the crown ether moiety where further functionalizations are possible.
N
NN
N
OO
NO
N
O R
HN
NNH
N
Br
N
NN
N
BrBr
N
NN
N
OO
NO
N
O R
OO
NO
N
OR
M
M
M = 2H
M = Zn
M = 2H
M
R = BOC
R = H
R = BOC
R = H
OO
HNO
NHO
1.
2. Boc2O
M = Zn
M = 2H R = BOC
R = H
R = BOC
R = H
M = Zn
OO
HNO
NHO
1.
2. Boc2O
Scheme 3-10: Synthesized crown ether-porphyrin building blocks.
46
25
44
45
54
53
51
52
50
49
48
47
46
Results
53
The reactions of these conjugates with bromomethyl porphyrins allow the
construction of systems with two, three, five and even more porphyrins connected via
crown ether bridges. The use of various metalloporphyrin precursors even allows the
assembly of oligomers with different metal centers. Such systems offer interesting
possibilities for the study of electron-transfer reactions or coordination properties.
Scheme 3-10 shows the synthesized building blocks. The reaction of the bromo
porphyrins 25, 44 or 45 with the diazacrown ether 46 and subsequent protection of
the free amino groups with BOC yielded the crown ether compounds 47, 49, 51, 53.
The implementation of the BOC
protecting groups was necessary
for an easier purification and for
stability reasons. The bisporphyrin
55 (see Scheme 3-11) was
obtained as a useful by-product of
the reaction of 25 with 46. After
the removal of the BOC-protecting
groups with TFA, the porphyrins
48, 50, 52 and 54 can be reacted
further with the bromoporphyrins
44, 45 and 25 to give dimeric,
trimeric or oligomeric porphyrin
arrays. In order to build up well-
defined structures, it is of course
necessary to do this step by step in a controlled manner. Scheme 3-12 shows the
synthesis of a porphyrin triade. By heating an excess of the azacrown ether-porphyrin
52 together with the bisbromoporphyrin 44 in toluene to reflux, the free base triade 57
could be detected in the crude mixture. Due to the high polarity it was not possible to
isolate a pure sample of the product at this stage by chromatography on silica. In
order to reduce the polarity of the system, the crude mixture was metallated with
nickel acetylacetonate. Afterwards, the purification became much easier and yielded
pure compound 58 after chromatography on silica as an orange powder. Nickel was
chosen because of its simple coordination chemistry, tending to form square planar
complexes being coordinatively saturated by the porphyrin core.
N
NN
N
OO
NO
N
O
N
N N
N
M
M
M = 2H
M = Zn2+i
Scheme 3-11: Porphyrin Dimers; i: MeOH, Zn(ac)2.
55
56
Results
54
N
NN
N
OO
NO
N
O
HN
NNH
N
BrBr
N
N N
N
N
N N
N
OO
NO
N
O
HN
NNH
N
OO
HNO
N
O
M
M
M
M = 2H
M = Ni2+ii
i
Scheme 3-12: Synthesis of a nickel porphyrin triade 58; i = toluene, NaHCO3, ii = Ni(acac)2, toluene 2h.
In order to investigate electron-transfer properties as well as the coordination
features of this system it is necessary to substitute the nickel center by a metal which
offers either redox behavior or exhibits at least one additional coordination site.
Metals like zinc, cobalt, and iron would match these requirements.
Figure 3-18 shows the 1H NMR spectrum of compound 58. The resonances of the
pyrrolic protons appear clearly resolved between 8.2 and 8.7 ppm as six signals with
a signal intensity of 2:4:2:4:4:8 (blue signals). At 2.9 and 2.8 ppm the signals of the
benzylic protons appear as two independent signals. The missing capability of the
nickel center to coordinate additional ligands can be best observed by the
resonances of the crown ether moieties. They show only minor shifts compared to
the resonances of the free crown ether (red signals). The Cs symmetry of the system
52 44
58
57
Results
55
can be recognized by the resonances of the t-butyl groups which appear as six
isolated signals with signal intensities of 2:4:2:2:1:1.
9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm
N
NN
N
OO
NO
N
O
N
N N
N
N
N N
N
OO
NO
N
O
Ni
Ni
Ni
Figure 3-18: 1H-NMR spectrum (CDCl3) of nickel triade 58.
Another proof for the purity of compound 58 is the signal at 3309 in the FAB mass
spectrum, which can be assigned to the molecular ion complexed with an additional
sodium.
The characteristic UV/Vis absorptions of 58 can be found at 416 nm and 530 nm.
Only the Soret band is slightly blue-shifted (2 nm) compared to the Soret band
absorption of the monomeric nickel crown ether porphyrin.
Results
56
3.1.6 Rare Earth Metal Porphyrins
Lanthanide porphyrins have provided interesting properties as biomimetic models of
photosynthetic reaction centers[141-143] and NMR shift reagents[144-146]. Attempts for
using lanthanide porphyrin complexes in catalytic reactions have also been
reported.[147, 148] Some species offer an interesting photochemistry which makes them
interesting as potential agents for the photodynamic therapy.[149-151] Especially
gadolinium complexes gain large attention as X-ray contrast agents[146] in computer
tomography, for MR imaging[152, 153] and radiation therapy[154]. In combination with
crown ethers their ability to act as receptors for aminoacids has been reported.[155]
The chemistry of the lanthanide porphyrins is dominated by the large size of the
metal ions. Therefore, coordination numbers of seven or eight are frequently
encountered. Often, lanthanides reach these coordination numbers in porphyrin
complexes by forming double- or triple-decker complexes.[156, 157] Beside their
spectroscopic and physical properties[158] their use in medicine has also gained an
increasing interest.
However, reports about lanthanide monoporphyrinato complexes are not found that
often. The main reason for that is probably the severe out of plane coordination of the
lanthanide ions due to their large ionic radii associated with an increased lability.
Additional ligands are always necessary to stabilize those monoporphyrinato
complexes. In many cases acetylacetone takes over this task and serves as co-
ligand.
With regard to the already mentioned porphyrin-crown ether conjugates it seemed
possible to use the internal crown ether as co-ligand to stabilize oxophilic lanthanide
ions. In this case the metal ion would be enclosed between the porphyrin core and
the crown ether like a “pearl in a clam”. Due to the additional chelating effect of the
crown these resulting complexes should have an increased stability and lower
tendency to form double-decker complexes. A similar behavior was already observed
for the cadmium complex.
The synthesis of the lanthanide complexes was performed by using the
acetylacetonate method. Heating the free base porphyrin 26 together with an excess
of the lanthanide(III) acetylacetonates (Gd(acac)3 ● xH2O or Eu(acac)3 ● xH2O) in
Results
57
1,2,4-trichlorobenzene to reflux yielded the corresponding lanthanide porphyrinates
59 and 60 in high yields (see Scheme 3-13).
MHN
NNH
N
OO
OO
N
O
N
NN
N
OO
OO
N
O
M = Gd3+
M(ac)2 orM(acac)3
TCB
M = Eu3+
Scheme 3-13: Synthesis of rare earth metall porphyrins.
The progress of the metallation reaction can be easily monitored via UV/Vis
spectroscopy. Figure 3-19 shows the UV/Vis spectra of the obtained compounds 59
and 60.
350 400 450 500 550 600 650 700-0,5
0,0
0,5
1,0
1,5
2,0
2,5
3,0
3,5
4,0
Abs
orpt
ion r
el
λ nm
Figure 3-19: UV/Vis spectra of compounds 59 and 60.
Due to the fact that both compounds are paramagnetic, no analysis by NMR-
spectroscopy was performed. Nevertheless, it was possible to obtain crystals of the
gadolinium compound 60 which were suitable for X-ray analysis by slow diffusion of
pentane into a toluene solution of gadolinium porphyrin (Figure 3-20). For a better
visualization the protons are omitted.
59
60
59 60
Results
58
CNGdO
Figure 3-20: Structure of gadolinium porphyrin 60 in the crystal.
Several structural details are noteworthy (Figure 3-20). First, in contradiction to the
expected intramolecular coordination and stabilization of the gadolinium by the crown
ether moiety an intermolecular coordination takes place. Here the crown ether binds
to the gadolinium center of a neighboring molecule and occupies two coordination
sites there. The crown ether of the second molecule binds back to the first one in the
same manner and a dimer is formed. As expected the gadolinium ion is coordinated
by the porphyrin core in a strong out-of-plane fashion. The two remaining
coordination sites are occupied by an acetate anion being formed during the
complexation process as a degradation product of the acetylacetonate. It also
compensates the remaining positive charge of the gadolinium-ion, and a neutral
complex is formed.
Three pentane molecules are also found in the unit cell but were omitted for reasons
of clarity.
Unfortunately, it has not been possible to obtain crystals of the corresponding Eu
species so far.
Results
59
Figure 3-21: Nettels and the algea chlorella.
3.2 The Photodynamic Therapy of Tumors – Construction of Multi-Pyropheophorbide-a-Fullerene Assemblies
3.2.1 Isolation of Pyropheophorbide-a 19
The first problem that had to be solved was the elaboration of an extraction
procedure to obtain the necessary gram-quantities of pyropheophorbide-a 19. There
are several procedures described in the literature,[159, 160] mainly for the extraction of
small quantities of the pigment. With some modifications, the procedures of
HYNNINEN and LÖTJÖNEN were applied for this project.[161-163]
The starting point was the extraction of the chlorophyll pigments from plant material
like dried nettles, frozen
spinach or the green algae
chlorella with an
acetone/water (8:2) mixture.
After the removal of the
magnesium center with 15 %
HCl, the methyl ester group in
position 10 was removed by refluxing the pigments in pyridine/water (see Scheme
3-14). Another method for the
removal of the methyl ester would
be the usage of collidine instead of
pyridine. The final step was the
cleavage of the phytyl ester with
30 % HCl, yielding the free
carboxylic acid group.[163]
During that step it was also
possible to remove the unwanted
pigment pyropheophorbide-b 62
because of its higher hydrochlorid
acid number.[164] If further
purification was necessary, the
crude pyropheophorbide-a 19 was
cleaned by FC on silica with CH2Cl2/MeOH 9:1 as the eluent (see Scheme 3-14).
O O
N
N N
N
OMeO2C
Chlorophyll-a
Mg
HO O
N
NH N
HN
O
1. HCl 15%2. pyridine/H2O3. HCl 30%
Pyropheophorbide-a
HO O
N
NH N
HN
O
O
Pyropheophorbide-b
XanthophyllPyropheophytin-aPyropheophytin-b
carotenes
xanthopyllspyropheophorbide-apyropheophorbide-b
Scheme 3-14: Isolation of chlorophyll-a and transformation to pyropheophorbide-a.
19 61
62
Results
60
Due to the inherent sensitivity of pyropheophorbide-a to light and oxygen all reactions
needed to be carried out with as little light as possible and under an inert
atmosphere.
3.2.1.1 Characterization of Pyropheophorbide-a 19 by 1H NMR- and UV/Vis-spectroscopy
The availability of high-field NMR-spectrometers and modern NMR techniques made
complete signal assignments for many chlorophylls and their degradation products
possible. In this thesis, assignments were made according to the NMR studies of
HYNNINEN and SMITH.[165-168] A good review was also given by ABRAHAM and ROWAN in
1991.[169]
9 8 7 6 5 4 3 2 1 0 -1 ppm
102b
2a
βγ
α
87
5a
4a
1a3a
7a/b
8a
4b
*
*
*
*
HO O
N
NH N
HN
O
11a2
2a2b
33a
44a
4b
5a5
10
77a7b
88a
α
βγ
* = solvent
Figure 3-22:1H NMR spectrum (CDCl3) of pyropheophorbide-a 19.
Figure 3-22 displays the 1H NMR spectrum of pyropheophorbide-a. The typical
absorptions of the meso-positions appear clearly resolved (9.40, 9.29 and 8.51 ppm)
as well as those for the vinylic protons (7.93, 6.17 ppm). Other characteristic signals
are the two dublets of the diastereotopic CH2-group (5.24, 5.07 ppm) adjacent to the
Results
61
keto group and the three singlets of the methyl groups (3.59, 3.36, 3.16 ppm) that are
directly attached to the porphyrin core.
The UV/Vis-spectrum of pyropheophorbide-a 19 (Figure 3-23) shows two main
absorptions at 413 nm and 669 nm respectively. In respect to the aims of this project
particularly the latter one is important. As it was already mentioned in the introduction
(see Chapter 1.3), the light penetration into the tissue increases in correspondence
with an increase of the absorption wavelength.
250 300 350 400 450 500 550 600 650 700 7500
20000
40000
60000
80000
100000
ε [M
ol-1
cm
-1]
λ nm
Figure 3-23: UV/Vis spectrum of pyropheophorbide-a 19 in CH2Cl2.
3.2.2 Synthesis of Fullerene-Pyropheophorbide-a Conjugates Carrying two Chromophoric Units
Before starting with the synthesis of multi-pyropheophorbide-a-fullerene systems, a
strategy had to be developed which was tested on much simpler, less substituted
compounds. The inherent sensitivity of pyropheophorbide-a 19 always required the
introduction of the dye molecules at a very late point of the synthesis.
For the construction of 71, C60 was reacted with a malonate unit connected to a
t-butyldimethylsilyl (TBDMS) mono-protected octane-1,8-diol 66. Utilizing well-known
fullerene chemistry,[106, 170] malonate 66 was attached to C60 and deprotected to give
the fullerene diol compound 70.
413 nm
669 nm
Results
62
OHOSi
ClO
ClO
EtOH / HCl
OO
OO O
O
NNH
NHN
O
OO
NNH
NHN
O
OHHO
HO OO
NNH
NHN
OCH2Cl2pyridine
CH2Cl2pyridine
EtOH / HCl
pyropheophorbide-a1-HOBT / EDCDMAP / THF
pyropheophorbide-a
1-HOBT / EDC DMAP / THF
pyropheophorbide-a1-HOBT / EDCDMAP / THF
TBDMS-ClTHFimidazole
7 7
OO
OO O
O
NNH
NHN
O
OO
NNH
NHN
O
7 7
OO
OO OO
7 7
OO
OO OHHO
7 7
OO
OO OO
7 7
OO
OO OHHO
7 7
SiSi
SiSi
Scheme 3-15: Synthesis of 64, 68 and 71.
Pyropheophorbide-a 19 was subsequently added to this monoadduct by a modified
SHEEHAN-coupling under very mild conditions, giving the desired bis-
pyropheophorbide-a-C60 system 71 (Scheme 3-15). The octyl-chain serves as a
spacer unit. Although it does not play a vital role for the synthesis of 71 (and 68) a
long spacer unit will become an integral part later because, in systems with much
higher dye contents the required space will also increase.
Compounds 64 and 68 were synthesized because they are potential metabolites of
71 and 75 in the human body and could serve as reference materials for
photophysical and photobiological studies.
63
71 70
69
68
67 66
65 64
Results
63
The UV/Vis spectrum of 71 accords well with a molecule that contains two chlorin
chromophores, having absorptions at 414, 508, 538, 609, and 667 nm with extinction
coefficients about twice as large as those of reference compound 64. Also, the typical
UV/Vis absorption of a monosubstituted C60 appears at 257 nm. The NMR spectra
are clearly resolved and thus show no interactions between the pyropheophorbide
units. If π-stacking was present, the resonances would show a significant line
broadening (Figure 3-24). Additionally, no major shifts can be observed that would
indicate a π-stacking of the pyropheophorbide-a units and the fullerene due to its
magnetic anisotropy above the five- and six-membered rings. As it was expected, a
photoinduced electron-transfer from the pyropheophorbide-a moieties to the C60 core
took place (see also Chapter 3.2.7).[171] Unfortunately 71 is not very stable in the
presence of light and/or oxygen.
C60 does not only possess one site for attaching addends but it can also take up to
six of these in an octahedral addition pattern which changes the physical/electronic
properties drastically.[172, 173]
O OO O
OSiOSi
ON
NH NHN
O
HO
EtOH / HCl
DCC / 1-HOBTCH2Cl2
OO
O
O
OO
O
O
OOOO
OO
O
O
OO
O
O
O OO
O
O
OSiSi
OO
O
O
OO
O
O
OOOO
OO
O
O
O
O
O
O
O OO
HO
O
OH
OO
O
O
OO
O
O
OOOO
OO
O
O
OO
O
O
O OO
OO
NNH
N HNO
O
OO
N HN
NNHO
EtO OEtOO
DMA / CBr4 / DBUtoluene
Scheme 3-16: Synthesis of the fullerene hexakisadduct 75.
72
73
74 75
Results
64
Furthermore, the biological behavior changes because the addends can deliver
either a more hydrophilic or a more lipophilic character to the whole molecule.[174-176]
Therefore the C60 hexakisadduct 75 that carries two pyropheophorbide-a units
(Scheme 3-16) was synthesized. The synthesis started with the monoadduct 72
which was then reacted with diethyl malonate, tetrabromomethane (CBr4) and 1,8-
diazabicyclo[5.4.0]undec-7-ene (DBU) taking advantage of the template effect of
9,10-dimethylanthracene (DMA) to give 73.[108] The hexaadduct 73 was deprotected
with a methanolic HCl-solution yielding the diol 74, which was coupled to
pyropheophorbide-a 19 giving the desired compound 75.
As a result of the drastically reduced electron accepting properties of the fullerene
core, the stability of the system as well as the singlet oxygen yield increases (see
3.2.7 and 3.2.8).[177]
Figure 3-24 shows the 1H NMR spectra of compounds 68, 71 and 75. When
comparing the resonances of pyropheophorbide-a 19 with those of 68, 71 and 75,
there are no major changes observable.
9 8 7 6 5 4 3 2 1 0 -1 -2 ppm
*
OO
OO O
O
NNH
NHN
O
OO
NNH
NHN
O
OO
OO O
O
NNH
NHN
O
OO
NNH
NHN
O
OO
O
O
OO
O
O
OOOO
OO
O
O
OO
O
O
O OO
OO
NNH
N HNO
O
OO
N HN
NNHO
Figure 3-24: 1H NMR spectra (*CDCl3) of compounds 68, 71 and 75.
68
75
71
71
75
68
Results
65
Again, the sharp absorptions in the 1H NMR spectrum of 75 indicate that no π-
stacking takes place although models suggest the possibility for such a behavior. The
absorptions of the protons at the meso-positions (9.23, 9.14, 8.47 ppm) as well as
those for the vinylic protons (7.82, 6.14 ppm) and the diastereotopic CH2-protons
(5.12 ppm) adjacent to the keto group of the pyropheophorbide moieties appear
clearly resolved. An integration of the signals shows that two chlorin units per
molecule are present.
The 13C NMR spectrum of compound 75 reveals the high symmetry of the
hexaadduct. Due to the more or less identical malonate substituents attached to the
fullerene, only 3 signals for the carbon atoms of the fullerene core (141.1, 145.8, 69.0
ppm) are found.
The UV/Vis spectrum of 75 proves the presence of two independent
pyropheophorbide-a moieties with the most prominent bands at 414 nm and 667 nm
which reach almost twice the extinction values of one single chromophore. The
typical absorptions of the fullerene hexaadduct at 271 nm and 281 nm which are
clearly visible for the precursor 73 are also found in the spectrum of 75. It is because
of the sixfold addition to C60 that the fullerene core does no longer act as a good
electron acceptor and therefore no photoinduced electron-transfer was observed.[177]
In contrast to the monoadduct 71, the hexaadduct 75 is an efficient singlet oxygen
generator in various solvents.[171, 177]
Results
66
3.2.3 Increasing the Number of Chromophores – Introduction of a Dendritic Unit
After gaining experience in the chemical and photophysical behavior of compounds
68, 71 and 75, the next logical step was to increase the number of
pyropheophorbide-a units 19 attached to the fullerene core. To achieve that goal
there are two possible methods to be considered.
The first possibility is to introduce an additional branching unit. As shown in Scheme
3-17, the NEWKOME-type dendrimer 78 was attached to a modified malonate which
was bound to C60. The synthesis of dendrimer 78 is well-established, and its use is
well documented in the literature.[178, 179] 78 exhibits three protected acid
functionalities where, after deprotection, further modifications are possible. By
reacting 78 with the deprotected malonate 79 via a DCC coupling, the malonate 80
with six t-butylester groups was obtained.
Malonate 80 was coupled to C60 utilizing the modified BINGEL-reaction and
successive deprotection with formic acid yielded the fullerene hexaacid compound
82. To attach the pyropheophorbide-a 19 units, it was necessary to introduce an
additional spacer unit. For this purpose diaminobutane in its mono-protected form
was first coupled to pyropheophorbide-a 19 via a DCC coupling resulting in
compound 83. Deprotection with trifluoroacetic acid (TFA) yielded the amino-
compound 84 which was subsequently coupled to the hexaacid-monoadduct 82 via
an EDC coupling giving the desired compound 85 with six chromophores.
As it was expected from previous results, the new compound 85 with six attached
pyropheophorbide-a units was not very stable (like compound 71) and showed a
strong electron-transfer from the chromophores to the C60 core. Nevertheless it was
possible to isolate a sample for characterization and photophysical investigations.
In order to study the photophysical and biological properties without the influence of
the fullerene core, the malonate 87 was synthesized as a reference compound
(Scheme 3-18).
The synthesis was performed in analogy to that of the monoadduct 85 without
performing the BINGEL-reaction step.
Results
67
O OO O
OO
OO
O OO O
OHO
HOO
O OO O H
NO
HN
O
OO
OO
OO
OO
OO
O O
O OO O
NHOOOOOO
O
HN OOOO O O
O
OHOO
ClO
ClO
CH2Cl2Pyridine
NH2
OO
OO
O O
+
O OO O
NHOOHOOHOOH
O
HN OHO
OHO OHO
O
O OO O
NHONH
ONHONH
O
O
N
NH N
HN
OHN
HN
HN
O NNH
NHN
O
O
NNH
NHN
O
HN ONH
OHN OHN
O
O
N
HNN
NH
O NH
HN
NH
ONNH
N HN
O
O
NHN
NNH
O
HCOOH
C60 / tolueneCBr4 / DBU
DCC / DMAP1-HOBT / CH2Cl2
HCOOH
EDC / DMAP1-HOBT
H2N
O
N
NH N
HN
OHN
BocHN
O
N
NH N
HN
OHN
TFA / CH2Cl2
Scheme 3-17: Synthesis of the monadduct 85 with 6 pyropheophorbide-a 19 units.
83 84
85
82 81
80
79 78
77 76
Results
68
O OO O H
NO
HN
O
OHO
OHO
OHO
OHO
HOOHO O
OO O
OHN
O
HN
O
HNO
HN
ONHO
O
NHN
N
NH
O
NH
NH
HN
O
N HN
NNHO
O
N
NH N
HN
O
NHO
HN
OHN O
O
NHN
N
NH
O
NH
HN
NH
O
NNH
N HN O
O
N
HNN
NH
O
O OO O H
NO
HN
O
OO
OO
OO
OO
OO
O O
EDC / DMAP1-HOBT / DMF
H2N
O
N
NH N
HN
OHN
HCOOH
Scheme 3-18: Synthesis of the malonate 87 with six chromophores.
Both compounds 85 and 87 show strong interactions between the pyropheo-
phorbide-a 19 moieties which leads to a drastically altered physical behavior.
The NMR spectra are no longer clearly resolved and exhibit a strong line broadening
(see also Figure 3-26).
Also, the absorptions in the UV/Vis spectra do not reach the values expected for a
molecule with six chromophores. In addition to that, the singlet oxygen yields and
fluorescence quantum yields are drastically reduced.
Beside the above-mentioned electron-transfer reaction between the
pyropheophorbide moieties and the fullerene core in compound 85, a possibly
massive π-stacking in both compounds would be an explanation for these
observations.
87
86
80
84
Results
69
3.2.3.1 Synthesis of a Hexakisadduct with Six Chromophores
In order to prevent the above-mentioned electron-transfer reaction between the
chromophores and the C60-core as well as to increase the chemical stability, the
conjugation of the fullerene π-system was broken up again. In analogy to compound
71 this was achieved by synthesizing the corresponding hexakisadduct 90 which
carries the malonate with six attached pyropheophorbide-a 19 units and five
additional diethyl malonates. Like hexakisadduct 75, compound 90 exhibits an
increased lipophilic character and has strongly altered physical, electronical and
biological properties compared to the monoadduct 85 (see 3.2.7 and 3.2.8).
The synthesis of 90 started from the monoadduct 81 which was reacted with diethyl
malonate, CBr4 and DBU, taking advantage of the already described template effect
of DMA, giving the hexakisadduct 88.
O OO O
NHOOOOOO
O
HN OOOO O O
O
OO
O
O
OO
O
O
OOOO
OO
O
O
OO
O
O
O OO O
NHOOHOOHOOH
O
HN OHO
OHO OHO
O
OO
O
O
OO
O
O
OOOO
OO
O
O
OO
O
O
O OO O
NHOOOOOO
O
HN OOOO O O
O
OO
O
O
OO
O
O
OOOO
OO
O
O
OO
O
O
O OO O
NHONH
ONHONH
O
O
N
NH N
HN
OHN
HN
HN
O NNH
NHN
O
O
NNH
NHN
O
HN ONH
OHN OHN
O
O
N
HNN
NH
O NH
HN
NH
ONNH
N HN
O
O
NHN
NNH
O
OOEt
OEtO
1. DMA / CH2Cl22. CBr4 / DBU
EDC / DMAP1-HOBT / DMFH2N
O
N
NH N
HN
OHN
+
HCOOH
Scheme 3-19: Synthesis of the fullerene hexakisadduct 90.
Successive deprotection with formic acid yielded the hexaacid 89 which was coupled
to pyropheophorbide-a-amine 84 giving the desired compound 90.
90
89 88
81
84
Results
70
Figure 3-25 shows a comparison between the UV/Vis spectra of the
hexapyropheophorbide (85, 87, 90) and bispyropheophorbide (68, 71, 75)
compounds. For all hexapyropheophorbide-a compounds the Soret band is slightly
split and a second peak has its maximum at 403 nm while the maximum of the
Q-band absorption is bathochromically shifted by 2 nm. In addition to that, their
extinction coefficients do not reach the values that would be expected for a
compound with six chromophores.
Figure 3-25 shows the extinction coefficients of the hexakisadducts 75 and 90 in
comparison to pyropheophorbide-a 19. The green line displays the theoretically
expected spectrum of a molecule with six attached pyropheophorbide molecules
without any interactions. Compounds 75 and 90 exhibit clearly reduced values
compared to the theoretically expected values.
300 350 400 450 500 550 600 650 700 7500
100000
200000
300000
400000
500000
ε l c
m-1
mol
-1
λ nm
Figure 3-25: UV/Vis spectra (CH2Cl2) of compounds 19 (blue), 75 (red) and 90 (black).
As already mentioned above, important changes can be observed in the 1H NMR
spectra of 85, 87 and 90. Although there are no major shifts visible, the signals are
no longer clearly resolved and show an intense line broadening, in particular for the
spectrum of 85. In Figure 3-26 the proton NMR spectrum of 90 is displayed. The
resonances of the pyropheophorbide-a 19 moieties are visible as broad lines (green
signals) whereas the resonances of the ethylester groups appear as sharp multiplets
90
19
75
19 x 6
Results
71
(red signals). Also, the resonances of the dendritic part can be assigned (blue
signals) although they overlap with the pyropheophorbide-a moieties.
9 8 7 6 5 4 3 2 1 0 -1 -2 ppm
CHCl3
OO
O
O
O
O
O
O
OOOO
OO
O
O
O
O
O
O
O OO O
NHONH
ONHONH
O
O
N
NH N
HN
OHN
HN
HN
O NNH
NHN
O
O
NNH
NHN
O
HN ONH
OHN OHN
O
O
N
HNN
NH
O NH
HN
NH
ONNH
N HN
O
O
NHN
NNH
O
Figure 3-26: 1H NMR spectrum (CDCl3) of hexakisadduct 90.
All these observed effects indicate strong interactions between the dye molecules in
the case of the hexapyropheophorbide-a compounds 85, 87 and 90. Due to the
additional spacer unit and high local density of the chromophores, π-stacking is very
likely and it would also be an explanation for the above-mentioned changes.
These interactions are also visible in the fluorescence spectra and other
photophysical measurements of the compounds (see Chapter 3.2.7).
90
Results
72
3.2.4 Hexa-Substituted C60-Systems as Multiplying Units
Our second concept for the accumulation of a high number of chromophores around
the C60-core was the usage the fullerene itself as the multiplying unit. In contrast to
the previously discussed systems 75 and 90, where only one malonate unit carries
the chromophores, it is also possible to construct a system where all six attached
malonic units act as carriers.
In this approach a highly symmetrical hexakisadduct with six identical malonates
carrying BOC-protected aminogroups in their periphery was synthesized.
Deprotection yielded twelve primary amino groups which afford the possibility for
further modifications.
The expansion of this concept is leading to the [5:1]-hexakisadducts. Such mixed
hexakisadducts offer the possibility to combine the coupling sites for the
chromophores as well as an additional coupling site for the attachment of an
addressing unit in the same molecule. As a highly selective addressing unit an
antibody or antibody fragment can be used which makes the targeting of tumor cells
possible.
3.2.4.1 Synthesis of a Dodecapyropheophorbide-a Compound
For the coupling of the pyropheophorbide-a chromophores to the malonic esters a
spacer unit was necessary. The easily accessible BOC-protected aminohexanol
seemed suitable for this purpose. The reaction of the protected aminoalcohol 91 with
malonic acid chloride gave the corresponding malonic ester 92 in good yields.
Successive coupling to C60, applying the already described template activation
method, led to the [6:0]-hexakisadduct 93 (42%). The subsequent cleavage of the
BOC-protecting groups with methanolic HCl yielded quantitatively the free
dodecaamine as its HCl-salt. In this form, it is stable and can be stored.
The last step of the synthesis of 96 was the attachment of the pyropheophorbide-a
19 units to the spacers via amide bonds. In order to prevent side reactions and for
reasons of purification, the dodecaamine was not directly coupled with
pyropheophorbide-a 19. Instead, the N-hydroxysuccinimide (NHS) active ester of
pyropheophorbide-a 95 was first prepared by an EDC-coupling and isolated.
Results
73
O
NNH
NHN O
HNO
NHN
NNHO
NH
OO
OO
O
NHN
NNHO
NHO
NNH
NHN O
HN
OO
OO
ON H
N
NNH
O
NH
O
N
NH N
HN
OHN
OO
O O
ON
NH
NHN
O
HN
O
N
NH N
HN
OHN
OO
O OO
NNH
N HN
O
HN
O
N
HNN
NH
ONH
OO
OO
ONH
N
NNH
O
NH
O
N
HNN
NH
ONH
OO
OO
ClH3N NH3Cl
OO
OO
NH3ClClH3N
OO
OO
NH3Cl
NH3Cl
OO
O O
NH3Cl
NH3Cl
OO
O OClH3N
ClH3N
OO
OO
ClH3N
ClH3N
OO
OO
O OHN
OONH
OO
OO
OONH
O OHN
OO
OO
OO
NH
O
OHN
OO
O O
OO
HN
O
OHN
OO
O OOO
HN
O
O NH
OO
OO
OO
NH
O
O NH
OO
OO
OHN O O
HN O
O O
O OO
HN OH
O OCl
OCl
CH2Cl2Pyridine
1. C60 / DMA / toluene2. Malonate / CBr4 /DBU
HCl
O O
N
NH N
HN
O
NOO
1. TEA
2.
Scheme 3-20: Synthesis of the dodecapyropheophorbide compound 96.
This NHS-active ester 95 was added to the dodecaamine 94, with a 1.5-fold excess
per amino group. The dodecaamine 94 was obtained from its HCl-salt 93 by adding
an excess of triethylamine, and the only by-product of the reaction was NHS. In order
to remove the excess of active ester 95 and the by-product NHS, SEC with
91 92
93 94
95
96
Results
74
BioBeads® SX3 was first used with CHCl3 as eluent. Subsequent chromatography
with BioBeads® SX1, again with CHCl3 as the eluent, gave the pure compound 96 in
excellent, 74 % yield.
The hexakisadducts 93 and 96 show a good solubility in chlorinated hydrocarbons
such as CHCl3, CH2Cl2 and C2H2Cl4. In THF and DMF, only moderate solubility is
observed, whereas toluene, acetonitrile, acetone and alcohols fail to dissolve these
compounds.
Figure 3-27 displays the MALDI-TOF spectrum of compound 96. It shows the
molecular ion peak at 8724.3 (calculated 8722.6) which unambiguously proves the
successful 12-fold amide formation.
4000 5000 6000 7000 8000 9000 m/z0
200
400
600
800
1000
1200
1400
a.i.
Figure 3-27: MALDI-TOF mass spectrum of compound 96.
In Figure 3-28, the 1H NMR spectra of the hexakisadducts 93, 96 and the NHS-ester
of pyropheophorbide-a 95 are compared. Not surprisingly, all resonances in the NMR
spectrum of compound 96 appear again as broad bands. This already indicates the
interaction of the chromophores, most likely in the form of π-stacking. Interestingly, all
pyropheophorbide-related resonances (green signals) are shifted to higher field when
Results
75
compared to those of 95 with an average shift of about -0.4 ppm. By using a ring-
current model for porphyrins,[180] it was possible to estimate an average distance of
the pyropheophorbide units of roughly 4-6 Å from each other, which is in good accord
with the value deduced from molecular modeling studies (see 3.2.7). Temperature-
dependent NMR spectroscopy with 96 was done in C2D2Cl4 at 0, 25, 50 and 70 °C
with a concentration of ~4 x 10-3 M (~5 x 10-2 M per dye) to see an exchange
broadening of the lines. No significant changes were visible in the spectra, which was
also true for lower concentrations (~2 x 10-3 M and ~0.4 x 10-3 M). Obviously, the side
arms are flexible, and the exchange rates are of intermediate order on the NMR time
scale. The concentration of the NMR sample of the monomeric compound 95 was
~5 x 10-3 M, which is, with regard to the dye content, the same as for the
hexakisadduct 96.
9 8 7 6 5 4 3 2 1 0 -1 -2 ppm
*
*
Figure 3-28: 1H NMR-spectra (CDCl3) of compounds 93, 95 and 96.
As sharp signals were found for the monomeric compound 95, we can draw the
conclusion that the broadening effects in the spectrum of 96 are attributed to internal
processes and not to intermolecular interactions. The resonances of the spacer units
of 96 (red lines) exhibit no shifts and appear in the same regions as in the spectrum
of 93 (top red spectrum).
93
95
96
Results
76
The 13C NMR spectrum of 96 is dominated by the resonances of the
pyropheophorbide-a units and shows no major changes compared to the spectra of
compounds 93 and 95.
Figure 3-29 shows the steady-state absorption spectra of the compounds 75, 90 and
96.
300 350 400 450 500 550 600 650 700 750 8000
100000
200000
300000
400000
500000
600000
700000
800000
900000
ε [M
-1 c
m-1]
λ nm
Figure 3-29: UV/VIS spectra (DMF) of 75, 90 and 96.
The electronic absorption spectrum of 96 shows the expected shape but has some
interesting features. Compared to the reference hexapyropheophorbide compound
90 the shape and spectral position of the absorption bands of 96 has practically not
changed. More visible are the changes in comparison to the spectrum of the
bispyropheophorbide compound 75. The maximum of the Q-band absorption of 96 is
shifted bathochromically by 1.5 nm, while the Soret band is split and a second peak
with lower absorbance has its maximum at 403 nm. The extinction coefficients of the
Soret band were determined to have values of 8.8 x 105, 4.1 x 105 and 1.6 x 105 M-1
cm-1 for 96, 90, and 75, respectively, at 414 nm in DMF. Obviously, neither does 90
reach the expected roughly 3-fold increase, nor does 96 attain the 6-fold level of the
extinction coefficient of the reference system 75.
All these effects already indicate the interactions between the dye molecules coupled
to one fullerene. However, the results of such interactions are more visible in the
75
90
96
Results
77
fluorescence spectra and will be discussed in more detail in chapter 3.2.7 (see
page 91).
