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2017 Functionalized BODIPY Dyes for Near-Infrared Emission of Lanthanide Complexes Rukshani Wickrama Arachchi Eastern Illinois University This research is a product of the graduate program in Chemistry at Eastern Illinois University. Find out more about the program.
Recommended Citation Arachchi, Rukshani Wickrama, "Functionalized BODIPY Dyes for Near-Infrared Emission of Lanthanide Complexes" (2017). Masters Theses. 2911. https://thekeep.eiu.edu/theses/2911
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Please submit in duplicate. BODIPY Functionalized dyes for Near-infrared Emission of Lanthanide Complexes
(TITLE)
BY
Rukshani Wickrama Arachchi
THESIS
SUBMITIED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF
Master of Science in Chemistry
IN THE GRADUATE SCHOOL, EASTERN ILLINOIS UNIVERSITY CHARLESTON, ILLINOIS
2017
YEAR
I HEREBY RECOMMEND THAT THIS THESIS BE ACCEPTED AS FULFILLING THIS PART OF THE GRADUATE DEGREE CITED ABOVE
7/t11/� l}'ESISCOMMITTEE CHAIR DATE DEPARTMENT/SCHOOL CHAIR DATE OR CHAIR'S DESIGNEE
THESIS COMMITTEE MEMBER 3iJJDATE /J7- THESIS COMMlfTEE MEMBER DATE
10/11 THESIS COMMITTEE MEMBER DATE THESIS COMMITTEE MEMBER DATE Functionalized BODIPY Dyes for Near-infrared Emission of Lanthanide Complexes
Rukshani Wickrama Arachchi
Research Advisor: Dr. Hongshan He
Eastern Illinois University
Chemistry Department TABLE OF CONTENTS
TABLE OF CONTENTS ...... i
ABBREVIATIONS ...... vi
ABSTRACT ...... vii
ACKNOWLEDGEMENTS ...... vii i
LIST OF FIGURES ...... ix
LIST OF TABLES ...... xiv
CHAPTER 1 INTRODUCTION 1 1.1. Overview 2 1.2. Luminescence 3 1.2.1. Fluorescence
1.2.1.1. Light-Matter Interaction 3
1.2.1.2. Emission Process 4
1.2.1.3. Excitation Spectrum 5
1.2.1.4. Emission Sp ectrum 5
1.2.1.5. Fluorescence Quantum Efficiency 7
1. 2.1. 6. Lifetime of Excited State 8
1.2. 1. 7. Factors Affecting fo r the Fluorescence Intensity 8
1.2.1. 8. limitations of Fluorescence 9
10 1.2.2. Phosphorescence 1 1.2.3. Fluorophores for Medical Diagnosis I 15 1.3. Fundamentals of Lanthaoides 16 1.3.1. Physical Properties l.3.2. Chemical Properties 16
1.3.3. Formation of Complexes 18
1.4. NIR Emitting Lanthanide Complexes 21
1.4.1. NIR Emission 21
1.4. 1. l. lanthanide NIR Emission 21
1.4.1.2. Energy Transfer Mechanism in lanthanide Organic Complex 23
1.4.2. Lanthanide Complexes 24
1.4.2.1. DOTA Complexes 24
1.4.2.2. Porphyrin Complexes 28
1.4.2.3. Phenanthroline Complexes 31
1.4.2.4. BOD/P Y Based Lanthanide Complexes 34
1.4.3. Applications of NIR Emitting Lanthanides 38
1.5. Motivation 41
1.6. Objectives 42
43 CHAPTER 2 SYNTHESIS & MEASUREMENTS 43 2.1. General 43 2.1.1. Materials 44 2.1.2. Instruments 45 2.2. Synthesis
2.2.1. Project 1 Synthetic Route 45
2.2.J. 1. Synthesis of RHl 46
2.2.1.2. Synthesis of RH2 47
2.2.1.3. Synthesis of RH3 48
ii 2.2. 1.4. Synthesis of RH4 49
2.2.1.5. Synthesis of RH5 50
2.2.J.6. Synthesis of RH6 51
2.2.1.7. Synthesis ofRH 7 52
2.2. 1.8. Synthesis of RHB 53
2.2. 1.9. Synthesis of RH9 54
2.2.2. Project 2 Synthetic Route 55
2.2.2.1. Synthesis of RHJO 56
2.2.2.2. Synthesis of RHII 57
2.2.2.3. Synthesis of RHI 2 58
2.2.2.4. Synthesis of RHl3 59
2.2.2.5. Synthesis of RHl4 60
2.2.2.6. Synthesis of Complex 1 61
2.2.2. 7. Synthesis of Complex 2 62
2.3. PhotophysicaJ Measurements 62
2.3.1. UV-VIS Absorption Spectra in Solutions 62
2.3.2. Fluorescence Spectra 63
2.3.2. 1. Excit ation and Emis sion Spect ra 63
2.3.2.2. Qu antum Yield Measu rements 64
2.3.3. Lifetime Measurements 65
2.3.3.J. Lifeti me Measu rement inthe Visib le Regio n 65
2.3.3.2. Lifetime Measu rement inthe NJR Region 65
2.3.4. Spectroscopic Titration 65
iii CHAPTER 3 RES UL TS AND DISCUSSION 67
3.1. Characterization 67
3.1.1. NMR Spectroscopy 67
3. 1.1.1. 1HNMR of Compound RH/ 67
3.1.1.2. 1HNMR of Compound RH2 68
3.1.1.3. 1HNMR of Compound RH3 69
3. 1. 1.4. 1HNMR of Compound RH4 70
3.1.1.5. 1H NM R of Compound RH5 71
3. 1. 1.6. 1HNMR of Compound RH6 72
3. 1. 1.7. 1HNMR of Compound RH7 73
3. 1.1.8. 1HNMR of Compound RH8 74
3. 1.1.9. 1HNMR of Compound RH9 75
3. 1.1.10. 1HNMR of Compound RHJO 76
3. 1.1.11. 1HNMR of Compound RH/ 1 77
3. 1.1. 12. 1HNM R of Compound RH12 78
3. 1. 1.13. 1HNMR o/Compound RHJ3 79
3. 1. 1. 14. 1HNM R of Compound RH14 80
3.1.2. X-Ray Crystallography 82
3.2. Photophysical Properties 89
3.2.1. Photophysical Properties of Ligands 89
3.2.1.1. Absorption Data 89
3.2.1.2. Fluorescence Data 89
3.2.1.3. Quantum Yield Measurements 90
iv 3.2. 1.4. lifetime Measurements 98
3.2.2. Photophysical Properties of Lanthanide Complexes 99
3.2.2.1. Interaction of RH12 with [Yb{TPP)(OAc)(MeOH)2] in CH2Cli 100
(formation of complex 1)
3.2.2.2. Interaction of RHJ4 with [Yb{TPP)(OAc)(MeOH)2] in CH2C'2 101
(formation of complex 2)
3.2.2.3. Spectroscopic Titrations with RH12 and RH14 Ligands 102
3.2.2.4. Lifetime at NIR Region 105
CHAPTER 4 CONCLUSION 107
REFERENCES 109
APPENDICES 118
v ABBREVIATIONS
I. BODIPY - boron-dipyrromethene
2. NIR - near-infrared
3. NMR-nuclear magnetic resonance
4. TMS - tetramethylsilane
5. DOTA -1,4,7,10-tetraazacyclododecane- I,4,7, I O-tetraacetic acid
6. DTPA - diethylenetriaminepentaacetic acid
7. TFA - trifluoroacetic acid
8. Ln - lanthanide
9. TCSCT - time-correlated single photon counting technique
10. CAFS - chemoselective alteration of fluorophore scaffolds
11. TRL - time-resolved luminescent
12. TRLM - time resolved luminescence microscopy
13. MRI - magnetic resonance imaging
14. PET - photoinduced electron transfer
15. LLBs - lanthanide luminescent bioprobes
16. TPP - tetraphenylporphyrin
17. HMRG - hydroxymethyl rhodamine green
18. TMOS - tetramethoxysilane
19. Por - Porphyrin
vi ABSTRACT
For many decades, fluorophores have been used to investigate natural systems, but autofluorescence and photobleaching diminish the detection capacity. Recently, BODIPY based lanthanide complexes have been synthesized and their photophysical properties have been investigated. These complexes exhibit higher emission efficienciesat NIR region with longer NIR lifetimes upon exciting at longer wavelength.
In this study, two BODIPY based lanthanide complexes were synthesized and their photophysical properties were evaluated. BODIPY based ligands were synthesized with phenanthroline as the ligand binding group. These ligands exhibit strong absorption at 527 nm and 532 nm, fluorescence at 540 nm and 548 nm with 0.55 (±0.0l) and 0.78 (±0.06) quantum yields. Upon exciting at 525 nm and by titrating with [Yb(TPP)(OAc)(MeOH)2], both ligands formed 1:1 ligand to metal complexes exhibiting strong NIR emissions at 977-
1006 nm with two characteristic peaks correspond to unique Yb(III) transition between
2 Fs12 ground state and 2F1112 excited state ofBODIPY based Yb(Ill) porphyrin complex.
The para substituted aromatic ring containing BODIPY demonstrated 4.97 ns excited state lifetimeand the ortho substituted aromatic ring containing BODIPY demonstrated 7 .56 ns in the visible region. In contrast, complex l and complex 2 possessed 12.24 µs and 8.86 µs respectively. Excitation scan of both complexes showed the evidence of higher excitations at 440 nm and 525 nm suggesting both porphyrin and BODIPY moieties contribute for the lanthanide sensitization process. Further studies are required to address the underline chemistry behind the energy transfer mechanism of both complexes and how the geometry of formed lanthanide complexes affect the sensitization process.
vii ACKNOWLEDGEMENTS
I would like to express my deepest gratitude to Dr. Hongshan He for his invaluable support and guidance during the course of the master's degree in chemistry. His patience and foresight greatly contributed to my learningexperience at Eastern Illinois University. The opportunity to learnfrom the wealth of knowledge and experience that was made available to the research group was a great privilege.
I would also like to thank Eastern lllinois University, and specifically the Chemistry
Department, the Graduate School, the College of Sciences, the EIU President's Fund for
Research and Creative Activity, the National Science Foundation, as well as the Chemistry
Department stock room for their contributions to this work. I would also like to thank my thesis committee; Dr. Daniel Sheeran, Dr. Jonathan Blitz, Dr. Edward Treadwell and Dr.
Zhiqing Yan for their assistance, guidance, and patience during the course of my degree.
I would like to acknowledge Dr. Kraig Wheeler for the X-ray diffraction analysis, and Dr.
Barbara Lawrence for the NMR training. Finally, I would like to express my deepest appreciation of my family, friends, and research colleagues for their continual positive support and encouragement.
viii LIST OF FIGURES
Figure 1.1. Excitation, emission spectra of a fluorophore with the stokes shift 7
Figure 1.2. Fluorescence and phosphorescence processes I 0
Figure 1.3. Small-molecule fluorophores with representative cores including 11 fluorescein, BODIPY (BODIPY-FL), rhodamine (rhodamine green), and cyanine (Cy5.5) cores
Figure 1.4. Structures of glutaryl phenylalanine hydroxyrnethyl rhodamine 12 green (gPhe-HMRG) probe is activated with chyrnotrypsin secreted in pancreatic juice and emits HMRG fluorescence
Figure 1.5. Nebraska red dyes 13
Figure 1.6. Chemoselective alteration offluorophore scaffolds 14
Figure 1.7. pH activatable BODIPY-based fluorescent probes 15
Figure 1.8. Synthetic strategies used to insert lanthanide ions into molecular 20 edifices
Figure 1.9. Characteristic lanthanide emission peaks of Tb(III), Sm(III), Dy( III), 22
Eu(III), Nd(III), Er(III), Ho(III), Tm(lll) and Yb(lll)
Figure 1.10. Energy transfer mechanism in lanthanide organic complexes 23
Figure 1.11. Gadolinium(III) DOTA complex functionalized with a BODIPY 26 derivative
Figure 1.12. pH Dependence of the europium emission spectrum showing the 26 two Eu(III) species CJ•ex= 380 nm, 298 K, 0. 1 M NaCl)
ix Figure 1.13. Ln2-Ll and Ln2-L2 Complexes and emission spectra for Ln2-L2 27
(red-Yb, blue-Nd, green-Er) measured in D20 using Aex= 400 nm.
Figure 1.14. Ytterbium tetraarylporphyrin complexes with asymmetrical 29 substitutes
Figure 1.15. Emission spectra for (RRR-R)-YbLl (295 K, 20% D20-CD 30D, 5 31 mM) in degassed solution (top), in aerated solution (bottom) and in the presence ofO.l mM [(CG)6]2,Aex= 529 nm
Figure 1.16 Lanthanide complexes of 2-thenoyltrifluoroacetonate complex 31
Figure 1.17. Emission spectra for the covalently linked [Ln(tta)3(phen)] 32
Aex complexes in the TMOS-DEDMS sol-gel bulk sample, 370 run.
Figure 1.18. Emission spectra for the covalently linked [Ln(tta)3(phen)] 32 complexes in the TMOS-DEDMS sol-gel bulk sample, Aex = 370 nm
Figure 1.19. Ytterbium(IID monoporphyrinate complexes 33
[Ln(Por)(BDL)(OAc)] complexes
Figure 1.20. BODIPY based lanthanide complexes 35
Figure 1.21. BODIPY based chromophores used to synthesized lanthanide 37 complexes
Figure 2.1. Synthesis of RHl. 46
Figure 2.2. Synthesis of RH2. 47
Figure 2.3. Synthesis ofRH3. 48
Figure 2.4. Synthesis of RH4 49
Figure 2.5. Synthesis ofRH5 50
Figure 2.6. Synthesis ofRH6 51
x Figure 2.7. Synthesis of RH7 52
Figure 2.8. Synthesis of RH8 53
Figure 2.9. Synthesis of RH9 54
Figure 2.10. Sy nthesis of RHlO 56
Figure 2.1 1. Synthesis ofRHl I 57
Figure 2.12. Synthesis ofRH12 58
Figure 2.13. Synthesis of RH13 59
Figure 2.14. Synthesis of RH14 60
Figure 2.15. Synthesis of Complex 1 61
Figure 2.16. Synthesis of Complex 2 62
Figure 3.1. ORTEP diagrams ofthe single-crystal st ruc tures of (a) RH4 and (b) 83
RH5 (c) RH6 with 50% thermal ellipsoid probability
Figure 3.2. ORTEP diagrams of the single-crystal structures of (a) RHlO and (b) 86
RH 13 with 50% the rmal ellipsoid probability
Figure 3.3. Abso rption spectra for RH12and RH14 ligands in CH2Ch 89
6 (1.6 x 10· M)
Figure 3.4. Excitation Spectra ofRH12 and RH 14 ligands in CH2Ch 90
6 ( 1.6 x 10· M)
Figure 3.5. Emission Spectra of RH 12 and RH 14 ligands in CH2Ch 90
6 ( 1.6 x 10· M)
Figure 3.6. Trial 1- UV-absorption spectra of RH 12 with four different 9 1 concentrations in CH2Ch
xi Figure 3. 7. Trial 1- Emission spectra of RH 12 with four different concentrations 92 in CH2Ch
Figure 3.8. Trial 1- Integrated fluorescence vs absorbance graph forquantum 92 yield determination of RH 12
Figure 3.9. Trial 1- UV-absorption spectra ofRH14 with four different 93 concentrations in CH2Ch
Figure 3.10. Trial 1- Emission spectra ofRH14 with four different 93 concentrations in CH2Ch
Figure 3.11. Trial 1- Integrated fluorescence vs absorbance graph for quantum 94 yield determination of RHl4
Figure 3.12. Trial 2- UV-absorption spectra of RH12 with fivedi fferent 95 concentrations in CH2Ch
Figure 3.13. Trial 2- Emission spectra of RH12 with fivedi fferent 95 concentrations in CH2Ch
Figure 3.14. Trial 2- Integrated fluorescence vs absorbance graph for quantum 96 yield determination ofRH12
Figure 3.15. Trial 2- UV-absorption spectra ofRH14 with five different 96 concentrations in CH2Ch
Figure 3.16. Trial 2- Emission spectra of RH 14 with five different 97 concentrations in CH2Ch
Figure 3.17. Trial 2- Integrated fluorescence vs absorbance graph forquantum 97 yield determination of RH14
xii Figure 3.18. Single Exponential Tail fitting of lifetime forRH12 in CH2Ch at 98 room temperature at 53 7 nm
Figure 3.19. Single Exponential Tail fitting of lifetime for RHl 4 in CH2Ch at 99 room temperature at 549 nm
Figure 3.20. (a) Excitation Spectrum (�m= 980 nm) (b)NIR emission under 101 different excitation wavelengths
Figure 3.21. Excitation vs NIR emission of complex 2 in CH2Ch l 02
Figure 3.22. NIR emission of complex 2 under different excitation wavelengths 102
Figure 3.23. Interaction of RH12 with [Yb(TPP)(OAc)(MeOH)z] in CH2Ch (a) 103 visible emission from RH12 (b) NIR emission from Yb(III)
Figure 3.24. Back titration by RH12, NIR emission from Yb(lll) at 977 nm and 103
1005 nm
Figure 3.25. Interaction ofRH14 with in CH2Ch (a) visible emission from 104
RH 14 (b) NIR emission from Yb(III)
Figure 3.26. Back titration by RH 1 4 , NIR emission from Yb(III) at 977 nm and 105
1005 nm
Figure 3.27. NIR decay curve for complex 1 recorded at 980 nm 106
Figure 3.28. NIR decay curve forcomplex 2 recorded at 980 nm I 06
xiii LIST OF TABLES
Table 1.1. Spectral luminescent characteristics of Yb(III) porphyrin complexes 30
Table 1.2. Photophysical data of synthesized complexes in toluene 34
Table 1.3. Photophysical properties of BODIPY based lanthanide complexes 36
Table 3.1. Crystal data and structural parameters for RH4, RH5 and RH6 84
Table 3.2. Selected Bond lengths for RH4, RH5 and RH6 85
Table 3.3. Selected Bond angles for RH4, RH5 and RH6 85
Table 3.4. Crystal data and structural parameters for RHlO and RH13 87
Table 3.5. Selected bond lengths RHlO and RH13 88
Table 3.6. Selected bond angles for RHIO and RH13 88
Table 3.7. Photophysical properties of RH12 and RH14 ligands 99 CHAPTER 1
INTRODUCTION
1.1. Overview
Understanding living systems in nature has long been a practice for different purposes, yet tracing biological systems effectively, efficiently, and accurately is a challenge.1 For many decades, fluorophores have been used to investigate these natural systems, but autofluoroscence arising from such systems diminish the detection capabilities resulting in lower selectivity and sensitivity. Also, the utilization of higher excitation energy sources to excite prevailing fluorophores causes photobleaching of analyzed samples. To overcome these mentioned detection barriers, lanthanide metal complexes have been synthesized and tested on physiological environments for the past 20 years.2
Lanthanide complexes show luminescence with unique emission properties in near infrared
(NIR) region but the emissions are poor due to lack of absorption capabilities. Owing to this problem, excitation of lanthanides requires strong light source (laser), which can also lead to photoblcaching of the analyte. To address this issue, organic chromophores can be synthesized with ligand binding groups. These chromophores are used to synthesize lanthanide complexes enabling them to excite at lower wavelengths (visible region). This sensitization process is an indirect excitation method which can enhance lanthanide NIR emission through the antenna effect. 3 So far, porphyrin, DOTA, and phenanthroline based lanthanide complexes have been synthesized that demonstrate NIR emissions; however,
8 they require 300-450 nm range energy sources to excite the complexes.4•5•6•7•
1 Recently, BODIPY (dipyrromethene) based lanthanide complexes have been synthesized and their photophysical properties have been investigated. These complexes exhibit higher
emission efficiencies at NIR region with longer NIR lifetimes upon exciting at longer
wavelengths. 9•10
This previous work suggests us to functionalizing BODIPY chromophores furtherto obtain well stabilized novel Yb(III) complexes with higher emissions and longer lifetimes at the
NIR region. These complexes can be evaluated as promising optical probes for medical diagnosis and imaging.
1.2. Luminescence
Luminescence is a phenomenon of light emission that indicates an excess over the thermal radiation and persists for a time beyond the period of electromagnetic oscillation. Luminescence is observed at any temperature while visible light from thermal radiation begins emitting at minimum temperatures of a few hundred degrees K, so luminescence is sometimes named as cold light. As luminescence lasts for a time beyond the period of electromagnetic oscillation, it can be differentiated from stray and reflected light. The luminescence includes a wide range of light emitting processes; their names originate from the diverse sources of energy that power them. Photoluminescence is one of the main categories in luminescence which further divides into fluorescence and phosphorescence.1•11
Photoluminescence, a luminescence enthused by light absorption in UV-Vis-NIR spectral region, denotes any process in which a material absorbs electromagnetic energy at a definitewavelength and then emits part of it at a longer wavelength transforming a part of absorbed light into luminescence, while the remainder dissipates as heat or molecular
2 vibrations. Since there are many reliable and inexpensive excitation sources available and the effectcan often be observed with the naked eye, photoluminescence is the most popular type of luminescence used for many applications. Usually the excitation source emits in the UV region and the photoluminescence occurs in Vis or NIR.1• 12
Due to the delay between the absorption of energy by the material and re-emission process, atoms or molecules can remain in an excited state for a short period, which is defined as the lifetime of excited state. Based on practical observations, this delay time can differ by many orders of magnitude fordifferent materials and two main types of photoluminescence processes were identified. Fluorescence has a shorter excited state ranging from 10-12 to
10-7 seconds ( s ), and phosphorescence has a much longer excited state up to a few hours and even days.1•13
1.2.1. Fluorescence
1.2.1.1. Light -Matter Interaction
Fluorescence is a "fast" photoluminescence phenomenon. Some atoms and molecules absorb light at a particular wavelength and consequently emit a longer wavelength range aftera short interlude. Every atom or molecule has a series of closely spaced energy levels.
