DESIGN AND APPLICATIONS OF LUMINESCENT RHENIUM...

DESIGN AND APPLICATIONS OF

LUMINESCENT RHENIUM(I) POLYPYRIDINE

COMPLEXES AS ION, BIOMOLECULAR,

AND CELLULAR PROBES

LOUIE MAN WAI

DOCTOR OF PHILOSOPHY

CITY UNIVERSITY OF HONG KONG

DECEMBER 2011

CITY UNIVERSITY OF HONG KONG

香港城市大學

Design and applications of luminescent rhenium(I)

polypyridine complexes as ion, biomolecular, and

cellular probes

具發光性錸多吡啶絡合物作為離子、生物分子及細胞

探測器之設計及應用 Submitted to

Department of Biology and Chemistry 生物及化學系

in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

哲學博士學位

by

Louie Man Wai 雷文威

DECEMBER 2011 二零一一年十二月

Abstract

This thesis describes the design, synthesis, photophysical and biological

properties of four classes of luminescent rhenium(I) polypyridine complexes.

Specifically, the use of these complexes as intracellular ion probes,

avidin-crosslinking and biotinylation reagents, glucose uptake indicators, and

fluorous-labeling reagents has been reported.

Zinc(II) and cadmium(II) ions are two biologically and environmentally

important metal ions. The development of zinc(II) and cadmium(II) ion

probes has relied on the use of fluorescent organic dyes and luminescent

lanthanide chelates as the reporters. Despite these reports, the possibility of

using luminescent transition metal complexes as zinc(II) and cadmium(II) ion

sensors has not been explored. In Chapter 2, three luminescent rhenium(I)

polypyridine complexes containing a tyramine-derived 2,2’-dipicolylamine

(DPAT) unit [Re(N^N)(CO)3(py-TU-DPAT)](CF3SO3) (py-TU-DPAT =

3-(2-(4-hydroxy-3-(2,2’-dipicolylaminomethyl)phenyl)ethylthioureidyl)pyridine;

N^N = 1,10-phenanthroline (phen) (1a),

3,4,7,8-tetramethyl-1,10-phenanthroline (Me4-phen) (2a),

4,7-diphenyl-1,10-phenanthroline (Ph2-phen) (3a)) and their DPAT-free

counterparts [Re(N^N)(CO)3(py-TU-Et)](CF3SO3) (py-TU-Et =

3-(ethylthioureidyl)pyridine; N^N = phen (1b), Me4-phen (2b), Ph2-phen (3b))

have been synthesized and characterized. Their electrochemical and

photophysical properties have been studied. Upon photoexcitation, all the

complexes exhibited triplet metal-to-ligand charge-transfer (3MLCT) (d(Re)

*(N^N)) emission in fluid solutions at 298 K and in low-temperature

I

alcohol glass at 77 K. The DPAT complexes showed lower emission

quantum yields and shorter emission lifetimes compared to those of the

DPAT-free analogues, indicative of the quenching properties of the appended

DPAT unit. The DPAT complexes also exhibited pH-dependent emission

with their emission intensities at pH < 3 being ca. 40 fold higher than those at

pH > 11. These complexes displayed emission enhancement and lifetime

elongation upon binding to zinc(II) and cadmium(II) ions. Additionally, the

cellular uptake of all the complexes by human cervix epithelioid carcinoma

(HeLa) cells has been examined by ICP-MS. The cytotoxicity of the

complexes of the DPAT complexes toward HeLa cells was higher that that of

the DPAT-free analogues, as revealed by the

3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay.

Furthermore, the cellular uptake of complexes 3a and 3b has been

investigated by laser-scanning confocal microscopy, and the results revealed

that both complexes were localized in the perinuclear region. The emission

intensity of HeLa cells stained with complex 3a was enhanced in the

presence of zinc(II) and cadmium(II) ions.

