Functionalized BODIPY Dyes for Near-Infrared Emission of

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Functionalized BODIPY Dyes for Near-InfraredEmission of Lanthanide ComplexesRukshani Wickrama ArachchiEastern Illinois UniversityThis research is a product of the graduate program in Chemistry at Eastern Illinois University. Find out moreabout the program.

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Recommended CitationArachchi, Rukshani Wickrama, "Functionalized BODIPY Dyes for Near-Infrared Emission of Lanthanide Complexes" (2017). MastersTheses. 2911.https://thekeep.eiu.edu/theses/2911

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Functionalized BODIPY 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

I N 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}'ESIS COMMITTEE CHAIR DATE DEPARTMENT/SCHOOL CHAIR

OR CHAIR'S DESIGNEE

3iJJ/J7-THESIS COMMITTEE MEMBER DATE THESIS COMMlfTEE MEMBER

10/11 THESIS COMMITTEE MEMBER DATE THESIS COMMITTEE MEMBER

DATE

DATE

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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . viii

LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .ix

LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiv

CHAPTER 1 INTRODUCTION

1.1. Overview

1.2. Luminescence

1.2.1. Fluorescence

1.2.1.1. Light-Matter Interaction

1.2.1.2. Emission Process

1.2.1.3. Excitation Spectrum

1.2.1.4. Emission Spectrum

1.2.1.5. Fluorescence Quantum Efficiency

1. 2.1. 6. Lifetime of Excited State

1.2. 1. 7. Factors Affecting for the Fluorescence Intensity

1. 2.1. 8. limitations of Fluorescence

1.2.2. Phosphorescence

1.2.3. Fluorophores for Medical Diagnosis

1.3. Fundamentals of Lanthaoides

1.3.1. Physical Properties

1

2

3

3

4

5

5

7

8

8

9

10

I 1

15

16

l .3.2. Chemical Properties

1.3.3. Formation of Complexes

1 .4. NIR Emitting Lanthanide Complexes

1.4.1. NIR Emission

1.4. 1. l. lanthanide NIR Emission

1.4.1.2. Energy Transfer Mechanism in lanthanide Organic Complex

1.4.2. Lanthanide Complexes

1.4.2.1. DOTA Complexes

1.4.2.2. Porphyrin Complexes

1.4.2.3. Phenanthroline Complexes

1.4.2.4. BOD/PY Based Lanthanide Complexes

1 .4.3. Applications of NIR Emitting Lanthanides

1.5. Motivation

1.6. Objectives

CHAPTER 2 SYNTHESIS & MEASUREMENTS

2.1. General

2.1.1. Materials

2.1.2. Instruments

2.2. Synthesis

2.2.1. Project 1 Synthetic Route

2.2.J. 1. Synthesis of RHl

2.2.1.2. Synthesis of RH2


16

18

2 1

21

2 1

23

24

24

28

3 1

34

38

41

42

43

43

43

44

45

45

46

47

48

ii

2.2. 1.4. Synthesis of RH4


2.2.J.6. Synthesis of RH6

2.2. 1 .7. Synthesis of RH 7

2.2. 1.8. Synthesis of RHB

2.2. 1 . 9. Synthesis of RH9


2.2.2.1. Synthesis of RHJO

2.2.2.2. Synthesis of RHI I

2.2.2.3. Synthesis of RHI 2

2.2.2.4. Synthesis of RHl3

2.2.2.5. Synthesis of RHl4

2.2.2.6. Synthesis of Co mp lex 1

2.2.2. 7. Synthesis of Co mp lex 2

2.3. PhotophysicaJ Measurements

2.3.1 . UV-VIS Absorption Spectra in Solutions

2.3.2. Fluorescence Spectra

2.3.2. 1. Excit at io n and E miss io n Spect ra

2.3.2.2. Qu antu m Yield Measu re me nts

2.3.3. Lifetime Measurements

2.3.3.J. Lifeti me Measu re me nt i n the Visib le Regio n

2.3.3.2. Lifetime Measu re me nt i n the N JR Region

2.3.4. Spectroscopic Titration

49

50

5 1

52

53

54

55

56

57

58

59

60

6 1

62

62

62

63

63

64

65

65

65

65

iii

CHAPTER 3 RES UL TS AND DISCUSSION

3.1. Characterization

3.1.1. NMR Spectroscopy

3. 1. 1 .1 . 1H NMR ofCompound RH/

3.1. 1.2. 1H NMR of Compound RH2


3. 1. 1.4. 1H NMR of Compound RH4

3.1.1.5. 1 HNMR of Compound RH5




3. 1. 1. 9. 1H NMR of Compound RH9

3. 1. 1.10. 1H NMR of Compound RHJO

3. 1. 1.11. 1H NMR of Compound RH/ 1


3. 1. 1.13. 1H NMR o/Compound RHJ3


3.1.2. X-Ray Crystallography

3.2. Photophysical Properties

3.2.1. Photophysical Properties of Ligands

3.2.1.1. Absorption Data

3.2.1.2. Fluorescence Data

3.2.1.3. Quantum Yield Measurements

67

67

67

67

68

69

70

7 1

72

73

74

75

76

77

78

79

80

82

89

89

89

89

90

iv

3.2. 1 .4. lifetime Measurements 98

3.2.2. Photophysical Properties of Lanthanide Complexes 99

3.2.2.1. Interaction ofRH12 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

(formation of complex 2)

3.2.2.3. Spectroscopic Titrations with RH12 and RH14 Ligands

3.2.2.4. Lifetime at NIR Region

CHAPTER 4 CONCLUSION

REFERENCES

APPENDICES

1 0 1

102

105

107

109

1 1 8

v

ABBREVIATIONS

I . BODIPY - boron-dipyrromethene

2. NIR - near-infrared

3. NMR- nuclear magnetic resonance

4. TMS - tetramethylsilane

5. DOTA - 1 ,4,7,1 0-tetraazacyclododecane- I ,4,7, I O-tetraacetic acid

6. DTP A - diethylenetriaminepentaacetic acid

7. TF A - trifluoroacetic acid

8. Ln - lanthanide

9. TCSCT - time-correlated single photon counting technique

10. CAFS - chemoselective alteration of fluorophore scaffolds

1 1 . TRL - time-resolved luminescent

12 . TRLM - time resolved luminescence microscopy

13 . MRI - magnetic resonance imaging

14. PET - photoinduced electron transfer

1 5 . LLBs - lanthanide luminescent bioprobes

16 . TPP - tetraphenylporphyrin

1 7. HMRG - hydroxymethyl rhodamine green

1 8. 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 efficiencies at 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 (±0.0 l) 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

2Fs12 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

lifetime and 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 learning experience at Eastern Illinois University. The

opportunity to learn from 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 1 1

fluorescein, BODIPY (BODIPY-FL), rhodamine (rhodamine green), and

cyanine (Cy5.5) cores

Figure 1.4. Structures of glutaryl phenylalanine hydroxyrnethyl rhodamine 1 2

green (gPhe-HMRG) probe i s activated with chyrnotrypsin secreted in

pancreatic juice and emits HMRG fluorescence

Figure 1.5. Nebraska red dyes 1 3

Figure 1.6. Chemoselective alteration o f fluorophore scaffolds 1 4

Figure 1.7. p H activatable BODIPY-based fluorescent probes 1 5

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 . 1 1 . 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 D 20 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 3 1

mM) in degassed solution (top), in aerated solution (bottom) and in the presence

of O. l mM [(CG)6]2,Aex= 529 nm

Figure 1 .16 Lanthanide complexes of 2-thenoyltrifluoroacetonate complex 3 1

Figure 1 . 17. Emission spectra for the covalently linked [Ln(tta)3(phen)] 32

complexes in the TMOS-DEDMS sol-gel bulk sample, Aex 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

