SYNTHESIS AND BIOASSAY STUDIES OF CATIONIC PORPHYRINS …eprints.utm.my/id/eprint/33842/1/KiewSiawFuiMFS2013.pdf · SYNTHESIS AND BIOASSAY STUDIES OF CATIONIC PORPHYRINS FOR GENE

SYNTHESIS AND BIOASSAY STUDIES OF CATIONIC PORPHYRINS FOR

GENE TRANSFECTION DELIVERY

KIEW SIAW FUI

A thesis submitted in fulfillment of the

requirements for the award of the degree of

Master of Science (Chemistry)

Faculty of Science

Universiti Teknologi Malaysia

JANUARY 2013

iii

To my beloved father, mother, brothers and dearest sister

iv

ACKNOWLEDGEMENT

First, I would like to express my deeply appreciation to my supervisor, Dr.

Mohd Bakri Bin Bakar for his valuable knowledge, patient guidance, enthusiastic

support and friendship towards the success of completion of this study. Besides, I

am very thankful to my co-supervisor, Dr. Razauden Mohamed Zulkifli for his

constructive advices, encouragement and generosity during the planning and

development of this research work.

I would also like to acknowledge the laboratory staff Pn Zahratulain, En

Azmi, Pn Nasrena, Pn Suhaini, En Amin, En Rasyidi, En Azidy, En Rahim, Ms

Norsyuhada and others for their technical support and assistance with the collection

of my data.

Special thanks to Ministry of Higher Education Malaysia (FRGS vot 78554),

MyMaster and Universiti Teknologi Malaysia (UTM) for the financial support as

well as the facilities provided by UTM.

My sincere appreciation also extends to all my colleagues and friends; Helmi,

Sayang, Nurhafizah, Ermi, Azhana, Syahda, Asita, Zuhairi, Ke Xin and Wen Yee for

their helps and willingness to share useful knowledge. Last but not least, I would

like to thank my family for their support and motivation both in spirit and financial.

v

ABSTRACT

Porphyrins are stable aromatic tetrapyrrolic macromolecules found in many

natural products such as hemin, chlorophylls and vitamin B12. Interaction ability of

cationic porphyrins with nucleic acid and their fluorescent properties for location

identification in cellular domain have promoted their uses as potential gene vectors

for gene therapy. In this study, basic cationic porphyrins bearing four positive

charges were synthesized. Besides, amphiphilic porphyrins anchored with both

hydrophobic and hydrophilic moieties were prepared to facilitate membrane

penetration and to give a higher cellular uptake. Polyamidoamine (PAMAM)-

porphyrin conjugate was also prepared to produce a complex with higher transfection

level but with low toxicity. Adler-Longo condensation method was mainly used to

synthesize these cationic porphyrin precursors. All cationic porphyrins were

obtained in high yield. All of the compounds were characterized using 1H-NMR,

13C-NMR, ultraviolet (UV) and infrared (IR) spectroscopies. Cytotoxicity and

cellular uptake of all cationic compounds were tested on Chinese Hamster Ovary

(CHO) cells to evaluate their potential uses as gene carriers. Results revealed that all

porphyrins show relatively low toxicity towards the cell even at high concentration

(100 µM). The 5,10,15,20-tetrakis(N-methyl-4-pyridyl)porphyrin and two

amphiphilic cationic porphyrins of 5-hexyl-10,15,20-tris(N-methyl-4-pyridyl)

porphyrin and 5-propyl-10,15,20-tris(N-methyl-4-pyridyl)porphyrin which contain

three positive charges on the periphery show the highest cellular uptake. It was also

found that the amphiphilic cis-porphyrins of 5,10-dipropyl-15,20-bis(N-methyl-4-

pyridyl)porphyrin and 5,10-dihexyl-15,20-bis(N-methyl-4-pyridyl)porphyrin

exhibited higher cellular uptake compared to their trans-isomers, 5,15-dipropyl-

10,20-bis(N-methyl-4-pyridyl)porphyrin and 5,15-dihexyl-10,20-bis(N-methyl-4-

pyridyl)porphyrin.

vi

ABSTRAK

Porfirina adalah makromolekul aromatik yang stabil dan boleh dijumpai

dalam banyak sebatian semula jadi seperti hemin, klorofil dan vitamin B12.