Atoms and molecules can go from low to high energy levels when they absorb light equal in energy to the difference between the ground and excited energy levels. Even though every atom and molecule possesses different energy levels, only few interact with light to undergo excitation and exhibit fluorescence.1 1
For polyatomic molecules, the orbital angular momentum of a particular state related to the
spin is given by M = 2.S. + I where M refers to multiplicity of the particular state. When
3 !.), polyatomic molecules have paired electrons S = 0(+ !.- and the multiplicity becomes 2 2
1 and is referred to as a singlet electronic state. When there are 2 unpaired electrons in a
1( + molecule, S = � + �) and multiplicity becomes 3 which is called a triplet state. When a discrete quantum of light is irradiating a molecule, it is absorbed within 10-15 s, and transitions to higher electronic states is observed. This absorption energy is unique to each molecule and it is used to characterize specific molecular structures by analyzing the ultraviolet and visible spectra due to the ground to singlet (SI, S2) electron transfer process.1 This absorption process typically starts from the lowest vibrational level of the ground state. Excited electrons stay in the excited state for a certain period of time. There are collisions between excited molecules resulting in energy loss leading those electrons to return to the excited singlet state; thereby, if further energy remains after collision, electrons return to the ground electronic state by releasing energy. This energy emission process is known as fluorescence (Figure l.2.).14
1.2.1.2. Emission Process
Fluorescence emission can take place in different ways. So far, a few main pathways are known to be related to fluorescence emission. Stokes fluorescence, which is observed in solutions, is related to reemission of photons having lower energy compared to absorbed energy. Anti-Stokes fluorescence occurs when thermal energy is provided to a compound or if it has many vibrational energy levels with higher populations, emitting more energy than it absorbed. Resonance fluorescence cannot be observed in a solution phase, which refers to reemission of photons containing the same energy as absorbed; thus, emission
wavelength is the same as excited wavelength. This phenomenon is called Rayleigh
4 scattering and occurs at all wavelengths. In terms of intensities related to emission and excitation they are varied, but are typically low intensity which cause problems when fluorescence is used for detection purposes. The Raman effect is another scattering form rdated to Rayleigh scattering, which occurs due to vibrational energy being added to, or deduced from, the excitation photons. Even though Raman scattering signals are weaker,
2- they are significant if high energy excitation sources are used.1. 1 13
1.2.1.3. Excitation Sp ectrum
A fluorescent molecule often exhibits two characteristic spectra. The excitation spectrum shows the relative efficiency of exciting radiation to cause fluorescence at different wavelengths. This spectrum is usually similar to the absorption spectrum of the same molecule obtained from a spectrophotometer, but may deviate due to different instrumental parameters. The excitation spectrum is often acquired from a fluorometer. To match the absorption spectrum to the excited spectrum, some corrections must be done such as changes in photomultiplier response, changes in the band width of the monochromator or light source, and consistency in the slit of fluorometer. The peak position of excitation spectrum is chosen to excite the molecule to observe fluorescence emission.
1.2.1.4. Emission Sp ectrum
Emission spectrum is the second characteristic spectrum of a fluorescent molecule that is recorded due to the reemission of radiation absorbed by the molecule. The shape of this spectrum and the quantum efficiency is independent from the excitation wavelength used to obtain the emission spectrum. When using a different wavelength than the excitation maxima (ft.ex) to excite the molecule, emission intensities in the emission spectrum can be
5 lower as absorption is lower at other wavelengths than A..:x. Excitation and emission spectra display mirror images of each other, which is useful to understand the transitions among first excited state or higher electronic levels.
The presence ofother peaks is an indication ofimpurities or Rayleigh or Tyndall scattering.
If the solution is very dilute, Raman scattering is also observed in the emission spectrum.
If the molecular structure is very complex and less symmetric, wider fluorescence bands could be observed in the emission spectrum. Ifone compound has several excitation peaks and emission peaks or two compounds have the same excitation peaks but different emission peaks or vice versa, these properties can be used to distinguish one molecule from the other, which are very useful parameters to develop analytical tools to identify such molecules. This ability of fluorescence spectroscopy is a major advantage over absorption spectroscopy, which improves the selectivity of analytical methods developed related to
fluorophores. The stoke shift is the physical constant that refers to the difference between the maximum wavelengths of excitation and emission spectra (Figure 1.1.). This constant represents the energy loss in between the excitation and emission processes before the molecule returns to ground state.
107 II II Stoke Shift= A..:x- Aem
A..:x=Corrected maximum wavelength for excitation (nm)
A.cm=Corrected maximum wavelength for emission (nm)
6 Stokes Shi.ft H
Excitation spectrum Emission spectrum
Figure 1.1. Excitation, emission spectra of a fluorophore with the stokes shift
1.2.1.5. Fluorescence Quantum Efficiency
The ratio of total energy emitted by any molecule per quantum of energy absorbed is
referred to as quantum efficiency, which is a measure of the probability of the excited state
being deactivated by fluorescence emission rather than the non-radiative mechanisms.15
Quantum yield in the visible region is measured using the following equation:
Gradx 0X = 0 [ ] [�]2 ST GradsT nsT
0x =Fluorescence quantum yield of compound
0sT = Standard fl uorescence quantum yield (A.ex= 480 nm)
Gradx = Gradient from the plot of the intergraded fluorescence intensity vs
absorbance of l.igands and complexes with diffe rent concentrations
GradsT =Gradient from the intcrgraded fluorescence intensity vs absorbance of
standard
nx =Refractive index of the solvent used to dissolve the compound
nsT = Refractive index of the compound used to dissolve the standard ln practice, it is quite difficult to measure the quantum yield due to some important
considerations. Concentration effects, solvent effects and validity of standard samples must
7 be considered when taking the measurements for a particular compound. Choosing a correct concentration range and obtaining data at different absorbance values can ensure the linearity, thereby, giving a reliable quantum yield value for a specific fluorophore.1
I. 2. 1. 6. Lifetime of Excited State
The mean lifetime that a fluorophore spends in the excited state before emitting photons and comes back to the ground state is known as lifetime of excited state. This is an intrinsic property of a fluorophore which does not depend on its concentration, sample thickness, method of measurement, absorption of the sample, fluorescence intensity, photo-bleaching, or the excitation intensity but can vary from picoseconds to nanosecond scales depending on the nature of the fluorophore. Lifetime can be measured either by frequency domain or by time domain methods. The fluorescence lifetime is measured from the slope of the decay curve. Many fluorescence detection methods are available related to time-correlated single photon counting (TCSPC). TCSPC enables simple data collection and improves the
l6 quantitative photon counting.1• 14•
1.2.1.7. Factors affecting/or thefluorescence intensity
There are three major factors that can affect the fluorescence intensity. The equation below reveals the concentration-fluorescence intensity relationship.
F = Fluorescence intensity € = Molar absorptivity
b = Path length of the cell 0 = Quantum efficiency
c =Molar concentration lo= Incident radiation power
8 If the quantum yield is high, that indicates the sample has higher fluorescence intensity.
According to the equation, more intense radiation power should give a higher fluorescence intensity, but practically it can cause photodecomposition of the sample. To emit fluorescence, molecules must first absorb radiation so if a compound has a higher molar absorptivity, fluorescence intensity will be higher. When the sample concentration of a particular fluorophore is higher, fluorescence intensity is decreased due to light scattering and inner cell effect. 1 • 15• 17
1.2.J.6. Limitations of fluorescence
There are some limitations associated with fluorescence detection and fluorophores. The major issue with fluorescence detection is that it is strongly dependent on environment factors such as pH, ionic strength, and temperature. Additional limitations arise when using ultraviolet light as the excitation source because it leads to photochemical decomposition of the fluorescent compound. Using longer wavelength radiation, measuring the fluorescence immediately after the excitation, and storing fluorescence active samples in amber color containers reduces the photochemical decomposition of these types of compounds. Next the viscosity of the medium can affect the fluorescence. If the viscosity is higher, higher fluorescence can be observed, so the fluorescence of most of the compounds can be increased by using viscous solvents such as glycerol or gelatin. This observation is due to fewer collisions in a viscous solvent and less energy reduction among the molecules in excited state. Fluorescence quenching due to temperature, oxygen, concentration, and impurities can also be observed as a result of the interaction of fluorophores with the surrounding.13• 18 1.2.2 Phosphorescence
Phosphorescence is a "slow" photoluminescence process. In contrast to fluorescence, it demonstrates itself as a glowing that lasts long after the excitation light is gone.
Phosphorescent materials are usually called "glow-in-the-dark". Phosphorescence is most likely to occur in molecules with restricted vibrational freedom such as aromatic molecules and their derivatives. The light emission process has a delay due to an electron undergoing a spin-flip to relapse to ground state, which normally takes I 0-3 to 102 s. The light emission is delayed long enough so that materials glow in the dark after exposure to light.
Phosphorescence usually begins from the lowest vibrational level of the lowest triplet state
(To) and terminates in any of several ground state vibrational levels. Spectroscopic study of phosphorescence provides information related to the triplet state of every type of molecule. Collisional deactivation by solvent molecules, photochemical reactions, energy transfer processes, and quenching by paramagnetic species impede the detection of phosphorescence in fluid media (Figure 1.2.).18-19
v absorption (1 ti S2 A : o-1ss) 2 F : fluorescence (1o-1 -10-ss) - - - ISC S1 phosphorescence (10-6-1 Os) ti ' P : tt A F .... Inter-System Crossing (ISC)
Vibrational relaxation ti So TITSo, Figure 1.2. Fluorescence and phosphorescence processes
10 1.2.3. Fluorophores for medical diagnosis
Among fluorescence and phosphorescence, fluorescence is used for various medical purposes. Fluorophores are merged on aromatic molecules that show fluorescence upon excitation. Recently, applications of fluorophores for medical diagnostic purposes have become a common practice with advanced detection tools. In molecular biology, planar and heterocyclic molecules exemplified by fluorescein (akaFAM), couma1in, and Cy3 are very commonly employed for different imaging purposes. Indocyanine green (ICG) and fluorescein have been widely used fluorophores for over 30 years.20 The PET
(photoinduced electron transfer) mechanism is applicable to a broad range of fluorophores such as BODIPYs, rhodamines, fluoresceins, cyanines, and so on (Figure 1.3.). Each of these molecules has a characteristic absorbance spectrum and a characteristic emission spectrum enabling them to act uniquely in the biological systems for sensing and imaging.21
HO
Figure 1.3. Small-molecule fluorophores with representative cores including
fluorescein,rhodamine (rhodamine green), cyanine (Cy5.5) and BODIPY (BODIPY -
FL) cores21
For example, indocyanine green (ICG) and fluorescein are used for the detection of bile leakages, fluorescence-navigated sentinel node biopsy, and NIR photodynamic therapy for human hepatocellular carcinoma cell line for tumors. 5-Aminolevulinic acid is used for photodynamic diagnosis of gastric cancers and for photodynamic detection of lymph node metastases in gastrointestinal cancers, while methylene blue and fluorescein are used to
11 ureter identification. The glutaryl phenylalanine hydroxymethyl rhodamine green (gPhe
HMRG) probe is a fluorescent probe which can be activated by chymotropsin secreted in pancreatic juice and emit HMRG florescence (Figure 1.4). gPhe-HMRG has been excited
at 490 run and emission was observed at 520 nm only when it is hydrolyzed by chymotrypsin which indicates the pancreatic leakages.22
I H:l\
Figure 1.4. Structures of glutaryl phenylalanine hydroxymethyl rhodamine green
(gPhe-HMRG) probe is activated with chymotrypsin secreted in pancreatic juice and
emits HMRG fluorescence22
Most fluoropbores for medical applications are organic molecules and emit in the visible region. In order to afford new probes and reagents for applications in chemical biology,
Stains et. al developed new near-infrared fluorophores that display remarkable photostability and brightness (Figure 1.5.). NIR fluorescence probes attribute deeper tissue penetration and mitigate issues related to autotluoroscence from native tluorophores in biological samples. The introduction of novel chemical functionality into these scaffolds allows for the tuning ofphysical properties such as cell pem1eabil ity and generation of self reporting small molecule delivery reagents. These scaffolds can be used to develop bioimaging reagents for proteins, small molecules, and post-translational modifications. In addition, these fluorophores have been explored as promising targeted drug delivery
12 reagents. These Nebraska red dyes exhibit excitation and emission above 600 run and show improved detection sensitivities. 23
Figure 1.5. Nebraska red dyes23
Stains et. al. also developed fluorophore scaffolds as a strategy to develop ratiometric chemodosimctcrs by slightly modifying the Nebraska red dyes. Phosphorus atom in
Nebraska red dyes were replaced with boron and silicon atoms where they could introduce novel NIR emitting fluoropbores that undergo oxidation in the presence of H202. (Figure
1.6.). These fluorophores have been developed as endogenous H202 sensors by overcoming the spectral overlap of the reactants and products before and after the oxidation by H202.
The fluorophores have also produced significant blue shifts (>66 nm) in excitation and
emission maxima enabling them to detect endogenous H202 in HeLa cells providing
evidence for the ability to rationally tune ratiomctric sensors using the chemoselective
alteration of fluorophore scaffolds (CAFS) approach. From the SiOH2R scaffold, researchers have first demonstrated a method to detect Tamao oxidation of a biologically
relevant signaling molecule.24
13 111nmshift
SiOH2R Figure 1.6. Chemoselective alteration of fluorophore scaffolds24
BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indaccnc) is another class of tluorophores frequently used as pl-I,peroxyn itrite, and nitric oxide sensors. A library of pl-I-activatable probes based on the BODIPY fluorophore has been created (Figure 1.7.), each activating at a specific pH; these were conjugated to a monoclonal antibody targeting a cancer- specific cell surface receptor. These pH activatablc probe-antibody conjugates were almost non fluorescent under neutral pH conditions; however, afier selectiveuptake by tumor cells, the conjugate was transported to the lysosomc to undergo degradation at pH 4-5. Upon
activation, the probe was highly fluorescent. The ex vivo imaging and in vivo imaging of human HER2-positive lung tumors in mice was performed with a humanized antibody against HER2 and trastuzumab, then conjugated with a BOOJPY-based pH-activatable small-molecule fluorophore. The probes had no fluorescence signal on the surface of
HER2-positive cells but internalized and activated the signal only in the lysosome in vitro and showed highly specific detection of tumors with minimal background signal in vivo.20
14 OH
Figure 1.7. pH activatable BODlPY-based fluorescent probes20
1.3. Fundamentals of Lanthanides
Due to the understanding of the biomedical applications, researchers have broadened their horizon of lanthanide chemistry to investigate the rare earth metals and their complexes addressing the spectral, magnetic and coordinative properties along with physical and chemical properties. 25
In 1937, J. H. van Vleck published a research paper titled as "The Puzzle of Rare-Earth
Sp ectra in Solids" which attracted the scientificcommunity to the unique lanthanide optical properties. This research work summarized the discovery of all the natural lanthanide elements between 1803 and 1907. The discovery of highly emissive Y203:Eu(III) material was one big step in understanding lanthanide luminescence. The other important findings were the discovery of purely inorganic neodymium YAG (Yttrium Aluminium Gamet) lasers in 1964 and Er-doped optical fibers for telecommunications in 1987.26
