SYNTHETIC STUDIES of Re(CO)3 BIOCONJUGATES, AZADIPYRROMETHENES, and FERROCENE-MODIFIED COMPOUNDS a Dissertation Presented To

SYNTHETIC STUDIES OF Re(CO)3 BIOCONJUGATES,

AZADIPYRROMETHENES, AND FERROCENE-MODIFIED COMPOUNDS

A Dissertation

Presented to

The Graduate Faculty at The University of Akron

In Partial Fulfillment

of the Requirements for the Degree

Doctor of Philosophy

Kullapa Chanawanno

December, 2016 SYNTHETIC STUDIES OF Re(CO)3 BIOCONJUGATES,

AZADIPYRROMETHENES, AND FERROCENE-MODIFIED COMPOUNDS

Kullapa Chanawanno

Dissertation

Approved: Accepted:

______Advisor Department Chair Dr. Christopher J. Ziegler Dr. Christopher J. Ziegler

______Committee Member Dean of the College Dr. Claire A. Tessier Dr. John C. Green

______Committee Member Dean of the Graduate School Dr. Sailaja Paruchuri Dr. Chand K. Midha

______Committee Member Date Dr. Wiley J. Youngs

______Committee Member Dr. Thein Kyu

ABSTRACT

+ The rhenium tricarbonyl (Re(CO)3 ) is one of the most commonly used organometallic cores for labeling various molecular types. The direct labeling of Re(I) via imine formation by the use of pyridine-2-carboxaldehyde (pyca) was achieved by

+ coordination to the fac-[Re(CO)3] . The Re(CO)3-aldehyde adduct reacts with a variety of aromatic/aliphatic amines and peptides resulting in Re(CO)3-conjugates.

In the second chapter, this thesis presents a study on Re(CO)3 pyridine–imine complexes with pendant phenol groups. The effects of the position of the hydroxyl group

(para, meta or ortho to the imine) were investigated. These compounds exhibit pH- dependent metal-to-ligand charge transfer bands. A series of Re(CO)3-imine with ferrocenyl units was presented in the third chapter. Hydrazine can be used as a linker to connect ferrocene and Re(CO)3, resulting in 1:1, 2:1, and 1:2 ferrocene:Re(CO)3 complexes. The electrochemistry and spectroscopy of the compounds were investigated and DFT and TDDFT calculations provided insight electronic structures of the conjugates. In the fourth chapter, this thesis presents a Re(CO)3-modified lysine that can

iii

be generated in a one-pot reaction. This Re(CO)3-modified lysine can be incorporated into four different peptides using solid phase peptide synthesis. And cellular uptake was monitored via the use of a fluorescein modified variant.

The number of studies of azadipyrromethenes (ADPM) and their BF2 complexes

(aza-BODIPY) has been growing due to their long-wavelength absorptions and high emissions observed in these compounds. Additionally, the discovery of the related novel fluorophore, BOPHY, by Ziegler’s group in 2014 has also initiated numerous studies focusing on the development of these compounds.

In the next chapter, this thesis presents a study of structures and photophysical of

ADPMs and several aza-BODIPYs. Both ADPMs and aza-BODIPYs are fluorescent, and the aza-BODIPYs exhibit red shifted emissions relative to their ADPMs analogs. In the subsequent chapter, the synthesis and characterization of the first ADPM and aza-

BODIPY complexes with ferrocene directly connected to the α-pyrrolic carbon is reported. The ferrocene units are electronically coupled to each other. DFT and TDDFT calculations are in agreement with the experimental data. In the last experimental chapter, the first organometallic BOPHY substituted with two ferrocene units is reported.

The study revealed an unprecedented long-range metal-metal coupling between the ferrocene-centered oxidation waves.

ACKNOWLEDGEMENTS

I would like to sincerely thank Dr. Christopher Ziegler, my great Ph.D. advisor and mentor. No words can honestly express my appreciation and describe how lucky I am to be one of his student. He provided support, understanding, and freedom for me to continue several projects, as well as the patience that he has, encourage me to be the chemist I am today. I would also like to extend my gratitude to Dr. Richard Herrick from

College of Holy Cross, Worcester, MA, who always has an insightful answer to my questions and he promptly guides me when I am in doubt.

I would also like to thank the members of my Ph.D. committee: Dr. Claire

Tessier, Dr. Wiley Youngs, Dr. Sailaja Paruchuri and Dr. Thein Kyu for their time and helpful advice. My acknowledgement goes to the UA department of Chemistry for not only accepting me into the program, but also financially supporting me during my Ph.D. career through teaching assistantship. I gratefully acknowledge the Royal Thai

Government for the financial support throughout my study.

I would also like to thank Dr. Victor Nemykin, University of Minnesota at

Duluth, Dr. Sailaja Paruchuri and Dr. Thomas Leeper, University of Akron, for their kind help and collaborations.

Friendship and support are those two things you wish for the most when being

9,000 miles away from home on a Ph.D. journey. Thank you Dr. Sasiwimon (Dao) foryour love and unconditional support. To Diane and Dr. Lakshman Negi, Diane Walter, and Charles ‘grandpa’ Ashley: I am very thankful for all your friendship and help during my ups and downs. I had some not-so-great years and I cannot imagine going through those without you.

To all my labmates: Ingrid, Laura, Abed, Allen, you are my good friends. These people are the best lab crew you could ask for. All of you make me love working in this lab.

Lastly, I would like to thank my family: my grandfather Somkid, my uncles Meta,

Metee, as well as Peter Chant who came to help me get set up during the first week in the

US, and my aunts Huan and Aood. Thank you for believing in this kid.

TABLE OF CONTENTS

Page

LIST OF TABLES ...... xi

LIST OF FIGURES ...... xiii

LIST OF SCHEMES...... xix

LIST OF ABBREVIATIONS ...... xxii

CHAPTER

I. INTRODUCTION AND BACKGROUND ...... 1

1.1 Coordination Chemistry of Re(I) Carbonyl Complexes ...... 1

+ The Rhenium Tricarbonyl Core (Re(CO)3 )...... 1

Pyridine-2-Carboxaldehyde Ligand Systems ...... 5

Re(CO)3 Complexes with Biologically Relevant Pyca-diimine Ligands ...... 10

Re(CO)3 Complexes with Non-Biologically Relevant Pyca-diimine Ligands ..... 16

Optical properties of Re(CO)3 with Pyca-diimine Phonolic Ligands...... 19

Re(CO)3-ferrocene Schiff base complexes ...... 23

+ 1.2 Re(CO)3 Peptide Conjugates ...... 26

Metal-peptide Conjugates ...... 26

Solid Phase Peptide Synthesis (SPPS) and Metal Precursor for SPPS...... 27

vii

Rhenium Metal-Peptide Conjugates and Single Amino Acid Chelator (SAAC) ...... 33

1.3 aza-BODIPY and BOPHY dyes ...... 45

Fluorescence ...... 45

Organic Fluorophores ...... 48

Azadipyrromethene ...... 49

BOPHY dyes ...... 61

II. THE SYNTHESIS AND pH-DEPENDENT BEHAVIOUR OF Re(CO)3 CONJUGATES WITH DIIMINE PHENOLIC LIGANDS ...... 71

Introduction ...... 71

Experimental ...... 73

Results and discussion ...... 85

Conclusions ...... 93

III. USING HYDRAZINE TO LINK FERROCENE WITH Re(CO)3: A MODULAR APPROACH ...... 94

Introduction ...... 94

Experimental ...... 96

Results and discussion ...... 109

Conclusions ...... 121

IV. FACILE RHENIUM–PEPTIDE CONJUGATE SYNTHESIS USING A

ONE-POT DERIVED Re(CO)3 REAGENT ...... 122

Introduction ...... 122

Experimental ...... 125

viii

Results and Discussion ...... 134

Conclusions ...... 143

V. THE SYNTHESIS AND STRUCTURES OF ARENE-SUBSTITUTED AZADIPYRROMETHENES ...... 145

Introduction ...... 145

Experimental ...... 147

Results and Discussion ...... 153

Conclusions ...... 166

VI. SYNTHESIS, REDOX PROPERTIES, AND ELECTRONIC COUPLING IN THE FIRST DIFERROCENE AZA-DIPYRROMETHENE AND AZABODIPY DONOR-ACCEPTOR DYAD WITH DIRECT FERROCENE-α- PYRROLE BOND ...... 168

Introduction ...... 168

Experimental ...... 170

Results and Discussion ...... 178

Conclusions ...... 187

VII. UNUSUALLY STRONG LONG-DISTANCE METAL-METAL COUPLING IN BIS(FERROCENE)-CONTAINING BOPHY: INTRODUCTION TO ORGANOMETALLIC BOPHYS ...... 189

Introduction ...... 189

Experimental ...... 191

Result and Discussion ...... 193

Conclusion ...... 205

VIII. SUMMARY ...... 206

REFERENCES ...... 210

APPENDIX: PERMISSION………...………………………………………………235

LIST OF TABLES

Table Page

2.1 Crystallographic data and structure refinement for 2...... 76

2.2 Crystallographic data and structure refinement for 3...... 77

2.3 Crystallographic data and structure refinement for 4...... 78

2.4 Crystallographic data and structure refinement for 5...... 79

2.5 Crystallographic data and structure refinement for 6...... 80

2.6 Selected bond lengths [Å] and angles [deg] for compounds 1-6...... 81

3.1 Crystallographic data and structure refinement for 4...... 100

3.2 Crystallographic data and structure refinement for 5...... 101

3.3 UV-visible absorption and FTIR data of all compounds……………...... ….……108

4.1 Absorption characteristics data of 2-6 in DMSO………………………...... ………136

4.2 Cellular uptake and accumulation percent values for compounds 4-6

in HUVECs………………..……………..……………….……………….……..…….. 142

5.1 Crystallographic data and structure refinement for 5b...... 158

5.2 Crystallographic data and structure refinement for 5d...... 159

5.3 Crystallographic data and structure refinement for 5f...... 160 xi

5.4 Crystallographic data and structure refinement for 6f...... 161

5.5 Absorption and emission data for compounds 5 and 6 (in CH2Cl2)………...... …....165

6.1 Crystallographic data and structure refinement for 3...... 176

6.2 Crystallographic data and structure refinement for 4...... 177

6.3 Redox potentials (V) of complexes 3 and 4 determined at room temperature in

DCM/0.05 M TBAF system…………………………………………………..……..… 180

xii

LIST OF FIGURES

Figure Page

1.1.1. Splitting energy and d-orbitals of metal in octahedral field...... 2

1.1.2. Molecular orbital diagram of an octahedral ML6 complex...... 3

1.1.3. Top: Molecular orbital of LnM(CO). Bottom: σ and π interactions between CO ligand and metal atom (M)...... 4

99 1.1.4. X-ray crystal structure of TcCl3(CO)3-pyridine-imine compound ...... 8

1.1.5. Crystal structure of Re(CO)3-imine complex a ...... 9

1.1.6. Crystal structure of Re-Br(CO)3-pyridyl-diimine complexes [Re- Br(CO)3(C5H4NCHNC6H5)] (left) and [Re- Br(CO)3(C5H4NCHNCH2CH2CH3)] (right)...... 10

1.1.7. Crystal structures of Re(CO)3Cl(pyca-β-Ala-OEt) (left) and Re(CO)3Cl(pyca- -Asp(OMe)-OMe) (right) ...... 11

1.1.8. Crystal structure (left) and structure drawing the H-bonding arrangement for [ReBr(CO)3(pyca–(CH2)2–COOH)] (right)...... 13

1.1.9. Crystal structure (acetone solvated, left) and packing structure showing the H-bonding arrangement for Re(CO)3Br-pyca-GlyVal (right) ...... 15

1 1.1.10. H NMR spectra of Re(CO)3Br-pyca-GlyGlyGly in acetone-d6 showing the NH proton shift upon addition of OPBu3...... 16

1.1.11. Diagram of electron transition in an octahedral diimine metal complex ...... 17

1.1.12. UV-Vis absorption spectrum (in dichloromethane, 298 K) and structure of [Re(CO)3(bpy)Cl]...... 19 xiii

1.1.13. Left: tilt angle between pyca and phenol planes of Re(I)(CO)3- diimine phenol. Right: calculated metrical data of Re(I)(CO)3-diimine phenolate anion relevant to planarization ...... 21

1.1.14. (a) UV/Vis/NIR absorption spectra of Re(CO)3-diimine phenol in THF upon addition of base P1-tBu and (b) color change of THF solution of Re(CO)3- diimine phenol upon addition of P1-tBu (a → c: depronation) and re-acidification with acetic acid (c → d → e: protonation) ...... 22

1.2.1. Representative examples of (a) bifunctional chelators with NxS4-x donors + and (b) Re(CO)3(dpa) ...... 34

1.2.2. Single Amino Acid Chelate (SAAC) concept scheme...... 35

+ 1.2.3. Structures of [(SAACQ-Re(CO)3) ]G complex (a), fMLF- + [(SAACQ-Re(CO)3) ]G peptide (b), and fluorescence microscopy image + of human leukocytes incubated with 1 nM of fMLF-[(SAACQ-Re(CO)3) ]G peptide (c) ...... 38

1.2.4. Fluorescent micrograph of a single-cell suspension of NSCs incubated with ReSAACQ-HIVTat for 2 hours at 37°C...... 41

1.3.1. Top: Jablonski diagram showing the electronic levels in common organic fluorophores and possible transitions. Bottom: Electrons in singlet and triplet states...... 45

1.3.2. General structure of a azadipyrromethene (aza-DIPY)...... 49

1.3.3. Some previously synthesized aza-BODIPYs ...... 55

1.3.4. Absorption characteristics of the aza-DIPY and aza-BODIPYs in DCM...... 56

1.3.5. Absorption characterisitcs of aza-BODIPYs in DCM with electron- donating substituents ...... 57

1.3.6. Some synthesized aza-BODIPY from literature...... 59

1.3.7. Hg2+ binding of pyridyl-aza-BODIPY and spectroscopic data ...... 60

1.3.8. X-ray crystal structure of BOPHY-2 ...... 64

xiv 1.3.9. BOPHY-5 structure and fluorescence spectra (pH 7, 5, 4, 3.5, 3, 2.8,

2.5, 2.2, 2.0, 1.5, 1 and 1 M, 2 M, 4 M, 6 M, 8 M of HCl, λemiss 490 nm) of 5 μM BOPHY-5 in CH3CN–H2O (1 : 1, v/v) ...... 66

1.3.10. Structures (top), and photophysical data (bottom) for BOPHY-6-BOPHY-11 and their optical data in dioxane ...... 67

2.1. Molecular structures of 2-6. Hydrogen atoms have been omitted for clarity. Crystallographic data, structure refinement, and selected bond lengths and angles are reported in Table 2.1-2.6...... 75

2.2. UV-visible spectra for complexes 1-6 in THF...... 87

2.3. Overlaid UV-visible absorption spectra of 4 (a) and 6 (b) in THF upon addition of 0.0-1.0 equivalent TMAH...... 90

2.4. Overlaid UV-visible absorption spectra of 1 (a) and 3 (b) in THF upon addition of 0.0-1.0 equivalent TMAH...... 91

3.1. Structures of compounds 1-9...... 96

3.2. Crystal structures of compounds 4 and 5 with 35% thermal ellipsoids. Hydrogen atoms have been omitted for clarity. Selected bond lengths and angles are reported in Table 3.1-3.2...... 99

3.3. The partial structure of compound 9...... 111

3.4. UV-visible spectra of compounds 4-9 in THF...... 112

3.5. Cyclic voltammograms of compounds 1, 3, 4, 6, and 8...... 114

3.6. Spectroelectrochemistry of compounds 4 (top) and 5 (bottom) upon oxidation (left) and reduction (right)...... 115

3.7. DFT predicted energy diagram of rhenium complexes 4-9. HOMOs and LUMOs are connected by a dotted line...... 116

3.8. Percent contribution to the frontier orbitals based on molecular fragment for compounds 1 and 3...... 117

xv 3.9. The DFT-PCM predicted isosurfaces of the HOMOs and LUMOs for ferrocene compounds 1 and 3 and conjugates 4-9...... 118

3.10. Percent contribution to the frontier orbitals based on molecular fragment for compounds 4-9...... 119

3.11. Experimental and TDDFT predicted UV-visible spectra for compounds 1, 3, 4, 6 and 8...... 120

4.1. Structures of Re(CO)3 conjugates with a neurotensin fragment (3), a nuclear localization sequence (RRRC, NLS, 4), a bombesin fragment (5), and a lutenizing hormone releasing hormone fragment (LHRH, 6)...... 130

4.2. The chromatograms of 4 (100% A → 100% B in 25 min): 19 min, 5 (100% A → 100% B in 30 min): 25 min, and 6 (100% A → 100% B in 32 min): 25 min with the MALDI-TOF spectra. (Chromatograms: blue trace: 280 nm, red trace: 254 nm detection)...... 133

4.3. FT-IR and UV-visible spectra of compounds 1, 2 and 4-6...... 136

4.4. Fluorescent images of peptides appended beads. Left column: GFP at 470 nm excitation. Right column: Texas red at 594 nm excitation for compound 4 (a), 5 (b), and 6 (c)...... 137

4.5. Fluorescent micrographs (20x magnification) of HUVECs incubated with 40 μM of compound 4 (first row), 5 (second row), and 6 (third row) for 24 hours. Images were achieved in GFP (a), DAPI (b), and merged (c) modes...... 140

4.6. Fluorescent micrographs (20x magnification) of HUVECs incubated with 5 μM of compound 4 (first row), 5 (second row), and 6 (third row) for 24 hours. Images were achieved in GFP (a), DAPI (b), and merged (c) modes...... 141

4.7. HUVECs incubated with 40 μM of 3, excited at 470 nm, showing uptake of the polypeptide...... 141

4.8. Rhenium emission intensity and rhenium uptake concentration calibration curve from ICP experiment. Relative standard deviations of replicate measurements (N = 3) of single calibration points are <6%...... 143

xvi 5.1. The structures of dipyrromethene (1, left) and azadipyrromethene (2, right)...... 146

5.2. Structures of 5b, 5d, 5f and 6f with 50% thermal ellipsoids. Non-ionizable hydrogen atoms have been omitted for clarity. Crystallographic data, structure refinement, and selected bond lengths and angles are reported in Table 5.1-5.4...... 157

5.3. Absorption (left) and emission (right) spectra in CH2Cl2 for compounds 5 and 6...... 164

6.1. X-ray crystal structures of 3 and two crystallographically independent molecules of 4...... 175

6.2. Experimental (CH2Cl2) and PCM-TDDFT predicted UV vis spectra of 3 (top) and 4 (bottom)...... 179

6.3. Room-temperature CV data on compounds 3 (left) and 4 (right) in CH2Cl2/0.05 M TBAF system...... 180

6.4. Room-temperature spectroelectrochemical oxidation of 4 at first (top) and second (bottom) oxidation potentials in CH2Cl2/0.15 M TBAF system...... 182

6.5. Reduction of compound 42+ to 4 under spectroelectrochemical conditions in DCM/0.15 M TBAF system...... 183

6.6. Stepwise oxidation of 3 to 3+ and 32+ under spectroelectrochemical conditions in DCM/0.15 M TBAF system...... 184

6.7. DFT-PCM (TPSSh/6-311G(d)) calculated orbital energies for complexes 3 and 4 with pictorial representation of the frontier MOs...... 185

6.8. Compositions of PCM-DFT orbitals for compounds 3 and 4...... 187

7.1. Experimental (top) UV-vis spectra of 1 and 2 in DCM and

TDDFT predicted UV-vis spectra of 2 for Ci and C2 geometries...... 195

xvii 7.2. Predicted-TDDFT of Ci and C2 Symmetry of BOPHY 2 in cm-1 energy scale...... 196

7.3. DFT-predicted frontier MOs of BOPHY 2 compound in Ci geometry...... 197

7.4. DFT-predicted frontier MOs of BOPHY 2 compound in C2 geometry...... 198

7.5. DFT-predicted energy diagram for Ci and C2 geometries of 2...... 200

7.6. DFT-predicted frontier MOs in 2 (Ci symmetry)...... 200

7.7. CV (top) and DPV (bottom) data for BOPHY 2 in DCM/0.05 M TBAF system...... 202

7.8. Changes in UV-vis spectra of BOPHY 2 during its transformation into [2]+ (A) and [2]2+ (B) under spectroelectrochemical conditions in DCM/0.15 M TBAF system...... 202

st nd 7.9. 1 and 2 single-electron oxidations of BOPHY 2 by Fe(ClO4)3...... 203

7.10. NIR band deconvolution analysis of BOPHY [2]+ generated under spectroelectrochemical (DCM/0.15M TBAF, A) and chemical (DCM, B) oxidation conditions...... 204

xviii LIST OF SCHEMES

Scheme Page

1.1.1. (a) General structure of Schiff base amino acid conjugates and

(b) synthesis scheme of new amino acid conjugate diimine M(CO)3 complexes...... 6

1.1.2. Synthesis of Tc(CO)3-Schiff base complex ...... 7

1.1.3. Labeling strategy using activation of pyridine-2-carboxaldehyde (pyca) ...... 8

1.1.4. Schiff base formation reaction between Re(CO)3-imine complexes and amines ...... 9

1.1.5. General synthesis of Re(I)-pyca amino acid complexes ...... 11

1.1.6. Reaction condition of [ReBr(CO)3(pyca–(CH2)2–COOH)] synthesis ...... 13

1.1.7. Synthesis of Re(CO)3Br-pyca-peptide complexes derived from dipeptide and tripeptide...... 14

1.1.8. (a) Deprotonation of a phenol-substituted diimine (catecholato)Pt(II) complex and (b) synthesis of Re(I)(CO)3-diimine phenol ...... 20

1.1.9. Synthesis (top) and crystal structures (bottom) of fac-[ReBr(CO)3(Fc-hydrazide)] complexes; teal, red, gray, purple, and brown colors represent Re, O, C, N, and Br atoms respectively...... 24

1.2.1. General solid-phase peptide synthesis (SPPS) using protected amino acids and metal precursor...... 28

1.2.2. Mechanisms of peptide coupling reaction by using HOBt-DCC (top) and HBTU (bottom) coupling reagents...... 30

1.2.3. The removal of Fmoc by using piperidine...... 31 xix + 1.2.4. Preparation of the lysine based compound [SAAC-Re(CO)3] ...... 36

+ 1.2.5. Solid-Phase Synthesis of fMLF[(SAAC-Re(CO)3) ]G peptide...... 37

+ 1.2.6. SPPS and structure of [SAACQ-Re(CO)3] -HIVTat-G (HIVTat = AcGRKKRRQRRR...... 40

1.2.7. Synthetic scheme and structure of an amyloid plaque imaging agent ε + K(N biotin)-[SAACQ-Re(CO)3] -LVFFAG ...... 43

1.3.1. Synthesis of the first tetraaryl-aza-DIPY ...... 50

1.3.2. Reagents and conditions of tetraarylazadipyrromethenes synthesis

(a)NH4COOH/neat, 30 min, yield 30%; (b) NH4OAc/neat, 1.5h, yield 47%; NH4OAC/EtOH, 24h, yield 33%; NH4OAC/BuOH, 24h, yield 39% ...... 51

1.3.3. Proposed mechanism for the transformation of γ-nitro-β-phenyl- butyrophenone (a) into tetraaryl-aza-DIPY (i)...... 52

1.3.4. (a) Synthesis of aza-BODIPY, aza-DIPY metal complexes and (b) the first synthesized aza-BODIPY dye...... 54

1.3.5. Synthesis (top) , and photophysical data (bottom) for

BOPHY and Me4BOPHY in CH2Cl2 ...... 62

1.3.6. Synthesis (top), and photophysical data (bottom) for BOPHY-1 and BOPHY-2 in THF...... 63

1.3.7. Synthesis (top), and photophysical data (bottom) for 2,7-bis-coupled BOPHY-3 and BOPHY-4 in THF...... 65

1.3.8. Preparation of BOPHY triplet sensitizers:

(I)NH2NH2·H2O, EtOH, AcOH, CH2Cl2, DIPEA and BF3·OEt2. (II) CHCl3, ICl and CH3OH. (III) aldehydes, toluene, p-TsOH and piperidine, 140 oC. 9,10-diphenylanthracene (DPA) was used as an acceptor of triplet-triple annihilation upconversion. diiodoBODIPY was used as a standard for singlet oxygen photosensitization...... 68

1.3.9. Synthesis of the tetraphenyl-containing BOPHYs and optical property of tetraphenylBOPHY1...... 69

xx 2.1. Syntheses of 1-6...... 73

2.2. Proposed electron flow for the compounds bearing para- (top) and ortho-OH-(bottom)...... 92

3.1. Synthesis of new compounds reported in this chapter...... 102

4.1. Top: General scheme for a one pot pyca Schiff base Re(CO)3X reaction. Bottom: Preparation of 1 and 2...... 126

4.2. Synthesis of peptide conjugates 3-6 where amides were formed using the HOBt/HBTU method and cleaved from the resin using TFA: i. Fmoc-deprotection, ii. Washing, iii. Coupling, iv. Repeat washing, v. TFA Cleavage...... 132

5.1. Synthetic pathways of ADPMs and their analogues, i: NaOH, rt in EtOH 24h; ii: CH3NO2, NEt3, reflux in EtOH 24-72 h; iii: NH4OAc, reflux in EtOH 48-72 h; iv: BF3·OEt2, DIPEA, room temp in EtOH 24 h...... 146

6.1. Preparation of the target azadipyrromethene 3 and azaBODIPY 4. Reagents and conditions: (i) PhCOCH3, NaOH/EtOH, rt/24 h, yield 68%; (ii) CH3NO2, NEt3/EtOH, heat/72 h, yield 70%; (iii) NH4OAc, EtOH, heat/72 h, yield 4.4%; (iv) BF3·Et2O DIPEA/CH2Cl2 heat/24 h, yield 47%...... 170

7.1. Preparation of the ferrocene-containing BOPHY 2...... 191

xxi LIST OF ABBREVIATIONS

MLCT – Metal-to-ligand charge transfer

LLCT – Ligand-to-ligand charge transfer

DMSO – Dimethyl sulfoxide

CDCl3 – Deuterated chloroform

DMF – Dimethylformamide

THF – Tetrahydrofuran

CH2Cl2/DCM – Dichloromethane

EtOH – Ethanol

NEt3 – Triethylamine

DIPEA – N,N-Diisopropylethylamine

TFA – Trifluoroacetic acid

HOMO – Highest occupied molecular orbital

LUMO – Lowest unoccupied molecular orbital

CV – Cyclic voltammetry

DFT – Density functional theory

TDDFT – Time-dependent density functional theory

BODIPY – Boron dipyrromethene xxii ADPM – Azadipyrromethenes

BOPHY – bis(difluoroboron)1,2-bis((1H-pyrrol-2-yl)methylene)hydrazine

SPPS – Solid phase peptide synthesis

Fmoc – Fluorenylmethoxycarbonyl

- HBTU (PF6 ) – (2-(1H-Benzotriazol-1-yl)-N,N,N’,N’-tetramethylaminium hexafluorophosphate

HOBt – Hydroxybenzotriazole

xxiii CHAPTER I

INTRODUCTION AND BACKGROUND

1.1 Coordination Chemistry of Re(I) Carbonyl Complexes

+ The Rhenium Tricarbonyl Core (Re(CO)3 )

+ The rhenium tricarbonyl core (Re(CO)3 ) is one of the most promising currently investigated organometallic cores for labeling of biomolecules. During the past decade,

+ many efforts have been undertaken to modify the Re(CO)3 core to facilitate conjugate formation with various types of molecules including chromophores, fluorophores, photoactive compounds and biologically relevant compounds. This monovalent Re(I) (d6) system receives attention for these types of applications for two main reasons.1 Firstly, it

+ has been hypothesized that the small size of Re(CO)3 core can decrease the likelihood that the biological activity of the conjugate will be altered. Secondly, the closed octahedral coordination sphere is an appropriate ligand system for a compact metal center. In such an environment, the metal center is effectively protected against further ligand attack or re-oxidation. Also, in addition to the thermodynamic stability of such a complex, the kinetic stability or inertness is also of great importance for application in

vivo. The metal centers are in a low spin oxidation state (+1), resulting in inert complexes.2

Crystal field and ligand field theory can be used to explain the inertness and

6 stability of low spin d octahedral Re(I) compounds. Both theories state that the Re(CO)3 unit, when present in an octahedral field, will be low-spin. The CO groups produce a large crystal-field splitting, which is defined by the increased splitting energy Δ0 (Figure

1.1.1).3

Figure 1.1.1. Splitting energy and d-orbitals of metal in octahedral field.

The nature of metal also plays a role in the size of Δ0. Metals utilizing 4d and 5d orbitals, including Re+ ([Xe]4f14, 5d5, 6s1), give rise to a large splitting energy. In octahedral coordination complexes with σ donor ligands, electrons from the ligands fill

six bonding molecular orbitals and electrons from the metal fill the nonbonding t2g and antibonding eg as shown in the molecular orbital diagram in Figure 1.1.2.

Figure 1.1.2. Molecular orbital diagram of an octahedral ML6 complex.

In case of the carbonyl (CO) ligand, the bonding of CO to a metal consists of two components: (a) the electron pair in HOMO donates electron density directly to an appropriate empty metal d orbital a σ-donor interaction and makes the metal more

electron rich. (b) in order to compensate for this increased electron density, a filled metal d-orbital interacts with empty π* orbitals (LUMOs) on CO to relieve electron density.

This second component is called π-backbonding or π-backdonation and is depicted in

Figure 1.1.3.

Figure 1.1.3. Top: Molecular orbital of LnM(CO). Bottom: σ and π interactions between CO ligand and metal atom (M).

Due to the synergistic behavior of σ-donor π-acceptor interactions, bonding between CO and metal is strong; hence CO is a strong field ligand. And as a result,

M(CO)n exhibits a large splitting t2g and eg and the large Δ0. This is the reason of for the

I inertness in Re (CO)3 compounds.

Pyridine-2-Carboxaldehyde Ligand Systems

One strategy for labeling of biomolecules with a metallic nuclide is to synthesize a bifunctional chelator.4–6 For this type of chelator, one part of the compound is designed for conjugation to a biomolecule and a second moiety is for coordination to the metal.