Keupayaan porfirina kationik untuk berinteraksi dengan acid nukleik dan sifat

pendarfluor porfirina yang dapat menentukan lokasi vektor di dalam sel

menyebabkan ianya berpotensi untuk digunakan sebagai vektor gen bagi aplikasi

terapi gen. Dalam kajian ini, porfirina kationik asas yang mengandungi empat cas

positif telah disintesis. Selain itu, porfirina amfifilik yang bersifat hidrofobik dan

hidrofilik telah disediakan untuk memudahkan penetrasi membran dan meningkatkan

pengambilan bahan oleh sel. Konjugat poliamidoamina (PAMAM)-porfirina juga

disediakan untuk menghasilkan komplek yang mempunyai tahap transfeksi yang

lebih tinggi dengan ketoksikan yang rendah. Kaedah kondensasi Adler-Longo

digunakan untuk mensintesis pelbagai bahan mula porfirina kationik. Semua

porfirina kationik diperolehi dengan hasil yang tinggi. Semua sebatian telah

dicirikan dengan menggunakan spektroskopi 1H-RMN,

13C-RMN, ultralembayung

(UL) dan inframerah (IM). Kajian toksisiti dan pengambilan sel untuk semua

sebatian kationik tersebut dilakukan dengan menggunakan sel Ovari Hamster Cina

(CHO) untuk menilai potensinya sebagai pembawa gen. Keputusan kajian

mendapati bahawa semua porfirina menunjukkan ketoksikan yang agak rendah

walaupun pada kepekatan yang tinggi (100 µM). 5,10,15,20-Tetrakis(N-metil-4-

piridil)porfirina dan dua porfirina kationik amfifilik, 5-heksil-10,15,20-tris(N-metil-

4-piridil)porfirina dan 5-propil-10,15,20-tris(N-metil-4-piridil)porfirina yang

mengandungi tiga cas positif menunjukkan pengambilan selular yang tinggi.

Dapatan kajian juga menunjukkan cis-porfirina amfifilik, 5,10-dipropil-15,20-bis(N-

metil-4-pyridil)porfirina dan 5,10-diheksil-15,20-bis(N-metil-4-piridil)porfirina

menunjukkan pengambilan selular yang lebih tinggi berbanding dengan trans-isomer,

5,15-dipropil-10,20-bis(N-metil-4-piridil)porfirina and 5,15-diheksil-10,20-bis(N-

metil-4-piridil)porfirina.

vii

TABLE OF CONTENTS

CHAPTER TITLE PAGE

TITLE i

DECLARATION ii

DEDICATION iii

ACKNOWLEDGEMENTS iv

ABSTRACT v

ABSTRAK vi

TABLE OF CONTENT vii

LIST OF TABLES xii

LIST OF FIGURES xiii

LIST OF SCHEMES xv

LISTS OF ABBREVIATIONS xvii

LIST OF APPENDICES xx

1 INTRODUCTION 1

1.1 Background of the Study 1

1.2 Problem Statement 4

1.3 Objectives of the Study 5

1.4 Scope of the Study 5

1.5 Significance of the Study 6

2 LITERATURE REVIEW 7

2.1 Porphyrins 7

2.1.1 The Electronic Structure and Properties 8

viii

of Porphyrins

2.1.2 NMR Spectroscopy of Porphyrins 8

2.1.3 UV Spectroscopy of Porphyrins 9

2.2 Synthesis of Porphyrins 11

2.2.1 Condensation of Pyrrole and Aldehydes 12

2.2.2 Synthetic Transformation of Porphyrins 18

2.2.3 Metallation of Porphyrin 19

2.3 Cationic Porphyrins 20

2.4 Amphiphilic Porphyrins 25

2.5 Cationic Dendrimer-Porphyrin Conjugates 29

2.5.1 Polyamidoamine (PAMAM) Dendrimer 29

2.6 Gene Therapy 34

3 RESULTS AND DISCUSSION 36

3.1 Synthesis of Symmetrical Porphyrins 37

3.1.1 Synthesis of 5,10,15,20-tetraphenyl 37

porphyrin (6)

3.1.2 Synthesis of 5,10,15,20-tetrakis(4-acetami 39

dophenyl)porphyrin (38)

3.1.3 Synthesis of 5,10,15,20-tetrakis(4-pyridyl) 43

porphyrin (15)

3.2 Synthesis of Asymmetrical Porphyrins 44

3.2.1 Synthesis of 5-propyl-10,15,20-tris 46

(4-pyridyl)porphyrin (39)

3.2.2 Synthesis of 5-hexyl-10,15,20-tris 49


3.2.3 Synthesis of 5-(carboxyphenyl)-10,15,20 51

-tris(4-pyridyl)porphyrin (53)

3.3 Synthesis of PAMAM G4-Porphyrin Conjugate 56

(54)

3.4 Synthesis of Basic Cationic Porphyrins 59

3.4.1 Synthesis of 5,10,15,20-tetrakis(N-methyl 59

-4-pyridyl)porphyrin (17)

ix

3.5 Synthesis of Amphiphilic Cationic Porphyrins 62

3.5.1 Synthesis of 5,15-dipropyl-10,20-bis 63

(N-methyl-4-pyridyl)porphyrin (57)





3.5.4 Synthesis of 5,10,15-trihexyl-20-(N-methyl 65


3.5.5 Synthesis of 5,15-dihexyl-10,20-bis 66






3.6 Synthesis of Cationic PAMAM G4-Porphyrin 69

Conjugate (64)