[Ln(Por)(BDL)(OAc)] complexes

33

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 RH l . 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 5 1

x




Figure 2.10. Synthesis of RHl O 56

Figure 2.1 1. Synthesis ofRHl I 57

Figure 2.12. Synthesis of RH1 2 58


Figure 2.14. Synthesis of RH 14 60

Figure 2.15. Synthesis of Complex 1 6 1

Figure 2.16. Synthesis of Complex 2 62

Figure 3.1. ORTEP diagrams o f the single-crystal st ructures of (a) RH4 and (b) 83

RH5 (c) RH6 with 50% thermal ellipsoid probability

Figure 3.2. OR TEP diagrams of the single-crystal structures of (a) RHlO and (b) 86

RH 1 3 with 50% the rmal ellipsoid probability

Figure 3.3. Abso rption spectra for RH1 2 and RH14 ligands in CH 2Ch

( 1 .6 x 10·6 M)

Figure 3.4. Excitation Spectra ofRH12 and RH 14 ligands in CH2Ch

( 1 .6 x 10·6 M)

Figure 3.5. Emission Spectra of RH 1 2 and RH 14 ligands in CH2Ch

( 1 .6 x 1 0·6 M)

Figure 3.6. Trial 1- UV-absorption spectra of RH 1 2 with four different

concentrations in CH2Ch

89

90

90

9 1

xi

Figure 3. 7. Trial 1- Emission spectra of RH 1 2 with four different concentrations 92

in CH2Ch

Figure 3.8. Trial 1- Integrated fluorescence vs absorbance graph for quantum 92

yield determination of RH 12

Figure 3.9. Trial 1- UV-absorption spectra ofRH 1 4 with four different 93


Figure 3.10. Trial 1- Emission spectra of RH 1 4 with four different


93

Figure 3.1 1 . Trial 1- Integrated fluorescence vs absorbance graph for quantum 94

yield determination of RHl4

Figure 3.12. Trial 2- UV-absorption spectra of RH 1 2 with five different 95


Figure 3.13. Trial 2- Emission spectra of RH12 with five different


95


yield determination of RH 1 2

Figure 3.15. Trial 2- UV-absorption spectra ofRH14 with five different 96


Figure 3.16. Trial 2- Emission spectra of RH 1 4 with five different


97


yield determination of RH14

xii

Figure 3.18. Single Exponential Tail fitting of lifetime for RH12 in CH2Ch at 98

room temperature at 53 7 nm

Figure 3.19. Single Exponential Tail fitting of lifetime for RH l 4 in CH2Ch at 99

room temperature at 549 nm

Figure 3.20. (a) Excitation Spectrum (�m= 980 nm) (b)NIR emission under 1 0 1

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 RH 1 2 with [Yb(TPP)(OAc)(MeOH)z] in CH2Ch (a) 103

visible emission from RH1 2 (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 of RH14 with in CH2Ch (a) visible emission from 104

RH 1 4 (b) NIR emission from Yb(III)

Figure 3.26. Back titration by RH 1 4, NIR emission from Yb(III) at 977 nm and 1 05

1005 nm

Figure 3.27. NIR decay curve for complex 1 recorded at 980 nm 106

Figure 3.28. NIR decay curve for complex 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

1 . 1 . Overview

CHAPTER 1

INTRODUCTION

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, DOT A, and phenanthroline based

lanthanide complexes have been synthesized that demonstrate NIR emissions; however,

they require 300-450 nm range energy sources to excite the complexes.4•5•6•7•8

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•1 0

This previous work suggests us to functionalizing BODIPY chromophores further to 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 •1 1

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

definite wavelength 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 effect can 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 for different materials and two main types of photoluminescence

processes were identified. Fluorescence has a shorter excited state ranging from 1 0-12 to

1 0-7 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

after a 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

molecule, S = 1( + � + �) 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,

they are significant if high energy excitation sources are used.1. 12-13

1.2.1.3. Excitation Spectrum

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 Spectrum

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 of other peaks is an indication of impurities 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. If one 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.

Stoke Shift= 107 II A..:x- II 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:

0X = 0 T [ Gradx ] [�]2 S GradsT nsT

0x =Fluorescence quantum yield of compound

0sT = Standard fluorescence quantum yield (A.ex= 480 nm)

Gradx = Gradient from the plot of the intergraded fluorescence intensity vs

absorbance of l.igands and complexes with different 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

quantitative photon counting.1• 14• l6

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

0 = Quantum efficiency

lo= Incident radiation power

€ = Molar absorptivity

b = Path length of the cell

c =Molar concentration

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

ti S2

ti S1

ti So

A

-- -

v

F

ISC

' tt

TIT, So

A : absorption (1 o-1ss)

F : fluorescence (1 o-12-10-ss)

P : phosphorescence (10-6-1 Os)

.... Inter-System Crossing (ISC)

Vibrational relaxation

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 (aka FAM), 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

1 1

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

al lows for the tuning of physical 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

111nm shift

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, peroxynitrite, 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 selective uptake 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 1 937, J. H. van Vleck published a research paper titled as "The Puzzle of Rare-Earth

Spectra in Solids" which attracted the scientific community 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 Y AG (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 �-<liketonates as bioprobes in time-resolved luminescent

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 1 5 lanthanide metals starting from Lanthanum (La, 57),

followed by Cerium (Ce, 58), Praseodymium (Pr, 59), Neodymium (Nd, 60), Promethium

(Pm, 6 1 ), 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

(Yb, 70), and Lutetium (Lu, 7 1 ).27

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 governs lanthanide

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

can be explained by the stabilizing effect of half (4f7) and completely-filled (4f4) shells of

those ions.27• 29

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. 1 8 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 ,4,7,1 0-tetraazacyclododecane-N,N',N",N"'-tetraaceticacid (DOTA) forms

the most stable Ln(lll) complexes (pLn :::::: 2 1 -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-fitted macrocycles 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) -@: ""ICIOJ,(-1

18-uown ..

({) l.,,)...) uypcanc:t (2.2.2)

a. Pre-organized macrocycles

Tb(trff),(solv) >

b. Pre-disposed macrocycles

Eu(Cl04),("10,. >

c. Podands

(Eu(HLJr

aubst. cyden

tripod

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 io n

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 of lanthanide 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( f l l) 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 energy transfer 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 reduction 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

1u•

'SC

A F p

\.

3 .. • � . . . . . .. .... Ln. bt

• • •

\;

CT -

p

...

A : absorption F : fluorescence

P : phosphorescence

... Inter-System Crossing (ISC)

.,_. Energy Transfer (ET) • • • • • Back Transfert (bt)

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, l 0-

tetraacetic acid (Gd(III)-DOT A,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,

tluoresceine, rhodamine, naphalimide) have also improved the sensitivity.34 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 coefficients and

fluorescence intensities that save them using as biological labeling. These dyes are stable

in biological systems and possess higher quantum yields.