Avidin-biotin technology is a useful tool for biological, medical, and

industrial applications. The development of luminescent avidin crosslinkers

and biotinylation reagents has attracted much attention. In Chapter 3, a new

class of luminescent rhenium(I) polypyridine bis-biotin complexes

[Re(N^N)(CO)3(pyridine)](CF3SO3) (N^N = 4,4’-bis((2-

(biotinamido)ethyl)aminocarbonyl)-2,2’-bipyridine, bpyC2B2 (1),

4,4’-bis((2-((6-(biotinamido)hexanoyl)amino)ethyl)aminocarbonyl)-2,2’-

bipyridine, bpyC2C6B2 (2)) and mono-biotin complexes

[Re(N^N)(CO)3(L)](PF6) (N^N = phen; L =

II

3-amino-5-(N-((2-biotinamido)ethyl)aminocarbonyl)pyridine, py-biotin-NH2

(4), 3-isothiocyanato-5-(N-((2-biotinamido)ethyl)aminocarbonyl)pyridine,

py-biotin-NCS (5), 3-ethylthioureidyl-5-(N-((2-biotinamido)ethyl)-

aminocarbonyl)pyridine, py-biotin-TU-Et (6)) has been synthesized and

characterized. A biotin-free complex [Re(N^N)(CO)3(pyridine)](CF3SO3)

(N^N = 4,4’-bis(n-butylaminocarbonyl)-2,2’-bipyridine, bpyC4 (3)) has also

been prepared. The bis-biotin complexes 1 and 2 can function as

luminescent crosslinkers for the protein avidin and the isothiocyanate

complex 5 can serve as a luminescent biotinylation reagent. The

electrochemical and photophysical properties of these complexes have been

investigated. All the complexes exhibited intense and long-lived 3MLCT

(d(Re) *(N^N)) emission in fluid solutions at room temperature and

alcohol glass at 77 K upon irradiation. The avidin-binding properties of the

biotin complexes have been studied by 4’-hydroxyazobenzene-2-carboxylic

acid (HABA) assays, emission titrations, and dissociation assays. The use

of the bis-biotin complexes as signal amplifiers for heterogeneous recognition

assays has been demonstrated using avidin-coated microspheres.

Additionally, the isothiocyanate complex 5 has been used to biotinylate a

model protein bovine serum albumin (BSA), resulting in the formation of a

bioconjugate, which exhibited intense and long-lived yellow emission upon

irradiation. The cytotoxicity of the mono-biotin and bis-biotin complexes

toward HeLa cells has been investigated by the MTT assay, and the results

revealed that the incorporation of one or more biotin units substantially

decreases the cytotoxicity of these complexes. Furthermore, laser-scanning

confocal microscopy revealed that the complexes were not homogeneously

distributed within the cytoplasm of HeLa cells but localized in the perinuclear

III

region, possibly bound to hydrophobic organelles such as Golgi apparatus.

Glucose is highly important in cellular metabolism and an energy source

for the growth of cells. The monitoring of glucose metabolism in cancer cells

has attracted much attention. In Chapter 4, three luminescent rhenium(I)

polypyridine complexes appended with an -D-glucose

[Re(N^N)(CO)3(py-3-glu)](PF6) (py-3-glu =

3-(N-(6-(N’-(4-(-D-glucopyranosyl)phenyl)thioureidyl)hexyl)thioureidyl)-

pyridine; N^N = phen (1), Me4-phen (2), Ph2-phen (3)) have been designed as

luminescent biomolecular and cellular probes. Their glucose-free

counterparts [Re(N^N)(CO)3(py-3-Et)](PF6) (py-3-Et =

3-(ethylthioureidyl)pyridine; N^N = phen (1a), Me4-phen (2a), Ph2-phen (3a))

have also been prepared for comparison studies. The electrochemical and

photophysical properties of the glucose complexes have been studied.

Upon photoexcitation, all these complexes exhibited 3MLCT (d(Re)

*(N^N)) emission in fluid solutions at 298 K and in low-temperature alcohol

glass at 77 K. The interactions of the glucose complexes with the lectin

concanavalin A (Con A) have been studied by emission titrations. These

complexes displayed emission enhancement and lifetime elongation upon

binding to Con A. The binding of complex 3 to the lectin FimH expressed in

Escherichia coli (E. coli) has also been investigated by laser-scanning

confocal microscopy. The lipophilicity of all these rhenium(I) complexes has

been determined by reversed-phase HPLC. Additionally, the cellular uptake

of the complexes by HeLa cells has been examined by ICP-MS. The

glucose complexes 1 3 were less cytotoxic toward HeLa cells than their

glucose-free counterparts and the cytotoxicity was closely related to the

lipophilicity and cellular uptake of the complexes, as revealed by the MTT

IV

http://en.wikipedia.org/wiki/Escherichia_coli

assay. Glucose-dependence, temperature-dependence, and chemical

inhibition experiments suggested that transport of complex 3 across

membrane barriers occurred via energy-requiring endocytosis and involved

glucose transporters (GLUTs). Also, the cellular uptake of this complex by

HeLa cells has been examined by laser-scanning confocal microscopy and

the results revealed that complex 3 was diffusely distributed in the cytoplasm

with enriched staining in the mitochondria. The complex showed much

higher resistance to photobleaching compared to a fluorescent organic

2-deoxyglucose derivative 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-

deoxy-D-glucose (2-NBDG).