These bifunctional chelates can be produced via two methods: prelabeling (where the metal chelation to ligand, or ‘pre-formed chelate, occurs first) and postlabeling (where the metal chelation to ligand occurs last, called ‘indirect labeling’). In the prelabeling strategy, complex formation is the first step followed by coupling of the complex to the biomolecule. For that purpose, the linking functionality must be activated, i.e. carboxylate group produced by cleavage of an ester formation. Hydrolysis of the activated group with water can compete with labeling, and hence yields are poor. It would be intriguing to bypass this problem by activating the linking group through the metal center itself. It is well known from organometallic catalysis that coordinated aldehydes or ketones are very active, and that they are also activated towards nucleophilic attack with amines for Schiff base formation.7 Schiff bases coordinate much more strongly to the metal center and render the process irreversible. If the amine is a functionality of the biomolecule, this method can be used for a direct labeling without

‘deactivation’ of the metal complex. 5

In 1999, Herrick prepared a novel chelating system that was able to incorporate amino acids in the coordination sphere of an organometallic complex. This chelator can be easily prepared, strongly binds to organometallic centers, and would have one end of the amino acid unbound which is available for further Schiff base formation. They reported the preparation and characterization of Group 6 (Cr, Mo, W) carbonyl complexes containing a new amino acid conjugate diimine ligand (Scheme 1.1.1) based on 2-pyridine-aldehyde (pyca).8 Similar work by Miguel9 will also be described in the following section.

Scheme 1.1.1. (a) General structure of Schiff base amino acid conjugates and (b) 8 synthesis scheme of new amino acid conjugate diimine M(CO)3 complexes.

In the same year, a report from Alberto presented a significant improvement for

99m + obtaining fac-[ Tc-(CO)3] radiolabeled biomolecules containing the pyca-Schiff base

unit (Scheme 1.1.2).10 4-(3-aminopropyl)-1-(2-methoxyphenyl)-piperazine, was reacted with 2-pyridine-carbaldehyde to yield the pyridine-imine Schiff base. Formation of the

99 - Tc(CO)3 Schiff base complex was then carried out by the reaction of [ TcCl3(CO)3]2 in methanol with 1 equiv. of pyridine-imine Schiff base at room temperature. The structure was elucidated by X-ray analysis and is shown in Figure 1.1.4. The strong d-orbital splitting induced by the CO ligands and d6 electronic configuration results in a very inert system, which is preferable in biological applications.

10 Scheme 1.1.2. Synthesis of Tc(CO)3-Schiff base complex.

99 Figure 1.1.4. X-ray crystal structure of TcCl3(CO)3-pyridine-imine compound; teal, green, gray, purple, and red colors represent Tc, Cl, C, N, and O atoms respectively.10

Later in 2003, Alberto and coworkers reported the preparation of Re(I) complexes containing a coordinated aldehyde or ketone along with their reactivity with aliphatic or aromatic amines.7 This allows a direct labeling of biomolecules with Re(I) via imine formation. The use of pyridine-2-carboxaldehyde (pyca) was exemplified in this strategy

+ by coordination to the ‘fac-[Re(CO)3] ’ core as depicted in Scheme 1.1.3. Compounds a and b were synthesized as precursors for subsequent Schiff base formation and the crystal structure of a is shown in Figure 1.1.5.

Scheme 1.1.3. Labeling strategy using activation of pyridine-2-carboxaldehyde (pyca).7

Figure 1.1.5. Crystal structure of Re(CO)3-imine complex a; teal, red, gray, purple, and brown colors represent Re, O, C, N, and Br atoms respectively.7

To show the versatility of Re(CO)3-aldehyde complexes a and b for metal assisted formation of Schiff base complexes, reactions with a variety of aromatic and aliphatic amines were explored. The reaction of a or b with aniline or 1-aminopropane in methanol or methanol-water (v:v, 4:1) rapidly produced the corresponding Schiff base complexes

[Re-Br(CO)3(C5H4NC(CH3)NC6H5)], [Re-Br(CO)3(C5H4NCHNC6H5)] and

[ReBr(CO)3(C5H4NCHNCH2CH2CH3)], respectively (Scheme 1.1.4). Two crystal structures of these complexes are presented in Figure 1.1.6.

Scheme 1.1.4. Schiff base formation reaction between Re(CO)3-imine complexes and amines.7 9

Figure 1.1.6. Crystal structure of Re-Br(CO)3-pyridyl-diimine complexes [Re- Br(CO)3(C5H4NCHNC6H5)] (left) and [ReBr(CO)3(C5H4NCHNCH2CH2CH3)] (right); teal, red, gray, purple, and brown colors represent Re, O, C, N, and Br atoms respectively.7

The pyridine group of the chelate coordinates strongly and induces concomitant activation of the carbonyl group toward primary amines. This strategy has been adopted in several reports in order to create a variety of fac-[Re(CO)3]-Schiff base complexes which reflect different arrays of properties. Some of the work in this field will be discussed below

Re(CO)3 Complexes with Biologically Relevant Pyca-diimine Ligands

Rhenium(I)-pyca compounds with amino ester derivatized diimine ligands,

Re(CO)3Cl(pyca-β-Ala-OEt) and Re(CO)3Cl(pyca-L-Asp(OMe)-OMe), have been synthesized and characterized (Scheme 1.1.5 and Figure 1.1.7).11 This chemistry is similar to the series of M(CO)4(pyca-Xxx-OR) compounds (M = Cr, Mo, W; Xxx represents amino acid) discussed above.8 Reactions were performed using one-pot conditions by stirring pyca and the appropriate amino ester hydrochloride salt in

methanol under nitrogen at room temperature for 20 min. Addition of Re(CO)5Cl followed by reflux for one hour produced the desired products in good yield (47-86%).

Scheme 1.1.5. General synthesis of Re(I)-pyca amino acid complexes.11

Figure 1.1.7. Crystal structures of Re(CO)3Cl(pyca-β-Ala-OEt) (left) and Re(CO)3Cl(pyca- -Asp(OMe)-OMe) (right); teal, red, gray, purple, and green colors represent Re, O, C, N, and Cl atoms respectively.11

Due to a stereogenic center metal center (pseudo-tetrahedral geometry if the three fac carbonyls are considered as one unit), the diastereomers can be formed when using chiral amino esters. Both 13C and 1H NMR analysis confirmed the presence of diastereomers. NMR analysis of Re(CO)3Cl(pyca-L-Asp(OMe)-OMe) showed duplication of some peaks that was consistent with the presence of unequal populations

of diasteromers. Separation by chromatography and fractional crystallization was attempted but was unsuccessful. The electronic absorption spectra of these complexes showed an intense dπ→π* (pyca) MLCT band at around 400 nm, similar to that observed

12–14 in the numerous Re(CO)3(diimine)Cl complexes. IR spectra displayed three metal

-1 carbonyl bands in the region of 2030–1900 cm . This is consistent with a pseudo-C3v symmetry of Re(CO)3-diimine unit with an a1 band and an e band split due to the reduction in symmetry.15

The organometallic Re(CO)3-diimine synthesis via pyca labeling increased over the next few years. The use of amino acids as well as peptides as ligands has been explored to find convenient ways to functionalize biological molecules. The Re(CO)3- pyca conjugate is a straightforward and effective precursor for preparation of diimine complexes under mild conditions. With this synthetic strategy, undesirable side reactions such as halide scrambling11 and racemization can be avoided. The commercially available halopentacarbonyl rhenium complexes (Re(CO)5X; X = Cl, Br) can be used as starting materials in the reaction without further modification.16

The synthesis of compound [ReBr(CO)3(pyca–(CH2)2–COOH)] is one example that demonstrates the use of pyca. The reaction can be carried out at room temperature but refluxing can enhance the reaction speed due to better solubility of amino acids at higher temperature.17 The synthesis of this compound is shown in Scheme 1.1.6.

17 Scheme 1.1.6. Reaction condition of [ReBr(CO)3(pyca–(CH2)2–COOH)] synthesis.

In the solid state, the compound showed interesting called centrosymmetric-dimer arrangement through H-bonding involving both the =O and –OH groups of the carboxylate unit as shown in Figure 1.1.8.

Figure 1.1.8. Crystal structure (left) and structure drawing the H-bonding arrangement for [ReBr(CO)3(pyca–(CH2)2–COOH)] (right); teal, red, gray, purple, and brown colors represent Re, O, C, N, and Br atoms respectively.17

Following the pioneering work by Alberto et al.,7 pyridine-2-carboxaldehyde was used as a convenient precursors to prepare iminopyridine complexes of amino esters, di- and tripeptides via κ2-(N,O) chelation by Miguel et al. in 2012.9 The synthesis was

depicted in Scheme 1.1.7 and the reactions can be carried out in refluxing methanol for 2 hours.

Scheme 1.1.7. Synthesis of Re(CO)3Br-pyca-peptide complexes derived from dipeptide and tripeptide.9

All products were produced in high yield (75-84%). The NMR spectrum of the crude reaction mixture of the GlyVal derivative showed the formation of diastereoisomers. This arises from the presence of two stereogenic centers which are the metal and the α-carbon of the -Val unit. Hydrogen bonding in Re(CO)3Br-pyca-GlyVal can be observed by means of crystal structure (Figure 1.1.9).

Figure 1.1.9. Crystal structure (acetone solvated, left) and packing structure showing the H-bonding arrangement for Re(CO)3Br-pyca-GlyVal (right); teal, red, gray, purple, and brown colors represent Re, O, C, N, and Br atoms respectively.9

In order to evaluate the hydrogen bonding capability of these complexes in solution, an NMR experiment in acetone-d6 using Re(CO)3Br-pyca-GlyGlyGly was carried out. The good H-bonding acceptor tributylphosphine oxide (OPBu3) was added to the sample and which interacts with the protons of amide (NH) and carboxylic acid group

1 (COOH). Figure 1.1.10 shows the H NMR titration spectra between Re(CO)3Br-pyca-

GlyGlyGly and OPBu3. The amide protons NH(a,b) are shifted to lower field upon addition of increasing equivalents of OPBu3. The larger shift of NH(a) can be attributed to the close proximity of this proton to the imine bonded to the metal, which is an electron withdrawing group, resulting in a better NHdonor in (a) rather than (b).9

1 Figure 1.1.10. H NMR spectra of Re(CO)3Br-pyca-GlyGlyGly in acetone-d6 showing 9 the NH proton shift upon addition of OPBu3. (figure reprinted with permission from Inorg. Chem. 2012, 51 (5), 2984–2996. Copyright © American Chemical Society)

Re(CO)3 Complexes with Non-Biologically Relevant Pyca-diimine Ligands

The use of pyca to link amines to Re(CO)3 unit is not limited to the synthesis of biologically relevant complexes. Re(I) complexes incorporating redox-active and/or photoactive ligands have also been investigated. These types of Re complexes are actively studied for potential applications in NLO materials,18 molecular devices19, photocatalysis and energy conversion,20 and chemical or biological sensors.21 The diimine-type ligand is one of the most widely used bidentate chelating moieties for those aforementioned applications. Rhenium complexes having π-acidic diimine (-N=C-C=N-)

functional ligands (such as polypyridyl) display exciting photochemical and photophysical properties, and have been applied in many applications.22,23 The interesting optical absorption of these complexes includes metal-to-ligand charge transfer (MLCT), ligand-to-ligand charge transfer (LLCT), and ligand-to-metal charge transfer (LMCT).24

Figure 1.1.11. Diagram of electron transition in an octahedral diimine metal complex.19

The diagram (Figure 1.1.11) shows the transitions responsible for the electronic absorption spectra of transition metal complexes with diamine type ligands. The πM MOs are filled with d electrons from the transition metal ion. There are many different allowed transitions between different electronic states which appear as bands in their absorption

spectra. The three basic types of electronic transitions are 12 metal-centered (MC)

(transitions localized on the central metal ion, also known as d-d transitions), ligand- centered (LC) or intraligand (transitions between MO localized on the ligands), and charge-transfer (CT) (transitions between MOs of different localization, resulting in displacement of charge typically either from the ligands to the metal (LMCT) or metal to ligand (MLCT)). In addition, there are two other less frequently occurred transitions that are not described in the above diagram. These transitions are ligand-to-ligand (LLCT) and metal-to-metal charge transfer (MMCT).

The charge transfer band discussed above can be observed in the absorption spectrum of [Re(CO)3(bpy)Cl] in Figure 1.1.12 which displays a number of different absorption bands. The higher energy absorption appeared at 295 nm is associated with a bipyridyl based (π-π*) transition.25,26 This ligand centered transition in the free ligand shows up at similar wavelength and exhibits very little change upon complexation to the rhenium. The broad band at 390 nm is assigned to Re → π*(bpy) MLCT transition. The

MLCT absorption band for Re(CO)3 complexes shifts to lower energy with decreasing solvent polarity due to a solvatochromic effect.27 The photochemical and photophysical properties of Re(CO)3 complexes can be fine-tuned by adjusting the ligands coordinated to the Re(CO)3 unit.

Figure 1.1.12. UV-Vis absorption spectrum (in dichloromethane, 298 K) and structure of 25 [Re(CO)3(bpy)Cl]. (figure reprinted with permission from J. Am. Chem. Soc. 1974, 96 (4), 998–1003. Copyright © American Chemical Society)

Optical properties of Re(CO)3 with Pyca-diimine Phonolic Ligands

The Re(CO)3 derivatives have been investigated as components of optical materials, in particular for the Re(CO)3-diimine species due to their novel photophysical

28 properties. The photoluminescent Re(CO)3 complexes have been studied for imaging applications.29–32 In addition, these compounds have been investigated for several applications such as electron transfer dyes in proteins,33 non-linear optical (NLO) materials,34 solar energy conversion20 and as chemical or biological sensors.33

Recent observations showed that a platinum(II) coordinated diimine ligand with a remote acidic phenol group (Scheme 1.1.8(a)) resulted in the unexpected observation of room temperature luminescence after deprononation. The emission, probably originating

from a ligand excited triplet state, appeared to be associated with the planarization of the phenolate-substituted diimine ligand.35

Scheme 1.1.8. (a) Deprotonation of a phenol-substituted diimine (catecholato)Pt(II) 35,36 complex and (b) synthesis of Re(I)(CO)3-diimine phenol.

In this case, the diimine-phenol ligands are considered as non-innocent ligands and can engage in various transformations which include deprotonation of hydroxyl side arms. The π systems of these ligands are readily modified upon the transformation and result in a hypsochromic shift of the catecholato → diimine LLCT bands. Similar behavior was observed in deprotonation of diimine ligands 2,2’-bipyridyl-5,5’- dicarboxylic acid)(3,4-tolyldithiolato)platinum(II) complex.37 Luminescence was also observed for deprotonated species which is possibly due to planarization and

rigidification of phenolate complexes.38 As a consequence, radiationless relaxation processes, such as molecular vibrations, are diminished and emission increases.

A Re(CO3)-diimine complex with phenolic ligand complexes was then synthesized (Scheme 1.1.8(b)) and studied for its pH-dependent optical behavior.36

Re(CO3)-diimine phenol exhibit an absorption band around 370–380 nm in THF which can be tentatively attributed to a combination of dπ(Re) → π*(diimine) and to π(C6H4–

OH) → π*(diimine) transitions. Deprotonation of Re(CO3)-diimine phenol with tert- butyliminotris(dimethylamino)phosphorane (P1-tBu) results in the decline of the 377 nm band and in the appearance of a new band at 589 nm, which is responsible for the blue color of deprotonated species (Figures 1.1.13-14). Two isosbestic points at 328 and 454 nm indicate a simple equilibrium between Re(CO3)-diimine phenol and its phenolate conjugate base. The new absorption band of phenolate conjugate base at 589 nm might arise from an intraligand π–π* charge transfer from the phenolate moiety to the Re(CO)3- diimine unit.

Figure 1.1.13. Left: tilt angle between pyca and phenol planes of Re(I)(CO)3-diimine phenol. Right: calculated metrical data of Re(I)(CO)3-diimine phenolate anion relevant to planarization; teal, red, gray, purple, and green colors represent Re, O, C, N, and Cl atoms respectively.36 21

Figure 1.1.14. (a) UV/Vis/NIR absorption spectra of Re(CO)3-diimine phenol in THF upon addition of base P1-tBu and (b) color change of THF solution of Re(CO)3-diimine phenol upon addition of P1-tBu (a → c: depronation) and re-acidification with acetic acid (c → d → e: protonation).36 (figure reprinted with permission from Dalton Trans. 2010, 39 (40), 9554-9564. Copyright © Royal Society of Chemistry)

The photochemical and electron transfer properties of Re+ (d6) complexes have been a subject of continuous interest due to their promising potential applications such as photocatalysts, photosensitizers, light emitters, or sensors.39–45 Among important classes of photoactive compounds, complexes of the type fac-[Re(X)(CO)3(diimine)] possess low lying excited states with metal-to-ligand charge transfer (MLCT) character46 that can be controlled by variations of the ligands X and diimines.47 These complexes also have capabilities as a building block for multinuclear complexes. The redox potentials of 22

binuclear complexes composed of two redox-active units, such as Re+, and a bridging group also afford a convenient means of assessing electronic interaction. The nature of the metal center and the type of ligands were found to affect the excited states properties of the complexes, which enable the tuning of photophysical and photochemical properties.

Re(CO)3-ferrocene Schiff base complexes

Hydrazone derivatives have been investigated for decades. Their main applications concern their biological activity such as antitumor and antimicrobial properties.48–51 Hydrazones also find applications acting as a building blocks for multidentate ligands with metals.52–54 Moreover, ferrocene (Fc) was found to be potentially useful as electron-donating groups for various type of assemblies which generate very interesting photophysical properties.55,56 Hydrazones are also very useful modules for Schiff base fabrication. With the incorporation of pyridine-2-carboxaldehyde as well as glyoxal (as diazabutadiene unit), hydrazone-Schiff base ligands can readily

57–61 bind to Re(CO)3X unit (X = halides). There are some reports in the past demonstrated the use of ferrocenecarboxaldehyde to produce ferrocenyl ligands which later be used in stepwise reactions with metal carbonyls such as Re, Cr, and Mo.62–64

In 2006, Barbazán and coworkers synthesized a ferrocenyl hydrazide ligand which readily binds to fac-[ReBr(CO)3(NCCH3)2] (Scheme 1.1.9). The resulting red solid

is stable in air. The incorporation of ferrocene (Fc) into the complex was expected to improve bioinorganic and catalytic properties. However, only synthetic methodology, characterization, and crystallographic data was studied and presented in the report and no electrochemical or calculation studies were presented.63 Later in 2009, the same research group reported s similar complex fac-[ReBr(CO)3(Fc-hydrazide)(phenol)] with hydroxyl group. (Scheme 1.1.9, bottom right).64

Scheme 1.1.9. Synthesis (top) and crystal structures (bottom) of fac-[ReBr(CO)3(Fc- hydrazide)] complexes; teal, red, gray, purple, and brown colors represent Re, O, C, N, and Br atoms respectively.63

Cyclic voltammetry results in the range -0.4–1.3 V for the ligand and its Re(CO)3 complex in DCM show the typical monoelectronic oxidation of the ferrocenyl group. The separation between waves corresponding to the anodic and cathodic process is similar to that shown by ferrocene itself under the same experimental conditions. Thus, the 24

electrochemical behavior is a typical quasi-reversible process. The cyclic voltammetric response of free ligand (0.554 V in DCM) is shifted with respect to free ferrocene (0.450

V), which implies a more difficult oxidation process. The N,O-coordination in rhenium complex fac-[ReBr(CO)3(Fc-hydrazide)(phenol)] results in the more positive oxidation of the ferrocene group (0.720 V).63

+ 1.2 Re(CO)3 Peptide Conjugates

Metal-peptide Conjugates

An important goal in drug development is to create optimal targeting systems in which drugs are delivered only to specific tissues or organs. Non-specificity can result in side effects ranging from mild to fatal.65,66 One promising strategy is to conjugate the potential drugs to biomolecules that can provide specificity for certain cells. These biomolecules often bind specific receptors at the cell surface and include amino acids,67 antibodies, proteins and peptides.68

A peptide strategy is an option for bioconjugate design that has much potential.

Peptides are oligomers of naturally-occurring -amino acids that are 5-50 units in length.

Peptides physiologically can function as messengers and signaling molecules in the body.

Peptides chemistry is well established and many peptides are commercially available, so a variety of reagents for their preparation are available.69 Solid phase peptide synthesis

(SPPS) is a convenient and well-established method for the preparation of peptide-based compounds and this technique has been widely applied to the synthesis of metal-peptide conjugates over the past two decades.70

The developments in the peptide conjugate field have led to specific delivery facilitated by improved receptor affinities and/or efficient cellular uptake.32,71 SPPS using insoluble resins as the solid support has been used to synthesize metal complexes based

on peptide backbone ligands. However, the chemistry of the SPPS, in particular the cleavage step, can often be incompatible with metal chemistry. An early attempt by

Gallop72 to use SPPS to obtain (2,2’-bispyridine) dichloro complexes of platinum(II) failed at the cleavage step. The lability of Pt(II)Cl2–ligand bonds in the building blocks relative to the typical C-C covalent bonds in organic molecules resulted in demetalation and the production of uncharacterized platinated species. Since this initial report, the solid-phase synthesis incorporating inorganic complexes, although is a rather new discipline, but has been developed by Metzler-Nolte, Zubieta, Valliant, Heinze and others.32,73–75

Solid Phase Peptide Synthesis (SPPS) and Metal Precursor for SPPS

Solid Phase Peptide Synthesis (SPPS) was invented in 1963 by Bruce Merrifield, who received the Nobel Prize in chemistry in 1984 for his methodology using a solid matrix.76 In SPPS, the peptide is assembled sequentially, amino acid by amino acid, on a solid support. In order to achieve highest selectivity, the incoming amino acid is N- terminally protected by a temporary protecting group (the one being most often used is the Fmoc (fluorenylmethoxycarbonyl) group), and all functional side chain residues are also protected and subsequently deprotected at the end of synthesis sequence. Upon completion, the peptide is cleaved from the resin and all side chain protecting groups are removed at the same time by using concentrated acids (Scheme 1.2.1). During the peptide

synthesis, all soluble reagents and reaction byproducts can be washed away from the peptide-solid support matrix by filtration and at the end of each coupling step.

Scheme 1.2.1. General solid-phase peptide synthesis (SPPS) using protected amino acids and metal precursor.

The coupling reaction, which is the formation of an amide bond (condensation reaction) between a carboxylic acid moiety and an amine group of two different amino acids, is the crucial step in peptide synthesis. The reaction consists of two consecutive steps: (a) activation of the carboxy moiety, and (b) acylation of the amino group. During the first step, the protected amino acid (or peptide) reacts with a “coupling reagent,” yielding a reactive intermediate which is ready for sequential steps. Several reagents that 28

activate the carboxyl groups of the amino acids are used for the coupling reaction. The most used now are uronium and phosphonium salts and hydroxybenzotriazole (HOBt) because of their improved reactivity, greater coupling yield and higher specificity than obtained in conventional systems, such as DCC/HOBt (dicyclohexyl-carbodiimide/1- hydroxybenzotriazol).77 HOBt, developed by W. König and R. Geiger in 1970,78 has been the most popular additive over the past few decades and remains one of the most effective suppressors of racemization reactions. The most important drawback of this additive lies in its explosive character. Another important coupling reagent currently

- being used is HBTU (PF6 ) (2-(1H-Benzotriazol-1-yl)-N,N,N’,N’-tetramethylaminium tetrafluoroborate/ hexafluorophosphate).79 HBTU use is widespread in solid-phase reactions. The mechanism of coupling reaction is shown in Scheme 1.2.2.

H H N N N N O R' O R' R O R O O N O N H H O O O N H N O N N DCC: N C N N N N HOBt: N N -DCC OH

O R' O R' H -HOBt R N R O N O N R'' O N N H H N O O H H N R''

O N N O O N N H H N DIPEA N N N + Fmoc OH Fmoc O CH3 N H C R1 R1 3 N O CH3 CH3 CH3 + PF6 N + H3C N DIPEAH PF6 CH3 HBTU CH3

O R H 2 N -HOBt O peptide NH2 O O

O R O H 2 H N N Fmoc O peptide N H O R1 O

Scheme 1.2.2. Mechanisms of peptide coupling reaction by using HOBt-DCC (top) and HBTU (bottom) coupling reagents.

Solid-phase peptide couplings using HOBt/HBTU require the presence of a base.

Diisopropylethylamine (DIPEA) and N-methylmorpholine (NMM) are the most commonly used bases in Fmoc-based SPPS. Due to the development of strategies based on orthogonal protection, Fmoc has become the most important base-labile N-protecting group. The important feature of the Fmoc group is that it is acid-stable and can withstand cleavage of Boc/tBu (TFA). The Fmoc group is removed via base-induced β-elimination

(usually by using secondary bases, such as piperidine) (Scheme 1.2.3). The Fmoc removal results in the formation dibenzofulvene and carbon dioxide which has to be removed rapidly from the peptide resin or scavenged by a secondary amine (piperidine) to avoid irreversible reaction with the liberated amino group.

Scheme 1.2.3. The removal of Fmoc by using piperidine.

In the final step, concentrated TFA (95% TFA/water) is the standard reagent to cleave the peptide from the resin. All side-chain protecting groups are removed concomitantly. During this cleavage, highly reactive carbocations are generated and they have to be trapped to avoid undesired reactions with sensitive amino acids such as Cys,

Met, Ser, Thr, Trp, Tyr. For this reason, carbocation scavengers are added to the cleavage solution. Water is a moderately efficient scavenger and can be used as single scavenger for the cleavage of peptides devoid of Cys, Met and Trp. Additionally, silane derivatives

(Trimethylsilane: TIS) exhibit more efficient carbocation quenching, especially in peptide sequences containing Arg and Trp residues.80

Over the past two decades, solid-phase peptide synthesis using insoluble resin solid support has been used to produce metal conjugates based on peptide chains. This versatile approach to incorporate metal modified amino acids into SPPS offers several advantages over solution phase or post-solid-phase peptide synthesis conjugation.81 This strategy provides the flexibility to incorporate a metal ion chelator with exclusive site specificity in any amino acid sequence.82 High yields of the conjugation reactions and purity of the conjugate are also advantages from SPPS technique. However, excess amounts of the metal complex precursor for a reaction as well as highly stable metal complexes that can survive all subsequent manipulations, including cleavage from the resin (as mentioned earlier, concentrated acid is needed in this final step), are required.

Due to the fact that many human tumor cells exhibit higher expression levels of small regulatory peptide receptors, these receptors are potential molecular targets for imaging and therapy in cancer treatment, and high affinity metal-peptide conjugates could be exploited for targeting tumor cells in vivo. Decoying the organometallic moiety with a small peptide can also help localize the metal complex to a tumor inside the body using high affinity receptors located on the tumor surface.83 As mentioned earlier, the

combination of cell-targeting peptides with cytotoxic metal complexes can lead to potential drug candidates with fewer adverse side effects.

Rhenium Metal-Peptide Conjugates and Single Amino Acid Chelator (SAAC)

The biological chemistry of the Re(CO)3 unit has been investigated and this fragment has frequently been attached to amino acids and peptides using either

BiFunctional Chelate Agents (BFCA)84 or Single Amino Acid Chelator (SAAC) strategies.31Solid-phase synthesis strategies have been employed to optimize the receptor binding affinity and biodistribution of Re(CO)3-labeled peptides and can be applied to the neighboring metals as technetium and manganese.85 Fluorescent Re complexes can be particularly useful for studying biological processes in vitro due to their long fluorescence lifetimes as well as their large Stokes shifts.32 The coordination chemistry of

+ the Re(CO)3 core and the known stability of the resulting chelate complexes are also suitable for use with the conditions employed in SPPS.74

The early approach to link metal nuclides to a molecule known to possess high affinity binding to a receptor is referred to as the BFCA method. Several previously explored ligands for labeling of peptide with Tc and Re cores are depicted in Figure

1.2.1. Many studies have shown that ligand systems containing nitrogen and thiol sulfur

(SH) positions are effective for the coordination of Tc and Re.86–88 These ligands offer

89,90 NxS4-x donors and –COOH that can be conjugated to the N-terminus of a biomolecule.

One common tridentate BFCA system for Re(CO)3 is also shown in the Figure 1.2.1(b).

The tridentate ligand bismethylpyridylamine (dpa) provides the structural basis for the 33

development of the bifunctional chelate design. The amine nitrogen provides a tethering site for peptide linking whereas the nitrogen atoms of pyridines provide the donor set in the appropriate geometry.91

Figure 1.2.1. Representative examples of (a) bifunctional chelators with NxS4-x donors + 74 and (b) Re(CO)3(dpa) .

Despite the advances in the development of peptide labeling methods and new generations of BFCAs, the ability to vary the position of the ligand within the backbone of the peptide remains a nontrivial challenge. Also, the ability to produce macroscopic amounts of Re complexes can be problematic. Thus, a new approach to producing labeled peptides remains an important goal. Amino acid based ligands that could be incorporated

+ into peptides based on the Re(CO)3 could address the limitations of existing systems such as BFCAs.

+ A good choice of nitrogen donors for Re(CO)3 core are aromatic N- heterocycles.92 These observations lead to the design of new type of chelators constructed

+ from amino acids to provide the donor set for the Re(CO)3 as well as the linker group(s) for peptide attachment. The ideal system should be based on a ligand system that forms stable complexes with the metal core and is able to be incorporated into a peptide at any position using conventional SPPS. The metal complex with the ligand should also be sufficiently stable and able to tolerate SPPS conditions. Consequently, Zubieta et al. created the single amino acid chelate (SAAC) concept, using a modified natural or synthetic amino acid incorporating a chelate terminus (A) and a terminus (C) for attachment to small peptides. The tether (B) can enable the variation of substituents at the amino position and carboxylic group (D) is available for amide bond formation in peptide chain growing process (Figure 1.2.2).74

Figure 1.2.2. Single Amino Acid Chelate (SAAC) concept scheme.74

Zubieta et al., reported the synthesis and characterization of a series of Re(I) complexes bearing tridentate chelates of two pyridines and one tertiary amine donor group.91 The ligands and their Re(I) complexes were prepared in high yield and exhibited a stability to a wide range of reaction conditions making them ideal synthons for SPPS.

With this in mind, Valliant and co-workers prepared an analogous ligand from Fmoc- lysine SAAC. This SAAC is a unique Re(I)-binding amino acid mimic, which has the potential to be incorporated into a peptide at any position using standard SPPS

93 + protocols. The synthesis of [SAAC-Re(CO)3] is shown in Scheme 1.2.4.