In 20 15 , 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 . 1 1 .). The

absorption spectrum of this adduct shows A.abs(max) at 503 nm in aqueous medium assigned

to the So-+S1 transition. 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 BODIPY dyes 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

mindfully alter 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 . 1 1 . 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 20 1 1 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 TF A 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-L l ; A.ex = 400 nm for Ln2-L2), resulted in

characteristic lanthanide emissions in the near-IR region.36

Ln2-Ll

Ln2-L2

1050 lZSO t 4SO wavelength / nm

1650

Figure 1.13. Ln2-L l and Ln2-L2 Complexes and emission spectra for Ln2-L2 (red-

Yb, blue-Nd, green-Er) measured in D20 using .?..ex= 400 nm.36

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 ( 1 3-20 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 l d complex (20 µs).37

29

Table 1.1. Spectral luminescent characteristics of Yb(Iffi porphyrin complexes Compound Luminescence Lifetime (µs)

Spectrum Amax nm (DMSO)

l a Yb( acac )-5-( 4-0 H- 982 16.3 Ph) 10, 1 5,20-( 4-COOCH3-Ph)3

l b Yb(acac)-5-(4-0H- 983 1 3.4 Ph) l 0, 1 5,20-( 4-COOCH3-Ph)3

l e Yb(acac)-5-(3-Py)- 10, 1 5,20- 983 14.8 ( 4-COOCH3-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, 1 5,20-( 4- 976 6-7 COOH-Ph)3

2c Yb(Im)2-5-(3-Py)-l 0, 1 5,20-( 4- 983 5 COOH-Ph)3

Id Yb(acac)-5, 1 0, 1 5,20- (4- 974 20 COOCH3-Ph)4

2d Yb(Jm)2-5 , 10, 1 5,20- (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 transfer efficiency

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 . 1 mM [(CG)6]2,Aex= 529 nm30

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 (cm·1) 16000 13000 10000 7000 12000 10000 8000

a

� b

-::> < -� LJI ·;;; c:: u -.5

600 800 1000 1200 1400 800 1000 1200 1<400

Figure 1 . 1 7. 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)

5/2-1 5/2 for this spectrum) and the different 6FJ levels ((J) 5/2-9/2).

(b) Ln) Nd(llI). All transitions start from the 4f 312 state and end at the 41J levels ((J)

9/2-1 3/2)38

Wavelength (nm)

1 1500 11000 10500 10000 9500 9000

d

850 900 950 1000 1050 1100 1150 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

(c) Ln) Er. The transition starts from the 411312 level to the 411s12 level. (d) Ln) Yb. The

transition starts from the 2Fs12 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 , 1 0- 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 1 538 nm with a l ifetime of 0.6

µs and at 980 nm with a lifetime of 6.6 µs, for erbium 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 ( 1 7 .29), 1 006 40

[Yb(TPP)( 0 Ac)( 4-MePhen)] 949, 978 ( 1 7.29), I 005 40

[Yb(TPP)( 0 Ac)( 4 7-MePhen)] 948, 978 ( 1 7 .82), I 005 40

[Yb(TPP)(OAc )(EPO-Phen)] 948,978 ( 1 4. 1 2), 1 006 40

[Yb(TClPP)(OAc)(5-NH2-Phen)] 980 ( l l .6),1 002 4 1

[Yb(TCIPP)(0Ac)(5-Me-Phen)] 948, 978 ( 1 9.37),1 006 4 1

[Er(TClPP)(OAc)(5-NH2-Phen)] 1538 4 1

[Er(TCIPP)(0Ac)(5-Me-Phen)] 1 539 4 1

[Yb(TPP)(OAc)(DPA)] 950, 975 ( 1 1 .60), 1006,1 023 40

1.4.2.4. BODIPY 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 ( 1 1 0,000 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

Ln(Ll)(NOJ)J

c

Ln

3 Ln(L3)3

eO

MeO

Ln(L21 or L22)3(tpy) R==H (L21) R=Br(L22)

d

I

Ln(L4)3

Yb

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 lifetime of 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 bl 1 3 Ph a e . . h . l otop tys1ca properties o f BODIPY b d l h "d ase ant am e comp exes Lanthanide Complex Aex Aem (NIR Lifetime Reference

(Excitation) Emission) /µs /nm /nm

Er(L l )(N03)3 528 1 527 - 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 1 530 - 9 Yb(L21)3(tpy) 583 978 - 9 Er(L22)3(tov) 583 1 530 - 9 Yb(L22)3(tpy) 583 978 - 9 Er(L3)3 522 1 382 - 1 0 Nd(L3)3 522 1060 - 1 0 Yb(L3)3 522 976,1003 19.78 1 0 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 20 1 1 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

gave a sharp peak at 975 run and a broader peak centering 1 000 nm due to typical 2Fs 2 -

2F112 transition 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 fitting of

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

Figure 1.21. BODlPY based chromophores used to synthesize lanthanide complexes44•45

[n 20 13 , research work was done to understand the recognition ability of different trivalent

lanthanide (La( J r r) , 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 20 1 4 lanthanide metal complexes with L6 (BODIPY with Bispidines) were synthesized

for dual imaging purposes (Figure 1 .2 1 .).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 BODIPY core 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 20 1 1 , Wong et al synthesized a mitochondria specific water-soluble porphyrinato

ytterbium complex linked to rhodamine B. This lanthanide complex exhibits strong two

photoninduced NIR emissions (A.em = 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 20 1 1 , 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-

1 000 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 201 4 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 for use in imaging and photodynamic

therapy.50

In 201 5, 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

6 1 5 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 of killing tumor cells was presented as a multi-modal

lanthanide-porphyrin PDT agent. Strong erbium NIR emission was observed at 654 run,

7 1 5 nm, and 1 5 3 1 nm along with specific affinity for bladder 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 for many 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 for immunoassays. 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. 57 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, functionalization 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

DOT A and phenanthroline ytterbium complexes.

41

1 .6. Objectives

Project 1

• Synthesize and functionalization ofBODIPY based DOTA-ytterbium complexes

./ Synthesize of parent BODIPY dye

Project 2

./ 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

• Synthesize and functionalization of BODIPY based phenanthroline-ytterbium

complexes

./ Synthesize of parent BODIPY dye

./ Synthesize of 5-amino-( 1 , 1 0-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 for intermediate and final

products

./ Record the NIR emission of final lanthanide complexes

42

2.1. General

2.1.1. Materials

CHAPTER 2

EXPERIMENT AL

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 , 1 0-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


A synthetic overview for the 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 o f ytterbium 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 ]difluoroboron)

OH

+ 2 O-N H

1. CH2Cl2, N2 atmosphere, r.t.

2. BF30Et2 , 3h

3. DDQ, l h

4 . NEt3

5. BF30Et2 , 2h

Figure 2.1. Synthesis of RHl

OH

This compound was synthesized according to a previously reported method.59 Under N2

atmosphere, to a solution of 4-hydroxybenzaldehyde (0.243 g, 2.0 mmol) and 2,4-

dimethyl-l H-pyrrole (0.5 mL, 5.0 mmol) in 1 50.0 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 1 5 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. 1 34 g (0.4

mmol, 1 9.8%). 1H NMR (400 MHz, CDCb): 8 = 7. 14 (d, 2Ha), 6.95 (d, 2Hb), 5.97 (s, 2H<l),

46


((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

Cs,C03,DMF r.t,12 h

Figure 2.2. Synthesis of RH2

o� Br

R H l (0.272 g, 0.8 mmol) was dissolved in 50.0 mL of DMF and Cs2C03 ( l .02 g, 3 . 1 3

mmol) was added to flask and stirring was continued for 1 5 minutes. Then 1 ,2-

dibromopropane (2.0 mL, 23. 1 mmol) was added to the reaction mixture. This mixture was

stirred for 1 2 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: 0.0 1 8 g (0.04 mmol, 1 0.0%). 1H NMR (400 MHz, CDCh):

� = 7.20 (d, 2Ha), 7.04 (d, 2Hb), 5.98 (s, 2Hd), 4.35 (t, 2H�, 3.69 (t, 2Hg), 2.55 (s, 6Hc),

1 .42 (s, 6Hc), 0.90 (m, 2Hh).