The science community looked forward to a bright future of luminescent coordination compounds starting from the mid-seventies after the introduction of bioprobes by Finnish researchers. They synthesized Eu(Ill) and Tb(Ill) (later also Sm(III) and Dy(III)) polyaminocarboxylates and �- 15 (TRL) immunoassays. Since then, lanthanide luminescence conquered many other techniques used for medical diagnosis. Nowadays, optimization ofbioconjugation methods for lanthanide luminescent chelates, homogeneous TRL assays, and time-resolved luminescence microscopy (TRLM) are some of the major applications of lanthanide luminescent bioprobes (LLBs) in many fields of biology and medicine.26 The finalized f-block contains 15 lanthanide metals starting from Lanthanum (La, 57), followed by Cerium (Ce, 58), Praseodymium (Pr, 59), Neodymium (Nd, 60), Promethium (Pm, 61), Samarium (Sm, 62), Europium (Eu, 63), Gadolinium (Gd, 64), Terbium (Tb, 65), Dysprosium (Dy, 66), Holmium (Ho, 67), Erbium (Er, 68), Thulium (Tm, 69), Ytterbium 27 (Yb, 70), and Lutetium (Lu, 71). 1.3.1. Physical properties Most of the lanthanide elements are silvery-white metals, which tarnish when they are exposed to air, forming oxides. These metals are relatively soft and their hardness barely increases barely with higher atomic numbers. Considering from left to right across the period, the radius of each 3+ ion steadily decreases, which is called "lanthanide contraction." Most of these rare earth metals are strongly paramagnetic and attributed with higher melting and boiling points. Under UV light, many rare earth compounds exhibit strong fluoresce. Lanthanide ions tend to be pale colors, due to weak, narrow, or forbidden f-f optical transitions.21-28 1.3.2. Chemical properties The lanthanide metals are highly electropositive and reactive. Except for Yb, lanthanide reactivity depends on their size. Lanthanide metals easily react with water and liberate 16 hydrogen slowly in cold water and quickly in hot water yielding hydrous oxides. Lanthanides commonly bind to water molecules. They react with diluted acidic media releasing hydrogen rapidly at ambient temperatures to give an aqueous solutions of Ln(Ill) salts. Many of them ignite and bum vigorously at elevated temperatures. They are strong reducing agents and many of their compounds are ionic. Lanthanides show higher coordination numbers, generally greater than 6. Usually observed coordination numbers are 8 and 9 but can be as high as 12. The most common oxidation state of these rare earth metals is + 3, and due to the large sizes of the Ln(III) ions, the bonding is predominantly ionic and the cations are the typical class-a acids, but +2 and +4 oxidation states can also be observed in some metal complexes. Although the + 3-oxidation state governslanthanide chemistry, Ln(II) are easily made reacting Ln(III) with an electron delocalizing ligand, generating oxidation states of +2 and +4. For instance, Ce(IV) and Eu(ll) ions are found to be very stable in water and are strongly oxidizing and strongly reducing respectively. Due to the stability of the 4f7 configuration, Tb(IV) ion also shows stability.25 Many trends linked with the ionic radii are observed across the lanthanide series. When Ln(III) radius decreases, the salts become slightly less ionic, and reduced ionic character in the hydroxide indicates a reduction in alkaline properties. Though Yb(OH)3 and Lu(OH)3 are alkaline, they can be barely solvated in hot concentrated NaOH. According to this change, the [Ln(H20)x]3+ ions have a potential to hydrolyze, and hydrolysis can only be avoided by using higher acidic media. However, solubility cannot be simply related to the cation radius. No consistent trends are apparent in aqueous or nonaqueous solutions, but a realistic dissimilarity can be seen between the lighter "cerium" lanthanide complexes and the heavier "yttrium" lanthanides. The occurrence of divalent state in Eu(II) and Yb(II) 17 f7 can be explained by the stabilizing effect of half (4 ) and completely-filled (4f4) shells of 9 those ions.27• 2 1.3.3. Formation of complexes Over the past years, several approaches have been developed to design mono- and polymetallic lanthanide-containing molecular and polymeric assemblies. Except for the utilization of polydentate chelating agents (e.g., P-diketonates, polyaminocarboxylates), strategies related to the making macrocyclic receptors, either (a) preorganized, (e.g., cryptands or crown ethers), or (b) predisposed, (e.g., large macrocycles or cyclen derivatives and calixarenes functionalized with pendant arms), (c) podands, or (d) self assembly processes have been tested to form lanthanide complexes (Figure 1.8.). (a) Pre-organized macrocycles Preorganization occurs according to the lock and key principle. Metal ions are encapsulated into preorganized receptors such as coronands or cryptands depending on the size match between the cavity and the metal ion (Figure 1.8.a.). The ionic radius of Ln(III) ions varies little along the series [� ionic radius (La-Lu) � 0. 18 A for coordination number 9; the range of ionic radius between consecutive Ln(Ill) ion is 0.01-0.02 A] while the receptors do not undergo any conformation changes during the complex formation step. This preserved conformation of free and bound receptors reduces the reorganization work on complexation. However, due to the practical difficulties of size matching between the receptor cavity and lanthanide ions, scientists introduced the strategy to use disposed macrocycles to synthesize lanthanide complexes. 18 (b). Pre-disposed macrocycles Owing to the conformation changes upon complexation, pre-disposed macrocycles form stable complexes with lanthanide ions by the enthalpy gained host-guest interaction. In this strategy, 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraaceticacid (DOTA) forms the most stable Ln(lll) complexes (pLn :::::: 21-22) (Figure 1.8.b) . These receptors form an induced cavity (known as Induced Fit Principle) with pendant arms that can wrap the metal center (e.g., cyclen or calixarene derivatives). Difficulties in synthesizing bidentate or tridentate arms Jed scientists to synthesize small anchors with aromatic rings. (c). Podands Podands allow the change number of donor atoms easily and they are less pre-disposed than the pendant arm-fittedmacrocycles yet require some conformational changes to form the metal complexes. This challenge was overcome by introducing weak noncovalent interactions, such as hydrogen bonding. This allows to organize the arms of the donor in a favorable conformation as depicted in Figure 1.8.c. A rare trifurcated hydrogen bonding of the receptor, forms the three coordinating units with a favorable conformation maximizing Ln(lll) ions interaction. ( d). Self-assembly processes Self-assembly process is governed by the strong ion-dipole interactions and the formation of host cavity due to noncovalent interactions between ligand stands. For instance, three benzimidazole moieties are arranged according to interstrand n:-n: interactions among aromatic rings and ion-dipole interactions (nine Ln-N ion-dipole bonds) as illustrated in Figure 1.8.d. Also number of other research work shows that, self-assembly processes 19 yield more favorable complex formation over the other mentioned strategies, enabling the synthesis of stable multimetallic edifices and devices in the future.25 (Nd(N0,h(Ha0)J) 18-uown.. ""ICIOJ,(-1 -@: l.,,)...) uypcanc:t({)(2.2.2) a. Pre-organized macrocycles Tb(trff),(solv)> aubst. cyden b. Pre-disposed macrocycles Eu(Cl04),("10,. > (Eu(HLJr tripod c. Podands 20 d. Self-assembly processes Figure 1.8 Synthetic strategies used to insert lanthanide ions into molecular edifices25 1.4. NJR Emitting Lanthanide Complexes 1.4.1. NIR emission 1.4.1.1. Lanthanide NIR emiss ion Considering the spectral properties of lanthanide complexes, due to their (Xe] 4r15s25p6 electronic configuration, luminescence can be observed from f-f transitions in the 4r1 shell (Figure 1.9.). This excitation can be hindered due to the shielding of 5s and 5p orbitals. This shielding can cause low absorptivity and thereby yield lower emissions and sharp narrow bands with low molar absorption coefficients. Not only that but also f-f transitions are both Laporte and spin forbidden.30 This fact explains the long-excited state lifetimes from milliseconds to the microseconds, enabling time-gated or time-resolved live cell or in vivo imaging applications.28 NIR luminescent lanthanide complexes show higher selectivity and sensitivity. The sharp emissions around NIR region avoid the misleading background fluorescence generated from biological systems, yielding sharp emission bands. The long luminescent lifetimes of these complexes enhance the assay sensitivity through time-resolved measurements and 21 reduce the overlapping autofluorescence. Most of tbe lanthanide complexes diminish photobleaching. The multiphoton excitation technique allows these complexes to excite in NIR region. Poor efficiencies and the requirement of higher exciting light sources have led scientists to develop NIR emining lanrhanide complexes with organic chromophores.31 In 1940, Weissman et al. revealed the utilization oflanthanide luminescence complexes of terbium and samarium metals for biomedical applications by introducing the antenna effect to sensitize lanthanide metal complexes.32 -Tb -Sm -Dy -Eu -Nd -Er -Ho -Tm - Yb 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 Wavelength/nm Figure 1.9 Characteristic lanthanide emission peaks of Tb( lll), Sm(III), Dy(III), Eu(Ill), Nd(lll), Er(lll), Ho(III), Tm(fll) and Yb(IIJ)32 22 1.4. 1.2. Energy transfer mechanism in lanthanide organic complex Indirect excitation or the antenna effect is the most accepted mechanism used to describe the energy transfer process of organic lanthanide complexes. The energytransfer process starts by exciting the singlet ground state (So) of lanthanide complex to its excited state (Si) due to the absorption of energy. Then, energy is transferred from Si to the triplet excited state (Ti) of the ligand through intersystem crossing followed by an intramolccular energy transfer process from Ti of the ligand to one of the excited 4fstates of the Ln (Ill) ion (Figure 1.10.). As lanthanide luminescence occurs due to intra-4f transitions within the ion, Dexter stated that for an efficient energy transfer, energy difference (till) between the triplet state of the organic ligand and resonance level of the LnfII ion plays a critical role in this proccss.32 When the energy gap is considerably larger, the energy transfer rate constant decreases due to the re duction of overlap between the donor and the acceptor energy levels in the organic lanthanide complex. There is also a possibility to observe energy back-transfer when the reverse condition happens in a complex, and to prevent such back-transfer processes, the energy gap must be at least I 0 ksT (2000 cm- i). In order to understand the underline chemistry behind the energy transfer process of organic lanthanide compounds, knowledge related to the donor triplet excited state of organic ligand and the accepting energy levels of the Ln(ITI) must be taken into account.33 A : absorption 1u• CT fluorescence - F : P : phosphorescence 'SC ...... 3 .... • Ln. • �• ... Inter-System Crossing (ISC) bt • .,_. Energy Transfer (ET) A F p • • • • Transfert (bt) p • Back Vibrational relaxation \. \; ... Lnl C,. Charge Transfer Figure 1.10 Energy transfer mechanism in lanthanide organic complexcs33 23 1.4.2. Lanthanide complexes 1.4.2.1. DOTA Complexes Magnetic resonance imaging (MRI) has been used for many years, therefore the demand of MRI contrasting agents increases day by day. Among the prevailing contrasting agents, gadolinium(III) chelates are clinically the most commonly used tissue specific marker. Due to the toxicity of free gadolinium(Ill) (LOSO = 0.2 mmol · kg-1 in mice), relatively high concentration of contrast agents is required for MRI scan (up to 0.3 mmol per kg body weight). To maintain a low free lanthanide ion concentration, and to ensure kinetic and thermostatic stability of the gadolinium(III)complexes, strong chelating agents are used.4 Currently, acyclic diethylenetriaminepentaacetic acid (Gd(Ill)-DTPA, Magnevist®, Bayer Healthcare, Berlin,Germany) and the cyclic 1,4, 7, 1O-tetraazacyclododecane- 1,4,7,l0- tetraacetic acid (Gd(III)-DOTA,Dotarem®, Guerbet, Hongkong, China) are the most commonly used contrasting agents for imaging techniques. Most of these complexes are eight coordinated and allow binding of one water molecule directly to the metal center yielding higher stability (logK =25.3 and 20.1 for DOTA and DOTA-propylamide respectively). 34 MRI is a technique with higher special resolution and depth penetration, yet it lacks higher sensitivity. Scientists have done some research work to combine other imaging techniques such as PET (photoinduced electron transfer) probes to enhance the sensitivity of MRI technique. This idea will open the understanding of morphology (MRI) and information about biochemical processes (PET) related to the human body. Optical imaging is another method which lacks spatial resolution and depth penetration but has a good sensitivity. Combining optical probes with DTP A, DOT A such as luminescent metal complexes, 24 luminescent polymers in lipophilic aggregates, dendrimers, and organic dyes (quinolone, 3 tluoresceine,rhodamine, naphalimide) have also improved the sensitivity. 4 BODIPY (4,4- difluoro-4-bora-3a,4a-diaza-s-indacene) is another organic dye that can be tuned to excite in the visible region. BODIPY dyes have comparatively higher absorption coefficientsand fluorescence intensities that save them using as biological labeling. These dyes are stable in biological systems and possess higher quantum yields. In 2015, Tatjana et al. functionalized BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s indacene) dyes by converting primary amines into azides via a copper flow reactor using an in-situ catalyst generated from the metallic copper to synthesize a Gd-DOTA-BODIPY derivative as a bimodal contrast agent for MRI and optical imaging (Figure 1.11.). The absorption spectrum of this adduct shows A.abs(max) at 503 nm in aqueous medium assigned to the So-+S1transition. There was another considerably weak peak around 360 nm, which was due to So-+S2 transition. This Ln-DOTA-BODIPY adduct was water soluble and emitted bright green fluorescence upon excitation at 366 nm. This complex shows excitation maxima at 503 nm and emission maxima at 523 nm. Since the optical properties of the BODIPYdyes can be altered by introducing conjugation via different substituents on BODIPY core, there is a potential to red shift main emission band, which is an advantage for biomedical applications. Tuning this complexes to optical imaging window from 665 to 900 nm can prevent hemoglobin, deoxyhemoglobin, and water absorptions thereby improving the sensitivity. However, care must be taken to mindfullyalter the BODIPY core as introducing more conjugated substituents can increase the hydrophobicity and reduce the solubility of Ln-DOT A-BODIPY complex in aqueous media.35 25 Figure 1.11. Gadolinium(III) DOTA complex functionalized with a BODIPY derivative35 In 2008, by introducing an azathioxanthone sensitizer and a pH dependent alkylsuJfonamide moiety to DOTA, two pH-responsive ratiometric Eu(III) complexes were synthesized (Figure 1.12.). Using 680/589 nm intensity ratio vs pH plot, scientists could determine the local pH within the cells. This emission intensity ratio vs pH plot gives accurate pH measurements within the range of pH 6-8 and allow to record intracellular pH by microscopy. The coordination environment around the Eu(TII) ion has a great impact on the spectral properties, circular polarization of emission and lifetime of these complexes. The pH-dependent ligation of a sulfonamide group associated with large changes in the t:J= 2 (around 620 nm) and !l.f= 4 transitions (680-700 nm). Figure 1.12. pH Dependence of the europium emission spectrum showing the two Eu(TTI) species (lcxc = 380 nm, 298 K, 0.1 M NaCl) 26 In 2011 Simon et al. synthesized bimodal, di metallic lanthanide complexes that bind to DNA (Figure 1.13). Fol.lowing a straightforward method of three consecutive steps two different DOTA-AQ ligands were synthesized. First diamino AQs (aromatic quinones) were converted to bis- chloroacetamides and attached to pre-formed cyclen units. Resulting hexa esters were hydrolyzed by TFA to obtain the free ligands. Di metallic complexes were obtained by the reaction with two equivalents of Ln(OTf)3. Irradiating visible light to the chromophores (ex = 440 nm for Ln2-Ll; A.ex = 400 nm for Ln2-L2), resulted in characteristic lanthanide emissions in the near-IR region.36 Ln2-Ll Ln2-L2 1050 lZSO t4SO 1650 wavelength / nm Figure 1.13. Ln2-Ll and Ln2-L2 Complexes and emission spectra for Ln2-L2 (red- 36 Yb, blue-Nd, green-Er) measured in D20 using .?..ex= 400 nm. 27 1.4.2.2 Porphyrin Complexes As porphyrins form stable lanthanide complexes that show intense emissions in NIR region, they play an important role when using them in medical and photochemical applications. By introducing different substituents in the meso- and/or �-positions of the porphyrin ring change physical and chemical properties of lanthanide porphyrin complexes, which is an advantage for using them in different fields in medicine. Since porphyrins accumulate in various types of tumor micro vessels and cancer cells, many different types of porphyrins have been synthesized. The first Yb-TPP (tetraphenylporphyrin) complex was synthesized in 1974. Then the Yb mesoporphyrine IX complex was synthesized and inserted to apomioglobin. Due to solubility issues with inorganic salts, under quite hard conditions, in the inert atmosphere and high-boiling solvents reactions were carried out with porphyrin macrocycle and ytterbium acetylacetonate for several hours to obtain soluble complexes. Asymmetric tetraarylporphyrin complexes containing ytterbium with three 4- methoxycarbonylphenyl groups, 4-hydroxyophenyl and 4-hydroxy-3-methoxyphenyl radicals or isomeric 3- and 4- pyridyl fragments as the fourth substitute (Figure 1.14.), and by alkyl hydrolysis, corresponding triacids were synthesized. 