O O NHFmoc NHFmoc O HO HO NHFmoc HO H N [NEt ] [Re(CO) Br ] O 4 2 3 3 N N

NaB(OAc)3H CH3OH NH2 N N N N Re OC CO Br CO

+ Scheme 1.2.4. Preparation of the lysine based compound [SAAC-Re(CO)3] .

Fmoc- -lysine was reacted with pyridine-2-carboxaldehyde (pyca) in the presence of sodium triacetoxyborohydride (NaB(OAc)3H). The resulting product was isolated in good yield and there was no epimerization of the lysine α proton. The Re complex was prepared by adding a stoichiometric amount of [NEt4]2[Re(CO)3Br3] in methanol to give

+ + [SAAC-Re(CO)3] . [SAAC-Re(CO)3] was then used in peptide preparation (Scheme

+ 1.2.5). To probe the utility of this SAAC system, a [SAAC-Re(CO)3] derivative of the well-studied fMLF peptide was prepared on a commercial automated peptide synthesizer.

The fMLF sequence has been used to target a formyl peptide receptors (FPR) found on the surface of neutrophils as a means of targeting infection and inflammation sites.94 In

+ this study, the [SAAC-Re(CO)3] was placed between a terminal glycine amino acid (G) and the fMLF targeting sequence.

+ Scheme 1.2.5. Solid-Phase Synthesis of fMLF[(SAAC-Re(CO)3) ]G peptide.

To begin the synthesis, a 4-fold excess of the Fmoc protected dipyridyl complex

[SAAC-Re(CO)3]Br was dissolved in DMF and coupled to the growing peptide chain.

The optimal duration of the coupling steps to afford the desired peptide was determined by exposing a small amount of resin bead samples taken from the reaction mixture to a solution containing ninhydrin. The free amine terminal (NH2) presented will react with ninhydrin and result in blue beads and indicates an incomplete coupling reaction.95 As a result, modification of standard peptide coupling protocols was not necessary.

HPLC analysis of the crude product enabled isolation of the major product as a

+ colorless solid which was confirmed to be fMLF[(SAAC-Re(CO)3) ]G by electrospray

+ mass spectroscopy. IR analysis of fMLF[(SAAC-Re(CO)3) ]G exhibited two distinct CO stretches at wavenumbers 1927 and 2032 cm-1 which is consistent with the proposed geometry of the Re(CO)3 unit and similar to the values reported for the Re complex

+ [SAAC-Re(CO)3] . Follow-up work from the same research group came out in the same year32 reporting more detailed studies and structural modification of fMLF[(SAAC-

+ + Re(CO)3) ]G. The analogous compound [(SAACQ-Re(CO)3) ]G was synthesized by using similar procedure and replacing pyridine-2-carboxaldehyde with quinoline-2-

+ aldehyde (Figure 1.2.3). The SPPS using [(SAACQ-Re(CO)3) ]G successfully generated

+ peptide fMLF-[(SAACQ-Re(CO)3) ]G.

+ Figure 1.2.3. Structures of [(SAACQ-Re(CO)3) ]G complex (a), fMLF-[(SAACQ- + Re(CO)3) ]G peptide (b), and fluorescence microscopy image of human leukocytes + 32 incubated with 1 nM of fMLF-[(SAACQ-Re(CO)3) ]G peptide (c). (microscopy image reprinted with permission from J. Am. Chem. Soc. 2004, 126 (28), 8598–8599. Copyright © American Chemical Society) 38

The fMLF sequence guides the metal nuclide to the formyl peptide receptor (FPR) being expressed on neutrophils. This strategy can serve as a means of imaging white

96 + blood cells. To demonstrate the used as a probe of fMLF-[(SAACQ-Re(CO)3) ]G, a fluorescence microscopy studies was performed by using a 1 nM solution of fMLF-

+ [(SAACQ-Re(CO)3) ]G and incubated with human leukocytes. The Re SAACQ peptide has the appropriate fluorescence properties to be used for in vitro microscopy studies and the uptake was clearly observed using a fluorescence microscopy.

+ Since multi-gram quantities of [(SAACQ-Re(CO)3) ]G can be readily obtained, additional application of peptide-labeling with this complex have been explored. The

SAAC technology was applied to potential peptide which can target NCS. Neural stem cells (NCS) have accrued significant interest for applications in neurodegenerative diseases for their ability to differentiate into neurons, astrocytes and oligodendrocytes.97

Despite their great promise, stem cell transplantations in animal models encounter difficulty in achieving accurate and reproducible graft placements. Despite the investigation of various strategies for monitoring cells with radioisotopes to monitor cell migration,98 these methods suffer from limitations such as high cost and low cell uptake.

One highly efficient and stable delivery SAAC vector is a peptide derived from HIV1-Tat

99 basic domain GRKKRRQRRR48–57. The GRKKRRQRRR48–57 SAAC was prepared and

+ first conjugated with [SAACQ-Re(CO)3] to provide a fluorescent probe for in vitro evaluation.100

1) 20% piperidine/DMF O 2) Wash cycle NHFmoc 3) HBTU, DIPEA, [Re(CO)3-SAACQ][Br] N 80 min. H NHFmoc O O

N N Re OC CO CO Br 1) Cycle 2-4: Fmoc-Arg(Pbf)-OH Cycle 5: Fmoc-Gln(Trt)-OH Cycle 6-7: Fmoc-Arg(Pbf)-OH Cycle 8-9: Fmoc-Lys(Boc)-OH Cycle 10: Fmoc-Arg(Pbf)-OH Cycle 11: AcGly-OH 2) TFA, TIS, EDT, H2O 24 hrs, ambient temp.

H2N NH H2N NH H2N NH H2N NH NH2 NH NH NH NH

O O O O O O H H H H H H N N N N N N OH N N N N N N H H H H H H O O O O O O O

HN O NH2 HN NH2 HN NH2 HN NH2 N

N N Re OC CO Br CO

+ Scheme 1.2.6. SPPS and structure of [SAACQ-Re(CO)3] -HIVTat-G (HIVTat = AcGRKKRRQRRR.

+ The peptides SAACQ-HIVTat and [SAACQ-Re(CO)3] -HIVTat-G were prepared by conventional Fmoc peptide synthesis protocols (Scheme 1.2.6). The peptides were released from the resin using a standard cleavage cocktail, and the products were purified

+ + by preparative HPLC. The presence of the [Re(CO)3] core in [SAACQ-Re(CO)3] -

HIVTat-G was also verified by IR spectroscopy from distinct CO stretches observed at

1 + 1933 and 2035 cm . The uptake of [SAACQ-Re(CO)3] -HIVTat-G in vitro was monitored using fluorescence microscopy. Suspensions of dissociated neurospheres were

+ incubated with [SAACQ-Re(CO)3] -HIVTat-G, and imaged by microscopy (Figure 40

+ 1.2.4). The [SAACQ-Re(CO)3] -HIVTat-G localized primarily in the cell nucleus with a smaller dispersion in the cytoplasm, which is similar to the results reported for a fluorescein isothiocyanate -labeled HIV-Tat analogue.101 The results clearly indicate that

+ the presence of the [SAACQ-Re(CO)3] complex in the peptide backbone did not show a detrimental effect on the cell-penetrating ability of the HIV-Tat sequence.

Figure 1.2.4. Fluorescent micrograph of a single-cell suspension of NSCs incubated with ReSAACQ-HIVTat for 2 hours at 37°C.100 (microscopy image reprinted with permission from Nucl. Med. Biol. 2008, 35 (2), 159–169. Copyright © Elsevier)

The application of SAAC labeling technique was expanded to the area of

Alzheimer’s disease, a progressive neurodegenerative affliction that involves the formation of plaques and neurofibril tangles which ultimately lead to neuronal degeneration.102 Since the formation of plaques has been correlated with the progression of the disease, plaques have emerged as a promising target for the treatment of AD.103

Senile plaques are composed primarily of β-amyloid peptides (Aβ1–40,42,43), naturally occurring peptides that form fibrils and plaques in AD patients. Maggio and co-workers

reported that Aβ1–40 labeled with 125I rapidly deposits on β-amyloid plaques in isolated tissue. However, the 125I-labeled peptide showed particularly poor brain uptake in vivo.104

The conjugation of an Aβ1–40-biotin derivative to streptavidin-tagged monoclonal antibody for the human insulin receptor results in effective transport across the blood brain barrier.105 It is noteworthy that the full Aβ1–40 peptide is not required for plaque binding or for inhibition of β-amyloid fibrillogenesis.106 Moreover, β-sheet breaker peptides containing four amino acids, LVFF, binds to plaques and inhibits fibril formation. Based on these observations, tracers based on β-breaker peptides and biotin should offer solutions to the blood brain barrier problem, a method for imaging β- amyloid plaques, and potential therapies. Stephenson et al.107 designed a peptide system that was capable of incorporating biotin as a linker for conjugation to carrier proteins and

+ of attaching a [SAACQ-Re(CO)3] complex. The synthetic scheme of peptide

ε + K(N biotin)-[SAACQ-Re(CO)3] -LVFFAG is shown in Scheme 1.2.7.

Cycle 2: Fmoc-D-Phe-OH O O Cycle 3: Fmoc-D-Phe-OH Cycle 1 H NHFmoc N Cycle 4: Fmoc-D-Val-OH O O NHFmoc 1) 20% piperidine/DMF O Cycle 5: Fmoc-D-Leu-OH + 2) wash Cycle 6: [SAACQ-Re(CO)3] 3) Fmoc-D-Ala-OH, HBTU, DIPEA Cycle 7: Fmoc-D-Lys(Biotin)-OH 4) wash O S HN H H HN 1) wash NH 2) 20% piperidine O O O O H H H H O 3) wash N N N N HO N N N NH2 H H H 4) 94% TFA, 2% EDT, O O O O 2% TIS, 2% H2O

O FmocNH N OH N N Re [SAACQ-Re(CO) ]+ = OC CO 3 N CO N N Re OC CO CO

O HN H NH O N Fmoc-D-Lys(Biotin)-OH = S NHFmoc COOH

Scheme 1.2.7. Synthetic scheme and structure of an amyloid plaque imaging agent ε + 107 K(N biotin)-[SAACQ-Re(CO)3] -LVFFAG.

ε + Peptide conjugate K(N biotin)-[SAACQ-Re(CO)3] -LVFFAG was tested for its in vitro ability to inhibit fibrillogenesis by using a thioflavin (ThT) standard essay. A unique 43

red shift in ThT’s absorption spectrum occurs when it binds to plaques, which is distinct from the unbound dye (the unbound dye has excitation and emission maxima of 385 and

445 nm, which shift to 450 and 485 nm, respectively).108 As a result, the formation of

Aβ1–40 fibrils can be quantitatively monitored in vitro by measuring the increase in fluorescence intensity at 485 nm wavelength.109 Zhang and co-workers reported the extent of amyloid formation as 76% when incubated with the D-analogue of klvff

108 ε + peptide. The rhenium analogue K(N biotin)-[SAACQ-Re(CO)3] -LVFFAG exhibited

43±2% inhibition when used in a 1:1 ratio with Aβ1–40. These results indicate that the presence of the metal complex and biotin derivative enhanced fibrillogenesis inhibition and did not have a detrimental impact on the ability of the peptide to bind to amyloid.

In conclusion, the Single Amino Acid Chelator (SAAC) method is a powerful peptide-labeling system that can be used to efficiently generate multiple Re- metallopeptides. It is conceivable to build Re-metallopeptides via this SAAC method either by automated peptide synthesis or manual peptide coupling. Various types of metallopeptides can be screened to identify lead agents in a time and resource efficient manner.

1.3 aza-BODIPY and BOPHY dyes

Fluorescence

Luminescence is the radiation emitted by a molecule after it absorbs energy that promotes an electron to an exited state. If the spin of the excited state can be determined, luminescence can be described as either fluorescence or phosphorescence. Compounds which exhibit fluorescent-type emission are called fluorophores.

Figure 1.3.1. Top: Jablonski diagram showing the electronic levels in common organic fluorophores and possible transitions. Bottom: Electrons in singlet and triplet states.

The electronic states of organic molecules can usually be divided into singlet states, where all electrons in the molecule are spin paired and thus the spin is zero, and triplet states, where a pair of electrons is unpaired and spin is equal to one (Figure 1.3.1

(bottom)). When a compound absorbs a photon, one of its valence electrons moves from the ground state to an excited state with conservation of the electron’s spin (singlet excited state). Emission of a photon from this singlet excited state to the singlet ground state is called fluorescence. In some cases an electron in a singlet excited state is transformed to a triplet excited state in which its spin becomes parallel with the electron in ground state. Emission between a triplet excited state and a singlet ground state is called phosphorescence.

Upon excitation of fluorophores with light of suitable wavelength, typically vibrational levels are also excited (as seen in Jablonski diagram, Figure 1.3.1 (top)). The probability of finding the molecule in one of the possible excited singlet states, Sn, depends on the transition probabilities and the excitation wavelength (λexcitation). Upon excitation to higher singlet states, Sn, molecules can relax through the process called internal conversion (IC) to vibrational levels of the first excited singlet state, S1, within

10-11-10-14 seconds (s). Also, molecules in higher vibrational levels will quickly fall to the lowest vibrational level of the same state via vibrational relaxation (VR; 10-10-10-12 s) by losing energy to other molecules through interactions such as collisions. From the lowest lying vibrational level of the first excited singlet state, the molecule can lose energy via non-radiative internal conversion (IC) followed by vibrational relaxation (VR). 110

In excited singlet states Sn, the electron in the excited orbital is paired with the second electron in the ground-state orbital. Consequently, return to the ground state is spin allowed and can occur rapidly often with emission of a photon, which is fluorescence. Typically, the emission rates of fluorescence are 108 s-1 and a typical fluorescence lifetime (τ, is the average time between its excitation and return to the ground state) is on the order of 10 ns (10 x 10–9 s).

The spin of an excited electron can also be flipped by intersystem crossing (ISC), which then converts the molecule to a triplet excited state, T1. Phosphorescence is emission of light from a triplet excited state, where the electron in the excited orbital has the same spin orientation as the ground-state electron. In most organic dyes, intersystem crossing is fairly inefficient because it is a spin forbidden transition, even though the triplet state is lower in energy than the excited singlet state. These forbidden transitions to the ground state and resultant emission rates are slow (103 to 10 s-1), so that phosphorescence lifetimes are typically longer than that of fluorescence (milliseconds to seconds). However, the presence of heavy atoms such as iodine and bromine or 4d and 5d transition metals can substantially accelerate ISC rate constants and decrease the emission lifetime.111 Conversely, even longer lifetimes are possible, especially in the solid state, and can be seen is seen from "glow-in-the-dark" materials in daily life.112

Organic Fluorophores

Fluorescent organic compounds have become indispensable materials in modern applications of science and technology.113 Their uses vary from fluorescent probes,114,115 sensors,116,117 fluorescent indicators,118,119 dyes and pigments,120 and light-emitting devices;121,122 all of these applications play important roles in scientific research and daily life.123 Fluorescent compounds consisting of organic skeletons have the following advantages, including the fine adjustability of their optical properties, their lightweight nature, and ease of synthesize.124–126 Many types of organic fluorescent compounds have been developed over the past few decades.127–129

Organic fluorophores usually exhibit a strong absorption and emission bands in the visible region of the electromagnetic spectrum. Suitable organic fluorescent dyes for most applications are distinguished by a high fluorescence quantum yield (ϕ), which is the ratio of photons absorbed to photons emitted through fluorescence. Quantum yield can be calculated by using equation 1.3.1130:

= Equation 1.3.1 where:

ϕx and ϕST = the fluorescence quantum yields of sample (x) and standard reference

(ST)

Grad = the gradient from the plot of integrated fluorescence intensity vs

absorbance of sample (x) and standard (ST)

η = the refractive index of the solvent of sample (x) and standard (ST)

In other words, the quantum yield shows the probability of the excited state electrons relaxing the ground state by emission rather than by non-radiative mechanisms.

Azadipyrromethene

Azadipyrromethenes (aza-DIPY) and their transition metal complexes or boron

(Figure 1.3.2) were first discovered by Rogers in the 1940s as unexpected dark blue colored products.131,132 Due to their long-wavelength absorptions as well as high emission, an increasing number experimental and theoretical studies have been focusing on aza-DIPYs.

Figure 1.3.2. General structure of a azadipyrromethene (aza-DIPY).

The tetraaryl-aza-DIPY (Figure 1.3.2.) was the first azadipyrromethene structure published.131 Rogers reacted γ-nitro-β-phenyl-butyrophenone (nitroketene) either with formamide under reflux or ammonium formate in a melt process (solvent-less) under the conditions of the Leuckart reaction. However, the conditions were harsh and only low yields of azadipyrromethene were obtained.

The main synthetic effort by the O'Shea group attempted to optimize the reaction conditions. The optimum synthetic conditions were found using of diaryl α,β-unsaturated ketones (chalcones) as the precursor, which can be readily made by an aldol condensation of the corresponding aldehyde and acetophenone (Scheme 1.3.1).133

Scheme 1.3.1. Synthesis of the first tetraaryl-aza-DIPY.131

The Michael addition of nitromethane to the α,β-unsaturated ketones, with triethylamine (TEA) as base, yields the 1,3-diaryl-4-nitrobutan-1-ones in high yields.

These two classical organic synthesis reactions efficiently provide the precursors to the tetraaryl-aza-DIPYs. O'Shea introduced ammonium acetate as an alternative ammonia source for the synthesis and discovered major improvements by the use of alcoholic solvents instead of solvent-less (neat) conditions (Scheme 1.3.2, condition b).134 50

Scheme 1.3.2. Reagents and conditions of tetraarylazadipyrromethenes synthesis (a) NH4COOH/neat, 30 min, yield 30%; (b) NH4OAc/neat, 1.5h, yield 47%; 134 NH4OAC/EtOH, 24h, yield 33%; NH4OAC/BuOH, 24h, yield 39%.

The reaction mechanism is not fully understood due to the lack of the observation of isolated intermediate products. However, a mechanism for this reaction was proposed135 and is shown in Scheme 1.3.3.

Scheme 1.3.3. Proposed mechanism for the transformation of γ-nitro-β-phenyl- butyrophenone (a) into tetraaryl-aza-DIPY (i).135

The key step is the ring closure of the ketimine (b) to the nitronate species which was activated by deprotonation of b. According to the Nef reaction,136 the species d formed can eliminate hyponitrous acid (H2N2O2, forming finally nitrous oxide) and water yielding f. With the rearrangement of f, the pyrrole g is formed.135,137 This pyrrole is nitrosated in situ to give the nitroso-pyrrole h, which readily condenses with another pyrrole unit (g) to form the azadipyrromethene i, as shown in Scheme 1.3.3.135

Aza-DIPY ligands are able to form stable complexes with a variety of metal or main group elements such as boron (Scheme 1.3.4). The metal complexes of aza-DIPY can be synthesized by using divalent metal ions such as Co2+ or Zn2+ and their structural

138,139 and spectroscopic properties were widely investigated. The boron difluoride (BF2) complexes, aza-BOron difluoride-chelated DIPYrromethene (aza-BODIPY), are important examples of aza-DIPY adducts. Aza-BODIPY was first synthesized by Boyer et al. in 1993.140 The most important property of aza-BODIPY is its intense long wavelength absorption (650-800 nm) and fluorescent emission (up to 840 nm). These spectroscopic characteristics are bathochromically shifted compared to the corresponding

BODIPY analogues. The near infrared (IR) spectroscopic emission is important for various applications.134

Scheme 1.3.4. (a) Synthesis of aza-BODIPY, aza-DIPY metal complexes and (b) the first synthesized aza-BODIPY dye.141

From 2002, continuous research by the O’Shea’s group has reignited interest in aza-BODIPY, and has resulted in the syntheses of compounds such as those shown in

Figure 1.3.2.134,142,143 It was found that electron donating substituent in the 3,5-position of tetraarylazadipyrromethenes (Scheme 1.3.4) tends to red shift the absorption. The substitution with dimethylamino groups (-N(CH3)2) in the para-positions of the 3,5- diphenyl rings leads to compound v which gives rise to NIR fluorescence. This

compound was investigated in a sensor application with a pH-responsive absorption and fluorescence change across a broad acidity range.143

H3CO OCH3

N N

N N N N B B F F F F

ii iii H3CO OCH3

CHCl3, 0.36 CHCl3, 0.23 N max abs = 688 nm max abs = 664 nm N N max emiss = 715 nm max emiss = 695 nm B = 85,000 M-1cm-1 = 78,000 M-1cm-1 F F Br Br i

CHCl3, 0.34 max abs = 650 nm N N max emiss = 672 nm -1 -1 79,000 M cm N N N N B B F F F F

iv v (H3C)2N N(CH3)2

CHCl3, 0.34 CHCl3, n/a max abs = 658 nm max abs = 799 nm max emiss = 680 nm max emiss = 823 nm = n/a = 87,000 M-1cm-1

Figure 1.3.3. Some previously synthesized aza-BODIPYs.134,142,143

The UV absorption maxima of the tetraarylaza-BODIPYs strongly depend on the nature of the aryl (Ar)-substituents. The para-electron donating groups on the 3- and 5 positions (see numbering in Scheme 1.3.4(a)) afford increased extinction coefficients (ε) and significant red shifts in the λmax abs (Figure 1.3.3: λmax abs for dimethylamino

substituted aza-BODIPY (compound v) is 799 nm and λmax abs for hydrogen-substituted aza-BODIPY (compound i) is 650 nm).143 Para-substitution with an electron donating group on the 1- and 7- positions has less impact, but still gives bathochromic shifts

(Figure 1.3.3: compound i vs. compound iii).134

Figure 1.3.4. Absorption characteristics of the aza-DIPY and aza-BODIPYs in DCM.144

For a better comparison, Figure 1.3.4 shows a set of aza-DIPY and their boron chelates aza-BODIPYs. A discussion of the absorption and will be presented here. The spectroscopic data of compounds 1a and 2a demonstrate that the λmax absorption is bathochromically shifted from the aza-DIPY free base (free ligand) 1a compared to the

aza-BODIPY 2a by about 60 nm. Simultaneously, extinction coefficients (ε) increase strongly upon the chelation of a boron difluoride unit.

Comparing the absorption behavior of the aza-BODIPYs 2a-2c with electron donating substituents at positions 3 and 5, an additional bathochromic shift is observed.

Going from the unsubstituted 2a to the methoxy-substituted aza-BODIPY 2b and to the dimethylamino-substituted aza-BODIPY 2c, the λmax absorption shifts from 650 nm to

680 nm up to 805 nm, respectively. The electron donating substituents in the 3- and 5- positions of the aza-BODIPY core lead to a significant bathochromic shift. An extinction coefficient of ~40,000 M-1cm-1 was measured for the aza-DIPY free ligand 1a and for the corresponding aza-BODIPY 2a, a much higher value of ~80,000 M-1cm-1 was reported.144

Figure 1.3.5. Absorption characterisitcs of aza-BODIPYs in DCM with electron donating substituents.144 57

To study the influence of the substituent position on the absorption characteristics, a set of aza-BODIPYs with dimethylamino groups in the para-position of the phenyl rings (Figure 1.3.5) were compared. All compounds exhibit two absorption bands in dichloromethane. The major bands appear around 800 nm and between 550-650 nm. In both 3,5-bis(dimethylaminophenyl)-substituted aza-BODIPYs 2c and 2e, the absorption maxima are approximately 800 nm, absorbing in the NIR. The most red-shifted absorption band of the 1,7-substituted 2d is at 785 nm and therefore blue-shifted compared to the other compounds 2c and 2e. This behavior was observed for several aza-

BODIPY compounds such as the methoxy-substituted variants, where the absorption is slightly blue-shifted with the substitution at the 1,7-position.135 The extinction coefficients are dependent on the –N(CH3)2 position. The lowest extinction coefficient was observed for compound 2d (1,7-substituted) at ~40,000 M-1cm-1. The extinction coefficient for the aza-BODIPY 2c (3,5-substituted) is nearly twice that of compound 2d at ~80,000 M-1cm-1. The highest extinction coefficient was found for the tetra-substituted compound 2e (1,3,5,7-substituted) at 92,000 M-1cm-1.

The introduction of electron-donating para-substituents such as –OMe and –

N(CH3)2 onto the phenyl rings at the 3,5-positions was found to result in a destabilization of both LUMOs and HOMOs of aza-BODIPYs due to the inductive effects associated with the para-substituents. The large MO contributions at the 3,5-positions result in a relative destabilization of the HOMO due to the mesomeric interaction between the aryl substituents and the aza-BODIPY core. The incorporation of electron-donating

substituents at the para-positions of 1,7-position phenyl substituents results in a smaller red-shift effect due to a smaller MO contribution at these positions in the HOMO.145

From Figure 1.3.6, compounds i and ii-iv exhibit moderate to high fluorescent quantum yields. In the case of compound iv, the bromine atom attached to the phenyl substituent did not significantly decrease the fluorescence of this compound. Fluorescent emission spectra of the aza-BODIPY dyes reported to date are also relatively insensitive to solvent polarity.134

Figure 1.3.6. Some synthesized aza-BODIPY from literature.146,147

Bathochromism is also observed in the heterocycle-substituted aza-BODIPYs.

The thiophene-substituted aza-BODIPYs shown in Figure 1.3.6 were synthesized and studied. It was found that a larger red-shift is observed for the absorption of 3,5- thiophene substituted aza-BODIPY (Figure 1.3.5, compound vi), than for the 1,7- thiophene substituted compound vii. The extinction coefficients observed for vi-viii differ markedly based on the substitution pattern. The absorption and emission spectra of these compounds are also not sensitive to solvent polarity.146,147

Figure 1.3.7. Hg2+ binding of pyridyl-aza-BODIPY and spectroscopic data.148

Aza-BODIPY dyes applications not only have been investigated in the area of imaging agents134,149 and photodynamic therapy (PDT),134,150 but also as chemosensors.

In Figure 1.3.7, compound ix is highly selective for binding mercury ions (Hg2+) that become bound between the 2-pyridyl groups at positions 1 and 7, which create a well- defined pocket for metal ion binding. A red-shift was observed upon Hg2+ complexation

148 for both λmax absorption (655 to 696 nm) and λmax emission (682 to 719 nm). However,

an attempt to introduce 2-pyridyl units at positions 3 and 5 reveals that the desired aza-

BODIPY is extremely unstable. The degradation of the product during purification was assumed to arise from the increased reactivity of the adjacent carbons of the N-bridge due to the conjugation of electron-deficient pyridine groups.151

BOPHY dyes

A new highly fluorescent pyrrole-BF2 chromophore bis(difluoroboron)1,2- bis((1H-pyrrol-2-yl)methylene)hydrazine (BOPHY) was synthesized based on a hydrazine-Schiff base linked bispyrrole.152,153 BOPHY can be synthesized via the coupling of pyrrole-2-carboxaldehyde with hydrazine followed by reaction with

BF3·OEt2 (Scheme 1.3.5).

Scheme 1.3.5. Synthesis (top) , and photophysical data (bottom) for BOPHY and 152 Me4BOPHY in CH2Cl2.

The resultant symmetric and dimeric tetracycle is composed of two BF2 units in six-membered chelate rings appended with pyrrole units on the periphery. The fluorescent quantum yields for BOPHY and Me4BOPHY in CH2Cl2 are 95 and 92% respectively. The absorption and emission data is shown in Scheme 1.3.5. These

BOPHYs are highly fluorescent in solution, films, and the solid state with excellent photostabilities versus the well-known commercialized 1,3,5,7-tetramethylBODIPY.153

After the discovery of BOPHYs in 2014, studies involving their photophysical properties were published154 as well as several structural modifications of the compounds.155–157

BOPHY can undergo the similar structural modification as BODIPY and aza-

BODIPY. The most common modifications are performed via Knoevenagel condensation113,135 and cross coupling reactions.158 Ziessel et al. reported the first functionalized BOPHY dyes at the 3,8 positions with vinyl-thiophene solubilizing chains and investigated their photophysical and optoelectronic properties as well as their performances as active materials in organic solar cells.155 The preparation of the target molecules is based on a double Knoevenagel condensation of thienyl aldehydes with 62

Me4BOPHY (Scheme 1.3.6). The reactions result in highly soluble dark violet BOPHY-1 and dark blue BOPHY-2 compounds.

O S

R F F F F 1 10 B 8 B N 9 N N N 2 7 N 4 N N N 3 B 5 6 piperidine, toluene, B F F p-TsOH,  F F R

BOPHY-1: R = S O S

S S BOPHY-2: R = S

Scheme 1.3.6. Synthesis (top), and photophysical data (bottom) for of BOPHY-1 and BOPHY-2 in THF.155

Figure 1.3.8. X-ray crystal structure of BOPHY-2; purple, pink, lime-green, gray, and yellow colors represent N, B, F, C, and S atoms respectively.155

The electronic absorption spectra of BOPHY-1 and BOPHY-2 dyes in THF solution exhibit broad absorption bands with bathochromic shifts compared with the starting Me4BOPHY compound. In both cases, excitation at the onset of the low energy absorption band gave structured emission profiles with maxima higher than 600 nm and quantum yields are lower than that of Me4BOPHY, being 13% and 10% for BOPHY-1 and BOPHY-2, respectively. Both new dyes exhibit the first oxidation and reduction processes that are quasi-reversible. The anodic shift of the oxidation wave of BOPHY-2 with respect to that of BOPHY-1 is likely due to the extended π-system of the dye. Bulk heterojunction solar cells assembled with these dyes and a fullerene derivative ([6,6]- phenyl C71 butyric acid methyl ester, PC71BM) as the electron acceptor give a power conversion efficiency as high as 4.3% over a broad range of wavelengths (580 to 720 nm), due to the increasing of electron density on the flat central core, which favors packing interactions with PC71BM domains in the solar cell system. Ziessel et al.156 also reported the modification of BOPHY that yields dyes displaying large Stokes

shifts. Compounds BOPHY-3 and BOPHY-4 were synthesized by palladium-catalyzed cross-coupling reactions with variety of alkynes, catalyst [Pd(dppf)Cl2]·CH2Cl2 (3 mol

%, dppf = 1,1′ bis(diphenylphosphino) ferrocene), CuI (6 mol %) as reductant, and triethylamine as the base (Scheme 1.3.7). Significant bathochromic shifts were observed for both BOPHY-3 and -4. However, the fluorescence of BOPHY-4 was heavily quenched.

