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
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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
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
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.
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
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.
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
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