3.2.4.2 Synthesis of a Tetraeicosapyropheophorbide-a Conjugate
In order to increase the number of photosensitizers attached to each fullerene core to
more than twelve, it is necessary to introduce an additional branching unit because it
is not possible to add more than six malonates to each fullerene core. Due to the
increasing steric hindrance as a consequence of the increasing size of the molecules,
a two-dimensional aromatic system was chosen.
Starting from the commercially available 3,5-diamino benzoic acid 97, the branched
bis-amide 98 was synthesized by applying an EDC-coupling (see Scheme 3-21). Due
to the reduced nucleophilicity of the aromatic amino groups, the yield of the amidation
reaction was clearly lower than that for an aliphatic amine. After the amidation
reaction, 98 was directly transformed into the corresponding NHS-active ester 99,
using EDC and NHS.
NHO
NNH
N HN O
O
N HN
NNHO
HN
ORO
N
NH N
HN
OHO
NH2H2N
OHO
1. EDC / 1-HOBTDMF
2.
R = OH
R = NHSEDC / NHS
Scheme 3-21: Synthesis of the two-dimensional-branching unit 99.
The purification of the active ester 99 is easier in comparison to the free acid 98, and
it was fully characterized. Looking at the proton NMR spectra, only minor changes in
the signal shapes and positions of the pyropheophorbide-a moieties can be
recognized compared to the spectrum of the mono-pyropheophorbide compound 95.
Nevertheless, all absorption bands in the steady-state absorption spectrum of 99 are
shifted bathochromically by 2 nm compared to the mono-pyropheophorbide
compound 95, and they also do not reach the expected 2-fold increase of the values.
98
99
97
Results
78
Successive coupling of the active ester 99 with a 1.3-fold excess per amino group to
the dodecaamine 94 gave the desired hexaadduct 100, carrying 24 pyropheo-
phorbide-a moieties in good yield (29 %) (see Scheme 3-22).
NHO O
HN
ORHN O OO
OO
O
O O
O
O
O
O
O
O
NHO
NNH
N HN O
O
N HN
NNHO
HN
ORHN
NH
O OHN
OHN
O NH
O
O
HN
O
NH
HNO
O
NHO
NH
O
HN
OO NH
O NH
O
NH
O OHN
OHN
OHN
O
O
HN
O
NHO
NHO
O
HN
OHN
ONH
O
O
NH
O
HN
OHN
O
O
NH
O
HN O
HNO
O
HN
ONH
O
NHO
O
NH O
HN
O
HN
OO NH
O NH
=
ClH3N NH3Cl
OO
OO
NH3ClClH3N
OO
OO
NH3Cl
NH3Cl
OO
O O
NH3Cl
NH3Cl
OO
O OClH3N
ClH3N
OO
OO
ClH3N
ClH3N
OO
OO
1. TEA NHO
NNH
N HN O
O
N HN
NNHO
HN
OHO2. Active ester
Scheme 3-22: Synthesis of hexakisadduct 100 carrying 24 pyropheophorbide-a moieties.
For the removal of the excess of 99 and the by-product NHS, the hexakisadduct 100
was purified by repeated SEC. First, Bio-Beads® SX3 was used with CHCl3 as eluent
and successive HPLC-SEC (Nucleogel® GFC 500-10, CHCl3) yielded the pure
tetraeicosapyropheophorbide-a conjugate 100.
The MALDI-TOF mass spectrum (Figure 3-30) of 100 shows the molecular ion peak
at 16539.19 (calculated 16519.86, ΔM=19.33 ≅ 0.12%), which proves the 12-fold
100
99
94
Results
79
amide formation with the active ester 99. A small peak around 13800 may be
assigned to a fragmentation product were one malonate is missing.
10000 12000 14000 16000 m/z0
50
100
150
200
250
300
350
a.i.
Figure 3-30: MALDI-TOF mass spectrum of 100.
Though the proton NMR spectrum of compound 100 shows an intensive line
broadening, most resonances can be assigned to the corresponding
pyropheophorbide-a protons. Interestingly, all signals are again shifted to higher field
for about 0.5 ppm. This indicates the close proximity between the pyropheo-
phorbide-a moieties. Surprisingly, the adding of roughly 20 % of MeOH-d4 to the
NMR probe was necessary to get any resolution in the spectra. This may be due to
the existence of intra- and intermolecular hydrogen bonds which have to be broken
up in order to get a good resolution.
Figure 3-31 shows the 13C NMR spectra of the protected hexakisadduct 93, the
pyropheophorbide-a active ester 75 and tetraeicosapyropheophorbide-a compound
100. The spectrum of 100 is dominated by the resonances of the pyropheo-
phorbide-a units. Additional resonances can be assigned to the fullerene, the spacer
units and the aromatic branching units.
Results
80
200 180 160 140 120 100 80 60 40 20 0 ppm
*
*
Figure 3-31: 13C NMR spectra of the 93, 75 and 100.
The UV/Vis spectra of 99 and the hexakisadduct 100 are compared in Figure 3-32.
Additionally shown (blue line) is the theoretically expected spectrum of a compound
with 12 bispyropheophorbide moieties.
The shape and spectral position of the absorption bands of 100 differ clearly from the
educt 99. The maximum absorption of the Qy-band of 100 is shifted bathochromically
by 3.5 nm compared to that of 99, while the Soret band of 100 is clearly broadened
with its maximum at 400 nm compared to 415 nm for 99. The extinction coefficients
of the Soret band maximum absorptions were determined to have values of 13.9 x
105 (400 nm), 1.3 x 105 (415 nm) and 15.8 x 105 (415 nm) for 100, 99 and 12 x 99 in
CH2Cl2. Obviously, these values do neither reach the expected roughly 24-fold
increase of the values for 100 nor the 2-fold increase for 99 compared to the values
obtained for pyropheophorbide-a (black spectrum).
All these effects again indicate strong interactions between the chromophores. This
excitonic coupling can already be seen for the bispyropheophorbide compound 99
and is, as expected, even more visible for compound 100.
93
75
100
Results
81
250 300 350 400 450 500 550 600 650 700 750 8000
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
2200000
ε [M
-1 c
m-1
]
λ [nm]
Figure 3-32: UV/Vis spectra (CH2Cl2) of 99, 100 and 19 as well as the theoretically expected spectra 12x99 and 24x19.
3.2.5 Synthesis of a Decapyropheophorbide-a-Antibody-Conjugate.
After a protocol for the synthesis of multi pyropheophorbide-a fullerene conjugates
was established, the focus turned to the attachment of an addressing unit to the
conjugate. The easiest way to accomplish this task was to modify the synthesis of the
dodecapyropheophorbide compound 96 by adding a coupling site for a monoclonal
antibody. From literature, there are several possible ways known for attaching large
proteins to organic molecules via, for example, amide and imide formation or disulfide
bridges.[93, 181-184]
The most frequently applied method is the amide coupling by reacting an activated
carboxylic acid species like an active ester or an isocyanate with one free amino
group of the protein. For our purpose, the active ester approach seemed to be more
suitable and was therefore chosen. Due to the reaction conditions applied during the
synthesis of the hexakisadducts and the inherent sensitivity of the
pyropheophorbide-a units, it was necessary to generate the coupling site for the
antibody by using very mild conditions. For synthetic reasons this should also be
done at a very late point of the synthesis. The large dimensions of monoclonal
antibodies (~ 145 kD) as well as the multipyropheophorbide systems, made it
96
10
12 x 99
19
24 x 19
Results
82
necessary to introduce a long spacer unit for the attachment of the antibody.
Starting from 1,20-eicosane diacid 101 (Scheme 3-23), 1,20-eicosanediol 102 was
obtained in high yields after a LAH-reduction. Successive coupling with
methylmalonylchloride yielded the corresponding malonate 103 in only 37%, which
may be due to the poor solubility of the diol compound 102. Malonate 103 was
attached to C60, by using a BINGEL reaction to give the monoadduct 104 in good
yields. The free alcohol group of 103 tolerates the BINGEL-reaction conditions and
does not interfere with the formation of the hexaadduct. Subsequent coupling of five
BOC-protected malonates 92 to the monoadduct 104 by taking advantage of the
aforementioned template activation method yielded the [5:1]-hexakisadduct 105.
HPLC was necessary to remove the pentakisadduct impurities. The overall yield after
the purification procedure was 24 %.
O O
OO
O
ONH
OO
HN
OO
OO
OO
NHO OHN
OO
O
O
OO NH
OO
HNOO
O
O
OO
HN OONH
OO
O
O
R
OO
NH
OOHN
O
OO O
O O OHO O
O O OHO O
HO OHHO OH
O
O
LAH
O ClO O
OHN O O
HN O
O
O O
O1.DMA2. DBU / CBr4
R=OH
R=OTosTsCl / TEA
R= N3NaN3 / DMF
Bingel
Scheme 3-23: Synthesis of the mixed [5:1]-hexakisadduct 107.
107
105
106
92
101 102
104 103
Results
83
Tosylation of the primary alcohol functionality in 105 followed by the substitution of
the tosyl group in compound 106 with sodium azide in DMF gave the azido species
107 in high yields. This azido group is stable during the deprotection conditions
applied for the liberation of the amines as well as the coupling reaction with the NHS-
ester 95. Deprotection of the amino groups with HCl in dioxane and subsequent
coupling with the NHS-active ester of pyropheophorbide-a 95 yielded the
decapyropheophorbide compound 108. 108 was isolated and purified in analogy to
96 by repeated SEC.
ONNH
NHN
O
HN
O
N
HNN
NH
O
NH
O
OOO
O NHN
NNH
O
NH
O
N
NHN
HN
O
HN
O
OO O
O
N HN
NNHO
NH
O
NNH
NHN
O
HN
O
OO
O
O O
OO
O
NNH
N HN O
HN
O
NHN
NNH
O
NH
O
OO
OO
N
HNN
NH
ONH
O
N
NH
NHN
O
HN
OO
OO
1. PMe3 / H2O
2.O O N
O
O
O
O
NO
O
O O
N
NH N
HN
O
NOO
2. TEA
3.
OO
HN
OO NH
O
OOO
OO
NH
OOHN
O
OO O
OONH
OO
HN
O
OO
O
O O
OO
O OHN
OO
NH
O
OO
OO
ONH
OO
HN
OO
OO
N N N
ONNH
NHN
O
HN
O
N
HNN
NH
O
NH
O
OOO
O NHN
NNH
O
NH
O
N
NHN
HN
O
HN
O
OO O
O
N HN
NNHO
NH
O
NNH
NHN
O
HN
O
OO
O
O O
OO
O
NNH
N HN O
HN
O
NHN
NNH
O
NH
O
OO
OO
N
HNN
NH
ONH
O
N
NH
NHN
O
HN
OO
OO
NH
O N
O
O
O
O
17N N N
17
1. HCl/Dioxane
Scheme 3-24: Synthesis of the decapyropheo species 111 with an additional coupling site for forming a connection to a monoclonal antibody.
108
107
111
109
110
95
Results
84
The transformation of the azido group of 108 into an amino group was performed
applying a modified STAUDINGER reaction with trimethylphosphine in THF/water.
The resulting amine 109 was neither isolated nor characterized. Nevertheless, the
MALDI-TOF mass spectrum of the crude mixture clearly shows the molecular ion
peak at 7807.20 (calculated 7794.88) as the main signal. The molecular ion peak of
the educt 108 appears as a small signal at 7836.22 (calculated 7820.88).
Subsequent reaction with a large excess of adipic acid-bis-NHS-ester 110 gave
compound 111 with one remaining activated carboxylic acid group. This active ester
group can be used to establish a covalent bond between the carrier system 111 and
a potential tumor addressing unit like a monoclonal antibody or other proteins,
bearing free amino functionalities. The raw mixture was purified by SEC to remove
the excess of bisactive ester 110.
All compounds except 109 have been characterized by NMR, mass spectrometry, IR
and UV/Vis-spectroscopy.
9 8 7 6 5 4 3 2 1 0 -1 -2 ppm
ONNH
NHN
O
HN
O
N
HNN
NH
O
NH
O
OOO
O NHN
NNH
O
NH
O
N
NHN
HN
O
HN
O
OO O
O
N HN
NNHO
NH
O
NNH
NHN
O
HN
O
OO
O
O O
OO
O
NNH
N HN O
HN
O
NHN
NNH
O
NH
O
OO
OO
N
HNN
NH
ONH
O
N
NH
NHN
O
HN
OO
OO
NH
O N
O
O
O
O
*
*
Figure 3-33: 1H NMR spectrum (CDCl3) of decapyropheophorbide compound 111.
Figure 3-33 exemplarily displays the proton NMR spectrum of 111. Compared to the
spectrum of the highly symmetrical dodecapyropheophorbide compound 96 (see
Results
85
Figure 3-28) the resonances appear no longer as broad singlets due to the reduced
symmetry. Unfortunately, the assignment of all signals of the NHS-ester 111 is not
possible. Therefore, the complete formation of the active ester 111 cannot be
guaranteed.
Nevertheless, the successful generation of the active ester 111 was proven by the
MALDI-TOF mass spectrum. The main signal at 8028.82 can be assigned to the
molecular ion peak (calculated 8019.95), whereas the smaller signal at 7802.34 can
be assigned to the unreacted amino species 109 (calculated 7794.88) or a
fragmentation product. This clearly proves the formation of 111 as the major
compound.
3.2.5.1 Synthesis of the Monoclonal Antibody Conjugate
As already mentioned above, one basic approach to the conjugation of organic
molecules with antibodies is the formation of amide bonds with free amino groups of
the antibody. Therefore, we synthesized a molecule bearing an NHS-active ester
which can address one amino group of the protein chain of the antibody.
The monoclonal antibody Rituximab was provided by Hoffmann La Roche and is
distributed under the commercial name MABTHERA®. This antibody is a genetically
engineered chimeric murine/human monoclonal antibody directed against the CD20
antigen found on the surface of normal and malignant B lymphocytes. The antibody
is an IgG1 kappa immunoglobulin containing murine light- and heavy-chain variable
region sequences and human constant region sequences. Rituximab is composed of
two heavy chains of 451 amino acids and two light chains of 213 amino acids (based
on cDNA analysis) and has an approximate molecular weight of 145 kD. It has a
binding affinity to the CD20 antigen on the cell surface of approximately 8.0 nM.[185]
The chimeric anti-CD20 antibody is produced by mammalian cell (Chinese Hamster
Ovary) suspension culture in a nutrient medium and is purified by affinity and ion
exchange chromatography. MABTHERA® is supplied as a sterile, preservative free
liquid concentrate for intravenous administration, at a concentration of 10 mg/ml in
100 mg (10 ml) vials. The product is formulated in 9.0 mg/ml sodium chloride, 7.35
mg/ml sodium citrate dihydrate, 0.7 mg/ml polysorbate 80, and sterile water. The pH
is adjusted to 6.5.
Rituximab is an already approved drug in several countries. It is indicated for the
treatment of patients with relapsed or refractory, low-grade or follicular, CD20-
Results
86
positive B-cell non-Hodgkin's lymphoma (NHL). More information can be found on
the website of Genentech Inc.[186]
The coupling reaction of the decapyropheophorbide system 111 with the antibody
was performed by dissolving the active ester 111 in a minimum amount of DMF and
successive addition of this solution to a stirred antibody-solution at pH 9. The
concentration of DMF in the final reaction mixture should not exceed 35%.
After 12 h at 4°C, the crude mixture was purified by SEC (Bio-Gel® P-60 eluent:
PBS/DMF 8/2) obtaining a light green fraction. UV/Vis analysis of the obtained green
solution proved that the fraction consists of approximately 60 % of the conjugate
together with approximately 40 % of the non-reacted antibody (see Figure 3-34). This
conclusion is based on the assumption that the coupled antibody does not alter the
absorption spectrum of the attached 111.
250 300 350 400 450 500 550 600 650 700 7500
100000
200000
300000
400000
500000
600000
700000
Abs
orpt
ion no
rm
λ nm
Figure 3-34: UV/Vis spectra (PBS) of Antibody+111 (blue line; theoretically expected) and the obtained conjugate (green line).
Figure 3-35 shows a HYPERCHEM[187] space filling model of a human IgG1 antibody
together with the covalently attached compound 111 (red part). The model of the
IgG1 antibody is a composite model built from F(ab')2 fragments and a Fc fragment.
Part of the hinge region and other details are theoretically modeled. The atomic
coordinate file (PDB) employed was kindly provided by Eduardo PADLAN of the
Antibody+111
obtained conjugate
Results
87
National Institutes of Health, Bethesda, MD, USA.[188] The data is available online at
http://www.umass.edu/microbio/rasmol/padlan.htm.
The attached decapyropheophorbide species 111 (red part) was calculated by using
HYPERCHEM (MM+ mode). It has to be admitted that this picture shows only one
possible conformation. Nevertheless, the model gives a good impression of the
proportions of the conjugate. Of course it is not possible to draw a conclusion about
the coupling site between both units because the active ester 111 does not
selectively address a certain amino group on the surface of the antibody.
Figure 3-35: Model of human IgG1 antibody with covalently attached compound 111.
In vitro investigations on the binding affinity and photodynamic activity of the isolated
conjugate are showing first promising results.
Results
88
3.2.6 Increasing the Solubility in Polar Solvents - Pyropheophorbide-a Derivatives with Polar Side Chains
All the above-mentioned multipyropheophorbide-a species are not soluble in water or
buffer solutions. However, solubility in water or buffer solutions are important
requirements of these compounds for achieving a good conjugation with
biomolecules. In addition to that, the bioavailability of these conjugates is strongly
affected by the solubility of the attached organic components. In order to increase the
solubility, it is necessary to introduce additional polar groups, either to the
pyropheophorbide-a moiety or to the carrier (fullerene). In the case of
pyropheophorbide-a, an easy way to do this is the functionalization of the vinylic side
chain in position 2. Another possibility would be the modification of the keto group in
position 9. Nevertheless, the latter case would strongly affect the UV/Vis spectrum of
the compound.
We decided to perform the introduction of a polar side chain in position 2a. Hereby,
we took advantage of the easy functionalization of the vinylic side chain. Another
positive effect is the almost unaffected UV/Vis-spectrum in which the PDT important
intensive Qy-band absorption around 660 nm is retained. The introduction of a
triethylene glycol chain should, at least in polar solvents, increase the solubility, and
also reduce the tendency of the compounds to form aggregates.
The major disadvantage of that approach is the formation of an additional sterogenic
center leading to the formation of diastereomers.
O
N
NH N
HN
HO O
Br
O
N
NH N
HN
R O
OOOO
O
N
NH N
HN
HO O
HBr /Acetic acid OHOOO
R = OH
R = NHS
R = OCH2CH2OCH2CH2OCH2CH2OCH3
NHS/EDC
Scheme 3-25: Synthesis of the ethyleneglycol-pyropheophorbide compounds 114 and 115.
The triethyleneglycol unit was introduced in a two step procedure, following the
method described by PANDEY et al..[189] First step was the hydro-bromination of the
vinylic double bond in 19, forming the bromo compound 112 by reacting 19 with
114 112 19
113
115
116
Results
89
anhydrous HBr in acetic acid. Subsequent addition of triethyleneglycol monomethyl
ether 113 in the second step gave rise to the glycol compound 114 in moderate
yields. One major by-product was identified as the corresponding ester 116. The
glycol compound 114 has a much higher solubility in polar organic solvents
compared to pyropheophorbide-a 19. With respect to a better conjugation with
proteins, this is a big advantage that should also increase the bioavailability. 114 is
still not soluble in water but fortunately in aqueous mixtures with small amounts of
DMF or ethanol. In order to synthesize conjugates containing higher amounts of 114 it was necessary
to prepare the corresponding active ester first. The NHS-active ester 115 was
obtained in analogy to the synthesis of 95 by the reaction of 114 with NHS and EDC
in CH2Cl2.
10 9 8 7 6 5 4 3 2 1 0 -1 -2 ppm
O
N
NH N
HN
O O
OOOO
NOO
O
N
NH N
HN
O O
NOO
Figure 3-36: 1H NMR (CDCl3) spectra of 95 and 115.
Figure 3-36 shows the 1H NMR spectra of 95 and 115. Compared to the active ester
95 there are some obvious changes visible. The resonances of the vinylic side chain
at 7.8 and 6.2 ppm disappeared, and instead a signal at 6.0 ppm appeared. The loss
of the exocyclic double bond in position 2 causes a highfield shift of the resonances
95
115
Results
90
of the α and β meso-protons whereas the δ-proton exhibits no shift. As expected for
an epimeric mixture, the resonances of the triethylene glycol ether side chain appear
as a multiplet between 3.45 and 3.95 ppm. Beside the new dublets at 2.13 ppm for
the methyl group in position 2b, no other major changes are observed.
The missing vinylic double bond has also some effects with respect to the UV/Vis
spectrum of 115 compared to the reference 95. Whereas the shape of the absorption
peaks is not altered, their positions have moved to higher energies. The Soret band
is hypsochromically shifted by 4 nm from 413 to 409 nm whereas the main Q-band
absorption is even more shifted by 7 nm from 667 to 660 nm. The hypsochromic shift
of the Q-band in particular is a little drawback in respect to the desired long
wavelength absorption of a good photosensitizer. Nevertheless, the disappearance of
the vinylic double bond causes only minor changes in the singlet oxygen quantum
yields.
Results
91
3.2.7 Photophysical Investigations
Photophysical investigations for several pyropheophorbide-a compounds have been
performed by Dr. Eugeny ERMILOV within the photobiophysics group of Prof. Dr.
Beate ROEDER at the Humboldt University of Berlin.
OO
OO O
O
NNH
NHN
O
OO
NNH
NHN
O
OO
OO O
O
NNH
NHN
O
OO
NNH
NHN
O
HOO
NNH
NHN
O
OO
O
O
OO
O
O
OOOO
OO
O
O
OO
O
O
O OO
OO
NNH
N HNO
O
OO
N HN
NNHO
OO
O
O
OO
O
O
OOOO
OO
O
O
OO
O
O
O OO O
NHONH
ONHONH
O
O
N
NH N
HN
OHN
HN
HN
O NNH
NHN
O
O
NNH
NHN
O
HN ONH
OHN OHN
O
O
N
HNN
NH
O NH
HN
NH
ONNH
N HN
O
O
NHN
NNH
O
OO O
OHN
O
HN
O
HNOH
NO
NHO
O
NHN
NNH
O
NH
NH
HN
O
N HN
NNHO
O
N
NH N
HN
O
NHOH
NOHN O
O
NHN
NNH
O
NH
HN
NH
O
NNH
N HN O
O
N
HNN
NH
O
O
NNH
NHN O
HNO
NHN
NNHO
NH
OO
OO
O
NHN
NNHO
NHO
NNH
NHN O
HN
OO
OO
ON H
N
NNH
O
NH
O
N
NH N
HN
OHN
OO
O O
ON
NH
NHN
O
HN
O
N
NH N
HN
OHN
OO
O OO
NNH
N HN
O
HN
O
N
HNN
NH
ONH
OO
OO
ONH
N
NNH
O
NH
O
N
HNN
NH
ONH
OO
OO
HO OO
NNH
NHN
O
Figure 3-37: Photophysically investigated compounds.
Amongst others, the analyzed parameters have been the fluorescence, fluorescence
lifetime and, in respect to the aims of the project most importantly the photosensitized
singlet oxygen quantum yield. Further details, especially on the experimental setups
can be found in the corresponding publications.[171, 177, 190-192]
19 64
68
71
75
87
90
96
Results
92
3.2.7.1 Photophysical properties of fullerene derivatives and reference compounds
Q, nm λem, nm τ, ns ε, M-1 cm-1 ϕ ΦΔ
19 668.0 - 0.52±0.05
64 667.5 674.5 7,0 ± 0,1 4.2⋅104 1 0.50±0.05
68 667.5 674.5 5,2 ± 0,1 8.3⋅104 0.77±0.02 0.43±0.05
71 668.5 674.5 4,7 ± 0,1
0,5 ± 0,1
4.0⋅104 0.09±0.02 0.03±0.05
75 667.5 674.5 5,2 ± 0,1 6.9⋅104 0.77±0.02 0.43±0.05
87 669.0 677 19.2⋅104 0.19±0.02 0.15±0.05
90 670.0 677 18.4⋅104 0.25±0.02 0.24±0.05
96 669.5 679 38.5⋅104 0.075±0.002 0.13±0.05
Table 3-4: Photophysical parameters of compounds 19, 64, 68, 71, 75, 87, 90 and 96 in DMF. The wavelength at the maximum of the last Q-band (Q), the wavelength at the maximum of the emission spectrum (λem), fluorescence lifetime (τ), the molar extinction coefficient (ε) at the maximum absorbance of the last Q band, the fluorescence quantum yields (φ) relative to 64 and the singlet oxygen quantum yields (ΦΔ) are reported.
Table 3-4 shows the photophysical parameters of compounds 19, 64, 68, 71, 75, 87,
90 and 96. Especially the values displayed in the last column are of importance for
this project. The highest singlet oxygen quantum yields are obtained for the
monomeric photosensitizers 19 and 64. Also, 68 and 75 with two
pyropheophorbide-a moieties each reach good values, whereas the monoadduct 71
stands out because of its very low yield.
Nevertheless, these results are expected because it is well-known from the
literature,[193, 194] that the conjugated π-system of the fullerene core (and also that of
the monoadducts) is a very good electron acceptor whereas porphyrins possess the
ability to act as electron donors. As a direct consequence, a strong reduction of the
fluorescence as well as of the singlet oxygen quantum yields was observed for 71
compared to those values of the reference 68. The main reason for that is the
domination of the photoinduced electron-transfer between the initially photoexcited
chromophores and the C60-core with its excellent electron accepting capabilities.
In order to prevent this electron-transfer reaction, it is necessary to break up the
conjugated π-system of the fullerene monoadduct. This is possible by the addition of
five malonate addends in the remaining octahedral positions forming a
hexakisadduct. In such systems the C60 moiety possesses a strongly reduced
Results
93
electron accepting ability and only acts as a neutral attachment. As it was expected,
the fluorescence and singlet oxygen quantum yields of the corresponding
hexakisadduct 75 are clearly higher and reach the same values as the reference
compound 68 without the fullerene.
Looking at the hexakisadduct 90 with six pyropheophorbide molecules, we noticed
that the fluorescence as well as the singlet oxygen quantum yields were reduced
compared to the values of the hexakisadduct 75. This result can be explained by
applying the model of energy traps formed by two closely located excitonically
interacting pyropheophorbide molecules (see Figure 3-38).[191] A similar behavior was
even more visible for hexakisadduct 96 with 12 pyropheophorbide-a molecules. The
strong reduction of the fluorescence as well as of the singlet oxygen quantum yields,
red shifted Q-absorption and fluorescence bands, and non-monoexponential
fluorescence decay of 96 offer unambiguous proof for intense interactions between
the pyropheophorbide chromophores within the fullerene-dye-complex.[192]
It was shown that stepwise intramolecular FÖRSTER energy-transfer between the
pyropheophorbide molecules coupled to one fullerene moiety causes a very fast and
efficient delivery of the excitation to an energy trap formed by two stacked and
excitonically interacting pyropheophorbide chromophores. As a direct result the
fluorescence as well as the singlet oxygen quantum yields of the hexakisadducts 90
and 96 are reduced compared to those values of the reference compound 75.[192]
Due to the higher local concentration of the pyropheophorbide moieties in compound
96 the interactions between pyropheophorbide chromophores should also be
stronger compared to 90. Molecular modelling studies (HYPERCHEM, MM+-method at
room temperature and in vacuum[187]) show that the average distance between two
neighbouring pyropheophorbide molecules belonging to the same fullerene moiety
(Ř) is shorter for 96 than for 90.
Figure 3-38 gives examples of two energetically optimized conformations for both
compounds 90 and 96.[192] It has to be mentioned that each of these pictures shows
just one possible conformation, but nevertheless they visualize an effect that was
visible for all performed calculations. The pyropheophorbide moieties within each
molecule have the strong tendency to stack with each other. Due to the higher local
concentration of pyropheophorbide molecules in 96, this stacking also has a higher
probability compared to 90. The value of Ř was estimated to be 6 Å for 96 and 14 Å
Results
94
for 90. These distances were also estimated from the analysis of the average shift of
resonances in the 1H NMR spectra (see chapter 3.2.4.1).
Figure 3-38: Energetically optimized conformations of 90 and for 96 at room temperature.
It should be mentioned that, since the calculations have been carried out in vacuum,
in solution the stacking effects should be reduced. In fact, the reductions of the
fluorescence as well as of the singlet oxygen quantum yields found experimentally
are not as high as the calculations predicted.
Due to the fact that the calculated FÖRSTER radius for dipole-dipole energy transfer
between the pyropheophorbide chromophores (52 Å) is larger than the average
trap 1
trap 2
Results
95
distance between neighbouring dye molecules attached to one fullerene moiety, the
stacking of just one pair of chromophores leading to excitonic interaction (and as a
result to the formation of the energy trap) is sufficient for a very efficient quenching of
the fluorescence of the whole complex. Because of the higher trap formation
probability for 96 compared to 90 – due to the above-mentioned higher local
concentration of pyropheophorbide chromophores – it is understandable that the
fluorescence of 96 is reduced compared to that of 90. Additionally, the delivery of the
excitation to the traps should occur faster.
It is known from the literature[195-200] that in special cases the formation of chlorophyll
and porphyrin dimers has changed the absorption and fluorescence spectra as well
as it has reduced the fluorescence quantum yields compared to those of the
monomeric compounds. The same effects could be observed for both 90 and 96. In
our case the Soret band was split and the Q-band was red shifted which is
characteristic for face-to-face dimer formation.[159]
It should be mentioned that two different types of energy traps were proposed to exist
in 90 (see Figure 3-38). One of them (Trap I) is formed via the face-to-face stacking
of two pyropheophorbide molecules. The second type of energy trap (Trap II) has an
oblique geometry of the interacting pyropheophorbide molecules.
Beside the changes of the photophysical properties mentioned before, there was
another positive effect noticed during the investigations of the hexakisadduct
systems. By increasing the dye content of the compounds they are getting more
stable under irradition with light. This behavior will be discussed in more detail in the
next paragraph.
3.2.7.2 Photostability of the Hexakisadducts 75, 90 and 96
The photostability of the hexaadduct compounds 75, 90 and 96 with two, six and
twelve pyropheophorbide moieties respectively was estimated through time-
dependent fluorescence measurements. (Figure 3-39) The analysis of the data
reveals an interesting effect: the higher the number of chromophores attached to the
fullerene, the higher the stability of the compound.
This behavior was most prominent for the hexaadduct 96 with twelve
pyropheophorbide-a units. Even after an illumination time of 90 minutes, a decrease
of the fluorescence quantum yield by only 10 % was observed, compared to 30 % for
90 and almost 40 % for 75. This behavior is the direct consequence of the above-
Results
96
mentioned energy traps which cause a very fast and efficient dispersion of the energy
within the molecule.
0 10 20 30 40 50 60 70 80 900,5
0,6
0,7
0,8
0,9
1
Fluo
resc
ence
quan
tum
yiel
d rel
Illumination time, min
759094
625 650 675 700 725 750 775 800
0,00,10,20,30,40,5
0,60,7
0,80,9
1,0
Nor
mal
ized
fluor
esce
nce,
a.u
.
Wavelength, nm
94 before94 after 90 min 75 after 90 min90 after 90 min
Figure 3-39: Fluorescence quantum yields and normalized fluorescence of the hexakisadducts 75, 90 and 96.
Table 3-4 shows the obtained photophysical parameters for the synthesized
hexaadduct compounds 75, 90 and 96. cflτ [ns]
Sample Soret
[nm]
Qa
[nm]
bmaxλ
[nm] 675 nm 707 m
dflΦ e
ΔΦ fISCΦ
75 414 668 674.5 [0.7]
5.7 (—)
(1.0) [0.7]
5.7 (—)
(1.0) 1 0.43 0.49
90 414
403 670 677
[0.071]
1.0
3.7
5.7
(0.40)
(0.11)
(0.39)
(0.1)
[0.071]
1.0
3.7
5.7
(—)
(0.29)
(0.62)
(0.09)
0.33 0.22 0.24
96 414
403 669.5 679
[0.023]
0.34
1.5
4.9
(0.56)
(0.19)
(0.20)
(0.05)
[0.023]
0.28
1.5
3.6
(0.50)
(0.20)
(0.23)
(0.07)
0.098 0.13 0.14
Table 3-5: Photophysical parameters of 75, 90 and 96 in DMF. a) Peak maxima of the absorption Q-band. b) Fluorescence maxima. c) Fluorescence decay times at different registration wavelengths. For all compounds the first decay times are shown in brackets because their values could not be estimated correctly with direct time-resolved fluorescence measurements (due to insufficient time resolution). These times were obtained by ps-TAS experiments. The relative amplitudes of the decay components are given in brackets. d) Fluorescence quantum yields (relative to 75). e) Quantum yields of photosensitized singlet oxygen generation. f) Intersystem-crossing quantum yields.
Results
97
3.2.8 Biological Investigations: In Vitro Experiments with Photosensitizer-Carrier-Systems; Uptake and Phototoxic Activity on Human Lymphoid Cells
In vitro investigations were performed by Fiorenza RANCAN in the group of Prof. Dr.
Fritz BÖHM at the Charitè University Hospital in Berlin. Cell culture experiments were
done with a special line of human T-lymphocytes (Jurkat cells: clone E 6-1, human
acute T-cell leukaemia, ACACC catalogue), human cervix carcinoma cells (HeLa
cells-data not shown) and human fibroblasts (Fi 301). The exact experimental setups
can be found in the corresponding publication and Ph. D. thesis[201, 202].
3.2.8.1 Intracellular Uptake of the Pyropheophorbide-a Compounds
The uptake of 19, 64, 68, 71, 75 and 90 by Jurkat cells was investigated with a
confocal laser scanning microscope and by measuring the fluorescence intensity of
cell extracts at the emission wavelength of pyropheophorbide-a 19 (Figure 3-40). All
compounds were imaged within the cells. The cells displayed a clear pattern of
intracellular fluorescence, which was detected in cytoplasmic compartments but not
within the nuclei. Fluorescence measurements of the cell extracts showed that the
intracellular concentrations of the fullerene complexes after 24 h of incubation are
approximately 27 times lower than the one of the free sensitizer 19 (Figure 3-40). The
kinetics of sensitizer intracellular uptake showed a high intracellular concentration for
19 already 6 h after incubation, while for compounds 64, 68, 71, 75, and 90 a longer
time was necessary to reach their maximal intracellular concentration.
The lower and slower intracellular accumulation of the fullerene derivatives and of 68
is probably due to the uptake mechanism. Lipophilic molecules with molecular
weights lower than 1000 Da normally diffuse through the plasma membranes while
larger molecules, like 68 and fullerene-sensitizer complexes 71, 75 and 90, can be
taken up only by mechanisms such as endocytosis or pinocytosis. These processes
have slower kinetics than passive diffusion through the cell membranes. Moreover,
endocytized molecules enter the lysosomal system and may be degraded by
digesting enzymes or released by exocytosis.
Results
98
FHP1
TTT
cell
extr
. con
c./c
elln
.
0
50
100
150
200
2506h16h24h
Figure 3-40: Intracellular uptake of some compounds. The images of transmitted light (T) and intracellular fluorescence of Jurkat cells were taken with a confocal scanning laser microscope (CLSM 510, Zeiss) equipped with a Helium Neon laser, using λexc=633 nm and λem > 655 nm. The graph reports the amount of photosensitizer equivalents uptaken by Jurkat cells after different incubation times with 19, 64, 68, 75, 90, and 71 at an incubation concentration of 2 µM.
a b c d
a' b’ c’ d’
a b c d
a' b’ c’ d’
Figure 3-41: Lysosomal localization of fullerene hexakisadduct 90 in Jurkat cells. Cells were incubated with a 1µM solution of 90 for 2 h (a,b,c,d) and 24 h (a’,b’,c’,d’). Cells were then incubated 2 h with the lysosome probe (LysoSensor-Green), washed twice and observed with a confocal laser microscope. The images represent: a) transmission picture, b) LysoSensor-Green green fluorescence, c) red fluorescence of 19 and d) superimposed fluorescence images. Lysosomes are the destination of all endocytized compounds. Therefore, beside their big size, the fact that the fullerene complexes are localized in lysosomes is a proof that they are up-taken by endocytosis.
