Fluorescent and photochromic fluorescent compounds for applications in optical nanoscopy [Elektronische Ressource] / vorgelegt von Svetlana Polyakova

Spolyak

Informations

• Publié par: georg-august-universitat_gottingen
• Publié le: 01 janvier 2009
• Nombre de lectures: 14
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Fluorescent and Photochromic Fluorescent Compounds
for Applications in Optical Nanoscopy




DISSERTATION


zur Erlangung des Doktorgrades
der Mathematisch-Naturwissenschaftlichen Fakultäten
der Georg-August-Universität zu Göttingen




vorgelegt von

Svetlana Polyakova

aus

Gatchina (Russische Föderation)





Göttingen 2009























D 7
Referent: Prof. Dr. Armin de Meijere
Korreferent: Prof. Dr. Stefan W. Hell
Tag der mündlichen Prüfung:

Die vorliegende Arbeit wurde in der Zeit von September 2006 bis September 2009 im
Max-Planck-Institut für biophysikalische Chemie Göttingen angefertigt.





























Hiermit möchte ich für die Überlassung des interessanten Themas und große Unterstützung
während meiner Promotionszeit Herrn Prof. Dr. Armin de Meijere, Herrn Prof. Dr. Stefan W.
Hell und Herrn Dr. Vladimir N. Belov ganz herzlich danken.

















to my family
Table of Contents
Preface............................................................................................................................................ 9
Chapter 1. Fluorescence modulation by the use of photochromic compounds..................... 11
1. Main principles of optical nanoscopy................................................................................... 11
2. Photochromism and types of photochromic compounds...................................................... 13
3. General strategies for the synthesis of photochromic diarylperfluorocyclopentenes ........... 16
4. Photochromic fluorescent 1,2-bis(3-thienyl)perfluorocyclopentenes................................... 20
4.1. Fluorescence modulation achieved by changes in conjugation ..................................... 20
4.2. Fluorescence modulation achieved by pcFRET ............................................................ 29
5. Photochromic diarylperfluorocyclopentenes with improved solubility in aqueous media... 34
Chapter 2. Synthesis, properties and microscopic applications of photochromic and
photochromic fluorescent compounds ...................................................................................... 39
1. Synthesis of the new photochromic and fluorescent photochromic 1,2-bis(3-
thienyl)perfluorocyclopentenes................................................................................................. 39
1.1. Model compounds.......................................................................................................... 39
1.2. Search for an optimal linker to attach the photochromic part to a fluorescent dye ....... 41
1.3. Synthesis of photochromic fluorescent compounds with rhodamine 101 as a fluorescent
unit ........................................................................................................................................ 50
1.4. Spectral properties of the new photochromic compounds and their adducts with
rhodamine 101 ...................................................................................................................... 51
2. Synthesis of fluorescent diarylethene derivatives with additional functionalities................ 56
2.1. Optimized fluorescent photochromic compounds with amino-reactive groups (NHS
ester) and imaging of the silica-gel nanoparticles with an incorporated fluorescent
photochromic compound ...................................................................................................... 56
2.2. A fluorescent photochromic compound with a maleimide fragment............................. 62
2.3. A fluorescent photochromic compound with an amino group....................................... 63
2.4. A fluorescent photochromic compound with a biotin fragment .................................... 64
3. Water-soluble photochromic and fluorescent photochromic compounds ............................ 65
3.1. Photochromic unit with a poly(ethyleneglycol) side chain............................................ 66
3.2. Introduction of sulfonic acid residues into the fluorescent part of the photochromic
assembly................................................................................................................................ 67
3.3. Introduction of sulfonic acid residues into the photochromic unit ................................ 69

Chapter 3. Fluorescent derivatives of the GM1 ganglioside and their use in single molecule
studies........................................................................................................................................... 82
1. Structure of ganglioside GM1 and the fluorescent ganglioside GM1 derivatives................ 82
2. Synthesis of ganglioside derivatives with α- or ω-amino- and ω-mercaptostearic acid
residues ..................................................................................................................................... 87
3. Synthesis of GM1 derivatives labelled with new fluorescent dyes ...................................... 91
4. Novel intermediates for selective twofold labelling of the natural GM1 skeleton............... 97
5. STED-FCS measurements of fluorescent GM1 lipid analogs .............................................. 98
Experimental Part..................................................................................................................... 100
Summary.................................................................................................................................... 173
References... 178
Spectral Data ............................................................................................................................. 186
Crystallographic Data .............................................................................................................. 200

Abbreviations
ACN acetonitrile;
9-BBN 9-borabicyclo[3,3,1]nonane;
Boc tert-butoxycarbonyl;
BODIPY 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene;
DCC dicyclohexylcarbodiimide;
DHP 3,4-dihydro-2H-pyran;
DIAD diisopropyl azodicarboxylate;
DIEA N,N-diisopropylethylamine;
DMAA N,N-dimethylacetamide;
DMAP 4-(N,N-dimethylamino)pyridine;
DMF N,N-dimethylformamide;
DMSO dimethyl sulfoxide;
DPPA O,O-diphenylphosphorylazide;
DTT dithiothreitol;
EDC 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide;
EDTA ethylenediaminetetraacetic acid;
Fmoc 9-fluorenylmethyloxycarbonyl;
FmocClethylchloroformiate;
HATU O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate;
HPLC high-pressure liquid chromatography;
HPTLC high performance thin layer chromatography;
Im imidazole;
LDA lithium diisopropylamide;
NBD 4-amino-7-nitrobenz-2-oxa-1,3-diazol;
NBS N-bromosuccinimide;
NCS N-chlorosuccinimide;
NHS N-hydroxysuccinimide;
NMR nuclear magnetic resonance;
PBS phosphate-buffered saline;
PMB 4-methoxybenzyl;
PPTS pyridinium p-toluenesulfonate;
Py pyridine;
7
TBDPS tert-butyldiphenylsilyl;
TFA trifluoroacetic acid;
THF tetrahydrofuran;
THP tetrahydro-2H-pyranyl;
TMEDA N,N,N′,N′-tetramethylethylenediamine;
TMP 2,2,6,6-tetramethylpiperidine;
TMS trimethylsilyl;
TsOH p-toluenesulfonic acid;
TSTU N,N,N′,N′-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate;
Z benzyloxycarbonyl.

8 Preface
______________________________________________________________________________
Preface
According to the Abbe principle, the optical resolution of a light microscope cannot be
better than one half of the wavelength of the used light. The Abbe law is no more valid
[1]nowadays, and the diffraction barrier in optical microscopy has been overcome. A diffraction-
unlimited resolution has been achieved using various physical concepts.
Reversible optical fluorescence modulations form a basis of a new physical concept in far-
[1a]field optical microscopy that allows one to achieve a resolution better than 100 nm.
Photochromic compounds may be reversibly interconverted by light between two isomers
with different absorption spectra, and therefore they are known to be good switchable units.
Differences in the absorption spectra may be used for modulation of a secondary function on a
molecular or supramolecular level. The switchable fluorescence signal is often used as a
parameter, which can easily be detected with high sensitivity.
Photochromic fluorescence resonance energy transfer (pcFRET) could be an attractive tool
for improving the optical resolution by imaging of objects bearing switchable fluorescent
[2]markers. Let us consider a label consisting of a photochromic compound and a fluorescent dye
connected with a linker (Scheme 1). If the fluorescent signal of the labelled object can be
modulated by re