47


((T-4)-[ 4-[ ( 4-ethyl-3,5-dimethyl- l H-pyrrol-2-yl-KN)( 4-ethyl-�,5-dimethyl-2H-pyrrol-2-

ylidene-KN)methyl]phenolato ]difluoroboron)

OH + 1b= N H

I. CH2Cl2, N2 atmosphere, r.t. 2. BF30Et2 , 3h 3. DDQ, lh 4. NEt3 5. BF30Et2 , 2h


OH

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 . 1 0 mL, 8. 1 5 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 ( 1 2.00 mL, 86.0 mmol) was added to the reaction mixture. After stirring the

reaction mixture for 1 5 minutes, Bf3.0Et2 (8.00 mL, 32.0 mmol) was added to it and


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: 0.3 1 0 g

(0.782 mmol, 1 9.6%). 1 H 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. Synthesis ofRH4

((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)

OH

1,2-Dibromocthanc K2C03, CH3CN 80°C, Sh


O�Br

RH3 (0.162 g, 0.41 mmol) was dissolved in 50.0 mL of CH3CN and anhydrous K2C03

( 1 . 0 1 g, 7 . 3 1 mmol) was added to the flask and stirring was continued for 1 5 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, 6 1 .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


( (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

1,3-Dibromopropane K2co,. CH3CN 80"C, Sh


o� Br

RH3 (0. 1 6 1 g, 0.41 mmol) dissolved in 50.0 mL ofCH3CN and anhydrous K1C03 ( I .O J g,

7.31 mmol) were added to flask and stirring was continued for 1 5 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:

0. 1 10 g (0.21 mmol, 5 1 .2%). 1 H NMR (400 MHz, CDCb): o = 7 . 1 8 (d, 2H3), 7.01 (d, 2Hb),

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


((T-4)-(2-[[4-(4-bromobutoxy)phenyl]( 4-ethyl-3,5-dimethyl-2H-pyrrol-2-ylidene-KN)

methyl]- 4-ethyl-3,5-dimethyl- l H-pyrrolato-KN]difluoroboron)

OH

I ,4·Dibromobutane K1C03, CH1CN 80"C, Sh


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. Synthesis of RH7

0�Br

+


0 (°lo1Bu

(N"'l 0,..,.._.N N'>=

�N-4uo 0 I � 1Bu0r(

0

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 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

fluorescence. Yield: 0. 1 50 g (0. 1 6 mmol, 41 .0%). 1H NMR (400 MHz, CDCh): o = 7 . 1 2

(d, 2H3), 7.08 (d, 2Hb), 4.28 (t, 2Hg), 3. 1 8 (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


O�Br

+


D03A (0.095 g, 0.20 mmol) was dissolved in 50.0 mL of CH3CN. Then K2C03 (2.01 g,

1 4.6 mol) was added to the reaction flask. Next RHS (0. 125 g, 0.24 mrnol) was added to




separate the target product. The isolated product was a bright orange solid with an orange

fluorescence. Yield: 0.088 g (0.09 mmol, 45.0%). 1H NMR (400 MHz, CDCh): () = 7 . 16

(d, 2Ha), 6.96 (d, 2Hb), 4.03 (t, 2Hg), 3.70- 3 . 10 (s, 2Hh), 3.70-2.84 (t, 2Hk), 3 .21-3 . 10 (br,

l 6HiJ), 2.53 (s, 6Hf), 2.30 (q, 2Hd), 2.00-2.05 (m, 2Hm), 1 .44-1.49 (m, 27H1), 1 .32 (s, 6Hc),

0.98 (t, 6He).

53

2.2. 1.9. Synthesis of RH9

O�Br

+ K2C03 •

CH3CN r.t l day


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. 1 25 g, 0.24 mmol) was added 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%). 1 H NMR (400 MHz, CDCb): 8 = 7 . 16

( d, 2H3), 6.97 ( d, 2Hb), 4.01 (t, 2Hg), 3.4 7 (t, 2Hh), 3.20-2.8 1 (s, 2Hk), 3.20-2.81 (br, 16Hij),

2.53 (s, 6Hf), 2.30 (q, 2 Hd), 1 .79 (m, 2Hm), 1 .78 (m, 2H"), 1 .44-1.47 (m, 27H1), 1 .33 (s,

54


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 ofRH 1 2 and RH14 (Complex 1

and Complex 2)

SS

2.2.2. 1. Synthesis of RHJO

( (T-4 )-[ 4-[ ( 4-ethyl-3 ,5-dimethyl- l H-pyrrol-2-yl-t<N)( 4-ethyl-3 ,5-dimethyl-2H-pyrrol-2-

ylidene-t<N)meth yl ]-benzenamina to] di fluoroboron)

+ 2'0= N H

I. CH2Cl2. N1 atmosphere, r.t. 2. BF30Eti , 3h 3. DDQ, lh 4. NEt3 5. BF30Et2 . 2h

Figure 2.10. Synthesis of RH I O

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 for 1 hour. Next

triethylamine (6.00mL, 43.0 mmol) was added to the reaction mixture. After stirring the

reaction mixture for 1 5 minutes, 8f3.0Et2 (8.00 mL, 32.0 mmol) was added to it and


black oily residue was purified using column chromatography on silica with CH2Cb first

and then with CH2Ch: Hexane ( 1 00: 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, CDCh): o = 7.74 (d, 2H3), 7.55 (d, 2Hb), 2.54 (s, 6H�, 2.33 (q, 2Hd), 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 2.1 1. Synthesis ofRH l 1

This compound was synthesized according to a previously reported method.60 To a

suspension of 5-amino- 1 , 1 0-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, l Ha),

9.28 (dd, lHh), 8.62 (dd, l Hc), 8.28 (dd, l Hr), 7.82 (s, l Hc), 7.80 (dd, l Hb), 7.72 (dd, I Hg).

57

2.2.2.3. Synthesis of RH I 2

(N- 1 , 1 O-phenanthrolin-5-yl-[ ((T-4)-[ 4-[( 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)

SCN +

Acetone, ,. r.t, 3 days

Figure 2.12. Synthesis of RH 1 2


suspension of RHlO (0.057g, 0. 1 4 mmol) in 50.0 mL of acetone, RH I 1 (0.053g, 0.22

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 purified on a column chromatography (silica gel, CH2Ch: CH30H = 1 0 : 1 , v/v) to give BDP as bright orange powder. Yield: 0.058 g, 65.5%. 1H NMR (400

MHz, CDCb): 9 . 12 (dd, l Hi), 9.0 1 (dd, 1 H0), 8.54 (dd, lHm), 8.23 (dd, l Hk), 7.99 (s, 1 H1),

7.75 (dd, I H"), 7.73 (dd, l Hj), 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. Synthesis of RHI 3

( (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]-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 of RHI3

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 1 5 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. 1 8 mmol, 9 . 1 %). 1H NMR ( 400 MHz,

CDCb): o = 7.25 (d, l Ha), 7.03 (d, l Hb), 6.88 (t, l Hc), 6.7 (d, I Hd), 2.55 (s, 6Hh), 2.33 (q,

3 Hr), 1 .4 1 (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 R H 1 4


suspension of RH l 3 (0.1 5g, 0.38 mmol) in 50.0 mL of acetone, RH l l (O.l Og, 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, 1 7.5%. 1H NMR

(400 MHz, CDCb): 8 = 9.22 (t, 2HP,q), 8.40 (dd, l Hi), 8.35 (dd, 1 H0), 8.28 (dd, l Hm), 7.77

{s, l Hk), 7.69 (m, 1 H0), 7.68 (m, l Hj), 7.62 (s, 1 H1), 7.53 (d, l H3), 7.49 (s, l Hb), 7.38 (dd,

l Hc), 7 . 1 2 (dd, l Hd), 2.30 (s, 6Hh), 2.0 1 (q, 2Hr), 1 .05 (s, 6He), 0.76 (t, 6Hg).