19 64 68
71 7590
19 64 68 71 7590
Results
99
Transmission image
Figure 3-41 shows the intracellular uptake and the localization of the
hexapyropheophorbide compound 90 in the Jurkat cells. The pictures clearly show
that this compound has the tendency to accumulate in the lysosomes which also
speaks for an uptake mechanism via endocytosis.
Beside their uptake by Jurkat
cells the compounds were
also tested with other cells.
Figure 3-42 shows
microscope images of the
intracellular uptake of 64, 68,
75, and 19 by human
fibroblasts. On the left side
the transmission image is
given, while the images on
the right side show the
fluorescence of the internalized photosensitizers. It is clearly visible that fibroblasts
also have the tendency to take up the pyropheophorbide compounds.
3.2.8.2 Photo-Induced Cytotoxicity– Apoptosis vs. Necrosis
In order to assess the effects of photosensitization, the cell membrane disruption, cell
morphology, nuclei fragmentation and caspase 3/7 activity were investigated.
Figure 3-44 shows the results of the compounds after irradiation with doses of
400 mJ/cm2 and 64 mJ/cm2. The rates of necrotic (trypan blue positive) and apoptotic
(fragmented nuclei) cells were determined 24 h after irradiation with a laser diode
(688 nm, 2.12 mW/cm2). After irradiation with a weak light dose (64 mJ/cm2), 100 %
of overall cell death was detected for the cells incubated with 19 and 64, while a very
low phototoxicity was observed for the compounds 68, 71, 75, and 90 (Figure 3-43).
In the case of a stronger irradiation dose (400 mJ/cm2) a higher phototoxicity for all
sensitizers was observed. At this light dose, samples incubated with 19 and 64 had
100 % of overall cell death and the ratio of necrotic cells increased to the detriment of
apoptotic ones. For 90, 75 and 68 the overall dead cell percentages were 76, 58 and
31, respectively.
No dark cytotoxicity was found towards Jurkat cells after 24 h and 48 h of incubation
with all studied sensitizers as well as after incubation with 0.5% DMF.
Figure 3-42: Intracellular uptake through human fibroblasts.
1964
6875
Results
100
400 mJ 64 mJ
020406080
100120
R
dead
cells
%total dead cellsnecrotic cellsapoptotis cells
020406080
100120
R
dead
cells
%
total dead cells
necrotic cells
apoptotis cells
Figure 3-43: Total number of dead cells (necrotic cells vs. apototic cells) under different illumination intensities; R = reference.
Figure 3-44 shows pictures of HeLa cells and Jurkat cells before and after PDT. For
the Jurkat cells a distinction between necrotic and apoptotic cells was made by
staining the cells with special dyes after the PDT: necrotic cells were stained with
trypan blue (TB) whereas apoptotic cells were stained with 4´,6-diamindino-2-
phenylindol dihydrochloride (DAPI).
Nuclei fragmentationapoptotic cells
HeLa cells(a) before and (b) after PDT
Jurkat cells stained with trypan blue(a) before and (b) after PDT
Cell morphology
an
b
a
l
a
b b
a
l
Jurkat cells stained with DAPI(a) before and (b) after PDT
Dye exclusionnecrotic cells
Figure 3-44: Distinction between necrotic and apoptotic cells.
The induction of apoptosis in cells incubated with the studied sensitizers after
irradiation was confirmed by the detection of caspase 3 and caspase 7 activities
(Figure 3-45).
Induction of caspase activity was detected for all investigated sensitizers. The degree
of caspase 3/7 activity resulted in a dependency on the applied light dose. For lower
19 64 68 7175 90 19 64 68 71 75 90
Results
101
illumination intensities, high levels of caspase 3 and 7 activities were detected in cells
incubated with compounds 19 and 64 but not for those incubated with the other
sensitizers. Contrary to that, caspase 3/7 activity was detected for the case in which
a higher light dose was applied and for samples treated with 68, 71, 75, and 90, but
not for those treated with 19 and 64. Actually, at this light dose, most of the cells
incubated with 19 and 64 underwent necrosis (Figure 3-43).
64 mJ/cm2 400 mJ/cm2
0255075
100
casp
ase
3/7
activ
ity%
0255075
100
casp
ase
3/7
activ
ity%
Figure 3-45: Caspase3/7 activity of Jurkat cells incubated with the investigated compounds (μM pyropheophorbide-a equivalent) and irradiated with laser light (668 nm). The diagrams show the dose dependency 4 h after irradiation. Cells incubated with staurosporine (1.5 μM) were used as positive control (St). Activity is expressed as a percentage of the positive control values 4 h after stimulation. R = reference (cells without photosensitzer).
The reason for this effect is probably the high photosensitizing efficiency of 19 and
64. In general, an enhancement of necrotic cells by the detriment of apoptotic ones is
correlated with a higher concentration of reactive oxygen species (ROS). These are
believed to either damage components of the apoptotic pathway preventing the
process or to induce such an extensive damage that cells undergo a rapid necrosis.
Different kinetics of caspase 3/7 activation were found for each sensitizer. Caspase
3/7 activity induced by 75 had a maximum 4 h after irradiation and lasted for further
20 h, while 68-induced caspase 3/7 activity reached its maximum 24 h after
irradiation. Applying a light dose of 64 mJ/cm2, the maximum caspase activity was
detected 4 h after irradiation for 19 and 8 h after irradiation for 64. The different
kinetics of caspase 3/7 activation can be related to the sensitizing efficiency of each
studied compound in the manner that a higher phototoxicity corresponded to a faster
kinetics of caspase 3/7 activation.
On the basis of all prior considerations, a row of increasing phototoxicity can be listed
as following: 71< 68< 75< 90< 64< 19. The hexakisadducts 90 and 75 resulted in
having a significant phototoxic activity while the monoadduct 71 had a very low
phototoxicity even at the highest used light dose. These results show that, even in a
cellular environment, compounds 90 and 75 can induce the production of singlet
oxygen leading to a type II photosensitization mechanism. The low phototoxicity of 71
19 64 68 71 75 90 R St 19 64 68 71 75 90 R St
Results
102
towards Jurkat cells can be attributed to its unfavorable photophysical properties and
its low intracellular uptake. Because of the very efficient photo-induced electron-
transfer from the pyropheophorbide singlet state to the fullerene moiety, 71 has a
very low intersystem crossing yield that results in a low singlet oxygen quantum yield
in polar and nonpolar organic solvents (see also chapter 3.2.7). In addition, the molar
extinction coefficient at 668 nm is much lower than that of the fullerene-free sensitizer
64 (~50%, Table 3-4). The lower absorption at the irradiation wavelength used, also
contributes to its lower phototoxic activity. The fullerene-sensitizer complexes 90 and
75 are less toxic than 19 and 64. This is mainly due to their lower intracellular
concentration, their lower molar extinction coefficient at 668 nm and also to their
lower singlet oxygen quantum yields (0.24 for 90, 0.43 for 75) in comparison to 19
and 64 (0.5). It is interesting to notice that 75 is more phototoxic than its
corresponding fullerene-free reference compound 68 despite having the same singlet
oxygen quantum yields in DMF and additionally, that 68 has a higher molar extinction
coefficient than 75. The reason for that may be the higher intracellular uptake of 75
with respect to 68 (~50% more) after 24 h of incubation. Anyway, these compounds
may have different singlet oxygen quantum yields in an intracellular environment. It
can be assumed that, in aqueous systems, compound 68 has a higher tendency to
form aggregates than compound 75. This may result in a lower singlet oxygen
quantum yield and could explain the lower phototoxicity of 68 with respect to 75.
3.2.8.3 Conclusion of Cell Experiments
Confocal laser scanning microscope images showed that fullerene–
pyropheophorbide-a complexes are incorporated by Jurkat cells, human cervix
carcinoma cells and human fibroblasts. A clear pattern of intracellular accumulation
could be visualized for all sensitizers. Fullerene complexes were found to be less
phototoxic than the fullerene-free sensitizers 64 and 19. This is mainly due to the
high molecular weight of the fullerene complexes. Because of their dimensions, cells
internalize them by non-receptor mediated endocytosis (or pinocytosis), a process
that, with respect to passive diffusion, leads to lower intracellular concentrations. The
introduction of a dendritic unit made it possible to increase the number of sensitizer
moieties coupled to each fullerene molecule. With this strategy a higher accumulation
of the photosensitizers in the cells was reached and the phototoxicity of the complex
was consequently improved. The hexapyropheophorbide-compound 90 was found to
Results
103
have reached the highest intracellular uptake and to have the highest phototoxic
activity of all tested fullerene-pyropheophorbide complexes. Still, the fullerene
complexes were found to be less phototoxic than the fullerene-free sensitizers 64 and
19. Nevertheless, it turned out to be that at high irradiation intensities the
hexakisadducts 75 and 90 favor the apototic way of cell destruction. In fact, that is a
very positive effect in respect to the photodynamic therapy because in apoptosis the
tumor cells are disposed of in an organized manner without extensive inflammation of
the surrounding tissue.
Summary/Zusammenfassung
104
4 Summary / Zusammenfassung The present work can be divided into two independent parts, both concerning
porphyrin chemistry.
In the first section of this thesis, the synthesis and characterization of crown ether-
porphyrin systems were picked up and expanded further, starting with motifs
obtained in my diploma thesis.
The synthesis of the parent system 26 and the precursor porphyrin 25 were
optimized in such a way that it is now possible to obtain both compounds in larger
amounts (1-3 g). Complexes with different transition-metals as well as some
lanthanoides were synthesized as soon as sufficient quantities of the crown ether-
porphyrin 26 were available. As central metals Zn2+,Co2+/3+, Ni2+, Fe3+, Cd2+, Eu3+ and
Gd3+ were chosen.
The influence of the crown ether on the kinetic stability of the corresponding
metalloporphyrins was investigated. This was done by the spectroscopic tracing of
the exchange of the cadmium center by a zinc metal. Compared to the reference
system 31 without the attached crown ether, this exchange reaction takes 3.6 times
longer in 29; clearly, a distinct stabilizing effect could be observed.
Another part of the thesis dealt with the ditopic properties of the zinc- 30 and the
cobalt-system 37. By UV/Vis experiments as well as by X-ray crystallography the
suitability of both systems for binding potassium salts in a ditopic fashion was clearly
proven. The ability of 30 and 37 of taking up solid potassium salts and transferring
them into the organic phase was of special interest. It was possible to obtain crystal
structures of different ditopic complexes of the zinc system 30 and the cobalt system
37. Remarkably, the zinc-KCN-complex 33 and the cobalt-KCN-complex 38 both
incorporate the cyanide ion between the zinc or cobalt atom and the potassium
center in the crown ether. Contrary to that, the structure of the cobalt system with
coordinated potassium thiocyanate 39 shows that the thiocyanate anion is no longer
fixed between both metal centers, even though a ditopic binding is existent. This
indicates that systems similar to 30 and 37 are specially suited for binding anions
consisting of two atoms.
As a potential application of such porphyrin-crown ether conjugates, the oxidation of
benzylic alcohol to benzaldehyde by using the zinc-porphyrin 30 as a phase-transfer
Summary/Zusammenfassung
105
catalyst and solid potassium superoxide as the oxidant was investigated. Indeed,
benzaldehyde was formed under these conditions and, interestingly, no benzoic acid
was found.
Because the systems so far reported are not soluble in water, the porphyrin 43
bearing six free carboxylic groups and a crown ether moiety was synthesized. The
starting point was the symmetric zinc-porphyrin 40 with four benzylic bromides. After
a successful monocoupling with 1-aza-18-crown 6-ether the remaining three benzylic
bromides were substituted by diethyl malonates to give compound 42. Successive
cleavage of the ester groups in 42 by ethanolic NaOH yielded 43 which is soluble in
water at pH-values >7.
Another project within the crown ether-porphyrin section was the construction of
oligomeric porphyrin-crown ether-conjugates. The combination of bisfunctional
porphyrins together with diaza crown ethers led to a library of monomeric building
blocks. These compounds offered the possibility for further functionalizations and the
selective construction of oligomeric systems could be achieved. To test the library,
species 57 was synthesized where three porphyrin units were connected via two
crown ether units. Isolation and full characterization of the corresponding nickel
complex 58 was possible.
The last part of the first section dealt with the synthesis of lanthanoid-metallo-
porphyrins. The goal was to obtain the monoporphyrin complexes 59 and 60 of
europium and gadolinium, respectively. It was possible to obtain the X-ray structure
of the gadolinium porphyrin 60. In the crystal, the crown ether moiety does not serve
as an intramolecular ligand. Instead, a dimer is formed where the crown ether serves
as an intermolecular ligand and occupies two coordination sites of the neighboring
gadolinium complex.
The development of these novel crown ether-porphyrin conjugates offers interesting
chances for further developments. In particular, the field of ditopic receptors can be
taken into the aqueous phase. The oligo-porphyrin-crown ether systems mentioned
before may lead to new materials with interesting electronic and even magnetic
properties. It is also likely that iron or manganese porphyrins which carry crown
ethers may act as oxidation catalysts allowing to take advantage of the cheap oxidant
potassium superoxide in non-polar solvents.
Summary/Zusammenfassung
106
The second section of this thesis dealt with the synthesis of pyropheophorbide-a-
fullerene-conjugates as new drug-delivery systems for the photodynamic tumor
therapy. Starting from the concept of modular drug carrier systems which comprises
of: drug (photosensitizer-pyropheophorbide-a) – multiplying unit (fullerene-dendrimer)
– addressing unit (antibody). The general idea was now to attach a large number of
pyropheophorbides to a tumor-affine antibody via a fullerene as multiplying unit.
Because larger quantities of pyropheophorbide-a (about 4 g per year) were
necessary for the construction of such conjugates, the development of a reliable
method for the isolation of the dye from plant material had to be established. For this
purpose, chlorophyll-a was extracted from different natural products (spinach, nettles,
chlorella algae) on a 100 g to 2 kg scale. The green algae chlorella turned out to be
the best material on a preparative scale and pyropheophorbide-a 19 was obtained in
1-2 g quantities from the plant extracts after several chemical transformations.
At first, comparatively simple systems were synthesized which combine two
pyropheophorbide-a molecules and one fullerene core. The monoadduct 71 was very
sensitive towards light and oxygen and the prevailing photophysical process was an
electron-transfer and not the generation of singlet oxygen. Therefore, the
corresponding hexakisadduct 75 with drastically reduced electron-accepting
properties was synthesized. As a result of the reduced electron-accepting properties,
75 produced singlet oxygen. As a reference for photophysical and photobiological
investigations the corresponding alcohol 64 and the malonate 68 were synthesized.
The next step was to increase the number of pyropheophorbide-a units attached to
the fullerene core. By introducing a dendritic part between the pyropheophorbide-a
moieties and the malonate-unit, it was possible to add six chromophores. The
corresponding monoadduct 87 was again very sensitive and an electron-transfer was
the prevailing process. Contrary to that, the mixed hexakisadduct 90 was much more
stable and had distinctly higher singlet-oxygen-quantum yields. As reference, the
corresponding malonate 85 was synthesized.
A second strategy for an increase of the number of attached pyropheophorbide-a
moieties abandoned the dendritic part and used the fullerene itself as an octahedral
multiplying unit. The addition of six malonates 92 bearing two BOC-protected amino-
functionalities yielded a highly symmetrical hexakisadduct 93 with 12 protected
amino-functionalities. Deprotection and successive coupling with pyropheophorbide-
a-active ester 95 led to 96 carrying 12 photosensitizers. By the additional insertion of
Summary/Zusammenfassung
107
diaminobenzoic acid as a branching unit it was possible, starting from 94, to construct
100 with 24 pyropheophorbide-a moieties. 96 and 100 were obtained in good yields
after SEC and were fully characterized despite their high molecular masses.
Expanding the successful concept using the fullerene as multiplying unit, a mixed
[5:1]-hexakisadduct was synthesized. This species carries 10 pyropheophorbide-a
photosensitizers as well as an additional anchor which is necessary for the coupling
to the antibody. The starting point was the synthesis of a non-symmetric malonate
103 carrying a long alkyl chain with a primary alcohol functionality. The
corresponding monoadduct 104 was synthesized followed by the construction of the
[5:1]-hexakisadduct 105. This system combined 10 BOC-protected amino
functionalities with one free alcohol group in the same molecule. Applying a two step
procedure, the alcohol was transformed into the azide 107 via its tosylate 106. After
the deprotection of the amino groups and successive coupling to the
pyropheophorbide-a moieties, this azido species 108 was reduced to the
corresponding amine 109 by applying the very mild reaction conditions of a
STAUDINGER reaction. The addition of an excess of the bis-NHS-active ester 110 led
to the active ester compound 111. The active ester functionality of 111 was
necessary for the formation of an amide bond between 111 and one amino group of
the antibody. As antibody, the already regulatory approved drug RITUXIMAB was used.
This antibody selectively addresses the CD20-antigen which is preferentially located
on the surface of lymphoma cells. The conjugate was separated from unreacted 111
by SEC and the successful conjugation was proven by UV/Vis spectroscopy. In
preliminary cell culture tests it was shown that the ability of the antibody to recognize
the tumor cells is maintained. Irradiation experiments with this conjugate show
promising results.
As all compounds synthesized so far are not soluble in water and because this is a
highly desirable property for the coupling with biomolecules, the next goal was to
increase the solubility in water. By adding a triethylene glycole unit in position 2a, a
distinct increase of the polarity of the pyropheophorbide-a-species 114 was achieved.
As a direct result the solubility of 114 in polar solvents was much higher compared to
the unsubstituted pyropheophorbide-a 19. The loss of the vinyl side chain had no
effect on the singlet oxygen quantum yields in polar solvents. Importantly, in very
polar solvent mixtures like ethanol/water, the 1O2 yields were significantly higher
compared to the parent system 19.
Summary/Zusammenfassung
108
For all synthesized fullerene-sensitizer-complexes as well as for the corresponding
reference systems photophysical investigations were performed by the
photobiophysics group of Prof. Dr. Beate RÖDER at the Humboldt-University of Berlin.
Beside the measurements of fluorescence, the most important factor was the
determination of singlet oxygen quantum yields.
In vitro cell-experiments with the compounds were performed in the group of Prof. Dr.
Fritz BÖHM at the Humboldt-University of Berlin. The cellular uptake as well as the
ability to act as a photosensitizer were determined. For this purpose, the mortality
rates in cell cultures after incubation and illumination with light were determined.
This project is still in progress and, quite obviously, needs still a lot of work to finally
come forward with a system that fulfills all requirements for a PDT drug. In particular,
the attachment of different carrier systems to antibodies must be developed further.
This thesis has set the foundation for future investigations, which was only possible
due to an intensive cooperation with physicists and physicians.
Summary/Zusammenfassung
109
Zusammenfassung
Die vorliegende Arbeit gliedert sich in zwei unabhängige Teilbereiche der Porphyrin-
Chemie.
Im ersten Abschnitt der Arbeit wurde die Synthese und Charakterisierung von
Kronenether-Porphyrin-Systemen, ausgehend von in der Diplomarbeit erhaltenen
Motiven aufgegriffen und weiter entwickelt.
Zunächst wurde die Synthese des Grundsystems 26 sowie des Vorläufer-Porphyrins
25 derart optimiert, dass es nun möglich ist, beide in größerenen Mengen (1-3 g)
herzustellen. Nachdem ausreichende Mengen des Kronenether-porphyrins 26 zur
Verfügung standen, wurden Komplexe mit verschiedenen Metallen der
Nebengruppen sowie der Lanthanoide hergestellt. Als Zentralmetalle wurden hierfür
Zn2+,Co2+/3+, Ni2+, Fe3+, Cd2+, Eu3+ und Gd3+ gewählt.
In kinetischen Experimenten wurde der Einfluß des Kronenethers auf die kinetische
Stabilität entsprechender Metalloporphyrine untersucht. Anhand des Cadium-
Kronenether-Porphyrins 29 konnte ein stabilisierender Effekt verifiziert werden.
Hierfür wurde die Austauschreaktion des Cadmium-Zentralmetalls in 29 durch Zink
UV/Vis-spektroskopisch verfolgt und mit dem entsprechenden Referenzsystem 31
ohne Kronenether verglichen. Hierbei konnte nachgewiesen werden, dass im
Kronenether-System 29 der Austausch 3.6 mal langsamer verläuft als im
Referenzsystem 31 ohne Kronenether.
Ein weiterer Teil der Arbeit befasste sich mit den ditopischen Eigenschaften des Zink-
30 sowie des Kobalt-Systems 37. Durch UV/Vis-Experimente sowie durch
Kristallstrukturanalysen konnte eindeutig die Eignung beider Systeme zur ditopischen
Koordination von Kaliumsalzen nachgewiesen werden. Besonders hervorzuheben ist
hierbei die Fähigkeit der Verbindungen, Salze aus dem Festkörper in die organische
Phase aufzunehmen. Im Verlauf der Arbeit gelang es, Kristallstrukturen
verschiedener ditopischer Komplexe des Zink-Systems 30 sowie des Kobalt-Systems
37 zu erhalten. Bemerkenswert sind zum einen der Zink-KCN-Komplex 33 sowie der
Kobalt-KCN-Komplex 38, welche beide das Zyanid-Ion fest zwischen dem Zink- bzw.
Kobaltatom und dem Kaliumatom im Kronenether einschließen. Die Kristallstruktur
des Kobalt-Systems mit koordiniertem Kaliumthiocyanat 39 zeigt hingegen, dass,
obwohl immer noch eine ditopische Bindung vorliegt, das Thiocyanation nicht mehr
Summary/Zusammenfassung
110
zwischen den beiden Metallzentren fixiert vorliegt. Dies demonstriert, dass das
vorliegende System besonders gut zweiatomige Anionen binden kann.
Als eine potentielle Anwendung dieser Verbindungsklasse wurde mit Hilfe des Zink-
porphyrins sowie festem Kaliumsuperoxids die Oxidationsreaktion von Benzylalkohol
zu Benzaldehyd in Cyclohexan untersucht. Hierbei wirkt das Kronenether-Porphyrin
als Phasentransferkatalysator und bringt das eigentlich unlösliche Superoxid in die
organische Phase, wo es schließlich als Oxidationsmittel wirkt.
Da die bisher untersuchten Systeme nicht wasserlöslich waren, sollte zudem ein
entsprechendes System aufgebaut werden, welches letztere Eigenschaft besitzt.
Ausgangspunkt war das symmetrische Zink-Porphyrin 40, welches vier benzylische
Bromide trägt. Nach erfolgreicher Monokopplung mit 1-Aza-18-krone-6 wurden die
verbleibenden drei benzylischen Bromide durch Diethylmalonat-Einheiten
substituiert. Im Anschluss wurden die sechs Estergruppen des Porphyrins 42 durch
ethanolische NaOH gespalten und das Porphyrin 43 mit sechs freien
Carboxylgruppen erhalten. Diese Verbindung ist nun in wässriger Umgebung bei
pH>7 löslich.
Ein weiteres Ziel war der Aufbau oligomerer Porphyrin-Kronenether-Konjugate. Unter
Verwendung bisfunktioneller Porphyrine sowie Diazakronenether gelang die
Synthese einer Bibliothek monomerer Bausteine, welche eine weitere
Funktionalisierung zulassen. Mithilfe derartiger Systeme ist der selektive Aufbau
oligomerer Strukturen möglich. Als Beispiel wurde ein Molekül 57 synthetisiert, in
welchem drei Porphyrin-Einheiten über zwei Bisazakronenether verbunden sind. Der
entsprechende Nickel-Komplex 58 konnte gereinigt und vollständig charakterisiert
werden.
Der letzte Abschnitt des ersten Teils befasste sich mit der Synthese von Lanthanoid-
Metalloporphyrinen. Ziel war es hierbei die Mono-Porphyrin-Komplexe des
Europiums 59 und Gadoliniums 60 zu erhalten. Vom Gadoliniumporphyrin konnte
eine Kristallstruktur erhalten werden. Hierbei trägt der Kronenether nicht wie erwartet
intramolekular zur Stabilisierung des Komplexes bei, sondern es bildet sich ein
Dimer. Der Kronenether fungiert hier als intermolekularer Ligand und belegt zwei
Koordinationsstellen des Gadoliniumions des Nachbarmoleküls.
Die Erforschung dieser neuen Kronenether-Porphyrin-Konjugate bietet viel Spielraum
für weitere Entwicklungen. Vor allem auf dem Gebiet der Ditopischen Rezeptoren
eröffnet der Übergang in die wässrige Phase neue Möglichkeiten. Auf dem Gebiet
Summary/Zusammenfassung
111
der vorher erwähnten oligo-Porphyrin-Kronenether-Systeme gelingt es vielleicht,
neue Materialien mit interessanten elektronischen oder sogar magnetischen
Eigenschaften zu finden. Weiterhin ist es vorstellbar, die entsprechenden
Kronenether-Eisen oder Mangan-Porphyrine als Oxidations-Katalysatoren
einzusetzen. Als Oxidationsmittel in unpolaren Lösungsmitteln währe das billige
Kalium Superoxids denkbar.
Der zweite Teil der vorliegenden Arbeit beschäftigte sich mit der Synthese von
Fulleren-Pyrophäophorbid-a-Konjugaten als neue Drug-Delivery-Systeme für die
Photodynamische Tumortherapie. Ausgangspunkt war das Konzept des Modularen
Carrier-Systems, welches aus folgenden drei Bausteinen besteht: Drug
(Photosensibilisator-Pyropheophorbid-a) - Multiplying Unit (Fulleren-Dendrimer) -
Addressing Unit (Antikörper). Ziel war es also, eine möglichst große Anzahl an
Photosensibilisator-Molekülen (Pyrophäophorbid-a) über ein Fulleren als
Verzweigungseinheit an einen tumoraffinen Antikörper zu binden.
Da für den Aufbau derartiger Konjugate größere Mengen (ca. 4 g pro Jahr) an
Pyrophäophorbid-a benötigt werden, war es zuerst notwendig, eine zuverlässige
Methode zur Isolierung aus Pflanzen zu entwickeln. Zu diesem Zweck wurde aus
verschiedenen Naturprodukten (Spinat, Brennessel, Grünalgen) im 100 g bis 2 kg
Maßstab Chlorophyll-a extrahiert wobei sich die Grünalge Chlorella als am besten
handhabbar erwies. In mehreren Folgeschritten wurde anschließend aus dem
erhaltenen Pflanzenextrakt das Pyrophäophorbid-a 19 in 1-2 g Mengen erhalten.
Zuerst wurden relativ einfache Systeme dargestellt, welche zwei Pyrophäophorbid-a
Moleküle gekoppelt an ein Fulleren enthalten. Da das erhaltene Monoaddukt 71 sich
als sehr empfindlich gegenüber Licht und Sauerstoff erwies und der primäre
photophysikalische Effekt ein Elektronentransfer und nicht die Produktion von
Singulett-Sauerstoff war, wurde das entsprechende gemischte Hexakisaddukt 75
synthetisiert. Dieses besitzt nur noch sehr verminderte Elektronenakzeptor-
Eigenschaften und produziert Singulett-Sauerstoff. Als Referenz für die
photophysikalischen und photobiologischen Untersuchungen wurden auch der
entsprechende Alkohol 64 sowie das Malonat 68 synthetisiert.
Der nächste Schritt war die Steigerung der Anzahl ans Fulleren gekoppelter
Pyrophäophorbid-a-Einheiten. Durch den Einbau einer dendritischen Gruppe
zwischen den Pyrophäophorbid-a-Molekülen und der Malonat-Einheit konnte die Zahl
Summary/Zusammenfassung
112
der Pyrophäophorbid-a-Substituenten auf sechs erhöht werden. Das entsprechende
Monoaddukt 87 war erneut nicht sehr stabil und zeigte einen starken
Elektronentransfer als dominierende Reaktion. Das gemischte Hexakisaddukt 90
hingegen, war deutlich stabiler und zeigte relativ hohe Singulett-Sauerstoff-
Ausbeuten. Zu Referenzzwecken wurde auch das entsprechende Malonat 85
synthetisiert.
Eine zweite Strategie zur Steigerung des Pyrophäophorbid-a-Anteils verzichtete auf
den dendritischen Teil und nutzte das Fulleren selbst in Form eines symmetrischen
Hexakisadduktes als oktahedrale Verzweigungseinheit. Durch die Verwendung eines
Malonsäurebisesters 92, welcher zwei BOC-geschützte Aminogruppen trägt, wurde
ein hochsymmetrisches Hexakisaddukt 93 mit 12 geschützten Kopplungsstellen
aufgebaut. Nach Entschützung der Aminofunktionalitäten und anschließender
Kopplung mit dem Pyrophäophorbid-a-Aktivester 95 wurde ein Molekül 96 mit 12
Pyrophäophorbid-a Einheiten erhalten. Unter Verwendung einer
Diaminobenzoesäure als zusätzliche Verzweigungseinheit konnte aus dem
Hexakisaddukt 94 ein System 100 mit 24 Pyropheophorbid-a-Einheiten synthetisiert
werden. 96 und 100 wurden über Größenausschluss-Chromatographie in guten
Ausbeuten rein erhalten und konnten trotz ihres hohen Molekulargewichts vollständig
charakterisiert werden.
Aufbauend auf dem erfolgreichen Konzept des Fullerens als Verzweigungseinheit
wurde nun ein gemischtes [5:1]-Hexakisaddukt synthetisiert, welches neben 10
Pyrophäophorbid-a-Einheiten eine zusätzliche Ankerkette enthält. Diese Ankerkette
ist essentiell, um eine Kopplung zwischen dem Multiplier-Molekül und dem Antikörper
zu erreichen.
Ausgangspunkt war hier die Synthese eines unsymmetrischen Malonsäureesters
103, welcher endständig an einer langen Alkylkette eine primäre Alkoholfunktion
trägt. Nach Darstellung des entsprechenden Monoaddukts 104 sowie anschließend
des [5:1]-Hexakisaddukts 105, erhielt man ein System mit 10 BOC-geschützten
Aminofunktionen und einer freien Alkoholfunktion. Letztere wurde in zwei Schritten
über das Tosylat 106 zum Azid 107 umgesetzt. Nach Entschützung und Kopplung
der Pyrophäophorbid-a-Einheiten wurde die Azido-Verbindung 108 unter den sehr
milden Bedingungen einer STAUDINGER-Reaktion zum entsprechenden primären
Amin 109 reduziert. Durch die Reaktion mit einem Überschuss des Bis-NHS-
Aktivesters 110 der Adipinsäure wurde die Verbindung 111 erhalten, welche eine
Summary/Zusammenfassung
113
aktivierte Säurefunktion trägt. Über diesen Aktivester erfolgte der Aufbau einer
Amidbindung zwischen einer Aminofunktion des Antikörpers und der Verbindung
111. Als Antikörper wurde das bereits gegen das Non-Hodgkin-Lymphom als
Arzneistoff zugelassene Rituximab verwendet. Dieser Antikörper richtet sich selektiv
gegen das CD20-Antigen, welches bevorzugt auf der Oberfläche von Lymphomzellen
auftritt. Nach erfolgter Kopplung wurde das Antikörper-Konjugat über
Größenausschluss-Chromatographie vom unumgesetzten Komplex 111 abgetrennt.
Über UV/Vis-Spektroskopie konnte die erfolgreiche Bildung des Konjugates
nachgewiesen werden. In ersten Zellkulturversuchen konnte gezeigt werden, dass
die Funktion des Antikörpers Tumor-Zellen zu erkennen, erhalten bleibt. In
Belichtungsversuchen zeigte das erhaltene Antikörper-Konjugat vielversprechende
Eigenschaften.
Da alle bisher synthetisierten Systeme in Wasser unlöslich waren, dies jedoch eine
Kopplung mit Biomolekülen überaus erleichtern würde, war die Erhöhung der
Löslichkeit in Wasser ein weiteres Ziel. Durch Einführung einer Trisethylenglykol-
Seitenkette an der Position 2a konnte die Polarität der Pyropheophorbid-a-
Verbindung 114 deutlich erhöht werden. Als Folge stieg die Löslichkeit von 114, im
Vergleich zum unsubstituierten Pyropheophorbid-a 19, in polaren Lösungsmitteln
deutlich an. Auf die Singulett-Sauerstoff-Ausbeute in unpolaren Lösungsmitteln hatte
der Verlust der Vinyl-Seitenkette keinen Einfluss. Im sehr polaren Ethanol-Wasser-
Gemisch hingegen war die Ausbeute sogar deutlich höher im Vergleich zum
Grundsystem 19.
Alle synthetisierten Fulleren-Sensibilisator-Komplexe sowie die entsprechenden
Referenzsysteme wurden im Arbeitskreis Photobiophysik bei Prof. Dr. Beate RÖDER
an der Humboldt-Universität zu Berlin auf ihre photophysikalischen Eigenschaften
untersucht. Neben Fluoreszenzmessungen wurde hierbei vor allem die Singulett-
Sauerstoff Quantenausbeute bestimmt.
Im Arbeitkreis von Prof. Dr. Fritz BÖHM an der Humboldt-Universität zu Berlin wurden
mit den synthetisierten Verbindungen Zellversuche durchgeführt. Hierbei wurde die
Aufnahme der Verbindungen in die Zellen sowie ihre Eignung als Photosensibilisator
zu wirken untersucht. Zu diesem Zweck wurde die Mortalitäts-Rate in Zellkulturen
nach Inkubation und Bestrahlung mit Licht bestimmt.
Das Projekt wird weiter bearbeitet und benötigt offensichtlich noch viel Arbeit, um
letztendlich ein System zu erhalten welches alle Voraussetzungen erfüllt, um einen
Summary/Zusammenfassung
114
guten Arzneistoff für die PDT darzustellen. Vor allem die Kopplung der
unterschiedlichen Carrier-Systeme an den Antikörper muss optimiert werden. Diese
Arbeit legt die Grundlagen für zukünftige Entwicklungen, was wiederum nur durch die
enge Zusammenarbeit mit den Physikern und Zellbiologen von der Humboldt
Universität zu Berlin erreicht wurde.
Experimental
115
5 Experimental Part
5.1 Chemicals and Instrumentation
Chemicals: Most reagents were purchased from Aldrich, Fluka, Sigma, Acros
Organics and Lancaster and, if not otherwise noted, used as purchased. C60 crude
mixture was provided by the Sanofi-Aventis AG (formerly Aventis/Hoechst AG) as a
crude mixture containing higher fullerenes. Purification was done by plug filtration.[203,
204] All analytical-reagent grade solvents were purified by distillation. If necessary, dry
solvents were prepared using customary literature procedures.[205, 206]
Thin Layer Chromatography (TLC): Riedel-de-Haën Silica gel F254. and Merck
Silica gel 60 F254. Detection by means of UV-lamp, H3[P(Mo3O10)4]/
Ce(SO4)2/H2SO4/H2O bath, KMnO4/H2O bath or iodine chamber.
Flash Chromatography (FC): ICN Silica 32-63, 60 Å from ICN Biomedicals.
Size Exclusion Chromatography (SEC): Bio-Beads® SX1, SX3 and Bio-Gel® P60,
from Bio-Rad, USA. The typical parameters e.g. for column diameter, loading,
optimum eluant mixtures, eluant flow rate etc. were determined according to the
handbook provided by the supplier.[207]
Analytical High Performance Liquid Chromatography (HPLC): Shimadzu Class-
LC10 consisting of Liquid Chromatographs LC-10AT, Communications Bus Module
CBM-10A, Diode Array Detector SPD-M10A, Auto Injector SIL-10A, Refractive Index
Detector RID-10A and Selection Valve FCV-10AL. Columns: Nucleosil 200 x 4 mm, 5
µm, Macherey-Nagel; Nucleogel GFC 500-5, Macherey-Nagel. Solvents were
purchased in HPLC grade from Acros Organics or SDS.