3.7 Cytotoxicity of Porphyrin Derivatives on CHO 70

Cells

3.8 Cellular Uptake of Porphyrin Derivatives 74

4 EXPERIMENTAL 80

4.1 General Instruments and Apparatus 80

4.2 Chemicals and Reagents 80

4.3 Synthesis of Symmetrical Porphyrins 81

4.3.1 Synthesis of 5,10,15,20-tetraphenyl 81

porphyrin (6)

4.3.2 Synthesis of 5,10,15,20-tetrakis(4-acetami 82

dophenyl)porphyrin (38)

4.3.3 Synthesis of 5,10,15,20-tetrakis(4-pyridyl) 83

porphyrin (15)

4.4 Synthesis of Asymmetrical Porphyrins 83


porphyrin (39)

x



4.4.3 Synthesis of 5-(methoxycarbonylphenyl) 88

-10,15,20-tris(4-pyridyl)porphyrin (48)

4.4.4 Synthesis of 5-(carboxyphenyl)-10,15,20 90

-tris(4-pyridyl)porphyrin (53)

4.5 Synthesis of PAMAM G4-Porphyrin Conjugate 91

(54)

4.6 Synthesis of Basic Cationic Porphyrins 92

4.6.1 Synthesis of 5,10,15,20-tetrakis(N-methyl 92


4.6.2 Synthesis of 5,10-bis(N-methyl-4-pyridyl) 92

-15,20-bis(4-pyridyl)porphyrin or 5,15-bis

(N-methyl-4-pyridyl)-10,20-bis(4-pyridyl)

porphyrin (55)

4.6.3 Synthesis of 5,10,15-tris(N-methyl-4- 93

pyridyl)-20-(4-pyridyl)porphyrin (56)

4.7 Synthesis of Amphiphilic Cationic Porphyrins 94







4.7.4 Synthesis of 5,10,15-trihexyl-20- 96








4.8 Synthesis of Cationic PAMAM G4-Porphyrin 98

Conjugate (64)

xi

4.9 Bioactivity Studies 99

4.9.1 Chemical and Instrumentation 99

4.9.2 Cell Culture and Subculture 100

4.9.3 Cytotoxicity or MTT Assay 101

4.9.3.1 Preparation of MTT Solution and 101

Cytotoxicity Assay Flow Chart

4.9.4 Cellular Uptake of Porphyrin Derivatives 104

5 CONCLUSIONS AND SUGGESTIONS 105

5.1 Conclusions 105

5.2 Suggestion for Future Work 106

REFERENCES 107

APPENDICES 115

LIST OF PUBLICATIONS 209

xii

LIST OF TABLES

TABLE NO. TITLE PAGE

3.1 Qualitative cellular uptake efficiency of porphyrin 77

derivatives with different distribution of substituents

xiii

LIST OF FIGURES

FIGURE NO. TITLE PAGE

1.1 Structure of porphyrin (1), chlorophyll (2) and hemin (3) 1

1.2 Gene therapy 3

2.1 Basic structure of porphyrin 8

2.2 UV-Vis Q band spectra of metal free porphyrin 10

2.3 UV-Vis Q band spectra for metalloporphyrin 11

2.4 meso-Tetrakis(4-N,N,N-trimethylanilinium)porphyrin (18) 22

2.5 meso-Substituted porphyrins (19) and β-substituted 23

porphyrins (20) with same charge position

2.6 meso-Tetrakis(N-methyl-4-pyridyl)porphyrin (17) and 23

meso-Tetrakis(N-methyl-2-pyridyl)porphyrin (21)

2.7 meso-Substituted porphyrin (22) and sapphyrin (23) 24

2.8 meso-Tetrakis(4-sulfonatophenyl)porphyrin (24) 24

2.9 meso-Substituted cationic porphyrins 27

2.10 Porphyrin (27) with two positive charges and porphyrin 28

(28) with four positive charges

2.11 Porphyrin (29) and porphyrin (30) 28

2.12 Structure of polyethylenimine (31) and polyamidoamine 29

(32)

3.1 1H NMR spectrum of monoalkylated porphyrin 42

3.2 Cell viability (%) versus various concentrations of 72

porphyrins compared with triton�R X-100

3.3 Endocytosis process of cell and phospholipid bilayer 74

3.4 Cellular uptakes of porphyrin derivatives under inverted 77

fluorescence microscope with 100X magnification.

xiv

4.1 General flow chart of cytotoxicity assay 102

4.2 Calculation formulas for percentage of cell inhibition and 102

cell growth

4.3 Experimental design for 96 wells microplate 103

4.4 General flow chart for cellular uptake of porphyrin 104

derivatives

xv

LIST OF SCHEMES

SCHEME NO. TITLE PAGE

2.1 Synthesis of tetraphenylporphyrin (6) 12

2.2 Synthesis of porphyrin (9) 13

2.3 One-flask synthesis of meso-substituted dipyrromethanes 13

(10)