F F terminal alkyne, F F B [Pd(dppf)Cl ], CuI B N N 2 N N I I Ar Ar N N NEt , THF N N B 3 B F F F F

BOPHY-3: R =

OC16H33

BOPHY-4: R = OC16H33

OC16H33

Scheme 1.3.7. Synthesis (top), and photophysical data (bottom) for 2,7-bis-coupled BOPHY-3 and BOPHY-4 in THF.156

Work involving BOPHY analogues are growing extensively in the past two years.

The research groups of Jiang and Li reported the studies of extended-conjugated BOPHY dyes and their optical properties, as seen in Figure 1.3.9-10. 65

Figure 1.3.9. BOPHY-5 structure and fluorescence spectra (pH 7, 5, 4, 3.5, 3, 2.8, 2.5, 2.2, 2.0, 1.5, 1 and 1 M, 2 M, 4 M, 6 M, 8 M of HCl, λemiss 490 nm) of 5 μM BOPHY-5 157 in CH3CN–H2O (1 : 1, v/v). (figure reprinted with permission from RSC Adv. 2015, 5 (22), 16735–16739. Copyright © Royal Society of Chemistry)

The fluorescence quantum yield of BOPHY-5 in CH3CN–H2O (1 : 1, v/v) is very low (ϕ = 0.01 at pH 7) due to the intramolecular charge transfer (ICT) effect from the donor (amine nitrogen atom) to that of the BOPHY fluorophore, causing fluorescence quenching. However, with decreasing pH, a dramatic increase in fluorescence intensity by 1200 fold at 532 nm (ϕ = 0.98 at 4 M HCl) was observed.157

F F F F B B N N N N R R R N N N N B B F F F F

BOPHY-6: R = t-Bu- BOPHY-9: R = t-Bu- BOPHY-7: R = C6H4- BOPHY-10: R = C6H4- BOPHY-8: R = p-MeOC6H4- BOPHY-11: R = p-MeOC6H4-

Figure 1.3.10. Structures (top), and photophysical data (bottom) for BOPHY-6-BOPHY- 11 and their optical data in dioxane.159

In Figure 1.3.10, compounds BOPHY-6-11 were synthesized by standard

Sonogashira coupling methodology to linearly extend BOPHY conjugation. The resulting compounds with ethynyl spacers showed red-shifted λem. These compounds were highly emissive in both solution and the solid state.159

The work involving BOPHY modification continues to grow. Triplet state properties of BOPHY photosensitizers was studied by Zhang and Zhao160 in 2016. These new BOPHY triplet photosensitizers were synthesized by attaching iodine atoms to the

BOPHY core in order to enhance intersystem crossing (ISC) process. The synthesis, structures, and photophysical data are presented in Scheme 1.3.8.

Scheme 1.3.8. Preparation of BOPHY triplet sensitizers: (I) NH2NH2·H2O, EtOH, AcOH, CH2Cl2, DIPEA and BF3·OEt2. (II) CHCl3, ICl and CH3OH. (III) aldehydes, toluene, p- TsOH and piperidine, 140 oC. 9,10-diphenylanthracene (DPA) was used as an acceptor of triplet-triple annihilation upconversion. diiodoBODIPY was used as a standard for singlet oxygen photosensitization.

The triplet-triplet annihilation upconversion with diiodoBOPHY C-2 as a triplet photosensitizer was studied. In the presence of a triplet acceptor DPA, the upconverted emission in the range of 390–475 nm was observed with the upconversion quantum yield of 2.8%. The triplet excited state lifetime was determined to be 177.2 μs. The singlet

1 oxygen ( O2) quantum yield of diiodoBOPHY C-2 was 0.58, which is only slightly lower

1 161 than that of diiodoBODIPY ( O2 quantum yield = 0.87 ), the traditional triplet state photosensitizer.

More BOPHY triplet sensitizer was synthesized and studied for singlet oxygen generation by Cui and coworkers.162 The similar approach to the work by Zhang and

Zhao160, which is attaching heavy atoms on BOPHY core, was adopted in this work.

However, the BOPHY core was slightly different from original BOPHY152 due to the addition of four phenyl units attached on positions 3- and 5- of pyrroles (see Scheme

1.3.9).

Scheme 1.3.9. Synthesis of the tetraphenyl-containing BOPHYs and optical property of tetraphenylBOPHY1.

The optical properties of tetraphenylBOPHY1 is comparable to those of the reported BOPHY dyes.152,156,157 The dibromo substituted tetraphenylBOPHY2 was able to generate the singlet oxygen as a photosensitizer. No photobleaching of

tetraphenylBOPHY2 was observed based on the λmax abs = 506 nm in toluene during the experiment.

In conclusion, BOPHY-based molecules are highly emissive fluorophores in solution and solid states. Their fluorescent quantum yields in solution are moderate to high and their emission wavelengths can be tuned by vertical or linear π-extension of the

BOPHY framework. It is notable that the remarkably red-shifted emission is achieved by the simple extension of the π system. Such interesting emission properties from simple organic molecules may lead to potential future applications.

70 CHAPTER II

THE SYNTHESIS AND pH-DEPENDENT BEHAVIOUR OF Re(CO)3 CONJUGATES

WITH DIIMINE PHENOLIC LIGANDS

The text of this chapter is a reprint of the material as it appears in: Chanawanno, K.; Engle, J. T.;

Le, K. X.; Herrick, R. S.; Ziegler, C. J. Dalton Trans. 2013, 42, 13679–13684.

Introduction

Recent years has seen a new-found interest in the chemistry of the d6 Re(CO)3 core, notable for the fact that so many of its derivatives are water and air stable. Re(CO)3

99m complexes have been investigated as models for Tc(CO)3 radiopharmaceutical imaging agents as well as for therapeutic agents using the β -emitting nuclides 186Re and

188Re.2,4,163 They have also been investigated as components of optical materials, in particular when the Re(CO)3 moiety is bound by a diimine. The Re(diimine)(CO)3 compounds exhibit metal-to-ligand charge transfer transitions, and are of interest due to their novel photophysical properties.28 Accordingly, these compounds have been investigated as electron transfer dyes in proteins,33 in non-linear optical

(NLO)34

71 materials, for solar energy conversion20 and as chemical or biological sensors.33

Compounds containing the photoluminescent Re(CO)3 core have been utilized for imaging applications as demonstrated in various studies.30,32,100,164

Several years ago, Heinze and coworkers35,36 observed that some rhenium, platinum and palladium complexes bearing diimine ligands with an acidic phenol group exhibit pH-sensitive behavior at room temperature. Upon deprotonation, these compounds undergo significant changes to their UV-visible spectra, and in particular

Re(CO)3Cl(pyca-C6H4-p-OH) (pyca = pyridine-2-carbaldehyde imine) shows intense absorptions above 550 nm upon loss of the phenol proton. We have been working on similar compounds incorporating the pyca ligand, and have synthesized a variety of related compounds as part of our investigations into the biologically relevant chemistry of

99m 11,61 M(CO)3, M = Tc(I)/Re(I). We observed that in many cases these compounds can be produced via a one-pot reaction, with Schiff base formation taking place at the metal ion.7

We decided to investigate how the identity of the halide and the position of the phenol group would affect the pH dependent UV-visible absorption properties of pyca- phenol Re(CO)3 compounds. Herein, we describe the reactions of Re(CO)5X (X = Cl or

Br) with pyridine-2-carboxaldehyde and ortho-, meta- or para-aminophenol, affording a series of Re(CO)3X(pyca-C6H4OH) compounds (1–6). The syntheses of these compounds are shown in Scheme 2.1. In addition to using the known method of generating the ligand prior to metal coordination, we were able to apply one-pot methods for the synthesis of most of the desired compounds. We observed that the relative position of the hydroxide 72 does affect the pH-dependent UV-Visible properties of the resultant complexes, but that the halide identity does not have an effect. We have structurally characterized all new compounds, and were able to compare structural parameters with the observed pH- dependent spectroscopic changes.

Scheme 2.1. Syntheses of 1-6.

Experimental

Materials and methods

Starting materials were obtained commercially and used without further purification. NMR spectra were recorded on a Varian 400 MHz spectrometer (for 1 and

3) and a Varian Mercury 300 MHz spectrometer (for 2, 4, 5 and 6). Chemical shifts were reported with respect to residual solvent peaks as internal standard (d6-DMSO, δ = 2.50 73

13 ppm; C: d6-DMSO, δ = 39.7 ppm). ATR-IR spectra for 1 and 3 were recorded on a

Perkin Elmer-Spectrum One spectrometer. FTIR spectra for 2, 4, 5 and 6 were recorded on a Nicolet iS5 spectrometer using NaCl disks. Elemental analyses were performed by

Atlantic Microlab of Norcross, GA 30091. Electrospray MS (ES-MS; positive mode) spectra were recorded using a Bruker HCT-ultra ETD II Ion Trap mass spectrometer.

X-ray data collection and structure determination

X-ray crystallographic analysis: single crystals of 2–6 were coated in Paratone-N

(Exxon) oil, mounted on a pin and placed on a goniometer head under a stream of nitrogen cooled to 100 K. The data were collected on either a Bruker SMART APEX I

CCD-based X-ray diffractometer system equipped with a Motarget X-ray tube (λ =

0.71073 Å) operated at 2000 W power (compounds 3–6) or a Bruker Kappa APEX II

DUO CCD-based diffractometer with Cu/Mo ImuS microfocus optics (compound 2). The frames were integrated with the Bruker SAINT software package using a narrow-frame algorithm. Data were corrected for absorption effects using the multiscan method

(SADABS) and the structure was solved and refined using the Bruker SHELXTL

Software Package until the final anisotropic full-matrix, least-squares refinement of F2 converged.165

Figure 2.1. Molecular structures of 2-6. Hydrogen atoms have been omitted for clarity. Crystallographic data, structure refinement, and selected bond lengths and angles are reported in Table 2.1-2.6.

Table 2.1 Crystallographic data and structure refinement for 2.

Compound 2

Empirical formula C15H10ClN2O4Re Formula weight 503.90 Crystal system Monoclinic Space group P2(1)/c a/ Å 21.5722(15) b/ Å 7.9755(5) c/ Å 18.3057(12) α(°) 90 β(°) 96.441(2) γ(°) 90 Volume (Å3) 3129.6(4) Z 8 Dc (Mg/m3) 2.139 µ (mm-1) 7.957 F(000) 1904 Reflections collected 39281 Data/Restraints/Parameters 10044/0/415 GOF on F2 0.904 2 R1 (on Fo , I > 2σ(I)) 0.0263 2 wR2 (on Fo , I > 2σ(I)) 0.0581 R1 (all data) 0.0407 wR2 (all data) 0.0653

Table 2.2 Crystallographic data and structure refinement for 3.

Compound 3

Empirical formula C15H10ClN2O4Re Formula weight 502.89 Crystal system Monoclinic Space group P2(1)/n a/ Å 13.005(3) b/ Å 8.8892(18) c/ Å 13.661(3) α(°) 90 β(°) 100.583(2) γ(°) 90 Volume (Å3) 1552.4(5) Z 4 Dc (Mg/m3) 2.152 µ (mm-1) 8.020 F(000) 948 Reflections collected 9681 Data/Restraints/Parameters 2393/8/246 GOF on F2 0.943 2 R1 (on Fo , I > 2σ(I)) 0.0286 2 wR2 (on Fo , I > 2σ(I)) 0.0616 R1 (all data) 0.0366 wR2 (all data) 0.0661

Table 2.3 Crystallographic data and structure refinement for 4.

Compound 4

Empirical formula C15H10BrN2O4Re Formula weight 548.36 Crystal system Monoclinic Space group P2(1)/n a/ Å 12.4869(15) b/ Å 9.2091(11) c/ Å 13.9549(16) α(°) 90 β(°) 100.1460(10) γ(°) 90 Volume (Å3) 1579.6(3) Z 4 Dc (Mg/m3) 2.306 µ (mm-1) 10.242 F(000) 1024 Reflections collected 12874 Data/Restraints/Parameters 3492/14/209 GOF on F2 0.811 2 R1 (on Fo , I > 2σ(I)) 0.0376 2 wR2 (on Fo , I > 2σ(I)) 0.0975 R1 (all data) 0.0443 wR2 (all data) 0.1038

Table 2.4 Crystallographic data and structure refinement for 5.

Compound 5

Empirical formula C15H10BrN2O4Re Formula weight 547.35 Crystal system Monoclinic Space group P2(1)/n a/ Å 12.577(3) b/ Å 9.011(2) c/ Å 14.165(3) α(°) 90 β(°) 99.942(3) γ(°) 90 Volume (Å3) 1581.3(6) Z 4 Dc (Mg/m3) 2.299 µ (mm-1) 10.231 F(000) 1020 Reflections collected 11591 Data/Restraints/Parameters 3075/34/220 GOF on F2 0.705 2 R1 (on Fo , I > 2σ(I)) 0.0467 2 wR2 (on Fo , I > 2σ(I)) 0.1518 R1 (all data) 0.0625 wR2 (all data) 0.1746

Table 2.5 Crystallographic data and structure refinement for 6.

Compound 6

Empirical formula C15H10BrN2O4Re Formula weight 548.36 Crystal system Monoclinic Space group P2(1)/n a/ Å 13.186(3) b/ Å 8.966(2) c/ Å 13.615(3) α(°) 90 β(°) 100.260(3) γ(°) 90 Volume (Å3) 1583.9(6) Z 4 Dc (Mg/m3) 2.300 µ (mm-1) 10.215 F(000) 1024 Reflections collected 11654 Data/Restraints/Parameters 2998/35/246 GOF on F2 0.867 2 R1 (on Fo , I > 2σ(I)) 0.0437 2 wR2 (on Fo , I > 2σ(I)) 0.1154 R1 (all data) 0.0622 wR2 (all data) 0.1321

Table 2.6 Selected bond lengths [Å] and angles [deg] for compounds 1-6.

Synthesis of 1: This compound has been previously synthesized via a two-step

method. For the current study, a one pot method was employed. Re(CO)5Cl (50 mg,

0.125 mmol) and one equivalent of 4-aminophenol (13.5 mg, 0.125 mmol) were combined in a round-bottom flask. One equivalent of pyridine-2-carboxaldehyde (0.011 mL, 0.125 mmol) was added after the addition of 5.0 mL of methanol. The clear, colorless solution turned yellow upon the addition of the aldehyde. The 4 h reflux resulted in a dark-red, clear solution. The solvent was then removed by rotary evaporation. The product was dried under vacuum, and collected as a red solid. Yield:

88%.

Synthesis of 2: Compound 2 was prepared in a similar fashion to 1. The product was dried under vacuum, and collected as a red solid. Crystals suitable for X-ray diffraction were obtained by slow evaporation in methanol. FTIR (cm 1): 3423 (br, w),

2022 (s), 1898 (s). Yield: 72%. Anal. Calc. for C15H10N2O4ReCl(C7H8)0.5(H2O)0.5; C,

1 39.71; H, 2.70; N, 5.01%. Found: C, 39.84; H, 2.98; N, 5.01%. H NMR (d6-DMSO): δ

9.98 (s, 1H, OH), 9.30 (s, 1H, H–C=N), 9.05 (d, 3J = 5.4 Hz, 1H, H on py), 8.34 (m, 2H,

H on py), δ 7.83 (m, 1H, H on py), 7.34 (t, 3J = 7.8 Hz, 1H, CH on phenol), 6.98–6.94

13 (m, 2H, CH on phenol), 6.88–6.84 (m, 1H, CH on phenol). C NMR (d6-DMSO): δ

198.2 (CO), 197.4 (CO), 188.1 (CO), 169.9 (C=N), 158.5 (C on py), 155.5 (C on py),

153.5 (C on py), 152.1 (C on py), 140.9 (C on py), 130.8 (C on phenol), 130.7 (C on phenol), 130.2 (C on phenol), 116.5 (C on phenol), 113.0 (C on phenol), 109.8 (C on phenol). ES-MS: m/z = calc. for C15H10N2O4ReNa: 527.00 found 526.80. UV-Vis (nm, ε

× 104 M 1 cm 1): 290 (2.2), 259 (2.8).

Synthesis of 3: Compound 3 was prepared in a similar fashion to 1. Crystal florets, suitable for X-ray diffraction, were obtained by vapor diffusion of hexane into a methylene chloride solution of the complex. Yield: 55%. Anal. Calc. for

C15H10N2O4ReCl; C, 36.92; H, 2.07; N, 5.24%. Found: C, 36.83; H, 2.02; N, 5.27%.

1 1 ATR-IR (cm ): 3269 (w), 2024 (s), 1903 (s). H NMR (d6-DMSO): δ 10.24 (s, 1H, OH),

9.29 (s, 1H, H–C=N), 9.04 (d, 3J = 5.6 Hz, 1H, H on py), 8.33 (m, 2H, H on py), 7.61 (m,

1H, H on py), 7.30 (m, 1H, CH on phenol), 7.21 (m, 2H, CH on phenol), 7.17 (m, 1H,

13 CH on phenol). C NMR (d6-DMSO): δ 171.9 (C=N), 155.2 (C on py), 153.6 (C on py),

148.8 (C on py), 141.0 (C on py), 130.6 (C on py), 138.5 (C on phenol), 130.2 (C on phenol), 129.7 (C on phenol), 124.6 (C on phenol), 119.4 (C on phenol), 117.2 (C on phenol). ES-MS: m/z calc. for C15H10N2O4ReNa: 527.00 found 526.80. UV-Vis (nm, ε

×103 M 1 cm 1): 411 (4.1), 290 (7.6), 240 (15.3).

Synthesis of 4: The diimine, pyca-C6H4-p-OH, was synthesized as described previously.36 The diimine (49 mg, 0.25 mmol) was refluxed with 50 mg, 0.125 mmol, of

Re(CO)5Br for 18 h in toluene. The orange solution was evaporated to produce an orange product 4 which was washed with diethyl ether. FTIR (cm 1): 3319 (w), 2021 (s), 1899

(s). Yield: 66%. Anal. Calc. for C15H10N2O4ReBr; C, 32.85; H, 1.84; N, 5.11%. Found:

1 C, 32.96; H, 1.68; N, 5.03%. H NMR (d6-DMSO): δ 10.02 (s, 1H, OH), 9.20 (s, 1H, H–

C=N), 9.05 (d, 3J = 5.4 Hz, 1H, H on py), 8.31 (m, 2H, H on py), 7.78 (m, 1H, H on py),

7.45 (d, 3J = 8.7 Hz, 2H, CH on phenol), 6.91 (d, 3J = 8.7 Hz, 2H, CH on phenol). 13C

NMR (d6-DMSO): δ 197.7 (CO), 197.1 (CO), 187.6 (CO), 167.8 (C=N), 159.0 (C on py),

155.8 (C on py), 153.5 (C on py), 143.1 (C on py), 140.8 (C on py), 130.4 (C on phenol),

129.7 (C on phenol), 124.4 (C on phenol), 116.2 (C on phenol). ES-MS: m/z calc. for

+ C15H10N2O4Re : 467.00 found 467.01, calc. for C15H10N2O4ReNa: 571.00 found 570.91.

UV-Visible (nm, ε × 104 M 1 cm 1): 348 (3.7).

Synthesis of 5: Re(CO)5Br (50 mg, 0.125 mmol) and pyridine-2-carboxaldehyde

(0.023 mL, 0.25 mmol) were mixed using a minimum amount of toluene (15 mL) as a solvent. After five minutes, the mixture turned purple and then 3-aminophenol (27 mg,

0.25 mmol) was added to the solution. The reaction was refluxed for 12 hours. The solution was evaporated producing a red solid product which was washed with diethyl ether. Yield: 96%. FTIR (cm 1): 3387 (w), 2021 (s), 1894 (s). Anal. Calc. for

C15H10N2O4ReBr(C7H8)0.25; C, 35.20; H, 2.12; N, 4.90%. Found: C, 35.12; H, 2.31; N,

1 3 4.92%. H NMR (d6-DMSO): δ 9.99 (s, 1H, OH), 9.27 (s, 1H, H–C=N), 9.07 (d, J = 5.4

Hz, 1H, H on py), 8.35–8.33 (m, 2H, H on py), δ 7.84–7.79 (m, 1H, H on py), 7.34 (t, 3J

= 8.1 Hz, 1H, CH on phenol), 6.99–6.94 (m, 2H, CH on phenol), 6.87–6.84 (m, 1H, CH

13 on phenol). C NMR (d6-DMSO): δ 197.8 (CO), 196.9 (CO), 187.5 (CO), 169.9 (C=N),

158.5 (C on py), 155.5 (C on py), 153.7 (C on py), 152.2 (C on py), 140.8 (C on py),

130.9 (C on phenol), 130.7 (C on phenol) 130.1 (C on phenol), 116.5 (C on phenol),

+ 113.1 (C on phenol), 109.8 (C on phenol). ES-MS: m/z calc. for C15H10N2O4Re : 467.00

4 found 467.01, calc. for C15H10N2O4ReNa: 571.00 found 570.93. UV-Vis (nm, ε × 10

M 1 cm 1): 298 (3.0), 254 (3.9).

Synthesis of 6: Compound 6 was prepared in a similar fashion to 5. Yield: 61%.

1 FTIR (cm ): 3397 (m), 2023 (s), 1904 (s). Anal. Calc. for C15H10N2O4ReBr(C6H7NO)0.5;

1 C, 35.85; H, 2.26; N, 5.81%. Found: C, 35.97; H, 2.18; N, 5.61%. H NMR (d6-DMSO): 84

δ 10.28 (s, 1H, OH), 9.30 (s, 1H, H–C=N), 9.10 (d, 3J = 7.5 Hz, 1H, H on py), 8.36 (m,

2H, H on py), 7.83 (m, 1H, H on py), 7.37 (d, 3J = 8.0 Hz, 1H, CH on phenol), 7.23 (t, 3J

= 8.0 Hz, 1H, CH on phenol), 7.05 (d, 3J = 8.0 Hz, 1H, CH on phenol), 6.94 (t, 3J = 8.0

13 Hz, 1H, CH on phenol). C NMR (d6-DMSO): δ 198.1 (CO), 197.2 (CO), 187.8 (CO),

171.8 (C=N), 155.3 (C on py), 153.8 (C on py), 148.8 (C on py), 140.8 (C on py), 138.8

(C on py), 130.6 (C on phenol), 130.1 (C on phenol) 129.7 (C on phenol), 124.7 (C on phenol), 119.4 (C on phenol), 117.2 (C on phenol). ES-MS: m/z calc. for

+ C15H10N2O4Re : 467.00 found 467.01, calc. for C15H10N2O4ReNa: 571.00 found 570.91.

UV-Visible (nm, ε × 104 M 1 cm 1): 374 (4.4).

Deprotonation studies

A 1.08 mM solution of TMAH in THF was prepared. Aliquots of this solution were added to THF solutions of compounds 1–6. UV-Visible spectroscopy was used to record changes that occurred upon titration. Upon addition of acid, the original spectra were obtained.

Results and discussion

Syntheses of 1–6

The synthesis of compound 1 made by refluxing the premade diimine ligand in

36 the presence of Re(CO)5Cl has been reported previously, by Liu and Heinze. We have prepared compound 1 along with 2, 3, 5 and 6 using one-pot reactions as shown in

Scheme 2.1. Re(CO)5Cl, pyridine-2-carboxaldehyde and the amino phenol were reacted at reflux in methanol or toluene for several hours. Compounds 1–3 were prepared after four hours of reaction in methanol. Compounds 5 and 6 were synthesized via a one-pot reaction in refluxing toluene for 12 hours, upon which the products precipitated from solution. Complex 4 was produced in a stepwise manner by the synthesis of the diimine ligand followed by addition of Re(CO)5Br. All compounds were isolated as pure compounds in good yield.

Spectroscopic characterization

Infrared spectra for each compound showed carbonyl peaks between 2030–1860

1 cm , typical of a pseudo-C3v symmetry resulting from a facial arrangement of carbonyls.

Phenolic OH stretches were observed between 3200–3400 cm 1. The 1H and 13C NMR spectra for 1–6 were as expected with diagnostic peaks in the 1H NMR spectrum at

∼10.00–10.15 ppm for the phenolic protons, at ∼9.2–9.3 ppm for the imine protons, and in the 13C NMR spectrum at 168–172 ppm for the exocyclic imine carbons. The UV- visible spectra of compounds 1–6 are shown in Figure 2.2.

Figure 2.2. UV-visible spectra for complexes 1-6 in THF.

X-ray data collection and structure determination

We were able to structurally characterize compounds 2–6, and compare them to the structure previously reported for 1.36 The structures of compounds 2–6 are shown in

Figure 2.1. Table 2.1-2.5 list data collection and structural parameters for compounds 2–

6; key bond lengths and angles for these complexes are shown in Table 2.6. Including the known structure of 1, all six complexes adopt slightly distorted octahedral geometries with the three carbonyls ligands occupying a facial geometry. The diimine ligand binds to the rhenium atom in a bidentate fashion via the two nitrogen atoms, forming a five- member metallocycle. All N1–Re1–N2 bite angles are ∼74–75° and are similar to those of previously reported metal complexes with pyridine imine ligands.73 The C–O bonds

+ 166 lengths are normal for Re(CO)3 species (∼1.14–1.16 Å). The Re–C bond lengths are

in agreement with typical ranges (∼1.87–1.95 Å), and the Re–X (Cl or Br) bond lengths are as expected. Re(CO)3–diimine complexes, including those reported in this work, show Re–N bonds ranging from ∼2.14–2.26 Å.167

For each compound the phenyl ring is rotated out of the plane of the diimine ligand. Compounds 1 and 4 have dihedral angles between pyridine–diimine and phenol planes of ∼56.8 and ∼55.4°, respectively, while the angle in compounds 3 and 6 is

∼57.5°. Compound 5, displays a dihedral angle of ∼53.7°, similar to those seen in the ortho and para analogues, but the tilts observed for compound 2 are ∼42.7 and ∼67.2° for the two molecules in the asymmetric unit. The structures of the unmodified phenyl variants exhibit tilt angles of ∼34.1° (X = Cl) and ∼35.2° (X = Br).7,168 In the para- chloro and para-iodo compounds (X = Br), the tilt angles are observed to be ∼44.0 and

∼46.2° respectively.169,170 It is unclear if packing in the crystal is the reason that 2 has different rotation angles than the other compounds. What is clear is that the para- and meta- derivatives should have unhindered rotation of the phenyl ring, while phenyl ring rotation for the ortho-derivative is restricted.

Deprotonation studies

Previously, the effect of phenolic deprotonation of 1 was investigated. UV-visible spectra were recorded following sequential addition of aliquots of the base t-butyliminotris(dimethylamino)phosphorane. A change to a blue color was observed upon addition of the base. Two isosbestic points were judged to be consistent with the phenoxide anion as the only other absorbing species in the solution.36

In order to examine the impact of moving the hydroxide group and varying the halide, we treated THF solutions of 1–6 with THF solutions of TMAH (tetramethyl ammonium hydroxide). Absorbance spectra were recorded after allowing reactions to equilibrate. The pairs of ortho-, meta-, and para-hydroxide compounds behaved similarly after treatment with TMAH. Compounds 1 and 4 showed a gradual decrease in intensity of a band near 380 nm and growth of a new band at 586 nm. Overlaid spectra following the titration of 4 with TMAH are shown in Figure 2.3. Isosbestic points were observed at

345 and 403 nm. This deprotonation was reversible upon the addition of acid to the blue solution.

Figure 2.3. Overlaid UV-visible absorption spectra of 4 (a) and 6 (b) in THF upon addition of 0.0-1.0 equivalent TMAH.

Compounds 3 and 6 (ortho-hydroxide derivatives) showed more modest changes with the increase of absorbance intensity, which corresponded with a slight darkening of the yellow color of these solutions (Figure 2.4). Notably, compounds 2 and 5, the meta-

hydroxide derivatives, showed no color change upon addition of base and no change in the absorbance spectrum.

Figure 2.4. Overlaid UV-visible absorption spectra of 1 (a) and 3 (b) in THF upon addition of 0.0-1.0 equivalent TMAH.

The dramatic spectral changes for 1 and 4 can be explained as arising from an extended conjugation system formed upon deprotonation. Similar behavior is observed

in phenolic azo dyes.171 Scheme 2.2 shows how deprotonation leads to an extended π- system that could result in the appearance of intense bands. Compounds 3 and 6 do not show dramatic spectral changes; it is possible that restricted rotation does not permit an extended conjugated system with the halide and the imine hydrogen preventing the phenyl ring from achieving a geometry that is co-planar with the diimine ligand.

Alternatively, it is possible that hydrogen bonding of the ortho-phenolic hydrogen with the halide could hinder deprotonation. With the negative charge localized in the meta position, compounds 2 and 5 are not able to extend their π-systems (Scheme 2.2).

Scheme 2.2. Proposed electron flow for the compounds bearing para- (top) and ortho- OH-(bottom).

Conclusions

Diimine rhenium(I) tricarbonyl species exhibit interesting photophysical properties due to their metal-to-ligand charge transfer transitions. By coupling this diimine with a base-sensitive phenol, we can generate compounds that exhibit pH dependent UV-visible transitions. Changes in color were observed for compounds 1, 3, 4 and 6 when tetramethylammonium hydroxide was added into a THF solution of each compound, and the degree of color change was dependent on the position of the phenol group. No change was observed for compounds 2 and 5, where the phenol is in the meta position. We can justify these changes based on a combination of resonance arguments and steric restrictions. None of the observed photophysical changes were dependent on the identity of the halide. We are continuing our investigations into rhenium tricarbonyl compounds both as mimics for technetium radiopharmaceuticals and as organometallic chromophores.

93 CHAPTER III

USING HYDRAZINE TO LINK FERROCENE WITH Re(CO)3:

A MODULAR APPROACH

The text of this chapter is a reprint of the material as it appears in: Chanawanno, K.; Rhoda, H.

M.; Hasheminasab, A.; Crandall, L. A.; King, A. J.; Herrick, R. S.; Nemykin, V. N.; Ziegler, C. J.