Preparative High Performance Liquid Chromatography (HPLC): Shimadzu Class-
LC10 with System Controller SCL-10AVP, Preparative Liquid Chromatographs LC-
8A, Communications Bus Module CBM-10A, UV/Vis Detector SPD-10A, Auto Injector
SIL-10A and Fraction Collector FRC-10A. Columns: Nucleosil 250 x 21 mm, 5 µm,
Experimental
116
Macherey-Nagel; Nucleogel GFC 500-10, Macherey-Nagel. Solvents were purchased
in analytical-reagent quality and purified by distillation.
UV/Vis Spectra: Shimadzu UV-3102 PC, UV-VIS-NIR Scanning Spectrophotometer.
The absorption maxima λmax are given in [nm], the extinction coefficients ε in
[M-1cm-1].
IR Spectra: Bruker FT-IR VECTOR 22. The spectra were measured as KBr pellets or
as thin films of the pure compound on NaCl plates.
ASI Applied Systems REACT IR®-1000 spectrometer (ATR-DiComp-detector). The
spectra were measured as pure solids on a diamond crystal.
All absorptions are given in wavenumbers ν~ [cm-1].
Mass Spectra: Micromass Zabspec FAB+ mode [3-Nitrobenzylalcohol (NBA) as
matrix];
Bruker Daltonics GmbH AUTOFLEX MALDI-TOF machine (2,5-Dihydroxybenzoic acid
as matrix).
NMR Spectra: JEOL JNM EX 400 and JEOL JNM GX 400 (1H: 400 MHz, 13C: 100.5
MHz), Bruker AVANCE 300 (1H: 300 MHz, 13C: 75.4 MHz), Bruker AVANCE 400 (1H:
400 MHz, 13C: 100.5 MHz). The chemical shifts are given in [ppm] relative to SiMe4
(TMS). The resonance multiplicities are indicated as s (singlet), d (dublet), t (triplet), q
(quartet) and m (multiplet), broad resonances as b. The raw data were processed
using the freeware program MestRe-C 2.3.[208]
The numbers in the schemes refer to the assignment of the NMR resonances and not
to nomenclature. In the second part the pyropheophorbide-a signals are indicated
with an asterisk*.
X-ray Crystallographic Analysis: MACH 3 diffractometer by Enraf-Nonius.
Calculations were carried out using SHELX software, the graphics were generated
using ORTEP-3.
Elementary Analysis: CE Instruments, EA 1110 CHNS.
Experimental
117
5.2 Synthetic Procedures
General Procedures (GP) for Ester- and Amide-formation
Several different methods to form esters or amides were used depending on possible
side reactions, reactivity and reaction conditions. Beside the formation of malonates
with malonyl dichloride and pyridine (GP1) and the formation of esters and amides by
activation with DCC(EDC)/DMAP/1-HOBT (GP2), the coupling of amines to
carboxylic acids via the activated ester method by NHS/EDC/DMAP (GP3) was used.
GP1: The formation of malonates by reaction of the corresponding acyl chloride in
CH2Cl2 / pyridine and the alcohol is achieved in good yields for starting compounds
without reactive or any functional groups. In the presence of other reactive groups
protection chemistry has to be used. The following procedure for 66 serves as a
general procedure for analog reactions:
A solution of 8-(t-butyldimethylsilyloxy)-1-octanol 65 (5.2 g, 20 mmol, 2.05 eq.) in dry
CH2Cl2 (250 ml) and pyridine (1.7 ml, 2.15 eq.) was cooled to 0°C under nitrogen.
Malonyl dichloride (0.95 ml, 9.8 mmol, 1 eq.) was diluted with dry CH2Cl2 (10 ml) and
added dropwise over a period of 1 h via a dropping funnel. The reaction mixture was
stirred for 6 h at room temperature and then washed with water (150 ml, three times).
After drying over MgSO4 the solvent was removed in vacuo. FC on silica (ethyl
acetate / hexane 1:1) yielded a colorless oil (5.18 g, 88%).
GP2: The formation of ester and amide bonds was achieved via in situ activation of
the carboxyl group with dicyclohexylcarbodiimide (DCC) or N-dimethylaminopropyl-
N’-ethyl-carbodiimide (EDC) and dimethylaminopyridine (DMAP). The following
procedure for the formation of an amide starting from bis-[5-(pentylcarboxyl)]
malonate 79 and the amine 78 via DCC-activation serves as general procedure for
both ester and amide formation of this reaction type:
Bis-[5-(pentylcarboxyl)] malonate 79 (850 mg, 2.5 mmol, 1 eq.) and the amino
compound 78 (2.55 g, 6 mmol, 2.4 eq.) were dissolved in 50 ml dry THF under N2
and cooled with an ice bath. DMAP (10 mol%, 0.2 eq.) and EDC (2 g, 7 mmol, 2.8
eq.) were added subsequently. After stirring the solution under N2 for 15 min at 0 C
and 2 h at room temperature, TLC control showed complete conversion. The reaction
Experimental
118
mixture was diluted with 150 ml CH2Cl2 and then washed with water (150 ml, three
times). After drying over MgSO4 the solvent was removed in vacuo. FC on silica
(ethyl acetate/CH2Cl2 2:1) yielded the desired amide 80 (2.14 g, 1.89 mmol, 76 %).
GP3: The formation of amide bonds was achieved in a two step procedure via
activation of the carboxyl group with EDC, DMAP and N-hydroxysuccinimide (NHS)
forming an active ester compound. The first step was the formation and isolation of
the NHS-active ester. The active ester could be obtained as a pure product and
stored under exclusion of moisture. By reacting the NHS-active ester with the amine,
the amide was formed with only NHS as a by-product.
The following procedure for the formation of pyropheophorbide-a-NHS active ester 95
serves as a general procedure for the formation of NHS-active esters.
Pyropheophorbide-a 19 (800 mg, 1.5 mmol, 1 eq.) was dissolved in dry CH2Cl2
(50 ml). NHS (213 mg, 1.8 mmol, 1.2 eq.), DMAP (24 mg, 0.2 mmol, 0.13 eq.) and
EDC (390 mg, 2 mmol, 1.35 eq.) were added under N2 at room temperature. The
solution was stirred for 12 h. Subsequent removal of the solvent in vacuo and FC on
silica (CH2Cl2/acetone, 9:1) yielded the desired NHS-ester 95 (420 mg, 0.6 mmol,
44 %).
General Procedure for the Formation of C60 Monoadducts (GP 4)
The synthesis of C60 monoadducts was performed using the modified BINGEL
reaction.[107] To obtain higher yields of the desired monoadduct and less multiple
adducts an excess of C60 was applied. The unreacted C60 could be recovered easily
by FC on silica with pure toluene. C60 (1.5 eq.) was dissolved in dry toluene (ca.
0.5 ml toluene per mg C60) resulting in a dark purple solution. Afterwards, CBr4
(1.1 eq.) and the malonate (1 eq.) were added. DBU (1.2-2.0 eq.) was diluted in
toluene and added dropwise over a period of 1 h to the stirred solution at room
temperature. After the solution was stirred for 12 h, TLC control showed the
remaining C60, the monoadduct and traces of bis- and trisadducts. The toluene was
removed in vacuo and then the reaction mixture was transferred to the FC column.
The pure monoadduct was obtained with 25-40 % as a typical yield depending on the
attached malonate. The essential characteristics for C60 monoadducts are found in
the UV/Vis spectrum [257 (ε = 120000), 326 (38000), 426 (3000), 481 (2000) nm)]
Experimental
119
and the 13C-NMR spectrum (16 sp2 resonances at 145-138 ppm, one sp3 resonance
at 71 ppm and one methano resonance at 52 ppm).
General Procedure for the Formation of [6:0]-, and [5:1]-Hexakisadducts of C60 (GP5)
The synthesis of Th-symmetrical hexakisadducts of C60 is similar for the two different
starting materials. The starting material (C60 or monoadducts of C60) determines the
addition pattern ([6:0] or [5:1]) of the obtained hexakisadduct. The following
procedure is based on the literature for [6:0]-hexakisadducts[108, 109, 174] and can be
adapted for [5:1]-hexakisadducts by division of the excess amounts of reacting
compounds by 1.2.
C60 (1 eq.) was dissolved completely in degassed toluene (dry or HPLC-grade) under
nitrogen. High dilutions should be avoided. A large excess of DMA (10-12 eq.) was
added to the solution and stirred for 12 h at room temperature. The malonate and
CBr4 were added subsequently in the same excess like DMA. After stirring for a few
minutes to allow complete dissolution, the double excess of DBU diluted in dry
toluene (10 ml) was added dropwise over the period of 1 h. The solution was stirred
for 1 to 3 days at room temperature under N2 until TLC control remains unchanged.
After removal of the solvent in vacuo a preliminary separation by FC on silica
followed to remove the non polar DMA and highly polar by-products. Subsequent
purification was done by HPLC which enables the separation of hexa- and
pentakisadducts. The yields for the pure [6:0]-hexakisadducts or [5:1]-hexakisadducts
ranged between 30 % and 40 % relative to the applied C60 compound.
The following compounds were prepared according to literature procedures. The
synthetic procedures as well as the characterization of these compounds are not
described in detail in this section.
52-(Bromomethyl)-54,104,154,204-tetra-t-butyl-56-methyl-5,10,15,20-tetraphenyl-
porphyrin 25[140]
52,56-Bis-(bromomethyl)-54,104,154,204-tetra-t-butyl-5,10,15,20-tetraphenylporphyrin
44[140]
Zinc-52,56,152,156-tetra-(bromomethyl)-54,104,154,204-tetra-t-butyl-5,10,15,20-
tetraphenylporphyrin 40[140]
Experimental
120
8-(t-Butyldimethylsilyloxy)-1-octanol 65[209]
6-(N-t-Butoxycarbonylamino)-1-hexanol 91 [210]
6-Hydroxyhexanoic acid t-butylester 76 [211, 212]
4-Amino-4-(2-t-butoxycarbonyl-ethyl)-heptanedioic acid di-t-butyl ester 78 [179]
52-[N-(4-aza-18-crown-6)methyl]-54,104,154,204-tetra-t-butyl-56-methyl-5,10,15,20-tetraphenylporphyrin 26
52-(Bromomethyl)- 54,104,154,204-tetra-t-butyl-
56-methyl-5,10,15,20-tetraphenylporphyrin 25
(950 mg, 1 mmol), 4-aza-18-crown-6 ether
(290 mg, 1.1 mmol) and NaHCO3 (92 mg,
1.1 mmol) were dissolved in 25 ml of dry
toluene and heated to reflux for 24 h. The
solvent was removed in vacuo and after FC
on silica (ethyl acetate/methanol 9:1) a violet
solid was obtained (yield: 914 mg, 0.81 mmol,
81 %)
1H NMR (400 MHz, CDCl3, 25 °C): δ = 8.91 (s, 4H, 12, 13), 8.87 (d, 3J = 4.7 Hz, 2H,
2), 8.67 (d, 3J = 4.7 Hz, 2H, 3), 8.22 (d, 3J = 8.1 Hz, 2H, 152, 156), 8.17 (d, 3J = 7.7 Hz, 4H, 102, 106, 202, 206), 7.87 (d, 4J = 1.7 Hz, 1H, 53), 7.79 (m, 6H, 103,
105, 153, 155, 203, 205), 7.53 (d, 4J = 1.7 Hz, 1H, 55), 3.19 (s, 2H, 52a), 3.16 (m, 4H,
C6), 3.14 (m, C5), 2.98 (m, 4H, C4), 2.92 (m, 4H, C3), 2.69 (t, 3J = 6.1 Hz, 4H, C2),
2.26 (t, 3J = 6.1 Hz, 4H, C1), 1.97 (s, 3H, 56a), 1.64 (s, 27H, 104b, 154b, 204b), 1.63 (s,
9H, 54b), -2.58 (s, 2H, NH). 13C NMR (100 MHz, CDCl3, 25 °C): δ = 150.9, 150.4, 147-145 (α-pyrr), 140.9, 139.3,
139.1, 138.9, 138.0, 134.5, 131-130 (β-pyrr), 124.6, 123.6, 123.6, 123.12, 120.2,
119.8, 117.5, 70.2 (C5, C6), 70.0 (C4), 69.7 (C3), 69.4 (C2), 59.1 (52a), 53.5 (C1), 34.9
(54a, 104a, 154a, 204a), 31.8 (104b, 154b, 204b), 31.7 (54b), 21.9 (56a).
IR (KBr): ν~ = 3316, 2957, 2902, 2865, 1474, 1362, 1350, 1108, 967, 801.
MS (FAB, NBA): m/z = 1129 [M+], 865 [M+-Aza crown ether].
UV/Vis (CH2Cl2): λmax (ε, M-1cm-1) = 421 (335000), 517 (13500), 552 (6770), 590
(4060), 646 (4060).
HN
NNH
N
OO
OO
N
O56
12
3 4 5
10
1112
1314
152
5252a
C4
C1
C3
C2
C5 C6
5454a
54b
56a
102 103
104
104a104b
5355
15151
153
154
154a
154b
105106
155156
Experimental
121
Elemental analysis: C74H89N5O5·2H2O (1163.71), calcd: C, 76.32; H, 8.05; N, 6.01,
found: C, 76.71; H, 7.72; N, 6.05.
Zinc-52-[N-(4-aza-18-crown-6)methyl]-54,104,154,204-tetra-t-butyl-56-methyl-5,10,15,20-tetraphenylporphyrin 30
52-[N-(4-aza-18-crown-6)methyl]- 54 ,104 ,154,
204-tetra-t-butyl-56-methyl-5 ,10 ,15 ,20- tetra-
phenylporphyrin 26 (200 mg, 0.18 mmol) and
zinc acetate (220 mg, 1 mmol) were dissolved
in 40 ml CH2Cl2/methanol (1:1) and stirred for
16 h at ambient temperature. The solvent was
removed in vacuo and FC on silica gel (ethyl
acetate/methanol 9:1) yielded a pink solid
(yield: 205 mg, 0.17 mmol, 96 %).
1H NMR (400 MHz, CDCl3, 25 °C): δ = 8.88 (s, 4H, 12, 13, 17, 18), 8.83 (d, 3J =
4.6 Hz, 2H, 2, 8), 8.66 (d, 3J = 4.6 Hz, 2H, 3, 7), 8.14 (m, 3H, 102, 152, 202), 8.04 (m,
3H, 106, 156, 206), 7.71 (m, 6H, 103, 105, 153, 155, 203, 205), 7.74 (s, 1H, 53), 7.52 (s,
1H, 55), 3.06 (s, 2H, 52a), 2.34 (bs, 4H, C1-6), 2.28 (bs, 4H, C1-6), 2.18 (bs, 4H, C1-6),
2.05 (s, 3H, 56a), 2.00 (bs, 4H, C1-6), 1.73 (bs, 4H, C1-6), 1.64 (bs, 4H, C1-6), 1.61 (s,
9H, 54b), 1.60 (s, 18H, 104b, 204b), 1.55 (s, 9H, 154b). 13C NMR (100 MHz, CDCl3, 25 °C): δ = 150.6, 150.1, 149.9, 149.6, 149.3, 140.2,
140.1, 138.9, 138.4, 134.5, 134.4, 134.2, 132.1, 131.6, 130.8, 124.6, 123.4, 123.2,
122.8, 120.7, 120.4, 118.4, 69.2, 68.9, 68.6, 68.5, 58.0 (1C, 52a), 53.1 (2C, C1), 34.9,
34.8 (54a, 104a, 154a, 204a), 31.7 (54b, 104b, 154b, 204b), 22.2 (54b).
IR (ATR): ν~ = 2953, 2903, 2868, 1478, 1459, 1363, 1339, 1204, 1112, 1065, 995,
810, 718.
MS (FAB, NBA): m/z = 1190 [M+], 927 [M+-crown ether].
UV/Vis (CH2Cl2): λmax (ε, M-1cm-1) = 429 (494500), 559 (16600), 603 (8100).
Elemental analysis: C74H87N5O5Zn·1.5H2O (1216.11), calcd: C 72.92, H 7.44,
N 5.75; found: C 72.92, H 7.47, N 5.59.
N
NN
N
OO
OO
N
O
Zn
56
12
3 4 5
10
1112
1314
152
5252a
C4
C1
C3
C2
C5 C6
5454a
54b
56a
102 103
104
104a104b
5355
15151
153
154
154a
154b
105106
155156
Experimental
122
Potassium cyanide complex of Zinc-52-[N-(4-aza-18-crown-6)methyl]-54,104,154,204-tetra-t-butyl-56-methyl-5,10,15,20-tetraphenylporphyrin 33
Zinc-52-[N-(4-aza-18-crown-6)methyl]-54 ,104 ,
154 ,204- tetra-t-butyl-56-methyl-5, 10, 15, 20-
tetraphenylporphyrin 30 (120 mg, 0.1 mmol)
was dissolved in 20 ml CH2Cl2 and solid
potassium cyanide (66 mg, 1 mmol) was
added. The pink solution was stirred for 24 h
at ambient temperature, filtered and the
residue was then subject to size exclusion
chromatography over Bio-Beads® SX3 with
CH2Cl2 as the eluent. The solvent was
removed in vacuo yielding a violet solid (yield: 118 mg, 0.09 mmol, 94 %).
1H NMR (400 MHz, CDCl3, 25 °C): δ = 8.73 (d, 3J = 4.2 Hz, 4H, 12, 13, 17, 18), 8.70
(d, 3J = 4.5 Hz, 2H, 2, 8), 8.53 (d, 3J = 4.5 Hz, 2H, 3, 7), 8.27 (dd, 3J = 7.9 Hz, 4J =
1.8 Hz, 2H, 102, 202), 8.20-8.05 (bs, 2H, 152, 156), 7.95 (dd, 3J = 7.9 Hz, 4J = 1.8 Hz,
2H, 106, 206), 7.70 (dd, 3J = 7.9 Hz, 4J = 2.0 Hz, 4H, 103, 203), 7.72-7.60 (bs, 2H, 153,
155), 7.62 (d, 3J = 7.9 Hz, 4J = 2.0 Hz, 2H, 105, 205), 7.56 (s, 1H, 53), 7.31 (s, 1H, 55),
3.15 (s, 2H, 52a), 3.16-2.50 (bm, 24H, C1-C6), 2.31 (s, 3H, 56a), 1.60 (s, 9H, 154b),
1.59 (s, 18H, 104b, 204b), 1.58 (s, 9H, 54b). 13C NMR (100 MHz, CDCl3, 25 °C): δ = 150.5, 150.3, 149.5, 149.4, 149.0, 148.4,
144.8 (CN-), 141.5, 140.6, 140.4, 140.0, 138.8, 134.8, 134.6, 134.3, 131.2, 131.0,
130.9, 129.4, 124.0, 123.0, 122.7, 122.6, 120.3, 120.2, 119.6, 115.5, 69.1, 69.0,
68.8, 67.2, 65.8, 52.6, 34.7, 34.6, 31.8, 31.7, 29.7, 22.4.
IR (ATR): ν~ = 2962, 2926, 2905, 2870, 1682, 1522, 1478, 1353, 1201, 1109, 990,
953, 769.
MS (FAB, NBA): m/z = 1228 [M+-CN], 927 [M+-crown ether-KCN].
UV/Vis (CH2Cl2): λmax (ε, M-1cm-1) = 438 (526700), 576 (15600), 620 (15300).
Elemental analysis: C75H87N6O5Zn·H2O, (1271.57) calcd: C 70.65, H 7.04, N 6.59;
found: C 70.53, H 7.06, N 6.34.
N
NN
N
OO
OO
N
O
Zn
56
12
3 4 5
10
1112
1314
152
5252a
C4
C1
C3
C2
C5 C6
5454a
54b
56a
102 103
104
104a104b
5355
15151
153
154
154a
154b
KNC
105106
155156
Experimental
123
Potassium superoxide complex of Zinc-52-[N-(4-aza-18-crown-6)methyl]-54,104,154,204-tetra-t-butyl-56-methyl-5,10,15,20-tetraphenylporphyrin 34
Zinc-52-[N-(4-aza-18-crown-6)methyl]-54, 104,
154, 204-tetra-t-butyl-56-methyl-5, 10, 15, 20-
tetraphenylporphyrin 30 (100 mg, 0.08 mmol)
was dissolved in 20 ml of dry benzene and
solid potassium superoxide (71 mg, 1 mmol)
was added. The greenish solution was stirred
for 24 h at ambient temperature and filtered.
The solvent was removed in vacuo yielding a
violet solid (yield: 98 mg, 0.07 mmol, 93 %).
1H NMR (300 MHz, C6D6, 25 °C): δ = 9.15 (s, 4H, 12, 13, 17, 18), 9.07 (d, 3J = 4.5
Hz, 2H, 2, 8), 8.86 (d, 3J = 4.5 Hz, 2H, 3, 7), 8.27 (d, 3J = 7.9 Hz, 2H, 102, 202), 8.32
(m, 1H, 152), 8.16 (d, 3J = 7.9 Hz, 3H, 106, 206, 156), 7.69 (s, 1H, 53), 7.64 (d, 3J = 7.9 Hz, 3H, 103, 153, 203), 7.52 (d, 3J = 7.9 Hz, 3H, 105, 155, 205),
7.38 (s, 1H, 55), 3.19 (s, 2H, 52a), 3.02-1.67 (bm, 24H, C1-C6), 2.63 (s, 3H, 56a), 1.59
(s, 9H, 154b), 1.45 (s, 18H, 104b, 204b), 1.40 (s, 9H, 54b). 13C NMR (75 MHz, C6D6, 25 °C): δ = 151.6, 151.5, 150.6, 149.7, 159.5, 149.2, 142.5,
142.3, 141.6, 141.4, 140.8, 140.6, 138.9, 135.4, 134.9, 132.1, 132.0, 131.9, 129.8,
129.2, 124.5, 123.7, 123.1, 121.3, 120.7, 120.0, 116.5, 71.2, 71.1, 69.4, 68.8, 68.4,
67.5, 53.7, 53.4, 34.7, 32.0, 31.8, 22.7.
UV/Vis (toluene): λmax (ε, M-1cm-1) = 340 (451500), 576 (21500), 618 (18000).
N
NN
N
OO
OO
N
O
Zn
56
12
3 4 5
10
1112
1314
152
5252a
C4
C1
C3
C2
C5 C6
5454a
54b
56a
102 103
104
104a104b
5355
15151
153
154
154a
154b
KO
105106
155156
O
Experimental
124
Cobalt(II)-52-[N-(4-aza-18-crown-6)methyl]-54,104,154,204-tetra-t-butyl-56-methyl-5,10,15,20-tetraphenylporphyrin 37
52-[N-(4-aza-18-crown-6)methyl]-54, 104, 154,
204-tetra-t-butyl-56-methyl-5, 10, 15, 20- tetra-
phenylporphyrin 26 (100 mg, 0.08 mmol) was
dissolved in 10 ml of dry THF and
cobalt(II)acetate (45 mg, 0.16 mmol) was
added. The solution was heated to reflux for
24 h. The solvent was removed in vacuo and
FC on silica (ethyl acetate/methanol 9:1)
yielded an orange solid (yield: 93 mg,
0.8 mmol, 98 %).
1H NMR (400 MHz, CDCl3, 25 °C): δ = 16.11, 15.79, 13.38, 12.38, 10.03, 9.98, 4.99,
4.66, 2.98 (s, 9H, t-Bu), 2.90 (s, 9H, t-Bu), 2.86 (s, 9H, t-Bu), 1.04, 0.97, 0.86, 0.58,
-0.33, -1.46. 13C NMR (100 MHz, CDCl3, 25 °C): δ = 153.8, 153.2, 153.0, 152.5, 152.0, 147.0,
145.8, 141.2, 127.7, 127.0, 126.1, 99.0, 98.5, 97.6, 74.6, 74.0, 72.8, 70.9, 70.2, 61.2,
53.4, 36.4, 36.3, 36.3, 33.3, 33.1, 25.9.
IR (ATR): ν~ = 2953, 2903, 2864, 1459, 1351, 1266, 1204, 1112, 1069, 999, 814,
718.
MS (FAB, NBA): m/z = 1185 [M]+, 922 [M-crown ether]+.
UV/Vis (CH2Cl2): λmax (ε, M-1cm-1) = 413 (257800), 530 (15100).
Elemental analysis: C74H87CoN5O5·2MeOH (1247.658): calcd. C 73.05, H 7.66, N
5.61; found: C 73.01, H 7.47, N 5.69.
N
NN
N
OO
OO
N
O
CoII
56
12
3 4 5
10
1112
1314
152
5252a
C4
C1
C3
C2
C5 C6
5454a
54b
56a
102 103
104
104a104b
5355
15151
153
154
154a
154b
105106
155156
Experimental
125
Potassium cyanide complex of Cobalt(III)-52-[N-(4-aza-18-crown-6)methyl]-54,104,154,204-tetra-t-butyl-56-methyl-5,10,15,20-tetraphenylporphyrin 38
Cobalt(II)-52-[N-(4-aza-18-crown-6)methyl]-54,
104,154,204-tetra-t-butyl-56-methyl-5,10,15,20-
tetraphenylporphyrin 37 (50 mg, 0.04 mmol)
was dissolved in 10 ml CH2Cl2 and solid
potassium cyanide (50 mg, 0.76 mmol) was
added. The orange solution was stirred for
24 h at ambient temperature, filtered and the
residue was then subject to size exclusion
chromatography over Bio-Beads® SX3 with
CHCl3 as the eluent. The solvent was
removed in vacuo yielding a brown solid (yield: 49 mg, 0.038 mmol, 96 %).
1H NMR (400 MHz, CDCl3, 25 °C): δ = 8.78 (d, 3J = 4.8 Hz, 2H, 13, 17), 8.76 (d, 3J =
4.8 Hz, 2H, 12, 18), 8.69 (d, 3J = 4.9 Hz, 2H, 2, 8), 8.55 (d, 3J = 4.9 Hz, 2H, 3, 7),
8.03 (bs, 6H, 102, 106, 152, 156, 202, 206), 7.64 (bm, 7H, 103, 105, 153, 155, 203, 205, 55), 7.07 (s, 1H, 53), 3.05 (s, 3H, 56a), 3.16-2.20 (bm, 24H, C1-C6), 2.31 (s, 2H, 52a),
1.60 (s, 9H, 154b), 1.59 (s, 18H, 104b, 204b), 1.58 (s, 9H, 54b). 13C NMR (100 MHz, CDCl3, 25 °C): δ = 150.0, 149.6, 142.8, 142.4, 140.9, 139.6,
139.3, 138.8, 138.7, 137.6, 134.3, 134.0, 133.9, 133.5, 133.0, 131.9, 130.6 (d, 2J =
54.78 Hz, CN), 124.5 (d, 2J = 54.78 Hz, CN) 123.3, 123.2, 120.5, 118.1, 118.0,
113.1, 109.2, 69.1, 69.1, 68.4, 68.3, 67.0, 53.8, 51.9, 34.8, 34.7, 34.5, 31.7, 31.7,
22.7.
IR (ATR): ν~ = 2953, 2902, 2867, 2079 (CN), 1473, 1351, 1268, 1106, 1007, 946,
813, 788, 707.
MS (FAB, NBA): m/z = 1277 [M+], 1250 [M+-CN], 1223 [M+-2CN], 922 [M+-crown
ether-KCN-CN].
UV/Vis (CH2Cl2): λmax (ε, M-1cm-1) = 453 (194100), 540 (4000), 587 (9300), 632
(16000).
Elemental analysis: C76H87CoKN7O5·H2O·CHCl3: calcd. C 65.41, H 6.42, N 6.93;
found: C 65.53, H 6.41, N 7.08.
N
NN
N
OO
OO
N
O
CoIII
56
12
3 4 5
10
1112
1314
152
5252a
C4
C1
C3
C2
C5 C6
5454a
54b
56a
102 103
104
104a104b
5355
15151
153
154
154a
154b
KNC
105106
155156
NC
Experimental
126
Potassium thiocyanate complex of Cobalt(III)-52-[N-(4-aza-18-crown-6)methyl]-54,104,154,204-tetra-t-butyl-56-methyl-5,10,15,20-tetraphenylporphyrin 39
Cobalt(II)-52-[N-(4-aza-18-crown-6)methyl]-54,
104,154,204-tetra-t-butyl-56-methyl-5,10,15,20-
tetraphenylporphyrin 37 (130 mg, 0.11 mmol)
was dissolved in 20 ml THF and solid
potassium thiocyanate (130 mg, 1.4 mmol)
was added. The orange solution was stirred
for 24 h at ambient temperature. The solvent
was removed in vacuo and the residue
redissolved in CH2Cl2. The orange brown
solution was filtered and purified by SEC (Bio-
Beads® SX3, CHCl3) yielding an orange brown solid (yield: 128 mg, 0.09 mmol,
87 %).
1H NMR (400 MHz, CDCl3, 25 °C): δ = 8.92 (m, 6H, 2, 8, 12, 13, 17, 18), 8.71 (d, 3J =
4.8 Hz, 2H, 3, 7), 8.08 (bs, 6H, 102, 106, 152, 156, 202, 206), 7.67 (m, 6H, 103, 105, 153, 155, 203, 205), 7.44 (s, 1H, 53, 55), 4.20-2.60 (bm, 24H, C1-C6), 2.85 (bs, 3H, 56a),
2.60 (s, 2H, 52a), 1.59 (s, 9H, 154b), 1.58 (s, 18H, 104b, 204b), 1.55 (s, 9H, 54b). 13C NMR (100 MHz, CDCl3, 25 °C): δ = 150.1, 149.9, 143.7, 143.4, 142.7, 142.3,
140.6, 139.3, 139.1, 138.7, 138.3, 134.4, 134.2, 133.9, 133.8, 132.6, 132.3, 124.6,
123.3, 120.7, 118.8, 118.1, 115.6, 70.3, 69.8, 69.2, 68.9, 68.8, 67.5, 67.1, 53.4, 53.0,
35.0, 34.8, 34.5, 31.9, 31.7, 31.7, 31.5, 22.6, 22.1.
IR (ATR): ν~ = 2960, 2903, 2868, 2078 (SCN), 1458, 1351, 1216, 1108, 1007, 951,
790, 750, 708.
MS (FAB, NBA): m/z = 1281 [M+-SCN], 1224 [M+-2SCN], 1186 [M+-KSCN-SCN], 922
[M+-crown ether-KSCN-SCN].
UV/Vis (CH2Cl2): λmax (ε, M-1cm-1) = 328 (20600), 442 (140900), 557 (9300), 598
(5900).
Elemental analysis: C76H87CoKN7O5S2·0.5CHCl3 (1398.48): calcd. C 65.61, H 6.30,
N 7.00, S 4.58; found: C 65.60, H 6.41, N 7.04, S 4.31.
N
NN
N
OO
OO
N
O
CoIII
56
12
3 4 5
10
1112
1314
152
5252a
C4
C1
C3
C2
C5 C6
5454a
54b
56a
102 103
104
104a104b
5355
15151
153
154
154a
154b
KSCN
105106
155156
SCN
Experimental
127
Nickel-52-[N-(4-aza-18-crown-6)methyl]-54,104,154,204-tetra-t-butyl-56-methyl-5,10,15,20-tetraphenylporphyrin 28
52-[N-(4-aza-18-crown-6)methyl]-54, 104, 154,
204-tetra-t-butyl-56-methyl-5, 10, 15, 20-tetra-
phenylporphyrin 26 (225 mg, 0.2 mmol) and
nickel(II)acetate (274 mg, 1.1 mmol) were
dissolved in 10 ml of dry DMF and heated for
24 h under reflux and nitrogen atmosphere.
The solvent was removed in vacuo and FC on
silica (ethyl acetate/methanol 9:1) yielded an
orange solid (yield: 201 mg, 0.17 mmol,
92 %).
1H NMR (400 MHz, CDCl3, 25 °C): δ = 8.84 (s, 4H, 12, 13, 17, 18), 8.81 (d, 3J = 4.9
Hz, 2H, 2, 8), 8.60 (d, 3J = 4.9 Hz, 2H, 3, 7), 8.01 (bs, 6H, 102, 152, 202, 106, 156,
206), 7.79 (s, 1H, 53), 7.73 (m, 6H, 103, 105, 153, 155, 203, 205), 7.52 (s, 1H, 55), 3.33
(bs, 4H, C3-6), 3.29 (bs, 4H, C3-6), 3.13 (bs, 4H, C3-6), 3.07 (s, 2H, 52a), 2.95 (bs, 4H,
C3-6), 2.79 (m, 4H, C2), 2.31 (m, 4 H, C1), 2.09 (s, 3H, 56a), 1.61 (s, 9H, 54b), 1.60 (s,
18H, 104b, 204b), 1.59 (s, 9H, 154b). 13C NMR (100 MHz, CDCl3, 25 °C): δ = 150.9, 150.5, 142.7, 142.6, 142.4, 140.2,
138.6, 137.9, 137.9, 136.8, 133.5, 132.5, 132.1, 132.0, 131.4, 124.7, 123.8, 123.3,
118.9, 118.7, 116.4, 70.3, 70.1, 69.7, 69.4 (C2-6), 58.5(52a), 53.4 (C1), 34.8 (54a, 104a,
154a, 204a), 31.7, 31.6 (54b, 104b, 154b, 204b), 21.7 (56a).
IR (KBr): ν~ = 2953, 2903, 2864, 1459, 1351, 1266, 1204, 1112, 1073, 1004, 814,
714.
MS (FAB, NBA): m/z = 1207 [M+Na]+, 1185 [M]+, 922 [M-crown ether]+.
UV/Vis (CH2Cl2): λmax (ε, M-1cm-1) = 417 (238200), 529 (18000).
Elemental analysis: C74H87NiN5O5·2MeOH: calcd. C 73.07, H 7.66, N 5.61; found: C
73.01, H 7.47, N 5.69.
N
NN
N
OO
OO
N
O
NiII
56
12
3 4 5
10
1112
1314
152
5252a
C4
C1
C3
C2
C5 C6
5454a
54b
56a
102 103
104
104a104b
5355
15151
153
154
154a
154b
105106
155156
Experimental
128
52-{[N-(1,10-diaza-18-crown-6)N´-t-butoxycarbonyl]methyl}-54,104,154,204-tetra-t-butyl-56-methyl-5,10,15,20-tetraphenylporphyrin 51
52-(Bromomethyl)-54, 104, 154, 204- tetra-t-
butyl-56-methyl-5, 10, 15, 20- tetraphenyl-
porphyrin 25 (950 mg, 1 mmol), 1,10-diaza-
18-crown-6 (570 mg, 2.2 mmol) and
NaHCO3 (92 mg, 1.1 mmol) were dissolved
in 60 ml dry toluene and heated to reflux for
24 h. The solution was cooled down to
room temperature and di-t-butyl-
dicarbonate (1 g, 4.5 mmol) was added.
After 12 h the solvent was removed in
vacuo and FC on silica (CHCl3/methanol 9:1) yielded a violet solid (yield: 705 mg,
0.57 mmol, 58 %)
1H NMR (400 MHz, CDCl3, 25 °C): δ = 8.86 (s, 4H, 12, 13, 17, 18), 8.81 (d, 3J = 4.7 Hz, 2H, 2, 8), 8.61 (d, 3J = 4.7 Hz, 2H, 3, 7), 8.14 (m, 6H, 102, 106, 152, 156,
202, 206), 7.80 (s, 1H, 53), 7.75 (d, 3J = 8.4 Hz, 6H, 103, 105, 153, 155, 203), 7.49 (s,
1H, 55), 3.61 (m, 4H, C1-C6), 3.59 (s, 2H, 52a), 3.50 (m, 4H, C1-C6), 3.15-2.65 (m, 8H,
C1-C6), 2.67 (t, 3J = 5.9 Hz, 2H, C1-C6), 2.50 (t, 3J = 5.9 Hz, 2H, C1-C6), 2.20 (m, 4H,
C1-C6), 1.91 (s, 3H, 56a), 1.60 (s, 27H, 104b, 204b), 1.59 (s, 9H, 154b), 1.45 (s, 9H,
54b), 1.32 (s, 9H, BOC-t-Bu),-2.64 (s, 2H, NH). 13C NMR (100 MHz, CDCl3, 25 °C): δ = 155.5, 155.3, 150.9, 150.5, 140.8, 139.3,
139.1, 138.9, 138.1, 134.4, 124.6, 123.6, 123.2, 120.2, 119.8, 117.4, 70.5, 70.0,
69.8, 69.4, 59.1, 53.2, 34.9, 47.9, 47.5, 47.4, 34.8, 31.7, 31.7, 28.5, 28.4, 21.9.
