| Abstract |
Aim of this study was the synthesis of red shifted azobenzenes in order for isomerization in
the visible range of the spectrum to be achieved, avoiding the toxic UV light. Azobenzenes
were chosen since they are pivotal molecules in photopharmacology, a clinical approach that
uses light as a mean for drug activation. In addition to this, we examined the possibility of
isomerization using bioluminescence, as it is widely applied in the pharmaceutical area and in
particular in bioluminescent imaging.
Our efforts to synthesize red shifted azobenzenes were initially based on
tetrahalogenated azobenzenes reported in literature, according to which the tetrha-orthofluoro azobenzene undergoes isomerization in the green region of the spectrum, while the
tetra-ortho-chloro azobenzene undergoes isomerization in the red region of the visible
spectrum. With the intention of synthesizing an azobenzene derivative that undergoes
isomerization in the yellow region of the visible spectrum, in order to investigate the
possibility of isomerization with firefly bioluminescence, we prepared two tetrahalogenated
derivatives bearing a combination of ortho- fluoro and ortho- chloro substituents. This study
led to the conclusion than when comparing tetrahalogenated azobenzene derivatives, the
fluoro substituent had a dominant effect over the chlorine in determining the red shifted
nature of azobenzenes. This conclusion was based on the fact that the azobenzenes 2 and 4
bearing two and three fluorine substituents respectively, exhibited isomerization solely in the
green part of the visible spectrum. For this reason, we decided to attempt the synthesis of a
trihalogenated azo compound bearing two chlorine substituents and just one fluorine
substituent for two reasons. Firstly, we aimed to investigate the red shifted nature of the
compound and secondly to attempt nucleophilic aromatic substitution reactions on the
fluorinated ring to investigate the effect of various nucleophiles.
As efforts at wavelength optimization in the yellow region of the spectrum weren’t
successful, we opted to synthesize azobenzene derivatives designed to undergo blue light
induced isomerization in order to investigate the possibility of isomerization using bacterial
bioluminescence. For this purpose, we synthesized a series of mono- and di- ortho substituted
azobenzenes as precursors for nucleophilic aromatic substitution to screen various
nucleophiles for red shifted behaviours. It was found in literature that ortho aminated
azobenzenes fitted our requirements, as these derivatives absorb in the visible range, possess
a small cis lifetime and are not reduced by glutathione. The ortho amination reactions,
however, were not straightforward as our attempt of nucleophilic aromatic substitution,
using pyrrolidine as a nucleophile, yielded a non-photostable azobenzene. Considering other
alternatives, we attempted ortho amination using a simpler amine, dimethylamine, in an
effort to test photostability. Ortho amination using dimethylamine was partly based on
literature, taking advantage of the hydroxide assisted thermal decomposition of DMF into
dimethylamine and formate. As the reaction did not proceed under the reported conditions,
we optimized both the apparatus and conditions to ortho aminated products for possible blue
light isomerization. Spectrophotometric studies proved that the ortho aminated derivative 16
displayed poor solubility in water. In an effort to increase overall solubility in water we studied
the substitution of the azo compound with a polar/water soluble group, such as piperazine.
We carried out various reactions including a combination of benzylic bromination,
nucleophilic aromatic substitution and benzylic SN2 reactions that did not result in the expected product.
In the final part of the Thesis, we investigated the possibility of bacterial luciferase
induced isomerization. For this purpose, we isolated blue light emitting bioluminescent
bacteria from fresh sea shrimp and cultivated them on agar plates containing LA medium. We
then proceeded with bioluminescent photometric studies by recording UV-Vis spectra before
and after irradiation with bioluminescent bacteria in order to gain a qualitative understanding
of the effect that bacterial bioluminescence has on azobenzene isomerization.
In summary, 14 new azobenzene derivatives were synthesized. The azobenzenes 2
and 4 showed isomerization under green light irradiation. The azobenzenes 15 and 16 showed
isomerization under blue light irradiation and specifically azobenzene 16 was shown to
undergo partial isomerization with bacterial bioluminescence.
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