J. Organomet. Chem. 2016, 818, 145–153.

Introduction

Organometallic compounds have been employed in a wide variety of applications, ranging from catalysis to materials science.172–175 Accordingly, much work has focused on the covalent attachment of organometallic fragments to other molecules to produce conjugate compounds, which continues in the recent literature.176–178 For example, over the past few decades there has been increasing interest in the linking of organometallic compounds to molecules of biological interest.179–181 Organometallic compounds are of interest in biochemistry and medicine as both therapeutic and diagnostic agents,182–184 and

94 as a probe to understand structure and function in biological macromolecules.185,186 As a result of this interest, there is a need for the development of new synthetic methodologies to produce these biologically relevant conjugates.

Two commonly used examples of organometallic moieties used as components of

187–189 2,190 conjugate molecules are ferrocene and the Re(CO)3 unit. Both of these groups are stable to water and dioxygen, and are robust enough to handle a variety of chemical manipulations. For example, both moieties are stable enough to append to biological or pharmacologically active compounds. Additionally, ferrocene and Re(CO)3 compounds can be readily incorporated into molecules for potential materials applications. In work from our laboratories, we have functionalized proteins and peptides with the Re(CO)3 unit,191,192 and have appended the ferrocene unit to chromophores like porphyrin, phthalocyanine, BODIPY, azaBODIPY and the recent BOPHY fluorophore.55,56

In this report, we present a series of ferrocene-Re(CO)3 conjugate compounds synthesized via a modular approach. These compounds are shown in Figure 3.1. In all cases, we can use hydrazine-derived Schiff base formation to produce 1:1 Fc:Re(CO)3 adducts as well as 2:1 and 1:2 Fc:Re(CO)3 systems. Both 1-acetylferrocene and 1,1’- diacetylferrocene react with hydrazine to afford the corresponding hydrazones. These hydrazones can then form a second C-N double bond via a Re(CO)3 mediated reaction involving a chelating aldehyde. In addition to the synthesis of these modular constructs, we have probed their spectroscopy and electrochemistry, and have investigated their electronic structures via DFT and TDDFT methods.

Figure 3.1. Structures of compounds 1-9.

Experimental

Materials and methods

All reagents were purchased from Strem, Acros Organics, TCI AMERICA or

Sigma-Aldrich and used as received without further purification. Diacetylferrocene (2) was synthesized by using a previously reported procedure.193 All solvents were purified by alumina and copper columns in the Pure Solve solvent system (Innovative

Technologies, Inc.) and were stored over molecular sieves. Syntheses were performed under nitrogen atmosphere with a Schlenk line apparatus equipped to a pre-drying column to minimize exposure to air and water. NMR spectra were recorded on a Varian

Mercury 300 MHz. Chemical shifts were reported with respect to residual solvent peaks

1 13 as internal standard ( H: CDCl3, δ = 7.26 ppm; C: CDCl3, δ = 77.2 ppm). Infrared spectra were collected on Thermo Scientific Nicolet iS5 which was equipped with iD5

ATR. Electronic absorption spectra were recorded on Hitachi U-2000 UV-vis spectrophotometer. Mass Spectrometric analyses were carried out at the University of

Minnesota Duluth.

X-ray data collection and structure determination

X-ray crystallographic analysis: Single crystal data for 4 were collected on a

Bruker SMART APEX I diffractometer. Samples were coated in Paratone-N (Exxon) oil, mounted on a pin and placed on a goniometer head under a stream of nitrogen cooled to

100 K. The detector was placed at a distance of 5.009 cm from the crystal. The data of compound 4 was collected on Mo-target X-ray tube (Mo Kα radiation, λ = 0.71073 Å) operated at 2000 W power. The data for compound 5 were collected on a Bruker APEX

II DUO system using a Cu source with ImuS microfocus optics (Cu Kα radiation, λ =

1.54178 Å). The frames for both crystals were integrated with the Bruker SAINT software package using a narrow-frame algorithm. Data were corrected for absorption effects using the multiscan method (SADABS) and the structure was solved and refined

using the Bruker SHELXTL Software Package until the final anisotropic full-matrix, least-squares refinement of F2 converged.194

Figure 3.2. Crystal structures of compounds 4 and 5 with 35% thermal ellipsoids. Hydrogen atoms have been omitted for clarity. Selected bond lengths and angles are reported in Table 3.1-3.2.

Table 3.1 Crystallographic data and structure refinement for 4.

Compound 4

Empirical formula C21H17ClFeN3O3Re Formula weight 636.88 Crystal system Triclinic Space group P-1 a/ Å 11.5372(9) b/ Å 13.4291(10) c/ Å 14.4988(11) α(°) 85.604(3) β(°) 88.865(3) γ(°) 67.679(3) Volume (Å3) 2071.8(3) Z 4 Dc (Mg/m3) 2.042 µ (mm-1) 6.695 F(000) 1224 Reflections collected 8377 Data/Restraints/Parameters 8377 / 7 / 489 GOF on F2 1.100 2 R1 (on Fo , I > 2σ(I)) 0.0530 2 wR2 (on Fo , I > 2σ(I)) 0.1447 R1 (all data) 0.0654 wR2 (all data) 0.1544

100

Table 3.2 Crystallographic data and structure refinement for 5.

Compound 5

Empirical formula C21H17BrFeN3O3Re Formula weight 681.34 Crystal system Triclinic Space group P-1 a/ Å 11.5739(9) b/ Å 13.3692(10) c/ Å 14.7085(11) α(°) 84.731(5) β(°) 88.560(5) γ(°) 68.617(4) Volume (Å3) 2110.2(3) Z 4 Dc (Mg/m3) 2.145 µ (mm-1) 19.018 F(000) 1296 Reflections collected 6728 Data/Restraints/Parameters 6728 / 66 / 534 GOF on F2 1.002 2 R1 (on Fo , I > 2σ(I)) 0.0783 2 wR2 (on Fo , I > 2σ(I)) 0.2495 R1 (all data) 0.0955 wR2 (all data) 0.2615

101

Synthetic procedures

Scheme 3.1. Synthesis of new compounds reported in this chapter.

Synthesis of 1: An ethanolic solution of acetylferrocene (1.00 g, 4.38 mmol) was slowly added to a reaction flask containing 2.56 mL (a 12-fold excess, ~53 mmol) of hydrazine hydrate. A catalytic amount (0.42 g, 2.19 mmol) of p-toluenesulfonic acid was then added. The reaction was stirred at room temperature for three days. Reaction completeness was monitored by thin layer chromatography (silica, 100% 102

dichloromethane). Upon completion, an excess of ice-cold DI water was added to the reaction flask and a golden crystalline solid started to form. The resultant crystals were

1 filtered and air-dried. 1: Yield 0.72 g (68%). H NMR (300 MHz, CDCl3, δ): 5.07 (s, 2H,

NH2), 4.50 (br s, 2H, C5H4) 4.26 (br s, 2H, C5H4), 4.15 (s, 5H, C5H5), 2.05 (s, 3H, CH3).

+ HR ESI MS: m/z = calc. for C12H14FeN: 243.0579 found 243.0724 [M+H] . UV-Vis

2 -1 -1 spectrum in THF λmax 436 nm ( = 6.7 x 10 M cm ).

Synthesis of 3: The synthesis and crystal structure of 3 was reported previously195,196 and we used a slightly modified synthetic method in this work.

Compound 2 (1.0 g, 3.7 mmol) was dissolved in ethanol and which was then added to a large excess amount (3.6 mL, 74 mmol) of hydrazine hydrate. The reaction was stirred at room temperature for 48 hours. The resultant orange precipitate was filtered and air-

1 dried. Yield 0.2 g (18%). H NMR (300 MHz, CDCl3, δ): 5.07 (s, 4H, NH2), 4.50 (m, 4H,

C5H4), 4.24 (m, 4H, C5H4), 1.96 (s, 6H, CH3). HR MS ESI: m/z = calc. for C14H18FeN4:

+ 2 299.0954 found 299.1123 [M+H] . UV-Vis spectrum in THF λmax 449 nm ( = 4.4 x 10

M-1 cm-1).

Synthesis of Re(CO)3-pyca-ferrocene complexes

Synthesis of 4 and 5: The procedure for 4 is representative of both compounds.

4: Re(CO)5Cl (50 mg, 0.14 mmol) and pyridine-2-carboxaldehyde (15 μL, 0.14 mmol) were refluxed in 15 mL of toluene for 30 minutes. The mixture turned purple as the reaction proceeded. Compound 1 (36 mg, 0.14 mmol) in toluene (20 mL) was then added to the purple reaction mixture and the combined solution was refluxed. The reaction was

103

monitored by TLC (silica, 2% MeOH/DCM). The reaction was complete in 4 hours according to the total consumption of compound 1 in TLC. The reaction was cooled and a red precipitate appeared. The solid was filtered, washed with ether, and dried under vacuum. Crystals suitable for X-ray diffraction were prepared by slow evaporation from

1 DCM. Yield 61 mg (68%). H NMR (300 MHz, CDCl3, δ): 9.02 (s, 1H, N=CH), 8.23 (m,

1H, H on py), 8.04 (m, 1H, H on py), 7.83 (m, 1H, H on py), 7.54 (m, 1H, H on py),

4.74-4.79 (m, 2H, C5H4), 4.51 (m, 2H, C5H4) 4.31 (s, 5H, C5H5) 2.42 (s, 3H, CH3) ppm.

13 C NMR (125 MHz CDCl3) 196.6, 195.6, 186.8, 165.9, 154.8, 157.4, 153.1, 139.1,

127.5, 126.8, 80.1, 71.4, 71.4, 69.9, 17.7 ppm. IR (CO stretch, cm-1): 2016 (m), 1916 (s),

1883 (s). HR MS ESI: m/z = calc. for C21H17FeN3O3ReClC4H8O: 709.0427 found

+ 3 -1 -1 709.0540 [M ]. UV-Vis spectrum in THF λmax 417 nm ( = 4.3 x 10 M cm ).

1 5: Yield 71 mg (80%). H NMR (300 MHz, CDCl3, δ): 9.05 (s, 1H, N=CH), 8.20

(m, 1H, H on py), 8.03 (m, 1H, H on py), 7.83 (m, 1H, H on py), 7.53 (m, 1H, H on py),

4.81-4.76 (m, 2H, C5H4), 4.52 (m, 2H, C5H4) 4.31 (s, 5H, C5H5) 2.43 (s, 3H, CH3) ppm.

13 C NMR (125 MHz CDCl3) 196.6, 195.6, 186.7, 166.0, 154.7, 154.4, 153.1, 138.9,

127.4, 126.7, 80.2, 71.4, 71.3, 69.9, 17.7 ppm. IR (CO stretch, cm-1): 2017 (m), 1918 (s),

1885 (s). HR MS ESI: m/z = calc. for C21H17FeN3O3ReBr: 680.9339 found 680.9350

+ 3 -1 -1 [M] . UV-Vis spectrum in THF λmax 427 nm ( = 3.6 x 10 M cm ).

Synthesis of 6 and 7: The procedure for 6 is representative of both compounds.

6: Re(CO)5Cl (50 mg, 0.14 mmol) and pyridine-2-carboxaldehyde (15 μL, 0.14 mmol) were refluxed in 15 mL of toluene for 30 minutes. The mixture turned purple as the reaction proceeded. Compound 3 (21 mg, 0.07 mmol) in toluene was then added to the 104

purple reaction mixture and the combined solution was refluxed. The reaction was monitored by TLC (silica, 2% MeOH/DCM). The reaction was complete in 1.5 hours according to the total consumption of compound 3. The reaction was cooled down and an orange precipitate appeared. The resultant solid was filtered, washed with ether, and dried

1 under vacuum. Yield 61 mg (80%). H NMR (300 MHz, d6-DMSO, δ): 9.01 (s, 2H,

N=CH), 8.84-8.81 (m, 2H, H on py), 8.31 (m, 2H, H on py), 8.19-8.17 (m, 2H, H on py),

7.76 (m, 2H, H on py), 4.98 (m, 4H, C5H4), 4.69 (m, 4H, C5H4) 2.38 (s, 6H, CH3) ppm.

13C NMR data could not be obtained due to decomposition of the compound. IR (CO stretch, cm-1): 2020 (s), 1909 (m), 1870 (s). HR MS ESI: m/z = calc. for

C32H24Cl2FeN6O6Re2C4H8O(CH3CN)2: 1265.0597 found 1265.0500. UV-Vis spectrum in

3 -1 -1 THF/NEt3 λmax 410 nm ( = 7.9 x 10 M cm ).

1 7: Yield 57 mg (74%). H NMR (300 MHz, d6-DMSO, δ): 9.02 (s, 1H, N=CH),

8.82-8.80 (m, 1H, H on py), 8.30 (m, 1H, H on py), 8.19 (m, 2H, H on py), 7.75 (m, 2H,

13 H on py), 4.99 (m, 4H, C5H4), 4.70 (m, 4H, C5H4) 2.38 (s, 6H, CH3) ppm. C NMR data could not be obtained due to decomposition of the compound. IR (CO stretch, cm-1):

2021 (s), 1909 (m), 1876 (s).HR MS ESI: m/z = calc. for C32H24Br2FeN6O6Re2N2:

+ 1203.8644 found 1203.9744 [M] . UV-Vis spectrum in THF/NEt3 λmax 418 nm ( = 8.2 x

103 M-1 cm-1).

Synthesis of 8 and 9: The procedure for 8 is representative of both compounds.

8: Re(CO)5Cl (50 mg, 0.14 mmol) and glyoxal (7 μL, 0.14 mmol) were refluxed in 10 mL of methanol for 1 hour. Compound 1 (71 mg, 0.28 mmol) in methanol was added to the reaction mixture and it was refluxed. The reaction was monitored by TLC (silica, 2% 105

MeOH/DCM). The reaction was complete in 3 hours based on the consumption of compound 1. The reaction was cooled down and reddish-brown precipitate appeared. The resulting solid was purified by column chromatography (silica, 2% MeOH/DCM). A red band was collected yielding a red solid that was filtered, washed with ether, and dried

1 under vacuum. Yield 45 mg (40%). H NMR (300 MHz, d6-DMSO, δ): 8.43 (s, 2H, glyoxal N=CH), 4.91 (br s, 4H, C5H4), 4.66 (br s, 4H, C5H4), 4.38 (br s, 10H, C5H5), 2.43

13 (br s, 6H, CH3). C NMR data cannot be obtained due to decomposition of the compound. HR MS ESI: m/z = calc. for C29H26Fe2N4O3ReCH3CN: 818.0521 found

+ 3 -1 -1 818.0501 [M] . UV-Vis spectrum in THF λmax 442 nm ( = 5.1 x 10 M cm ).

1 9: Yield 42 mg (38%). H NMR (300 MHz, d6-DMSO, δ): 8.32 (s, 2H, glyoxal

N=CH), 4.83 (m, 4H, C5H4), 4.57 (m, 4H, C5H4), 4.30 (s, 10H, C5H5), 2.34 (s, 6H, CH3).

13C NMR data cannot be obtained due to decomposition of the compound. IR (CO

-1 stretch, cm ): 2010 (m), 1884 (s). HR MS ESI: m/z = calc. for C29H26Fe2N4O3ReBr:

+ 3 -1 855.9439 found 855.9236 [M] . UV-Vis spectrum in THF λmax 454 nm ( = 4.4 x 10 M cm-1).

Spectroscopy: The UV-visible absorption maxima, extinction coefficients, and infrared carbonyl stretching vibrations are listed in Table 3.2. All UV-vis spectra were recorded using Hitachi U-2000 UV-vis spectrophotometer and JASCO-720 instruments.

Electrochemistry: Electrochemical measurements were conducted using a CHI-

620C electrochemical analyzer utilizing the three-electrode scheme. Unless stated otherwise, platinum working, platinum auxiliary, and Ag/ AgCl pseudo-reference

106

electrodes were employed in a 0.1 M solution of TBAP in DMF for electrochemical experiments. In all cases, the redox potentials are referenced to the FcH/FcH+ couple using decamethylferrocene as an internal standard.

Spectroelectrochemistry: Spectroelectrochemical data were collected on a

JASCO-720 spectrophotometer at room temperature. The experiments were conducted using a CHI-620C electrochemical analyzer using a custom-made 1 mm cell with a platinum mesh-working electrode. In order to suppress overtones in the NIR region of the optical spectra, measurements were conducted in 0.3 M TBAP in DMF and in 0.05 M

TFAB in DCM.

Computational: The starting geometries of all compounds were adopted from X- ray structures. All were optimized using the TPSSh exchange–correlation functional197,198 coupled with the Wachter's full-electron basis set199 for the Fe atom and the 6-311G(d) basis set200 for the remaining atoms. Energy minima in optimized geometries were confirmed by frequency calculations. DMF was used as a solvent in all of the single point

DFT-PCM and TDDFT-PCM calculations; solvent effects were calculation using the polarized continuum model (PCM).201 The first 50 states of each compound were calculated in all TDDFT-PCM calculations. All DFT calculations were conducted using the Gaussian 09 software package,202 and the QMForge program203 was used for the molecular orbital analysis.

Table 3.3 UV-visible absorption and FTIR data of all compounds

107

3 -1 -1 -1 Compound λmax (nm) ε (x10 M cm ) ν C≡O (cm )

1 436 0.67 n/a

3 449 0.44 n/a

2016 (m) 4 417 4.3 1916 (s) 1883 (s)

2017 (m) 5 424 3.6 1918 (s) 1885 (s)

2020 (s) 6* 410 7.9 1909 (m) 1870 (s)

2021 (s) 7* 418 8.2 1909 (m) 1876 (s)

8 446 4.3 2018 (m) 1888 (s)

9 454 4.4 2010 (m) 1884 (s)

* For UV-vis experiments, a few drops of triethylamine (NEt3) were added to this solution prior to the experiment to prevent oxidation.

108

Results and discussion

Previously, we have used hydrazine to synthesize dimeric compounds via Schiff base formation with aldehydes. For example, we have used hydrazine with pyridine-2- carboxaldehyde to afford dimeric Re(CO)3 compounds that exhibit strong coupling between the metal-diimine units.58 In a separate system, we also used hydrazine to produce the precursor to the dimeric BF2 chromophore BOPHY, where the hydrazine unit links two pyrrole-2-carboxyaldehyde units to form the boron-chelating ligand.152,154

Although aldehydes work well making symmetric dimeric ligands with hydrazine, we have found that using this bridge to link different aldehydes does not result in the formation of asymmetric diimines; rather, mixtures of the symmetric products result from the reaction of hydrazine and different aldehydes. We hypothesized that using the differential reactivity of aldehydes and ketones towards hydrazine might be useful for producing asymmetric systems.

Acetyl ferrocene and diacetyl ferrocene both readily react with an excess of hydrazine to afford the corresponding hydrazone compounds, as shown in Scheme 3.1.

The bis-hydrazone compound 3 has been previously synthesized.195,196 Although we did not structurally elucidate either the mono or bis hydrazone modified ferrocenes, all of the spectroscopy on compound 1 is in good agreement with the proposed structures, and our characterization of compound 3 is in agreement with literature values. For both 1 and 3, half of the hydrazine reacts to form Schiff bases with the ketones, leaving the remaining

NH2 group to form a second Schiff base which can coordinate to a Re(CO)3 center. Thus,

109

we can readily generate a connection to a Re(CO)3 center using metal mediated Schiff base formation, as shown in Figure 3.1. We and others have used this reaction to append a wide variety of molecules to the Re(CO)3, including aryl rings, fluorescent compounds, peptides and isomerizable groups like azobenzene.59 These reactions can be carried out using one-pot conditions, and thus for the synthesis of conjugate systems is an attractive way to obtain a variety of structures.

All of the 1:1, 2:1, 1:2 ferrocene:Re(CO)3 conjugate complexes have been fully characterized; we were able to structurally elucidate the 1:1 conjugates (compounds 4 and

5), which are shown in Figure 3.2. For both 4 and 5, there are two molecules per asymmetric unit. With regard to connectivity and overall bond length patterns, the structures of 4 and 5 are identical with the exception of the identity of the halide. The C-

N bonds of the hydrazone unit have double bond character (1.289(9) and 1.285(9) Å for 4 and 1.258(17) and 1.253(18) Å for 5). The N-N bonds, however, are single in character, with lengths of 1.402(8) and 1.434(8) Å for 4 and 1.422(15) and 1.374(17) Å for 5. The ferrocene units and the Re-diimine planes are neither co-planar nor orthogonal; the complexes in the structures of 4 and 5 exhibit angles between these planes ranging from

~71 to ~78°. The remaining 2:1 and 1:2 conjugates were not structurally elucidated, however NMR and mass spectrometric characterization was in complete agreement with the expected structures. We did obtain crystals of 9, but the quality was poor and we were not able to get a converged solution to the structure. However, a partial solution did provide confirmation of connectivity that is in agreement with the proposed structure; this partial structure can be found in Figure 3.3.

110

Figure 3.3. The partial structure of compound 9.

Since both ferrocene and Re(CO)3 complexes have been investigated for their uses as optical and electronic materials, we probed electronic structures of the six new conjugates presented in this report using spectroscopy, electrochemistry and theoretical methods. The UV-visible maxima and extinction coefficients are listed in Table 3.3, and the UV-visible spectra of the conjugate compounds are shown in Figure 3.4. All of the compounds presented in this report are colored, resulting from both ferrocene and

Re(CO)3 diimine based transitions. For compounds 1 and 3, ferrocene hydrazones exhibit low molar absorbtivites in the range of hundreds of units. Re(CO)3 diimine complexes exhibit metal to ligand charge transfer (MLCT) transitions that show much higher molar absorbtivity (>5 times higher for 4 and 5, and ~20 times higher for 6 and 7).

The molar absorptivities of 4 and 5 were comparable to those observed in mono-rhenium tricarbonyl Schiff base compounds with absorptivities around 3,000-4,000 M-1cm-1.59,204

Complexes (6 and 7) show stronger absorbtivities in the range of 7,000-8,000 M-1cm-1, about twice as much as those of monomeric Re(CO)3 Schiff base complexes, which is 111

consistent with their dimeric structures.57,58 All of the transitions occur between 400 and

500 nm, which is as expected. Compounds 7 and 8 exhibit a significant amount of bathochromic shifting; we observed similar trends in other dimeric Re(CO)3 complexes.57,58 All of the conjugates display diagnostic CO stretching frequencies in their IR spectra; these correspond to the a1 and e type modes that result from the facial

Re(CO)3 unit.

Figure 3.4. UV-visible spectra of compounds 4-9 in THF.

The electrochemistry of both ferrocene and Re(CO)3 diimine complexes have been extensively investigated; the two metal complexes can exhibit reversible oxidations and reductions respectively. In conjugates with either two ferrocenes or two Re(CO)3 diimine centers, we have previously observed coupling and the formation of mixed 112

valence behavior.57,58 We probed the precursor molecules 1 and 3 as well as the 1:1 conjugate compounds 4 and 5 using cyclic voltammetry. Both ferrocenes and the chloride analog data 4 are shown in Figure 3.5. The voltammograms for the bromide analog are found in the supplementary information. Unlike unmodified ferrocene, these substituted variants do not show reversible oxidations, clearly due to the oxidative reactivity of the hydrazone functional groups. The 1:1 complexes 4 and 5 show slightly more reversible oxidations of the ferrocenes, as well as exhibit quasi-reversible reduction waves attributable to the Re(CO)3 diimine units. In these complexes, reduction of the

Re(CO)3 diimine unit is a ligand based processes; we have reported this phenomenon previously and this conclusion is supported by our DFT-PCM calculations.

For the conjugates with either two ferrocenes or two rhenium centers, we were interested in possibly observing coupling between the peripheral units on these compounds. The cyclic voltammagrams of the 1:2 ferrocene:rhenium compound 6 as well as the 2:1 ferrocene:rhenium compound 8 are also shown in Figure 3.5. The bromide analog voltammograms can be found in the supplementary information.

Compounds 6 and 7 exhibit irreversible oxidation waves, and quasi-reversible reduction processes. Unlike our previously reported Re(CO)3 dimer compounds, we do not observe any separation of the reduction wave into two peaks, and thus do not coupling across the ferrocene unit. In compounds 8 and 9, we observe the opposite trend from 6 and 7: quasi reversible oxidations and irreversible reductions. In these cases as well, we do not observe any evidence for coupling between the ferrocene units in the cyclic voltammetry experiments.

113

Figure 3.5. Cyclic voltammograms of compounds 1, 3, 4, 6, and 8.

In addition to using cyclic voltammetric behavior, the presence of a mixed valence system also can be detected via the presence of intervalence charge transfer bands upon either one electron oxidation or reduction. Previously, we have used this to confirm the presence of mixed valence systems in both ferrocene and Re(CO)3 systems.57,58 Figure 3.6 shows the oxidative and reductive spectroelectrochemistry of compound 4 and 5, the 1:1 compounds. We observe, upon oxidation, the formation of a new band at ~580 nm, which corresponds to the expected charge transfer transition seen for the ferricenium cation. Unfortunately, compounds 6-9 are unstable in solution during the time course of the spectroelectrochemistry experiment, so we were not able to observe any intervalence charge transfer bands.

114

Figure 3.6. Spectroelectrochemistry of compounds 4 (top) and 5 (bottom) upon oxidation (left) and reduction (right).

We also probed the electronic structures of using DFT methods. The energy levels of the frontier orbitals for compounds 4-9 are shown in Figure 3.7. The percent contribution to the frontier orbitals based on molecular fragment for compounds 1, 3, and

4-9 are shown in Figures 3.8-3.9. Graphs of molecular composition and a diagram showing the energy levels of the frontier orbitals of 1 and 3 compared to the other compounds presented in this report can be found in the supplementary information. For 115

compounds 1 and 3, we observe that the HOMO orbital is primarily composed of iron and cyclopentadienyl orbital character, while the LUMO has a significant contribution from the hydrazone orbitals. Compound 3, with two hydrazone functional groups, has approximately twice as much of this contribution to the LUMO (Figure 3.8). The structures of the HOMOs and LUMOs for ferrocenes 1 and 3 and conjugates 4-9 can be seen in Figure 3.9.

Figure 3.7. DFT predicted energy diagram of rhenium complexes 4-9. HOMOs and LUMOs are connected by a dotted line.

116

Figure 3.8. Percent contribution to the frontier orbitals based on molecular fragment for compounds 1 and 3.

117

Figure 3.9. The DFT-PCM predicted isosurfaces of the HOMOs and LUMOs for ferrocene compounds 1 and 3 and conjugates 4-9.

With regard to the conjugate complexes, they exhibit similar patterns in their frontier orbital energies. For the 1:1 conjugates 4 and 5, the HOMO and HOMO-1 are ferrocene based, and the LUMO is localized on the ligand. The same trends are also observed for the conjugate compounds 6-9. The rhenium d orbital contributions are lower in energy than the occupied ferrocene frontier orbitals, starting at HOMO-3 for compounds 4-7, and HOMO-5 for 8 and 9. For all of the conjugate complexes, replacement of chloride with bromide results in slight destabilization of the frontier orbitals (Figure 3.10).

118

Figure 3.10. PercentPercent contribuributioncontcontributiontion to the to frontierthe frontier orbitals orbitals based based on molecularmolecula on molecula fragmentagmentrr fr fragment for for compounds 4-9.

119 We also used TDDFT-PCM method to predict the optical spectra of the compounds presented in this report, as can be seen in Figure 3.11. We observe good agreement between calculated and observed spectra for the ferrocene compounds 1 and 3.

For the conjugates 4-9 (the chloride analogs are shown in the figure) we see a red shift to our calculated spectra versus the observed. The UV-visible transitions between 400 and

460 in these conjugates can be characterized as MLCT bands (metal d orbital to diimine).

For the bis-rhenium conjugate systems, we observe a red shifting and expansion of the primary visible absorption band to lower wavelengths; this may result from long range coupling interactions between the rhenium centers, and we have observed this phenomenon previously. Our calculations do not predict such behavior, possibly because the model does not adequately predict such interactions.

Figure 3.11. Experimental and TDDFT predicted UV-visible spectra for compounds 1, 3, 4, 6 and 8. 120

Conclusions

In conclusion, hydrazine can be used a linking reagent to connect the two well studied organometallic fragments ferrocene and Re(CO)3. A modular approach to producing conjugates was developed, resulting in 1:1 as well as 2:1 and 1:2 ferrocene:Re(CO)3 By first reacting hydrazine with acyl ferrocenes, we can produce hydrazones that can be subsequently used in Re(CO)3 mediated Schiff base formation reactions. The resultant conjugates have been fully characterized, but the 1:2 and 2:1 complexes exhibit stability problems in solution. We investigated their electrochemistry and spectroscopy of the compounds presented in this report; we do not observe fully reversible oxidations or reductions in these conjugates, most likely due to the redox sensitivity of the Schiff base bridge. Finally, DFT and TDDFT calculations provided insight into the electronic structures of the ferrocene precursor compounds as well as the conjugate systems. We are continuing our investigations into the covalent coupling of organometallic moieties to both biologically relevant compounds as well as chromophore systems.

121

CHAPTER IV

FACILE RHENIUM–PEPTIDE CONJUGATE SYNTHESIS USING A ONE-POT

DERIVED Re(CO)3 REAGENT

The text of this chapter is a reprint of the material as it appears in:

(a) Chanawanno, K.; Caporoso, J.; Kondeti, V.; Paruchuri, S.; Leeper, T. C.; Herrick, R. S.;

Ziegler, C. J. Dalton Trans. 2014, 43, 11452–11455

(b) Chanawanno, K.; Kondeti, V.; Caporoso, J.; Paruchuri, S.; Leeper, T. C.; Herrick, R. S.;

Ziegler, C. J. Dalton Trans. 2016, 45, 4729–4735.

Introduction

The functionalization of biological molecules with organometallic groups continues apace.181 Investigations into this chemistry have been driven by both older applications and new developments in bioinorganic chemistry.205–210

Organometallic compounds have been used as phasing agents for protein structure elucidation, and compounds containing the Tc(CO)3 moiety are used for SPECT 211– imaging applications. 213 More recently, metal carbonyl compounds have been employed as electron transfer

122 reagents as well as probes for protein dynamics via vibrational relaxation studies.