28 COOCHj HOOC COOH COOCH;! COOH 1. a. R = 4- OH-Ph 2. a. R = 4- OH-Ph b. R =4-Py b. R=4-Py c. R = 3-Py c. R = 3-Py d. R = 4-COOCH3-Ph d. R = 4-COOH-Ph Figure 1.14. Ytterbium tetraarylporphyrin complexes with asymmetrical substitutes A significant difference was observed in the excited state lifetimes of these ytterbium complexes of porphyrin ethers and acids as shown in Table 1.1. According to the results, ytterbium complexes with hydrophobic tri- and tetra-methyl ethers showed higher excited states lifetimes (13-20 µs) than hydrophilic complexes. It is thought that hydrogen bonds formed by hydrophilic ytterbium complexes with water can quench the luminescence of these hydrophilic complexes. Incorporation of a pyridyl fragment led to a decrease the molecular symmetry, thereby lowering the lifetime compared to corresponding ld complex (20 µs).37 29 Table 1.1. Spectral luminescent characteristics of Yb(Iffiporphyrin complexes Compound Luminescence Lifetime (µs) Spectrum Amax nm (DMSO) la Yb( acac )-5-(4-0 H- 982 16.3 Ph) 10, 1 5,20-(4-COOC H3-Ph)3 lb Yb(acac)-5-(4-0H- 983 13.4 Ph) l 0, 1 5,20-(4-COOCH3-Ph )3 le Yb(acac)-5-(3-Py)-10, 1 5,20- 983 14.8 (4-COOCH 3-Ph)3 2a Yb(Im)2-5-(4-0H-Ph)- 982 3 l 0, 1 5,20(4-COOH-Ph )3 2b Yb(Im)2-5-(4-Py)- 10, 15,20-( 4- 976 6-7 COOH-Ph)3 2c Yb(Im)2-5-(3-Py)-l 0, 15,20-(4- 983 5 COOH-Ph)3 Id Yb(acac)-5,10,15,20- (4- 974 20 COOCH3-Ph)4 2d Yb(Jm)2-5,10,15,20- (4- 980 8 COOH-Ph)4 In 2000, Williams et al, synthesized a palladium porphyrin complex which was covalently linked to a chiral lanthanide complex and successfully sensitized NIR emission from Yb(III) and Nd(III) at 529 nm (Figure 1.15.). Enhanced sensitization was observed in the absence of oxygen and in the presence of a nucleic acid. The highest emission was observed in deuterated solvents (a factor of 2.5 for Nd(lll) and a factor of 4 for Yb(III)), revealing OH oscillation alters the sensitivity of the excited ion. Degassed samples of Yb(Ill) and Nd(Ill) emitted at 980 nm and l 064 or 870 nm respectively. The energy transfereffi ciency observed for Nd(llI) is higher than for Yb(III), due to better spectral overlap.30 30 2400I 1IOll •1•eoo 7110 1GO 100 1- 1100 1200 W•,,...ICllhlnm Figure 1.15. Emission spectra for (RRR-R)-YbLI (295 K, 20% D20-CD30D, 5 mM) in degassed solution (top), in aerated solution (bottom) and in the presence of 0 0.1 mM [(CG)6]2,Aex=529 nm3 1.4.2.3. Phenanthroline Complexes 3 Figure 1.16. Lanthanide complexes of 2-thenoyltrifluoroacetonate complex38 Lanthanide complexes of 2-thenoyltrifluoroacetonate complexes (Pr( III), Nd(III), Sm(lll), Eu(III), Dy(III), Ho(III), Er(III), Tm(III), Yb(III)) in an organic-inorganic hybrid material were synthesized by embedding the complexes in a solid matrix via 2-substituted imidazo[4,5-f]-l,l 0-phenanthroline moieties (Figure 1.16.). High resolution luminescence spectra (Figure 1.17., 1.18.) and luminescence decay times were recorded for each complex.38 31 Wavenumber (cm"1) Wavenumber m·1 (c ) 16000 13000 10000 7000 12000 10000 8000 a b - ::> <- � � ·;;; c:: -u .5 LJI 600 800 1000 1200 1400 800 1000 1200 1<400 Figure 1.17. Emission spectra for the covalently linked [Ln(tta)3(phen)] complexes in the TMOS-DEDMS sol-gel bulk sample, A.ex 370 nm. (a) Ln) Sm(III). The transitions start from the 4Gs12 state and end at the 6HJ levels ((J) 6 5/2-15/2 for this spectrum) and the different FJ levels ((J) 5/2-9/2). (b) Ln) Nd(llI). All transitions start from the 4f312 state and end at the 41J levels ((J) 9/2-13/2)38 1150011 000 10500 10000 9500 9000 d 850 900 950 1000 1050 1100 1150 Wavelength (nm) Wavelength (nm) Figure 1.18. Emission spectra for the covalently linked [Ln(tta)3(phen)] complexes in the TMOS-DEDMS sol-gel bulk sample, Aex = 370 nm 32 4 (c) Ln) Er. The transition starts from the 11312 level to the 411s12 level. (d) Ln) Yb. The 2 transition starts from the Fs12 level to the 2F112 level38 [Ln(Por)(BDL)(OAc)] Figure 1.19. Ytterbium(Ill) monoporphyrinate complexes [Ln(Por)(BDL)(OAc)] complexes40 In 2009, He et al performed a study on [Yb(TPP)(OOCCH3)(CH30H)2] and neutral bidentate NN ligands and were able to observe the formation of monoporphyrinate ytterbium(III) complexes (Figure 1.19.).39 In 2010, same research group synthesized eight coordinated erbium(III) and ytterbium(III) monoporphyrinate complexcs([Ln(Por)(BDL)(OAc)]) and hybridized into silica xerogel framework via the coordination of 5-(N,N-bis(3-propyl)ureyl-1 , 10- phenanthroline.40 All the complexes show NIR emission upon exciting with visible light. lo their first work, eight-coordinate ytterbium complexes exhibited longer lifetimes and stronger NIR emission than the seven- coordinate complexes (Table 1.2.). But there was a decrease in the lifetimes after these complexes were combined into organic polymer PMMA. In their second study, lanthanide hybridized films resulted in characteristic NIR emissions at 1538 nm with a lifetime of 0.6 µs and at 980 nm with a lifetime of 6.6 µs, forerbium and ytterbium respectively. There 33 was a 50 to 70% reduction in lifetime compared to corresponding parent complexes in toluene.40-4t Table 1.2. Photophysical data of synthesized complexes in toluene. The excitation wavelength was 532 run. The number in parenthesis is the lifetime Compound Near-infrared (�m, nm) (µs) Reference [Yb(TPP)(OAc)(Phen)] 980 ( 17.29), 1006 40 [Yb(TPP)(0 Ac)( 4-MePhen)] 949, 978 (17.29), I 005 40 [Yb(TPP)(0 Ac)( 4 7-MePhen)] 948, 978 ( 17.82), I 005 40 [Yb(TPP)(OAc )(EPO-Phen)] 948,978 (14.12), 1006 40 [Yb(TClPP)(OAc)(5-NH2-Phen)] 980 (l l.6),1002 41 [Yb(TCIPP)(0Ac)(5-Me-Phen)] 948, 978 (19.37),1006 41 [Er(TClPP)(OAc)(5-NH2-Phen)] 1538 41 [Er(TCIPP)(0Ac)(5-Me-Phen)] 1539 41 [Yb(TPP)(OAc)(DPA)] 950, 975 (1 1.60), 1006,1023 40 1.4.2.4. BODIP Y Based Lanthanide Complexes Though indirect lanthanide sensitization has been done with different ligand binding chromophores, applications of those lanthanide complexes are lacking due to their demand of high excitation wavelengths and emission efficiencies. BODIPY ( 4,4-difluoro-4-bora- 3a,4a-diaza-s-indacene) dyes along with ligand binding groups show remarkable spectral properties with high extinction coefficients ( 110,000 M"1cm-1) and high fluorescence quantum yields (60-90%). To harvest thest! beneficial properties, scientists have 34 functionalized the BODIPY core with different ligand binding groups and studied lanthanide NIR emission capabilities of these BODIPY based lanthanide complexes (Figure 1.20.).10 a b eO MeO Ln(Ll)(NOJ)J Ln(L21 or L22)3(tpy) R==H (L21) R=Br(L22) c d Ln Yb 3 3 I Ln(L3)3 Ln(L4)3 Figure 1.20. BODIPY based lanthanide complexes10.44 Considering the photophysical properties of prevailing BODIPY based lanthanide complexes, all complexes were excited using visible light, but some complexes showed low emission efficiencies in NIR region {Table 1.3.). The Yb(Ll )(N03)3 complex synthesized by Btinzli et al. in 2006 showed lifetimeof 9.7 µs with higher NIR emissions than corresponding Nd(III) and Er(ITI) complexes (Figure 1.20.a), suggesting the higher potential of Yb(III) using as the most suitable lanthanide ions to observe indirect excitation with the LI ligand.42 35 . T a bl e 1 ..3 Ph otop h tys 1cal properties o f BODIPY base d lant ham "d e comp exes Lanthanide Complex Aex Aem (NIR Lifet ime Reference (Excitation) Emission) /µs /nm /nm Er(L l )(N03)3 528 1527 - 43 Nd(L l )(N03)3 528 865 0.2 43 Yb(L 1)(N03) 3 528 978 9.7 43 Er(L21)3(tpy) 583 1530 - 9 Yb(L21)3(tpy) 583 978 - 9 Er(L22)3(tov) 583 1530 - 9 Yb(L22)3(tpy) 583 978 - 9 Er(L3)3 522 1382 - 10 Nd(L3)3 522 1060 - 10 Yb(L3)3 522 976,1003 19.78 10 Yb(L4)3 543 975, 1 000 95.0 44 In 201 1 two different ligands (Ln(L21)3(tpy), R=H and Ln(L22)3(tpy), R=Br) (Figure 1.20., b) were synthesized that altered the 2,6 positions and contained two methoxyphenyl substituents in the 1,7 position in the BODIPY core. Regardless of the incorporation of bromine into the BODIPY core, lanthanide complexes formed by both ligands showed very similar photophysical properties. Further investigations showed no triplet state emissions from Ga(III) for both ligands due to the lack of efficient intersystem crossing because of unfavorable molecular conformations. But, longer wavelengths in the visible range can be used to excite these types of complexes, which is favorable for biological applications.9 The research work done by He and coworkers in 2011 on BODIPY modified 8- hydroxylquinoline ligand (8-HOQ-BODIPY(L3)) (Figure 1.20., c) and the following related work on 8- HOQ-BODIPY-31 (L4)(Figure 1.20., d) showed strong NIR emissions for ytterbium complexes.10· 43 The first work with 8-HOQ-BODIPY ligand yielded stable Yb(III), Nd(III) and Er(III) complexes, however in contrast to ytterbium complex, weak emissions were observed for neodymium and erbium complexes upon excitation at 522 nm10. In their second study, under UV and visible excitation [Yb(8- HOQ-BODIPY-31)3] 36 2 gave a sharp peak at 975 run and a broader peak centering 1000 nm due to typical Fs 2 - 2F112tra nsition for Yb(III) ion.43 The major difference between the iodized and non-iodized ligand containing ytterbium complexes is the NIR lifetimes. The non-exponential fittingof its emission decay curve at 975 nm of iodized complex was 95.0 µs which is much longer than non-iodized complex and clearly shows the heavy atom effect associated with iodine in the L4 ligand that facilitated to yield higher population in ligand triplet state. This longer lifetime of [Yb(8- HOQ-BODIPY-31)3) is favorable for probing and imaging applications in biological systems. LS L6 4445 Figure 1.21. BODlPY based chromophores used to synthesize lanthanide complexes • [n 2013, research work was done to understand the recognition ability of different trivalent lanthanide (La( J rr), Gd(III), Tb(III), Dy( lII), Er(Ill) and Yb(Hl)) ions by calixa[4]crown ether appended BODIPY fluorescent probes (L5) (Figure 1.21.). Ertul et al synthesized this L5 ligand which contained a calix[4]azacrown derivative capped with a two amide bridges with cone conformation bearing two BODIPY groups. The fluorescence intensity change was monitored upon complexation to understand the sensitivity of different 37 lanthanide ions.44 Results revealed that the metal ion binding caused to increase in the florescence intensity of L5 which is due to an intramolecular energy transfer process. In addition, selectivity experiments showed the ability of Yb(III) to recognize L5 was independent and not affected by other lanthanide ions. Therefore, these types of ligands are effectively used as chemical sensors, phase transfer catalysts or ion-binding processes. In 2014 lanthanide metal complexes with L6 (BODIPY with Bispidines) were synthesized for dual imaging purposes (Figure 1.21.).45 Lanthanide ions (Tb(llI), Eu(III), Nd(III)) and BODIPY exhibited emissions enabling dual emission detection in both NIR and visible region respectively. NIR emission for these lanthanide complexes were observed when they were excited at 260 nm suggesting there is no contribution from the BODIPYcore for lanthanide indirect sensitization. The bispidine subunit along sensitizes the lanthanide ions according to Dexter mechanism as T 1 is higher than lanthanide excited state. 1.4.3. Applications of NIR emitting Lanthanides Lanthanides and their complexes are a demanding field of study for the development of different biomedical tools.46 Among them are the development of lanthanide analytical probes as tools for biological labeling, imaging (MRI, optical) and therapy (PDT for cancer). The utilization of lanthanide ions as luminescent probes could show higher potential of successful applications.47 In 2011, Wong et al synthesized a mitochondria specific water-soluble porphyrinato ytterbium complex linked to rhodamine B. This lanthanide complex exhibits strong two (A.em photoninduced NIR emissions = 650 nm, porphyrinate ligand n - n* transition; A.em = 1060 nm, Yb(Ill) 5Fs12 - 5f712 transitions; in DMSO) in aqueous solution with enhanced 38 Yb(III) NIR quantum yield (1% at A.ex= 340 nm; 2.5% at A.ex= 430 nm). Due to the minimal cytotoxicity (ICso = 1.2 mM) and the enhanced NIR emission properties of this ytterbium complex, it is used as a new time-resolved imaging probe enabling excitation and imaging in the NIR region.48 In 2011, Cyclin A specific europium complexes with modified synthetic peptides and chromophores were synthesized by the same research group that demonstrated enhanced europium emission around 560-700 nm upon exciting in the biological window (700 nm- 1000 nm). These lanthanide complexes are being tested as a new generation of bio-tracer for Cyclin A and applicable as potential candidates for both Cyclin A detection and inhibition of the cell division.49 In 2014 Wong et al also did some research work related to a photo selective, functional and Golgi apparatus specific,amphiphilic water-soluble ytterbium complex as a responsive dual probe capable of sensitizing emission within the biological window (660 and 750 nm), producing the singlet oxygen (UD = 0.45) and triggering local cell damage only upon exposure to visible and near-infrared laser excitation. The Yb(III) complex had a low cytotoxicity and highly emissive two-photon induced emission in several carcinoma cell lines along with long resident lifetime with a low dosage requirement, and demonstrated a strong NIR emission in Golgi apparatus enabling it foruse in imaging and photodynamic therapy.50 In 2015, a cisplatin-linked europium-cyclen complex was introduced as a light -responsive antitumor agent. This modified structure controls the release of cisplatin by linear/two photon excitation in vitro. Photo-dissociation of cisplatin leads to detect Eu emission at 615 nm upon excitation at 325 nm, allowing the real-time monitoring of the cisplatin 39 discharge. This allows use of the cisplatin-linked europium-cyclen complex as an antitumor chemotherapeutic agent (prodrug) as well as a luminescence imaging probe.51 In 2017, a porphyrin moiety capable ofkilling tumor cells was presented as a multi-modal lanthanide-porphyrin PDT agent. Strong erbium NIR emission was observed at 654 run, 715 nm, and 1531 nm along with specific affinity forbladder cancer cells by specifically targeting the integrin avb3 isoform. The results obtained by flow cytometry and in vitro imaging proved the selective uptake of complexes by cancer and the ability to interrupt the growth of bladder cancer cells via specific binding to the "integrin avb3 isoform".52 Lanthanide complexes have been synthesized to detect nucleic acid in such roles as heterogeneous DNA analysis, homogeneous assays, intercalation assays, real time PCR assays, and genotyping assays. 53 Eu(III) complexes have been used formany years in DNA analysis assays. Tb(III) and Eu(III) typically exhibit emission lifetimes in the millisecond range, therefore these long lifetimes are useful to detect biomolecules labeled with lanthanide ions with zero background measurements with higher sensitivities.54 Tuning of the emission wavelengths from 520-680 nm and lifetimes from 50-500 µs by introducing larger conjugation systems in the ligand of the lanthanide metal complexes has broadened the horizon of their applications as contrasting agents.34 Dipicolinic acids (DPA) and its derivatives have been demonstrated intense Tb(III) emissions showing useful properties forimmunoassays. Upon excitation at 800 nm, Eu(III) and Tb(Ul) derivatives of dipicolinic acid fuctionalized with polyoxyethylene pendant arms and terminal groups containing complexes show luminescence which is useful to use these as immune probes.55 As Lanthanides possess the ability to bind active sites of proteins and metalloproteins, there is a potential to use these ions to explore the 40 composition and structure of those biomolecules, by obtaining the data related to excitation and emission spectra.56 Tb(III)and Eu(III) are used for this application as they can replace Ca(II), Zn(II), Mg(II), Mn(II), Fe(II), and Fe(III) from some metalloproteins. For the treatment of treatment of age-related macular degeneration (AMD) and for atherosclerotic plaque in coronary heart disease, lutetium texaphyrin was tested as photo activated agent.5 7 Furthermore, texaphyrins complexes were tested for photodynamic therapy as they showed emissions at longer wavelengths (700 nm) facilitating higher tissue penetration. For cancer treatment using radio- and chemo-sensitization applications, a Gd texaphyrin complex, motexafin gadolinium (MGd, Xcytrin) was tested based on its remarkable redox properties. 58 1.5. Motivation To address the major shortcomes related to narrow excitation window, low emission efficiency, low solubility, poor functionality, low sensitivity, and selectivity due to autofluorescence arise from biological environments, and photobleaching by prevailing fluorescent materials, there is a thirst of introducing novel compounds to clinical trials and medical diagnosis purposes with higher functionality. To accomplish above mentioned goal, fu nctionalization of BODIPY dyes to synthesize lanthanide complexes is a solution to come up with better emission efficiencies in NIR region with low background noise. The choice of BODIPY as the chromophore allows to excite the novel complexes in visible region, minimizing photobleaching. Functionalizing the BODIPY core with conjugated ligand binding group enhance the stable complex formation and energy transfer efficiencies. Therefore, research work has been done to synthesize novel BODIPY based DOTA and phenanthroline ytterbium complexes. 41 1.6. Objectives Project 1 • Synthesize and functionalization ofBODIPY based DOTA-ytterbium complexes ./ Synthesize of parent BODIPYdye ./ Functionalization with different lengths of alkyl chains on the meso position of the BODIPY core ./ Combine the BODIPY derivative with DOT A moiety ./ Characterization of final products via 1H NMR and X-ray crystallography data Project 2 • Synthesize and functionalization of BODIPY based phenanthroline-ytterbium complexes ./ Synthesize of parent BODIPY dye ./ Synthesize of 5-amino-( 1,10-phenanthroline) ./ Functionalization of the BODIPY core with phenanthroline ligand binding group ./ Characterization of intermediates and final products via 1 H NMR and X-ray crystallography data • Photophysical studies of BODIPY based phenanthroline ytterbium complexes ./ Record the absorption, excitation, emission spectra of intermediate and final products ./ Obtain the quantum yield, lifetime forintermediate and final products ./ Record the NIR emission of final lanthanide complexes 42 CHAPTER2 EXPERIMENTAL 2.1. General 2.1.1. Materials All reagents and solvents were purchased from commercial sources and employed without further purification unless otherwise stated. The procedure includes analytical grade chemicals that were used as received. Dichloromethane, methanol, hexane, chloroform, acetonitrile, triethylamine, and toluene were purchased from Fisher Chemical Scientific. 2,3-Dichloro-5,6-dicyano-1 ,4-benzoquinone (DDQ) was supplied by Biosynth International, Inc. Tetrahydrofuran (THF), dimethylformamide (DMF), deuterated chloroform, boron trifluoride diethyl etherate, 2,4-dimethyl-3-ethylpyrrole, 4- hydroxybenzaldehyde, anhydrous K2C03, 1,2-dibromoethane and thiophosgene (CSCh) were purchased from ACROS Organics. Anhydrous Na2C03 was purchase from Fisher science education. 1,3-Dibromopropane, and 1,4-dibromobutane, were purchased from Tokyo Chemical Industry Co. Ltd. D03A was purchase from Areva Med.Company. 2- Aminobenzaldehyde and 4-aminobenzaldehyde were purchased from Santa-Cruz Biotechnology Inc. 5-Amino-1,10-phenanthroline was purchased from Aldrich. Acetone was purchased from Sherwin Williams. Nitrogen gas was supplied by Geno Welding (Mattoon, IL). Column chromatography was conducted using 70-230-mesh silica gel. 43 2.1.2. Instruments A 400 MHz Bruker Avance II-NMR spectrometer was used to obtain 1H NMR spectra, using ACROS Organics chloroform-d 99.8% D, containing 0.03% (v/v) TMS. NMR spectra were processed using Bruker's TopSpin software. The chemical shifts were reported in parts per million (ppm). For the signal splitting, the following abbreviations are used: s, singlet; d, doublet; t, triplet; m, multiplet; br, broad peaks; dd, doublet of doublets. UV-Vis absorption spectra were performed on a Cary 100 Series UV-Vis Dual Beam Spectrophotometer over a range of 200-800 nm. Steady state fluorescence spectra were obtained from FS5 fluorimeter (Edinburgh Instruments, Inc.). 44 2.2. Synthesis 2.2.1. Project 1 Synthetic Route A synthetic overview forthe synthesis of the dyes is illustrated below (Scheme 2.1.). The details of the synthesis are presented in sections 2.2. 1.3-2.2.1.9. OH RH3 n• 2.3.• 11•2JOW n•3RHS D•4Rlf6 -l Scheme 2.1. Synthesis ofytterbium complexes ofRH7, RH8, and RH9. 