IR (ATR): ν~ = 2960, 2904, 2867, 1690, 1461, 1403, 1364, 1245, 1150, 1109, 1069,
967, 800, 733.
MS (FAB, NBA): m/z = 1228 [M+], 867 [M+-crown ether].
UV/Vis (CH2Cl2): λmax (ε, M-1cm-1) = 421 (420300), 486 (6400), 517 (19900), 553
(12200), 592 (8100), 648 (7700).
Elemental analysis: C79H98N6O6·0.5CHCl3·0.5H2O (1289.43): calcd. C 73.66,
H 7.74, N 6.48; found: C 73.79, H 7.65, N 6.60.
HN
NNH
N
OO
NO
N
O56
12
3 4 5
10
1112
1314
152
5252a
C4
C1
C3
C2
C5C6
5454a
54b
56a
102 103
104
104a104b
5355
15151
153
154
154a
154b
105106
155156
OO
Experimental
129
N,N´-Bis[52-methyl(54,104,154,204-tetra-t-butyl-56-methyl-5,10,15,20-tetraphenyl-zinc porphyrin)]-(1,10-diaza-18-crown-6)ether] 56
52 - (Bromomethyl) - 54, 104,
154, 204 -tetra-t-butyl- 56-
methyl-5, 10, 15, 20-tetra-
phenylporphyrin 25 (190 mg,
0.2 mmol), 1,10-diaza-18-
crown-6 ether (35 mg, 0.12
mmol) and NaHCO3 (10 mg,
0.1 mmol) were dissolved in
dry toluene (25 ml) and
heated to reflux for 48 h.
The solvent was removed
and the residue redissolved
in THF (20 ml). Zinc acetate
(440 mg, 2 mmol) was
added and the residue
heated to reflux for 24 h. The solvent was removed in vacuo and after FC on silica
(CHCl3/ethanol 5:1) a violet solid was obtained (yield: 155 mg, 0.07 mmol, 73 %)
1H NMR (400 MHz, CDCl3/Pyridine-d5, 25 °C): δ = 8.78 (d, 3J = 4.6 Hz, 4H, 13, 17),
8.76 (d, 3J = 4.6 Hz, 4H, 12, 18), 8.67 (d, 3J = 4.4 Hz, 4H, 2, 8), 8.44 (d, 3J = 4.4 Hz,
4H, 3, 7), 8.19 (s, 1H, 53), 7.97 (m, 12H, 102, 106, 152, 156, 202, 206), 7.75 (s, 2H, 53),
7.57 (m, 12H, 103, 105, 153, 155, 203, 205), 7.39 (s, 1H, 55), 3.45 (m, 16H, C2-3), 2.85
(bs, 4H, 52a), 2.63 (bs, 8H, C2-3), 1.93 (bs, 8H, C1), 1.46 (s, 18H, 154b), 1.43 (s, 36H,
104b, 204b), 1.40 (s, 18H, 54b), -2.58 (s, 2 H, NH). 13C NMR (100 MHz, CDCl3/Pyridine-d5, 25 °C): δ = 155.1, 150.1,149.8, 149.7,
149.6, 149.1,140.3, 140.2, 138.9, 138.3, 134.2, 131.7, 131.2, 130.0, 122.9, 120.3,
119.8, 117.0, 70.2, 70.1, 69.9, 69.5, 68.9, 58.3 (52a), 53.0 (C1),47.7, 47.6, 34.4, 34.4
(54a, 104a, 154a, 204a), 31.4, 31.3 (54b, 104b, 154b, 204b), 28.1(56a).
IR (ATR): ν~ = 2958, 2904, 2867, 1694, 1461, 1362, 1337, 1268, 1204, 1109, 1065,
996, 810, 796, 720.
N
N N
N
OO
NO
N
O
N
NN
N
Zn
Zn
56
12
3 4 5
10
1112
1314
152
5252aC1
C3
C25454a
54b
56a
102 103
104
104a104b
5355
15151
153
154
154a
154b
105106
155156
Experimental
130
MS (FAB, NBA): m/z = 2120 [M+], 927 [Zn-porphyrin]+.
UV/Vis (CH2Cl2/pyridine): λmax (ε, M-1cm-1) = 430 (830900), 527 (5200), 564
(31000), 604 (18900).
Elemental analysis: C136H150N10O4Zn2·EtOH·CHCl3 (2278.99): calcd. C 73.06, H
6.93, N 6.13; found: C 73.21, H 7.08, N 6.08.
Nickel-porphyrin triade 58
Dibromoporphyrin 44
(51 mg, 0.05 mmol),
crown ether porphyrin
52 (129 mg, 0.11
mmol) and NaHCO3 (10
mg, 0.11 mol) were
heated to reflux in
toluene (50 ml) for
72 h. Nickel
acetate·4H2O (124 mg,
0.5 mmol) was added
and the solution heated
to reflux for 2 h again.
The solvent was
removed in vacuo and
FC of the orange
residue on silica (1.
CHCl3, 2.CHCl3/MeOH
10:1) yielded the pure
orange compound 58
(yield: 40 mg, 0.012 mmol, 24%)
1H NMR (400 MHz, CDCl3, 25 °C): δ = 8.69 (m, 8H, 12, 13, 17, 18), 8.66 (m, 4H, 2*,
3*, 7*, 8*), 8.67 (d, 3J = 4.9 Hz, 4H, 2, 8), 8.54 (d, 3J = 4.9 Hz, 4H, 12*, 18*), 8.40 (d, 3J = 4.9 Hz, 4H, 3, 7), 8.29 (d, 3J = 4.9 Hz, 4H, 13*, 17*), 7.85 (m, 18H, ArH), 7.61
(m, 22H, ArH), 7.34 (s, 2H, 153*, 155*), 2.90 (s, 4H, 56a), 2.83 (s, 4H, 15*2a, 15*6a),
N N
NN
O
O N
O
N O
NN
N N
O
O N
O
N O
NN
N N
Ni
Ni
Ni56
123
4
5
10 11 1213
14 152
52
52a
C4
C1
C3
C2
C5
C6
54
54a
54b
56a
102
103104
104a
104b
53
55
15151
153
154 154a154b
105
106
155156
5*6
1*2*3*4*
5*
10* 11*12*13*
14*15*2a
5*25*4
5*4a
5*4b
10*210*3
10*410*4a
10*4b
5*3
5*5
15*15*1
153
15*4 15*4a15*4b
10*5
10*6
15*515*6
Experimental
131
2.59 (m, 32H, C2-5), 2.01 (m, 16H, C1, C6), 1.87 (s, 6H, 52a), 1.51 (s, 18H, tBu), 1.48
(s, 36H, tBu), 1.46 (s, 18H, t-Bu), 1.42 (s, 18H, t-Bu), 1.37 (s, 9H, t-Bu), 1.23 (s, 9H,
t-Bu). 13C NMR (100 MHz, CDCl3, 25 °C): δ = 150.8, 150.4, 142.6, 142.3, 140.3, 138.5,
138.0, 137.9, 136.6, 133.5, 132.4, 132.3, 132.0, 131.3, 124.5, 123.7, 123.7, 123.0,
118.6, 116.3, 69.9, 69.5 (16C, C2-C5), 58.5 (4C, 56a, 156a*), 53.3 (8C, C1, C6), 34.8,
34.8, 34.7, 31.9, 31.6, 29.7, 29.4, 21.6.
IR (ATR): ν~ = 2956, 2904, 2867, 1551, 1461, 1351, 1268, 1204, 1109, 1071, 1003,
996, 814, 797, 714.
MS (FAB, NBA): m/z = 3311 [M+Na]+, 927 [Ni-porphyrin]+.
UV/Vis (CH2Cl2+pyridine): λmax (ε, M-1cm-1) = 416 (595000), 530 (48500).
Elemental analysis: C210H236N16Ni3O8·CHCl3, (3401.58): calcd. C 74.37, H 7.01, N
6.58; found: C 74.23, H 7.36, N 6.37.
Zinc-52,56-{bis-[N-(1,10-diaza-18-crown-6)-N´-t-butoxycarbonyl]methyl}-54,104,154,204-tetra-t-butyl-5,10,15,20-tetraphenylporphyrin 49
Zinc-52, 56- bis- (bromomethyl)-54, 104,
154, 204-tetra-t-butyl-5, 10, 15, 20-tetra-
phenylporphyrin 45 (125 mg, 0.12
mmol) and 1,10-diaza-18-crown-6 (120
mg, 0.46 mmol) were dissolved in
20 ml dry toluene and heated to 50°C
for 72 h. NaHCO3 (15 mg) was added,
followed after 30 minutes at room
temperature by Boc2O (150 mg, 0.68
mmol). Stirring the solution for 12 h at
room temperature followed by removal of the solvent in vacuo and FC on silica
(CH2Cl2/methanol 97:3), yielded a violett solid (yield: 93 mg, 0.064 mmol, 57 %)
1H NMR (400 MHz, CDCl3, 25 °C): δ = 8.91 (s, 4H, 12, 13, 17, 18), 8.85 (d, 3J = 4.6 Hz, 2H, 2, 8), 8.64 (d, 3J = 4.6 Hz, 2H, 3, 7), 8.13 (bs, 6H, 102, 106, 152, 156,
202, 206), 7.73 (m, 8H, 53, 55, 103, 105, 153, 155, 203, 205), 3.26 (bs, 4H, 52a, 56a),
2.77, 2.67 (m, C2-C5), 2.57 (m, C2-C5), 2.47 (bs, 8H, C6), 2.38(m, C2-C5), 2.30 (m, C2-
N
NN
N
OO
NO
N
O
12
3 4 5
10
1112
1314
152
5252a
C4
C1
C3
C2
C5C6
5454a
54b
102 103
104
104a104b
53
15151
153
154
154a
154b
105106
OO
NO
N
O OOO O
Zn
Experimental
132
C5), 2.06 (bs, 8H, C1), 1.61 (s, 9H, 154b), 1.60 (s, 18H, 104b, 204b), 1.57 (s, 9H, 54b),
1.24 (s, 18H, Boc-t-Bu). 13C NMR (100 MHz, CDCl3, 25 °C): δ = 155.4, 154.9, 150.6, 150.4, 150.1, 149.9,
149.8, 149.6, 146.7, 140.2, 140.1, 138.9, 134.4, 132.1, 131.7, 130.9, 130.5,
127.6,124.8, 123.3, 123.0, 120.7, 120.4, 117.6, 79.2, 70.4, 70.2, 69.5, 69.2, 68.4,
59.2 (2C, 52a), 53.1 (2C, C1), 47.2 (2C, C6), 35.1, 34.9, 34.8, 31.5, 28.2.
IR (ATR): ν~ = 2956, 2902, 2867, 1692, 1459, 1407, 1364, 1245, 1109, 1063, 996,
797, 719.
MS (FAB, NBA): m/z = 1652 [M+], 925 [M+-2x aza-crown ether].
UV/Vis (CH2Cl2): λmax (ε, M-1cm-1) = 430 (467200), 559 (18400), 600 (8300).
Elemental analysis: C96H128N8O12Zn·2H2O (1684.915): calcd. C 68.33, H 7.88,
N 6.64; found: C 68.40, H 7.63, N 6.60.
Zinc-52-[N-(1-aza-18-crown-6)methyl]-56,152,156-tris-(brommethyl)-54,104,154,204-tetra-t-butyl-5,10,15,20-tetraphenylporphyrin 41
Zinc-52, 56, 152,156-tetra-(brommethyl)-54,104,
154, 204-tetra-t-butyl-5, 10, 15, 20-tetraphenyl-
porphyrin 40 (877 mg, 0.69 mmol) and 1-aza-
18-crown-6 ether (300 mg, 1.1 mmol) were
dissolved in 50 ml dry toluene and heated to
70°C for 48 hours. The solvent was removed
and after FC on silica (CHCl3/methanol 9:1) a
violett solid was obtained (yield: 377 mg, 0.26
mmol, 37 %)
1H NMR (400 MHz, CDCl3, 25 °C): δ = 8.88 (m, 4H, 12, 13, 17, 18), 8.68 (d, 3J = 4.5
Hz, 2H, 2), 8.61 (d, 3J = 4.5 Hz, 2H, 3), 8.13 (m, 4H, 102, 106, 202, 206), 7.85 (m, 3H,
55, 153, 155), 7.73 (m, 5H, 53, 103, 105, 203, 205), 4.32 (s, 2H, 56a), 4.24 (s, 2H, 152a),
3.88 (s, 2H, 156a), 3.20 (s, 2H, 52a), 2.51 (s, 4H, C1-6), 2.42 (s, 4H, C1-6), 2.20 (s, 4H,
C1-6), 2.15 (s, 4H, C1-6), 1.60 (bs, 8H, C1-6), 1.62 (s, 9H, 54b), 1.61 (s, 9H, 154b), 1.59
(s, 18H, 104b, 204b). 13C NMR (100 MHz, CDCl3, 25 °C): δ = 152.4, 151.6, 150.5, 150.4, 150.1, 149.8,
149.6, 141.5, 139.9, 139.5, 139.1, 138.7, 138.1, 137.8, 134.7, 134.4, 134.1, 132.1,
N
NN
N
OO
OO
N
O56
12
3 4 5
10
1112
1314
152
5252a
C4
C1
C3
C2
C5C6
5454a
54b
56a
102 103
104
104a104b
5355
15151
153
154154a
154b
105106
155156
Br
Br Br156a 152a
Zn
Experimental
133
131.3, 131.2, 129.0, 128.2, 127.2, 126.8, 125.3, 125.1, 123.5, 123.2, 120.9, 116.6,
112.8, 69.3, 69.1, 68.8, 68.4, 68.3, 66.9, 57.7, 53.5, 35.1, 35.0, 34.8, 34.7, 33.4,
32.7, 32.4, 31.7, 31.7, 31.5, 24.9, 21.4.
IR (ATR): ν~ = 2956, 2904, 2869, 1478, 1461, 1362, 1337, 1202, 1109, 1065, 994,
801, 795, 719.
MS (FAB, NBA): m/z = 1456 [M+], 949 [M+-aza-crown ether].
UV/Vis (CH2Cl2): λmax (ε, M-1cm-1) = 428 (309200), 558 (14700), 606 (4600).
Elemental analysis: C76H88Br3N5O5Zn·H2O·CHCl3 (1587.288): calcd. C 58.02, H
5.75, N 4.39; found: C 58.09, H 5.91, N 4.16.
Zinc-52-[N-(1-aza-18-crown-6)methyl]-56,152,156-[tris-(2,2-diethoxycarbonyl-ethyl)]-54,104,154,204-tetra-t-butyl-5,10,15,20-tetraphenylporphyrin 42
Zinc-52-[N-(1-aza-18-crown-6)methyl]-56, 152,
156-tris-(brommethyl)-54, 104, 154, 204-tetra-t-
butyl-5,10,15,20-tetraphenylporphyrin 41 (377
mg, 0.26 mmol) was dissolved in 25 ml dry
DMF. A freshly prepared solution of diethyl
malonate (1 ml, 6.6 mmol) and potassium
hydride (220 mg, 5.48 mmol) in DMF was
added and heated to 55°C for 16 h.
Afterwards the solution was poured into an
ice-cold NH4Cl-solution (200 ml) and filtered.
The residue was dried and FC on silica (ethyl acetate/methanol 9:1) yielded a violett
solid (yield: 200 mg, 0.12 mmol, 45 %)
1H NMR (400 MHz, CDCl3, 25 °C): δ = 8.88 (m, 4H, 2, 8, 12, 18), 8.68 (m, 4H, 3, 7,
13, 17), 8.14 (m, 4H, 102, 106, 202, 206), 7.85 (s, 1H, 55), 7.73 (m, 5H, 53, 103, 105,
203, 205), 7.45 (m, 2H, 153, 155), 3.65 (bm, 12H, OCH2), 3.15 (s, 2H, 52a), 3.12 (m,
1H, 56b), 3.00-2.70 (m, 16H, C3-4, 56a, 152a, 152b, 156a, 156b), 2.65 (bs, 4H, C2),2.35
(m, 4H, C1), 1.96 (bs, 2H, C5-6), 1.75 (bs, 2H, C5-6), 1.59 (s, 18H, 104b, 204b), 1.56 (s,
9H, 154b), 1.52 (s, 9H, 54b), 1.34 (bs, 4H, C5-6), 0.91 (t, 3J = 7.0 Hz, 6H, OCH2CH3),
0.82 (t, 3J = 7.2 Hz, 6H, OCH2CH3), 0.74 (t, 3J = 7.0 Hz, 6H, OCH2CH3).
56
12
3 4 5
10
1112
1314
152
5252a
C4
C1
C3
C2
C5C6
5454a
54b
56a
102 103
104
104a104b
5355
15151
153
154154a
154b
105106
156
156a 152a
N
NN
N
OO
OO
N
OOO
EtO OO
OEtO
OEtO
OEt
OEt
OEt
Zn
156b
56b
152b
Experimental
134
13C NMR (100 MHz, CDCl3, 25 °C): δ = 168.9, 168.7, 168.6, 150.9, 150.8, 150.4,
150.3, 150.0, 149.8, 141.6, 140.0, 139.5, 138.7, 134.5, 134.4, 132.6, 132.4, 130.9,
130.6, 124.6, 124.3, 123.9, 123.4, 123.3, 120.5, 115.4, 70.5, 69.8, 69.4, 68.8, 68.6,
68.4, 66.7, 61.8, 60.9, 60.8, 60.8, 53.6, 53.4, 52.7, 52.3, 34.9, 34.8, 34.0, 33.9, 33.8,
31.7, 31.6, 13.7, 13.6, 13.5.
IR (ATR): ν~ = 2954, 2904, 2869, 1748, 1733, 1465, 1366, 1221, 1146, 1111, 1030,
997, 797, 722.
MS (FAB, NBA): 1716 [M++Na], 1695 [M+], 1430 [M+-aza-crown ether].
UV/Vis (CH2Cl2): λmax (ε, M-1cm-1) = 427 (496000), 556 (20700), 596 (6000). Elemental analysis: C97H121N5O17Zn·2H2O (1727.826): calcd. C 67.33, H 7.28,
N 4.05; found: C 67.41, H 7.27, N 3.86.
Zinc-52-[N-(1-aza-18-crown-6)methyl]-56,152,156-[tris-(2,2-dicarboxylethyl)]-54,104,154,204-tetra-t-butyl-5,10,15,20-tetraphenylporphyrin 43
Zinc-52-[N-(1-aza-18-crown-6)methyl]-56, 152,
156-tris-(2,2-diethoxycarbonylethyl)-54, 104,
154, 204-tetra-t-butyl-5, 10, 15, 20-tetraphenyl-
porphyrin 42 (145 mg, 0.08 mmol) was
dissolved in 100 ml ethanol and 50 ml of an
ethanolic NaOH-solution (5 N) was added.
The solution was stirred for 3 h at 50°C,
filtrated, and the residue redissolved in
methanol. The addition of Et2O gave a pink
precipitate which was filtrated and dried in
vacuo (yield: 112 mg, 0.7 mmol, 85 %)
1H NMR (400 MHz, MeOH-d4/D2O, 25 °C): δ = 8.25 (m, 6H, β-Pyrrol), 8.19 (m, 2H,
β-Pyrrol), 7.65 (m, 2H, Ar-H), 7.49 (m, 2H, Ar-H), 7.40 (s, 1H, Ar-H), 7.27 (m, 5H,
Ar-H), 7.20 (s, 1H, Ar-H), 7.14 (s, 1H, Ar-H), 2.86 (m, 5H, 52a, 56b, 152b, 156b), 2.82
2.86 (m, 6H, 56a, 152a, 156a), 2.54 (m, 2H, C1-C5), 2.44 (m, 4H, C1-C5), 2.34 (m, 4H,
C1-C5), 2.25 (m, 4H, C1-C5), 2.08 (m, 2H, C1-C5), 1.87 (bs, 4H, C1-C5), 1.14 (s, 27H,
t-Bu), 1.08 (s, 9H, t-Bu), 0.66 (bs, 2H, C1-C5), 2.30 (bs, 2H, C1-C5).
56
12
3 4 5
10
1112
1314
152
5252a
C4
C1
C3
C2
C5C6
5454a
54b
56a
102 103
104
104a104b
5355
15151
153
154154a
154b
105106
156
156a 152a
N
NN
N
OO
OO
N
OOO
HO OO
OHO
OHO
OH
OH
OH
Zn
56b
56b
Experimental
135
13C NMR (100 MHz, MeOH-d4/D2O, 25 °C): δ = 181.0, 180.06, 179.9, 179.8, 170.0,
151.8, 151.7, 151.5, 151.2, 151.0, 150.9, 141.6, 140.1, 139.8, 135.5, 135.5, 132.9,
132.8, 124.3, 124.1, 123.8, 121.0, 119.6, 68.4, 67.9, 67.6, 60.3, 58.3, 53.5, 42.9,
37.0, 35.8, 35.7, 35.6, 32.3, 32.1, 31.9, 24.3, 18.2.
IR (ATR): ν~ = 2954, 2904, 2869, 1561, 1407, 1351, 1337, 1202, 1109, 1065, 994,
797, 722.
UV/Vis (MeOH): λmax (ε, M-1cm-1) = 427 (420600), 560 (16200), 599 (6500).
Elemental analysis: C85H91Na6N5O17·11NaOH (2135,42): calcd. C 47.75, H 4.86, N
3.28; found: C 47.90, H 5.05, N 3.16.
Europium-52-[N-(4-aza-18-crown-6)methyl]-54,104,154,204-tetra-t-butyl-56-methyl-5,10,15,20-tetraphenylporphyrin 59
52-[N-(4-aza-18-crown-6)methyl]-54, 104, 154,
204-tetra-t-butyl-56-methyl-5, 10, 15, 20-tetra-
phenylporphyrin 26 (100 mg, 0.08 mmol) and
europium(III)acetylacetonate hydrate
(100 mg, 0.2 mmol) were dissolved in 2 ml of
dry trichlorobenzene (TCB) and heated for
24 h under reflux and a slow stream of
nitrogen. The solvent was removed in vacuo
and subsequent chromatography with SEC
(BioBeads SX3; CH2Cl2) yielded a ruby
colored solid (yield: 90 mg, 0.07 mmol, 85 %).
IR (KBr): ν~ = 2961, 2907, 2868, 1590, 1517, 1393, 1200, 1262, 1108, 988, 799,
722.
MS (FAB, NBA): m/z = 1279 [M]+, 1129 [M-Eu]+, 1016 [M-crown ether]+.
UV/Vis (CH2Cl2): λmax (ε, M-1cm-1) = 425 (228000), 559 (13500), 599 (5300).
Elemental analysis: C76H90EuN5O7·2CH2Cl2 (1505.51): calcd. C 62.15, H 6.29, N
4.65; found: C 62.42, H 6.06, N 4.41.
N
NN
N
OO
OO
N
O
EuIII
56
12
3 4 5
10
1112
1314
152
5252a
C4
C1
C3
C2
C5 C6
5454a
54b
56a
102 103
104
104a104b
5355
15151
153
154
154a
154b
105106
155156
OO
Experimental
136
Gadolinium-52-[N-(4-aza-18-crown-6)methyl]-54,104,154,204-tetra-t-butyl-56-methyl-5,10,15,20-tetraphenylporphyrin 60
52-[N-(4-aza-18-crown-6)methyl]-54, 104, 154,
204-tetra-t-butyl-56-methyl-5, 10, 15, 20-tetra-
phenylporphyrin 26 (100 mg, 0.08 mmol) and
gadolinium(III)acetylacetonate hydrate
(100 mg, 0.2 mmol) were dissolved in 2 ml of
dry TCB and heated for 24 h under reflux and
a slow stream of nitrogen. The solvent was
removed in vacuo and subsequent
chromatography with SEC (BioBeads SX3;
CHCl3) yielded a ruby colored solid (yield:
201 mg, 0.07 mmol, 88 %).
IR (ATR): ν~ = 2958, 2904, 2869, 1555, 1403, 1362, 1260, 1200, 1109, 1023, 986,
799, 724.
MS (FAB, NBA): m/z = 1283 [M]+, 1020 [M-crown ether]+.
UV/Vis (CH2Cl2): λmax (rel) = 430 (2.053), 560 (0.108), 598 (0.047).
Elemental analysis: C76H90GdN5O7·0.5CHCl3 (1401.55): calcd. C 65.51, H 6.50, N
4.99; found: C 65.81, H 6.76, N 4.24.
N
NN
N
OO
OO
N
O
GdIII
56
12
3 4 5
10
1112
1314
152
5252a
C4
C1
C3
C2
C5 C6
5454a
54b
56a
102 103
104
104a104b
5355
15151
153
154
154a
154b
105106
155156
OO
Experimental
137
Bis-[8-(t-butyldimethylsilanyloxy)-octyl] malonate 66
The malonate 66 was
synthesized according to
general procedure GP1
(page 117) with malonyl dichloride and pyridine. The reaction with 8-t-butyldimethyl-
silyloxy-1-octanol 65 (5.20 g, 20 mmol) gave 66 (5.18 g, 17.6 mmol, 88 %) after FC
on silica gel (ethyl acetate/hexane 1:1) as a colorless oil.
1H NMR (400 MHz, CDCl3, 25 °C) : δ = 4.10 (t, 3J = 6.7 Hz, 4H, 3), 3.58 (t, 3J = 6.6
Hz, 4H, 10), 3.33 (s, 2H, 1), 1.60 (m, 4H, 4), 1.47 (m, 4H, 9), 1.27 (m, 16H, 5-8), 0.86
(s, 18H, 13), 0.01 (s, 12H, 11). 13C NMR (100 MHz, CDCl3, 25 °C): δ = 166.6 (2C, 2), 65.6 (2C, 10), 63.2 (2C, 3),
41.6 (2C, 1), 32.7, 29.3, 29.2, 29.1, 25.9 (6C, 13), 25.7, 18.3 (2C, 12), -5.3 (4C, 11).
IR (NaCl): ν~ = 2931, 2857, 2120, 1740 (C=O), 1471, 1254, 1099, 836, 775.
MS (FAB, NBA): m/z = 590 [M]+, 531 [M-t-Bu]+.
Bis[1´-(8-(t-butyldimethylsilyloxy)-octyloxycarbonyl]-1,2-methano[60]-fullerene 69
The synthesis of
monoadduct 69 was
performed according to
general procedure GP4
(page 118). C60 (2.10 g,
2.9 mmol, 1.16 eq.) and
malonate 66 (1.49 g, 2.5 mmol, 1 eq.) were reacted with CBr4 (843 mg, 2.55 mmol,
1.02 eq.) and DBU (418 μl, 2.8 mmol, 1.12 eq.). FC on silica gel (toluene) yielded a
brown powder, which was dried in vacuo (yield: 1320 mg, 1 mmol, 40 %).
1H NMR (400 MHz, CDCl3, 25 °C): δ = 4.45 (t, 3J = 6.7 Hz, 4H, 4), 3.57 (t, 3J = 6.6
Hz, 4H, 11), 1.81 (dt, 3J = 6.7 Hz, 3J = 6.7 Hz, 4H, 10), 1.49 (m, 4H, 5), 1.30 (m,
16H, 6-9), 0.88 (s, 18H, 14), 0.02 (s, 12H, 12). 13C NMR (100 MHz, CDCl3, 25 °C): δ = 163.7 (2C, 3), 145.4, 145.2, 145.2, 144.9,
144.7, 144.6, 144.6, 143.9, 143.1, 143.0, 143.0, 142.2, 141.9, 140.9, 139.0 (58C,
1
3O OO O
45
67
89
OSiOSi11
1012
13142
O OO O
12 3
45
67
8OSiOSi
109
111213
Experimental
138
C60-sp2), 71.6 (2C, 1), 67.4 (2C, 4), 63.2 (2C, 11), 52.4 (1C, 2), 32.8, 29.4, 29.2, 28.6,
26.0, 25.9, 25.8, 18.4, -5.24 (2C, 11).
IR (KBr): ν~ = 2925, 2852, 1742, 1460, 1427, 1230, 1095, 833, 772, 525.
MS (FAB, NBA): m/z = 720 [C60]+.
UV/Vis (CH2Cl2): λmax, (ε, M-1cm-1) = 258 (120000), 325 (37300), 425 (3200).
Bis[1´-(8-hydroxyoctyloxycarbonyl)]-1,2-methano[60]fullerene 70
Bis[1´-(8-(t-butyldimethylsilyloxy)-
octyloxycarbonyl]- 1,2- methano-
[60]fullerene 69 (300 mg, 0.22
mmol) was dissolved in a mixture
of 50 ml of ethanol and 20 ml of
CH2Cl2. 1 ml of concentrated HCl
was added and the mixture was stirred for 2 h at room temperature. After
neutralization with saturated aqueous NaHCO3 solution and drying over MgSO4 the
solvent was removed in vacuo to give a brownish material (yield: 235 mg, 0.21 mmol,
95 %).
1H NMR (400 MHz, CDCl3, 25 °C): δ = 4.46 (t, 3J = 6.6 Hz, 4H, 4), 3.62 (t, 3J = 6.5
Hz, 4H, 11), 1.82 (dt, 3J = 6.7 Hz, 3J = 6.7 Hz 4H, 10), 1.55 (dt, 3J = 7.1 Hz, 4H, 5),
1.34 (m, 16H, 6-9). 13C NMR (100 MHz, CDCl3, 25 °C): δ = 163.6 (2C, 3), 145.3, 145.2, 145.2, 145.1,
145.1, 145.1, 144.8, 144.6, 144.6, 144.5, 143.8, 143.0, 143.0, 142.9, 142.1, 141.8,
140.9, 139.9 (58C, C60 sp2), 71.6 (2C, 1), 67.4 (2C, 4), 62.9 (2C, 11), 52.3 (1C, 2),
32.7, 29.3, 29.2, 28.5, 25.9, 25.7 (12C, 5-10).
IR (KBr): ν~ = 2923, 2851, 1741, 1461, 1427, 1266, 1231, 1205, 1055, 525.
MS (FAB, NBA): m/z = 1079 [M]+, 720 [C60]+.
UV/Vis (CH2Cl2): λmax, (ε, M-1cm-1) = 257 (111000), 325 (34500), 425 (2300).
1
3O OO O
45
67
89
OHHO11
1012
2
Experimental
139
Pyropheophorbide-a diester of bis[1´-(8-hydroxyoctyloxycarbonyl)]-1,2-methano[60]fullerene 71
OO
OO O
O
NNH
NHN
O
OO
NNH
NHN
O
11a
22a 2b
33a
44a 4b
5a56
77a7b
7c88a 11 12
109
13
141516
17
18α
βγ
δ
19
20
21
22
23
24
25
2627
28
29
The esterification of the monoaddukt 70 with pyropheophorbide-a 19 was performed
according to general procedure GP2 (page 117). Bis[1´-(8-hydroxyoctyloxycarbony)]-
1,2-methano[60]fullerene 70 (50 mg, 0.046 mmol), pyropheophorbide-a 19 (73 mg,
0.137 mmol) and 1-hydroxybenzotriazole (1-HOBT) (30 mg, 0.22 mmol) were reacted
with N-(3-dimethylaminopropyl)-N-ethylcarbodiimid (EDC) (43 mg, 0.22 mmol) and
dimethylamino pyridine (DMAP) (16 mg, 0.12 mmol) in 10 ml of dry THF. FC on
silica gel (CH2Cl2/Methanol 19:1) yielded the desired dark green product as the first
fraction (yield: 42 mg, 0.02 mmol, 44 %).
1H NMR (400 MHz, CDCl3, 25 °C): δ = 9.25* (s, 2H, β), 9.17* (s, 2H, α), 8.47* (s, 2H,
δ), 7.86* (m, 2H, 2a), 6.12* (m, 4H, 2b), 5.10*(m, 4H, 10), 4.42* (d, 3J = 7.1 Hz, 2H,
7), 4.34 (t, 3J = 6.4 Hz, 4H, 26), 4.22* (m, 2H, 8), 3.96 (m, 4H, 19), 3.53* (s, 6H, 5a),
3.52* (m, 4H, 4a), 3.32* (s, 6H, 1a), 3.08* (s, 6H, 3a), 2.63* (m, 2H, 7b), 2.51* (m,
2H, 7b), 2.27* (m, 4H, 7a), 1.77* (d, 6H, 3J = 7.4 Hz, 8a), 1.65 (m, CH2), 1.58* (t, 3J =
7.4 Hz, 6H, 4b), 1.45 (m, CH2), 1.15 (m, CH2), 0.22* (bs, 2H, NH), -1.89* (s, 2H, NH). 13C NMR (100 MHz, CDCl3, 25 °C): δ = 196.1 (2C, 9), 173.1 (2C, 7c), 171.2, 163.5
(2C, 27), 160.2, 155.0, 150.6, 148.8, 144.8, 144.5, 144.3, 144.1, 143.8, 143.8, 143.7,
143.6, 142.9, 142.2, 143.0, 141.4, 141.1, 140.2, 138.4, 137.7, 136.0, 135.9, 135.6,
131.4, 130.3, 129.1, 128.7, 128.1, 122.4, 105.9 (2C, γ), 103.9 (2C, β), 97.1 (2C, α),
92.9 (2C, δ), 71.7, 68.0, 67.1, 64.6, 53.4, 52.2, 51.7, 49.9, 48.0, 32.7, 31.9, 31.2,
30.0, 29.8, 29.7, 29.4, 28.9, 28.5, 28.4, 27.1, 25.8, 25.7, 23.1, 22.7, 19.8, 19.3, 17.4,
14.1, 12.1, 11.9, 11.1.
IR (KBr): ν~ = 2959, 2924, 2853, 1734, 1698, 1616, 1222, 1161, 978, 733.
Experimental
140
MS (FAB, NBA): m/z = 2113 [M]+, 720 [C60]+.
UV/Vis (CH2Cl2): λmax, (ε, M-1cm-1) = 257 (82900), 324 (53300), 414 (134000), 508
(13400), 538 (12400), 609 (10600), 667 (47000).
Pyropheophorbide-a-(8-hydroxyoctyl)ester 64
The formation of the pyropheo-
phorbide-a-8-hydroxyoctylester 64
was performed according to general
procedure GP2 (page 117).
Pyropheophorbide-a 19 (160 mg,
0.3 mmol), octane-1,8-diol 20
(87 mg, 0.6 mmol) and 1-hydroxybenzotriazole (1-HOBT) (53 mg, 0.39 mmol) were
reacted with EDC (76 mg, 0.4 mmol) and DMAP (13 mg, 0.1 mmol) in 30 ml of dry
THF. After chromatography on silica gel (CH2Cl2/ethyl acetate 1:1) a dark green
product was obtained (yield: 127 mg, 0.19 mmol, 64 %).