Organometallic compounds have also been used as drugs that can interact with proteins, as seen in their use as kinase inhibitors214,215 or as carbon monoxide releasing molecules

(CORMs).1,216–218

Another active area of investigation is the covalent attachment of organometallic units to biomolecules via conjugate formation.219–224 The bifunctional chelate method

(BFCA)84,225 has frequently been used, which involves the construction of an organic linker that both chelates the metal unit and forms a covalent bond to the biological molecule.226–228 Accordingly, this strategy has been applied to biomolecules such as polypeptides. This has been successfully implemented to attach units such as the Re(CO)3 moiety to peptides and can be applied to solid phase peptide synthesis (SPPS) methods.69,75,229,230 However, the construction of such linkages requires several steps,31,231,232 that may limit their usefulness in radiopharmaceutical applications, especially with isotopes of limited lifetimes.

Neurotensin and other small polypeptides have been used for the fabrication of

233–235 BFCA compounds incorporating the Re(CO)3 fragment. Previously, Metzler-Nolte et al. appended a rhenium complex to neurotensin(8-13) using a bis(phenanthridinyl- methyl)amine-based chelate attached to the amine terminus of the fragment.164

Zagermann and co-workers synthesized tungsten nodified-neurotensin(8-13),236 and

Mn(CO)3(cymantrene) was attached to neurotensin using the SPPS method as presented in the work by N’Dongo et al.237 A similar strategy was recently employed to append the same moiety to a bombesin-based peptide.238 123 For several years we have been interested in the fundamental chemistry of the

Re(CO)3 unit, and have used this chemistry to functionalize biological molecules with this group. Originally use to model the chemistry of Tc(CO)3 compounds, Re(CO)3 now also relevant to many alternate uses of organometallic compounds in biological

1,216–218 systems. One such method to append biological molecules to Re(CO)3 is to use metal-mediated Schiff base formation, which can be readily accomplished using pyridine-2-carboxaldehyde (pyca) as a reagent. We and others have used Re(CO)5X or

+ Re(CO)3(H2O)3 with pyca and primary amines to produce Re(CO)3 diimine conjugate complexes (Scheme 3.1).10,34–61 This method is broadly applicable to a wide variety of primary amines and can be carried out using one-pot methods. We have used this reaction to synthesize amino acid Re(CO)3 conjugates and surmised that we would be able to use the same process to generate a sidechain-modified amino acid that could be incorporated into SPPS methods.

In this chapter we present the one pot synthesis of a sidechain-modified lysine using pyca and Re(CO)5X to afford an Nε substituted Re(CO)3 complex. The resultant modified lysine is quite stable and can be used in SPPS methods, and we have incorporated it into four biologically active peptides: neurotensin (NEU: RRPYIL), bombesin (BBN: MLHGVAWQ), lutenizing hormone releasing hormone (LHRH:

EHWSYGLRPG), and a nuclear localization sequence (NLS: KRRRC). Additionally, we exposed these rhenium-modified peptides to human umbilical vascular endothelial cells

(HUVECs), and demonstrated uptake using a fluorescent label as well as rhenium elemental analysis. We observed that the identity of the peptide affects the degree of

124 uptake in HUVECs, which supports the hypothesis that this uptake is at least in part mediated by receptors on the surface of the cells.

Experimental

Materials and methods

All single amino acid loaded resins, Fmoc-amino acids, HOBt

(hydroxybenzotriazole), HBTU (O-henzotriazole-N,N,N’,N’-tetramethyluronium hexafluorophosphate) and N-methylmorpholine, and other chemicals were purchased and used without further purification. NMR spectra were recorded on a Varian Mercury 300

MHz and Varian NMRS 500 MHz (2 channel with automation) spectrometers. Chemical shifts were reported with respect to residual solvent peaks as internal standard (1H:

13 DMSO-d6, δ = 2.50 ppm; C: DMSO-d6, δ = 39.7 ppm). MALDI-TOF mass spectra were carried out on a Bruker Ultraflex-III TOF/TOF mass spectrometer (Bruker Daltonics,

Inc., Billerica, MA) equipped with a Nd:YAG laser (355 nm). All spectra were measured in positive reflection mode. UV-visible spectroscopy was carried out on a Hitachi U-3010 spectrometer.

125 X O Re(CO) X OC CO N 5 Re + R NH2 N N R CO

H2N OC CO O Re O N N N OH Re(CO)5X O O NH + CO OH O , MeOH, 8 hours O NH O 1: X = Cl 2: X = Br

Scheme 4.1. Top: General scheme for a one pot pyca Schiff base Re(CO)3X reaction. Bottom: Preparation of 1 and 2.

Synthesis of 1 and 2

Pyridine-2-carboxaldehyde (0.012 mL, 0.125 mmol) and Fmoc-Lys-OH (46 mg,

0.125 mmol) were heated in methanol (15 mL) for 0.5 hour. Upon reaction the solution’s colour changed from very pale yellow to light brown, Re(CO)5Cl (45 mg, 0.125 mmol) was then added into reaction mixture. The solution was further refluxed for 8 hours. The resulting orange solution was allowed to cool down to room temperature. The solvent was removed and the orange solid was dried under vacuum for a few days. The orange solid was repeatedly washed with ether and air-dried. Compound 1 was obtained in 76% yield (66 mg, 0.088 mmol). Compound 2 can be synthesized by the same method using

Re(CO)5Br and was obtained as orange solid in 79% yield (80 mg, 0.099 mmol).

1 1: H-NMR (500 MHz in DMSO-d6): 9.23 (s, 1H, HC=N), 8.98 (m, 1H, H on py),

8.19-8.28 (m, 2H, H on py), 7.88 (m, 2H, H on py and NH), 7.60-7.76 (m, 4H, H on

126

Fmoc), 7.28-7.43 (m, 4H, H on Fmoc), 4.25 (m, 2H, O-CH2), 4.20 (m, 1H, HC-NH), 4.03

(m, 2H, CH2-CH-NH), 3.95 (m, 1H, H on Fmoc), 1.90-1.95 (m, 2H, CH2-CH2-CH-NH),

13 1.69-1.75 (m, 2H, =N-CH2), 1.42 (m, 2H, =N-CH2-CH2). C-NMR (125 MHz in DMSO- d6): 198.2, 197.9, 188.2, 174.3, 170.0, 156.6, 155.2, 153.4, 144.3, 141.2, 129.7, 128.1,

127.5, 125.7, 120.5, 66.1, 64.3, 54.2, 47.1, 30.7, 29.2, 23.1 ppm

1 2: H-NMR (500 MHz in DMSO-d6): 9.21 (s, 1H, HC=N), 9.02 (m, 1H, H on py),

8.23-8.28 (m, 2H, H on py), 7.89 (m, 1H, H on py), 7.87 (s, 1H, NH), 7.31-7.76 (m, 8H,

H on Fmoc), 4.20 (m, 1H, H on Fmoc), 4.26 (m, 2H, O-CH2), 3.95 (m, 1H, HC-NH),

4.05 (m, 2H, CH2-CH-NH), 1.87-2.01 (m, 2H, CH2-CH2-CH-NH), 1.30-1.45 (m, 2H, =N-

13 CH2), 1.66-1.81 (m, 2H, =N-CH2-CH2). C-NMR (125 MHz in DMSO-d6): 197.7, 197.5,

187.5, 174.3, 169.9, 156.6, 155.3, 153.6, 144.3, 141.2, 129.6, 128.1, 127.5, 125.7, 120.6,

66.1, 64.4, 54.3, 47.1, 30.7, 29.3, 23.1 ppm.

Peptide synthesis

Rhenium-peptide conjugates were synthesized using standard Fmoc solid phase peptide synthesis methods. The completeness of each coupling reaction was monitored by the Kaiser Test.43 Fmoc removal between each cycle was performed by 30-minute treatment with 20% piperidine in dimethyl formamide (DMF).

Each synthesis was initiated by using 100 milligrams of Fmoc-Xxx-Wang resin.

The resin was soaked in DMF for 1-2 h to swell the resin beads. Prior to coupling, Fmoc groups were removed using 20% piperidine in DMF for 30 minutes followed by washing with DMF, methanol and dichloromethane several times. Five equivalents of Fmoc-Xxx- 127

OH along with 4.5 equivalents of HBTU and HOBt and 10 equivalents of 4- methylmorpholine were used in this synthesis. The coupling process was performed for 2 hours followed by washing and the Kaiser test was carried out prior to the next cycle. All reactions were performed on a mechanical shaker. In cases where free NH2 termini remained present on the resin, the coupling process was repeated. After completion of all cycles with Fmoc-Xxx(s), 5 equivalents of the rhenium complex 2 were coupled overnight using HBTU/HOBt 4-methylmorpholine in the amounts described above. The coupling reaction using 5(6)-carboxyfluorescein was performed in a similar manner. The coupled resin turned yellow/orange after completion of the last cycle and was washed with DMF. In each case the resin was moved to a clean round bottom flask and air-dried.

Methylene chloride was added into the flask and the resin was left until swollen with solvent (1 hour). Methylene chloride was removed under vacuum and the TFA

(trifluoroacetic acid) cleavage method was started immediately.

The TFA/water/triisoproplysilane (95:2.5:2.5% v/v/v) cocktail solution (10 mL: 1 mL per 10 mg resin) was added to dry resins. The suspension was gently stirred for three hours. The mixture was filtered and the resin was washed twice with a small amount of fresh TFA cocktail. The filtrate was kept and the volume was reduced to be around ¼ of original volume. An excess amount of cold ether (> 10 times the initial TFA cocktail volume) was added causing the crude yellow peptide to precipitate. The suspension was cooled and centrifuged for 5 minutes at 5000 rpm and the supernatant was discarded. The peptide was solubilized in acetonitrile/water/TFA (50:50:0.1% v/v/v) and was lyophilized to give yellow-orange powder.

128

Conjugates 3-6 were purified by HPLC. The purification process was performed on a GE Äkta Purifier equipped with a UV900 detector using a semipreparative Vydac

218TP C18 reversed-phase column. The solvent system used was 99.9% water/0.1% TFA

(v/v) and 95% acetonitrile/5% water/0.1% TFA v/v/v. The detector was set to detect absorption at 254 and280 nm. Mass spectra of the purified sample fractions showed the expected m/z values for all conjugates.

129

Figure 4.1. Structures of Re(CO)3 conjugates with a neurotensin fragment (3), a nuclear localization sequence (RRRC, NLS, 4), a bombesin fragment (5), and a lutenizing hormone releasing hormone fragment (LHRH, 6).

Cell studies

Human umbilical vascular endothelial cells (HUVEC) were maintained in EBM2

TM medium with SingleQuots and 10% fetal bovine serum (FBS) in a humidified 5% CO2 environment at 37oC. For staining experiments, cells were seeded on 24-well 0.1% 130

gelatine coated plates (75,000 cells/ well) and incubated for 24 h prior to incubation with the rhenium-peptide conjugates at different concentrations.

Incubation of compounds 3-6 was carried out on adherent HUVECs.

Concentrated DMSO stock solutions of each compound were diluted using cell culture medium to achieve desired final concentrations of 80, 40, 20, 10, and 5 μM. The amount of DMSO used in each dilution was lower than 1% to prevent cytotoxicity from DMSO itself.44 After incubation, the cells were washed three times with phosphate buffered saline (PBS) (pH 7.4), fixed in 2% paraformaldehyde (PFA), for 15 minutes at room temperature and washed again three times with PBS to remove residual PFA. Nuclei were stained using 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI).

Rhenium uptake studies

The uptake of rhenium by HUVECs was measured using inductively coupled plasma optical emission spectroscopy (ICP-OES). The cells were seeded on 6-well plates and were incubated with each compound as described above. After 24 hours, cells were washed thoroughly with PBS several times, lysed with lysis buffer (RIPA, 100 μL per well), at 4°C. The adherent cells were scraped off the dish using a plastic cell scraper and cell lysates were stored at -20°C for ICP-OES analysis.

The Agilent 700 series ICP-OES with Expert II software was used to confirm the cellular uptake of rhenium. Rhenium standard solutions were prepared using commercially available BDH® ARISTAR® single element standards Re (1,000 µg/mL in

131

3% HNO3). All measurements were conducted in triplicate and the results were found to be reproducible (with <6%RSD).

Scheme 4.2. Synthesis of peptide conjugates 3-6 where amides were formed using the HOBt/HBTU method and cleaved from the resin using TFA: i. Fmoc-deprotection, ii. Washing, iii. Coupling, iv. Repeat washing, v. TFA Cleavage. 132

Figure 4.2. The chromatograms of 4 (100% A → 100% B in 25 min): 19 min, 5 (100% A → 100% B in 30 min): 25 min, and 6 (100% A → 100% B in 32 min): 25 min with the MALDI-TOF spectra. (Chromatograms: blue trace: 280 nm, red trace: 254 nm detection).

133

Results and Discussion

For many years, researchers have looked to append the Re(CO)3 unit to peptides.

Historically, these studies aimed to produce new methods for generating radionuclide conjugates that could be directed to specific tissues or cell types. Frequently, organic metal-bridging ligands have been employed to couple the Re(CO)3 unit to a polypeptide, however recently several groups have focused on modifying the Nε of lysine to convert it into a metal binding unit. In 2004, Stephenson et al.45,46 reported a new strategy for the synthesis of a Re(CO)3 complex with a lysine-derived bis(pyridyl) amine ligand. This strategy was referred to as the single amino acid chelate (SAAC) method. N-α-Fmoc-L- lysine was reacted with either pyridine or quinoline aldehyde to give the bifunctional ligand that was subsequently reacted with [NEt4]2[Re(CO)3Br3], and formed a

Re(I)/Tc(I)-binding amino acid mimic as a result. This inert SAAC complex with a

M(CO)3+ core (M = Re, Tc) can be incorporated into peptides in a similar way as a natural amino acid. The SAAC technology was used to make a variety of Tc/Re(CO)3 biomolecules.31,232 Cyclic peptides for the urokinase plasminogen activator receptor were prepared by using a series of N-α-Fmoc-lysine derivatives with two heterocyclic donor groups at the -amine.31 Moreover, a SAAC-Re complex was integrated within fMLF, a targeting sequence guiding radionuclides to the formyl peptide receptor (FPR). The peptide conjugate internalized into human leukocytes and can be fluorescently monitored.45 Alberto el at.242 reported the work in which Fmoc- -Lys(Dap(Boc))

(Dap=2,3-diamino propionic acid) was used to conveniently post-label Tc(CO)3 and

Re(CO)3 units to BBN peptide. 134

For several years, we have been reporting on Re(CO)3 mediated Schiff base formation using pyca and primary amines.57,60,204,243 This chemistry is broadly applicable and can be used to synthesize Re(CO)3 pyridine-2-carboxaldimines with a variety of groups off of the imine nitrogen. Additionally, this chemistry can either be carried out sequentially after initial formation of a pyridine-2-carboxaldehyde Re(CO)3 complex, or via one pot conditions. We decided to use the latter method to produce a sidechain- modified lysine, as shown in Scheme 4.1. By reacting Re(CO)5X with the N-terminal protected lysine using one pot conditions, we were able to isolate the Nε functionalized pyridine-2-carboxaldimine complex. This reaction can be out with either Re(CO)5Cl or

Re(CO)5Br as the source of the Re(CO)3 group, and yields of these reactions were approximately 80%. It is important to note that the metal centre is chiral and that both enantiomers are produced in this reaction. Compounds 1 and 2 exhibit spectra that are typical for (diimine)Re-(CO)3X(diimine) compounds. As can be seen in Figure 4.1 for compound 2, two key spectroscopic features can be observed. First, in the vibrational spectra, the localized C3v symmetry of the Re(CO)3 moiety affords two IR active CO stretching vibrations corresponding to the a1 and e modes. Another key spectroscopic feature is the presence of metal to ligand charge transfer (MLCT) bands in the UV-visible spectra of 1 and 2, with absorption maxima at ∼394 nm with extinction coefficients near

3600 M 1 cm 1 (Figure 4.3).

135

Figure 4.3. FT-IR and UV-visible spectra of compounds 1, 2 and 4-6.

Table 4.1 Absorption characteristics data of 2-6 in DMSO

λ 3 -1 -1 Compound max ε (x10 M cm ) 2 394 3.6 3 380 2.0 4 379 2.1 5 364 3.9 6 372 2.9

136

Figure 4.4. Fluorescent images of peptides appended beads. Left column: GFP at 470 nm excitation. Right column: Texas red at 594 nm excitation for compound 4 (a), 5 (b), and 6 (c).

We hypothesized that the kinetically inert nature of the d6 Re(I) center in

Re(CO)3diimine compounds would make them useful as SSPS reagents. Incorporating

Re(CO)3/Tc(CO)3 groups into polypeptides has been actively pursued since the 1990s.

For the Re(CO)3 unit, a good portion of this work has focused on integrating the moiety into a peptide via a tridentate-ligand linker that could either be incorporated into the end of the polypeptide or in a modified lysine or alternate sidechain.

As a proof of principle for our approach, we used compound 2 to synthesize several polypeptides via standard SPPS methods. The peptides constructed for this study are shown in Figure 4.1. Initially, we reported in a communication the synthesis of a

137

42 neurotensin Re(CO)3 derivative, and then extended this to three additional peptides, including bombesin, LHRH, and NLS. All four sequences have been used to transfer metal ions into cells, but each does so via a different mechanism. After synthesis of each of the peptides, we were able to confirm the identity of the peptide conjugates via mass spectrometric methods and the purity via HPLC (Figure 4.2). Once the peptides were successfully appended on beads, fluorescent images of the beads were observed as shown in Figure 4.4.

Additionally, we were able to observe two distinct spectroscopic features in these rhenium-peptide conjugates that establish the presence of a Re(CO)3 diimine unit. First, all four systems show IR bands that result from the facial coordination mode of the three carbonyl ligands (Figure 4.3). The pattern of the carbonyl stretching frequency results from the localized C3v symmetry present in the Re(CO)3 unit, producing bands between

-1 2040 and 1880 cm that correlate to the a1 and e modes. The second key feature is a metal-to-ligand charge transfer (MLCT) band that is diagnostic for Re(CO)3 diimine compounds. These bands appear at 370-380 nm and exhibit extinction coefficients ranging from 2000 to 3900 M-1cm-1 (Table 4.1, Figure 4.3). Due to the charge transfer nature of these transitions, they are solvent dependent, exhibiting shifts in the absorption maxima with changes in solvent polarity. In the peptide conjugates 4-6, we also observe similar charge transfer bands in their UV-visible spectra and the presence of fluorescein absorbance bands that appear as shoulders on the MLCT band. Additionally, all of the three new peptide conjugates exhibit the expected carbonyl stretching modes in their IR spectra.

138

Previously, we showed that our rhenium pyridine-2-carboxaldimine compounds were taken up by human vascular endothelial cells (HUVECs).43 It is known that

HUVECs express NTSR1, which is the receptor for neurotensin binding and was chosen in our prior work since conjugates that contain neurotensin(8-13) are able to bind to this receptor.244 We wanted to use the same cell line for the current study, and these cells also have receptors that can bind bombesin and LHRH based peptides. Bombesin, a homologue of mammalian gastrin-releasing peptide (GRP), possesses specific binding ability to the G-coupled protein receptors expressed on breast, lung, prostate and pancreatic cancers.245,246 It was observed that a bombesin-like peptide called neuromedin

B can enter and stimulate the growth of HUVECs.247 The presence of luteinizing hormone/human choriogonadotropin receptors (LH/hCG) expressing in HUVECs has also confirmed and hCG mediated angiogenic effects can take place through interaction with this receptor.248

We extended this to the three new peptides presented in the current study. As in the original study with peptide 3, we appended a fluorescein unit to the end of the polypeptide chain 4, 5 and 6 to more easily monitor uptake. Figures 4.5, 4.6 and 4.7 show fluorescent micrographs of HUVECs treated with these peptides. The left column shows whole cell fluorescence, the middle shows DAPI staining, and the right shows merged images. All the three sets of micrographs show the presence of fluorescein in the cells, which we can attribute to uptake of the rhenium modified peptides. Uptake is seen at both the higher concentration (Figure 4.5 and 4.8, 40 μM) and at an eight fold smaller concentration (Figure 4.6, 5 μM). Fluorescence is detected both in the cytoplasm as well

139

as the nuclei of these cells. In the more concentrated sample, minimal difference between the three peptide sequences was observed. This might arise either from similar degrees of uptake, or from lysis of the fluorescein unit within the cells. At low concentrations, we observed differential uptake, with 5 showing the least uptake.

Figure 4.5. Fluorescent micrographs (20x magnification) of HUVECs incubated with 40 μM of compound 4 (first row), 5 (second row), and 6 (third row) for 24 hours. Images were achieved in GFP (a), DAPI (b), and merged (c) modes.

140

Figure 4.6. Fluorescent micrographs (20x magnification) of HUVECs incubated with 5 μM of compound 4 (first row), 5 (second row), and 6 (third row) for 24 hours. Images were achieved in GFP (a), DAPI (b), and merged (c) modes.

Figure 4.7. HUVECs incubated with 40 μM of 3, excited at 470 nm, showing uptake of the polypeptide (20x magnification).

141

We can quantify the amount of peptide conjugate uptake in the HUVECs by use of rhenium elemental analysis via ICP OES (Figure 4.8 and Table 4.2). One of the advantages of this method is that there is no naturally abundant rhenium in cells, and therefore it can provide an absolute measure of peptide uptake. Table 4.2 shows the results of our analysis. All three peptides investigated do show uptake, however we observe significantly higher uptake for the peptide 4, which contains the nuclear localization sequence. Peptide conjugate 4 shows more than four times greater uptake as measured by rhenium analysis, clearly showing that the identity of the peptide sequence affects this process. This is not too surprising since K/R rich peptides are often also used as cell penetrating peptides for efficiently shuttling various cargos into cells.47

Table 4.2 Cellular uptake and accumulation percent values for compounds 4-6 in

HUVECs.

Loaded peptide Cellular uptake Peptide Accumulation (%) %RSD (ppm) (ppm) 4 2.68 0.237 8.85 5.6 5 2.98 0.049 1.66 3.0 6 3.38 0.054 1.61 1.1

142

Figure 4.8. Rhenium emission intensity and rhenium uptake concentration calibration curve from ICP experiment. Relative standard deviations of replicate measurements (N = 3) of single calibration points are <6%.

Conclusions

In conclusion, we have synthesized a new Re(CO)3-modified lysine that can be generated in one step via a one-pot reaction from commercially available reagents.

Although other methods have been used to append the Re(CO)3 unit to the sidechain of lysine, the pyca-Schiff base forming reaction affords a one-pot/one step method for carrying out this chemistry. We have shown that this modified amino acid can be incorporated into a polypeptide using solid phase peptide synthetic methods, and is robust to standard TFA cleavage cocktail procedures. To demonstrate the flexibility of this new modified lysine reagent, we incorporated it into four different peptide sequences, and monitored cellular uptake via use of a fluorescein modified variant. We are continuing 143

our work on the functionalization of proteins and polypeptides with the Re(CO)3 moiety and other transition metal ions.

144

CHAPTER V

THE SYNTHESIS AND STRUCTURES OF ARENE-SUBSTITUTED

AZADIPYRROMETHENES

The text of this chapter is a reprint of the material as it appears in: Chanawanno, K.;

Hasheminasab, A.; Engle, J. T.; Ziegler, C. J. Polyhedron 2015, 101, 276–281.

Introduction

The dipyrromethene and azadipyrromethene families of compounds (1 and 2,

Figure 5.1) can be considered as “half-macrocycle” bidentate chelate analogs of porphyrins and azaporphyrins respectively. Both types of chelates can be used to coordinate a variety of main group and transition metal ions,249–251 and their chemistry with boron has been extensively explored, such as in the BODIPY family of fluorophores.252–254 The dipyrromethenes can be synthesized from pyrroles and aldehydes, the same reagents used to make porphyrins. Azadipyrromethenes (ADPMs) were first synthesized in the 1940s by Rogers131,132 and are produced via very different synthetic methodologies than the structurally similar phthalocyanines. One common route

145 for generating these compounds is via chalcone intermediates (Scheme 5.1), which involves the synthesis of a chalcone via condensation, followed by a Michael addition to the chalcone and formation of the desired chelate using ammonium acetate.

Figure 5.1. The structures of dipyrromethene (1, left) and azadipyrromethene (2, right).

Scheme 5.1. Synthetic pathways of ADPMs and their analogues, i: NaOH, rt in EtOH 24h; ii: CH3NO2, NEt3, reflux in EtOH 24-72 h; iii: NH4OAc, reflux in EtOH 48-72 h; iv: BF3·OEt2, DIPEA, room temp in EtOH 24 h.

146 The chelating properties of the ADPMs and in particular the photophysical

255–258 properties of their BF2 adducts has spurred synthetic work on these ligand systems.

However, in contrast to the dipyrromethenes, less work has been carried out on ADPM systems, and the chalcone-based syntheses exhibit different challenges. In this report, we present a study into the synthesis of several arene substituted azadipyrromethenes

(compounds 5) and their difluoroboron adducts (compounds 6). The arene groups, positioned at the 2 and 4 positions on the pyrroles, include pyridines and phenyl groups both with and without substitutions. The 2-pyridyl ADPM compounds, also recently reported by Bessette et al.,151 often rearrange to form terpyridines, but can be stabilized with an electron donating group.259 For the remaining compounds, some have been synthesized but not structurally characterized via X-ray methods, and others have not been probed for their photophysical properties. All compounds and intermediates have been fully characterized, and we report the structures of several intermediate compounds along with the resultant ADPMs and their BF2 complexes.

Experimental

Materials and General Procedures

Chemicals were obtained commercially and used without further purification.

Basic alumina (50 200 μm) was purchased from Sorbent Technologies. NMR spectra were recorded on a Varian Mercury 300 MHz and Varian NMRS 500 MHz (2 channel 147 with automation) spectrometers. Chemical shifts were reported with respect toresidual

1 solvent peaks as internal standard ( H: CDCl3, δ = 7.26 ppm, d6-DMSO, δ = 2.50 ppm;

13 C: CDCl3, δ = 77.36 ppm, d6-DMSO, δ = 39.7 ppm). Electrospray MS (ES-MS; positive mode) spectra were recorded using a Bruker HCT-ultra ETD II Ion Trap mass spectrometer. UV-visible spectroscopy was carried out on a Hitachi U-3010 spectrometer. Fluorescence measurements were made on a Horiba Jobin Yvon

FluoroMax-4 spectrofluorometer. Absorption and emission measurements were carried out using standard 1 cm pathlength quartz cuvettes. Oxazine 170 was used as the standard for quantum yield measurements (ϕ = 63% in methanol).260 Due to possible decomposition of compounds, all spectroscopic experiments used freshly prepared solution samples.

Syntheses

3a-f: Chalcone synthesis: All chalcones syntheses were carried out using a similar procedure, and the following is representative of the synthesis of 3a-f: An ethanolic solution (15 mL) of both acetylpyridine (10 mmol) and benzaldehyde or p- substituted-benzaldehyde (10 mmol) were mixed in a 250-mL Erlenmeyer flask and 2 mL of a 30% aqueous NaOH solution was then added dropwise (using a Pasteur pipet). In most cases, the reaction mixture turned yellow or orange immediately after NaOH was added. After 12 hours, an excess of cold water was added to the flask while the mixture was stirred vigorously until a solid precipitate was observed. The solid was collected,

148 washed with cold water, and air-dried. The purity of the crude product was determined at this point using 1H NMR spectroscopy. In all cases, the spectroscopic characteristics were in good agreement with those found in the literature and the percent yield above 60% was achieved for all chalcones, which is similar to those reported earlier.261 All chalcones could be used immediately in the next step.

4a-f: Nitroketone synthesis: All nitroketone syntheses were carried out using a similar procedure and the following is representative of the synthesis of 4a-f: The chalcone (3.9 mmol), nitromethane (20 mmol), and triethylamine (35 mmol) were combined in 30 mL of ethanol and heated to reflux for 48 h. The ethanol was removed, and the resulting oil was portioned between CH2Cl2 (50 mL) and water (50 mL). The organic layer was washed with DI water (2 × 50 mL), and the organic fractions were dried over anhydrous magnesium sulfate. The solvent was removed by rotary evaporation, and the resulting oily brown product was use in next process without further purification. Compounds 4b, 4c and 4f are new. Single crystals of 4b were grown by slow evaporation from ethanol. All nitroketones were produced in moderate to good yield: 4a: 1.02 g, 39%, 4c: 1.12 g, 44%, 4d: 2.03 g, 71%, 4e: 1.46 g, 42%, 4f: 1.50 g,

35%.

1 4b: Yield: 1.70 g, 68%. H (CDCl3) δ (ppm) 2.92 (s, 6H, CH3), 3.59 (dd, 1H, J =

18,9 Hz, CH2); 3.81 (dd, 1H, J = 18,9 Hz, CH2); 4.17 (quintet, 1H, J = 9 Hz, CH); 4.63

(dd, 1H, J = 18,9 Hz, CH2), 4.75 (dd, 1H, J = 18,9 Hz, CH2); 6.67 (d, 2H, J = 9 Hz, H in benzene); 7.17 (d, 2H, J = 9 Hz, H in benzene), 7.45-7.50 (m, 1H, H in pyridine), 7.82

149 (td, 1H, J = 9,3 Hz, H in pyridine), 7.99 (dt, 1H, J = 9,3 Hz, H in pyridine), 8.67 (d, 1H, J

= 9 Hz, H in pyridine).

5a-f Azadipyrromethene synthesis: All azadipyrromethenes were synthesized using a similar method as follows: An ethanolic solution of the nitroketone (10 mmol) and ammonium acetate (0.30 mol) were refluxed for 48-72 h. After the solution was cooled to room temperature, the solvent was removed by rotary evaporation and crude product was dissolved in CH2Cl2 and washed with water. The CH2Cl2 layer was collected, dried over Na2SO4 and evaporated to dryness. The resulting green or blue oil was purified by column chromatography using basic alumina and 5% methanol/CH2Cl2 as the mobile phase. Compounds 5a, 5c, 5d, 5e and 5f were isolated as light blue solids and 5b as dark blue solid. The absorption/emission data of all compounds are summarized in Table 5.5. Single crystals of 5b, 5d, and 5f were grown by slow evaporation from dichloromethane.