Although the functionalization of organic fluorophore and lanthanide

chelates with a fluorous unit has been reported, the design and applications

of luminescent transition metal fluorous complexes have been unexplored.

In Chapter 5, a series of luminescent biological labeling reagents derived from

rhenium(I) polypyridine fluorous complexes [Re(N^N)(CO)3(py-Rf-NCS)](PF6)

(py-Rf-NCS = 3-isothiocyanato-5-(N-((3-perfluorooctyl)propyl)amino-

carbonyl)pyridine; N^N = phen (1b), Me4-phen (2b), Ph2-phen (3b)) has been

synthesized and characterized. These complexes have been synthesized

from the reaction of the precursor amine complexes

[Re(N^N)(CO)3(py-Rf-NH2)](PF6) (py-Rf-NH2 = 3-amino-5-(N-

((3-perfluorooctyl)propyl)aminocarbonyl)pyridine; N^N = phen (1a), Me4-phen

(2a), Ph2-phen (3a)) with thiophosgene in acetone at 298 K. The

isothiocyanate complexes 1b 3b have been reacted with ethylamine, which

acts as a model substrate, yielding the thiourea complexes

[Re(N^N)(CO)3(py-Rf-TU-Et)](PF6) (py-Rf-TU-Et = 3-ethylthioureidyl-

5-(N-((3-perfluorooctyl)propyl)aminocarbonyl)pyridine; N^N = phen (1c),

V

Me4-phen (2c), Ph2-phen (3c)). The electrochemistry and photophysics of

all the fluorous complexes have been studied. Upon irradiation, all these

fluorous complexes exhibited intense and long-lived 3MLCT (d(Re)

*(N^N)) emission in fluid solutions at 298 K and low-temperature alcohol

glass at 77 K. The fluorous isothiocyanate complexes 1b 3b have been

used to label the peptide glutathione (GSH) and the protein BSA. The

photophysical properties of the resultant bioconjugates have been

investigated. The lipophilicity and cellular uptake of the amine and thiourea

complexes have been studied. Additionally, the isolation of the three

luminescent rhenium(I) fluorous GSH conjugates from a mixture of twenty

amino acids has been accomplished using fluorous solid-phase extraction

(FSPE). Also, the cytotoxicity of the amine and thiourea complexes toward

HeLa cells has been examined by the MTT assay and the results showed that

the less lipophilic fluorous complexes exhibited higher cytotoxicity. The

cellular uptake of complex 3c has been investigated by laser-scanning

confocal microscopy, and the complex was enriched in the mitochondria of

HeLa cells.

In summary, four classes of luminescent rhenium(I) polypyridine

complexes have been designed, synthesized, and characterized. These

complexes showed interesting electrochemistry, photophysics, ion- or

protein-binding properties, cytotoxicity, and cellular uptake. The results in

this thesis are expected to form the basis of the design and development of

ion, biomolecular, and cellular probes.

VI

Abbreviations ABC avidin-biotin complex

acedan 2-acetyl-6-dimethylaminonaphthalene

2-appt 2-amino-4-phenylamino-6-(2-pyridyl)-1,3,5-triazine

ATP adenosine triphosphate

BODIPY 4,4’-difluoro-4-bora-3a,4a-diaza-s-inacene

bpm bis(phenanthridinylmethyl)amine

bpy 2,2’-bipyridine

bpyC2B2 4,4’-bis((2-(biotinamido)ethyl)-aminocarbonyl)-2,2’-

bipyridine

bpyC2C6B2 4,4’-bis((2-((6-(biotinamido)hexanoyl)amino)ethyl)