2.4 One-flask synthesis of a bis-dipyrromethanes (12) 14

2.5 Six porphyrins formed from mixed condensation 14

2.6 Synthesis of trans-substituted porphyrins using four types 15

of dipyrromethanes

2.7 MacDonald 2+2 Condensation 16

2.8 “2+2” route to ABCD-porphyrins 16

2.9 “3+1” approach to cis-A2-substituted porphyrin 17

2.10 Direct synthesis of tripyrrane 17

2.11 Synthesis of A3- and A2B-type porphyrins via SNAr 18

reactions

2.12 Preparation of formylated porphyrin via Vilsmeier 19

reaction

2.13 Bromination of meso-tetramesitylporphyrin 19

2.14 Metallation of porphyrin 20

2.15 Synthetic route of TMPyP4 (17) 21

2.16 Synthesis ethylenediamine (EDA) core PAMAM 30

2.17 Schematic representations of PAMAM-TAMCPP 33

conjugate (37) and structure of PAMAM G4

3.1 Synthesis of 5,10,15,20-tetraphenylporphyrin (6) 37

3.2 Synthesis of 5,10,15,20-tetrakis(4-acetamidophenyl) 39

porphyrin (38)

3.3 Synthesis of cationic porphyrin (18) 42

xvi

3.4 Synthesis of 5,10,15,20-tetrakis(4-pyridyl)porphyrin (15) 43

3.5 Attempted synthesis of dipyrromethanes (8) 45

3.6 Synthesis of 5-propyl-10,15,20-tris(4-pyridyl)porphyrin 46

(39) and its derivatives

3.7 Synthesis of 5-hexyl-10,15,20-tris(4-pyridyl)porphyrin 50

(43) and its derivatives

3.8 Synthesis of 5-(methoxycarbonylphenyl)-10,15,20-tris 52

(4-pyridyl) porphyrin (48) and its derivatives

3.9 Synthesis of 5-(carboxyphenyl)-10,15,20-tris(4-pyridyl) 55

porphyrin (53)

3.10 Synthesis of PAMAM G4-porphyrin conjugate (54) 57

3.11 Synthesis of 5,10,15,20-tetrakis(N-methyl-4-pyridyl) 60

porphyrin (17)

3.12 Synthesis of 5,10-bis(N-methyl-4-pyridyl)-15,20-bis 61

(4-pyridyl)porphyrin (55a) or 5,15-bis(N-methyl-4-pyridyl)

-10,20-bis(4-pyridyl)porphyrin (55b) and 5,10,15-tris(N-

methyl-4-pyridyl)-20-(4-pyridyl)porphyrin (56)

3.13 Synthesis of 5,15-dipropyl-10,20-bis(N-methyl-4-pyridyl) 63

porphyrin (57)

3.14 Synthesis of 5,10-dipropyl-15,20-bis(N-methyl-4-pyridyl) 64

porphyrin (58)

3.15 Synthesis of 5-propyl-10,15,20-tris(N-methyl-4-pyridyl) 65

porphyrin (59)

3.16 Synthesis of 5,10,15-trihexyl-20-(N-methyl-4-pyridyl) 66

porphyrin (60)

3.17 Synthesis of 5,15-dihexyl-10,20-bis(N-methyl-4-pyridyl) 67

porphyrin (61)

3.18 Synthesis of 5,10-dihexyl-15,20-bis(N-methyl-4-pyridyl) 68

porphyrin (62)

3.19 Synthesis of 5-hexyl-10,15,20-tris(N-methyl-4-pyridyl) 68

porphyrin (63)

3.20 Synthesis of cationic PAMAM G4-porphyrin conjugate 70

3.21 Formation of purple formazan (66) by metabolically 70

active cell

xvii

LIST OF ABBREVIATIONS

ArCHO - Aromatic aldehyde

ANOVA - Analysis of variance

BF3.O(Et)2 - Boron trifluoride etherate

br - Broad

13C - Carbon-13

CHO - Chinese Hamster Ovary

COSY - H-H correlation spectroscopy

CSCl2 - Thiophosgene

d - Doublet

DCC - Dicyclohexylcarbodiimide

dd - Doublet of doublet

DDQ - 2,3-Dichloro-5,6-dicyano-p-benzoquinone

DiTMPyP - 5,10-Bis(N-methyl-4-pyridyl)-15,20-bis(4-pyridyl)porphyrin

or 5,15-bis(N-methyl-4-pyridyl)-10,20-bis(4-pyridyl)porphyr-

in (55)