1 5a: Yield: 5.0 mg, 0.11%. H (CDCl3) δ (ppm) 8.55 (d, 1H, J = 6.6 Hz, H in pyridine); 8.50 (d, 1H, J = 6.6 Hz, H in pyridine); 8.01 (br t, 1H, J = 6.6 Hz, H in pyridine); 7.71-7.69 (m, 2H, H in pyridine and benzene); 7.55-7.62 (m, 4H, H in

13 benzene); 7.34 (s, 1H, H in pyrrole). C (CDCl3) δ (ppm) 143.5, 141.2, 135.1, 135.0,

130.7, 129.7, 128.9, 128.5, 127.4, 120.7, 120.0, 114.0, 109.9. m/z (ESI) calcd [M-C4H5

+ (pyridine dissociation)] for C26H16N5 398.1, found 398.3.

1 5b: Yield: 57 mg, 1.1%. H (CDCl3) δ (ppm) 8.63 (d, 1H, J = 6.0 Hz, py); 8.06 (d,

1H, J = 6.0 Hz, H in pyridine); 7.96 (d, 2H, J = 9.0 Hz, H in benzene); 7.70 (m, 1H, H in

150

pyridine); 7.18 (s, 1H, H in pyrrole); 7.17 (m, 1H, H in pyridine); 6.68 (d, 2H, J = 9.0 Hz,

13 H in benzene); 2.94 (s, 6H, 2xCH3). C (CDCl3) δ (ppm) 174.2, 154.9, 150.9, 150.2,

149.8, 143.0, 136.5, 130.3, 123.4, 122.7, 121.6, 112.6, 112.0, 40.4. m/z (ESI) calcd

+ [M+H] for C34H31N7 538.7, found 538.2.

1 5c Yield: 6 mg, 0.13%. H (CDCl3) δ (ppm) 8.54 (d, 1H, J = 6.6 Hz, H in pyridine); 8.49 (d, 1H, J = 6.6 Hz, H in pyridine); 7.99 (br t, 1H, J = 6.6 Hz, H in pyridine); 7.60-7.52 (m, 3H, H in pyridine and benzene); 7.39-7.42 (m, 3H, H in benzene

13 and pyrrole); 2.46 (s, 3H, CH3). C (CDCl3) δ (ppm) 143.5, 141.3, 135.1, 134.8, 130.4,

128.8, 128.5, 127.8, 124.4, 120.6, 120.0, 113.9, 109.5, 21.6. m/z (ESI) calcd [M-C4H5

+ (pyridine dissociation)] for C28H20N5 426.2, found 426.3.

1 5d Yield: 250 mg, 5.2% H (CDCl3) δ (ppm) 7.99 (d, 1H, J = 5 Hz, H in benzene); 7.79 (d, 1H, J = 5 Hz, H in benzene); 7.37-7.42 (m, 3H, H in benzene); 7.26-

7.28 (m, 3H, H in benzene and pyrrole); 7.02 (d, 2H, J = 5 Hz, H in benzene). 13C NMR data cannot be obtained due to solubility issues.

1 5e Yield: 479 mg, 7.9% H (CDCl3) δ (ppm) 7.96 (d, 1H, J = 7.0 Hz, H in benzene); 7.70 (d, 2H, J = 7.0 Hz, H in benzene); 7.59 (br d, 2H, J = 7.0 Hz, H in benzene); 7.53 (br d, 1H, J = 7.0 Hz, H in benzene); 7.35 (t, 1H, J = 7.0 Hz, H in

13 benzene); 7.25-7.31 (m, 2H, H in benzene); 7.08 (s, 1H, H in pyrrole). C (CDCl3) δ

(ppm) 175.8, 132.4, 132.0, 131.0, 129.0, 128.3, 127.8, 127.4, 124.5, 114.9, 105.0.

151 1 5f Yield: 111 mg, 1.4% H (CDCl3) δ (ppm) 8.15-8.26 (m, H in benzene); 7.80

(s, 1H, H in pyrrole); 7.72-7.83 (m, 3H, H in benzene); 7.49-7.55 (m, 3H, H in benzene).

13C NMR data cannot be obtained due to solubility issues.

6d-f Boron aza-dipyrromethenes synthesis: The azadipyrromethene (0.15 mmol, 1 eq.) was dissolved in dichloromethane (15 mL) and diisopropylethylamine (0.27 mL, 1.50 mmol, 10 eq.) and BF3·OEt2 (0.29 mL, 2.25 mmol, 15 eq.) were added dropwise. The solution was stirred at room temperature for 24 h. The organic layer was evaporated under vacuum. The crude product was purified by chromatography on silica gel (1:1 CH2Cl2: hexane) to afford dark green solids. Single crystals of 6f were grown by slow evaporation from dichloromethane.

1 6d Yield: 36.7 mg, 50% H (CDCl3) δ (ppm) 8.16 (d, 2H, J = 7.2 Hz, H in benzene); 7.87 (dd, 1H, J = 7.2, 1.2 Hz, H in benzene); 7.48 (m, 2H, H in benzene); 7.40-

7.44 (m, 2H, H in benzene); 7.25 (s, 1H, H in pyrrole); 7.12 (t, 1H, J = 7.2 Hz, H in

13 benzene); 7.03 (d, 1H, J = 7.2 Hz, H in benzene). C (CDCl3) δ (ppm) 155.9, 149.8,

142.2, 133.4, 132.4, 129.3, 129.0, 128.7, 126.6, 125.5, 120.9, 120.2, 118.9, 113.3.

1 6e Yield: 55 mg, 56% H (CDCl3) δ (ppm) 8.05-8.08 (m, 2H, H in pyridine); 7.92

(dd, 1H, J = 7.0, 1.0 Hz, H in benzene); 7.87 (s, 1H, H in pyrrole); 7.74 (d, 1H, J = 7.0

Hz, H in benzene); 7.63-7.66 (m, 2H, H in benzene); 7.55 (t, 1H, J = 7.0 Hz, H in

13 benzene); 7.46-7.51 (m, 1H, H in benzene). C (CDCl3) δ (ppm) 156.4, 150.7, 138.1,

131.9, 131.9, 129.4, 129.2, 128.7, 127.2, 123.7, 117.2.

152

1 6f Yield: 51 mg, 42% H (CDCl3) δ (ppm) 8.01-8.05 (m, 3H, H in benzene); 7.73

(br t, 1H, H in benzene); 7.50-7.52 (m, 3H, H in benzene); 7.44 (br t, 1H, H in benzene);

7.05 (s, 1H, H in pyrrole). 13C NMR data cannot be obtained due to solubility issues.

Results and Discussion

Synthesis

Work on ADPMs has primarily focused on the end products, and thus research into their fundamental properties has been sporadic. The most comprehensive study was carried out by Bessette and coworkers, who examined a series of arene substituted

ADPMs, which included the unmodified tetraphenyl and bis-pyridyl compounds.151,259

We became interested in pyridine-substituted ADPMs as possible tetradentate chelates, but we found that these compounds were unstable and thus unsuitable for metal chelation.

Bessette and coworkers reported the same issues, as will be noted below. We have also investigated bis-phenol substituted ADPMs with greater success and have carried out some structural studies. Additionally, there has been interest in bromine-substituted

ADPMs, since they can be readily functionalized via C-C bond forming reactions.

The synthesis of ADPMs was performed by starting with the synthesis of chalcone derivatives via a Claisen-Schmidt condensation of an aldehyde and acetophenone using base as a catalyst.133 The desired chalcones can be produced in good yields from the reaction at room temperature in ethanol. The chalcones were then used in 153 a Michael addition reaction with nitromethane to yield the corresponding nitroketones.

Both the chalcones and the nitroketones were isolated as stable crystalline materials; we were able to elucidate the structures of two of the intermediate structures (compounds 3b and 4b) and both exhibit the expected features (see supplemental information).

ADPMs were subsequently obtained by reaction of the nitroketones with ammonium acetate in alcoholic solvent under reflux.262 A relatively long reaction time

(up to three days) is required in most cases. All ADPMs can be purified with column chromatography. Basic alumina gel was a better stationary phase than silica gel, in particular for the pyridine compounds, due to their lack of mobility on silica. In general, low yields (less than 10%) of 5a-5c were observed, which is comparable with literature observations.134,143 Thus, despite the facility of the chalcone synthetic method, the low yield of the final step can limit the usefulness of this strategy. Moreover, the instability of 5a and 5c preclude us from synthesizing their aza-BODIPY analogues. The yields of both nitroketones as well as ADPMs likely depends on the identity of the R groups attached to phenyl rings (5a; R=H, 5b; R=N(CH3)2, 5c; R=CH3). Strongly electron donating groups, such as R=N(CH3)2, resulted in the highest yields for compound 4b and

5b. This implies the ability of electron donating/withdrawing ability of R can induce the production efficiency of these ADPMs.

All compounds showed moderate to good solubility in several organic solvents e.g. chloroform, dichloromethane, acetone, and alcohols. The NMR spectra of all

ADPMs were in accordance with the data reported in the literature.263,264 The aromatic proton signals were found in the range between 6.0 ppm and 9.0 ppm. The diagnostic 154 peak for the pyrrolic C-H group appears as a singlet and found around 7.20-7.30 ppm.

The solvent signal from CDCl3 can possibly overlap with this peak, however. In solution phase, all compounds tend to decompose over time as observed by the gradual disappearance of 1H signals in NMR. Bessette and coworkers have demonstrated that the similar compound as 5a containing proximal pyridines underwent decomposition especially in solution phase.151 We observed degradation to form tripyrranes, which would often crystallize out of solutions of 5a. The degradation of this product possibly resulted from the increased reactivity of the adjacent carbons of the N-bridge due to conjugation with electron-poor pyridine substituents. However, an electrochemistry study on a free azadipyrromethene with electron-rich anisole groups also reported similar instability.252

The more stable compounds 5d-5f were further reacted with BF3·OEt2 and compounds 6d-6f were obtained. Of these compounds, compound 6d and 6e have been prepared previously; only 6f is a new compound. Unlike the ADPM freebase syntheses, reactions to insert BF2 proceed reasonably easily and afford products in decent yield; our observations on these compounds are in agreement with literature values.

X-Ray crystallography

Both the ADPMs and their BF2 complexes readily form single crystals and can often be elucidated by X-ray crystallography. Of the compounds that we investigated, the structure of 6e has been previously presented in the literature,265 and a disordered structure of 5a was also elucidated by Bessette and coworkers.151 We were also able to

155

obtain a structure of compound 5a, but encountered the same problems and were not able to get an improved structure. However, we were able to obtain good X-ray data for the dimethylaminophenyl analog compound 5b. The bis-phenol ADPM 5d was structurally characterized and is similar to several systems also presented by Bessette and coworkers.151,259 In addition, we were able to elucidate the structure of the tetrabromo compound 5f and its BF2 analog 6f. The structures of these four compounds are shown in

Figure 5.2 and the X-ray data collection and structure parameters can be found in Table

5.1-5.4.

156

Figure 5.2. Structures of 5b, 5d, 5f and 6f with 50% thermal ellipsoids. Non-ionizable hydrogen atoms have been omitted for clarity. Crystallographic data, structure refinement, and selected bond lengths and angles are reported in Table 5.1-5.4.

157 Table 5.1 Crystallographic data and structure refinement for 5b.

Compound 5b

Empirical formula C34H31N7 Formula weight 537.66 Crystal system orthorhombic

Space group Pna21 a/ Å 18.7433(5) b/ Å 14.6652(4) c/ Å 10.1077(3) α(°) 90.00 β(°) 90.00 γ(°) 90.00 Volume (Å3) 2778.35(13) Z 4 Dc (Mg/m3) 1.285 µ (mm-1) 0.617 F(000) 1136.0 Reflections collected 16683 Data/Restraints/Parameters 4544/1/374 GOF on F2 1.193 2 R1 (on Fo , I > 2σ(I)) 0.0325 2 wR2 (on Fo , I > 2σ(I)) 0.0779 R1 (all data) 0.0389 wR2 (all data) 0.0806

158

Table 5.2 Crystallographic data and structure refinement for 5d.

Compound 5d

Empirical formula C32H23N3 O2 Formula weight 481.53 Crystal system monoclinic Space group C2/c a/ Å 40.455(4) b/ Å 3.8342(4) c/ Å 29.733(3) α(°) 90.00 β(°) 93.714(8) γ(°) 90.00 Volume (Å3) 4602.3(8) Z 8 Dc (Mg/m3) 1.390 µ (mm-1) 0.699 F(000) 2016.0 Reflections collected 20736 Data/Restraints/Parameters 3610/6/336 GOF on F2 1.085 2 R1 (on Fo , I > 2σ(I)) 0.0396 2 wR2 (on Fo , I > 2σ(I)) 0.1083 R1 (all data) 0.0433 wR2 (all data) 0.1167

159 Table 5.3 Crystallographic data and structure refinement for 5f.

Compound 5f

Empirical formula C32H19Br4N3 Formula weight 765.14 Crystal system Triclinic Space group P-1 a/ Å 7.2063(3) b/ Å 13.4467(6) c/ Å 14.3807(7) α(°) 79.159(2) β(°) 79.864(3) γ(°) 85.742(2) Volume (Å3) 1346.05(11) Z 2 Dc (Mg/m3) 1.888 µ (mm-1) 7.520 F(000) 744 Reflections collected 13997 Data/Restraints/Parameters 4213/0/352 GOF on F2 1.044 2 R1 (on Fo , I > 2σ(I)) 0.0456 2 wR2 (on Fo , I > 2σ(I)) 0.1226 R1 (all data) 0.0485 wR2 (all data) 0.1267

160 Table 5.4 Crystallographic data and structure refinement for 6f.

Compound 6f

Empirical formula C33H20BBr4Cl2F2N3 Formula weight 897.87 Crystal system Triclinic Space group P-1 a/ Å 10.8200(15) b/ Å 12.1002(15) c/ Å 13.393(2) α(°) 83.532(5) β(°) 66.530(4) γ(°) 78.096(3) Volume (Å3) 1572.9(4) Z 2 Dc (Mg/m3) 1.896 µ (mm-1) 8.166 F(000) 872 Reflections collected 17304 Data/Restraints/Parameters 4949 / 24 / 406 GOF on F2 1.136 2 R1 (on Fo , I > 2σ(I)) 0.0368 2 wR2 (on Fo , I > 2σ(I)) 0.1096 R1 (all data) 0.0369 wR2 (all data) 0.1097

161 For compound 5b, the examination of the molecular structure revealed an orthorhombic system with the space group Pna21. As shown in Figure 2, the dipyrrole unit is planar and each of the phenyl rings is slightly tilted from that plane with the angles being ~29 and 34o. The pyridine rings are coplanar with the dipyrrole unit.

Intramolecular hydrogen bonding was observed between two pyrrole rings with an N-N distance of ~2.83 Å. The crystal packing exhibited a zig-zag pattern with no significant strong intermolecular hydrogen bonds.

Compound 5d, which was solved in the monoclinic C2/c space group, also showed hydrogen bonding, but in this case the strongest bonding was observed between the phenyl hydroxyl groups and the pyrrole nitrogen atoms. Both interactions measure

~2.65 Å. Weaker hydrogen bonding interactions are also seen between the two nitrogen positions and between the two oxygen atoms, measuring ~2.93 and ~2.96 Å respectively.

No intermolecular hydrogen bonding is observed in the packing structure, which exhibits a herringbone type pattern. The planes of the two phenyl rings are slightly tilted from the plane of the dipyrrole, with angles of ~29 and ~24°. For the phenol rings, one is co-planar with the dipyrrole while the second exhibits a slight deviation of ~8°.

The tetrabrominated compound 5f was solved in the triclinic P-1 space group and forms layers in the extended packing structure. The hydrogen bonding between the pyrrolic nitrogen positions is a bit stronger than in 5b, measuring ~2.72 Å, and there are no intermolecular hydrogen bonding contacts. In 5f, all four of the phenyl rings are tilted away from the planar dipyrrolic unit, with the brominated rings tilted at ~27 and ~21° away from the plane, and the unsubstituted phenyls tilted at ~20 and ~27°. 162

We were also able to elucidate the structure of the tetrabromo ADPM BF2 adduct

6f. Like 5f, this compound adopts the triclinic P-1 space group and forms layers in the solid. In this structure, there is a dicholormethane solvent molecule in the void space. All four phenyl rings are tilted away from the plane of the dipyrrole unit, as seen in the free base ADPMs: the two brominated phenyl rings form angles of ~27 and 44° away from this plane and the two unusbstituted phenyls are at ~12 and ~29°. Due to the BF2 binding at the core, no intra- or intermolecular H-bond is observed in this structure.

Photophysical properties

In spite of the interest in free base ADPMs and their boron complexes for photonic applications, the number of fundamental studies on these simple chromophores has been limited. The most comprehensive study to date was reported in the Bassette paper, which examined absorption characteristics and carried out DFT calculations to provide insight into the nature of these transitions. We probed both the absorption and emission properties of both the free base and BF2 ADPMs synthesized for this work.

With regard to absorbance, our observations are in good agreement with the results and conclusions from the Bessette manuscript. All of the azadipyrromethenes synthesized exhibited strong absorbance bands around 600 nm and higher (Figure 5.3). Compounds 5 show absorptions with moderate molar absorptivities ranging from ~15,000 to 40,000 M-

1cm-1. In addition, all of the compounds exhibited spectra with similar lineshapes with the exception of compound 5b, which displays significant broadening and loss of intensity of the primary absorption band.

163

Figure 5.3. Absorption (left) and emission (right) spectra in CH2Cl2 for compounds 5 and 6.

164 Table 5.5 Absorption and emission data for compounds 5 and 6 (in CH2Cl2).

ε (x104 M- Stokes shift compound λ (nm) λ (nm) ϕ (%)* abs 1cm-1) emis (nm) 5a 635 3.63 655 20 6.5 297 2.44 5b - - - 631 1.77 5c 640 3.64 670 30 11 307 1.43 5d 643 29 0.029 614 3.19 313 3.65 5e 653 49 0.033 604 3.94 304 3.51 5f 646 48 0.064 598 3.44 325 8.03 6d 498 1.57 741 17 4.3 724 8.83 317 30.16 6e 683 26 23 657 8.60 315 1.87 6f 684 27 7.6 657 6.37

* The percentage values were achieved by fluorescent study using oxazine 170 as the reference (ϕMeOH = 63%).

The free base ADPMs exhibited fluorescent emission above 640 nm when excited at their primary absorption band, with the exception of compound 5b, which exhibited no fluorescence. The absorption/emission characteristics of both 5a and 5c are very similar to the previously reported heterocycles-containing ADPM (thiophene-ADPM).147

Compounds 5a and 5c exhibit quantum yields of ~6.5 and ~11 percent, respectively. The quantum yield values of the aryl substituted ADPMs 5d-5f, with the exocyclic bromide and hydroxide substitutents, are much smaller than in 5a and 5c, and are only a fraction 165 of a percent. One notable quality seen in the free base ADPM compounds are the magnitude of their Stokes shifts, which are larger than that of similarly structured

BODIPY compounds such as tetramethyl-BODIPY (which is less than 10 nm).266 In particular, very large Stokes shifts were seen in the brominated analogs 5e (49 nm) and 5f

(48 nm).

The aza-BODIPY compounds 6d-6f exhibited more intense fluorescence emissions that are bathochromically shifted relative to the free base chelates.

Compounds 6e and 6f exhibit similar energies of emission (~684 nm), but the flurorescence from 6d is even lower in energy at ~741 nm, which follows the trends seen in the free base absorption and emission spectra. The Stokes shifts, while larger than those seen in BODIPYs, are smaller than in the free base compounds.

Conclusions

In conclusion, we have synthesized a series of ADPM compounds and several

BF2 adducts, and present a study into their structures and photophysical attributes.

Although the chalcone method for synthesis of ADPMs is readily accessible via easily prepared starting materials, this strategy can suffer from low yields in the final step. The stability of the resultant ADPM materials is highly dependent on the identity and substitution of the phenyl groups, however, when stable, free base ADPMs can be readily structurally elucidated. Both ADPMs and their BF2 complexes are fluorescent, and in 166

particular the BF2 adducts exhibit red shifted emissions relative to their BODIPY analogs. Our observations are in good agreement with previously reported studies.

167 CHAPTER VI

SYNTHESIS, REDOX PROPERTIES, AND ELECTRONIC COUPLING IN THE

FIRST DIFERROCENE AZA-DIPYRROMETHENE AND AZABODIPY DONOR-

ACCEPTOR DYAD WITH DIRECT FERROCENE-α-PYRROLE BOND.

The text of this chapter is a reprint of the material as it appears in: Ziegler, C. J.; Chanawanno,

K.; Hasheminasab, A.; Zatsikha, Y. V.; Maligaspe, E.; Nemykin, V. N. Inorg. Chem. 2014, 53,

4751 4755.

Introduction

The preparation of new functional materials that are highly efficient for solar energy conversion is of fundamental interest.267–270 Donor acceptor dyads or donor antennae acceptor triads that can generate long-lived highly energetic charge-separated (CS) states have been proposed for applications as soft light-harvesting materials.271–275 Because of their well-known absorption properties in the visible electromagnetic spectrum, a large variety of porphyrins and their analogues coupled with organometallic or organic donor groups have been tested as donor acceptor dyads for organic photovoltaics (OPVs) and dye-sensitized solar cells (DSSCs).276–281 Ferrocene

168 substituents were found to be potentially useful as electron-donating groups for light- harvesting assemblies. Not surprisingly, photophysical as well as redox properties of ferrocene-porphyrins, subphthalocyanines, phthalocyanines, and tetraazaporphyrins have also been intensively investigated in recent years.282–305 More recently, dipyrromethenes, azadipyrromethenes, and their borylated derivatives BODIPYs and azaBODIPYs have attracted significant attention because of their specific photophysical properties.113,124,135,147,306–317 During the past decade, several ferrocene-

(aza)dipyrromethenes and ferrocene-(aza)BODIPY conjugates in which ferrocene substituent is connected to the chromophoric unit via spacer located at α-, β-pyrrolic, or meso-position were reported.318–324 To the best of our knowledge, however, no reports are available on azadipyrromethenes and azaBODIPYs with ferrocene groups connected directly to the heterocyclic chromophore. Thus, in this chapter, we report preparation and characterization of the ferrocenyl-containing azadipyrromethene and azaBODIPY donor acceptor dyads 3 and 4 (Scheme 6.1) with direct ferrocene-α-pyrrole bond.

169 NO2 COCH3 O O ii i Fe Fe Fe 1 2

iii

iv N N NN B NHN Fe Fe F F Fe Fe 4 3

Scheme 6.1. Preparation of the target azadipyrromethene 3 and azaBODIPY 4. Reagents and conditions: (i) PhCHO, NaOH/EtOH, rt/24 h, yield 68%; (ii) CH3NO2, NEt3/EtOH, heat/72 h, yield 70%; (iii) NH4OAc, EtOH, heat/72 h, yield 4.4%; (iv) BF3·Et2O DIPEA/CH2Cl2 heat/24 h, yield 47%.

Experimental

Materials: Chemicals were obtained commercially and used without further purification. Basic alumina (50 200 μm) was purchased from Sorbent Technologies. The tetrabutylammonium tetrakis-(pentafluorophenyl)borate (TBAF) was used in anhydrous

DCM for electrochemical studies, after preparation according to literature procedures.325

Instrumentation: UV vis data were obtained on Jasco-720 spectrophotometer.

Electrochemical measurements were conducted using a CH Instruments electrochemical analyzer utilizing a three electrode scheme with platinum working, auxiliary, and

Ag/AgCl reference electrodes in a 0.05 M solution of TBAF in DCM with redox potentials corrected using an internal standard (decamethylferrocene, FcH*) in all cases. 170 The redox potentials were then corrected to ferrocene using appropriate oxidation

+ potentials for FcH*/FcH* versus FcH/FcH+ in the CH2Cl2/0.05 M TBAF system. NMR spectra were recorded on a Varian Mercury 300 MHz and Varian NMRS 500 MHz spectrometers with a 500 MHz frequency for protons and 125 MHz for carbon. Chemical shifts were reported in parts per million (ppm) with respect to residual solvent peaks as

1 13 internal standard ( H CDCl3, δ = 7.26 ppm, DMSO-d6, δ = 2.50 ppm; C CDCl3, δ =

77.36 ppm, DMSO-d6, δ = 39.7 ppm) and referenced to tetramethylsilane [Si(CH3)4].

FTIR spectra were recorded on a Nicolet iS5 spectrometer using NaCl disks. Elemental analyses were performed by Atlantic Microlab of Norcross, GA 30091. Electrospray MS

(ES-MS; positive mode) spectra were recorded using a Bruker HCT-ultra ETD II Ion

Trap mass spectrometer.

Computational aspects: All computations were performed using Gaussian09 software running under Windows or UNIX OS.202 Molecular orbital contributions were compiled from single point calculations using the VMOdes program.326 In all calculations, TPSSh hybrid (10% of Hartree Fock exchange)327 exchange correlation functional was used because it was found in a set of model gas-phase calculations that it is superior over standard GGA (BP86)12 and hybrid B3LYP197,198 exchange correlation functionals. Indeed, use of hybrid B3LYP exchange-correlation functional results in

HOMO, which is purely azaBODIPY centered MO in disagreement with experimental data, while calculated using BP86 exchange-correlation functional vertical excitation energies for MLCT transitions were found to be severely underestimated. In all calculations, Wachter’s full-electron basis set for iron and 6-311G(d) basis set for all

171 other atoms were employed.328 Solvent effects were modeled using PCM approach.329 In all TDDFT calculations, the lowest 40 excited states were calculated to cover experimentally observed transitions in UV vis region.

Synthesis

Compound 1: This compound has been previously synthesized.261 An ethanolic solution of 1.00 g of acetylferrocene (4.4 mmol) and 0.44 mL of benzaldehyde (4.4 mmol) were mixed in a 250 mL Erlenmeyer flask and 2 mL of a 30% aqueous NaOH solution was added dropwise after mixing. The reaction mixture turned dark orange upon addition of the base. After 24 h of reaction, an excess amount of cold water was added with vigorous stirring to the flask until a solid precipitate was observed. The solid was collected, washed with cold water, and air-dried. The purity of the crude product was determined at this point using TLC and 1H NMR spectroscopy. The spectral characteristics were in good agreement with those found in the literature. Yield: 1.3 g

1 (68%). H (CDCl3): δ (ppm) 7.82 (d, 1H, J =15 Hz, trans H); 7.67 (s, 2H, H on benzene);

7.43 (s, 3H, H on benzene); 7.15 (d, 1H, J = 15 Hz, trans H); 4.93 (s, 2H, Cp on ferrocene); 4.61 (s, 2H, Cp on ferrocene); 4.23 (s, 5H, Cp on ferrocene).

Compound 2: Compound 1 (1.00 g, 2.4 mmol), nitromethane (0.64 mL, 12 mmol), and triethylamine (3.01 mL, 21.6 mmol) were combined in 30 mL of ethanol and heated to reflux for 72h. The ethanol was removed, and the resulting oil extracted with

CH2Cl2 (50 mL) and water (50mL). The organic layer was washed with DI water

(2×50mL), and the organic fractions were dried over anhydrous magnesium sulfate. The

172

solvent was removed, and the resulting brown oily product used in the next step without further purification. Yield: 0.81 g (70%).

Compound 3: An ethanolic solution of 2 (2 g, 4.14 mmol) and ammonium acetate (9.57 g, 124.2 mmol) was refluxed for 48 h. After the solution was cooled to room temperature, the solvent was removed and crude product was dissolved in CH2Cl2 and washed with water. The CH2Cl2 layer was collect, dried over Na2SO4, and evaporated to dryness. The collected dark solution was purified by column chromatography using basic alumina and CH2Cl2 as the mobile phase. The pure product is purple solid. Yield 0.12 g

1 (4.35%, based on 1). Purple crystals were obtained from CH2Cl2 solution. H NMR

(CDCl3) δ: 8.09 (d, 2H, J = 6 Hz), 7.35 7.43 (m, 3H), 6.82 (s, 1H), 4.89 (s, 2H, Cp on ferrocene), 4.61 (s, 2H, Cp on ferrocene), 4.23 (s, 5H, Cp on ferrocene). 13C NMR

(CDCl3) δ: 67.6, 70.3, 71.2, 75.4, 111.7, 127.6, 128.2, 128.9, 134.1, 140.4, 149.1, 156.2.

Anal. Calcd for 3·0.7H2O (C40H31Fe2N3(H2O)0.7): C, 70.86; H, 4.82; N, 6.20%. Found: C,

70.73; H, 4.66; N, 6.24%

Compound 4: Compound 3 (0.04 g, 0.06 mmol) was dissolved in CH2Cl2 (40 mL). Diisopropylethylamine (0.021 mL, 0.12 mmol) and boron trifluoride diethyl etherate (0.015 mL, 0.12 mmol) were added and the mixture was stirred at room temperature for 24 h. The mixture was washed with water and the organic layer was separated, dried over Na2SO4, and evaporated to dryness. The residue was purified by column chromatography (basic alumina) with CH2Cl2 to give the product as a green solid.

1 Yield: 0.02 g (47%). Green crystals were obtained from CH2Cl2/hexane solution. H

NMR (CDCl3) δ: 8.10 (d, 2H, J = 6 Hz), 7.44 7.47 (m, 3H), 6.95 (s, 1H), 5.40 (s, 2H, Cp 173 on ferrocene), 4.83 (s, 2H, Cp on ferrocene), 4.27 (s, 5H, Cp on ferrocene). 13C NMR

(CDCl3) δ: 71.1, 71.3, 73.5, 74.3, 117.7, 127.8, 128.5, 128.9, 133.0, 139.3, 145.2, 157.9.

Anal. Calcd for 4·0.26H2O·1.8(CH2Cl2) (C40H30Fe2N3BF2(H2O)0.26(CH2Cl2)1.8): C, 57.73;

H, 3.96; N, 4.83%. Found: C, 57.82; H, 4.13; N, 4.64%.

X-ray Crystallography: X-ray intensity data for compound 3 was measured ona

CCD-based X-ray diffractometer system equipped with a Mo-target X-ray tube (Mo Kα radiation, λ = 0.71073 Å) operated at 2000 W power. Crystals were mounted on a cryoloop using Paratone oil and placed under a steam of nitrogen at 100 K. The detector was placed at a distance of 5.009 cm from the crystal. A specimen of 3 approximate dimensions 0.22 mm × 0.13 mm × 0.09 mm was used for the X-ray crystallographic analysis. Data were acquired using three sets of Omega scans at different Phi settings.

The frame width was 0.5°. The structure was solved and refined using the Bruker

330 SHELXTL Software Package, using the space group P2(1)/c, with Z = 4 for the formula unit, C40H31Fe2N3. Hydrogen atoms were assigned ideal positions and refined isotropically as riding atoms.