aminocarbonyl)-2,2’-bipyridine

bpyC4 4,4’-bis(n-butylaminocarbonyl)-2,2’-bipyridine

bpyC6B 4-((6-(biotinamido)hexyl)aminocarbonyl)-4’-methyl-

2,2’-bipyridine

bpy-C6-est 4-(N-(6-(4-(17-ethynylestradiolyl)benzoylamino)-

hexyl)aminocarbonyl)-4’-methyl-2,2’-bipyridine

bpy-en-biotin 4-(N-((2-biotinamido)ethyl)aminomethyl)-4’-methyl-

2,2’-bi-pyridine

bpy-ind 4-((2-(indol-3-yl)ethyl)aminocarbonyl)-4’-methyl-

2,2’-bipyridine

bpyNB 4-(N-(-(5,5-dimethylborinan-2-yl)benzyl)-N-(methyl

amino)methyl)-4’-methyl-2,2’-bipyridine

bpy-Ph-est 5-(4-(17-ethynylestradiolyl)phenyl)-2,2’-bipyridine

X

bpy-Rf 4-(N-((3-perfluorooctyl)propyl)aminocarbonyl)-4’-

methyl-2,2’-bipyridine

bpy-TEG-biotin 4-((13-biotinamido-4,7,10-trioxa)tridecylaminocarbo

nyl)-4’-methyl-2,2’-bipyridine

bpy-Y 4’-methyl-2,2’-bipyridine-4-tyrosine

BRAB bridged avidin-biotin

5-Br-phen 5-bromo-1,10-phenanthroline

BSA bovine serum albumin

tBu2-bpy 4,4’-bis-tert-butyl-2,2’-bipyridine

tBu3terpy 4,4’,4’’-tri-tert-butyl-2,2’:6’,2”-terpyridine

bzimpy 2,6-bis(benzimidazo-2-yl)pyridine

C^N cyclometalating ligand

carboxy-H2DCFDA, AM 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate,

di(acetoxymethyl ester)

CCCP carbonyl cyanide 3-chlorophenylhydrazone

5-Cl-phen 5-chloro-1,10-phenanthroline

Con A concanavalin A

CRP cAMP receptor protein

DAPI 4’,6-Diamidino-2-phenylindole

DCC N,N’-dicyclohexylcarbodiimide

DHR dihydrorhodamine 123

DMEM Dulbecco’s modified Eagle’s medium

dpa dipyridin-2-ylamine

DPA 2,2’-dipicolylamine

DPAT tyramine-derived 2,2’-dipicolylamine

XI

dpe 1,2-di(4-pyridyl)ethylene

dppn benzo[i]dipyrido[3,2-a:2’,3’-c]phenazine

dppz dipyrido[3,2-a:2’,3’-c]phenazine

ds-DNA double-stranded DNA

DTPA diethylenetriaminepentaacetic acid

E. coli Escherichia coli

EDC 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide

hydrochloride

EDTA ethylenediaminetetraacetic acid

EPA ether/isopentane/ethanol

ER estrogen receptor

ESI electrospray ionization

EtBr ethidium bromide

F-18 FDG [18F]-2-fluoro-2-deoxyglucose

Fast MCS fast multichannel scaler

FBS fetal bovine serum

5-FOAF 5-(perfluorooctylthio)acetamidofluorescein

FPR formyl peptide receptor

FR folate acid receptors

FRET fluorescence resonance-energy transfer

FSPE Fluorous Solid-Phase Extraction

GLUTs glucose transporters

GSH glutathione

gly glycine

H2DCFDA 2’,7’-dichlorodihydrofluorescein diacetate

XII

HABA 4’-hydroxyazobenzene-2-carboxylic acid

HSA human serum albumin

Hbsb 2-((1,1’-biphenyl)-4-yl)benzothiazole

Hbzq 7,8-benzoquinoline

HDAC histone deacetylases

Hdcbpy 4-carboxy-2,2’-bipyridine-4’-carboxylate

Hdfpy 2-(2,4-difluorophenyl)pyridine

His histidine

Hmppy 2-(4-methylphenyl)pyridine

Hmppz 3-methyl-1-phenylpyrazole

Hpba 4-(2-pyridyl)benzaldehyde

Hpiq 1-phenyl-isoquinoline

Hppy 2-phenylpyridine

HppyC6B 2-(4-(N-(6-(biotinamido)hexyl)aminomethyl)phenyl)-

pyridine

Hppz 1-phenylpyrazole

Hpq 2-phenylquinoline

HRMS high-resolution mass spectroscopy

ICP-MS inductively coupled plasma mass spectrometry

IL intraligand

LAB labeled avidin biotin

LLCT ligand-to-ligand charge-transfer

5,6-Me2-phen 5,6-dimethyl-1,10-phenanthroline

5-Me-phen 5-methyl-1,10-phenanthroline

2,9-Me2-4,7-Ph2-phen 2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline

XIII



mbpy-C6-est 4-(N-(6-(4-(17-ethynylestradiolyl)benzoylamino)