45 2.2.1.1 Synthesis of RHl ( (T-4 )-[ 4-[ (3 ,5-Dimethyl-l H-pyrrol-2-yl-KN)(3 ,5-dimethyl-2H-pyrrol-2-ylidene- KN)methyl]phenolato]difl uoroboron) OH OH 1. CH2Cl2, N2 atmosphere, r.t. + 2 2. BF30Et2 , 3h O- 3. DDQ, lh N 4. NEt3 H 5. BF30Et2 , 2h Figure 2.1. Synthesis of RHl 59 This compound was synthesized according to a previously reported method. Under N2 atmosphere, to a solution of 4-hydroxybenzaldehyde (0.243 g, 2.0 mmol) and 2,4- dimethyl-lH-pyrrole (0.5 mL, 5.0 mmol) in 150.0 mL of CH2Ch, 0.5 mL Bf3·0Eti was added at room temperature. This solution was stirred overnight. Then 2,3-dichloro-5,6- dicyano-1,4-benzoquinone (0.900 g, 4 mmol) was added and stirred for 1 hour. Next triethylamine (6.00 mL, 43.0 mmol) was added to the reaction mixture. After stirring the reaction mixture for 15 minutes, BFrOEti (8.00 mL, 32.0 mmol) was added to it and stirring was continued for 3 hours. Then the solvent was removed under vacuum and the black oily residue was purified using column chromatography on silica with CH2Ch. The isolated product was a bright orange solid with an orange fluorescence. Yield: 0.134 g (0.4 1 b 46 2.2.1.2. Sy nthesis of RH2 ((T-4)-[2-[[4-(3-bromopropoxy)phenyl](3,5-dimethyl-2H-pyrrol-2-ylidene-KN)methyl]-3, 5-dimethyl-l H-pyrrolato-KN]difluoroboron) OH o� Br Cs,C03,DMF r.t,12 h Figure 2.2. Synthesis of RH2 RHl (0.272 g, 0.8 mmol) was dissolved in 50.0 mL of DMF and Cs2C03 (l.02 g, 3.13 mmol) was added to flask and stirring was continued for 15 minutes. Then 1,2- dibromopropane (2.0 mL, 23. 1 mmol) was added to the reaction mixture. This mixture was stirred for 12 hours under N2 atmosphere at room temperature. Then the solvent was removed by vacuum and the orange crude product was purified using column chromatography on silica with CH2Ch. The isolated product was a bright orange solid with an orange fluorescence. Yield: 0.018 g (0.04 mmol, 10.0%). 1H NMR (400 MHz, CDCh): 2Hb), 6Hc), � = 7.20 (d, 2Ha), 7.04 (d, 5.98 (s, 2Hd), 4.35 (t, 2H�, 3.69 (t, 2Hg), 2.55 (s, 1.42 (s, 6Hc), 0.90 (m, 2Hh). 47 2.2.J.3. Sy nthesis of RH3 ((T-4)-[4-[ (4-ethyl-3 ,5-dimethyl- lH-pyrrol-2-yl-KN)(4-et hyl-�,5-dimethyl-2H-pyrrol-2- ylidene-KN)methyl]phenolato]dif luoroboron) OH OH I. CH2Cl2, N2 atmosphere, r.t. 2. BF30Et2 , 3h + 3. DDQ, lh 1 4. NEt3 N b=H 5. BF30Et2 , 2h Figure 2.3. Synthesis of RH3 This compound was synthesized according to a previously reported method. 59 Under N2 atmosphere, to a solution of 4-hydroxybenzaldehyde (0.487 g, 4.0 mmol) and 3-ethyl-2,4- dimethyl-l H-pyrrole (1.10 mL, 8.15 mmol) in 300.0 mL ofCH2Ch, 0.5 mL Bf3·0Etiwas added at room temperature. This reaction mixture was stirred overnight.Then 2,3-dichloro- 5,6-dicyano-1 ,4-benzoquinone (1.80 g, 7.93 mmol) was added and stirred for 1 hour. Next triethylamine (12.00 mL, 86.0 mmol) was added to the reaction mixture. After stirring the reaction mixture for 15 minutes, Bf3.0Et2 (8.00 mL, 32.0 mmol) was added to it and stirring was continued for 3 hours. Then the solvent was removed under vacuum and the black oily residue was purified using column chromatography on silica with CH2Clz. The isolated product was a bright orange solid with an orange fluorescence. Yield: 0.310 g (0.782 mmol, 19.6%). 1H NMR (400 MHz, CDCb): 8 = 7.12 (d, 2Ha), 6.95 (d, 2Hb), 2.52 (s, 6Hf), 2.30 (q, 2Hd), 1.34 (s, 6Hc), 0.98 (t, 6He) 48 2.2. 1.4. Sy nthesis of RH4 ((T-4)-[2-[[4-(2-bromoethoxy)phenyl]( 4-ethyl-3,5-dimethyl-2H-pyrrol-2-ylidene-KN) methyl]- 4-ethyl-3,5-dirnethyl-l H-pyrrolato-KN]difluoroboron) Br OH O� 1,2-Dibromocthanc K2C03, CH3CN 80°C, Sh Figure 2.4. Synthesis of RH4 RH3 (0.162 g, 0.41 mmol) was dissolved in 50.0 mL of CH3CN and anhydrous K2C03 ( 1.01 g, 7 .31 mmol) was added to the flask and stirring was continued for 15 minutes. Then 1,2-dibromoethane (2.0 mL, 23.1 mmol) was added to the reaction mixture. This mixture was stirred for 5 hours under N2 atmosphere at 80°C. Then the solvent was removed by vacuum and the orange crude product was purified using column chromatography on silica with CH2Ch. The isolated product was a bright orange solid with an orange fluorescence. Yield: 0.127 g (0.25 mmol, 61.0%). 1H NMR (400 MHz, CDCb): 8 = 7.20 (d, 2H3), 7.03 (d, 2Hb), 4.36 (t, 2H&), 3.70 (t, 2Hh), 2.53 (s, 6Hr), 2.30 (q, 2Hd), 1.32 (s, 6Hc), 0.98 (t, 6He). 49 2.2.1.5. Syn thesis of RH5 ( (T-4)-[2-[[ 4-(3-bromopropoxy) phenyl]( 4-ethyl-3 ,5-dimethyl-2H-pyrrol-2-ylidene-KN) methyl]- 4-ethyl-3 ,5-dimethyl-I H-pyrrolato-KN]difluoroboron) OH o� Br 1,3-Dibromopropane K2co,. CH3CN 80"C, Sh Figure 2.5. Synthesis of RH5 RH3 (0.161 g, 0.41 mmol) dissolved in 50.0 mL ofCH3CN and anhydrous K1C03 (I.OJ g, 7.31 mmol) were added to flask and stirring was continued for 15 minutes. Then 1,2- dibromopropane (2.4 rnL, 23. l rnrnol)was added to the reaction mixture. This mixture was stirred for 5 hours under N1 atmosphere at 80°C. Then the solvent was removed by vacuum and the orange crude product was purified using column chromatography on silica with CH2Ch. The isolated product was a bright orange solid with an orange fluorescence. Yield: b 0. 110 g (0.21 mmol, 51.2%). 1H NMR (400 MHz, CDCb): o = 7.18 (d, 2H3), 7.01 (d, 2H ), 4.17 (t, 2Hg), 3.65 (t, 2Hh), 2.53 (s, 6Hr), 2.37 (m, 2Hi), 2.30 (q, 2Hd), 1.33 (s, 6Hc), 0.98 50 2.2.J.6. Sy nthesis of RH6 ((T-4)-(2-[[4-(4-bromobutoxy)phenyl]( 4-ethyl-3,5-dimethyl-2H-pyrrol-2-ylidene-KN) methyl]- 4-ethyl-3,5-dimethyl-lH-pyrrolato-KN]difluoroboron) OH I ,4·Dibromobutane K1C03, CH1CN 80"C, Sh Figure 2.6. Synthesis of RH6 RH3 (0.163 g, 0.41 mmol) was dissolved in 50.0 mL of CH3CN and anhydrous K1C03 (l.06 g, 7.67 mmol) was added to flask and stirring was continued for 15 minutes. Then 1,2-dibromobutane (2.0 mL, 16.8 mmol) was added to the reaction mixture. This mixture was stirred for 5 hours under N2 atmosphere at 80°C. Then the solvent was removed by vacuum and the orange crude solid was purified using column chromatography on silica with CH2Ch. The isolated product was a bright orange solid with an orange fluorescence. Yield: 0.126 g (0.24 mmol, 58.5%). 1H NMR (400 MHz, CDCb): 8 = 7.17 (d, 2H3), 7.00 (d, 2Hb), 4.08 (t, 2Hg), 3.52 (t, 2Hh), 2.53 (s, 6H\ 2.30 (q, 2Hd), 2.12 (m, 2Hi), 2.00 (m, 2IP), 1.33 (s, 6Hc), 0.98 (t, 6He). 51 2.2.J.7. Sy nthesis of RH7 0 (°lo1Bu (N"'l ,..,.._.N N'>= 0�Br 0 0 I� 1Bu0r(�N-4uo + 0 Figure 2.7. Synthesis of RH7 D03A (0.185 g, 0.39 mmol) was dissolved in 50.0 mL of CH3CN. Then K2C03 (2.02 g, 14.6 mol) was added to the reaction flask. Next RH4 (0.204 g, 0.41 mmol) was added to the reaction flask and stirred for one day at room temperature. Next day the solvent was removed and the orange color residue was purifiedusing column chromatography on silica. At first CH2Ch was used as the solvent. Then CH2Ch: Methanol (300:20) was used to separate the target product. The isolated product was a bright orange solid with an orange 1 fluorescence. Yield: 0. 150 g (0. 16 mmol, 41 .0%). H NMR (400 MHz, CDCh): o = 7.12 (d, 2H3), 7.08 (d, 2Hb), 4.28 (t, 2Hg), 3.18 (s, 2Hk), 3.07 (t, 2Hh), 2.91 (br, 16Hij), 2.52 (s, 6Hr), 2.30 (q, 2Hd), l.40-1 .46 (m, 27H1), 1.30 (s, 6Hc), 0.98 (t, 6Hc). 52 2.2.1.8. Synthesis of RH8 O�Br + Figure 2.8. Synthesis of RH8 D03A (0.095 g, 0.20 mmol) was dissolved in 50.0 mL of CH3CN. Then K2C03 (2.01 g, 14.6 mol) was added to the reaction flask. Next RHS (0. 125 g, 0.24 mrnol)was added to the reaction flask and stirred for one day at room temperature. Next day the solvent was removed and the orange color residue was purified using column chromatography on silica. At first CH2Ch was used as the solvent. Then CH2Ch: Methanol (300:20) was used to separate the target product. The isolated product was a bright orange solid with an orange 0.088 (0.09 45.0%). 1H (400 MHz, = 7.16 fluorescence. Yield: g mmol, NMR CDCh): () k (d, 2Ha), 6.96 (d, 2Hb), 4.03 (t, 2Hg), 3.70- 3.10 (s, 2Hh), 3.70-2.84 (t, 2H ), 3.21-3.10 (br, m l 6HiJ), 2.53 (s, 6Hf), 2.30 (q, 2Hd), 2.00-2.05 (m, 2H ), 1.44-1.49 (m, 27H1), 1.32 (s, 6Hc), 0.98 (t, 6He). 53 2.2. 1.9. Sy nthesis of RH9 �Br O K2C03 + • CH3CN r.t l day Figure 2.9. Synthesis of RH9 D03A (0.089g, 0. 188 rnmol)was dissolved in 50.0 mL of CH3CN. Then K2C03 (1.04 g, 7.53 mot) was added to the reaction flask. Next RH6 (0. 125 g, 0.24 mmol) was added to the reaction flask and stirred for one day at room temperature. Next day the solvent was removed and the orange color residue was purifiedusing column chromatography on silica. At first CH2Ch was used as the solvent. Then CH2Ch: Methanol (300:20) was used to separate the target product. The isolated product was a red solid with an orange fluorescence. Yield: 0.127 g (0.132 mmol, 70.2%). 1H NMR (400 MHz, CDCb): 8 = 7.16 h ( d, 2H3), 6.97 ( d, 2Hb), 4.01 (t, 2Hg), 3.4 7 (t, 2H ), 3.20-2.81 (s, 2Hk), 3.20-2.81 (br, 16Hij), 2.53 (s, 6Hf), 2.30 (q, 2Hd), 1.79 (m, 2Hm), 1.78 (m, 2H"), 1.44-1.47 (m, 27H1), 1.33 (s, 54 2.2.2. Project 2 Synthetic Route A synthetic overview for the synthesis of the dyes is illustrated below (Scheme 2.2). The details of the synthesis are presented in sections 2.2.2. 1-2.2. J. 7. Scheme 2.2. Project 2, synthesis of ytterbium complexes ofRH12 and RH14 (Complex 1 and Complex 2) SS 2.2.2. 1. Sy nthesis of RHJO ( (T-4 )-[4-[ ( 4-ethyl-3 ,5-dimethyl- l H-pyrrol-2-yl-t I. CH2Cl2. N1 atmosphere, r.t. + 2. BF30Eti , 3h 3. DDQ, lh 2 N 4. NEt3 '0=H 5. BF30Et2 . 2h Figure 2.10. Synthesis ofRHIO This compound was synthesized according to a previously reported method.59 Under N2 atmosphere, to a solution of 4-aminobenzaldehyde (0.244 g, 2.0 mmol) and 3-ethyl-2,4- dimethyl-I H-pyrrole (0.6 mL, 4.4 mmol) in 300.0 mL of CH2Cb, 0.5 mL Bf 3·0Et2 was added at room temperature. This reaction mixture was stirred overnight. Then 2,3-dichloro- 5,6-dicyano-l ,4-benzoquinone (0.90 g, 3.86 mmol) was added and stirred for1 hour. Next triethylamine (6.00mL, 43.0 mmol) was added to the reaction mixture. After stirring the reaction mixture for 15 minutes, 8f3.0Et2 (8.00 mL, 32.0 mmol) was added to it and stirring was continued for 3 hours. Then the solvent was removed under vacuum and the black oily residue was purified using column chromatography on silica with CH2Cb first and then with CH2Ch: Hexane (100: I 0). The isolated product was a bright orange solid with an orange fluorescence. Yield: 0.081 g (0.205 mmol, 10.25%). 1H NMR (400 MHz, b � d CDCh): o = 7.74 (d, 2H3), 7.55 (d, 2H ), 2.54 (s, 6H , 2.33 (q, 2H ), 1.33 (s, 6Hc), 0.99 (t, 56 2.2.2.2. Synthesis of RHJ I (5-Isothiocyanato-l,1 0-phenanthroline) CSC12, Na2C03 Acetone r.t. , ., N2 atmosphere Figure 2.11. Synthesis ofRHl 1 This compound was synthesized according to a previously reported method.60 To a suspension of 5-amino-1,10-phenanthroline (0.204g, 1.0 mmol) in 100.0 mL of acetone, Na2C03 (0.44g, 4.1 mmol) was added. To the same reaction mixture CSCh (0.35 mL, 4.57 mmol) was added and stirring was continued at ambient temperature for 22 �ours under N2 atmosphere. Then the solvent was removed under reduced pressure. A pale-yellow solid was obtained. Yield: 0.201g (84.8%). 1H NMR (400 MHz, CDCb): 8 = 9.33 (dd, lHa), 9.28 (dd, lHh), 8.62 (dd, lHc), 8.28 (dd, lHr), 7.82 (s, lHc), 7.80 (dd, lHb), 7.72 (dd, IHg). 57 2.2.2.3. Sy nthesis of RHI 2 (N- 1, 1 O-phenanthrolin-5-yl-[((T-4)-[ 4-[(4-ethyl-3,5-dimethyl- lH-pyrrol-2-yl-KN)(4- ethyl-3,5-dimethyl-2H-pyrrol-2-ylidene-KN)methyl]-N-phenyl]difluoroboron)thiourea) Acetone, ,. SCN r.t, 3 days + Figure 2.12. Synthesis ofRH12 This compound was synthesized according to a previously reported method.61 To a suspension of RHlO (0.057g, 0. 14 mmol) in 50.0 mL of acetone, RHI 1 (0.053g, 0.22 mmol) was added. The reaction mixture was furtherstirred at ambient temperature for three days. The reaction progress was monitored with TLC. After three days, the solvent was u removed and the final product purified on a col mn chromatography (silica gel, CH2Ch: CH30H = 10:1, v/v) to give BDP as bright orange powder. Yield: 0.058 g, 65.5%. 1H NMR (400 MHz, CDCb): 9.12 (dd, lHi), 9.01 (dd, 1H0), 8.54 (dd, lHm), 8.23 (dd, lHk), 7.99 (s, 1H1), 7.75 (dd, IH"), 7.73 (dd, lHj), 7.67 (d, 2H3), 7.65 (d, 2Hb), 2.52 (s, 6H�, 2.29 (q, 2Hd), l.30 (s, 6Hc), 0.96 (t, 6He). 58 2.2.2.4. Sy nthesis of RHI 3 ((T-4 )-(2-( ( 4-ethyl-3 ,5-dimethyl-lH-pyrrol-2-yl-KN)(4-ethyl-3 ,5-dimethyl-2H-pyrrol-2- ylidene-KN)methyl]-benzenaminato ]difluoroboron) !. Cfi:iCl2.N2 atmosphere,r.t. 2. BF;f?Et2,3h 3. DDQ,lh 4.NE.t:3 5. BFJ0Eli . 2h Figure 2.13. Synthesis ofRHI3 This compound was synthesized according to a previously reported method.59 Under N2 atmosphere, to a solution of 2-aminobenzaldehyde (0.244 g, 2.0 mmol) and 3-ethyl-2,4- dimethyl-I H-pyrrole (0.6 mL, 4.4 mmol) in 300.0 mL of CH2Ch, 0.5 mL Bf 3.0Eti was added at room temperature. This reaction mixture was stirred for overnight. Then 2,3- dichloro-5,6-dicyano-l,4-benzoquinone (0.90 g, 3.86 mmol) was added and stirred for 1 hour. Next triethylamine (6.00mL, 43.0 mmol) was added to the reaction mixture. After stirring the reaction mixture for 15 minutes, Bf3·0Eti (8.00 mL, 32.0 mmol) was added to it and stirring was continued for 3 hours. Then the solvent was removed under vacuum and black oily residue was purified using column chromatography on silica with CH2Ch first and then with CH2Ch: Hexane ( 100: 10). The isolated product yielded as a bright orange solid with an orange fluorescence. Yield: 0.072 g, (0.18 mmol, 9 .1%). 1H NMR ( 400 MHz, b d CDCb): o = 7.25 (d, lHa), 7.03 (d, lH ), 6.88 (t, lHc), 6.7 (d, IH ), 2.55 (s, 6Hh), 2.33 (q, 3Hr), 1.41 (s, 6He), 1.03 (t, 6Hg). 59 2.2.2.5. Synthesis of RHJ4 (N-1,1 O-phenanthrolin-5-yl-[ ((T-4)-[2-((4-ethyl-3 ,5-dimethyl- l H-pyrrol-2-yl-KN)(4- ethyl-3,5-dimethyl-2H-pyrrol-2-ylidene-KN)methyl]-N-phenyl]difluoroboron)thiourea) Acetooe, r.t, 3 days Figure 2.14. Synthesis of RH14 This compound was synthesized according to a previously reported method.61 To a suspension ofRHl3 (0.15g, 0.38 mmol) in 50.0 mL of acetone, RHl l (O.lOg, 0.42 mmol) was added. The reaction mixture was further stirred at ambient temperature for three days. The reaction progress was monitored with TLC. After three days, the solvent was removed and the final product was purified on a column chromatography (silica gel, CH2Ch: CH30H = 10: 1, v/v) to give BDP as bright orange powder. Yield: 0.042 g, 17.5%. 1H NMR (400 MHz, CDCb): 8 = 9.22 (t, 2HP,q), 8.40 (dd, lHi), 8.35 (dd, 1H0), 8.28 (dd, lHm), 7.77 {s, lHk), 7.69 (m, 1H0), 7.68 (m, lHj), 7.62 (s, 1H1), 7.53 (d, lH3), 7.49 (s, lHb), 7.38 (dd, lHc), 7.12 (dd, lHd), 2.30 (s, 6Hh), 2.01 (q, 2Hr), 1.05 (s, 6He), 0.76 (t, 6Hg). 60 2.2.2.6. Sy nthesis of Complex 1 Figure 2.15. Synthesis of Complex I To a suspension of RH12 (0.010 g, 0.016 mmol) m 25.0 mL toluene, [Yb(TPP)(OAc)(MeOH)2] (0.014 g, 0.015 mmol) was added. The reaction mixture was stirred at ambient temperature overnight. The solvent was removed and the final product was purified on a column chromatography (silica gel, first with CH2Ch: then CH30H = 100:1, v/v) to give dark purple powder. Yield: 0.023 g, 86. l l %. 61 2.2.2. 7. Sy nthesis of Complex 2 Figure 2.16. Synthesis of Complex 2 To a suspension of RH14 (0.004 g, 0.006 mmol) m 25.0 mL toluene, [Yb(TPP)(OAc)(MeOH)2] (0.006 g, 0.006 mmol) was added. The reaction mixture was stirred at ambient temperature, overnight. The solvent was removed and the final product was purified on a column chromatography (silica gel, first with CH2Ch: then CH30H = l 00: 1, v/v) to give dark purple powder. Yield: 0.005 g, 87.68%. 2.3. Photophysical measurements 2.3.1 UV-VIS Absorption spectra in solutions Absorption spectra for synthesized ligands and complexes were measured using HP Agilent 5439 UV-Visible spectrometer with 1.0 cm quartz cell. The stock solution concentration of the samples was 2.0x I 0-4 M, and 20 µL of each was added to 2.5 mL of dichloromethane in a 1.0 cm quartz cuvette. The spectra were recorded from 400-800 nm. The final concentration of each dye was l .6x 10 6 M. The final concentr�tion for each dye 62 in the cuvette (C2) was determined fust by determining the concentration of the stock solution (M (stock)= C 1 (stock)) using the molarity formula, M (stock)= C1 (stock) = Moles of dye/ Liters of solvent where C1 is the concentration of the stock solution, V1 is the volume of the stock solution added, and V2 is the total volume in the cuvette. The molar extinction coefficient was calculated for each ligand and complex using Beer Lambert law, A=ccl where A is the maximum absorption of ligand or the complex, E is absorption coefficient, c is the analyte concentration and l is the path length of the incident light. 2.3.2. Fluorescence Spectra 2.3.2.J. Excitation and Emission Sp ectra Excitation and emission spectra for RH12 and RH 14 were recorded using steady-state excitation with a xenon arc lamp. Both RH12and RH14 ligands were excited at 500 nm and data was collected from 450-575 nm range to obtain the emission spectra. The excitation spectra were recorded by setting the emission at 540 nm and spectra were recorded from 510-700 range. Concentrations of both ligands were kept the same as l .6x 1 o-6 M in the cuvette. 63 2.3.2.2. Quantum yield measurements The quantum yield of ligands was evaluated according to the below equation. 0x = 0sT r Gradx] r�12 GradsTe nsT where 0x is the fluorescence quantum yield of compound, 0sT is the standard fluorescence quanttim yield (0x= 0.95, A.ex =480 nm for rhodamine 6G), Gradx is the gradient from the plot of the integrated fluorescence intensity vs absorbance of ligands with different concentrations, GradsT is the gradient from the integrated fluorescence intensity vs absorbance with different rhodamine 6G standard in ethanol, nx and nsT are refractive indexes of the solvents used to dissolve ligands and standard rhodamine 6G samples, respectively. Initial concentration of rhodamine 6G was 3 .34 x 104 M. A stock solution of rhodamine 6G with 3.34 x 104 M concentration (in ethanol) was prepared and 0, 50, 100, 150, 185 and 225 µL of it was added to 2.5 mL of CH2Ch in a cuvette. For each sample, both absorption (400-800 nm range) and fluorescence emission spectra(excited at 480 nm, emission measured at 500-700 nm range, 1 repeat, 0.5 dwell time, 1.0 emission and excitation bandwidth) were recorded separately. Standard 10 mm path length fluorescence cuvette was used for the measurements so that above mentioned volumes were chosen below 0.1 absorbance at 480 nm. The quantum yields of RH12 and RH14 ligands were measured using above method. A stock solution ofRH12 with 2.16 x 104M concentration (20.00 mL in CH2Ch) was used to determine the quantum yield. 10, 20, 40, 60, 80, 90 µL volumes from the stock solution were added to 2.5 mL of CH2Ch in a fluorescence cuvette. RH14 with 1.93 x 10·4 M 64 concentration stock solution (20.00 mL in CH2Ch) was prepared and 20, 45, 70, 95, 120 µL were added from the stock solution into 2.5 mL of CH2Ch in a fluorescence cuvette and absorption and emission spectra were recorded with above mentioned parameters. Using an integrated fluorescence intensity vs absorbance graphs for standard rhodamine 6G and ligands, quantum yields were calculated substituting the gradients of the graphs to above mentioned equation. 