DMAP - 4-(Dimethylamino)pyridine

DMSO - Dimethylsulfoxide

DNA - Deoxyribonucleic acid

DNases - Deoxyribonuclease

Et3N - Triethylamine

FTIR - Fourier Transform Infrared Spectrometer

G4 - Generation four

h - Hour

1H - Proton

IR - Infrared

J - Coupling constant

m-py - meta-Pyridyl

m - Multiplet

xviii

M - Molar

MeOH - Methanol

mg - Milligram

mL - Milliliter

mp - Melting point

MTT - 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

N2 - Nitrogen

NBS - N-bromosuccinimide

NCS - N-chlorosuccinimide

NHS - N-hydroxysuccinimide

nm - Nanometer

NMR - Nuclear Magnetic Resonance

o-py - ortho-Pyridyl

ODN - Oligodeoxyribonucleotide

OLED - Organic light emitting diode

PAMAM - Polyamidoamine

PDT - Photodynamic cancer therapy

PEI - Polyethylenimine

PLG - Poly(lactide-co-glycolide)

ppm - Part per million

PTSA - p-Toluenesulfonic acid

Rf - Retention factor

RNA - Ribonucleic acid

rt - Room temperature

SPSS - Statistical package for the social science version 16.0

STD - Standard deviation

TAPP - 5,10,15,20-Tetrakis(4-acetamidophenyl)porphyrin (38)

TFA - Trifluoroacetic acid

THF - Tetrahydrofuran

TLC - Thin layer chromatography

TMPyCOPAMAM- Cationic PAMAM G4-porphyrin conjugate (64)

TMPyP4 - meso-Tetrakis(N-methyl-4-pyridyl)porphyrin

TMPyHP2nd - 5,10,15-Trihexyl-20-(N-methyl-4-pyridyl)porphyrin (60)

xix

TMPyHP3rd - 5,15-Dihexyl-10,20-bis(N-methyl-4-pyridyl)porphyrin (61)

TMPyHP4th - 5,10-Dihexyl-15,20-bis(N-methyl-4-pyridyl)porphyrin (62)

TMPyHP5th - 5-Hexyl-10,15,20-tris(N-methyl-4-pyridyl)porphyrin (63)

TMPyP - 5,10,15,20-Tetrakis(N-methyl-4-pyridyl)porphyrin (17)

TMPyPP2nd - 5,15-Dipropyl-10,20-bis(N-methyl-4-pyridyl)porphyrin (57)

TMPyPP3rd - 5,10-Dipropyl-15,20-bis(N-methyl-4-pyridyl)porphyrin) (58)

TMPyPP4th - 5-Propyl-10,15,20-tris(N-methyl-4-pyridyl)porphyrin (59)

TPP - Tetraphenylporphyrin

TriTMPyP - 5,10,15-Tris(N-methyl-4-pyridyl)-20-(4-pyridyl)porphyrin (56)

UV - Ultraviolet

Zn(OAc)2 - Zinc acetate

q - Quartet

s - Singlet

t - Triplet

v/v - Volume per volume

µm - Micrometer

µM - Micromolar

µL - Microliter

δ - Chemical shift

λ - Lambda

% - Percent

xx

LIST OF APPENDICES

APPENDIX TITLE PAGE

1 1H NMR of 5,10,15,20-tetraphenylporphyrin (6) 116

2 13

C NMR of 5,10,15,20-tetraphenylporphyrin (6) 117

3 UV–Vis Spectrum of 5,10,15,20-tetraphenylporphyrin (6) 118

4 IR Spectrum of 5,10,15,20-tetraphenylporphyrin (6) 119

5 1H NMR of 5,10,15,20-tetrakis(4-acetamidophenyl)porphyrin (38) 120

6 13

C NMR of 5,10,15,20-tetrakis(4-acetamidophenyl)porphyrin (38) 121

7 UV-Vis spectrum of 5,10,15,20-tetrakis(4-acetamidophenyl) 122

porphyrin (38)

8 IR spectrum of 5,10,15,20-tetrakis(4-acetamidophenyl) 123

porphyrin (38)

9 1H NMR of 5,10,15,20-tetrakis(4-pyridyl)porphyrin (15) 124

10 13

C NMR of 5,10,15,20-tetrakis(4-pyridyl)porphyrin (15) 125

11 UV-Vis spectrum of 5,10,15,20-tetrakis(4-pyridyl)porphyrin (15) 126

12 IR spectrum of 5,10,15,20-tetrakis(4-pyridyl)porphyrin (15) 127

13 1H NMR of 5-propyl-10,15,20-tris(4-pyridyl)porphyrin (39) 128

14 UV-Vis spectrum of 5,10-dipropyl-15,20-bis(4-pyridyl)porphyrin 129

(42), 5,10,15,20-tetrakis(4-pyridyl)porphyrin (15), 5-propyl-10,15,

20-tris(4-pyridyl)porphyrin (39) and 5,15-dipropyl-10,20-bis(4-

pyridyl)porphyrin (41)