60

2.2.2.6. Synthesis of Complex 1

Figure 2.15. Synthesis of Complex I

To a suspension of RH1 2 (0.0 1 0 g, 0.016 mmol) m 25.0 mL toluene,

[Yb(TPP)(OAc)(MeOH)2] (0.014 g, 0.0 1 5 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. Synthesis 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 1 0 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 Spectra

Excitation and emission spectra for RH 1 2 and RH 1 4 were recorded using steady-state

excitation with a xenon arc lamp. Both RH 1 2 and 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 5 1 0-700 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 1 04 M concentration (in ethanol) was prepared and 0, 50, 100,

1 50, 1 85 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 1 0 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 ofRH 1 2 with 2 . 1 6 x 1 04 M 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. RH1 4 with 1 .93 x 1 0·4 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 1 2 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 field monochromator

(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 10-4 M. The initial concentrations of RH 1 2 and

RH 1 4 were 2. 1 6 x 1 0-4 M and 1 .93 x 1 0-4 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 ( 1 00 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 1 2 and

RH 14. These two approaches were used to confirm the stoichiometry between the ligand

and the Yb(III) ion in both complexes.

66

3.1. Characterization

3.1.1. NMR Spectroscopy

CHAPTER 3

RESULTS AND DISCUSSION

3. 1 . 1 . 1. 1H NMR of Compound RHJ

OH

1H NMR (400 MHz, CDCb): 8 = 7 . 14 (d, 2H8), 6.95 (d, 2Hb), 5.97 (s, 2Hd), 2.55 (s, 6He),

1 .44 (s, 6Hc).

The 1 H 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 NMR of Compound RH2

f I o� h

e

1H NMR (400 MHz, CDCb): 8 = 7.20 (d, 2H3), 7.04 (d, 2Hb), 5.98 (s, 2Hct), 4.35 (t, 2Hf),

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 of methyl groups on the BODIPY

core, labeled c, appeared as a singlet at 1.42 ppm.

68

3. l. l.3. 1H NMR ofCompoundRH3

OH

e

1H NMR (400 MHz, CDCb): 8 = 7 . 1 2 (d, 2H3), 6.95 (d, 2Hb), 2.52 (s, 6Hr), 2.30 (q, 3H<l),

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 . 1 2 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 -CH3 protons in the ethyl group bound to BODIPY

core appeared at 0.98 ppm.

69


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


1H NMR (400 MHz, CDCb): o = 7 . 18 (d, 2Ha), 7.01 (d, 2Hb), 4 . 17 (t, 2Hg), 3.65 (t, 2Hh),

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 l inked 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


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


e

f

g j o�Br i h

1 H NMR (400 MHz, CDCb): 8 = 7. 1 7 (d, 2Ha), 7.00 (d, 2Hb), 4.08 (t, 2H&), 3.52 (t, 2Hh),

2.53 (s, 6Hr), 2.30 (q, 3 Hd), 2. 1 2 (m, 2Hi), 2.00 (m, 2Hi), 1 .33 (s, 6Hc), 0.98 (t, 6He).

The 1 H NMR spectrum for RH6 is i llustrated 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


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 NMR of Compound RH7

1 H NMR (400 MHz, CDCb): 8 = 7. 1 2 (d, 2Ha), 7.08 (d, 2Hb), 4.28 (t, 2Hg), 3 . 1 8 (t, 2Hk),

b . . f d I 3.07 (t, 2H ), 2.91 (br, 1 6H1J), 2.52 (s, 6H ), 2.30 ( q, 3H ), 1 .40- 1 .46 (s, 27H ), 1 .30 (s,

6Hc), 0.98 (t, 6He).

The 1 H 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 . 1 2 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 DOT A moiety. The other protons in the DOT A 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

1H NMR (400 MHz, CDCb): o = 7 . 1 6 (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. 1 0 (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


7 . 1 6 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 difficult to assign due to peak overlapping. The methyl groups

74

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 .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

1 H NMR (400 MHz, CDCb): o = 7. 1 6 (d, 2H3), 6.97 (d, 2Hb), 4.01 (t, 2Hg), 3.47 (t, 2Hh),

3.20-2.81 (t, 2Hk), 3.20-2 .8 1 (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 1 H NMR spectrum for RH9 is illustrated in the appendix (Figure A.9). The phenol


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

1H NMR (400 MHz, CDCb): o = 7.74 (d, 2Ha), 7.55 (d, 2Hb), 2.54 (s, 6Hr), 2.33 (q, 3H<l),

1 .33 (s, 6Hc), 0.99 (t, 6He).

The 1 H NMR spectrum for RH I O 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 NMR (400 MHz, CDCb): 8 = 9.33 (dd, l H3), 9.28 (dd, l Hh), 8.62 (dd, l Hc), 8.28 (dd,

I H�, 7.82 (s, l He), 7.80 (dd, l Hb), 7.72 (dd, I Hlt).

The 1H NMR spectrum for RHI 1 is illustrated in the appendix (Figure A. 1 1 ). 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


I

1 H NMR (400 MHz, CDCh): 9 . 12 (dd, ! Hi), 9.01 (dd, 1 H0), 8.54 (dd, l Hm), 8.23 (dd, I Hk),

7.99 (s, l H1), 7.75 (dd, l H"), 7.73 (dd, I Hj), 7.67 (d, 2H3), 7.65 (d, 2Hb), 2.52 (s, 6H\ 2.29

(q, 3Hd), 1 .30 (s, 6Hc), 0.96 (t, 6Hc).

The 1 H NMR spectrum for RH 1 2 is illustrated in the appendix (Figure A . 1 2). Thirteen

major peaks were produced. Seven signals were observed in this spectrum corresponding

to protons in 2-9 positions in I , 1 0-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 RH 13

b

1H NMR (400 MHz, CDCb): o = 7.25 (d, l Ha), 7.03 (d, l Hb), 6.88 (t, l Hc), 6.7 (d, l Hd),

2.55 (s, 6Hh), 2.33 (q, 3H�, 1 .4 1 (s, 6He), 1.03 (t, 6Hg).

The 1H NMR spectrum for RH13 is illustrated in the appendix (Figure A . 1 3) . 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 quartet at 2.33 ppm corresponds to the -CH2 in the ethyl

group bound to BODIPY core. At 1 .4 1 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

i

k j

h

1 H NMR (400 MHz, CDCb): o = 9.22 (t, 2HM), 8.40 (dd, I Hi), 8.35 (dd, I H0), 8.28 (dd,

l Hm), 7.77 (s, l Hk), 7.69 (m, l H"), 7.68 (m, l Hj), 7.62 (s, 1 H1), 7.53 (d, l H3), 7.49 (s, l Hb),

7.38 (dd, I He), 7 . 12 (dd, l Hd), 2.30 (s, 6Hh), 2.01 (q, 3H1, 1.05 (s, 6He), 0.76 (t, 6Hg).