1H NMR (400 MHz, CDCl3, 25 °C): δ = 9.19* (s, 1H, β), 9.10* (s, 1H, α), 8.50* (s, 1H,
δ), 7.84* (dd, 3J = 11.6 Hz, 17.8 Hz, 1H, 2a), 6.18* (dd, 2J = 1.5 Hz, 3J = 17.8 Hz, 1H,
2b), 6.08* (dd, 2J = 1.5 Hz, 3J = 11.6 Hz, 1H, 2b), 5.24* (d, 2J = 19.7 Hz, 1H, 10),
5.08* (d, 2J = 19.7 Hz, 1H, 10), 4.47* (dq, 3J = 7.3 Hz, 3J = 2.0 Hz, 1H, 8), 4.27* (dt,
1H, 3J = 8.4 Hz, 3J = 2.7 Hz, 7), 4.01 (m, 2H, 19), 3.53* (s, 3H, 5a), 3.52 (t, 3J = 6.7
Hz, 2H, 26), 3.44* (m, 2H, 4a), 3.33* (s, 3H, 1a), 3.02* (s, 3H, 3a), 2.67* (m, 1H, 7b),
2.54* (m, 1H, 7b), 2.27* (m, 2H, 7a), 1.82* (d, 3J = 7.3 Hz, 3H, 8a), 1.58* (t, 3J = 7.6
Hz, 3H, 4b), 1.46 (m, 4H, 20, 25), 1.20 (m, 8H, 21-24), 0.14* (bs, 1H, NH), -1.88* (s,
1H, NH). 13C NMR (100 MHz, CDCl3, 25 °C): δ = 196.1 (1C, 9), 173.1 (1C, 7c), 171.2, 160.1,
154.9, 150.5, 148.8, 144.6, 141.4, 137.6, 135.9, 135.7, 135.5, 131.4, 130.2, 129.0,
128.0 (1C, 2a), 122.3 (1C, 2b), 105.8 (1C, γ), 103.7 (1C, β), 96.9 (1C, α), 92.8 (1C,
δ), 64.6 (1C, 26), 62.8 (1C, 19), 51.6 (1C, 7), 49.9 (1C, 8), 48.0 (1C, 10), 32.6, 31.1,
29.8, 29.1, 29.0, 28.4, 25.7, 25.5, 23.0, 19.2 (1C, 4a), 17.3, 12.0, 11.9, 11.0 (1C, 3a).
IR (KBr): ν~ = 3389, 2962, 2927, 2852, 1730, 1690, 1620, 1496, 1262, 1092, 1024,
802, 673.
MS (FAB, NBA): m/z = 663 [M]+.
HO OO
NNH
NHN
O
11a
22a 2b
33a
44a 4b
5a5
6
77a7b
7c88a 11 12
109
13
141516
17
18α
βγ
δ
19
20
21
22
23
24
25
2627
Experimental
141
UV/Vis (CH2Cl2): λmax, (ε, M-1cm-1) = 414 (89700), 508 (9000), 538 (8200), 609
(7200), 667 (36900).
Malonic acid-bis[(8-hydroxyoctyl)-pheophorbide-a-ester] 68
OO
OO O
O
NNH
NHN
O
OO
NNH
NHN
O
11a
22a 2b
33a
44a 4b
5a5
6
77a7b
7c88a 11 12
109
13
141516
17
18α
βγ
δ
19
20
21
22
23
24
25
2627
28
The formation of the diester 68 was performed according to general procedure GP2
(page 117). Pyropheophorbide-a 19 (254 mg, 0.5 mmol), bis(8-hydroxyoctyl)-
malonate 67 (71 mg, 0.2 mmol) and 1-HOBT (110 mg, 0.8 mmol) were reacted with
EDC (114 mg, 0.6 mmol) and DMAP (14 mg, 0.12 mmol) in 30 ml of dry THF. After
chromatography on silica gel (CH2Cl2/ethyl acetate 1:1) a dark green product was
obtained (yield: 133 mg, 0.13 mmol, 64 %).
1H NMR (400 MHz, CDCl3, 25 °C): δ = 9.20* (s, 2H, β), 9.12* (s, 2H, α), 8.49* (s, 2H,
δ), 7.82* (dd, 3J = 11.5 Hz, 17.8 Hz, 1H, 2a), 6.17* (dd, 2J = 1.4 Hz, 3J = 17.9 Hz, 1H,
2b), 6.06* (dd, 2J = 1.4 Hz, 3J = 11.9 Hz, 1H, 2b), 5.23* (d, 2J = 19.8 Hz, 1H, 10),
5.07* (d, 2J = 19.8 Hz, 1H, 10), 4.46* (dq, 3J = 7.4 Hz, 3J = 1.9 Hz, 1H, 8), 4.26* (m,
2H, 7), 4.05 (t, 3J = 6.6 Hz, 4H, 26), 3.96* (m, 4H, 19), 3.52* (s, 6H, 5a), 3.46* (q, 3J =
2.2 Hz, 4H, 4a), 3.33* (s, 6H, 1a), 3.30 (s, 2H, 28), 3.04* (s, 6H, 3a), 2.66* (m, 2H,
7b), 2.55* (m, 2H, 7b), 2,27* (m, 4H, 7a), 1.81* (d, 3J = 7.1 Hz, 6H, 8a), 1.58* (t, 3J =
7.7 Hz, 6H, 4b), 1.53 (m, 8H, 20, 25), 1.16 (m,16H, 21-24), 0.21* (s, 2H, NH), -1.90*
(s, 2H, NH). 13C NMR (100 MHz, CDCl3, 25 °C): δ = 196.1 (2C, 9), 173.1 (1C, 7c), 171.2 (1C,
27c), 163.6, 160.1, 154.9, 150.5, 148.8, 144.7, 141.3, 137.6, 135.9, 135.7, 135.5,
131.3, 130.3, 129.0, 128.8, 128.0 (1C, 2a), 122.3 (1C, 2b), 105.8 (1C, γ), 103.7 (1C,
β), 96.9 (1C, α), 92.8 (1C, δ), 128.0 (1C, 2a), 65.5 (1C, 26), 64.6 (1C, 19), 49.9, 48.0,
41.5, 31.0, 29.7, 28.9, 28.9, 28.4, 28.3, 25.7, 25.6, 23.0, 19.2, 17.3, 12.0, 11.9, 11.0,
11.0.
IR (KBr): ν~ = 2925, 2856, 1731, 1692, 1616, 1498, 1220, 1161, 1025, 979, 673.
MS (FAB, NBA): m/z = 1394 [M]+, 461 [Pyropheo-a]+.
Experimental
142
UV/Vis (CH2Cl2): λmax, (ε, M-1cm-1) = 414 (192000), 508 (15800), 538 (14600), 609
(12600), 667 (73700).
[5:1]-Hexakisadduct: 1,2-{Bis [(8-(t-butyldimethy-silanyloxy)-octyloxy-carbonyl] methano}-18,36: 22,23: 27,45: 31,32: 55,60-pentakis [di(ethyloxy-carbonyl) methano]-1,2:18,36:22,23:27,45:31,32:55,60-dodecahydro[60]fullerene 73
The synthesis of the [5:1]-hexakisaddukt
73 was performed according to general
procedure GP5 (page 119). Monoadduct
72 (220 mg, 0.168 mmol, 1 eq.) was
reacted with 9,10-dimethyl anthracene
(DMA) (350 mg, 1.68 mmol, 10 eq.),
diethyl malonate (255 μl, 1.68 mmol,
10 eq.) CBr4 (557 mg, 1.68 mmol, 10 eq.)
and diluted DBU (251 μl,1.68 mmol,
10 eq.) in dry CH2Cl2. Pre-cleaning on
silica gel (toluene/ethyl acetate 5:1) and
subsequent purification by preparative
HPLC (Nucleosil 5 μm, toluene/ethyl acetate 49:1) gave a yellow solid (yield: 54 mg,
0.025 mmol, 15.3 %).
1H NMR (400 MHz, CDCl3, 25 °C): δ = 4.30 (q, 3J = 7.1 Hz, 20H, 18), 4.21 (t, 3J = 6.8
Hz, 4H, 4), 3.56 (t, 3J = 6.6 Hz, 4H, 11), 1.65 (dt, 3J = 6.2 Hz, 3J = 6.7 Hz, 4H, 12),
1.47 (m, 8H, 5, 9), 1.30 (m, 12H, 6-8), 1.30 (t, 3J = 7.1 Hz, 30H, 19), 0.86 (s, 18H,
15), 0.02 (s, 12H, 13). 13C NMR (100 MHz, CDCl3, 25 °C): δ = 163.9 (2C, 3), 163.8 (10C, 16), 145.8 (24C,
C60 sp2), 141.1 (24C, C60 sp2), 69.1 (2C, 1), 69.0 (10C, 1), 67.0 (2C, 11), 63.2 (2C, 4),
62.8 (10C, 18), 45.4 (1C, 2), 45.3 (5C, 16), 32.8, 29.2, 29.2, 28.4, 25.9, 25.7(12C, 5-12), 18.3 (6C, 15), 14.0 (10C, 19), -5.3 (4C, 13).
IR (KBr): ν~ = 2979, 2931, 2855, 1745, 1367, 1264, 1218, 1094, 1079, 1017, 835,
714, 528.
MS (FAB, NBA): m/z = 2097 [M]+, 2052 [M-3CH3]+, 720 [C60]+.
OO
O
O
OO
O
O
OOOO
OO
O
O
OO
O
O
O O
1
23
1 16 17 18
19
O
O 11
129
87
65
4O
OSiSi 13
1415
Experimental
143
UV/Vis (CH2Cl2): λmax, (ε, M-1cm-1) = 244 (95600), 271 (72600), 280 (76500), 315
(47800), 332 (38200).
Deprotected [5:1]-Hexakisadduct: 1,2-{Bis[(8-(hydroxyoctyloxycarbonyl]-methano}-18,36: 22,23: 27,45: 31,32: 55,60- pentakis[di-(ethyloxycarbonyl)-methano]-1,2: 18,36: 22,23: 27,45: 31,32: 55,60-dodecahydro[60]fullerene 74
54 mg (0.026 mmol) of the protected [5:1]-
hexakisadduct 73 were dissolved in 40 ml of
ethanol. 0.5 ml concentrated HCl was added
and the mixture stirred for 2 h at room
temperature. After neutralization with
saturated NaHCO3 solution and drying over
MgSO4 the solvent was removed in vacuo to
give a brownish material (yield: 45 mg,
0.024 mmol, 93 %).
1H NMR (400 MHz, CDCl3, 25 °C): δ = 4.30 (q, 3J = 7.1 Hz, 20H, 15), 4.22 (t, 3J =
6.6 Hz, 4H, 4), 3.60 (t, 3J = 6.6 Hz, 4H, 11), 1.64 (m, 8H, 5, 10), 1.53, 1.30 (m, 16H,
6-9), 1.30 (t, 30H, 3J = 7.1 Hz, 16). 13C NMR (100 MHz, CDCl3, 25 °C): δ = 163.9 (2C, 3), 163.8 (10C, 14), 145.8, 145.7
(24C, C60 sp2), 141.1, 141.0 (24C, C60 sp2), 69.1 (2C, 1), 69.0 (10C, 1), 67.0, 62.9
(2C, 4), 62.8 (10C, 15), 45.4 (1C, 2), 45.3 (5C, 13), 32.7, 29.2, 29.1, 28.9, 28.4, 25.7,
25.6, 14.0 (10C, 16).
IR (KBr): ν~ = 2980, 2931, 2855, 1744, 1367, 1264, 1220, 1079, 1017, 715, 528.
MS (FAB, NBA): m/z = 1869 [M]+, 1711 [M-diethyl malonate]+, 720 [C60]+.
UV/Vis (CH2Cl2): λmax, (ε, M-1cm-1) = 244 (91700), 271 (70200), 281 (70200), 316
(46400), 332 (36900).
OO
O
O
OO
O
O
OOOO
OO
O
O
OO
O
O
O O
1
23
1 13 14 15
16
O
HO 11
109
87
65
4O
OH12
Experimental
144
Pyropheophorbide-a diester of 1,2-{Di-[(8-(hydroxyoctyloxycarbonyl] methano}-18.36:22,23:27,45:31,32:55,60-pentakis[di(ethyloxycarbonyl)-methano]-1,2: 18,36:22,23:27,45:31,32:55,60-dodecahydro[60]fullerene 75
The formation of the bis-
pyropheophorbide-a-ester
75 was performed
according to general
procedure GP2 (page
117). Fullerene [5:1]-
hexakisadduct 74 (48 mg,
0.025 mmol) and
pyropheophorbide-a 19
(42 mg, 0.079 mmol) were
reacted with EDC (18 mg,
0.09 mmol) and DMAP
(8 mg, 0.06 mmol) in 30 ml of dry DMF at 0°C. After addition of 50 ml of CHCl3, the
mixture was washed twice with 50 ml of dilute acetic acid, twice with 50 ml of a
saturated NaHCO3 solution and twice with 50 ml of brine. After drying over Na2SO4,
the solvent was removed in vacuo and the residue pre-cleaned by FC on silica gel
(CH2Cl2/ethyl acetate 5:1). The second fraction was subjected to preparative HPLC
(Nucleosil 5 μm, CH2Cl2/ethyl acetate 47:3) giving a dark greenish material (yield:
47 mg, 0.016 mmol, 64 %).
1H NMR (400 MHz, CDCl3, 25 °C): δ = 9.23* (s, 2H, β), 9.14* (s, 2H, α), 8.47* (s, 2H,
δ), 7.82* (dd, 3J = 11.5 Hz, 3J = 17.8 Hz, 2H, 2a), 6.20* (dd, 2J = 1.5 Hz, 3J = 17.8 Hz,
2H, 2b), 6.10* (dd, 2J = 1.5 Hz, 3J = 11.5 Hz, 2H, 2b), 5.12* (d, 4H, 2J = 19.8 Hz, 10),
4.43* (m, 2H, 8), 4.33* (m, 2H, 7), 4.33 (m, 20H, 32), 4.16 (t, 3J = 6.8 Hz, 8H, 26),
3.52* (s, 6H, 5a), 3.49* (m, 4H, 4a), 3.31* (s, 6H, 1a), 3.06* (s, 6H, 3a), 2.63* (m, 2H,
7b), 2.50* (m, 2H, 7b), 2,24* (m, 4H, 7a), 1.78* (d, 3J = 7.3 Hz, 6H, 8a), 1.58* (t, 3J =
7.6 Hz, 6H, 4b), 1.31 (m, 24H, 20-25), 1.31 (m, 30H, 33), 0.10* (bs, 2H, NH), -1.92*
(s, 2H, NH).
OO
O
O
OO
O
O
OOOO
OO
O
O
OO
O
O
O O
29
2827
2930 31 32
33
O
OO
NNH
N HNO
11a
2 2a
2b
3 3a4
4a4b
5a5
6
77a
7b7c
88a
11
1210
913
1415
1617
18
a
b
g
d
19
2021
2223
2425
26O
OO
N HN
NNHO
3435
Experimental
145
13C NMR (100 MHz, CDCl3, 25 °C): δ = 195.9 (2C, 9), 173.0 (2C, 7c), 171.2, 163.8
(2C, 27), 163.7 (10C, 31), 160.2, 154.8, 150.4, 148.8, 146.2, 146.0, 145.7 (24C, 35),
144.7, 144.6, 141.4, 141.3, 141.1 (24C, 34), 137.7, 137.6, 137.5, 136.0, 135.9,
135,8. 135.7, 135.6, 135.5, 131.4, 131.3, 130.3, 129.1, 129.0, 128.7, 128.6, 128.2,
128.1, 122.4, 122.3, 105.9 (2C, γ), 103.8 (2C, β), 96.9 (2C, α), 92.9 (2C, δ), 69.0
(12C, 29), 66.8, 64.6, 62.8, 51.6, 49.9, 48.0, 45.4, 45.3, 31.0, 29.8, 29.7, 29.6, 29.0,
28.9, 28.4, 28.3, 25.7, 25.6, 23.0, 19.2, 19.1, 17.3, 14.0 (10C, 33), 12.0, 11.9, 11.8,
11.1, 11.0.
IR (KBr): ν~ = 2957, 2925, 2854, 1744, 1696, 1618, 1366, 1263, 1218, 1080, 1019,
715, 528.
MS (FAB, NBA): m/z = 2903 [M]+, 720 [C60]+.
UV/Vis (CH2Cl2): λmax, (ε, M-1cm-1) = 242 (90000), 271 (71900), 281 (75700), 319
(62800), 335 (55600), 414 (134000), 508 (13900), 538 (12800), 610 (10800), 667
(52900).
Bis-[5-(t-butoxycarbonyl)pentyl] malonate 77
The malonate 76 was synthesized
according to general procedure GP1
(page 117) with malonyl dichloride
(3.21 ml, 33 mmol) and pyridine (5.34 ml, 66 mmol). The reaction with 6-hydroxy-
hexanoic acid-t-butylester 76 (12.01 g, 66 mmol) in dry CH2Cl2 yielded the malonate
77 (yield: 7.52 g, 16.8 mmol, 51 %) after FC on silica gel (hexane/ethyl acetate 4:1)
as a white solid.
1H NMR (400 MHz, CDCl3, 25 °C): δ = 4.10 (t, 3J = 6.6 Hz, 4H, 3), 3.32 (s, 2H, 1),
2.18 (t, 3J = 7.7 Hz, 4H, 7), 1.60 (m, 8H, 4, 6), 1.41 (m, 4H, 5), 1.41 (s, 18H, 10). 13C NMR (100 MHz, CDCl3, 25 °C): δ = 172.8 (2C, 8), 166.6 (2C, 2), 80.1 (2C, 9),
65.3 (2C, 3), 41.5 (1C, 1), 35.3 (2C, 7), 28.2, 28.1, 25.3, 24.6.
IR (ATR): ν~ = 2976, 2868, 1731, 1458, 1367, 1256, 1152, 848.
MS (FAB, NBA): m/z = 445 [M]+, 389 [M-t-Bu]+, 333 [M-2t-Bu]+.
O OO O
OO
OO 1
2 34
56
78 9
10
Experimental
146
Bis-[5-(pentylcarbonyl)] malonate 79
Bis-[5-(t-butoxycarbonyl)pentyl] malonate
77 (3200 mg, 7 mmol) was dissolved in
30 ml of formic acid and stirred at room
temperature for 24 h. The solvent was removed in vacuo and the residue dried under
high vacuum. There was no further purification necessary (yield: 2260 mg,
6.85 mmol, 98 %).
1H NMR (400 MHz, CDCl3, 25 °C): δ = 11.26 (bs, 2H, 9), 4.13 (m, 4H, 3), 3.35 (m,
2H, 1), 2.33 (m, 4H, 7), 1.63 (m, 8H, 4, 6), 1.39 (m, 4H, 5). 13C NMR (100 MHz, CDCl3, 25 °C): δ = 179.4 (2C, 8), 166.4 (2C, 2), 65.3 (2C, 3),
41.7 (1C, 1), 32.9 (2C, 7), 28.2, 25.3, 24.3 (6C, 4-6).
IR (ATR): ν~ = 3550, 2946, 1746, 1696, 1590, 1412, 1258, 1191, 1150, 933.
MS (FAB, NBA): m/z = 333 [M]+, 289 [M-CO2]+.
6-Cascade: dihydromethane-[2]:(2-aza-9-oxa-3,10-dioxodecylidyne) :propanoic acid t-butylester 80
The amide 80 was
synthesized
according to general
procedure GP2
(page 117). Bis-[5-
(pentylcarbonyl)]
malonate 79 (509 mg, 1.53 mmol), 4-amino-4-[2-t-butoxycarbonyl)ethyl]heptanoic
acid di-t-butylester 78 (1430 mg, 3.4 mmol) and 1-HOBT (436 mg, 3.2 mmol) were
reacted with DCC (721 mg, 3.5 mmol) in 150 ml of dry CH2Cl2 at 0°C. FC on silica gel
(ethyl acetate/CH2Cl2 2:1) yielded the amide 80 (1305 mg, 1.16 mmol, 76 %) as a
white solid.
1H NMR (400 MHz, CDCl3, 25 °C): δ = 5.90 (s, 2H, NH), 4.10 (t, 3J = 6.6 Hz, 4H, 3),
3.33 (s, 2H, 1), 2.18 (t, 3J = 7.3 Hz, 12H, 12), 2.08 (t, 3J = 7.4 Hz, 4H, 7), 1.93 (t, 3J = 7.3 Hz, 12H, 11), 1.61 (m, 8H, 4, 6), 1.40 (s, 54H, 15), 1.33 (m, 4H, 5).
O OO O
OHO
HOO 1
2 34
56
78
9
O OO O H
NO
HN
O
OO
OO
OO
OO
OO
O O
12 3
45
67
89
10 1112
13
1415
Experimental
147
13C NMR (100 MHz, CDCl3, 25 °C): δ = 172.7 (6C, 13), 171.9 (2C, 8), 166.4 (2C, 2),
80.6 (6C, 14), 65.3 (2C, 3), 57.3 (2C, 10), 41.6 (1C, 1), 37.3 (2C, 7), 30.1 (6C, 12),
29.9 (6C, 11), 28.3 (2C, 4), 28.1 (18C, 15), 25.6, 25.4 (4C, 5, 6).
IR (ATR): ν~ = 2981, 1727, 1519, 1459, 1258, 1146, 949, 849.
MS (FAB, NBA): m/z = 1150 [M+Na]+, 1128 [M]+, 791 [M-6x t-Bu]+.
6-Cascade: dihydromethane -[2]:(2-aza-9-oxa-3,10-dioxodecylidyne) :propanoic acid 86
6-Cascade: dihydro-
methane-[2]:(2-aza-9-
oxa-3,10-dioxodecyli-
dyne):propanoic acid t-
butylester 80 (1.25 g,
1.1 mmol) was dissolved in 30 ml of formic acid and stirred at room temperature for
24 h. The solvent was removed and the residue dried in vacuo (yield: 856 mg, 1.08
mmol, 98 %).
1H NMR (400 MHz, THF-d8, 25 °C): δ = 10.25 (bs, 6H, 14), 6.58 (s, 2H, NH), 4.07 (t, 3J = 6.6 Hz, 4H, 3), 3.33 (s, 2H, 1), 2.21 (t, 3J = 7.4 Hz, 12H, 12), 2.11 (t, 3J = 7.2 Hz,
4H, 7), 1.96 (t, 3J = 7.4 Hz, 12H, 11), 1.60 (m, 8H, 4, 6), 1.37 (m, 4H, 5). 13C NMR (100 MHz, THF-d8, 25 °C): δ = 174.6 (6C, 13), 172.4 (2C, 8), 166.9 (2C, 2),
65.7 (2C, 3), 57.8 (2C, 10), 42.0 (1C, 1), 37.2 (2C, 7), 30.0 (6C, 12), 29.4, 28.7
(6C, 11), 26.6, 26.5.
IR (ATR): ν~ = 2991, 1710, 1416, 1279, 1181, 1042, 920, 855.
MS (FAB, NBA): m/z = 791 [M]+.
O OO O H
NO
HN
O
OHO
OHO
OHO
OHO
HOOHO O
12 3
45
67
89
10 1112
13
14
Experimental
148
6-Cascade: 1,2-Methano-1,2-dihydro[60]-fullerene [2]:(2-aza-9-oxa-3,10-dioxo-decylidyne):propanoic acid t-butylester 81
The synthesis of
monoadduct 81 was
performed according to
general procedure GP4
(page 118). C60 (950 mg,
1.3 mmol, 1.3 eq.) and
malonate 80 (1127 mg,
1 mmol, 1 eq.) were reacted
with CBr4 (331 mg, 1 mmol,
1 eq.) and DBU (164 μl,
1.1 mmol, 1.1 eq.) FC on
silica gel (toluene/ethyl acetate 3:1) yielded a brown powder, which was dried in
vacuo (yield: 719 mg, 0.39 mmol, 39 %).
1H NMR (500 MHz, THF-d8, 25 °C): δ = 5.94 (s, 2H, NH), 4.49 (t, 3J = 6.7 Hz, 4H, 4),
2.22 (t, 3J = 7.8 Hz, 12H, 13), 2.14 (t, 3J = 7.5 Hz, 4H, 8), 1.97 (t, 3J = 7.8 Hz, 12H,
12), 1.86 (q, 3J = 7.4 Hz, 4H, 5), 1.70 (q, 3J = 7.6 Hz, 4H, 7), 1.46 (m, 4H, 6), 1.44 (s,
54H, 16). 13C NMR (100 MHz, THF-d8, 25 °C): δ = 172.8 (6C, 14), 172.0 (2C, 9), 163.5 (2C, 3),
145.4, 145.3, 145.2, 144.9, 144.7, 144.6, 143.9, 143.4, 143.1, 143.0, 143.0, 141.2,
141.9, 140.9, 139.0 (58C, C60 sp2), 80.6 (6C, 15), 71.7 (2C, 1), 67.2 (2C, 4), 57.4
(2C, 8), 52.4 (1C, 2), 37.1, 30.0 (6C, 13), 29.8 (6C, 12), 28.4, 28.1, 27.9 (18C, 16),
25.6, 25.2 (4C, 6, 7).
IR (KBr): ν~ = 2975, 2933, 1731, 1680, 1654, 1537, 1456, 1391, 1234, 848, 527.
MS (FAB, NBA): m/z = 720 [C60]+.
UV/Vis (CH2Cl2): λmax (ε, M-1cm-1) = 258 (116100), 325 (36100), 425 (2200), 489
(1400).
O OO O
NHOOOOOO
O
HN OOOO O O
O
1
2 34
5 67 8 9 10
111213
14
15
16
Experimental
149
6-Cascade: 1,2-Methano-1,2-dihydro[60]-fullerene [2]:(2-aza-9-oxa-3,10-dioxo-decylidyne):propanoic acid 82
The monoadduct 81 (207 mg,
0.11 mmol) was dissolved in
10 ml toluene and 5 ml TFA were
added. After stirring for 2 h at
room temperature the solvent
was removed in vacuo yielding a
brown solid (yield: 163 mg, 0.11
mmol, 97 %).
1H NMR (400 MHz, THF-d8, 25 °C): δ = 9.90 (bs, 6H, 15), 6.61 (s, 2H, NH), 4.49 (t, 3J = 6.7 Hz, 4H, 4), 2.23 (m, 12H, 13), 2.14 (m 4H, 8), 1.97 (m 12H, 12), 1.86 (m, 4H,
5), 1.70 (m, 4H, 7), 1.46 (m, 4H, 6). 13C NMR (100 MHz, THF-d8, 25 °C): δ = 174.7 (6C, 14), 172.7 (2C, 9), 163.7 (2C, 3),
147.3, 147.0, 146.7, 145.4, 145.3, 145.2, 144.9, 144.6, 144.2, 144.1, 143.8, 143.7,
143.4, 143.2, 142.9, 142.7, 141.6, 139.9 (58C, C60-sp2), 73.9 (2C, 1), 67.8 (2C, 4),
57.7, 53.4, 37.1, 30.2, 30.0, 29.3, 28.5, 26.3, 25.6.
IR (KBr): ν~ = 3356, 2975, 2932, 1731, 1680, 1537, 1456, 1427, 1391, 1368, 1234,
1154, 848, 527.
MS (FAB, NBA): m/z = 720 [C60]+, 1509 [M]+.
UV/Vis (THF): λmax (ε, M-1cm-1) = 258 (81300), 325 (31300), 425 (2100), 489 (1300).
O OO O
NHOOHOOHOOH
O
HN OHO
OHO OHO
O
1
2 34
5 67 8 9 10
111213
14
15
Experimental
150
N-(4-N´-t-Butoxycarbonyl-aminobutyl)-pyropheophorbide-a-amide 83
The pyropheophorbide-a-amide 83
was synthesized according to
general procedure GP2 (page 117).
Pyropheophorbide-a 19 (300 mg,
0.56 mmol), t-butyl-4-aminobutyl-
carbamat (230 mg, 1.2 mmol) and
1-HOBT (76 mg, 0.56 mmol) were reacted with EDC (110 mg, 0.57 mmol) in 30 ml of
dry DMF at 0°C. After addition of 50 ml of CHCl3, the mixture was washed twice with
40 ml of dilute HCl, twice with 40 ml of a saturated NaHCO3 solution and twice with
40 ml of brine. FC on silica gel (ethyl acetate/methanol 9:1) yielded the amide 83
(367 mg, 0.52 mmol, 93 %) as a black green solid.
1H NMR (400 MHz, CDCl3, 25 °C): δ = 9.18* (s, 1H, β), 8.93* (s, 1H, α), 8.47* (s, 1H,
δ), 7.85* (dd, 3J = 11.5 Hz, 17.8 Hz, 1H, 2a), 6.18* (dd, 2J = 1.3 Hz, 3J = 17.8 Hz, 1H,
2b), 6.07* (dd, 2J = 1.3 Hz, 3J = 11.5 Hz, 1H, 2b), 5.53 (bs, 1H, NH), 5.05 (d, 2J =
19.6 Hz, 1H, 10), 4.91 (d, 2J = 19.6 Hz, 1H, 10), 4.54 (bs, 1H, NH), 4.42* (m, 1H, 8),
4.18* (m, 1H, 7), 3.41* (m, 2H, 4a), 3.32* (s, 3H, 5a), 3.09 (s, 6H, 1a, 3a), 2.90 (m,
2H, 20), 2.85 (m, 1H, 24), 2.46-1.77* (m, 4H, 7a/7b), 1.73* (d, 3J = 7.3 Hz, 3H, 8a),
1.51* (t, 3J = 7.5 Hz, 3H, 4b), 1.30 (s, 9H, 27), 1.17 (m, 4H, 21, 22), 0.22* (bs, 1H,
NH), -1.83* (s, 1H, NH). 13C NMR (100 MHz, CDCl3, 25 °C): δ = 196.1, 172.2, 171.7, 160.4, 155.9, 154.9,
150.4, 148.7, 144.8, 141.4, 137.4, 135.8, 135.6, 131.4, 130.0, 128.5, 127.8, 122.4,
105.8, 103.6, 96.9, 92.9, 78.9, 72.2, 51.6, 50.2, 49.9, 47.9, 39.9, 38.9, 32.7, 30.2,
28.7, 28.6, 28.4, 28.4, 28.3, 27.4, 26.3, 22.9, 19.4, 19.2, 17.3, 12.0, 11.5, 11.1.
IR (ATR): ν~ = 3272, 2960, 2923, 2865, 1621, 1551, 1497, 1347, 1123, 978, 893.
MS (FAB, NBA): m/z = 705 [M]+.
UV/Vis (CH2Cl2): λmax, (ε, M-1cm-1) = 322 (18700), 414 (89600), 508 (9200), 538
(7900), 609 (6800), 667 (37800).
NNH
NHN
O
HNN
HO
O
O
1 2
34
56
7
8
1a 2a 2b
3a
4a
5a9
10
7a7b
8a
4b
α
β
δ
γ
11 1213
141516
17
187c
19
20212223
24
252627
Experimental
151
Pyropheophorbide-a hexaamide of 6-Cascade: 1,2-Methano-1,2-dihydro[60]-fullerene [2]:(2-aza-9-oxa-3,10-dioxodecylidyne):propanoic acid 85
O OO O
NHONH
ONHONH
O
O
N
NH N
HN
OHN
HN
HN
O NNH
NHN
O
O
NNH
NHN
O
HN ONH
OHN OHN
O
O
N
HNN
NH
O NH
HN
NH
ONNH
N HN
O
O
NHN
NNH
O1
1a2
2a2b
33a
44a
4b
5a5
6910
788a
7a7b7c
11
12 13
1415
1617
18
192021
222324
25262728
293031
32333435
3637
38
N-(4-N´-t-butoxycarbonyl-4-aminobutyl)-pyropheophorbide-a-amide 83 (169 mg,
0.24 mmol) was dissolved in CH2Cl2 (30 ml) and TFA (5 ml) was added. The solution
was stirred for 2 h at room temperature and then neutralized using a saturated
NaHCO3-solution. After drying over MgSO4 and evaporation of the solvent, the dark
green powder 84 was used without further purification and characterization.
Monoadduct 82 (40 mg, 0.026 mmol) and N-(4-aminobutyl)-pyropheophorbide-a-
amide 84 (140 mg, 0.18 mmol) were dissolved in dry DMF (10 ml) and cooled to 0°C.
EDC (85 mg, 0.45 mmol), 1-HOBT (35 mg, 0.26 mmol) and DMAP (22 mg,
0.18 mmol) were added and the mixture was stirred for 12 h at room temperature.
After 24 h, more N-(4-aminobutyl)-pyropheophorbide-a-amide 83 (77 mg, 0.24 mmol)
was added. The solvent was removed in vacuo and the residue cleaned by SEC
(Biobeads SX3, CH2Cl2) yielding a dark green residue (yield: 47 mg, 0.009 mmol,
37%).
1H NMR (400 MHz, CDCl3, 25 °C): δ = 8.84* (bs, 12H, α, β), 8.30* (bs, 6H, δ), 7.49*
(bs, 6H, 2a), 7.16 (bs, NH), 5.93* (bs, 12H, 2b), 4.90* (bs, 6H, 10), 4.68* (bs, 6H, 10),
4.24* (bs, 10H, 8, 35), 3.96* (bs, 6H, 7), 2.97* (bs, 90H, 4a, 5a, 1a, 3a, 20, 23), 2.05*
(bs, 18H, 7a/b, 26, 31), 1.58 (bs, 18H, 8a), 1.33 (bs, 78H, 4b, 21, 22, 32-34), -0.18*
(bs, 6H, NH), -2.21* (s, 6H, NH).
Experimental
152
13C NMR (100 MHz, CDCl3, 25 °C): δ = 196.2, 192.8, 173.3, 172.9, 172.7, 172.5,
172.3, 172.1, 171.7, 160.6, 159.4, 155.0, 154.8, 154.7, 150.2, 148.5, 147.5, 146.5,
146.4, 145.7, 145.4, 144.2, 144.3, 144.1, 143.9, 143.4, 143.2, 143.0, 142.8, 142.1,
141.3, 140.9, 140.0, 139.999, 137.1, 136.9, 136.2, 135.9, 135.6, 134.9, 131.4, 131.3,
130.3, 129.6, 129.3, 129.2, 128.8, 128.5, 128.2, 127.6, 127.2, 122.3, 122.2, 122.1,
121.9, 118.5, 113.8, 107.5, 102.7, 96.8, 96.7, 93.0, 90.3, 60.6, 51.5, 51.3, 49.8, 47.9,
47.7, 39.5, 39.0, 38.8, 38.6, 37.0, 33.5, 33.1, 31.4, 31.2, 31.1, 30.6, 30.1, 29.7, 28.3,
28.0, 27.9, 27.5, 27.3, 27.1, 26.8, 26.2, 26.0, 25.7, 25.3, 23.8, 23.4, 22.8, 22.4, 19.3,
19.0, 19.3, 19.0, 17.2, 17.0, 11.9, 11.5, 10.9.
IR (ATR): ν~ = 2954, 2925, 2867, 1648, 1619, 1536, 1497, 1451, 1366, 1347, 1219,
1027, 978, 671.
MS (Maldi-TOF, 2,5-dihydroxy benzoic acid): m/z = 5037.9 (calc. 5030.1) [M]+,
4432.6 (calc. 4423.0) [M-(pyropheo+spacer)]+, 3826.4 (calc. 3819.7)
[M-2 (pyropheo+spacer)]+.
UV/Vis (DMF): λmax, (ε, M-1cm-1) = 322 (119100), 413 (325400), 509 (43900), 539
(35000), 613 (27800), 669 (127500).
Elemental analysis: C317H304N38O24·5CHCl3 (5615.95), calcd: C, 68.73; H, 5.54;
N, 9.46, found: C, 68.40; H, 5.88; N, 9.69.
6-Cascade: 18.36: 22,23: 27,45: 31,32: 55,60-pentakis[di(ethyloxy-carbonyl)-methano]-1,2: 18,36: 22,23: 27,45: 31,32: 55,60-dodecahydro-1,2-methano-[60] fullerene [2]:(2-aza-9-oxa-3,10-dioxodecylidyne) :propanoic acid t-butylester 88
The synthesis of the [5:1]-
hexakisaddukt 88 was
performed according to general
procedure GP5 (page 119).
Monoadduct 81 (1.38 g, 0.75
mmol, 1 eq.) was reacted with
9,10-dimethyl anthracene
(DMA) (1.59 g, 7.7 mmol, 10.2
eq.), diethyl malonate (1.6 ml,
10 mmol, 13.3 eq.) CBr4 (3.31 g,
10 mmol, 13.3 eq.) and dilute
OO
O
O
OO
O
O
OOOO
OO
O
O
OO
O
O
O OO O
NHOOOOOO
O
HN OOOO O O
O
1
23
45 6
7 8 9 10
1112
1314 15
16
1718
1920
21
Experimental
153
DBU (1.6 ml, 10.7 mmol, 14.2 eq.) in dry CH2Cl2. Pre-cleaning on silica gel
(toluene/ethyl acetate 1:1) and subsequent purification by preparative HPLC
(Nucleosil 5 μm, toluene/ethyl acetate 39:11) gave a yellow solid (yield: 330 mg,
0.125 mmol, 16.6 %).