15 IR spectrum of 5-propyl-10,15,20-tris(4-pyridyl)porphyrin (39) 130

16 1H NMR of 5,15-dipropyl-10,20-bis(4-pyridyl)porphyrin (41) 131

17 1H NMR of 5,10-dipropyl-15,20-bis(4-pyridyl)porphyrin (42) 132

18 1H NMR of 5-hexyl-10,15,20-tris(4-pyridyl)porphyrin (43) 133

19 UV-Vis spectrum of 5,15-dihexyl-10,20-bis(4-pyridyl)porphyrin 134

(46), 5,10-dihexyl-15,20-bis(4-pyridyl)porphyrin (47) and

5-hexyl-10,15,20-tris(4-pyridyl)porphyrin (43)

xxi

20 IR spectrum of 5-hexyl-10,15,20-tris(4-pyridyl)porphyrin (43) 135

21 1H NMR of 5,10,15-trihexyl-20-(4-pyridyl)porphyrin (45) 136

22 1H NMR of 5,15-dihexyl-10,20-bis(4-pyridyl)porphyrin (46) 137

23 IR spectrum of 5,15-dihexyl-10,20-bis(4-pyridyl)porphyrin (46) 138

24 1H NMR of 5,10-dihexyl-15,20-bis(4-pyridyl)porphyrin (47) 139

25 IR spectrum of 5,10-dihexyl-15,20-bis(4-pyridyl)porphyrin (47) 140

26 1H NMR of 5-(methoxycarbonylphenyl)-10,15,20-tris(4-pyridyl) 141

porphyrin (48)

27 13

C NMR of 5-(methoxycarbonylphenyl)-10,15,20-tris(4-pyridyl) 142

porphyrin (48)

28 UV-Vis spectrum of 5-(methoxycarbonylphenyl)-10,15,20-tris 143

(4-pyridyl)porphyrin (48), 5,10-di(methoxycarbonylphenyl)-

15,20-bis(4-pyridyl)porphyrin (52), 5,15-di(methoxycarbonyl

phenyl)-10,20-bis(4-pyridyl)porphyrin (51), 5,10,15-tri(methoxy

carbonylphenyl)- 20-(4-pyridyl)porphyrin (50)

29 IR spectrum of 5-(methoxycarbonylphenyl)-10,15,20-tris 144


30 1H NMR of 5,10,15,20-tetra(methoxycarbonylphenyl)porphyrin 145

(49)

31 IR spectrum of 5,10,15,20-tetra(methoxycarbonylphenyl) 146

porphyrin (49)

32 1H NMR of 5,10,15-tri(methoxycarbonylphenyl)-20-(4-pyridyl) 147

porphyrin (50)

33 IR spectrum of 5,10,15-tri(methoxycarbonylphenyl)-20-(4-pyridyl) 148

porphyrin (50)

34 1H NMR of 5,15-di(methoxycarbonylphenyl)-10,20-bis(4-pyridyl) 149

porphyrin (51)

35 IR spectrum of 5,15-di(methoxycarbonylphenyl)-10,20-bis 150


36 1H NMR of 5,10-di(methoxycarbonylphenyl)-15,20-bis(4-pyridyl) 151

Porphyrin (52)

37 IR spectrum of 5,10-di(methoxycarbonylphenyl)-15,20-bis 152


38 1H NMR of 5-(carboxyphenyl)-10,15,20-tris(4-pyridyl)porphyrin 153

(53)

xxii

39 UV-Vis spectrum of PAMAM G4, PAMAM G4-porphyrin 154

Conjugate (54), 5-(carboxyphenyl)-10,15,20-tris(4-pyridyl)

porphyrin (53)

40 IR spectrum of 5-(carboxyphenyl)-10,15,20-tris(4-pyridyl) 155

porphyrin (53)

41 1H NMR of PAMAM G4-porphyrin conjugate (54) 156

42 13

C NMR of PAMAM G4-porphyrin conjugate (54) 157

43 IR spectrum of PAMAM G4-porphyrin conjugate (54) 158

44 1H NMR of PAMAM G4 159

45 1H NMR of 5,10,15,20-tetrakis(N-methyl-4-pyridyl)porphyrin (17) 160

46 1H-

1H

COSY NMR of 5,10,15,20-tetrakis(N-methyl-4-pyridyl) 161

porphyrin (17)

47 13

C NMR of 5,10,15,20-tetrakis(N-methyl-4-pyridyl)porphyrin (17) 162

48 UV-Vis spectrum of 5,10,15,20-tetrakis(N-methyl-4-pyridyl) 163

porphyrin (17)

49 IR spectrum of 5,10,15,20-tetrakis(N-methyl-4-pyridyl)porphyrin 164

(17)