hexyl)aminomethyl)-4’-methyl-2,2’-bipyridine

Me2-bpy 4,4’-dimethyl-2,2’-bipyridine

Me4-phen 3,4,7,8-tetramethyl-1,10-phenanthroline

MLCT metal-to-ligand charge-tranfer

MPO

MOPS

2-mercaptopyridine-N-oxide

3-morpholinopropanesulfonic acid

MTT 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium

bromide

MV2 methyl viologen

N^N diimine ligand

NBD 7-nitrobenz-2-oxa-1,3-diazole

2-NBDG 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-

deoxyglucose

6-NBDG 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-6-

deoxyglucose

NEM N-ethylmaleimide

NHS N-hydroxysuccinimide

5-NO-phen 5-nitro-1,10-phenanthroline

NIR near-infrared

NMR nuclear magnetic resonance

PAMAM polyamidoamine

PEG poly(ethylene glycol)

XIV

PEI poly(ethylene imine)

phe phenylalanine

phen 1,10-phenanthroline

phen(C6H4SO3)2 bathophenanthroline sulfate

phen-CIA 5-chloroacetamido-1,10-phenanthroline

phen-IAA 5-iodoacetamido-1,10-phenanthroline

phen-mal 5-maleimido-1,10-phenanthroline

phen-NCS 5-isothiocyanato-1,10-phenanthroline

Ph2-phen 4,7-diphenyl-1,10-phenanthroline

5-Ph-phen 5-phenyl-1,10-phenanthroline

PI propidium iodide

PMC N-(2-pyridinylmethylene)-2,3,5,6,8,9,11,12-octa-

hydro-1,4,7,10,13-benzopen-taoxacyclononadecan-

16-ylamine

pta 1,3,5-triaza-7-phosphatricyclo[3.3.1.1]decane

py pyridine

py-3-glu 3-(N-(6-(N’-(4-(-D-glucopyranosyl)phenyl)thio-

ureidyl)hexyl)thioureidyl)pyridine

py-3-mal N-(3-pyridyl)maleimide

py-3-NCS N-(3-pyridyl)isothiocyanate

py-4-COOH isonicotinic acid

py-4-Et 4-ethylpyridine

pyAm-Mepy N-(4-pyridyl)--(N-methylpyridinium-3-yl)acrylamide

py-An N-(1-anthraquinonyl)-N’-(4-pyridinylmethyl)thiourea

py-az 1-(4-pyridinylformyl)aza-15-crown-5

XV

py-biotin-NH2 3-amine-5-(N-((2-biotinamido)ethyl)-aminocarbonyl)