1H NMR (400 MHz, CDCl3, 25 °C): δ = 5.92 (s, 2H, NH), 4.30 (q, 3J = 7.1 Hz, 20H,
20), 4.23 (t, 3J = 6.5 Hz, 4H, 4), 2.17 (t, 3J = 7.3 Hz, 12H, 13), 2.07 (t, 3J = 7.3 Hz, 4H,
8), 1.93 (t, 3J = 7.3 Hz, 12H, 12), 1.68-1.55 (m, 12H, 5-7), 1.39 (s, 54H, 16), 1.30 (t, 3J = 7.1 Hz, 30H, 21). 13C NMR (100 MHz, CDCl3, 25 °C): δ = 172.8, 172.2, 163.9, 163.8, 146.1, 146.0,
145.8, 145.7, 145.5, 141.3, 141.1, 141.1, 141.0, 96.3, 80.5, 69.1, 66.7, 62.9, 62.9,
62.8, 57.3, 53.4, 45.4, 45.4, 37.0, 29.8, 29.7, 29.3, 28.3, 28.1, 25.6, 25.2, 14.0.
IR (KBr): ν~ = 2979, 2936, 1744, 1679, 1528, 1458, 1367, 1264, 1220, 1154, 1018,
715, 529.
MS (FAB, NBA): m/z = 2637[M]+, 2300 [M-6 t-Bu]+, 720 [C60]+.
UV/Vis (CH2Cl2): λmax (ε, M-1cm-1) = 245 (93200), 282 (75100), 314 (46900), 334
(37500).
6-Cascade : 18.36: 22,23: 27,45: 31,32: 55,60-pentakis[di(ethyloxy-carbonyl)-methano]-1,2: 18,36: 22,23: 27,45: 31,32: 55,60-dodecahydro-1,2-methano-[60] fullerene [2] :(2-aza-9-oxa-3,10-dioxodecylidyne) :propanoic acid 89
Hexakisadduct 88 (247 mg,
0.09 mmol) was dissolved in
50 ml of dry CH2Cl2. TFA (5 ml)
was added and the mixture
stirred for 24 h at room
temperature. The mixture was
washed twice with 40 ml of brine,
dried over NaSO4 and the solvent
was removed in vacuo yielding
an orange solid. (yield: 211 mg,
0.09 mmol, 98%).
OO
O
O
OO
O
O
OOOO
OO
O
O
OO
O
O
O OO O
NHOOHOOHOOH
O
HN OHO
OHO OHO
O
1
23
45 6
7 8 9 10
1112
1314
15
1617 18 19
20
Experimental
154
1H NMR (400 MHz, THF-d8, 25 °C): δ = 6.60 (s, 6H, 15), 4.31 (m, 24H, 4, 19), 2.22 (t, 3J = 7.1 Hz, 12H, 13), 2.12 (t, 3J = 6.8 Hz, 4H, 8), 1.98 (t, 3J = 7.9 Hz, 12H, 13), 1.69
(m, 4H, 7), 1.61 (m, 4H, 5), 1.38 (m, 4H, 6), 1.29 (t, 3J = 7.1 Hz, 30H, 20). 13C NMR (100 MHz, THF-d8, 25 °C): δ = 174.9 (6C, 14), 172.7 (2C, 9), 163.9 (12C, 3,
18), 146.6, 146.5, 146.5, 142.0, 141.9, 141.9 (all C60-sp2), 69.9, 63.4, 63.3, 57.7,
46.6, 46.4, 36.9, 30.5, 30.2, 29.1 28.6, 26.4, 26.1, 14.2, 14.2.
IR (KBr): ν~ = 2981, 1743, 1396, 1265, 1222, 1043, 715, 529.
MS (FAB, NBA): m/z = 2339 [M+2Na]+, 2323 [M+Na]+, 2300 [M]+, 720 [C60]+
UV/Vis (CH2Cl2): λmax, (ε, M-1cm-1) = 223 (125600), 244 (84600), 281 (68900), 315
(43200), 334 (34600).
Pyropheophorbide-a hexaamide of 6-Cascade: 18.36: 22,23: 27,45: 31,32: 55,60-pentakis[di(ethyloxycarbonyl)methano]-1,2: 18,36: 22,23: 27,45: 31,32: 55,60-dodecahydro-1,2-methano-[60]fullerene [2]: (2-aza-9-oxa-3,10-dioxodecylidyne) :propanoic acid 90
N-(4-N´-t-butoxycarbonyl-aminobutyl)-pyropheophorbide-a-amide 83 (132 mg,
0.18 mmol) was dissolved in CH2Cl2 (30 ml) and TFA (5 ml) was added. The solution
was stirred for 2 h at room temperature and then neutralized with saturated NaHCO3-
solution. After drying over MgSO4 and evaporation of the solvent, the dark green
OO
O
O
OO
O
O
OOOO
OO
O
O
OO
O
O
O OO O
NHONH
ONHONH
O
O
N
NH N
HN
OHN
HN
HN
O NNH
NHN
O
O
NNH
NHN
O
HN ONH
OHN OHN
O
O
N
HNN
NH
O NH
HN
NH
ONNH
N HN
O
O
NHN
NNH
O1
1a2
2a2b
33a
44a
4b
5a5
6910
788a
7a7b7c
11
12 13
1415
1617
18
192021
222324
25262728
293031
32333435
3637
38
39
4041 42
43
Experimental
155
residue was used without further purification and characterization.
Hexaacid 89 (35 mg, 0.015 mmol) and N-(4-aminobutyl)-pyropheophorbide-a-amide
84 (113 mg, 0.18 mmol) were dissolved in dry DMF (10 ml) and cooled to 0°C. EDC
(85 mg, 0.45 mmol), 1-HOBT (35 mg, 0.26 mmol) and DMAP (22 mg, 0.18 mmol)
were added and the mixture was stirred for 12 h at room temperature. After 70 h,
additional EDC (83 mg, 0.43 mmol) was added and stirring prolonged for 4h at room
temperature. CHCl3 (50 ml) was added, the mixture was washed twice with dilute HCl
(50 ml), twice with a saturated NaHCO3 solution (50 ml) and twice with brine (50 ml).
The organic layer was dried over Na2SO4, the solvent removed in vacuo and FC on
silica (CHCl3/ethanol 7:3) yielded a dark green powder (yield: 37 mg, 0.006 mmol,
49 %).
1H NMR (400 MHz, CDCl3, 25 °C): δ = 8.83 (bs, 6H, β), 8.63 (bs, 6H, α), 8.25 (bs,
6H, δ), 7.52 (bs, 6H, 2a), 6.97 (bs, NH), 5.92 (m, 12H, 2b), 4.88 (bs, 6H, 10), 4.65
(bs, 6H, 10), 4.30 (m, 24H, 35, 42), 4.10 (bs, 6H, 8), 3.82 (bs, 6H, 7), 3.09 (bs, 24H,
20, 23), 2.91 (s, 18H, 5a), 2.87 (bs, 30H, 1a, 4a), 2.85 (s, 18H, 3a), 2.16 (bs, 16H,
26, 31), 2.16 (bs, 12H, 7a/b), 1.92 (bs, 12H, 7a/b), 1.52 (bs, 18H, 8a), 1.36 (bs, 18H,
4b), 1.30 (m, 30H, 43), 1.25 (m, 12H, 32-34), 1.09 (m, 24H, 21, 22), -0.18* (bs, 6H,
NH), -2.21* (s, 6H, NH). 13C NMR (100 MHz, CDCl3, 25 °C): δ = 196.0, 172.7, 171.8, 163.7, 150.0, 148.5,
145.8, 145.7, 145.7, 144.7, 144.4, 141.5, 141.1, 141.0, 137.2, 135.7, 135.4, 131.3,
129.7, 129.0, 128.7, 127.3, 122.1, 105.6, 103.2, 96.5, 92.8, 69.0, 69.0, 66.7, 62.8,
51.6, 49.7, 47.8, 45.4, 45.3, 45.2, 38.9, 36.4, 32.9, 30.5, 29.6, 29.4, 28.1, 26.5, 25.4,
22.7, 19.0, 17.1, 14.0, 13.7, 11.9, 11.5, 10.9.
IR (KBr): ν~ = 2926, 2864, 1744, 1653, 1545, 1498, 1450, 1366, 1347, 1262, 1220,
1123, 1024, 786, 750, 673.
MS (Maldi-TOF, 2,5-dihydroxy benzoic acid): m/z = 5821.4 (calc. 5820.8) [M]+,
5216.7 (calc. 5217.5) [M-(pyropheo+spacer)]+, 4612.1 (calc. 4613.1)
[M-2 (pyropheo+spacer)]+.
UV/Vis (DMF): λmax, (ε, M-1cm-1) = 264 (206200), 320 (155300), 413 (479500), 471
(23500), 509 (51000), 539 (36100), 668 (208500).
Elemental analysis: C352H354N38O44·3CHCl3 (6170.41), calcd: C, 69.01; H, 5.82;
N, 8.61, found: C, 68.99; H, 5.97; N, 8.31.
Experimental
156
Pyropheophorbide-a hexaamide of 6-Cascade: dihydromethane -[2]:(2-aza-9-oxa-3,10-dioxodecylidyne):propanoic acid 87
N-(4-N´-t-butoxycarbonyl-aminobutyl)-pyropheophorbide-a-amide 83 (200 mg,
0.28 mmol) was dissolved in CH2Cl2 (30 ml) and TFA (5 ml) was added. The solution
was stirred for 2 h at room temperature and then neutralized with saturated NaHCO3-
solution. After drying over MgSO4 and evaporation of the solvent, the dark green
residue was used without further purification and characterization.
Hexaacid 86 (23 mg, 0.03 mmol) and N-(4-aminobutyl)-pyropheophorbide-a-amide
84 (170 mg, 0.28 mmol) were dissolved in dry DMF (10 ml) and cooled to 0°C.
1-HOBT (40 mg, 0.29 mmol), DMAP (20 mg, 0.16 mmol) and EDC (106 mg,
0.55 mmol) were added and the mixture stirred for 24 h at room temperature. CHCl3
(50 ml) was added, the mixture was washed twice with dilute HCl (50 ml), twice with
a saturated NaHCO3 solution (50 ml) and twice with brine (50 ml). The organic layer
was dried over NaSO4, the solvent removed in vacuo and FC on silica
(CHCl3/ethanol 7:3) yielded a dark green powder (yield: 68 mg, 0.016 mmol, 72 %).
1H NMR (400 MHz, CDCl3, 25 °C): δ = 8.80* (bs, 6H, β), 8.53* (bs, 6H, α), 8.23* (bs,
6H, δ), 7.49* (m, 6H, 2a), 6.84 (bs, 6H, NH), 5.90* (d, 3J = 17.7 Hz, 6H, 2b), 4.81* (d,
OO O
OHN
O
HN
O
HNOH
NO
NHO
O
NHN
NNH
O
NH
NH
HN
O
N HN
NNHO
O
N
NH N
HN
O
NHOH
NOHN O
O
NHN
NNH
O
NH
HN
NH
O
NNH
N HN O
O
N
HNN
NH
O
11a
22a
2b
3a34a
4b
45a
5
109
7
8a8
7a7b7c
11
1213
1415
1617
18
α
β
δ
γ
19202122
232425
2627
28
29
3031
32
33
34
35
3637
Experimental
157
6H, 3J = 11.4 Hz, 6H, 2b), 4.83* (d, 6H, 2J = 19.5 Hz, 10), 4.57* (d, 6H, 2J = 19.5 Hz,
10), 4.12* (bs, 6H, 8), 3.82* (m, 10H, 7, 35), 3.04* (bs, 42H, 4a, 20, 23), 2.82* (bs,
54H, 5a, 1a, 3a), 2.40-1.70* (m, 52H, 7a/b, 26, 31), 1.50* (m, 18H, 8a), 1.30 (t, 18H, 3J = 7.0 Hz, 4b), 1.22 (bs, 12H, 32-34), 1.10 (bs, 32H, 21, 22), -0.19* (bs, 6H, NH),
-2.21* (s, 6H, NH). 13C NMR (100 MHz, CDCl3, 25 °C): δ = 196.1, 173.2, 172.7, 171.6, 166.4, 160.3,
154.7, 150.1, 148.6, 148.4, 144.5, 137.0, 135.7, 135.5, 135.2, 131.2, 129.5, 129.1,
128.7, 128.3, 127.2, 122.0, 105.5, 103.2, 96.5, 92.7, 65.8, 65.1, 64.6, 57.1, 51.4,
49.7, 47.7, 41.9, 38.9, 36.5, 32.9, 30.5, 30.2, 29.7, 27.9, 27.8, 26.6, 25.2, 22.6, 18.9,
17.2, 15.3, 11.8, 11.3, 10.8.
IR (ATR): ν~ = 3309, 2981, 1731, 1519, 1416, 1264, 1235, 1040, 1001, 920.
MS (FAB, NBA): m/z = 4312 [M]+.
UV/Vis (CH2Cl2): λmax, (ε, M-1cm-1) = 280 (75400), 326 (114300), 401 (369600), 413
(368800), 510 (39700), 540 (33600), 614 (30300), 670 (160200).
Elemental analysis: C257H306N38O24·4CHCl3 (4780.05), calcd: C, 65.46; H, 6.52;
N, 11.11, found: C, 65.57; H, 6.58; N, 11.04.
Bis-(6-N-t-butoxycarbonylamino-1-hexanol)-malonate 92
The BOC-protected malonate
92 was synthesized
according to general
procedure GP1 (page 117) with malonyl dichloride (2.34 ml, 25 mmol) and pyridine
(4.43 ml, 55 mmol). The reaction with t-butyl-6-hydroxyhexylcarbamate 91 (10.86 g,
50 mmol) and DMAP (612 mg, 5 mmol) in dry CH2Cl2 yielded the malonate 92 (yield:
8.30 g, 16.8 mmol, 66 %) after FC on silica gel (CH2Cl2/ethyl acetate 7:3) as a white
solid.
1H NMR (400 MHz, CDCl3, 25 °C): δ = 4.56 (bs, NH), 4.09 (t, 3J = 6.6 Hz, 4H, 3),
3.32 (s, 2H, 1), 3.07 (q, 3J = 6.5 Hz, 4H, 8), 1.61 (dt, 3J = 6.8 Hz, 3J = 6.7 Hz, 4H, 4),
1.43 (dt, 3J = 7.2 Hz, 3J = 6.9 Hz, 4H, 7), 1.40 (s, 18H, 12), 1.31 (m, 8H, 5). 13C NMR (100 MHz, CDCl3, 25 °C): δ = 166.6 (2C, 2), 155.9 (2C, 10), 79.0 (2C, 11),
65.4 (2C, 3), 41.6 (1C, 1), 40.4 (2C, 8), 29.9 (2C, 7), 28.7 (2C, 4), 28.4 (6C, 12), 26.3
(2C, 5), 25.5 (2C, 6).
12 3
4 5 6 7 8
910 11 12O
HN O O
HN O
O O
O O
Experimental
158
IR (KBr): ν~ = 3368, 2935, 2861, 2249, 1703, 1518, 1391, 1250, 1172, 733.
MS (FAB, NBA): m/z = 503 [M]+, 403 [M-BOC]+, 347 [M-BOC -t-Bu]+.
Elemental analysis: C25H48N2O8 (502.33), calcd: C, 59.74; H, 9.22; N, 5.57, found:
C, 59.47; H, 9.22; N, 5.65.
BOC-protected [6:0]-Hexakisaddukt: 1,2:18,36:22,23:27,45:31,32:55,56-Hexakis {[di-(6-N-BOC-aminohexyloxycarbonyl)]-methano}-1,2:18,36:22,23:27,45:31,32: 55,56-dodecahydro[60]fullerene 93
The synthesis of the [6:0]-
hexakisaddukt 93 was performed
according to general procedure GP5
(page 119). C60 (900 mg, 1.25 mmol,
1 eq.) was reacted with DMA (2.60 g,
12.5 mmol, 10 eq.), 6-N-t-butoxy-
carbonylamino-1-hexyl malonate 92
(6.30 g, 12.5 mmol, 10 eq.), CBr4
(4.15 g, 12.5 mmol, 10 eq.) and diluted
DBU (2.24 ml, 15 mmol, 12 eq.) in dry
toluene. Pre-cleaning on silica gel
(CH2Cl2/ethyl acetate 3:2) and
subsequent purification by preparative HPLC (Nucleosil 5 μm, CH2Cl2/ethyl acetate
3:1) gave a yellow solid (yield: 1.95 g, 0.52 mmol, 42 %).
1H NMR (400 MHz, CDCl3, 25 °C): δ = 4.76 (bs, NH), 4.22 (t, 3J = 6.5 Hz, 24H, 6),
3.06 (dt, 3J = 6.3 Hz, 3J = 6.5 Hz, 24H, 11), 1.64 (m, 24H, 7), 1.45 (m, 24H, 10) 1.40
(s, 108H, 15), 1.31 (m, 48H, 8, 9). 13C NMR (100 MHz, CDCl3, 25 °C): δ = 163.8 (12C, 5), 156.0 (12C, 13), 145.7 (C60-
sp2), 141.1 (C60-sp2), 78.9 (12C, 14), 69.1 (12C, 3), 66.9 (12C, 6), 45.4 (6C, 4), 40.5
(12C, 11), 29.9, 28.4 (36C, 15), 28.3, 26.4, 25.6.
IR (KBr): ν~ = 3414, 2932, 2858, 1746, 1714, 1521, 1365, 1265, 1169, 715.
MS (FAB, NBA): m/z = 3724 [M]+, 3667 [M-t-Bu]+, 3624 [M-BOC]+, 3568 [M-2 t-Bu]+,
3525 [M-2BOC]+, 3424 [M-3BOC]+, 3324 [M-4BOC]+, 3224 [M-5BOC]+,
3124 [M-6BOC] +.
O OHN
OONH
OO
OO
OONH
O OHN
OO
OO
OO
NH
O
OHN
OO
O O
OO
HN
O
OHN
OO
O OOO
HN
O
O NH
OO
OO
OO
NH
O
O NH
OO
OO
1 2
3
45
67
89
1011 12
13
1415
Experimental
159
UV/Vis (CH2Cl2): λmax (ε, M-1cm-1) = 243 (97700), 281 (77000), 439 (1500).
Elemental analysis: C210H264N12O48 (3724.39), calcd: C, 67.72; H, 7.14; N, 4.51,
found: C, 67.15; H, 7.25; N, 4.52.
Pyropheophorbide-a-N-hydroxysucinimid ester 95
The NHS-active ester 95 was prepared according to
general procedure GP3 (page 118).
Pyropheophorbide-a 19 (800 mg, 1.5 mmol, 1 eq.) was
dissolved in dry CH2Cl2 (50 mL). NHS (213 mg,
1.8 mmol, 1.2 eq.), DMAP (24 mg, 0.2 mmol, 0.13 eq.)
and EDC (390 mg, 2 mmol, 1.35 eq.) were added under
N2 at room temperature. The solution was stirred for
12 h at room temperature. Subsequent removal of the
solvent in vacuo and FC on silica (CH2Cl2/acetone 9:1) yielded the desired NHS-
ester (yield: 420 mg, 0.6 mmol, 44 %).
1H NMR (400 MHz, CDCl3, 25 °C): δ = 9.25 (s, 1H, β), 9.15 (s, 1H, α), 8.50 (s, 1H, δ),
7.84 (dd, 3J = 11.5 Hz, 17.8 Hz, 1H, 2a), 6.18 (dd, 2J = 1.5 Hz, 3J = 17.8 Hz, 1H, 2b),
6.08 (dd, 2J = 1.5 Hz, 3J = 11.5 Hz, 1H, 2b), 5.17 (d, 2J = 19.9 Hz, 1H, 10), 5.09 (d, 2J = 19.9 Hz, 1H, 10), 4.45 (dq, 3J = 7.3 Hz, 3J = 2.0 Hz, 1H, 8), 4.35 (dt, 3J = 9.8 Hz, 3J = 2.0 Hz, 1H, 7), 3.55 (s, 3H, 5a), 3.49 (q, 3J = 8.5 Hz, 2H, 4a), 3.34 (s, 3H, 1a),
3.06 (s, 3H, 1a), 2.80 (m, 2H, 7a/b), 2.80 (bs, 4H, 20), 2.60 (m, 1H, 7a/7b), 2.20 (m,
1H, 7a/7b), 1.79 (d, 3J = 7.3 Hz, 3H, 8a), 1.60 (t, 3J = 7.7 Hz, 3H, 4b), 0.23 (bs, 1H,
NH), -1.90 (s, 1H, NH). 13C NMR (100 MHz, CDCl3, 25 °C): δ = 196.1 (1C, 9), 170.9 (1C, 7c), 169.0 (1C, 19),
168.2 (1C, 19), 159.5, 154.9, 150.5, 148.8, 144.7, 141.4, 137.6, 135.9, 135.8, 135.6,
131.4, 130.4, 130.0, 129.0 (1C, 2a), 128.1, 122.3 (1C, 2b), 105.9 (1C, γ), 103.8 (1C,
β), 97.0 (1C, α), 92.9 (1C, δ), 50.9 (1C, 7), 49.7 (1C, 8), 47.8 (1C, 10), 29.5 (1C, 7a),
28.1 (1C, 7b), 25.5 (2C, 20), 23.0 (1C, 8a), 19.2 (1C, 4a), 17.3 (1C, 4b), 12.0 (1C,
5a), 11.9 (1C, 1a), 11.0 (1C, 3a).
IR (ATR): ν~ = 2966, 2929, 2861, 1808, 1785, 1739, 1617, 1499, 1349, 1210, 1061,
980, 820.
MS (FAB, NBA): m/z = 632 [M]+.
O O
N
NH N
HN
O
NOO
11a2
2a2b
33a
44a
4b
5a5
6910
77a7b7c
88a
11
12 13
1415
1617
18
1920
Experimental
160
UV/Vis (CH2Cl2): λmax (ε, M-1cm-1) = 322 (20600), 413 (108300), 476 (4000), 508
(10600), 538 (9500), 609 (8000), 666 (46000).
Elemental analysis: C37H37N5O5·CH2Cl2 (716.65), calcd: C, 63.69; H, 5.49; N, 9.77,
found: C, 63.35; H, 5.89; N, 10.31.
1,2:18,36:22,23:27,45:31,32:55,56-Hexakis{[di-(6-N-pyropheophorbide-a-amino-hexyloxycarbonyl)]-methano}-1,2:18,36:22,23:27,45:31,32:55,56-dodecahydro [60]fullerene 96
The deprotected
hexakisadduct 94
(55 mg, 0.015 mmol)
was dissolved in 50 ml
of brine. CH2Cl2
(50 ml) and TEA
(100 μl) were added.
The organic layer was
separated and
washed with brine (2x
20 ml). After drying
over Na2SO4, pyro-
pheophorbide-a-
hydroxysuccinimid-
ester 95 (170 mg,
0.27 mmol) was
added and the solution stirred for 72 h at room temperature. Evaporation of the
solvent and subsequent SEC (1. Bio-beads SX3 2. Bio-beads SX1, CHCl3) yielded a
black green solid (yield: 97 mg, 0.011 mmol, 74 %).
1H NMR (400 MHz, CDCl3, 25 °C): δ = 8.81* (bs, 12H, β), 8.49* (bs, 12H, α), 8.29*
(bs, 12H, δ), 7.46* (dd, 3J = 11.6 Hz, 17.8 Hz, 12H, 2a), 6.17 (bs, NH), 5.88* (d, 3J =
17.8 Hz, 12H, 2b), 5.81* (d, 3J = 11.6 Hz, 1H, 2b), 4.90* (d, 2J = 19,4 Hz, 12H, 10),
4.65* (d, 2J = 19,4 Hz, 12H, 10), 4.21* (bs, 12H, 7), 3.94* (bs, 12H, 8), 3.94 (bs, 24H,
25), 3.13* (bs, 24H, 4a), 2.99* (s, 36H, 5a), 2.91* (bs, 36H, 1a), 2.78 (bs, 24H, 20),
O
NNH
NHN O
HNO
NHN
NNHO
NH
OO
OO
O
NHN
NNHO
NHO
NNH
NHN O
HN
OO
OO
ON H
N
NNH
O
NH
O
N
NH N
HN
OHN
OO
O O
ON
NH
NHN
O
HN
O
N
NH N
HN
OHN
OO
O OO
NNH
N HN
O
HN
O
N
HNN
NH
ONH
OO
OO
ONH
N
NNH
O
NH
O
N
HNN
NH
ONH
OO
OO
11a
22a
2b
33a 4 4a
4b
5a
56
910
788a
7a7b
7c
11
1213
1415
1617
18
1920
21 2223
2425
2627
28
Experimental
161
2.68* (bs, 36H, 3a), 2.38* (m, 12H, 7a/b), 2,06* (m, 24H, 7a/b), 1,85* (m, 12H, 7a/b),
1.53* (d, 3J = 6,9 Hz, 36H, 8a), 1.32* (t, 3J = 7,3 Hz, 36H, 4b), 1.32 (bs, 24H, CH2),
0,98 (bs, 72H, CH2), -0.22* (bs, 12H, NH), -2.21* (s, 12H, NH). 13C NMR (100 MHz, CDCl3, 25 °C): δ = 195.8 (12C, 9), 172.3, 171.5, 166.9, 163.6,
160.4, 154.5, 150.0, 148.4, 145.6, 144.4, 141.0, 137.0, 135.6, 135.4, 131.1, 131.1,
129.5, 128.7, 127.3, 122.0, 105.6, 103.2, 96.5, 92.7, 69.0, 66.7, 51.6, 49.6, 47.8,
45.5, 39.1, 32.8, 31.4, 30.2, 29.0, 28.0, 26.2, 25.3, 22.7, 18.9, 17.1, 11.8, 11.2, 10.8.
IR (ATR): ν~ = 2962, 2925, 2861, 1742, 1683, 1617, 1497, 1260, 1218, 1057, 978,
795, 671.
MS (Maldi-TOF, 2,5-dihydroxy benzoic acid): m/z = 8724.3 (calc. 8722.6).
UV/Vis (CH2Cl2): λmax (ε, M-1cm-1) = 279 (251000), 323 (309000), 400 (868000), 413
(858000), 510 (99200), 540 (83700), 613 (78900), 669 (395000).
Elemental analysis: C546H552N60O48·4CHCl3 (9200.11), calcd: C, 71.80; H, 6.09; N,
9.13, found: C, 71.85; H, 5.93; N, 9.21.
Benzoic acid-[3,5-pyropheophorbide-a-bis amide]-sucinimidyl ester 99
Pyropheophorbide-a 19 (250 mg,
0.47 mmol) was dissolved in CH2Cl2
(20 ml) and stirred together with
oxalylchloride (330 μl) for 12 h at
room temperature. The solvent was
removed in vacuo and the residue
dissolved in CH2Cl2 (5 ml). The
green solution was added dropwise
to a solution of diamino benzoic acid
97 (33 mg, 0.21 mmol) in pyridine
(20 ml) at 0°C and stirred for 48 h at room temperature. The solvent was removed in
vacuo. The residue was redissolved in CH2Cl2 (15 ml) and N,N´-Disuccinimidyl
carbonate (256 mg, 1 mmol) and DMAP (10 mg, 0.08 mmol) were added. The
solution was stirred for 12 h at room temperature. After evaporation of the solvent FC
on silica (CHCl3/THF 70:30) yielded the succinimidyl active ester 99 (yield: 180 mg,
0.14 mmol, 67 %).
NHO
NNH
N HN O
O
N HN
NNHO
HN
OO
1
1a
22a
2b
33a4
4a4b
5
5a
109
6
7 7a7b
8a8
11
1213
14 15
1617
18
α
β
γ
δ
7c
1920
2122
23
24
25
NO O25
Experimental
162
1H NMR (400 MHz, CDCl3, 25 °C): δ = 9.11 (s, 2H, β), 8.52 (s, 2H, α), 8.37 (bs, 3H,
δ, 25), 7.77 (m, 2H, 2a), 7.64 (bs, 1H, 21), 7.77 (bs, 1H, 21), 6.08 (d, 3J = 17.8 Hz,
2H, 2b), 5.98 (d, 3J = 11.8 Hz, 2H, 2b), 5.10 (m, 2H, 10), 4.87 (m, 2H, 10), 4.35 (m,
2H, 8), 4.14 (m, 2H, 7), 3.26 (bs, 4H, 4a), 3.18 (s, 12H, 1a, 5a), 3.04 (s, 6H, 3a),
2.70-1.80 (m, 4H, 7a/b), 2.46 (m, 4H, 25), 1.58 (m, 6H, 8a), 1.36 (bs, 6H, 4b), 0.22
(s, 1H, NH).-1.74 (s, 1H, NH). 13C NMR (100 MHz, CDCl3, 25 °C): δ = 196.6, 172.0, 171.3, 160.9, 160.4, 154.9,
150.2, 148.7, 144.8, 141.4, 139.2, 137.1, 136.1, 135.5, 131.6, 129.5, 128.9, 127.5,
124.5, 122.3, 115.0, 106.3, 103.4, 98.4, 96.6, 93.1, 72.5, 70.5, 69.9, 67.4, 66.7, 62.6,
61.8, 51.4, 49.9, 48.1, 33.2, 29.7, 29.4, 24.2, 24.1, 23.3, 22.4, 19.0, 17.2, 15.1, 12.0,
11.1.
IR (ATR): ν~ = 3296, 2961, 2876, 1733, 1668, 1617, 1552, 1451, 1366, 1204, 1065,
980, 907.
MS (FAB, NBA): m/z = 1282 [M]+.
UV/Vis (CH2Cl2): λmax (ε, M-1cm-1) = 324 (32900), 415 (131700), 474 (6600), 510
(14100), 540 (11800), 611 (10800), 669 (56800).
Elemental analysis: C77H75N11O8·1.5CHCl3 (1458.5), calcd: C, 64.51; H, 5.28; N,
10.54, found: C, 64.74; H, 5.73; N, 11.03.
Tetraeicosapyropheophorbide-a-[6:0]-fullerene hexakisadduct 100
O OO
OO
O
O O
O
O
O
O
O
O
NHR
ONHR
O
RHN
O
NHR
ONHRO
RHN
ORHN
ORHN O
NHR
O
RHN
O
RHN
R=NH
O
NNH
N HN O
O
N HN
NNHO
HN
O
1
1a
22a
2b
33a4
4a4b
5
5a
109
6
7 7a7b
8a8
11
1213
14 15
1617
18
α
β
γ
δ
7c
1920
2122
23
25
24
2526
2728
2930
3132
33
3435
NHR
Experimental
163
The deprotected hexakisadduct 94 (27 mg, 0.009 mmol) was dissolved in brine
(30ml). CH2Cl2 (20 ml) and TEA (100 μl) were added and the organic layer was
washed twice with brine (20 ml). After drying over Na2SO4, benzoic acid-[3,5-
pyropheophorbide-a-bis amide]-sucinimidyl ester 99 (230 mg, 0.18 mmol) was added
and the solution stirred for 72 h at room temperature. Evaporation of the solvent and
subsequent SEC (1. Bio-Beads® SX3, CH2Cl2, 2. Bio-Beads® SX1, CHCl3, 3. HPLC-
CHCl3) yielded a black green solid (yield: 43 mg, 0.0026 mmol, 29 %). 1H NMR (400 MHz, CDCl3/MeOH-d4, 25 °C): δ = 8.42 (bs, 48H, α, β), 7.95 (bs, 36H,
δ, 25), 7.50 (bs, 24H, 21), 7.18 (bs, 24H, 2a), 5.64 (bs, 48H, 2b), 4.65 (bs, 24H, 10),
4.38 (bs, 24H, 10), 4.03 (bs, 48H, 8, 30), 3.57 (bs, 24H, 7), 3.30-2.40 (m, 288H, 1a,
3a, 4a, 5a, 25), 2.40-1.90 (bm, 96H, 7a, 7b), 1.24 (bs, 240H, 4b, 8a, 26-29), -2.43
(bs, 24H, NH). 13C NMR (100 MHz, CDCl3/MeOH-d4, 25 °C): δ = 196.4, 171.8, 171.4, 167.6, 163.7,
160.0, 154.5, 149.8, 148.0, 145.6, 144.2, 141.0, 140.7, 138.9, 136.5, 135.3, 135.1,
134.7, 131.0, 128.9, 128.4, 126.8, 121.6, 113.7, 105.1, 102.9, 96.0, 92.4, 69.0, 66.8,
53.4, 51.2, 47.4, 45.5, 39.9, 33.5, 29.6, 28.9, 28.1, 26.3, 25.3, 22.2, 18.7, 16.9, 11.5,
10.9, 10.5.
IR (ATR): ν~ = 2958, 2927, 2867, 1742, 1671, 1617, 1551, 1497, 1445, 1347, 1219,
1027, 978, 731, 716.
MS (Maldi-TOF, 2,5-dihydroxy benzoic acid): m/z = 16539.19 (calc. 16519.86).
UV/Vis (CH2Cl2): λmax (ε, M-1cm-1) = 328 (569300), 400 (1389500), 511 (162100),
543 (148100), 616 (142300), 672 (585800).
20-Hydroxy-eicosyl-methylmalonate 103
The malonate 103 was
synthesized according to
general procedure GP1
(page 117). The reaction of methyl malonyl chloride (1.4 ml, 13 mmol) with
eicosandiol 102 (4.128 g, 13 mmol) and pyridine (4 ml, 52 mmol) in try THF (500 ml)
yielded after FC on silica gel (1. CHCl3 2. CHCl3/ethyl acetate 95:5) a white solid
(yield: 1.96 g, 4.7 mmol, 37 %).
O O OHO O
1 23
4 56
78
910
1112
1314
1516
1718
1920
2122
2324
Experimental
164
1H NMR (400 MHz, CDCl3, 25 °C): δ = 4.11 (t, 3J = 6.7 Hz, 2H, 5), 3.72 (s, 3H, 1),
3.61 (t, 3J = 6.6 Hz, 2H, 24), 3.36 (s, 2H, 3), 2.61 (dt, 3J = 6.8 Hz, 3J = 6.8 Hz, 4H,
23), 1.63 (dt, 3J = 6.8 Hz, 3J = 6.7 Hz, 4H, 6), 1.39 (bs, 32H, 7-22). 13C NMR (100 MHz, CDCl3, 25 °C): δ = 176.1 (1C, 4), 166.6 (1C, 2), 65.7 (1C, 24),
63.1 (1C, 5), 52.5 (1C, 1), 41.4 (1C, 3), 32.8, 29.7, 29.6, 29.5, 29.4, 29.2, 28.4, 26.2,
25.7, 25.7, 23.5.
IR (ATR): ν~ = 3331, 2918, 2849, 1737, 1463, 1339, 1200, 1154, 1061, 1031, 729.
MS (FAB, NBA): m/z = 416 [M]+.
Elemental analysis: C24H46O5 (414.34), calcd: C, 69.52; H, 11.18, found: C, 69.74;
H, 10.98.
[1-(Methyloxycarbonyl)-1´-(20-hydroxy-eicosyloxycarbonyl]-1,2-methano[60]-fullerene 104
The synthesis of
monoadduct 104 was
performed according to
general procedure GP4
(page 118). C60 (2.5 g,
3.47 mmol, 1.02 eq.) and
Malonate 103 (1.4 g, 3.37 mmol, 1 eq.) were reacted with CBr4 (1.7 g, 5.1 mmol,
1.5 eq.) and DBU (800 μl, 5.2 mmol, 1.54 eq.). FC on silica gel (1. toluene, 2.
toluene/CHCl3 19:1) yielded a brown powder, which was dried in vacuo (yield: 1.65 g,
1.45 mmol, 42 %).