2.3.3. Lifetime measurements 2.3.3.J. Lifetime measurement in the visible region Luminescence decay curves were generated using pulsed laser excitation by a laser diode EPL 375 (Edinburgh Instruments, Inc) with 375 nm light source. Decay curves of the sample in the visible region were obtained by a time-correlated single photon counting technique (TCSCT). The lifetimes were measured by exponential fitting of the deconvoluted decay data and tail fitting of the data for visible emission. The lifetime of RH 12 was measured at 537 nm while RH14 lifetime was measured at 549 nm. 2.3.3.2. Lifetime measurement in the NIR region NIR decay curves were acquired using an optical parametric oscillator (OPTEK Opelette) as an excitation source. NIR emission was detected using a 0.3 m flat fieldmonochromator (Jobin Yvon TRIAX 320) equipped with a NIR-sensitive photomultiplier tube (Hamamatsu R2658P). 2.3.4 Spectroscopic titration The spectroscopic titration curves in the visible and NIR region were obtained by titrating RH12 and RH14 solutions with [Yb(TPP)(OAc)(MeOH)2] solution in CH2Ch to 65 understand the stoichiometry of the ligand and Yb(III) ion. The initial concentration of [Yb(TPP)(OAc)(MeOH)2] was 2.34 x 10-4M. The initial concentrations of RH12 and RH 14 were 2.16 x 10-4 M and 1.93 x 10-4 M in CH2Ch respectively. The titrations were carried out until the molar ratio of Yb(III) ions to ligand becomes 1:I. For both ligands spectroscopic titrations were done in two different methods. First the stoichiometry was determined, titrating the ligand ( 100 µL from the stock solution of ligand was added to 2.5 mL of CH2Ch in a cuvette) by Yb(III) ions, by adding 0, 20, 40, 60, 80, 100 µL from the stock solution. Secondly, back titrations were done, titrating Yb(III) ions (100 µL from the stock solution of [Yb(TPP)(OAc)(MeOH)2] was added to 2.5 mL of CH2Ch in a cuvette) with the ligand, by adding 0, 20, 40, 60, 80, 100 µL from the stock solutions of RH 12 and RH14. These two approaches were used to confirm the stoichiometry between the ligand and the Yb(III) ion in both complexes. 66 CHAPTER 3 RESULTS AND DISCUSSION 3.1. Characterization 3.1.1. NMR Spectroscopy 3.1.1.1. 1H NMR of Compound RHJ OH d = b 1H NMR (400 MHz, CDCb): 8 7.14 (d, 2H8), 6.95 (d, 2H ), 5.97 (s, 2H ), 2.55 (s, 6He), 1.44 (s, 6Hc). The 1H NMR spectrum for RHl is illustrated in the appendix (Figure A. l ). Five major peaks were produced. The phenol substituent's aromatic protons, labeled a and b, resulted in the most deshielded peaks at 7.14 ppm and 6.95 ppm, respectively. The two aromatic protons on the pyrrole groups, labeled d, produced a singlet at 5.97 ppm. The methyl groups bound to the pyrrole adjacent to the nitrogen atoms, labeled e, produced a singlet at 2.55 ppm. The second set of methyl groups on the BODIPY core, labeled c, appeared as a singlet at 1.44 ppm. 67 3.1.1.2. 1H NMRof Compound RH2 o�f I h e 1 b ct f H NMR (400 MHz, CDCb): 8 = 7.20 (d, 2H3), 7.04 (d, 2H ), 5.98 (s, 2H ), 4.35 (t, 2H ), 3.69 (t, 2Hg), 2.55 (s, 6He), 1.42 (s, 6Hc), 0.90 (m, 2Hh) The 1H NMR spectrum for RH2 is illustrated in the appendix (Figure A.2). Eight major peaks were produced. The phenol substituent's aromatic protons, labeled a and b, resulted in the most deshielded peaks at 7.20 ppm and 7.04 ppm, respectively. The two aromatic protons on the pyrrole groups, labeled d, produced a singlet at 5.98 ppm. Protons in the three-carbon alkyl chain labeled as f, g and h appeared at 4.35 ppm, 3.69 ppm and 0.90 ppm respectively. Two triplets were observed corresponding to -CH2 protons bound to oxygen and bromine which are appeared more downfield due to the neighboring electro- negative atoms. The methyl groups bound to the pyrrole adjacent to the nitrogen atoms, labeled e, produced a singlet at 2.55 ppm. The second set ofmethyl groups on the BODIPY core, labeled c, appeared as a singlet at 1.42 ppm. 68 3. l.l.3. 1H NMRofCompoundRH3 OH e 1.34 (s, 6Hc), 0.98 (t, 6He). The 1H NMR spectrum for RH3 is illustrated in the appendix (Figure A.3). Six major peaks were produced. The phenol substituent's aromatic protons, labeled a and b resulted in the most deshielded peaks at 7.12 ppm and 6.95 ppm, respectively. The methyl groups bound to the BODIPY core adjacent to the nitrogen atoms, labeled as f, experienced a downfield shift to 2.52 ppm. The quartet at 2.30 ppm corresponds to the -CH2 in the ethyl group bound to BODIPY core. At 1.34 a singlet appeared which corresponds to the other methyl group in the BODIPY core. The most shielded -CH3protons in the ethyl group bound to BODIPY core appeared at 0.98 ppm. 69 3. 1.1.4. 1H NMR of Compound RH4 g e 1H NMR (400 MHz, CDCh): 8 = 7.20 (d, 2Ha), 7.03 (d, 2Hb), 4.36 (t, 2Hg), 3.70 (t, 2Hh), 2.53 (s, 6Hr), 2.30 (q, 3Hd), 1.32 (s, 6Hc), 0.98 (t, 6He). The 1 H NMR spectrum for RH4 is illustrated in the appendix (Figure A.4). Eight major peaks were produced. The phenol substituent's aromatic protons, labeled a and b resulted in the most deshielded peaks at 7.20 ppm and 7.03 ppm, respectively. The two-carbon alkyl chain linked to the phenolic group produced two signals. One signal with a triplet was observed at 4.36 ppm corresponding to -CH2 protons bound to oxygen and the other one is at 3. 70 ppm which is bound to the bromine atom in the other end. The methyl groups bound to the BODIPY core adjacent to the nitrogen atoms, labeled as f, experienced a downfield shift to 2.53 ppm. The quartet at 2.30 ppm corresponds to the -CH2 in the ethyl group bound to BODIPY core. At 1.32 ppm, a singlet appeared which corresponds to the other methyl group in the BODIPY core. The most shielded -CH3 protons in the ethyl group bound to BODIPY core appeared at 0.98 ppm. 70 3.1.1.5. 1H NMRof Compound RH5 1 b h H NMR (400 MHz, CDCb): o = 7.18 (d, 2Ha), 7.01 (d, 2H ), 4.17 (t, 2Hg), 3.65 (t, 2H ), 2.53 (s, 6Hf), 2.37 (m, 2Hi), 2.30 (q, 3Hd), 1.33 (s, 6Hc), 0.98 (t, 6He). The 1H NMR spectrum for RH5 is illustrated in the appendix (Figure A.5). Nine major peaks were produced. The phenol substituent's aromatic protons, labeled a and b resulted in the most deshielded peaks at 7.18 ppm and 7.01 ppm, respectively. The three-carbon alkyl chain linked to the phenolic group produces three signals. One signal with a triplet was observed at 4.17 ppm corresponding to -CH2 protons bound to oxygen and the other one is at 3.65 ppm which is bound to the bromine atom in the other end. The middle two protons in the three-carbon alkyl chain appeared at 2.37 ppm. The methyl groups bound to the BODIPY core adjacent to the nitrogen atoms, labeled as f, experienced a downfield shift to 2.53 ppm. The quartet at 2.30 ppm corresponds to the -CH2 in the ethyl group bound to BODIPY core. At 1.33 ppm, a singlet appeared which corresponds to the other methyl group in the BODIPY core. The most shielded -CH3 protons in the ethyl group bound to BODIPY core appeared at 0.98 ppm. 71 3. 1.1.6. 1H NMRof Compound RH6 o�g j Br i h e f = h 1H NMR (400 MHz, CDCb): 8 7.17 (d, 2Ha), 7.00 (d, 2Hb), 4.08 (t, 2H&), 3.52 (t, 2H ), 2.53 (s, 6Hr), 2.30 (q, 3Hd), 2.12 (m, 2Hi), 2.00 (m, 2Hi), 1.33 (s, 6Hc), 0.98 (t, 6He). The 1 H NMR spectrum for RH6 is illustrated in the appendix (Figure A.6). Ten major peaks were produced. The phenol substituent's aromatic protons, labeled a and b resulted in the most deshielded peaks at 7.17 ppm and 7.00 ppm, respectively. The four-carbon alkyl chain linked to the phenolic group produced four signals. One signal with a triplet was observed at 4.08 ppm corresponding to -CH2 protons bound to oxygen and the other one is at 3.52 ppm which is bound to the bromine atom in the other end. The other four protons in the four-carbon alkyl chain appeared at 2.12 ppm and 2.00 ppm. The methyl groups bound to the BODIPY core adjacent to the nitrogen atoms, labeled as f, experienced a downfield shift to 2.53 ppm. The quartet at 2.30 ppm corresponds to the -CH2 in the ethyl group bound to BODIPY core. At 1.33 ppm, a singlet appeared which corresponds to the other methyl group labeled as c in the BODIPY core. The most shielded-CH3 protons in the ethyl group bound to BODIPY core appeared at 0.98 ppm. 72 3. 1. 1. 7. / H NMRof Compound RH7 b 1 H NMR (400 MHz, CDCb): 8 = 7. 12 (d, 2Ha), 7.08 (d, 2H ), 4.28 (t, 2Hg), 3.18 (t, 2Hk), . . 3.07 (t, 2H b ), 2.91 (br, 16H1J), 2.52 (s, 6Hf ), 2.30 ( q, 3Hd ), 1.40-1.46 (s, 27H I ), 1.30 (s, 6Hc), 0.98 (t, 6He). The 1H NMR spectrum for RH7 is illustrated in the appendix (Figure A.7). The phenol substituent's aromatic protons, labeled a and b resulted in the most deshielded peaks at 7 .12 ppm and 7 .08 ppm, respectively. The two-carbon alkyl chain linked to the phenolic group produced two signals. One signal with a triplet was observed at 4.28 ppm corresponding to -CH2 protons bound to oxygen and the other one is at 3.07 ppm which bound to the nitrogen atom in the DOTA moiety. The other protons in the DOTA moiety appeared at 3.18 ppm, 2.91 ppm and 1.40-1.46 ppm labeled as k, i, j and I. The methyl groups bound to the BODIPY core adjacent to the nitrogen atoms, labeled as f, experienced a downfield shift to 2.52 ppm. The quartet at 2.30 corresponds to the -CH2 in the ethyl group bound to BODIPY core labeled as d. At 1.30 ppm, a singlet appeared which corresponds to the other methyl group in the BODIPY core labeled as c. The most shielded 73 -CH3 protons in the ethyl group bound to BODIPY core appeared at 0.98 ppm represented as e. 3.1.1.8. 1H NMR of Compound RHB 1 H NMR (400 MHz, CDCb): o = 7.16 (d, 2Ha), 6.96 (d, 2Hb), 4.03 (t, 2Hg), 3.70- 3.10 (t, 2Hh), 3.70-2.84 (t, 2Hk), 3.21-3.10 (br, 16Hij), 2.53 (s, 6Hr), 2.30 (q, 3Hd), 2.00 (m, 2Hm), 1.44-1 .49 (s, 27H1), 1.32 (s, 6Hc), 0.98 (t, 6He). The 1H NMR spectrum for RH8 is illustrated in the appendix (Figure A.8). The phenol substituent's aromatic protons, labeled a and b resulted in the most deshielded peaks at 7 .16 ppm and 6.96 ppm, respectively. The three-carbon alkyl chain linked to the phenolic group produced three signals. One signal with a triplet was observed at 4.03 ppm corresponding to -CH2 protons bound to oxygen and the other one is at 3.7-3.10 ppm range which bound to the nitrogen atom in the DOT A moiety. The more shielded protons between g and h protons labeled as m, appeared at 2.0 ppm as a multiplet. The other protons in the DOTA moiety appeared at 3.70-2.84 ppm, 3.2 1-3.10 ppm range and 1 .40-1 .46 ppm labeled as k, i,j and l which are quite difficultto assign due to peak overlapping. The methyl groups 74 bound to the BODIPY core adjacent to the nitrogen atoms, labeled as f, experienced a downfieldshift to 2.53 ppm. The quartet at 2.30 corresponds to the -CH2 in the ethyl group bound to BODIPY core labeled as d. At 1.32 ppm, a singlet appeared which corresponds to the other methyl group in the BODIPY core labeled as c. The most shielded -CH3 protons in the ethyl group, bound to BODIPY core appeared at 0.98 ppm represented as e. 3.1.J.9. 1 H NMR of Compound RH9 b 1H NMR (400 MHz, CDCb): o = 7. 16 (d, 2H3), 6.97 (d, 2H ), 4.01 (t, 2Hg), 3.47 (t, 2Hh), 3.20-2.81 (t, 2Hk), 3.20-2.81 (br, 16Hij), 2.53 (s, 6H�, 2.30 (q, 3Hct), l .79 (m, 2Hm), 1.78 (m, 2H"), 1.44-1.47 (s, 27H1), 1.33 (s, 6Hc), 0.98 (t, 6He). The 1H NMR spectrum for RH9 is illustrated in the appendix (Figure A.9). The phenol substituent's aromatic protons, labeled a and b resulted in the most deshielded peaks at 7.16 ppm and 6.97 ppm, respectively. The four-carbon alkyl chain linked to the phenolic group produced four signals. One signal with a triplet was observed at 4.01 ppm corresponding to -CH2 protons bound to oxygen and the other one is at 3.47 ppm which 75 bound to the nitrogen atom in the DOTA moiety. More shielded four protons between g and h protons labeled as m and n appeared at 1.79 and 1.78 respectively as multiplets. The other protons in the DOTA moiety appeared at 3.20-2.81 ppm, and 1.40-1.46 ppm labeled as k, i, j, and I. The methyl groups bound to the BODIPY core adjacent to the nitrogen atoms, labeled as f, experienced a downfield shift to 2.53 ppm. The quartet at 2.30 corresponds to the -CH2 in the ethyl group bound to BODIPY core labeled as d. At 1.33 ppm, a singlet appeared which corresponds to the other methyl group in the BODIPY core labeled as c. The most shielded -CH3 protons in the ethyl group bound to BODIPY core appeared at 0.98 ppm represented as e. 3. 1.1.10. 1H NMR a/ Compound RHJO 1 b r), 1.33 (s, 6Hc), 0.99 (t, 6He). The 1 H NMR spectrum for RHIO is illustrated in the appendix (Figure A. l 0). Six major peaks were produced. The phenyl protons, labeled a and b resulted in the most deshielded peaks at 7.74 ppm and 7.55 ppm, respectively. The methyl groups bound to the BODIPY core adjacent to the nitrogen atoms, labeled as f, experienced a downfield shift to 2.54 ppm. 76 The quartet at 2.33 ppm corresponds to the -CH2 in the ethyl group bound to BODIPY core. At 1.33 ppm, a singlet appeared which corresponds to the other methyl group in the BODIPY core. The most shielded -CH3 protons in the ethyl group labeled as e, bound to BODIPY cum appeared at 0.99 ppm. 3. 1.1.11. 1H NMR a/ Compound RH11 e f I h 1 h H NMR (400 MHz, CDCb): 8 = 9.33 (dd, lH3), 9.28 (dd, lH ), 8.62 (dd, lHc), 8.28 (dd, IH�, 7.82 (s, lHe), 7.80 (dd, lHb), 7.72 (dd, IHlt). The 1H NMR spectrum for RHI 1 is illustrated in the appendix (Figure A.11). Seven signals were observed in this spectrum corresponding to protons in 2-9 positions in 1,10- phenanthroline. The most deshielded protons were observed at 9.33 ppm and 9.28 ppm represent 2 and 9 positions of phenanthroline. These are closer to the nitrogen atoms present in the phenanthroline group. Protons labeled as c and f were the next most deshielded protons corresponding 4 and 7 positions of the phenanthroline group appeared at 8.62 and 8.28. A singlet appeared at 7 .82 ppm labeled as e corresponds to the proton closer to -NCS group. Two protons in 3 and 8 positions of phenanthroline labeled as b and g appeared at 7.80 ppm and 7.72 ppm respectively. 77 3.1.1.12. 1H NMR of Compound RH12 I 1H NMR (400 MHz, CDCh): 9.12 (dd, !Hi), 9.01 (dd, 1H0), 8.54 (dd, lHm), 8.23 (dd, I Hk), 7.99 (s, lH1), 7.75 (dd, l H"), 7.73 (dd, I Hj), 7.67 (d, 2H3), 7.65 (d, 2Hb), 2.52 (s, 6H\ 2.29 d (q, 3H ), 1.30 (s, 6Hc), 0.96 (t, 6Hc). The 1H NMR spectrum for RH12 is illustrated in the appendix (Figure A.12). Thirteen major peaks were produced. Seven signals were observed in this spectrum corresponding to protons in 2-9 positions in I,10 -phenanthroline. The most deshielded protons were observed at 9.12 ppm and 9.01 ppm represent 2 and 9 positions of phenanthroline labeled as i and o in the spectrum. Protons labeled as m and k were the next most deshielded protons corresponding 4 and 7 positions of the phenanthroline group appeared at 8.54 ppm and 8.23 ppm respectively. A singlet appeared at 7.82 ppm labeled as 1 corresponds to the proton closer to -NH group in the bridge between the BODIPY core and the phenanthroline group. Two protons in 3 and 8 positions of phenanthroline labeled as n and j appeared at 7.75 ppm and 7.73 ppm respectively. The phenyl protons, labeled a and b resulted in the downfield at 7 .67 ppm and 7 .65 ppm, respectively. The methyl groups bound to the 78 BODIPY core adjacent to the nitrogen atoms, labeled as f, experienced a downfield shift to 2.52 ppm. The quartet at 2.29 ppm corresponds to the -CH2 in the ethyl group bound to BODIPY core. At 1.30 ppm, a singlet appeared which corresponds to the other methyl group in the BODIPY core labeled as c in the spectrum. The most shielded -CH3 protons in the ethyl group labeled as e bound to BODIPY core appeared at 0.96 ppm. 3. I. l. I 3. 1 H NMR of Compound RH13 b d 1H NMR (400 MHz, CDCb): o = 7.25 (d, lHa), 7.03 (d, lHb), 6.88 (t, lHc), 6.7 (d, lH ), 2.55 (s, 6Hh), 2.33 (q, 3H�, 1.41 (s, 6He), 1.03 (t, 6Hg). The 1H NMR spectrum for RH13 is illustrated in the appendix (Figure A.13). Eight major peaks were produced. The phenyl protons, labeled a, b, c, and d resulted in the most deshielded peaks at 7.25 ppm, 7.03 ppm, 6.88 ppm, and 6.7 ppm respectively. The methyl groups bound to the BODIPY core adjacent to the nitrogen atoms, labeled as f, experienced a downfield shift to 2.55 ppm. The quartetat 2.33 ppm corresponds to the -CH2 in the ethyl group bound to BODIPY core. At 1.41 ppm, a singlet appeared which corresponds to the other methyl group in the BODIPY core. The most shielded -CH3 protons in the ethyl group, bound to BODIPY core appeared at 1.03 ppm. 79 3.1.1.14. 1H NMR of Compound RH14 h k j i CDCb): 1H NMR (400 MHz, o = 9.22 (t, 2HM), 8.40 (dd, I Hi), 8.35 (dd, IH0), 8.28 (dd, lHm), 7.77 (s, lHk), 7.69 (m, lH"), 7.68 (m, lHj), 7.62 (s, 1H1), 7.53 (d, lH3), 7.49 (s, lHb), d h 7.38 (dd, I He), 7.12 (dd, lH ), 2.30 (s, 6H ), 2.01 (q, 3H1, 1.05 (s, 6He), 0.76 (t, 6Hg). The 1H NMR spectrum for RH 14 is illustrated in the appendix (Figure A.14). Sixteen major peaks were produced. The most deshielded protons were observed at 9.22 ppm corresponding to two -NH groups present in the ligand. Seven signals were observed in this spectrum corresponding to protons in 2-9 positions in l,10 -phenanthroline. The deshielded protons observed at 8.40 ppm and 8.35 ppm represent 2 and 9 positions of phenanthroline labeled as i and o in the spectrum. Protons labeled as m and k were the next most deshielded protons corresponding 4 and 7 positions of the phenanthroline group appeared at 8.28 ppm and 7.77 ppm respectively. Two protons in 3 and 8 positions of phenanthroline labeled as n and j appeared at 7.69 ppm and 7.68 ppm respectively. A singlet appeared at 7.62 ppm labeled as I corresponds to the proton closer to -NH group in 80 the bridge between the BODIPY core and the phenanthroline group. The amme substituent's aromatic protons in the BODIPY core, labeled a, b, c, and d resulted in down field at 7.53 ppm, 7.49 ppm, 7.38 ppm, and 7.12 ppm respectively. The methyl groups bound to the BODIPY core adjacent to the nitrogen atoms, labeled as h, experienced a downfield shift to 2.30 ppm. The quartet at 2.01 ppm labeled as f corresponds to the -CH2 in the ethyl group bound to BODIPY core. At 1.05 ppm, a singlet appeared which labeled as e corresponds to the other methyl group in the BODIPY core. The most shielded -CH3 protons in the ethyl group labeled as g bound to BODIPY core appeared at 0.76 ppm. 81 3.1.2 X-Ray Crystallography Using slow evaporation technique, single crystals of the BODIPY ligands (RH4, RH5, RH6, RHIO, RH13) were obtained. Dichloromethane-methanol solvent system was used to crystalize the ligands at room temperature. The crystals were mounted on glass fibers before data collection. Diffraction measurements were made on a CCD-based commercial X-ray diffractometer using Cu-Ka radiation ().. = 0.15418 A 0). The obtained single crystal structures of RH4, RH5, RH6, RHIO and RH13 are illustrated Figure 3.1 and Figure 3.2 below. The structural parameters of five BODIPY ligands are listed in Table 3.1 and Table 3.4, and selected bond lengths and bond angles are shown in Table 3.2, Table 3.3, Table 3.5, and Table 3.6. Comparing the crystal structures of RH4, RH5 and RH6, it is noteworthy that RH4 has a triclinic crystal system with P-1 space group while RH5 and RH6 both possess monoclinic crystal system with P21 and P2i/c space groups respectively. The R values were below 5% for both RH4 and RH6 crystalsindicating the good quality ofrefinement for both RH4 and RH6 crystals, but R value was just above 5% for RH5 (5.3%) which was still in the acceptable level. Two different conformation isomers were observed for RH4 and four different conformation isomers were observed for RH5 while only one conformation structure was observed for RH6. The ratio of number of reflections/number of parameters for RH4, RH5 and RH6 were 14.25, 14.5 and 15.28 respectively. These values further confirmed the quality of data were in a good level. In RH4 crystal structure one conformational isomer showed disorder around the bromine atom. Solvent molecule (CH2Ch) was also present in RH4 crystal structure. 