50 1H NMR of 5,10-bis(N-methyl-4-pyridyl)-15,20-bis(4-pyridyl) 165

porphyrin (55a) or 5,15-bis(N-methyl-4-pyridyl)-10,20-bis

(4-pyridyl)porphyrin (55b)

51 UV-Vis spectrum of 5,10-bis(N-methyl-4-pyridyl)-15,20-bis(4- 166

pyridyl)porphyrin (55a) or 5,15-bis(N-methyl-4-pyridyl)-10,20-bis


52 IR spectrum of 5,10-bis(N-methyl-4-pyridyl)-15,20-bis(4-pyridyl) 167

porphyrin (55a) or 5,15-bis(N-methyl-4-pyridyl)-10,20-bis


53 1H NMR of 5,10,15-tris(N-methyl-4-pyridyl)-20-(4-pyridyl) 168

porphyrin (56)

54 UV-Vis spectrum of 5,10,15-tris(N-methyl-4-pyridyl)-20- 169


55 IR spectrum of 5,10,15-tris(N-methyl-4-pyridyl)-20-(4-pyridyl) 170

Porphyrin (56)

56 1H NMR of 5,15-dipropyl-10,20-bis(N-methyl-4-pyridyl) 171

porphyrin (57)

xxiii

57 1H-

1H COSY NMR of 5,15-dipropyl-10,20-bis(N-methyl-4- 172

pyridyl)porphyrin (57)

58 13

C NMR of 5,15-dipropyl-10,20-bis(N-methyl-4-pyridyl) 173

porphyrin (57)

59 UV-Vis spectrum of 5-propyl-10,15,20-tris(N-methyl-4-pyridyl) 174

porphyrin (59) , 5,15-dipropyl-10,20-bis(N-methyl-4-pyridyl)

porphyrin (57), 5,10-dipropyl-15,20-bis(N-methyl-4-pyridyl)

porphyrin (58)

60 IR spectrum of 5,15-dipropyl-10,20-bis(N-methyl-4-pyridyl) 175

porphyrin (57)

61 1H NMR of 5,10-dipropyl-15,20-bis(N-methyl-4-pyridyl) 176

porphyrin (58)

62 13

C NMR of 5,10-dipropyl-15,20-bis(N-methyl-4-pyridyl) 177

porphyrin (58)

63 1H NMR of 5-propyl-10,15,20-tris(N-methyl-4-pyridyl) 178

porphyrin(59)

64 IR spectrum of 5-propyl-10,15,20-tris(N-methyl-4-pyridyl) 179

porphyrin (59)

65 1H NMR of 5,10,15-trihexyl-20-(N-methyl-4-pyridyl)porphyrin 180

(60)

66 UV-Vis spectrum of 5,10,15-trihexyl-20-(N-methyl-4-pyridyl) 181

porphyrin (60)

67 IR spectrum of 5,10,15-trihexyl-20-(N-methyl-4-pyridyl) 182

porphyrin (60)

68 1H NMR of 5,15-dihexyl-10,20-bis(N-methyl-4-pyridyl) 183

porphyrin (61)

69 13

C NMR of 5,15-dihexyl-10,20-bis(N-methyl-4-pyridyl) 184

porphyrin (61)

70 UV-Vis spectrum of 5-hexyl-10,15,20-tris(N-methyl-4-pyridyl) 185

porphyrin (63), 5,10-dihexyl-15,20-bis(N-methyl-4-pyridyl)

porphyrin (62), 5,15-dihexyl-10,20-bis(N-methyl-4-pyridyl)

porphyrin (61)

71 IR spectrum of 5,15-dihexyl-10,20-bis(N-methyl-4-pyridyl) 186

porphyrin (61)

xxiv

72 1H NMR of 5,10-dihexyl-15,20-bis(N-methyl-4-pyridyl) 187

porphyrin (62)

73 13

C NMR of 5,10-dihexyl-15,20-bis(N-methyl-4-pyridyl) 188

porphyrin (62)

74 IR spectrum of 5,10-dihexyl-15,20-bis(N-methyl-4-pyridyl) 189

porphyrin (62)

75 1H NMR of 5-hexyl-10,15,20-tris(N-methyl-4-pyridyl) 190

porphyrin (63)

76 13

C NMR of 5-hexyl-10,15,20-tris(N-methyl-4-pyridyl) 191

porphyrin (63)

77 IR spectrum of 5-hexyl-10,15,20-tris(N-methyl-4-pyridyl) 192

porphyrin (63)

78 1H NMR of cationic porphyrin-PAMAM (G4) conjugate (64) 193

79 13

C NMR of cationic porphyrin-PAMAM (G4) conjugate (64) 194

80 UV-Vis spectrum of cationic porphyrin-PAMAM (G4) 195

conjugate (64)

81 IR spectrum of cationic porphyrin-PAMAM (G4) conjugate (64) 196

A Cytotoxicity assay 197

B ANOVA and Post Hoc Tests 203

CHAPTER 1

INTRODUCTION

1.1 Background of the Study

Porphyrins (1) are important cofactor that can be found in massive amounts

of natural products such as chlorophyll (2) and hemin (3) [1]. They are the central

regulatory effectors in many biochemical processes. Over- or underproduction of

porphyrins will result in significant health problems ranging from mental illnesses

like schizophrenia, leukemia to physical symptoms such as, port-coloured urine.