pyridine

py-biotin-NCS 3-isothiocyanato-5-(N-((2-biotinamido)ethyl)amino-

carbonyl)pyridine

py-biotin-TU-Et 3-ethylthioureidyl-5-(N-((2-biotinamido)ethyl)amino-

carbonyl)pyridine

pybz 2-pyridyl-benzimidazole

py-C6-est

4-(N-(6-(4-(17-ethynylestradiolyl)benzoylamino)-

hexanoyl)aminomethyl)pyridine

py-C6-ind 4-(N-(6-N-(2-indol-3-yl-ethyl)hexanamido)amido)-4’-

methyl-2,2’-bipyridine

pyCONH-cat N-(2,3-dihydroxylphenyl)-isonicotinamide

(pyNHCO)2py N,N’-dipyridin-4-ylpyridine-2,6-dicarboxamide

py-O5-CM coumarin-3-carboxylic acid 1-(4-pyridyloxy)-3,6,9-

rioxaundecane-11-yl ester

py-Ph N-(1-phenyl)-N’-(4-pyridinylmethyl)thiourea

py-PTZ 10-(4-picolyl)phenothiazine

py-Rf-NCS 3-isothiocyanato-5-(N-((3-perfluorooctyl)propyl)-

aminocarbonyl)pyridine

py-Rf-NH2 3-amino-5-(N-((3-perfluorooctyl)propyl)amino-

carbonyl)pyridine

py-Rf-TU-Et 3-ethylthioureidyl-5-(N-((3-perfluorooctyl)propyl)-

aminocarbonyl)pyridine

py-TU-DPAT 3-(2-(4-hydroxy-3-(2,2’-dipicolylaminomethyl)-

phenyl)ethylthioureidyl)pyridine

XVI

py-TU-Et 3-(ethylthioureidyl)pyridine

quqo 2-(2-quinolinyl)quinoxaline

RET resonance-energy transfer

ROS reactive oxygen species

r-SAv recombinant streptavidin

SAv streptavidin

SDS-PAGE sodium dodecyl sulfate polyacrylamide gel

electrophoresis

TBAP tetra-n-butylammonium hexafluorophosphate

TCSPC time correlated single photon counting

TFA trifluoroacetic acid

TMS tetramethylsilane

TPEF two-photon excited fluorescence

TPEN N,N,N’,N’-tetra(2-picolyl)ethylenediamine

tpphz tetrapyrido[3,2-a:2’,3’-c:3”,2”-h:2’’’,3’’’-j]phenazine

TTHA triethylenetetraamine hexaacetic acid

TEG triethylene glycol

trp tryptophan

uv ultraviolet

VSMC vascular smooth muscle cells

XVII

Table of Contents

Abstract I

Acknowledgements VI

Declaration VIII

Abbreviations IX

Chapter 1 Introduction

1.1. Luminescent Biological Labels and Probes 1

1.1.1. Fluorescent Organic Labels and Probes 4

1.1.2. Luminescent Lanthanide Labels and Probes 10

1.1.3. Luminescent Transition Metal Complexes as Labels and

Probes

15

1.1.3.1. Luminescent Transition Metal Complexes as

Ion Probes

15


Labels and Probes for Proteins

22


Cellular Probes

32

1.2. Luminescent Rhenium(I) Polypyridine Complexes 41

1.2.1. Luminescent Rhenium(I) Polypyridine Complexes as Ion

Probes

49

1.2.2. Luminescent Rhenium(I) Polypyridine Complexes as

Labels and Probes for Proteins

54

1.2.3. Luminescent Rhenium(I) Polypyridine Complexes as 60

XVIII

Cellular Probes

1.3. Objectives 65

Chapter 2 Rhenium(I) Polypyridine Dipicolylamine Complexes

2.1. Background 70

2.2. Experimental 87

2.2.1. Materials and Reagents 87

2.2.2. Synthesis and Characterization 87

2.2.3. Physical Measurements and Instrumentation 93

2.2.4. pH- and Ion-dependent Emission Studies 97

2.2.5. Cellular Studies 98

2.3. Results and Discussion 101


2.3.2. Electrochemical Properties 103

2.3.3. Photophysical Properties 103

2.3.4. pH-dependent Emission 125

2.3.5. Ion-dependent Emission 129

2.3.6. Cellular Uptake 140

2.3.7. Cytotoxicity 143

2.3.8. Live-cell Confocal Microscopy 144

2.3.9. Cellular Ion-binding Studies 148

2.4. Summary 152

Chapter 3 Rhenium(I) Polypyridine Mono-biotin and Bis-biotin

Complexes

XIX

3.1. Background 153





3.2.4. Avidin-binding Studies 184

3.2.5. Biotinylation Studies 186






3.3.4. HABA Assays 203

3.3.5. Emission Titrations 206

3.3.6. Dissociation Assays 212

3.3.7. Signal Amplification 212

3.3.8. Biotinylation of BSA with the Isothiocyanate Complex 5 216


3.3.10. Live-cell Confocal Imaging 220

3.4. Summary 227

Chapter 4 Rhenium(I) Polypyridine Glucose Complexes

4.1. Background 228



XX



4.2.4. Determination of Lipophilicity 247

4.2.5. Con A-Binding Studies 248

4.2.6. Bacteria Binding Studies 248






4.3.4. Con A Binding 263

4.3.5. Bacteria Binding 270

4.3.6. Lipophilicity 270



4.3.9. Cell Line Dependence 280

4.3.10. Glucose Dependence 285

4.3.11. Taxol Inhibition 288

4.3.12. Live-cell Confocal Imaging 288

4.3.13. Photostability 290

4.4. Summary 296

Chapter 5 Rhenium(I) Polypyridine Fluorous Complexes

5.1. Background 297


XXI

XXII




5.2.4. Labeling of BSA with the Isothiocyanate Complexes 1b

3b

318

5.2.5. Labeling of GSH with the Isothiocyanate Complexes 1b

3b

318

5.2.6. Isolation of Rhenium(I)-GSH Conjugates from a Mixture

of Amino Acids with FSPE

319






5.3.4. Fluorous-labeling Properties 341

5.3.5. Isolation and Analysis of a Labeled Peptide by Fluorous

Solid-Phase Extraction

345

5.3.6. Lipophilicity 351



5.3.9. Confocal Imaging 354

5.4. Summary 362

Chapter 6 Conclusions 363

References 368

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