82 (a) (b) F2 (c) Figure 3.1. OR TEP diagrams of the single-crystal structures of (a) RH4, (b) RH5 and (c) RH6 with 50% thennal ellipsoid probability. Hydrogen atoms were omitted for clarity. 83 Table 3.1. Crystal data and structural parameters for RH4, RH5 and RH6 RH4 RHS RH6 Crystal data Chemical formula C2sH30BBrF2N20 C26HnBBrF2N20 C21Ih.1BBrF2N20 Mr 503.22 517.26 531.28 Crystal system, Triclinic, P-1 Monoclinic, P21 Monoclinic, P2tfc space group Temperature (K) 100 173 173 c (A) a, h, 13.1235 (4), 15.0693 (4), 13.9134 (4), 10.7963 (6), 22.1 893 (13), 15.4029 (4) 16.9277 (4), 11.4150 (6) 20.8463 (6) ex, p, Y (o) 108.293 (2), 111.712 (2), 90, 91.108 (2), 90 90, 99.561 (3), 90 I 01.152 (2) V(A3) 2516.13 (13) 4908.8 (2) 2696.6 (3) z 2 8 4 µ (mm-1) 3.50 2.58 2.36 Crystal size (mm) 0.38 x 0.22 x 0.05 Not Reported 0.35 x 0.22 x 0.08 Data collection 0.564, 0.753 Not Reported 0.565, 0.753 T mnh T max No. of measured. 56696, 9049, 7829 7295 1, 17614, 32276, 4784, 3927 independent and 15028 observed [J > 2cr(J)] reflections Rini 0.051 0.065 0.093 (sin Ot?..)max cA-1) 0.603 0.604 0.602 Refinement R[F2 > 2cr(P2)), 0.048, 0.131, 1.05 0.053, 0.146, 1.05 0.045, 0.1 78, 1.06 wR(F2),S No. of reflections 9049 17614 4784 No. of parameters 635 1214 313 84 Table 3.2. Selected Bond lengths for RH4, RH5 and RH6 Bond Lengths (A) Bond RH4 RHS RH6 B-Fl 1.395(5) 1.397(6) 1.401(4) B-F2 l .389(3) 1.406(7) 1.400(4) B-Nl 1.549(4) 1.551(6) 1.554(4) B-N2 1.544(4) 1.546(7) l.553(5) Table 3.3. Selected Bond angles forRH4, RH5 and RI16 Bond Angles (0) Bond RH4 RHS RH6 Nl-B-N2 106.7(2) 106.8(4) 107.2(2) Fl-B-F2 109.7(2) 109.1(4) 109.1(3) Nl-B-FI 110.0(2) 111.4(4) 110.2(3) N2-B-F2 110.7(2) I 09.8(4) 110.4(3) N2-B-FI 109.7(2) 110.6(4) l 09.8(3) Nl-B-F2 110.0(2) 109.1(4) 110.0(3) - Most of the crystal parameters of RH4, RH5 and RH6 including bond lengths and bond angles were similar to each other as listed in Table 3.2. and Table 3.3. This confirmed that changing the length of alkyl group bound to phenolic substituent does not affect the conjugated ring system in the BODIPY core and it docs not deviate from the tetrahedral geometry around the boron atom. However, the two planes between the aromatic substituent and the BODIPY core arc not exactly perpendicular to each other forall three ligands. RH5 showed the highest tilting by 12.27° while RH4 and RH6 showed 6.68° and 4.88° respectively. This was the major considerable diffe rence among the crystal structures of RH4, RH5 and RH6 ligands. 85 (a) (b) Figure 3.2. ORTEP diagrams of the single-crystal structures of (a) RH IO and (b) RH13 with 50% thermal ellipsoid probability. Considering the crystal data obtained for RHI 0 and RH I 3, both showed monoclinic crystal system belonging to the C2/c and P21 space groups respectively. The R values were below 5% for both RHlO and RH 13 crystals indicating the good quality of refinement. No conformational isomers were observed any of the crystals. The ratio of number of reflections/number of parameters for RH I 0 and RH 13 were I 3.13 and 13.58 respectively. These values further confirmed the quality of refinement is good for both crystals. Due to the -NH2 group present in the aromatic substituent in the BODIPY core, two planes were tilted by 21.74°in RH10 and 9. 11° in RH 13 which resulted as the major difference among these two crystal structures. This large tilt of the aromatic substituent could be due to the hydrogen bonding interaction present between the hydrogens of para amino group and the F atoms in an adjacent BODIPY core. Hydrogen bonds are present in the ortho amino substituted aromatic ring containing RH I 3 as well yet the orientation has not been changed significantly due to the hydrogen bonding directionality is favorable with the more two plane perpendicular structure of the RH 13 I igand. 86 Table 3.4. Crystal data and structural parameters for RHIO and RH13 RHlO RH13 Crystal data Chemical formula CnH2&BF2N3 C23H2i;BF�N3 395.29 395.29 Mr Crystal system, space group Monoclinic, Cllc Monoclinic, P21 Temperature (K) 100 273 b, c (A) 17.3936 (7), 12.2974 7.5651 (6), 17.4444 (13), a, (5), 11.8024 (5) 8.0325 (6) 126.519 (2) 94.221 (5) a. p, Y (o) V(A3) 2028.83 ( 15) 1057.16 (14) z 4 2 µ(mm ') 0.59 0.69 0.34 0.31 0.20 Crystal size (mm) Not Reported x x Data collection Not Reported Not Reported T mm, T mu No. of measured, independent 21247, 1865, 1772 14432,3722, 3256 and observed [/ > 2o(J)] reflections R;n1 0.030 0.053 1 (sin 0/A.)mnx (A ) 0.608 0.602 Refinement R[F2> 2o(F2)],wR(F2), S 0.034, 0.093, 1.04 0.043, 0. 131, 1.09 No. of reflections 1865 3722 No. of parameters 142 274 87 Table 3.5. Selected bond lengths RH 10 and RH 13 Bond Lengths (A) Bond RHlO RH13 B-Fl 1.409 1.400(3) B-F2 1.409 1.408(4) B-Nl 1.548 1.542(4) B-N2 1.548 1.548(4) Table 3.6. Selected bond angles forRH I 0 and RH 13 Bond Angles (0) Bond RH lO RH13 Nl-B-N2 107.5 107.1(2) Fl-B-F2 108.2 108.7(2) N 1-B-Fl 110.5 110.6(2) N2-B-F2 11O. l I 09.7(2) N2-B-Fl 11O. l 110.7(2) Nl-B-F2 110.5 I IO. l (2) Bond lengths and bond angles of RH I 0 and RH 13 arc very similar to each other. Both ligands showed bond angles closer to I 09° indicating the tetrahedral geometry around boron atom in the BODIPY core. However, N1-B-N2 bond angle (106°) is quite smaller than l 09° identical bond angle in all fivecrystal structures. 88 3.2. Photophysical Properties 3.2.1. Photophysical properties of ligands 3.2. 1.1. Absotplion data Absorption spectra for both RH l 2 and RH 14 are shown in Figure 3.3 and detailed spectral data are listed in Table 3.7. Absorbance Vs Wavelength/ nm 0.1 0.08 (,) RHl4 u 0.06 - c: co 004 RH l 2 .D..... - 0Vl .D 0.02 RH l2 RH14 CH2Ch ·6 Figure 3.3. Absorption spectra for and ligands in ( l .6 x t0 M) RH 14 RHI Absorption maxima for is 532 nm which is quite higher than for 2 observed around 527 nm. 3.2. 1.2. Fluorescence data Excitation and emission spectra for both RH 12 and RH 14 are shown in Figure 3.4 and Figure 3.5. The detailed spectral data arc listed in Table 3.7. The maximum excitation of RH12 was observed around 526 nm but it was observed at 531 nm for RH14. Emission maxima for RH 12 and RH14 were observed at 540 nm and 548 nm respectively. Higher 89 stokes' shift wasobserved for RH14than RH12. With the same concentration, both RH12 and RH 14 showed more or less equal fluorescence intensities. Intensity Vs Wavelength/ nm 9.00E+OS 8.00E+OS 7.00E+OS - RI-114 6.00E+OS >- S.OOE+OS - RI-112 ·�s:: 2 4.00E+OS s:: ,...... 3.00E+05 2.00E+05 l.OOE-105 O.OOE+OO 450 470 490 510 530 550 570 Wavelength/ run 6 Figure 3.4. Excitation Spectra of RHl 2 and RH14 ligands in CH2Ch (1.6 x 10- M) Intensity Vs Wavelength/ nm 2.00E+05 l.50E+05 - RH14 .£ -RH12 :g l.OOE+OS ....4) ..s S.OOE+04 O.OOE+OO 500 520 540 560 580 600 620 640 660 680 700 Wavelength/ nm 6 Figure 3.5. Emission Spectra of RH12 and RH14 ligands in CH2Ch (1.6 x 10- M) 3.2.1.3. Quantum yield measurements As listed in the Table 3.7 quantum yields were calculated by plotting an integrated fluorescence intensity vs ahsorhance graph. Two trials were done for the reference 90 rhodamine 6G, RH 12 and RH 14 samples and average quantum yields were calculated for each sample. Figure 3.6, Figure 3.7, and Figure 3.8 illustrate the UV-absorption spectra, fluorescence spectra, and integrated fluorescence intensity vs absorbance graph of diffe rent concentrations of RH 12 for the trial I measurements. Figure 3.9, Figure 3.10, and Figure 3.11 illustrate the UV-absorption spectra, fluorescencespectra, and integrated fluorescence intensity vs absorbance graph of different concentrations of RH 14 for trial l measurements. Absorbance vs Wavelengths/ nm 0.6 -- IOuL 0.5 40 uL --60uL 0.4 90 uL Q) -- u c 0.3 "' ..0 i... 0.2 en0 ..0 Figure 3.6. Trial 1- UY-absorption spectra of RH12 with four different concentrations in 91 Intensity vs Wavelengths/ nm 9.00E+05 8.00E+05 7 OOE+05 --IOuL 6.00E+05 .,,,>. 40 uL 5.00E+05 · ;;; 60 ul c B 4 OOE+05 90 uL .5 3 OOE+05 -- 2.00E+05 l.OOE+OS O.OOE+OO 490 510 530 550 570 590 610 630 650 670 690 Wavelengths/ nm Figure 3.7. Trial I- Emission spectra of RH12with four different concentrations in Integrated fluorescence vs absorbance graph fortrial I RHl2 0 40000000 en c: .,,,0 35000000 •• c: y = 6E+08x - 228956 ... ··· 30000000 = · 0 R2 0.9968 . .. ·· () . . c: . ·· 0 25000000 .. ·· () en .. ... � 20000000 ... 0 . ... ::s .. . 15000000 .. c;:: .. .. . "O ... 0 10000000 .... (ii So 5000000 .....0 .s 0 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 Absorbancc Figure 3.8. Trial 1- Integrated fluorescence vs absorbance graph for quantum yield determination of RH 12 92 Absorbance vs Wavelengths/ run 0.45 0.4 0.35 --45 uL Cl) 0.3 (.) 70uL 0.25 -- coc: ..D 95 uL .... 0.2 0en 120 uL ..D 0.15 <( 01 0.05 0 -0.05 390 410 430 450 470 490 510 530 550 570 wavelength/ nm Figure 3.9. Trial I- UV-absorption spectra of RH14 with four different concentrations in Absorbancc vs Wavelengths/ nm l.OOE+06 9.00E+05 8.00E+05 45 uL o.> 7.00E+OS -- (.) � 6.00E+05 70 uL 5.00E+05 -2 95 uL 0 4.00E+05 J5 -- 120 uL � 3.00E+05 2.00E+05 1 OOE+05 O.OOE+OO 490 540 590 640 690 Wavelengths/ nm Figure 3.10. Trial I- Emission spectra of RH14 with four different concentrations in 93 Integrated fluorescence vs absorbance graph for trial l RH 14 0 45000000 V) Cl) 40000000 = ...... y 7E+08x + IE+07 .. . ·····• . S 35000000 · . · R2 = 0.9995 Cl) . · .... ·· ··· u . . · c: 30000000 ...... Cl) .... C,) 25000000 ... V) •... Cl) ....·· i... 20000000 . 0 · ::s .. ······ c;:;. 15000000 •····· "O 10000000 Cl) ...... "' 5000000 Cl)5h 0 ...., 0 0.01 O.D2 O D3 ..s . 0.04 0.05 Absorbance Figure 3.1 1. Trial /- Integrated fluorescence vs absorbance graph forquantum yield determination of RH 14 Figure 3.12, Figure 3.13, and Figure 3.14 illustrate the UV-absorption spectra, :fluorescencespectra, and integrated fluorescence intensity vs absorbance graph of different concentrations ofRH12 for the trial 2. Figure 3.15, Figure 3.16, and Figure 3.17 illustrate the UV-absorption spectra, fluorescence spectra, and integrated fluorescence intensity vs absorbance graph of different concentrations of RHl 4 for trial 2 measurements. 94 Absorbance vs Wavelength/ nm 4 50E-Ol 4.00E-01 3.SOE-01 --20 uL 3.00E-01 -40uL 60 uL (l) 2.50E-01 (.) c: 80 uL Cl3 2.00E-01 90uL .D'- -- l.50E-01 Vl0 .D l.OOE-01 < 5.00E-02 0.00E+OO 4oo 420 440 460 480 500 520 540 560 580 -5.00E-02 wavelength/ nm Figure 3. 12. Trial 2- UV-absorption spectra of RH 12 with five different concentrations Intensity vs Wavelength/ nm 9.00E+05 8.00E+05 7.00E+05 20uL -- 6.00E+OS 40 uL C -- . 5.00E+05 iii 60 uL c:: � 4.00E+05 80 uL 3.00E+05 ..S --90uL 2.00E+05 l.OOE+OS O.OOE+OO 490 510 530 550 570 590 610 630 650 670 690 Wavelength/ nm Figure 3.13. Trial 2- Emission spectra of RH 12 with five different concentrations in A RH 12 stock solution with 2.16 x I 0-4 M concentration was used to record above absorption and emission spectra. Integrated fluorescence vs absorbance emission graph 95 was plotted using above absorption and emission spectra as illustrated in Figure 3.14. The gradient was used to calculate the quantum yield of RH 12. Integrated fluorescence vs absorbance graph for trial 2 RH12 40000000 . v 35000000 y = 5E+08x + 470430 . ••• (.) . . .. ·· § 30000000 R2 = 0.9994 · . (.) · .. Cl) · . e 25000000 . . · 0 ··. ... ·· ·· � 20000000 .. . -0 ....·· ·...... � 15000000 ·· ro . · . � 10000000 . ...·· · ..... � 5000000 0 0 0.01 O.D2 0.03 0.04 0.05 0.06 0.07 Absorbance Figure 3.14. Trial 2- Integrated fluorescence vs absorbance graph for quantum yield determination ofRH12 0.6 Absorbance vs Wavelength/ nm 0.5 --2o uL 4 04 -- 5 uL v (.) 70 uL c:: «:! 0.3 ..0 95 uL ..... 0 Cl) 0.2 -- 120uL ..0 0 400 420 440 460 480 500 520 540 560 580 ·0.1 Wavelength/ nm Figure 3.15. Tr ial 2- UV-absorption spectra of RH 14 with fivedif fe rent concentrations 96 Intensity vs Wavelength/ nm 1.20E+06 1 OOE+06 --20uL -- 45uL 8.00E+05 O 70 uL . ;;; 6.00E+05 c 95 uL Cl) .... -- 1 20 uL ..S 4.00E+05 2.00E+05 o.ooE�oo 490 540 590 640 690 Wavelength/ nm Figure 3.16. Trial 2- Emission spectra of RH 14 with five different concentrations in Integrated fluorescence vs absorbance graph for trial 2 60000000 RH l4 Cl) 0 50000000 5 y = 7E+08x + 4E+06 0 ...... V> . .. R2 = 0.9926 . 40000000 .. · � . · ..· .. · ::s .. · . 30000000 · · .. · � ... · ·. -0Cl) · .. · .. · . 20000000 · · .. · .. � · • . Cl) .. .. .s 10000000 .. 0 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 Absorbancc 2- Figure 3.17. Trial lntegrated fluorescence vs absorbancc graph for quantum yield determination of RH14 A stock solution of RH 14 with 1.93 x 10-4M concentration was used to record above ahsorption and emission spectra. Integrated fluorescence vs absorbance emissions graph 97 was plotted using above absorption and emission spectra as illustrated in Figure 3.17. From integrated fluorescence vs absorbance plots obtained fortwo trials, two quantum yield values were calculated and average values are reported in Table 3.7. Refractive index of CH2Cli was used as 1 .4244 and for ethanol 1.36 17at 20°C. lt turned out that the quantum yield of RH 14 was 20% higher than RH 12. 3.2.1.4. Lifetime measurements The lifetime (vis) of RH14 was higher than the lifetime of RH l2 as listed in Table 3.7. This higher lifetime could be an advantage in indirect energy transferprocess of RH 14 compared to RH 12. Figure 3.18. Single Exponential Tail titting of lifetime for RH 12 in CH2C'2 at room temperature at 537 nm 98 10 (/) c :::; 1 o· 0 u , . . ·' . . •, , . . ,.:.,.,, .... 10 10 0 10 20 30 40 50 60 70 T1rne1ns (/) 49 CV _, v 00 C/l O'.'.Q) -4 9 Figure 3.19. Single Exponential Tail fitting of lifetime for RH 14 in CH2Ch at room temperature at 549 nm . Ta bl e 3 . 7. Photop h 1y s1ca1 properties o f RH 12 and RH 14 I'1ga n ds Asto cs Ligand AAbs (nm) Aex(nm) �m (nm) k Lifetime/ Quantum (nm) (vis) ns Yield RH 12 527 526 540 14 4.97 0.55 (±0.0 I) RH14 532 531 548 17 7.56 0.78 (±0.06) 3.2.2. Photophysical properties of lanthanide complexes Complex formation was studied and confirmed by NIR emission signals corresponding to the Yb(III) ion around 980 and I 010 nm range. With both RH12 and RH 14 ligands, strong emissions were observed in the NIR region confirming complex formation. In this study (Yb(TPP)(OAc)(McOHh] complex was used to synthesize the lanthanide complex. Porphyrin alone can sensitize Yb(III) ion to emit around NIR region. To understand the contribution from each conjugated system to sensitize Yb(IIr) NlR emission, an excitation spectrum was recorded monitoring the emissions at 980 nm. 99 3.2.2.1. Interaction of RH/ 2 with [Yb(TPP)(OAc)(MeOH)2} in CH2Ch (fo rmation of complex I) First, excitation spectrum was recorded for the complex I to investigate the excitation maxima of the complex I Figure 3.20. (a). The highest excitation was observed at 440 nm and the second highest excitation was observed at 527 nm. These are compatible with the excitation maxima of porphyrin and BODIPY unit respectively. At 440 nm, the porphyrin unit can be excited to sensitize the lanthanide ion and at 525 nm BODIPY unit can be excited to sensitize the lanthanide ion to emit in the NIR region. Then emission in the NIR region was recorded at 440 nm and 525 nm along with two other wavelengths at 375 nm and 550 nm as illustrated in Figure 3.20. (b ). For both 440 nm and 525 nm emission intensities, the NIR region was more or less similar and it was higher than when the other two wa¥elengths were used, concluding the sensitization process is achieved by both porphyrin and BODIPY units in the complex l. Maximum NIR emission peaks were observed at 977 nm and l 005 nm. These can be 2 2 assigned to typical Yb(III) transition between Fs12 ground state and F1112 excited state of the BODIPY based Yb(III) porphyrin complex. 100 RH12 432 +Yb 1005 - 375 nm I- lntensitYl 440 n 977 525 nm -550nm 527 350 400 450 500 550 600 650 70(900 950 1000 1050 1100 1150 Wavelength (nm) Wavelength (nm) (a) (b) Figure 3.20. (a) Excitation Spectrum (A.cm= 980 nm) (b)NIR emission under different excitation wavelengths for complex l 3.2.2.2. Interaction of RH/ 4 with {Yb(TPP)(OAc)(MeOHh} in CH2Cl2 (fo rmation of complex 2) Similar results were observed for complex 2 ([Yb(TPP)(OAc) RH14]) as illustrated in Figure 3.21. but the highest emission was observed at 440 nm and the second highest emission was observed at 527 nm. In contrast to complex I, complex 2 NIR emissions were higher at 550 nm as depicted in Figure 3.22. Maximum NlR emission peaks were observed at 978 nm and I 006 nm. These can be assigned to typical Yb(HI) transition between 2Fs12 ground state and 2F1112 excited state of BODTPY based Yb( Ill) porphyrin complex. 101 < IOO MO E � .c IOO f i• "° ! olOO ii: MO toO 1000 ,. 1100 1150 W-.gtl lnm (a) Contour view (b) 30 view Figure 3.21. Excitation vs NIRemission of complex 2 in CH2Cli 1006 375 nm 978 440 nm S?S nm 550nm 900 950 1000 1050 1100 Wavelength (nm) Figure 3.22. NTR emission of complex 2 under different excitation wavelengths 3.2.2.3. Sp ectroscopic ti/rations with RH/2 and RJl/4/igands The RH12ligand shows strong fluorescence in the visible region but when it forms the complex with Yb(III) fluorescence was almost completely diminished as shown in Figure 3.23 (a). Upon addition of Yb(III) ions to the RH 12 solution, the fl uorescence intensity at 540 nm gradually decreased until the molar ratio of RH 12 to Yb(IU) becomes I: I. Further addition of Yb(HI) ions did not exhibit considerable level of fluorescence decrement. f n contrast, emission intensity at 977 nm increased by increasing the Yb(IID during the 102 titration process. This observation fu rther confirmed the indirect lanthanide sensitization process by BODIPY based ligands. liLi7iYbil Yb·MeOH by RH12 �o0.2o; -L:I 060.4 �01.0.8 Ic-- 1�. 