X-ray intensity data for 4 was collected on a CCD-based diffractometer withdual

Cu/Mo ImuS microfocusoptics (Cu Kαradiation, λ = 1.54178 Å). Crystals were mounted on a cryoloop using Paratone oil and placed under a steam of nitrogen at 100 K. A specimen of 4 approximate dimensions 0.26 mm × 0.32 mm × 0.59 mm, was used for the

X-ray crystallographic analysis. The integration of the data using a triclinic unit cell yielded a total of 35640 reflections to a maximum θ angle of 62.99° (0.87 Å resolution), of which 9559 were independent (average redundancy 3.728) The structure was solved 174 and refined using the Bruker SHELXTL Software Package,330 using the space group P1̅ with Z = 4 for the formula unit, C40H30BF2Fe2N3. Hydrogen atoms were assigned ideal positions and refined isotropically as riding atoms. Crystal data and structure refinement parameters for 3 and 4 are summarized in Tables 6.1 and 6.2. CCDC reference numbers:

CCDC 986211 (3) and CCDC 986212 (4).

Figure 6.1. X-ray crystal structures of 3 and 4.

175 Table 6.1 Crystallographic data and structure refinement for 3.

Compound 3

Empirical formula C40 H31 Fe2 N3 Formula weight 665.38 Crystal system Monoclinic Space group P2(1)/c a/ Å 14.221(4) b/ Å 15.845(5) c/ Å 14.164(4) α(°) 90 β(°) 111.228(4) γ(°) 90 Volume (Å3) 2975.1(15) Z 4 Dc (Mg/m3) 1.486 µ (mm-1) 1.011 F(000) 1376 Reflections collected 20852 Data/Restraints/Parameters 5244 / 0 / 406 GOF on F2 1.006 2 R1 (on Fo , I > 2σ(I)) 0.0451 2 wR2 (on Fo , I > 2σ(I)) 0.1042 R1 (all data) 0.0679 wR2 (all data) 0.1192

176 Table 6.2 Crystallographic data and structure refinement for 4.

Compound 4

Empirical formula C40 H28 B F2 Fe2 N3 Formula weight 711.16 Crystal system Triclinic Space group P-1 a/ Å 12.9677(4) b/ Å 14.0822(4) c/ Å 18.6401(6) α(°) 89.8390(10) β(°) 81.1100(10) γ(°) 66.1370(10) Volume (Å3) 3068.84(16) Z 4 Dc (Mg/m3) 1.539 µ (mm-1) 7.973 F(000) 1456 Reflections collected 36399 Data/Restraints/Parameters 9988 / 0 / 865 GOF on F2 1.011 2 R1 (on Fo , I > 2σ(I)) 0.0376 2 wR2 (on Fo , I > 2σ(I)) 0.1165 R1 (all data) 0.0414 wR2 (all data) 0.1200

177 Results and Discussion

The precursor to the azadipyrromethene modified with ferrocene at the α- pyrrolic positions is the chalcone 1, which can be synthesized via an aldol condensation with ferrocenecarbaldehyde. The chalcone 1 was further modified with nitromethane by using a Michael addition to afford compound 2, which is then reacted with ammonium acetate in refluxing ethanol to produce azadipyrromethene 3. The corresponding ferrocene-containing azaBODIPY 4 can be generated by reaction of 3 with BF3·Et2O in the presence of a base (DIPEA). Organometallic azadipyrromethene 3 and azaBODIPY 4 are stable in the solid state in air and can be purified by conventional chromatographic methods, but slowly degrade upon prolonging standing in solution under ambient atmosphere. Structures of the target compounds 3 and 4 were confirmed by the X-ray diffraction analysis (Figure 6.1 and Tables 6.1-6.2) and further elucidated by 1H and 13C

NMR spectroscopy, UV vis spectra (Figure 6.2), and elemental analyses.

Experimental crystal structures of 3 and 4 are shown in Figure 6.1, while their detailed descriptions are provided in Tables 6.1-6.2. In the X-ray crystal structures of both complexes 3 and 4, ferrocene groups were found in a syn conformation. The torsion angles between the ferrocene and pyrrole fragments vary between 7.63° and 20.25° in 3, whereas they are significantly smaller in the case of complex 4 (~1.25 4.54°). The torsion angles between the phenyl and pyrrole groups follow the same trend: they are larger in complex 3 (~19.81 21.25°) compared to those in complex 4 (~12.07 20.38°).

178 Because of the larger ferrocene to pyrrole torsion angle, the crystallographic Fe Fe distance in complex 3 is smaller (~6.83 Å) than those in 4 (~7.13 7.40 Å).

Figure 6.2. Experimental (CH2Cl2) and PCM-TDDFT predicted UV vis spectra of 3 (top) and 4 (bottom).

In agreement with the other ferrocene-BODIPY dyads, complexes 3 and 4 have two major bands in the visible region with the lower energy band being broader

179 compared to the higher energy band (Figure 6.2). These bands in ferrocenyl-containing azadipyrromethene derivative 3 (546 and 673 nm) were observed at significantly higher energies compared to those in azaBODIPY 4 (623 and 850 nm). Steady-state fluorescence measurements in 3 and 4 are indicative of complete quenching in both systems and agree well with the standard quenching mechanism in ferrocene-containing compounds.331–338 According to this mechanism, electron-transfer from the low-spin iron(II) center in ferrocene to the photoexcited azadipyrromethene or azaBODIPY core is predominantly responsible for the fluorescence quenching.

Figure 6.3. Room-temperature CV data on compounds 3 (left) and 4 (right) in CH2Cl2/0.05 M TBAF system.

Table 6.3 Redox potentials (V) of complexes 3 and 4 determined at room temperature in DCM/0.05 M TBAF system.a

Compound Ox2 Ox1 Red1 3 +0.35 +0.01 -1.42 (irreversible) 4 +0.45 -0.01 -1.15 aAll potentials are given with respect to FcH/FcH+ couple. 180 The redox properties of diiron compounds 3 and 4 were studied using electrochemical CV and DPV methods (Figure 6.3 and Table 6.3). In both cases two reversible oxidations of ferrocene substituents were observed. The difference in potential between the first and the second oxidations waves is 340 and 460 mV for complexes 3 and 4, respectively, in CH2Cl2/0.05 M TBAF system (TBAF = tetrabutylammonium tetrakis(pentafluorophenyl)borate, which is indicative of their electronic coupling (Figure

6.3).325,339–344 Although electronic coupling in one diferrocene BODIPY system has been previously described,320 the magnitude of electronic communication between the ferrocene units in compounds 3 and 4 is unprecedented. Oxidation of the azaBODIPY core was not observed for both compounds 3 and 4 within electrochemical window, while irreversible (compound 3) and reversible (compound 4) reduction was observed at 1.42 and 1.15 V, respectively.

181

Figure 6.4. Room-temperature spectroelectrochemical oxidation of 4 at first (top) and second (bottom) oxidation potentials in CH2Cl2/0.15 M TBAF system.

To clarify the nature of redox-active species in oxidized forms of 3 and 4, we have conducted spectroelectrochemical oxidation and reduction experiments (Figures

6.4-6.6). In the case of azaBODIPY 4, during the first oxidation process, both major bands in visible region lose their intensity and three new bands at 639, 760, and 996 nm appear in the spectrum. In addition, a new broad NIR band appears in the spectrum

(Figure 6.4). This band spans between ∼1700 and 2650 nm and is centered outside of the accessible range for our spectrometer. The appearance of the NIR bands in 4+ is clearly

182 suggestive of its mixed-valence character and resembles the ferrocenyl-containing mixed- valence porphyrins discussed earlier.293–296 During the second oxidation, all NIR bands lose their intensities and new intensive band at 697 nm appears in the visible region

(Figure 6.4). Similar behavior has been also observed for 3 → 3+ → 32+ transformation under spectroelectrochemical conditions (Figure 6.6). It is important to note that 42+ cation generated under spectroelectrochemical oxidation conditions can be easily reduced back to the neutral 4.

Figure 6.5. Reduction of compound 42+ to 4 under spectroelectrochemical conditions in DCM/0.15 M TBAF system.

183 Figure 6.6. Stepwise oxidation of 3 to 3+ and 32+ under spectroelectrochemical conditions in DCM/0.15M TBAF system.

184 Figure 6.7. DFT-PCM (TPSSh/6-311G(d)) calculated orbital energies for complexes 3 and 4 with pictorial representation of the frontier MOs.

The electronic structure and the vertical excitation energy calculations for compounds 3 and 4 (Figures 6.2 and 6.7) correlate well with the experimental results.

First, DFT calculations suggest that the energies and electronic structures of syn and anti conformations of 3 and 4 are very close to each other. In agreement with the electrochemical data, HOMO to HOMO 5 MOs in complexes 3 and 4 are predominantly ferrocene-centered, whereas HOMO 6 and HOMO 7 are π-orbitals centered on heterocyclic core. In addition, HOMO and HOMO 4 in 3 and 4 have substantial

(∼20 40%) π-character. The LUMO in both compounds is predominantly heterocycle- centered π*-orbital and energetically well separated from the LUMO+1. Such electronic structure results in appearance of numerous low-energy ferrocene-to-chromophore

185 MLCT transitions predicted by the TDDFT calculations (Figure 6.2). MLCT transitions, which originate from almost pure ferrocene MOs (HOMO 1 to HOMO 3) to LUMO are predicted to have low intensities, while similar transitions from the mixed ferrocene chromophore HOMO and HOMO 4 to LUMO are predicted to be intense (Figure 6.8).

TDDFT calculations also correctly predict low-energy shifts of the major bands in

UV vis spectra going from 3 to 4 (Figure 6.2). The major reason for such a shift is the substantial stabilization of LUMO in 4 compared to 3.

186 Figure 6.8. Compositions of PCM-DFT orbitals for compounds 3 and 4.

Conclusions

In conclusion, we have synthesized and characterized the first azadipyrromethene and azaBODIPY complexes with ferrocene substituents directly connected to the α- pyrrolic carbon atom position. The ferrocene units in these compounds are electronically 187 coupled to each other. The DFT and TDDFT calculations on target systems 3 and 4 are in reasonable agreement with the experimental data and suggesting of the predominantly ferrocene-centered MOs in the HOMO region and chromophore π*-character LUMO.

188 CHAPTER VII

UNUSUALLY STRONG LONG-DISTANCE METAL-METAL COUPLING IN

BIS(FERROCENE)-CONTAINING BOPHY: INTRODUCTION TO

ORGANOMETALLIC BOPHYS

The text of this chapter is a reprint of the material as it appears in: Rhoda, H. M.; Chanawanno,

K.; King, A. J.; Zatsikha, Y. V.; Ziegler, C. J.; Nemykin, V. N. Chem. Eur. J. 2015, 21, 18043 –

18046.

Introduction

Standard organic chromophores are important for imaging, bio-imaging, light harvesting, and medicinal applications.345–352 In addition to traditional dyes such as porphyrins,353–357 phthalocyanines,284,358,359 and their analogues,360–364 a new cohort of non-macrocyclic chromophores recently immerged in the field. In particular, the boron dipyrromethene (BODIPY)113,135,314,316,365 and boron azadipyrromethene (aza-

BODIPY)315,366–370 families were shown to have both outstanding and tunable optical, photophysical, and redox properties. Success of these two new families of chromophores inspired the search for new non-macrocyclic organic chromophoric systems. As a result 189 of this search, in 2014, Ziegler and coworkers introduced a new fully conjugated tetracyclic BOPHY system that exhibits outstanding fluorescence properties.152 Later, the parent BOPHY platform was complimented by systems with alkyl and aryl functional groups, as well as, specifically designed electron donating and withdrawing substituents.153,154,156,157 Similar to BODIPY and aza-BODIPY platforms, the BOPHY system can be functionalized starting from substituted pyrroles or by the

Knoevenagel reaction and by the standard coupling reactions.152–154,156,157 None of the currently known BOPHY systems, however, have an organometallic group conjugated to the tetracyclic chromophore. In this communication, we present the first organometallic

BOPHY 2 functionalized with ferrocene substituents. This compound exhibits both long- range metal-metal coupling as well as unusual photophysical properties.

For several years we have investigated electron transfer and redox properties of ferrocene substituted porphyrins and their analouges,189,292–294,305,371–375 as well as

BODIPYs and aza-BODIPYs.55,318,319,321,376–383 In 2014, we reported an aza-BODIPY with an exceptional electron coupling between two ferrocene groups.55 Our next target was to investigate the degree of electronic coupling in the symmetric ferrocene containing BOPHY system. In order to prepare a symmetric bis(ferrocene)-containing

BOPHY, in which the ferrocene groups are linked to the BOPHY chromophore via vinyl bridges, we used the earlier reported tetramethyl substituted system 1, which can be modified via a Knoevenagel reaction with ferrocene carboxaldehyde to form the target

BOPHY 2 (Scheme 7.1). This new complex can be purified using standard

190 chromatographic techniques and is stable in air in the solid state and to some extent in solutions of nonpolar or chlorinated solvents for prolonged times.

Scheme 7.1. Preparation of the ferrocene-containing BOPHY 2.

Experimental

Materials: All commercial reagents were ACS grade and were used without further purification. Tetrahydrofuran (THF) was distilled over sodium metal and dichloromethane was dried over CaH2. Tetrabutylammonium

384,385 tetrakis(pentafluorophenyl)borate (TBAF, (NBu4)[B(C6F5)4]) and tetramethyl

BOPHY 1152 were prepared according to the literature procedure.

Instrumentation: All UV-Vis data was obtained on a Jasco-720 spectrophotometer at room temperature. Steady-state fluorescence data was obtained from using Cary

Eclipse fluorimeter at room temperature. Electrochemical measurements were conducted using a CHI-620C electrochemical analyzer using the three-electrode scheme. The three electrodes include a platinum working, a platinum auxiliary, and an Ag/AgCl reference electrode. A solution of 0.05 M TBAF in DCM was used as the liquid medium. The 191 solution of DCM and TBAF was purged under pure nitrogen gas. All redox potentials

+ were referenced to FcH/FcH couple using decamethylferrocene (Me10Fc) as the internal standard. The spectroelectrochemical data was collected using a custom-made 1mm cell, a platinum mesh working electrode, a platinum auxiliary, and an Ag/AgCl reference electrode. A 0.15 M solution of TBAF in DCM was used as the liquid medium. Chemical titrations were conducted with a stock solution of ~0.01 M iron (III) perchlorate

-3 (Fe(ClO4)3) and ~5 x 10 M solution of commercially available “magic blue” oxidant.

Each of the oxidants was added in 1-5 μL increments.

Computational Details: All computations were performed using the Gaussian 09 software package running under UNIX OS.202 The BOPHY 2 compound was optimized with both the Ci and the C2 symmetry to model the experimental geometry. For both the

327 Ci and C2 symmetries of BOPHY 2, the TPSSh hybrid exchange-correlation functional was used along with 6-311+G199 basis set for the iron atoms and the 6-311G(d)386 basis set for all other atoms for all calculations. Solvation effects were modeled by the PCM approach201 using DCM for the solvent in all calculations. Frequencies were calculated for all optimized geometries in order to ensure that final geometries represent a minima on the potential energy surface. PCM-TDDFT calculations were conducted for the first

50 excited states. Molecular orbital contributions were compiled from single point calculations using the program QMForge.203

Synthesis: Compound 1152 (80 mg, 0.24 mmol) and ferrocenecarboxaldehyde

(102 mg, 0.48 mmol) were mixed in 20 mL toluene. Piperidine (1 mL) and a catalytic amount of p-toluenesulfonic acid (17 mg, 0.10 mmol) were added. The solution was 192

refluxed for 48 hrs in a round-bottomed flask and reaction was monitored by TLC. Upon the disappearance of 1, the resulting mixture was cooled to room temperature and solvent was removed under vacuum. The crude product was purified via chromatography (silica gel, CH2Cl2/hexane =1/1, v/v) to afford the desired compound 2 in 4% yield (8 mg).

1 H NMR (500 MHz, CDCl3, δ (ppm): 2.30 (s, 6H, CH3-H), 4.20 (s, 10H, Cp-H), 4.44 (br s, 4H, β-Cp-H), 4.64 (br s, 4H, α-Cp-H), 6.63 (s, 2H, β-pyrr-H), 6.98 (s, 2H, meso-H),

7.12 (d, J = 16.0 Hz, vinyl-H), 7.25 (d, J = 16.0 Hz, vinyl-H)

1 H NMR (500 MHz, DMSO-d6, δ ppm): 7.57 (s, 2H, meso-H), 7.37 (d, 7.37 Hz, 2H, vinyl-H), 7.01 (d, 7.37 Hz, 2H, vinyl-H), 6.89 (s, 2H, β-pyrr-H), 4.62 (s, 4H, β-Cp-H),

19 4.53 (s, 4H, β-Cp-H), 4.21 (s, 10H, Cp-H), 2.30 (s, 6H, CH3-H). F NMR (300 MHz,

13 CDCl3, δ ppm): -139.77 (q, J B-F = 21.0 Hz) C NMR (500 MHz, CDCl3, δ ppm): 11.35,

68.11, 69.68, 70.45, 82.21, 115.14, 115.34, 117.21, 135.21, 136.44, 139.15, 153.45. Anal.

Calcd for BOPHY 2·4C7H8·1.6CH2Cl2, C36H32B2F4Fe2N4·(C7H8)4·(CH2Cl2)1.6: C, 63.83;

H, 5.49; N, 4.54%. Found: C, 64.18; H, 5.14; N, 4.24%.

Result and Discussion

The absorption spectra of the parent tetramethyl BOPHY 1 and the bis(ferrocene)- containing 2 are shown in Figure 7.1. The parent BOPHY 1 has two absorption maxima at 444 and 467 nm while the organometallic 2 has a broad NIR absorption peak with the prominent band at 694 nm, lower intensity band at 564 nm and an additional band at 343 193 nm. Thus, the addition of two conjugated ferrocene substituents to the BOPHY chromophore results in a more than 200 nm bathochromic shift of the lowest energy band. The profile of the low-energy band is fairly unusual compared to the ferrocene containing BODIPYs and aza-BODIPYs due to the absence of a broad low-energy low- intensity metal to ligand charge transfer (MLCT) band in the NIR region of the spectrum.320,323,387 In order to clarify the nature of the broad band observed in the visible region of the spectrum of BOPHY 2, we conducted Density Functional Theory (DFT) and Time-Dependent DFT (TDDFT) calculations (Figures 7.1-7.6). DFT calculations suggested two close in energy geometries (Ci and C2 symmetries) for Fc-BOPHY 2.

Indeed, the energy difference between Ci and C2 structures is only 1.51 kcal/mol. Both Ci and C2 isomers have only minor differences in their electronic structures and vertical excitation energies. For both geometries, the HOMO to HOMO-10 molecular orbitals were predicted to be predominantly ferrocene-centered while the highest energy BOPHY localized π orbitals were calculated as HOMO-11 and HOMO-12. Unlike in the parent

BOPHY 1, we found no substantial contribution from the bridge in N-N group in the

HOMO region. Despite being predominantly ferrocene-centered, the HOMO, HOMO-4,

HOMO-5, HOMO-7, and HOMO-8 orbitals have substantial contribution from the

BOPHY chromophore (Figure 7.6). Moreover, if one would separate contributions from

FeII and Cp ligands, HOMO, HOMO-4, and HOMO-7 can be viewed as π-orbitals with

FeII contribution varying between ~33 and 44%. On the contrary, the HOMO-1 to

HOMO-3 and HOMO-5 and HOMO-6 are almost pure ferrocene-centered MOs with predominant contribution from FeII centers. The DFT predicts that the LUMO and

LUMO+1 are BOPHY chromophore-centered orbitals. The LUMO is ~1 eV lower in 194 energy than LUMO+1 and the LUMO+1 orbital is also well energetically separated from the rest of the unoccupied orbitals.

Figure 7.1. Experimental (top) UV-vis spectra of 1 and 2 in DCM and TDDFT predicted UV-vis spectra of 2 for Ci and C2 geometries.

195

-1 Figure 7.2. Predicted-TDDFT of Ci and C2 Symmetry of BOPHY 2 in cm energy scale.

196 Figure 7.3. DFT-predicted frontier MOs of BOPHY 2 compound in Ci geometry.

197 Figure 7.4. DFT-predicted frontier MOs of BOPHY 2 compound in C2 geometry.

198 The TDDFT-predicted UV-vis spectra for BOPHY 2 are in good agreement with the experimental data (Figure 7.1) for both geometries and explain its unusually broad profile in the low-energy region. The TDDFT predicts that the low-energy region of the

UV-vis spectrum of 2 can be described by two main contributions from excited states 1 and 5 for both C2 and Ci geometries. Excited state 1 is almost pure HOMO→LUMO single electron transition with a very high TDDFT-predicted oscillator strength. This transition can be viewed as predominantly π-π* in nature, complimented by significant

MLCT character (~37 - 39% of FeII character in HOMO). The TDDFT-predicted energy for the first excited state (701-713 nm) correlates very well with the observed 694 nm peak in the UV-visible spectrum of BOPHY 2. TDDFT nature of excited state 5 is dominated by a HOMO-4→LUMO single electron transition, which also can be viewed

II as a mixture of π-π* and MLCT characters (~33 - 35% of Fe character in HOMO-4).

The TDDFT calculations also predict that the lowest energy predominantly BOPHY- localized π-π* transitions (HOMO-11, HOMO-12→LUMO) in 2 should be observed at

~330-350 nm region.

199 Figure 7.5. DFT-predicted energy diagram for Ci and C2 geometries of 2.

View 1 View 2

88% BOPHY LUMO

40% BOPHY/60% Fc (38% Fe) OMO H

38% BOPHY / 62% Fc (38% Fe) HOMO-4

Figure 7.6. DFT-predicted frontier MOs in 2 (Ci symmetry).

200 The redox properties of the new organometallic BOPHY 2 were investigated by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) methods (Figure 7.7).

Electrochemical data reveal three reversible single-electron oxidation processes in

BOPHY 2 observed at 32, 236, and 906 mV potentials with respect to Fc/Fc+ couple. No reduction processes were observed in the electrochemical window. The first two processes were assigned as ferrocene-centered on a basis of chemical and spectroelectrochemical oxidation data as well as their observed potentials. The third oxidation process was assigned to the BOPHY core. The separation between the

3 ferrocene-centered first and the second oxidation process (~200 mV, Kc = 2.41x10 ) is quite remarkable taking into consideration the DFT-predicted Fe-Fe distance (~17.2 Å).

Taking into consideration the topology of 2, it is safe to conclude that the -system of

BOPHY facilitates through-bond long-range metal-metal coupling. The ion-pairing properties of electrolyte can affect the electrostatic repulsion between metal centers and thus separation between redox waves in electrochemical experiments.325,388 Thus, to prove the metal-metal coupling in 2, we conducted additional spectroelectrochemical and chemical oxidation experiments (Figure 7.8-7.10).

201 Figure 7.7. CV (top) and DPV (bottom) data for BOPHY 2 in DCM/0.05M TBAF system.

Figure 7.8. Changes in UV-vis spectra of BOPHY 2 during its transformation into [2]+ (A) and [2]2+ (B) under spectroelectrochemical conditions in DCM/0.15 M TBAF system.

202

st nd Figure 7.9. 1 and 2 single-electron oxidations of BOPHY 2 by Fe(ClO4)3.

The results from both methods correlate well with each other. For instance, during the first one-electron oxidation of BOPHY 2 into the mixed-valence [2]+, under spectroelectrochemical and chemical oxidation conditions, the most intense band at 694 nm loses intensity. The band at 343 nm transforms into a lower energy band at 390 nm and new bands at 619 nm, 993 nm, and a very broad NIR band (~2500 nm) appear in the spectrum (Figure 7.8A and Figure 7.9). During the second one-electron oxidation process, a new sharp band at 662 nm appears in the spectrum of [2]2+, the 390 nm band transforms into a 308 nm peak, and the NIR band at 993 nm decreases in intensity and shifts to 902 nm. The far NIR band ~2500 nm decreases in intensity and a new broad NIR band centered at 1410 nm appears in the spectrum (Figure 7.8B and Figure 7.9). Band deconvolution analysis of the NIR part of the spectrum of [2]+ generated by chemical oxidation or bulk electrolysis revealed presence of at least 5 peaks, one of which (peak 2) was assigned as the inter-valence charge transfer (IVCT) in nature, while the other peaks

203

can be attributed to the LMCT (BOPHY-to-FeIII) and d-d transitions (Figure 7.10). IVCT band parameters are in the range of weakly-coupled Class II (in the Robin-Day classification)389 systems and similar to those observed in the other mixed-valence poly(ferrocenyl)-containing compounds.390–394 Because the center of band 1 is located outside of the NIR detector range, however, the IVCT parameters in [2]+ should be treated with caution.

The BOPHY chromophores are known for their exceptional fluorescence properties. Organometallic substituents in 2, however, quench the fluorescence in this compound and similar behavior was earlier observed for the ferrocene-substituted subphthalocyanines,395–397 BODIPYs,189,292–294,305,371–375 and aza-BODIPYs.55,318,319,321,376–

383

Figure 7.10. NIR band deconvolution analysis of BOPHY [2]+ generated under spectroelectrochemical (DCM/0.15M TBAF, A) and chemical (DCM, B) oxidation conditions.

204 Conclusion

In conclusion, we have synthesized and characterized the first organometallic

BOPHY chromophore substituted with two ferrocene fragments via vinyl bridges. The ferrocene-containing BOPHY 2 has a broad absorption band centered around 700 nm, which, according to TDDFT calculations, has a mixed BOPHY-centered π-π* and ferrocene-to-BOPHY core charge-transfer character. In addition, electrochemical, spectroelectrochemical, and chemical oxidation data reveal unprecedented long-range metal-metal coupling in 2 with 200 mV separation between the ferrocene-centered oxidation waves. The spectroscopic signatures of the mixed valence [2]+ were revealed using spectroelectrochemical and chemical oxidation methods and are indicative of the

Class II weakly coupled system.

205 CHAPTER VIII

SUMMARY

In Chapter 2, a study of a series of Re(CO)3 pyridine-imine complexes with pendant phenol groups was presented. The effects of the position of the phenol hydroxyl group (para, meta or ortho to the imine) on the steric and electronic characteristics of a series of Re(CO)3X(pyca-C6H4OH) compounds, where X = Cl, Br and pyca = pyridine-2-carbaldehyde imine were investigated. These compounds can be generated either via ligand synthesis followed by metal chelation (compound 4) or via a one-pot method (compounds 2, 3, 5 and 6). All six compounds show striking differences in pH-dependent UV-visible absorption based on the position of the phenol hydroxyl group.

Acetyl ferrocene and diacetyl ferrocene both readily react with an excess of hydrazine to afford the corresponding hydrazone compounds, as reported in

Chapter 3. These compounds can then be linked to Re(CO)3 via a metal mediated

Schiff base reaction, resulting in a series of ferrocene-Re(CO)3 conjugates with different stoichiometries. Conjugates with 1:1, 1:2 and 2:1 ferrocene:

Re(CO)3 ratios can be produced via this “modular” type synthesis approach.

Several examples of these conjugates were structurally characterized, and their spectroscopic, electrochemical, 206 spectroelectrochemical behaviors were investigated. The electronic structures of these compounds were also probed using DFT and TDDFT calculations.

In Chapter 4, the synthesis of two Re(CO)3-modified lysine complexes where the metal is attached to the amino acid at the Nε position, via one-pot Schiff base formation reactions was presented. These compounds can be used in the solid phase synthesis of peptides, and to date four conjugate systems incorporating neurotensin, bombesin, leutenizing hormone releasing hormone, and a nuclear localization sequence have been synthesized. Uptake into human umbilical vascular endothelial cells as well as differential uptake depending on peptide sequence identity, as characterized by fluorescence and rhenium elemental analysis, was observed.

In Chapter 5 the syntheses of several azadipyrromethenes with four peripheral arene units were presented, which can be readily generated via the well-established chalcone synthetic method. The stability and yields of these azadipyrromethenes is highly dependent on the nature of the arene substituent, with the bis-pyridine systems exhibiting the highest degree of instability. The structures of several of the compounds and their BF2 analogues are presented; intramolecular hydrogen bonding is observed in the free base ADPMs. We also discussed a study in to the absorption and emission properties of the ADPMs and their BF2 compounds and observe that they are also highly dependent on the identity of the substituents.

As reported in Chapter 6, 3,3′-diferrocenylazadipyrromethene (3) and the corresponding difluoroboryl (azaBODIPY) complex (4) were synthesized in several steps

207 from ferrocenecarbaldehyde, following the well-explored chalcone-type synthetic approach. The novel diiron complexes, in which ferrocene groups are directly connected to the α-pyrrolic positions were characterized by a variety of spectroscopic techniques, electrochemistry, spectroelectrochemistry, and X-ray crystallography, while their electronic structure, redox properties, and UV vis spectra were correlated with density functional theory (DFT) and time-dependent DFT calculations.

In Chapter 7, the first organometallic BOPHY containing two ferrocene substituents was prepared via a Knoevenagel condensation between tetramethyl substituted BOPHY and ferrocene carboxaldehyde. An unprecedentedly strong long- range (~17.2 Å) metal-metal coupling in this new complex was investigated using electrochemical, spectroelectrochemical, and chemical oxidation methods.

Electrochemical data is indicative of a 200 mV separation between the first and the second ferrocene-centered oxidation processes. Formation of the mixed valence states and appearance and disappearance of two NIR bands were observed during stepwise oxidation of the first organometallic BOPHY. The electronic structure and the nature of the excited states in this new chromophore were studied by DFT and TDDFT calculations.

Possible future research in the Ziegler group will be related to the development of new compounds series using Re(CO)3halide(pyca) in the synthetic approach. With the excellent chelating ability of pyridine-2-carboxaldehyde introduced in Chapter 1.3, the group can continue using this methodology to synthesize and study for biologically-, photophysically-, and electrochemically-active Re(CO)3halide(pyca) compounds. The 208 fluorophores aza-dipyrromethenes as well as BOPHYs can serve as interesting starting materials for various modifications, such as halogenations, cross-coupling reactions, and condensations. The modified species can be expected to exhibit some fascinating photophysical and electrochemical properties, due to the interesting properties of parent compounds.

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