The 1 H NMR spectrum for RH 1 4 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 , 1 0-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 . 154 18 A 0). The obtained single crystal

structures of RH4, RH5, RH6, RHIO and RH 1 3 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 crystals indicating 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 1 5.28 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 5 1 7.26 5 3 1 .28

Crystal system, Triclinic, P-1 Monoclinic, P21 Monoclinic, P2tfc space group

Temperature (K) 100 173 1 73

a, h, c (A) 1 3 . 1 235 (4), 1 5.0693 (4), 13.9134 (4), 1 0.7963 (6), 22.1 893 ( 13), 1 5 .4029 (4) 16.9277 (4), 1 1 .4 1 50 (6)

20.8463 (6)

ex, p, Y (o) 1 08.293 (2), 1 1 1 .7 1 2 (2), 90, 9 1 . 1 08 (2), 90 90, 99.561 (3), 90 I 0 1 . 152 (2)

V (A3) 2 5 1 6 . 1 3 ( 1 3) 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

T mnh T max 0.564, 0.753 Not Reported 0.565, 0.753

No. of measured. 56696, 9049, 7829 7295 1 , 17614, 32276, 4784, 3927 independent and 1 5028 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 . 1 3 1 , 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 1 2 1 4 3 1 3

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 .55 1(6) 1 .554(4)

B-N2 1 .544(4) 1 .546(7) l .553(5)

Table 3.3. Selected Bond angles for RH4, RH5 and RI 16

Bond Angles (0)

Bond RH4 RHS RH6

N l -B-N2 1 06.7(2) 1 06.8(4) 107.2(2)

F l -B-F2 1 09.7(2) 109. 1(4) 1 09.1(3)

N l -B-FI 1 1 0.0(2) 1 1 1 .4(4) 1 1 0.2(3)

N2-B-F2 1 1 0.7(2) I 09.8(4) 1 1 0.4(3)

N2-B-FI 109.7(2) 1 1 0.6(4) l 09.8(3)

N l -B-F2 1 1 0.0(2) 109. 1(4) 1 1 0.0(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 for all three

ligands. RH5 showed the highest tilting by 1 2.27° while RH4 and RH6 showed 6.68° and

4.88° respectively. This was the major considerable difference 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 I O and (b) RH13

with 50% thermal ellipsoid probability.

Considering the crystal data obtained for RH I 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 1 3 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 1 3 were I 3 . 1 3 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 2 1 .74°in RH10 and 9. 1 1 ° 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 R H I O and R H 1 3

RHlO RH13

Crystal data

Chemical formula CnH2&BF2N3 C23H2i;BF�N3

Mr 395.29 395.29

Crystal system, space group Monoclinic, Cllc Monoclinic, P21

Temperature (K) 100 273

a, b, c (A) 1 7.3936 (7), 1 2.2974 7.5651 (6), 17.4444 ( 1 3), (5), 1 1 .8024 (5) 8.0325 (6)

a. p, Y (o) 1 26.5 1 9 (2) 94.221 (5)

V (A3) 2028.83 ( 15) 1057.16 ( 1 4)

z 4 2

µ (mm ') 0.59 0.69

Crystal size (mm) Not Reported 0.34 x 0.31 x 0.20

Data collection

T mm, T mu Not Reported Not Reported

No. of measured, independent 2 1 247, 1865, 1772 14432,3722, 3256 and observed [/ > 2o(J)] reflections

R;n1 0.030 0.053

(sin 0/A.)mnx (A 1) 0.608 0.602

Refinement

R[F2 > 2o(F2)], wR(F2), S 0.034, 0.093, 1 .04 0.043, 0. 1 3 1 , 1 .09

No. of reflections 1865 3722

No. of parameters 142 274

87

Table 3.5. Selected bond lengths RH 1 0 and RH 1 3

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 for RH I 0 and RH 1 3

Bond Angles (0) Bond RH l O RH13

N l -B-N2 107.5 107.1(2)

F l -B-F2 108.2 108.7(2)

N 1 -B-F l 1 1 0.5 1 1 0.6(2)

N2-B-F2 1 1 O. l I 09.7(2)

N2-B-Fl 1 1 O. l 1 1 0.7(2)

Nl -B-F2 1 1 0.5 I I O. 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, N 1 -B-N2 bond angle ( 1 06°) is quite smaller

than l 09° identical bond angle in all five crystal 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.

0.1

0.08 (,) u 0.06 c: co .D 004 ..... 0 Vl .D 0.02 <!'.

0 450

·O.D2

Absorbance Vs Wavelength/ nm

470 490

- R H l 4

- RH l 2

510 530 550 570 590

Wavelength/ run

Figure 3.3. Absorption spectra for RH l 2 and R H 1 4 ligands in CH2Ch ( l .6 x t 0·6 M)

Absorption maxima for RH 14 is 532 nm which is quite higher than for RH I 2 observed

around 527 nm.

3.2. 1.2. Fluorescence data

Excitation and emission spectra for both RH 1 2 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

RH 1 2 was observed around 526 nm but it was observed at 531 nm for R H 1 4. Emission

maxima for RH 1 2 and R H 1 4 were observed at 540 nm and 548 nm respectively. Higher

89

stokes' shift was observed for R H 1 4 than RH12. With the same concentration, both RH12

and RH 14 showed more or less equal fluorescence intensities.

9.00E+OS

8.00E+OS

7.00E+OS

>- 6.00E+OS

·� S.OOE+OS s:: 2 4.00E+OS s::

,...... 3.00E+05

2.00E+05

l.OOE-105

O.OOE+OO

450

Intensity Vs Wavelength/ nm

470 490 510 530

Wavelength/ run

- RI-1 1 4

- RI-1 1 2

550 570

Figure 3.4. Excitation Spectra of RHl 2 and RH14 ligands in CH2Ch ( 1 .6 x 10-6 M)

2.00E+05

l.50E+05

.£ :g l.OOE+OS 4) ....

..s S.OOE+04

O.OOE+OO

Intensity Vs Wavelength/ nm

- RH14

- RH12

500 520 540 560 580 600 620 640 660 680 700

Wavelength/ nm

Figure 3.5. Emission Spectra of RH12 and RH14 ligands in CH2Ch ( 1 .6 x 10-6 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 1 2 and RH 1 4 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 different

concentrations of RH 12 for the trial I measurements. Figure 3.9, Figure 3.10, and Figure

3.1 1 illustrate the UV-absorption spectra, fluorescence spectra, and integrated fluorescence

intensity vs absorbance graph of different concentrations of RH 14 for trial l measurements.

0.6

0.5

Q) 0.4 u c 0.3 "' ..0 i... 0 0.2 en ..0

<!'. 0 l

0 400

·0.1

Absorbance vs Wavelengths/ nm

420 440 460 480 500 520

Wavelengths/ nm

-- I O uL

40 uL

--60uL

-- 90 uL

540 560 580

Figure 3.6. Trial 1 - UY-absorption spectra of RH12 with four different concentrations in

91

9.00E+05

8.00E+05

7 OOE+05

>. 6.00E+05 .,,, · ;;; 5.00E+05 c B 4 OOE+05

.5 3 OOE+05

2.00E+05

l.OOE+OS

O.OOE+OO

Intensity vs Wavelengths/ nm

--IO uL 40 uL 60 ul

--90 uL

490 510 530 550 570 590 610 630 650 670 690

Wavelengths/ nm

Figure 3.7. Trial I- Emission spectra of RH 1 2 with four different concentrations in

Integrated fluorescence vs absorbance graph for trial I 0 RH l 2

40000000 en c: 0 35000000 •• .,,, y = 6E+08x - 228956 c: ... ··· 0 30000000 R2 = 0.9968 .. .. ··· () .. ·· c: 0 25000000 .. ·· ()

..... en � 20000000 ... 0 .... ::s .. ..

c;:: 15000000 .. ... "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 1 2

92

0.45

0.4

0.35

Cl) 0.3 (.) c: co 0.25

..D 0.2 .... 0 en

0.15 ..D <(

0 1

0.05

0

-0.05 390

Absorbance vs Wavelengths/ run

--45 uL

-- 70 uL

95 uL

120 uL

410 430 450 470 490 510 530 550 570

wavelength/ nm

Figure 3.9. Trial I- UV-absorption spectra of RH 14 with four different concentrations in

l.OOE+06

9.00E+05

8.00E+05

o.> 7.00E+OS (.) � 6.00E+05

-2 5.00E+05 0 J5 4.00E+05

� 3.00E+05

2.00E+05

1 OOE+05

O.OOE+OO

490

Absorbancc vs Wavelengths/ nm

540 590

Wavelengths/ nm

-- 45 uL

70 uL

95 uL

-- 1 20 uL

640 690

Figure 3.10. Trial I- Emission spectra of RH 14 with four different concentrations in

93

0 V) Cl) ......

. S Cl) u c: Cl) C,) V) Cl) i... 0 ::s

c;:;.