1H NMR (400 MHz, CDCl3, 25 °C): δ = 4.48 (t, 3J = 6.5 Hz, 2H, 6), 4.07 (s, 3H, 2),
3.61 (t, 3J = 6.7 Hz, 2H, 25), 1.81 (dt, 3J = 6.6 Hz, 3J = 6.7 Hz, 2H, 7), 1.54 (m, 2H,
24), 1.44 (m, 2H, 8), 1.22 (bs, 30H, 9-23). 13C NMR (100 MHz, CDCl3, 25 °C): δ = 164.5 (1C, 5), 163.9 (1C, 3), 145.4, 145.4,
145.4, 145.1, 144.9, 144.9, 144.1, 143.3, 143.3, 142.4, 142.2, 142.1, 141.2, 139.4,
139.1 (58C, C60-sp2), 71.5 (2C, 1), 67.5 (1C, 6), 63.2 (1C, 25), 53.9 (1C, 4), 52.4
(1C, 2), 32.6, 29.5, 29.4, 29.3, 29.0, 28.4, 25.8, 25.58.
IR (KBr): ν~ = 2921, 2851, 1741, 1462, 1430, 1267, 1233, 1187, 1060, 749.
MS (FAB, NBA): m/z = 1132 [M]+, 720 [C60]+.
O O OHO O
1
23
45
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
2526
Experimental
165
UV/Vis (CH2Cl2): λmax, (ε, M-1cm-1) = 257 (113000), 325 (35000), 425 (2100).
Elemental analysis: C84H44O5·0.5CHCl3 (1191.3), calcd: C, 85.08; H, 3.76; found:
C, 85.58; H, 3.63.
BOC protected decaamino-monoalcohol-[5:1]-fullerene hexakisadduct 105
O O
OO
O
ONH
OO
HN
OO
OO
OO
NHO OHN
OO
O
O
OO NH
OO
HNOO
O
O
OO
HN OONH
OO
O
O
OH5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
OO
NH
OOHN
O
OO O
1
23425
26
27
28
29
30313233
343536
37
38
39
The synthesis of the [5:1]-hexakisadduct 105 was performed according to general
procedure GP5 (page 119). Monoadduct 104 (1.37 g, 1.2 mmol, 1 eq.) was reacted
with DMA (2.50 g, 12 mmol, 10 eq.), malonate 92 (6.10 g, 12.1 mmol, 10 eq.), CBr4
(4.02 g, 12.1 mmol, 10 eq.) and diluted DBU (3.18 ml, 21 mmol, 17.5 eq.) in dry
toluene. Pre-cleaning on silica gel (CH2Cl2/ethyl acetate 6:4) and subsequent
purification by preparative HPLC (Nucleosil 5 μm, CH2Cl2/ethyl acetate 74:26) gave a
yellow solid (yield: 1.068 g, 0.29 mmol, 24.1 %).
1H NMR (400 MHz, CDCl3, 25 °C): δ = 4.76 (bs, 10H, 36), 4.22 (t, 3J = 6.3 Hz, 20H,
30), 4.22 (t, 3J = 6.3 Hz, 2H, 5),3.83 (s, 3H, 1), 3.60 (t, 3J = 6.6 Hz, 2H, 24), 3.06 (m,
20H, 35), 1.66 (m, 22H, 6, 31), 1.54 (m, 2H, 23), 1.40 (s, 90H, 39), 1.50-1.20 (m,
94H, 7-22, 32-34).
Experimental
166
13C NMR (100 MHz, CDCl3, 25 °C): δ = 164.3 (2C, 2, 4), 163.8 (10C, 29), 156.0
(10C, 37), 145.7 (C60-sp2), 141.1 (C60-sp2), 78.9 (10C, 38), 69.1 (C60-sp3), 66.9 (11C,
5, 30), 63.0 (1C, 24), 53.6 (1C, 1), 45.5 (6C, 3, 28), 40.5 (10C, 35), 32.8 (1C, 23),
29.9, 29.7, 29.6, 29.4, 28.4 (30C, 39), 28.3, 26.4, 25.7, 25.6.
IR (ATR): ν~ = 2975, 2930, 2856, 1743, 1689, 1516, 1365, 1244, 1212, 1165, 714.
MS (FAB, NBA): m/z = 3635 [M]+, 3579 [M-t-Bu]+, 3536 [M-BOC]+, 3479 [M-BOC-
t-Bu]+, 3435 [M-2 BOC]+, 3336 [M-3 BOC]+, 3235 [M-4 BOC]+, 3135 [M-5 BOC]+.
UV/Vis (CH2Cl2): λmax, (ε, M-1cm-1) = 244 (93600), 280 (73100), 496 (500).
Elemental analysis: C209H264N10O45·2CH2Cl2 (3801.8), calcd: C, 66.58; H, 7.10;
N, 3.68, found: C, 66.65; H, 7.08; N, 3.67.
BOC protected decaamino-monotosylato-[5:1]-fullerene hexakisadduct 106
O O
OO
O
ONH
OO
HN
OO
OO
OO
NHO OHN
OO
O
O
OO NH
OO
HNOO
O
O
OO
HN OONH
OO
O
O
O S5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
OO
NH
OOHN
O
OO O
1
23428
29
30
31
32
33343536
373839
40
41
42
O
O
25 26
27
The hexakisadduct 106 (460 mg, 0.126 mmol), trimethylamine hydrochloride
(102 mg, 1.07 mmol) and TEA (2 ml, 14 mmol) were dissolved in 150 ml of dry
CH2Cl2. The reaction mixture was cooled to 0°C using an ice bath. Tosyl chloride
(182 mg, 1 mmol) was dissolved in CH2Cl2 (30 ml) and added dropwise over the
period of 1 h, keeping the temperature below 5°C. After the mixture was stirred for 12
h at room temperature the solvent was evaporated. FC on silica (1. CH2Cl2, 2.
Experimental
167
CH2Cl2/ethyl acetate 7:3) yielded the product as a orange solid (yield: 380 mg, 0.1
mmol, 79 %).
1H NMR (400 MHz, CDCl3, 25 °C): δ = 7.75 (d, 3J = 8.2 Hz, 2H, 25), 7.31(d, 3J = 8.3
Hz, 2H, 26),4.75 (bs, 10H, 39), 4.22 (t, 3J = 6.3 Hz, 20H, 33, 5), 3.98 (t, 3J = 6.5 Hz,
2H, 24),3.82 (s, 3H, 1), 3.06 (m, 20H, 38), 2.42 (s, 3H, 27), 1.65 (m, 22H, 6, 34), 1.61
(m, 2H, 23), 1.40 (s, 90H, 39), 1.50-1.20 (m, 94H, 7-22, 35-37). 13C NMR (100 MHz, CDCl3, 25 °C): δ = 164.3 (2C, 2, 4), 163.8 (10C, 32), 156.0
(10C, 40), 145.7 (C60-sp2), 144.6, 141.1 (C60-sp2), 133.2, 129.7 (2C, 25), 127.8 (2C,
26), 78.9 (10C, 38), 70.7 (1C, 24), 69.1, 69.0 (12C, 28), 67.1, 66.8 (11C, 5, 33), 53.5
(1C, 1), 45.5 (6C, 3, 31), 40.5 (10C, 38), 29.9, 29.7, 29.6, 29.5, 29.4, 29.2, 28.9,
28.8, 28.4 (30C, 42), 28.3, 28.1, 27.9, 26.4, 25.7, 25.6, 25.3, 21.6 (1C, 27).
IR (ATR): ν~ = 2976, 2930, 2857, 1742, 1692, 1515, 1365, 1242, 1214, 1167, 1043,
753, 714.
MS (FAB, NBA): m/z = 3789 [M]+, 3733 [M-t-Bu]+, 3690 [M-BOC]+, 3633 [M-BOC-
t-Bu]+, 3490 [M-3 BOC]+, 3390 [M-4 BOC]+, 3289 [M-5 BOC]+, 3188 [M-6 BOC]+
UV/Vis (CH2Cl2): λmax, (ε, M-1cm-1) = 244 (90100), 280 (69000), 496 (420).
Elemental analysis: C216H270N10O47S·2CH2Cl2 (3955.8), calcd: C, 66.11; H, 6.97;
N, 3.54; S. 0.81, found: C, 65.83; H, 7.14; N, 3.56; S, 0.47.
Experimental
168
BOC protected decaamino-monoazido-[5:1]-fullerene hexakisadduct 107
O O
OO
O
ONH
OO
HN
OO
OO
OO
NHO OHN
OO
O
O
OO NH
OO
HNOO
O
O
OO
HN OONH
OO
O
O
N N N5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
OO
NH
OOHN
O
OO O
1
23425
26
27
28
29
30313233
343536
37
38
39
The hexakisadduct 106 (202 mg, 0.053 mmol) and sodium azide (60 mg, 0.9 mmol)
were dissolved in 4 ml of dry DMF and stirred for 16 h at room temperature. The
solvent was removed in vacuo. FC on silica (CH2Cl2/ethyl acetate 7:3) yielded the
product as an orange solid (yield: 160 mg, 0.044 mmol, 82 %).
1H NMR (400 MHz, CDCl3, 25 °C): δ = 4.76 (bs, 10H, 36), 4.22 (t, 3J = 6.5 Hz, 20H,
30), 4.14 (m, 2H, 5),3.83 (s, 3H, 1), 3.22 (t, 3J = 6.9 Hz, 2H, 24), 3.06 (m, 20H, 35),
1.66 (m, 22H, 6, 31), 1.56 (m, 2H, 23), 1.40 (s, 90H, 39), 1.45-1.20 (m, 94H, 7-22,
32-34). 13C NMR (100 MHz, CDCl3, 25 °C): δ = 164.3 (2C, 2, 4), 163.8 (10C, 29), 156.0
(10C, 37), 145.7 (C60-sp2), 141.1 (C60-sp2), 78.9 (10C, 38), 69.1 (12C, 25), 66.9
(11C, 5, 30), 53.6 (1C, 1), 51.5 (1C, 24), 45.4 (6C, 3, 28), 40.5 (10C, 35), 29.9, 29.7,
29.7, 29.6, 29.6, 29.5, 29.4, 29.2, 28.8, 28.3 (30C, 39), 28.3, 26.7, 26.4, 25.7, 25.6.
IR (ATR): ν~ = 2976, 2930, 2856, 2097, 1743, 1691, 1514, 1365, 1244, 1213, 1166,
753, 715.
MS (FAB, NBA): m/z = 3682 [M+Na]+, 3659 [M]+, 3603 [M-t-Bu]+, 3561 [M-BOC]+,
3504 [M-BOC -t-Bu]+, 3359 [M-2BOC]+, 3259 [M-3BOC]+, 3160 [M-4BOC]+.
Experimental
169
UV/Vis (CH2Cl2): λmax, (ε, M-1cm-1) = 243 (95400), 270 (70900), 280 (72500).
Elemental analysis: C209H263N13O44·CH2Cl2 (3742.82), calcd: C, 67.33; H, 7.13;
N, 4.86, found: C, 67.36; H, 7.30; N, 5.00.
Decapyropheophorbide-a-monoazido-[5:1]-fullerene hexakisadduct 108
ONNH
NHN
O
HN
O
N
HNN
NH
O
NH
O
OOO
O NHN
NNH
O
NH
O
N
NHN
HN
O
HN
O
OO O
O
N HN
NNHO
NH
O
NNH
NHN
O
HN
O
OO
O
O O
OO
O
NNH
N HN O
HN
O
NHN
NNH
O
NH
O
OO
OO
N
HNN
NH
ONH
O
N
NH
NHN
O
HN
OO
OO
N
1
1a
22a
2b3
3a 4 4a
4b
5a5
6
910
78
8a7a
7b7c
11
1213 14
15
161718
1920
2122
2324
25
2627
28
N N29
30
313233
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
The hexakisadduct 107 (160 mg, 0.04 mmol) was dissolved in methanol (30 ml) and
hydrochloric acid (5 ml, 4 N in dioxane) was added. After stirring for 3 h at room
temperature the solvent was removed in vacuo. The orange residue was used
without any further purification.
The deprotected hexakisadduct (55 mg, 0.018 mmol) was dissolved in 50 ml of brine.
CHCl3 (50 ml) and TEA (200 μl) were added and the organic layer was washed twice
with brine. After drying over Na2SO4, pyropheophorbide-a-NHS ester 95 (170 mg,
0.27 mmol) was added and the solution stirred for 72 h at room temperature.
Evaporation of the solvent and subsequent SEC (1. Bio-Beads® SX3 2. Bio-Beads®
SX1, CHCl3) yielded a black green solid. Additional FC with silica (CHCl3/methanol,
93.5:7.5) yielded the pure compound 108 (yield: 85 mg, 0.011 mmol, 74 %).
Experimental
170
1H NMR (400 MHz, CDCl3, 25 °C): δ = 8.83* (m, 10H, β), 8.54* (m, 10H, α), 8.20* (m,
10H, δ), 7.48* (m, 10H, 2a), 6.21* (bs, NH), 6.11* (bs, NH), 5.86* (m, 10H, 2b), 4.91*
(m, 10H, 10), 4.64* (m, 10H, 10), 4.22* (m, 10H, 7), 3.96* (m, 10H, 8), 3.96 (m, 22H,
25, 34), 3.64 (s, 3H, 30), 3.25-2.60* (m, 132H, 4a, 5a, 1a, 20, 3a, 53), 2.40* (m, 10H,
7a/b), 2,10* (m, 20H, 7a/b), 1,88* (m, 10H, 7a/b), 1.54* (m, 30H, 8a), 1.35* (m, 54H,
4b, 24, 35), 1.24-1.03 (m, 94H, 21-23, 36-52), -0.15* (bs, 10H, NH), -2.16* (s, 10H,
NH). 13C NMR (100 MHz, CDCl3, 25 °C): δ = 195.9, 172.4, 171.5, 164.1, 163.6, 160.3,
154.7, 150.2, 148.4, 146.0, 144.5, 141.0, 140.8, 137.1, 135.7, 135.5, 131.1, 129.5,
129.5, 128.7, 127.2, 121.9, 107.9, 105.5, 105.5, 103.2, 96.5, 92.6, 69.0, 67.6, 66.7,
51.6, 51.4, 49.6, 49.4, 47.8, 45.4, 39.1, 32.8, 31.9, 30.2, 29.6, 29.5, 29.4, 29.1, 28.7,
28.1, 26.6, 26.2, 25.3, 23.9, 22.7, 18.9, 17.2, 11.8, 11.2, 11.1, 10.9.
IR (ATR): ν~ = 2961, 2923, 2855, 2095, 1743, 1688, 1617, 1498, 1367, 1260, 1218,
1057, 978, 793.
MS (Maldi-TOF, 2,5-dihydroxy benzoic acid): m/z = 7833 (calc. 7820).
MS (FAB, NBA): m/z = 7821 [M]+.
UV/Vis (CH2Cl2): λmax (ε, M-1cm-1) = 281 (184600), 324 (220900), 400 (623200), 413
(621300), 509 (708000), 540 (60600), 613 (55900), 669 (287600).
Elemental analysis: C489H503N53O44·1.5CHCl3 (7997.75), calcd: C, 73.59; H, 6.35;
N, 9.27, found: C, 73.42; H, 6.44; N, 9.90.
Experimental
171
Decapyropheophorbide-a-[5:1]-fullerene hexakisadduct-NHS-active ester 111
ONNH
NHN
O
HN
O
N
HNN
NH
O
NH
O
OOO
O NHN
NNH
O
NH
O
N
NHN
HN
O
HN
O
OO O
O
N HN
NNHO
NH
O
NNH
NHN
O
HN
O
OO
O
O O
OO
O
NNH
N HN O
HN
O
NHN
NNH
O
NH
O
OO
OO
N
HNN
NH
ONH
O
N
NH
NHN
O
HN
OO
OO
NH
1
1a
22a
2b3
3a 4 4a
4b
5a5
6
910
78
8a7a
7b7c
11
1213 14
15
161718
1920
2122
2324
25
2627
28
29
30
313233
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53 OO
ON
O
O54
5556
57
58
59 60
61 62
To a solution of the hexakisadduct 108 (25 mg, 0.003 mmol) in THF (2 ml) and water
(0.2 ml), trimethyl phosphine (1 ml, 1 M solution in toluene) was added. After stirring
for 16 h at room temperature, the solvent was removed in vacuo. The dark green
residue was used without any further purification.
The obtained amino hexakisadduct 109 (25 mg, 0.003 mmol) was dissolved in 5 ml of
dry CH2Cl2 and adipic acid bis-hydroxysuccinimid diester 110 (50 mg, 0.14 mmol)
was added. Stirring for 16 h at room temperature and subsequent SEC (1.
Bio-Beads® SX3 CH2Cl2) yielded a black green solid (yield: 22 mg, 0.0027 mmol,
91 %).
1H NMR (400 MHz, CDCl3, 25 °C): δ = 8.83* (m, 10H, β), 8.53* (m, 10H, α), 8.19* (m,
10H, δ), 7.48* (m, 10H, 2a), 6.18* (bs, NH), 6.10* (bs, NH), 5.81* (m, 10H, 2b), 4.91*
(m, 10H, 10), 4.63* (m, 10H, 10), 4.21* (m, 10H, 7), 3.96* (m, 10H, 8), 3.96 (m, 22H,
25, 34), 3.64 (m, 3H, 30), 3.30-2.60* (m, 136H, 4a, 5a, 1a, 20, 3a, 53, 62), 2.40* (m,
10H, 7a/b), 2,10* (m, 20H, 7a/b), 1,88* (m, 10H, 7a/b), 1.54* (bs, 30H, 8a), 1.35* (bs,
Experimental
172
52H, 4b, 24, 35), 1.24-1.03 (m, 98H, 21-23, 36-52), -0.14* (bs, 10H, NH), -2.18* (s,
10H, NH). 13C NMR (100 MHz, CDCl3, 25 °C): δ = 195.9, 172.3, 171.4, 163.6, 160.3, 154.7,
150.2, 148.4, 145.6, 144.5, 141.0, 137.1, 135.7, 135.5, 135.1, 131.1, 129.6, 129.5,
128.7, 127.2, 121.9, 105.6, 103.2, 96.6, 92.7, 69.1, 66.7, 51.6, 49.7, 47.8, 45.5, 39.1,
32.9, 30.2, 29.5, 29.4, 29.1, 28.1, 26.2, 25.4, 22.8, 19.0, 17.2, 11.8, 11.3, 10.9.
IR (ATR): ν~ = 2963, 2921, 2865, 1741, 1685, 1617, 1497, 1410, 1259, 1218, 1082,
1014, 866, 792.
MS (Maldi-TOF, 2,5-dihydroxy benzoic acid): m/z = 8028.57 [M]+ (calc. 8019.95),
7803, 7394.
UV/Vis (CH2Cl2): λmax (ε, M-1cm-1) = 279 (175900), 322 (211900), 399 (592980), 413
(588100), 510 (67200), 540 (57500), 613 (53100), 669 (273300).
Pyropheophorbide-a-2a-triethyleneglycol monomethylether 114
Pyropheophorbide-a 19 (320 mg, 0.6 mmol)
was dissolved in dry CH2Cl2 (5 ml) and HBr
(5.4 M in glacial acetic acid) (4 ml) was
added at room temperature. After the
solution was stirred for 4 h, the solvent was
removed in vacuo and triethyleneglycol
monomethyl ether (2 ml, 12 mmol) was
added together with CH2Cl2 (10 ml). The solution was stirred for further 14 h at room
temperature. Removal of the solvent in vacuo and subsequent FC on silica
(CHCl3/methanol, 9:1) yielded the desired trisethylenglycol-ether derivative 114
(yield: 220 mg, 0.32 mmol, 53 %).
1H NMR (400 MHz, CDCl3, 25 °C): δ = 9.72 (s, 1H, β), 9.29 (s, 1H, α), 8.50 (s, 1H, δ),
5.99 (q, 3J = 6.7 Hz, 1H, 2a), 5.22 (d, 2J = 19.7 Hz, 1H, 10), 5.08 (d, 2J = 19.7 Hz, 1H,
10), 4.44 (dq, 3J = 7.2 Hz, 3J = 2.0 Hz, 1H, 8), 4.35 (m, 1H, 7), 3.94-3.35 (m, 15H, 2c, 2e), 3.57 (q, 3J = 8.5 Hz, 2H, 4a), 3.37 (s, 3H, 5a), 3.26 (s, 3H, 1a), 3.25 (s, 3H, 3a),
2.70-2.10 (m, 4H, 7a/b), 2.12 (q, 3J = 6.6 Hz, 3H, 2b), 1.78 (d, 3J = 6.7 Hz, 3H, 8a),
1.67 (t, 3J = 7.6 Hz, 3H, 4b), -1.72 (s, 1H, NH).
11a2 2a
2b
33a
44a
4b
5a56
910
77a7b7c
88a
11
12 1314
15
1617
18
O
N
NH N
HN
HO O
OOOO2c2e
2c
2c
2c 2c
2c
Experimental
173
13C NMR (100 MHz, CDCl3, 25 °C): δ = 196.5, 177.6, 171.4, 160.2, 155.2, 148.9,
144.9, 141.3, 141.3, 139.0, 138.9, 137.6, 136.2, 135.5, 135.5, 132.6, 132.5, 130.1,
128.2, 107.9, 105.8, 104.0, 97.7, 92.6, 73.1, 73.1, 71.7, 70.8, 70.5, 70.5, 68.6, 67.6,
58.9, 51.4, 49.9, 47.9, 30.7, 29.5, 29.1, 24.6, 23.8, 23.1, 19.4, 17.4, 11.9, 11.3, 11.0.
IR (ATR): ν~ = 3390, 2960, 2923, 2866, 1730, 1688, 1619, 1499, 1367, 1220, 1089,
978, 678.
MS (FAB, NBA): m/z = 699 [M]+, 535 [M-glycol ether]+.
UV/Vis (CH2Cl2): λmax (ε, M-1cm-1) = 318 (19800), 409 (98600), 472 (3400), 505
(9000), 535 (9000), 604 (7500), 660 (46300).
Elemental analysis: C40H50N4O7·1.5H2O (725.38), calcd: C, 66.19; H, 7.36; N, 7.72,
found: C, 66.06; H, 7.17; N, 7.27.
Pyropheophorbide-a-2a-triethyleneglycol monomethylether succinimidylester 115
Pyropheophorbide- a- 2a- triethyleneglycol-
monomethylether 114 (220 mg, 0.32 mmol),
EDC (90 mg, 0.47 mmol) and NHS (54 mg,
0.47 mmol) were dissolved in dry CH2Cl2
(20 ml) and stirred for 24 h at room
temperature. The solvent was removed in
vacuo and subsequent FC on silica
(CHCl3/acetone, 8:2) yielded the desired
succinimidyl active ester derivative 115 (yield: 184 mg, 0.23 mmol, 73 %).
1H NMR (400 MHz, CDCl3, 25 °C): δ = 9.76 (s, 1H, β), 9.48 (s, 1H, α), 8.55 (s, 1H, δ),
6.01 (m, 1H, 2a), 5.20 (d, 2J = 19.9 Hz, 1H, 10), 5.13 (d, 2J = 19.9 Hz, 1H, 10), 4.49
(m, 1H, 8), 4.40 (m, 1H, 7), 3.90-3.40 (m, 15H, 2c, 2e), 3.63 (m, 2H, 4a), 3.39 (s, 3H,
5a), 3.27 (s, 3H, 1a), 3.26 (s, 3H, 3a), 2.92-2.20 (m, 4H, 7a/b), 2.79 (m, 4H, 20), 2.13
(q, 3J = 6.6 Hz, 3H, 2b), 1.81 (d, 3J = 7.3 Hz, 3H, 8a), 1.70 (t, 3J = 7.6 Hz, 3H, 4b),
0.38 (s, 1H, NH), -1.75 (s, 1H, NH). 13C NMR (100 MHz, CDCl3, 25 °C): δ = 196.1, 171.0, 169.0, 168.9, 159.6, 155.1
150.8, 148.9, 145.0, 141.3, 141.3, 139.1, 139.1, 137.7, 136.2, 135.5, 135.4, 132.6,
132.5, 130.5, 128.3, 106.1, 104.1, 97.8, 92.7, 73.1, 73.1 (1C, 2a), 71.8, 70.9, 70.9,
11a2 2a
2b
33a
44a
4b
5a56
910
77a7b7c
88a
11
12 1314
15
1617
18
O
N
NH N
HN
O O
OOOO2c2e
2c
2c
2c 2c
2c
NOO19
20
Experimental
174
70.6, 70.5, 70.4, 68.6, 54.9, 50.9 (1C, 7), 49.8 (1C, 8), 47.9 (1C, 10), 29.6, 28.1,
25.5, 25.5, 24.6, 23.0, 19.4, 17.4, 12.0, 11.3, 11.0.
IR (ATR): ν~ = 3394, 2961, 2927, 2869, 1783, 1736, 1688, 1619, 1499, 1367, 1202,
1082, 979, 751.
MS (FAB, NBA): m/z = 796 [M]+, 632 [M-glycol ether]+.
UV/Vis (CH2Cl2): λmax (ε, M-1cm-1) = 318 (20200), 409 (103800), 472 (3500), 504
(9400), 536 (9500), 604 (7800), 660 (47700).
Elemental analysis: C44H53N5O9·0.5CHCl3 (854.34), calcd: C, 62.47; H, 6.30;
N, 8.19, found: C, 62.57; H, 6.21; N, 8.09.
Crystal Structures
175
6 Crystal Structures
Crystal Structure Data of Crown ether-Porphyrin-Zinc-Complex with KCN
Figure 6-1: Crystall structure data of 33.
General crystallographic data of 33
Formula C75H87KN6O5Zn·3THF
Formula weight 1472.28
Diffractometer Nonius KappaCCD
Temperature [K] 173(2)
Wavelength λ(MoKα)[Å] 0.71073
Crystal system monoclinic
Space group P21/c
a [Å] 17.4963(3)
b [Å] 24.3943(5)
Crystal Structures
176
c [Å] 20.1222(5)
α [°] 90
β [°] 110.3290(10)
γ [°] 90
V [Å3] 8053.4(3)
Z 4
ρcalcd [g cm-3] 1.214
Absorpt. coeff. [mm-1] 0.415
F(OOO) 3148
Crystal size [mm3] 0.20×0.20×0.20
2θmax [°] 50.08
Index range (h, k, l) -20 to 20; -29 to 26; -23 to 23
Reflections collected 25408
Independent reflections 14211
Reflections [I>2σ(I)] 9291
Data / restraint / parameters 14211 / 0 / 928
Goodness-of-fit on F2 1.007
Final R indices [I>2σ(I)] R1 = 0.0554; wR2 = 0.1424
R indices (all data) R1 = 0.0971; R2 = 0.1663
largest diff. peak and hole [e Å-3] 0.501 and -0.462
The structure of 33·3THF was solved by direct methods (SHELXS-97); parameters
were refined with all data by full-matrix least-squares on F2 (SHELXL-97).
Crystal Structures
177
Crystal Structure Data of Crown Ether-Porphyrin-Cobalt-Complex with KCN
Figure 6-2: Crystall structure data of 38.
General crystallographic data of 38
Formula C76H87CoKN7O5x5THFx2H2O
Formula weight 1700.11
Diffractometer Nonius KappaCCD
Temperature [K] 173(2)
Wavelength λ(MoKα)[Å] 0.71073
Crystal system monoclinic
Space group C2/c
a [Å] 38.5830(4)
b [Å] 19.8730(3) Å
c [Å] 30.1790(3) Å
Crystal Structures
178
α [°] 90
β [°] 124.6161(5)
γ [°] 90
V [Å3] 19043.7(4)
Z 8
ρcalcd [g cm-3] 1.185
Absorpt. coeff. [mm-1] 0.286
F(OOO) 7288
Crystal size [mm3] 0.30×0.20×0.20
2θmax [°] 55.0
Index range (h, k, l) -49 to 50; -25 to 25; -39 to 39
Reflections collected 41847
Independent reflections 21841
Reflections [I>2σ(I)] 15947
Data / restraint / parameters 21841 / 2 / 1073
Goodness-of-fit on F2 1.168
Final R indices [I>2σ(I)] R1 = 0.0883; wR2 = 0.2738
R indices (all data) R1 = 0.1123; R2 = 0.3026
largest diff. peak and hole [e Å-3] 1.945 and -1.882
The structure of 38·5THF·2H2O was solved by direct methods (SHELXS-97);
parameters were refined with all data by full-matrix least-squares on F2 (SHELXL-97).
Crystal Structures
179
Crystal Structure Data of Crown Ether-Porphyrin-Zinc-Complex with KSCN 39
Figure 6-3: Crystall structure data of 39.
General crystallographic data of 39
Formula C76H87CoKN7O5S2·4THF·H2O
Formula weight 1646.10
Diffractometer Nonius KappaCCD
Temperature [K] 100(2)
Wavelength λ(MoKα)[Å] 0.71073
Crystal system monoclinic
Crystal Structures
180
Space group P2(1)/n
a [Å] 15.481(3)
b [Å] 28.149(3) Å
c [Å] 20.875(1) Å
α [°] 90
β [°] 103.15(1)
γ [°] 90
V [Å3] 8858(2)
Z 3
ρcalcd [g cm-3] 1.234
Absorpt. coeff. [mm-1] 0.349
F(OOO) 3516
Crystal size [mm3] 0.40×0.19×0.14
2θmax [°] 51.36
Index range (h, k, l) -18 to 18; -34 to 32; -25 to 25
Reflections collected 89896
Independent reflections 16565
Reflections [I>2σ(I)] 9291
Data / restraint / parameters 16565 / 0 / 1031
Goodness-of-fit on F2 1.0397
Final R indices [I>2σ(I)] R1 = 0.0617; wR2 = 0.1544
R indices (all data) R1 = 0.1041; R2 = 0.1814
largest diff. peak and hole [e Å-3] 0.678 and -0.652
The structure of 39·4THF·H2O was solved by direct methods (SHELXTL NT 6.12);
parameters were refined with all data by full-matrix least-squares on F2 (SHELXTL
NT 6.12).
Crystal Structures
181
Crystal Structure Data of Gadolinium porphyrin 60
Figure 6-4: Crystall structure data of 60.
General crystallographic data of 60
Formula C152 H180 Gd2 N10 O14·5C5H12
Formula weight 2901.498 (1451.00)
Diffractometer Nonius KappaCCD
Temperature [K] 173(2)
Wavelength λ(MoKα)[Å] 0.71073
Crystal system triclinic
Space group P-1
a [Å] 15.6169(2)
b [Å] 18.5220(2) Å
c [Å] 28.4300(4) Å
α [°] 81.9230(10)
β [°] 76.5070(10)
Crystal Structures
182
γ [°] 81.3050(10)
V [Å3] 7857.16(17)
Z 4
ρcalcd [g cm-3] 1.227
Absorpt. coeff. [mm-1] 0.899
F(OOO) 3056
Crystal size [mm3] 0.40×0.20×0.20
2θmax [°] 55.18
Index range (h, k, l) -18 to 20; -24 to 23; -36 to 36
Reflections collected 60411
Independent reflections 35338
Reflections [I>2σ(I)] 25614
Data / restraint / parameters 35338 / 18 / 1666
Goodness-of-fit on F2 1.137
Final R indices [I>2σ(I)] R1 = 0.0519; wR2 = 0.1533
R indices (all data) R1 = 0.0802; R2 = 0.1720
largest diff. peak and hole [e Å-3] 1.628 and -1.3542
The structure of 60·3pentane was solved by direct methods (SHELXS-97);
parameters were refined with all data by full-matrix least-squares on F2 (SHELXL-97).
Publications
183
7 Publications Matthias Helmreich, Eugeny A. Ermilov, Matthias Meyer, Norbert Jux*, Andreas
Hirsch*, Beate Roeder*
Dissipation of Electronic Excitation Energy within a C60 [6:0]-Hexaadduct Carrying
12 Pyropheophorbide a Moieties
J. Am. Chem. Soc., 2005, 127, 8376-8385.
Matthias Helmreich, Andreas Hirsch, Norbert Jux*
Synthesis of novel pyropheophorbide a-fullerene conjugates
J. Porphyrins Phtalocyanines, 2005, 9, (2), 130-137.
Fiorenza Rancan*, Matthias Helmreich, Andreas Mölich, Norbert Jux, Andreas
Hirsch, Beate Röder, Christian Witt and Fritz Böhm Fullerene-pyropheophorbide a complexes as sensitizer for photodynamic therapy:
Uptake and photo-induced cytotoxicity on Jurkat cells
J. Photochem. Photobiol., B, 2005, 80, 1-7.
Eugeny A. Ermilov, Steffen Hackbarth, Saleh Al-Omari, Matthias Helmreich, Norbert
Jux, Andreas Hirsch, Beate Roeder.
Trap formation and energy transfer in the hexapyropheophorbide a - fullereneC60
hexaadduct molecular system.
Opt. Commun., 2005, 250, 95-104.
Saleh Al-Omari; Eugeny A. Ermilov; Matthias Helmreich; Norbert Jux; Andreas
Hirsch; Beate Roeder.
Transient absorption spectroscopy of a monofullerene C60-bis-(pyropheophorbide a)
molecular system in polar and nonpolar environments.
Applied Physics B: Lasers and Optics, 2004, 79, 617-622.
Eugeny A. Ermilov; Saleh Al-Omari; Matthias Helmreich; Norbert Jux; Andreas
Hirsch; Beate Roeder.
Photophysical properties of fullerene-dendron-pyropheophorbide supramolecules.
Chem. Phys. 2004, 301, 27-31.
Publications
184
Eugeny A. Ermilov; Saleh Al-Omari; Matthias Helmreich; Norbert Jux; Andreas
Hirsch; Beate Roeder.
Steady-state and time-resolved studies on the photophysical properties of fullerene-
pyropheophorbide a complexes in polar and nonpolar solvents.
Opt. Commun., 2004, 234, 245-252.
Conference poster contributions
Third International Conference on Porphyrins and Phthalocynaines (ICPP-3) in
New Orleans, USA
Matthias Helmreich, Eugeny Ermilov, Fiorenza Rancan, Fritz Böhm*, Beate Roeder*,
A.Hirsch*,Norbert Jux*
Synthesis and Photophysics of Fullerene-Dendrimer-Pyropheophorbide-Conjugates
Matthias Helmreich, Norbert Jux*
Novel crown ether porphyrin conjugates
SFB-Symposium on Redoxactive Metall complexes – Control of Reactivity via
Molecular Architecture at the University of Erlangen
Matthias Helmreich, Norbert Jux*
Novel crown ether porphyrin conjugates
References
185
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Lebenslauf
Persönliche Daten Name: Matthias Helmreich Geburtsdatum: 17.06.1976 Geburtsort: Bamberg Familienstand: ledig
Akademische Ausbildung 09/05 – 04/02 Promotion am Institut für Organische Chemie der Friedrich-
Alexander-Universität Erlangen-Nürnberg unter Anleitung von Dr. Norbert Jux / Prof. Dr. Andreas Hirsch
Thema: Crown Ether-Metalloporphyrins as Ditopic Receptors and Pyropheophorbide-a Conjugates for the Photodynamic Therapy of Tumors
07/02 – 06/04 Promotionsstipendium des Verbands der Chemischen Industrie (VCI)
09/01 – 03/02 Diplomarbeit am Institut für Organische Chemie der Friedrich-
Alexander-Universität Erlangen-Nürnberg unter Anleitung von Dr. Norbert Jux / Prof. Dr. Andreas Hirsch
Thema: Konjugate aus cyclischen Azachelatliganden und Metallo-
porphyrinen 08/01 – 11/96 Studiengang Diplom Chemie an der Friedrich-Alexander-
Universität Erlangen-Nürnberg
Ersatzdienst 08/96 – 08/95 Alten- und Pflegeheim St. Otto Bamberg
Schulbildung 06/95 – 09/86 Clavius Gymnasium Bamberg 07/86 – 09/82 Grundschule Breitengüßbach