950 1050 1100 1150 500 550 600 650 900 1000 Wavelength (nm) Wavelength (a) (b) Figure 3.23. Interaction of RH 12 with [Yb(TPP)(OAc)(MeOHh] in CH2Ch (a) visible emission from RH 12 (b) NIR emission from Yb(Ill). Titration ofYb(HI) by RH12 2.00E+03 -- Oul -20ul 1 50f+03 - 40 ul � - GOuL c: ;:s --soul 0 1.00E+03 u -- lOO ul S.00Et02 O.OOE+OO 9 1000 1050 1150 ·5.00E+02 Wavelength/ nm Figure 3.24. Back titration by RH 12, NTR emission from Yb(III) at 977 nm and l 005 nm 103 To confirm the stoichiometric ratio between Yb(TII) and RH 12 ligand, back titration was done (Figure 3.24.). (Yb(TPP)(OAc)(McOH)2] was titrated by RH 12 ligand and NTR emission by Yb(III) was monitored with added ligand volumes. When increasing the ligand concentration, NIR emission by Yb(ITI) increased and increment was continued until the molar ratio becomes I: l. This observation further confirms the [Yb(TPP)(0Ac)t3 forms 1:1 complex with RH 12. RH14+Yb-MeOH RH14+Yb 1006 :;j :;j .i � ?:- � -;;; u; c c Q) £ E$ 500 550 600 650 900 950 1000 1050 1100 1150 Wavelength (nm) Wavelength (nm) Figure 3.25. Interaction of RH14 with in CH2C'2 (a) visible emission from RH14 (b) NIR emission from Yb(Jll). Like RH 12, the RH 14 ligand shows strong fluorescence in the visible region but when it forms the complex with Yb(Ill) the fluorescence was diminished as shown in Figure 3.25 (a). Upon addition of Yb(lll) ions to the RH14 solution, the fluorescence intensity at 548 nm gradually decreased until the molar ratio of RH 14 to Yb(lll) becomes I: I. Considerable change in the emission spectra could not be observed by further addition of Yb(lll) ions. Considering the emission intensity at 977 nm, it increased with increasing Yb(III) concentration during the titration process. This observation also supports the idea of indirect lanthanide sensitization mechanism by BODlPY based ligands. 104 Titration ofYb-OH by RHl4 2.00E+03 -- Oul - 20uL l.SOE+03 --4oul - 60ul - s0 l.00Et03 80uL -- lOO uL u -- llOul S.OOE+02 O.OOE+OO 90 -S.OOE+02 Wavelength/ nm Figure 3.26. Back titration by RH14, NTR emission from Yb(III) at 977 nm and I 005 nm For fu rther confirmation of the stoichiometric ratio between Yb(III) and RH 14 ligand, back titration was done. [Yb(TPP)(OAc)(MeOH)2] was titrated by RH 14 ligand and NIR emission by Yb(III) was monitored with varying ligand concentrations. When increasing the ligand concentration, NIR emission by Yb( III) increased and increment was continued until the molar ratio becomes 1:1. This observation further confirms the [Yb(TPP)(OAc)] tJ forms I: I complex with RH14. 3.2.2.4. lifetime at NIR region NIR decay curves were acquired using an optical parametric oscillator. For the complex I, the NTR lifetime was 12.2394 µs. For the complex 2, the NIR lifetime was 8.8636 µs. Even though the quantum yield, visible lifetime and NlR emission intensity were higher for RH 14 by itself, when it forms the lanthanide complex, a lower NIR lifetime was found than the corresponding complex formed with RH 12. 105 3000 RH12 Equation y = A1 "exp(-xlt1) + yO 2500 Adj. R-Squ 0.99898 - 2000 Value Standard E Intensity yO 43.39678 1.2306 «I� A 1 6.26845 3.69581E9 - 1500 Intensity Intensity 11 12.23947 0.04175 �(/) c 1000 c:Q) 500 0 200 250 300 Time (µs) Figure 3.27. NIR decay curve for complex I recorded at 980 nm 3000 RH14 • 2500 Equaiont A1'exp(- xlt1)y = +y0 • Adj. R-Squ 0.98241 2000 Value Standard Er Intensity yO 52.82228 2.96469 :::i 8.9719E12 � 1500 Intensity A1 2.70238E � Intensity 11 8.8636 0.12404 ·u; c 1000 Q) c: 500 0 200 250 300 Time (µs) Figure 3.28. NIR decay curve for complex 2 recorded at 980 nm 106 CHAPTER 4 CONCLUSION As a remedy for prevailing analytical techniques with fluorophores, BODTPY based lanthanide complexes have been synthesized for the past decade. BODIPY molecules are low molecular weight fluorophores that show narrow emission bandwidths with high extinction coefficients. Furthermore, alterations of the absorbance and emission spectra is possible by introducing substituents to the BODIPY core. They are photostable and possess relatively longer excited-state lifetimes compared to many other fluorophores. Functionalizing the BODIPY core with ligand binding groups allow it to form stable lanthanide complexes. These BODIPY based lanthanide complexes show unique emission bands at NIR region. Synthesizing such complexes and utilize the selective and sensitive emission properties of lanthanide ions and harvesting the benefits to analyze biological systems is one promising area in recent medical diagnostic studies. As a contribution to such diagnostic studies, in this study, two new BODIPY functionalized ligands were synthesized and their structural and photophysical properties were evaluated. A ligand with a phenanthroline group attached to ortho position of the aromatic meso substituent (RH14)demonstrated higher quantum yield and visible range lifetime than the ligand with phenanthroline group attached to para position of the aromatic substituent (RH12). Both ligands emit strong fluorescence; however, fluorescence was quenched upon the addition of [Yb(TPP)(OAc)(MeOH)2]. In the meantime, NIR emission by Yb(III) around 977-1006 nm range was gradually increased exhibiting a lanthanide metal and ligand interaction. Lanthanide ions formed 1:1 metal to ligand complex showing a dextre 107 type sensitization mechanism. However, excitation at different wavelengths suggested both porphyrin and BODIPY moieties responsible for the sensitization process of these complexes. Results showed the capability ofBODIPY functionalized with phenanthroline can function as an efficient chromophore for the indirect sensitization of lanthanide complexes. However, NIR emission was hindered when using [Yb(hfa)3 (H20)2] as the ytterbium source for both synthesized ligands. Therefore, further studies should be carried out to understand the underlining chemistry behind the NIR emission efficiency of both complexes formed by [Yb(TPP)(OAc)(MeOH)2] and BODIPY ligands (complex l and complex 2). 108 REFERENCES 1. Guilbault, G. G., Practical.fluorescence 211d ed. CRC Press: 1990; Vol. 3. Chapter 3, 110-340. 2. Kagan, H. B., Introduction: frontiers in lanthanide chemistry. ACS Publications: 2002, I 02( 6), 1 805-6 3. Zhang, J.; Badger, P. D.; Geib, S. J.; Petoud, S., Sensitization ofNear-lnfrared-Emitting Lanthanide Cations in Solution by Tropolonate Ligands. Angewandte Chemie 2005, 44( 17), 2508-25 12. 4. Tallec, G.; Imbert, D.; Fries, P. H.; Mazzanti, M., Highly stable and soluble bis-aqua Gd, Nd, Yb complexes as potential bimodal MRl/NIR imaging agents. Dalton Transactions 2010, 39 (40), 9490-9492. 5. Bulach, V.; Sguerra, F.; Hosseini, M. W., Pmphyrin lanthanide complexes for NIR emission. Coordination Chemistry Reviews 2012, 256 (15), 1468-1478. 6. Khalil, G. E.; Thompson, E. K.; Gouterman, M.; Callis, J. B.; Dalton, L. R.; Turro, N. J.; Jockusch, S., NIR luminescence of gadolinium porphyrin complexes. Chemical Physics Letters 2007, 435 (1), 45-49. 7. Feng, J.; Zhang, H.-J.; Song, S.-Y.; Li, Z.-F.; Sun, L.-N.; Xing, Y.; Guo, X.-M., Syntheses, crystal structures, visible and near-IR luminescent properties of ternary lanthanide (Dy 3+, Tm 3+) complexes containing 4, 4, 4-trifluoro-1-phenyl- l, 3- butanedione and 1, 10-phenanthroline. Journal of Luminescence 2008, 128 (12), 1957- 1964. 8. Xu, H.-B.; Shi, L.-X.; Ma, E.; Zhang, L.-Y.; Wei, Q.-H.; Chen, Z.-N., Diplatinum alkynyl chromophores as sensitisers for lanthanide luminescence in Pt 2 Ln 2 and Pt 2 109 Ln 4 (Ln= Eu, Nd, Yb) arrays with acetylide-functionalized bipyridine/phenanthroline. Chemical Communications 2006, (15), 1601-1603. 9. Ryu, J. H.; Eom, Y. K.; Bilnzli, J.-C. G.; Kim, H. K., Ln (Ill)-cored complexes based on boron dipyrromethene (Bodipy) ligands for NlR emission. New Journal of Chemistry 2012, 36 (3), 723-73 1 . 10. Zhong, Y.; Si, L.; He, H.; Sykes, A. G., BODIPY chromophores as efficient green light sensitizers for lanthanide-induced near-infrared emission. Dalton Transactions 201 1, 40 (43), 11389-1 1395. 11. Albani, J. R., Principles and applications of fluorescence spectroscopy. John Wiley & Sons: 2008. 44-67. 12. Valeur, B.; Berberan-Santos, M. N., Molecular Fluorescence: Principles and Applications. John Wiley & Sons: 2012. 78-10 l. 13. Elson, E. L.; Magde, D., Fluorescence correlation spectroscopy. I. Conceptual basis and theory. . Biopolymers 1974, 13 (1), 1-27. 14. Albani, J. R., Fluorescence spectroscopy principles. Principles and Applications of Fluorescence Spectroscopy. Springer: 2008, 88-1 14. 15. Melhuish, W.H., Quantum efficiencies of fluorescence of organic substances: effect of solvent and concentration of the fluorescent solute. The Journal of Physical Chemistry 1961, 65 (2), 229-235. 16. Pinsky, B. G.; Ladasky ·J r, J. J., Apparatus and method fo r fluorescent lifetime measurement. 1994. U.S. Patent 5,315,122. 110 17. Li, Y.-H.; Chan, L.-M.; Tyer, L.; Moody, R. T.; Himel, C. M.; Hercules, D. M., Solvent effects on the fluorescenceof 1-(dimethylamino )-5-naphthalenesulfonic acid and related compounds. Journal of the American Chemical Society 1975, 97 (11), 3118-3 126. 18. Pringsheim, P., Fluorescence and phosphorescence. 1949. lnterscience Publishers; !51 Edition, 280-292 19. (a) Oldham, P. B.; McCarroll, M. E.; McGown, L. B.; Warner, I. M., Molecular fluorescence, phosphorescence, and chemiluminescence spectrometry. Analytical chemistry 2000, 72 (12), 197-210; (b) Powe, A. M.; Fletcher, K. A.; St. Luce, N. N.; Lowry, M.; Neal, S.; Mccarroll, M. E.; Oldham, P. B.; McGown, L. B.; Warner, I. M., Molecular fluorescence, phosphorescence, and chemiluminescence spectrometry. Analytical Chemistry 2004, 76 (16), 4614-4634. 20. Kobayashi, H.; Ogawa, M.; Alford, R.; Choyke, P. L.; Urano, Y., New strategies for fluorescent probe design in medical diagnostic imaging. Chemical Reviews 2009, 110 (5), 2620-2640. 21. Loudet, A.; Burgess, K., BODIPY dyes and their derivatives: syntheses and spectroscopic properties. Chemical Reviews 2007, 107 ( 11), 4891-4932. 22. Yamashita, S.; Sakabe, M.; Ishizawa, T.; Hasegawa, K.; Urano, Y.; Kokudo, N., Visualization of the leakage of pancreatic juice using a chymotrypsin-activated fluorescent probe. British Journalof Surgery 2013, 100 (9), 1220-1228. 23. Zhou, X.; Lai, R.; Beck, J. R.; Li, H.; Stains, C. I., Nebraska Red: a phosphinate-based near-infrared fluorophore scaffold for chemical biology applications. Chemical Communications 2016, 52 (83), 12290-12293. 111 24. Zhou, X.; Lesiak, L.; Lai, R.; Beck, J. R.; Zhao, J.; Elowsky, C. G.; Li, H.; Stains, C. I., Chemoselective Alteration of Fluorophore Scaffolds as a Strategy forthe Development of Ratiometric Chemodosimeters. Angewandte Chemie International Edition 2017, 56 (15), 4197-4200. 25. Biinzli, J.-C. G., Benefiting from the unique properties of lanthanide ions. Accounts of Chemical Research 2006, 39 (1), 53-61 . 26. Eliseeva, S. V.; Biinzli, J.-C. G., Lanthanide luminescence for functional materials and bio-sciences. Chemical Society Reviews 2010, 39 (1), 189-227. 27. Cotton, S., Lanthanide and Actinide Chemistry. John Wiley & Sons: 2013, 292-320. 28. Edelstein, N. M., Lanthanide and Actinide Chemistry and Sp ectroscopy. American Chemical Society, 1980, 131, 232-334. 29. Sturza, C. M.; Boscencu, R.; Nacea, V., The Janthanides: physico-cherpical properties relevant for their biomedical applications. FA RMACIA - 2008, 56 (3), 326. 30. Beeby, A.; Dickins, R. S.; FitzGerald, S.; Govenlock, L. J.; Maupin, C. L.; Parker, D.; Riehl, J. P.; Siligardi, G.; Williams, J. A. G., Porphyrin sensitization of circularly polarised near-IR lanthanide luminescence: enhanced emission with nucleic acid bindingElectronic supplementary information (ESI) available: emission spectra. See http://www. rsc. org/suppdata/cc/b0/b002452j. Chemical Communications 2000, (13), 1183-1184. 31. Evans, C. H., Interesting and useful biochemical properties of lanthanides. Trends in Biochemical Sciences 1983, 8 (12), 445-449. 32. Biinzli, J.-C. G., Lanthanide luminescence for biomedical analyses and imaging. Chemical Reviews 2010, 110 (5), 2729-2755. 112 33. Gon9alves e Silva, F. R.; Malta, 0. L.; Reinhard, C.; Gtidel, H.-U.; Piguet, C.; Moser, J. E.; Btinzli, J.-C. G., Visible and near-infrared luminescence of lanthanide-containing dimetallic triple-stranded helicates: energy transfer mechanisms in the Smlll and Yblll molecular edifices. The Jo urnal of Physical Chemistry A 2002, I 06 (9), 1670-1677. 34. Caravan, P.; Ellison, J. J.; McMurry, T. J.; Lauffer, R. B., Gadolinium (III) chelates as MRI contrast agents: structure, dynamics, and applications. Chemical Reviews 1999, 99 (9), 2293-2352. 35. Ceulemans, M.; Nuyts, K.; De Borggraeve, W. M.; Parac-Vogt, T. N., Gadolinium (III) DOTA complex functionalized with BODIPY as a potential bimodal contrast agent for MRI and optical imaging. lnorganics 2015, 3 (4), 516-533. 36. Jones, J. E.; Amoroso, A. J.; Dorin, I. M.; Parigi, G.; Ward, B. D.; Buurrna,N. J.; Pope, S. J.A., Bimodal, dimetallic lanthanide complexes that bind to DNA: the nature of binding and its influence on water relaxivity. Chemical Communications 201 1 , 47 (12), 3374-3376. 37. Rumyantseva, V.D.; Gorshkova, A.S.; Mironov, A.F., Ytterbium and its porphyrin complexes. Fine Chemical Technologies 2014, 9 (1). 38. Lenaerts, P.; Storms, A.; Mullens, J.; D'Haen, J.; Gorller-Walrand, C.; Binnemans, K.; Driesen, K., Thin Films of highly luminescent lanthanide complexes covalently linked to an organic- inorganic hybrid material via 2-substituted imidazo [4, 5-f]-1, 10- phenanthroline groups. Chemistry of Materials 2005, 17 (20), 5194-5201. 39. He, H.; Sykes, A. G.; May, P. S.; He, G., Structure and photophysics of near-infrared emissive ytterbium (III) monoporphyrinate acetate complexes having neutral bidentate ligands. Dalton Transactions 2009, (36), 7454-7461. 113 40. He, H.; Dubey, M.; Sykes, A. G.; May, P. S., Hybridization of near-infrared emitting erbium (III) and ytterbium (Ill) monoporphyrinate complexes with silica xerogel: synthesis, structure and photophysics. Dalton Transactions 2010, 39 (28), 6466-6474. 41. He, H., Near-infrared emitting lanthanide complexes of porphyrin and BODIPY dyes. Coordination ChemistryReviews 2014, 273, 87-99. 42. Ziessel, R. F.; Ulrich, G.; Charbonniere, L.; Imbert, D.; Scopelliti, R.; Bunzli, J. C. G., NIR lanthanide luminescence by energy transfer from appended terpyridine boradiazaindacene dyes. Chemistry-A European Journal 2006, 12 ( 19), 5060-5067. 43. He, H.; Si, L.; Zhong, Y.; Dubey, M., Iodized BODIPY as a long wavelength light sensitizer for the near-infrared emission of ytterbium (iii) ion. Chemical Communications 2012, 48 (13), 1886-1888. 44. Bayrakc1, M.; Kursunlu, A. N.; GUier, E.; Ertul, S., A new calix [4] azacrown ether based boradiazaindacene (Bodipy): Selective fluorescence changes towards trivalent lanthanide ions. Dyes and Pigments 2013, 99 (2), 268-274. 45. Stephan, H.; Walther, M.; Fahnemann, S.; Ceroni, P.; Molloy, J. K.; Bergamini, G.; Reisig, F.; Muller, C. E.; Kraus, W.; Comba, P., Bispidines for dual imaging. Chemistry-A European Journal 2014, 20 (51), 17011-17018. 46. Biiozli, J.-C. G., Lanthanides in Biological Labeling, Imaging, and Therapy. In Encyclopedia of Metalloproteins, Springer: 2013; pp 1110-1 1 18. 47. Horrocks, Jr, W. D.; Sudnick, D. R., Lanthanide ion luminescence probes of the structure of biological macromolecules. Accounts of Chemical Research 1981, 14 ( 12), 384-392. 114 48. Zhang, T.; Zhu, X.; Cheng, C. C. W.; Kwok, W.-M.; Tam, H.-L.; Hao, J.; Kwong, D. W.J.; Wong, W.-K.; Wong, K.-L., Water-soluble mitochondria-specific ytterbium complex with impressive NIR emission. Journal of the American Chemical Society 2011, 133 (50), 20120-20122. 49. Kong, H.-K.; Chadbourne, F. L.; Law, G.-L.; Li, H.; Tam, H.-L.; Cobb, S. L.; Lau, C. K.; Lee, C.-S.; Wong, K.-L., Two-photon induced responsive f-f emissive detection of Cyclin A with a europium-chelating peptide. Chemical Communications 201 1, 47 (28), 8052-8054. 50. Zhang, J.-X.; Li, H.; Chan, C.-F.; Lan, R.; Chan, W.-L.; Law, G.-L.; Wong, W.-K.; Wong, K.-L., A potential water-soluble ytterbium-based porphyrin-cyclen dual bio probe for Golgi apparatus imaging and photodynamic therapy. Chemical Communications 2012, 48 (77), 9646-9648. 51. Li, H.; Lan, R.; Chan, C.-F.; Jiang, L.; Dai, L.; Kwong, D. W.J.; Lam, M. H.-W.; Wong, K.-L., Real-time in situ monitoring via europium emission of the photo-release of antitumor cisplatin from a Eu-Pt complex. Chemical Communications 2015, 51 (74), 14022-14025 . 52. Zhou, Y.; Chan, C.-F.; Kwong, D. W.J.; Law, G.-L.; Cobb, S.; Wong, W.-K.; Wong, K.-L., cx.vP3-lsoform specific erbium complexes highly specific for bladder cancer imaging and photodynamic therapy. Chemical Communications 2017, 53 (3), 557-560. 53. (a) Btinzli, J.-C. G., Lanthanide luminescent bioprobes (LLBs). Chemistry Letters 2008, 38 (2), 104-109; (b) Bunzli, J.-C. G., Lanthanides in Nucleic Acid Analysis. Encyclopedia of Metalloproteins 2013, 1 1 I8-1 123. 115 54. Tweedle, M.F.; Eaton, S.M.; Eckelman, W.C.; Gaughan, G.T.; Hagan, J.J.; Wedeking, P.W.; Yost, F.J., Comparative chemical structure and phannacokinetics ofMRI contrast agents. Investigative Radiology 1988, 23, S236-S239. 55. Eliseeva, S. V.; Aubock, G.; Van Mourik, F.; Cannizzo, A.; Song, B.; Deiters, E.; Chauvin, A.-S.; Chergui, M.; Biinzli, J.-C. G., Multiphoton-excited luminescent lanthanide bioprobes: two-and three-photon cross sections of dipicolinate derivatives and binuclear helicates. The Journalof Physical ChemistryB 2010, I 14 (8), 2932-2937. 56. Kennedy, M. L.; Gibney, B. R., Metalloprotein and redox protein design. Current Op inion in Structural Biology 2001, I I (4), 485-490. 57. Sigel, H., Metal Jons in Biological Systems: Volume 40: The Lanthanides and Their Interrelations with Biosystems. CRC Press: 2003, 202-622. 58. (a) Young, S. W.; Sidhu, M. K.; Qing, F.; Muller, H. H.; Neuder, M.; Zanassi, G.; Mody, T. D.; Hemmi, G.; Dow, W.; Mutch, J. D., Preclinical Evaluation of Gadolinium (III) Texaphyrin Complex: A New Paramagnetic Contrast Agent for Magnetic Resonance Imaging. Investigative radiology 1994, 29 (3), 330-338; (b) Young, S. W.; Fan, Q., Imaging of human colon cancer xenograft with gadolinium-texaphyrin. Investigative Radiology 1996, 31 (5), 280-283. 59. Nepomnyashchii, A. B.; Broring, M.; Ahrens, J.; Bard, A. J., Synthesis, photophysical, electrochemical, and electrogenerated chemiluminescence studies. Multiple sequential electron transfers in BODIPY monomers, dimers, trimers, and polymer. Journal of the American Chemical Society 2011, I 33 (22), 8633-8645. 60. Khimich, N.N.; Obrezkov, N.P.; Koptelova, L.A., Synthesis of 5-isothiocyanato-1, 10- phenanthroline. Russian Journal of Organic Chemistry 2006, 42 (4), 555-557. 116 61. Bessette, A.; Nag, S.; Pal, A. K.; Derossi, S.; Hanan, G. S., Neutral Re (I) complexes for anion sensing. Supramolecular Chemistry 2012, 24 (8), 595-603. 117 Appendix I • .. 0- • CU.• • u • ·� • • Figure A.1. 118 Figure A.2. 119 IQ � iD"o· oa·o- c It') c:i 9 · G,) ( 6 o .l r a- D 96'0 es· i �- u DO" vc· l- � - e a:· N "a z -f ez·z 5z·z IC) lc·z cc·z z9·z- N :15 �� IC) ::r: ..,: 0 �i Cl Lr) '+- - 0£'9 IC) Lr) 0 Ii;;) LO tQ .a -ID: <:I m ,...; .l - gz·.l gz· �0 IC) ,...; � � CJ) co ,... � It') • M N - - c:i c:i c:i 0 c:i c:i c:i t:i c:i e Al!BU9µJI paZ!IBWCN Figure A.3. 120 0 IO 0 ... O :J 0 «> u ic 1 - ������--��� ...... ---= � O0 :J ... I() .. 0 N .r :a 't- ... M 0 &9 <:-·��� --��-- --�� oe=::::::!� '� :I rt • 0t!S ....K .e � "! � • C"I_ N0 :1 I O E · 6 Ng ::J IO "" 0 t6 '° 16 0 .; IO � 0 • - 0 8 Jg !I .. N .... 8 ou- ....,Ng :r ti'9! l � O'I w I': � If! ... 0 c:i 0 0 0 0 0 Attue1u1 P•ZlllWION Figure A.4. 121 R g h s ... N D -� .... 1 i I - 09 Cl� t . 1 I c hi-:... .// I I 08 :I 07 ! 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