"O Cl) ...... "' 5h Cl) ....,

..s

Integrated fluorescence vs absorbance graph for trial l RH 1 4

45000000

40000000

35000000

30000000

25000000

20000000

15000000

10000000

5000000

0

0

y = 7E+08x + I E+07 R2 = 0.9995

•····· .. ·······

0.01

... ··

... ... .... ... •...

..... ··

O.D2 O.D3

Absorbance

.... ······• ..... ······

0.04 0.05

Figure 3.1 1. Trial /- Integrated fluorescence vs absorbance graph for quantum yield

determination of RH 14

Figure 3.12, Figure 3.13, and Figure 3.14 il lustrate the UV-absorption spectra,

:fluorescence spectra, 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

(l) (.) c: Cl3 .D '-0 Vl .D

<

4 50E-Ol

4.00E-01

3.SOE-01

3.00E-01

2.50E-01

2.00E-01

l.50E-01

l.OOE-01

5.00E-02

0.00E+OO

-5.00E-02 4oo

Absorbance vs Wavelength/ nm

420

--20 uL

-40uL

60 uL

80 uL

-- 90uL

440 460 480 500 520 540 560 580

wavelength/ nm

Figure 3. 12. Trial 2- UV-absorption spectra of RH 12 with five different concentrations

9.00E+05

8.00E+05

7.00E+05

C 6.00E+OS

. iii 5.00E+05 c:: � 4.00E+05

..S 3.00E+05

2.00E+05

l.OOE+OS

O.OOE+OO

Intensity vs Wavelength/ nm

--20 uL

--40 uL

60 uL

80 uL

--90uL

490 510 530 550 570 590 610 630 650 670 690

Wavelength/ nm

Figure 3.13. Trial 2- Emission spectra of RH 1 2 with five different concentrations in

A RH 1 2 stock solution with 2 . 1 6 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 il lustrated in Figure 3.14.

The gradient was used to calculate the quantum yield of RH 1 2.

40000000 v 35000000 (.) § 30000000 (.) Cl) e 25000000 0 � 20000000

-0 � 15000000 ro � 10000000 .....

� 5000000 0

0

Integrated fluorescence vs absorbance graph for trial 2 R H 1 2

y = 5E+08x + 470430 R2 = 0.9994 ..

.. ···

.. ····

·

....

0.01

... ..

. ··

.... ····

......

... ·······

O.D2 0.03 0.04 0.05 Absorbance

.....

. •••

0.06 0.07

Figure 3.14. Trial 2- Integrated fluorescence vs absorbance graph for quantum yield

determination ofRH 1 2

0.6

0.5

0 4 v (.) c:: 0.3 «:! ..0 ..... 0 0.2 Cl) ..0 <t'. 0 1

0 400

·0.1

Absorbance vs Wavelength/ nm

--2o uL --45 uL

70 uL 95 uL

-- 120 uL

420 440 460 480 500 520 540 560 580

Wavelength/ nm

Figure 3.15. Trial 2- UV-absorption spectra of RH 14 with five different concentrations

96

1.20E+06

1 OOE+06

O 8.00E+05

. ;;; c 6.00E+05 Cl) ....

..S 4.00E+05

2.00E+05

o.ooE�oo

490

Intensity vs Wavelength/ nm

540 590

Wavelength/ nm

--20uL

-- 45uL

70 uL

95 uL

-- 1 20 uL

640 690

Figure 3.16. Trial 2- Emission spectra of RH 14 with five different concentrations in

60000000 Cl) 0 5 50000000 0 V> � 40000000

::s � 30000000 -0 Cl) � 20000000

Cl) .s 10000000

0

Integrated fluorescence vs absorbance graph for trial 2 RH l 4

0

y = 7E+08x + 4E+06 R2 = 0.9926

...

0.01

... .. · .... · · ·

... · · · .. ·

•.... ·· .... ···

0.02 0.03 0.04

Absorbancc

.. · ... ··

0.05

.......... .. ·

0.06 0.07

Figure 3.17. Trial 2- lntegrated fluorescence vs absorbancc graph for quantum yield

determination of RH14

A stock solution of RH 14 with 1 .93 x 10-4 M 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 il lustrated in Figure 3.17.

From integrated fluorescence vs absorbance plots obtained for two 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 1 7 at 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 l 2 as listed in Table 3.7.

This higher lifetime could be an advantage in indirect energy transfer process of RH 1 4

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

1 0

(/) c :::; 1 o· 0

u , . . ·' . . •, , . . ,. : ., . , , ....

1 0

1 0 0 1 0 20 30 40 50 60 70

T1rne1ns (/)

4 9 CV _, v 0 0 C/l Q) -4 9 O'.'.

Figure 3.19. Single Exponential Tail fitting of lifetime for RH 14 in CH2Ch at room

temperature at 549 nm

T bl 3 7 Ph a e . . h . 1 otop 1ys1ca properties o Ligand AAbs (nm) Aex (nm) �m (nm)

RH 1 2 527 526 540 RH14 532 531 548

f RH 1 2 d RH 1 4 I' d an 1gan s Astokcs Lifetime/ Quantum (nm) (vis) ns Yield

14 4.97 0.55 (±0.0 I ) 1 7 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 0 1 0 nm range. With both RH 1 2 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 (formation 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 i llustrated 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

assigned to typical Yb(III) transition between 2Fs12 ground state and 2F1112 excited state of

the BODIPY based Yb(III) porphyrin complex.

100

432

I- lntensitYl

527

350 400 450 500 550 600 650 70(900

Wavelength (nm) (a)

RH12 +Yb

977

950

1005

1000 1050

Wavelength (nm)

(b)

- 375 nm 440 n 525 nm

- 550nm

1100 1150

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 (formation 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( Il l) porphyrin complex.

101

toO 1000 ,. W-.gtllnm 1100

(a) Contour view

1150

< IOO

MO E � .c IOO f • "° i ! olOO ii:

MO

(b) 30 view

Figure 3.21. Excitation vs NIR emission of complex 2 in CH2Cli

1006

978

900 950 1000

Wavelength (nm) 1050

375 nm

440 nm

S?S nm

550nm

1100

Figure 3.22. NTR emission of complex 2 under different excitation wavelengths

3.2.2.3. Spectroscopic ti/rations with RH/2 and RJl/4 /igands

The RH 1 2 ligand 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 fluorescence intensity at

540 nm gradually decreased until the molar ratio of RH 1 2 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 further confirmed the indirect lanthanide sensitization

process by BODIPY based ligands.

500 550 600 Wavelength (nm)

(a)

liLi7iYbil �o.o; L:0.2 I 0.4 -06 �0.8� I 1.0 c-- 1 .

650

Yb·MeOH by RH12

900 950 1000 1050 1100 1150 Wavelength

(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 --O ul

-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 1 2 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

:;j .i ?:--;;; c Q) £

500 550 600 650

Wavelength (nm)

:;j � � u; c $ E

RH14+Yb

900 950

1006

1000 1050 1100 1150 Wavelength (nm)

Figure 3.25. Interaction of RH 14 with in CH2C'2 (a) visible emission from RH 1 4 (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 RH 14 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

2.00E+03

l.SOE+03

</)

s 0 l.00Et03

u

S.OOE+02

O.OOE+OO

90

-S.OOE+02

Titration ofYb-OH by RHl4

-- O ul

- 20uL

--4oul

- 60ul

-80uL -- lOO uL

-- llOul

Wavelength/ nm

Figure 3.26. Back titration by RH 14, NTR emission from Yb(III) at 977 nm and I 005 nm

For further 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 1 4 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 l ifetime was 8.8636 µs. Even

though the quantum yield, visible lifetime and NlR emission intensity were higher for

RH 1 4 by itself, when it forms the lanthanide complex, a lower NIR lifetime was found than

the corresponding complex formed with RH 12.

105

3000

2500

- 2000 � «I

- 1500 � (/) c Q) 1000 c:

500

0

200

RH12

Equation y = A1"exp(-xlt1) + yO

Adj. R-Squ 0.99898

Intensity yO Intensity A 1

Intensity 11

250

Value Standard E 43.39678 1.2306

6.26845 3.69581E9 12.23947 0.04175

300

Time (µs)

Figure 3.27. NIR decay curve for complex I recorded at 980 nm

3000 • RH14

2500 Equa tion y = A1'exp(-xlt1) +y0

• 2000

Adj. R-Squ 0.98241 Value Standard Er

:::i Intensity yO 52.82228 2.96469 � 1500 Intensity A1 2.70238E 8.9719E12

� ·u; Intensity 11 8.8636 0.12404

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

(RH 12). 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 977-1 006 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

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Appendix

I •

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•

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Figure A.1.

118

Figure A.2.

119

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u vc· l-

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0

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gz· .l

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f cc· z

0£' 9 -

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Al!BU9µJI pa Z!IBWCN

Figure A.3.

IQ � c It') c:i

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e N

IC) N

: 1 5 � �

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Cl Lr)

IC) Lr)

0 Ii;;)

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e

120

� • - l o u-

8 .. 8 9! ti'

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...

u O :J «> 0 ic 1 - ��--�� ....... ---=� O :J ...

O'I c:i

't- .r :a ...

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w 0 I': � If! ... 0 0 0 0

Attue1u1 P•ZlllWION

Figure A.4.

•

• 0 :1 N

g::J N

Jg !I N ....,g :r N 0

0 IO 0 0

I() ..

0 N

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0 .... t!S K .e �

"! � C"I _ I O E · 6 IO ""

0 t6

'° 16

0 .;

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121

g h R

s ...

1 Di -� N .... I

0 91 Cl� - I t . c :I hi-:... .// I I 0 8

0 7 f o.sj �r ,� I I !

N N h 1 ',.../ • .,., 'E <iii' i 0 5] f f f I I 1 e

c .... � '!> J� VI J 0 41 I� : �00131 I! �I� g h 0.3� a b '1 0 t:= � i i2 .... "'" e "" M C') 0_21 ...: \ I ID I · � I 0 ,

0 1 r -.. 99 2.00 "' "' I"' I 1111 '"I., ii

7 5 1 0 &.5 0 0 5.5 � o '4 5 4.0 �. 5 3.0 2 5 20 1 5 1 .0 0 5 0

f--" Chemic.I Shift (ppm)

N N

...... N w

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1.0

0.9

0.8

0.7

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(ti E � 0.41

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0

Br-�BODIPY (RW011 }001.esp

g j o�Br i h

e f

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g «> � 'o;f 11.0

cola a . • 'o;f 'o;f �

h N � (Y) I

a 'o;f � � (Y) (Y) ; "'

4.0 3.5 3.0 Chemical Shin (ppm)

(Y} � N I

f

d O>

..- N � c-.i N I

i j ,_ N O N O cr> �<'-! ..- ..- a a co � N c-.j N N N � N ff-'-:.....S I "-

;:::..:. ,.:; . "L._, 6.044.05 2.142.13

u bl bl bl 2.5 2.0 1 .5

(Y) (Y} ..... I

c

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e

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z .

0.3

0.2

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8'-C2

o'eu r· � \.\ 0 u N I i N g N )

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f F F

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7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1 .5 1.0 0.5 Chemical Shi� (ppm)

� 0 --0.5

I-' N V'1

"Tl �· c: ... ID l> ?>

C3.-BODIPY with 003A finsl.001 .esp

1.0

0.9

0.8

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z

0.3

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0.1

�oc13I a b Pi! � � lB �

�..:,� ;µ 0 Jn ......

c

0 otsu--f g h H>k o� N� o

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C'i I f

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M i0 1 M M '-, \ \ ' 'f C'i I I � I

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1 .6 1 1 270.740.9914 058 284 352 0527 03>.95 6.06 � ""'"' " "" u " '-' ""'"' " �

E5 ci I

�tsl

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 .().5 Chemical Shift (ppm)

...... N O'l

..,, ciQ' c � � )> io

1.0

0.9

0.8

0.7

. � I!! 0.6 .. c il 0.5· .ti iii E 0 0 4 z .

0 3

0.2

0.1

0

0

C4-BODIFY ,yith D02A(RWOO 10) 001 esp o'eu� /".... \_.o'eu l;t

c

�:�1 CD -.:I" b � fq ..... ;:: lii! �

,.,; ,,... ...... ..__, 9 a)

.... I ('N j_l g n

N ) o�(_, N�oBu �.

0

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c;; IC) I

ia I f

d

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B l!ii ... 0

�-- ,...> . -,.....::.i('-·L.,�L....-..:; 1 70 0 7• 0 .8413.67e 27 4.141 7 1 1 7926 91·6.<l� 23 1-1 1-1 11 11 11 1..1 1-l l-I U i..1

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....... N -....I

00·0-------------------:::::

'Jl"L-�------------------.i�----.:=::;::

en 0

00 c:i ..... � � .. "'

0 0 0 0 c:i .... c:i 0

�Rl8llJI P82!1BWJON

Figure A.11.

128

00()-

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1 - j • :a •

. , ...

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M

eio� .c e I r� � · -·-

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fl-

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i · -.:Ill. '

t E ff �

� 0 .,. • � Cl "' • "' ... 0 0 0 0 0 0 0 0 0 0

AllUJll Ptl!.-WJON

Figure A.12.

129

I-' OJ 0

.,., o"Q" c: "' l'D '"!> .... w

0.45

0.4-0

0.35

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0.15

0.10

0.05

0

RH 1�tal sarnple.00 U>Ol 1 r.s p i-.: I

a b e d R iri I

c

h

7.5 7.0 6.5 6.0 5.5 5.0

b

� C'"i I

h fR 7 f

P.l R � N N � . /-" . N 1 N

4.5 4.0 3.5 3.0 2.5 2.0 ChemicaF Shift {ppm)

� ....

e

� .... g

8 ti I

Elbi ..-jl l; � r d

�� 1.5 1.0 0.5

8 d I

.... ,q o o d '

0

(D'Q- 0

VL'O J"� :S bO 9'.'0- 81. 0 l(l o ,.. ..... ., ti()'�- cS

99·�-.,. J" O :J N � o -.s::. oc·z- cS

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Figure A.14.

131

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