N

NH N

HN

N

N N

N

C20H39O2COH3CO2C

Mg

N

N N

N

Fe

HOOC COOH

(1) (2) (3)

Figure 1.1: Structure of porphyrin (1), chlorophyll (2) and hemin (3)

Porphyrins can be accessed through laboratory synthesis and they are

designed based on two porphyrins families; the β-substituted porphyrins that

resemble naturally occurring porphyrins and the meso-substituted porphyrins.

Porphyrins and their derivatives are usually synthesized based on condensation

reactions between pyrroles and aldehyde derivatives since it represents a facile and

straightforward synthetic method. Besides, different synthetic strategies also have

been employed to obtain various porphyrin macrocycles. For example, MacDonald

2

reaction (2+2 acid-catalysed condensation) [2], Vilsmeier reaction, nucleophilic

aromatic substitution reaction (SNAr reaction) [3], electrophilic substitution reaction

(SEAr reaction) [4] and transition-metal catalysed reactions [5].

Porphyrins which consist of large aromatic macrocyles possess important

chemical properties such as photochemical (energy and excitation transfer), redox

(electron transfer, catalysis) [6] and coordination properties (metal and axial ligand

binding) [7] which make these tetrapyrrole macrocyles play a crucial role in

disparate areas like photodynamic cancer therapy [8-9], artificial photosynthesis [10],

oxidation catalyst, sensors [11] and nanomaterial [12].

In addition, porphyrins and porphyrin-related macrocycles are found to have

ability to bind to lysosomes, mitochondria and plasma membrane [8]. Cationic

porphyrins have been reported for their ability to form strong electrostatic interaction

with DNA [13] and used as nucleic acid transporting agents. For example, Kralova

et al. reported that cationic porphyrin derivatives were efficient in transporting

antisense oligodeoxyribonucleotides (ODNs) to primary leukemia cells [14].

Therefore, the electronic interaction with nucleic acids [15] and fluorescence

properties has made porphyrins become promising delivery agents in gene therapy

[16].

Gene therapy has been developed extensively as it provides a unique

approach in treating both inherited and acquired disease. It involves the process of

transferring genetic materials (DNA or RNA) into human cells to replace, correct or

modify a mutated gene. To date, gene therapy has been shown as a successful tool to

cure diseases such as, cystic fibrosis [17-18], severe combined immune deficiency

(SCID) [19], haemophilia [20-21] and muscular dystrophies [22] as well as

Parkinson disease [23]. Gene transfection process requires to surpass several barriers,

starting from repulsion of negatively charged DNA by the negatively charged cell

membrane, enzyme degradation of the DNA trafficks into endosome and finally

internalization of the DNA through the nuclear pore into nucleus (Figure 1.2) [24].

Therefore, the gene delivery process has imposed formidable challenge to the

development of gene therapy [14, 16].

3

Figure 1.2 Gene therapy [24]

Retroviruses and adenoviruses have been used as viral vectors in gene

therapy that may present higher gene transfection efficiency compared to their non-

viral counterparts. However, viral vectors may trigger specific immune response and

under certain circumstance could result in death. Their clinical trials indicated

several concerns, such as mutagenesis [24], inflammatory properties [25] and high

cost [22]. On the other hand, cationic polymers such as, polyethylenimine (PEI),

poly-L-lysine, cationic dendrimers and hydrophobic polymers such as poly(lactide-

co-glycolide) (PLG) or polyanhydrides that form polyplexes with DNA are one of

the primary non-viral vectors systems that have been examined for the optimization

of gene delivery [26]. These cationic polymers give protection to DNA from

deoxyribonuclease (DNases) degradation as well as enhancing the intracellular

uptake [27]. However, the clinical trials of these non-viral delivery vectors are

limited by the issues of transfection efficiency, biocompatibility [28], toxicity [29],

low targeting specificity, cellular uptake and tend to aggregate in the blood [24].

These drawbacks have stimulated the search for non-viral gene carriers with

high biocompability and cellular uptake but give low aggregation and minimal safety

concern. In this study, various cationic porphyrin derivatives were synthesized and

their cytotoxicity and intracellular uptake were tested as an evaluation for their

potential uses as gene delivering agent.

Negatively charged

cell membrane

EXTRACELLULAR

Nucleus

DNA and gene carrier complexNegatively charged DNA

- -

Positively charged

gene carrier

Endocytosis

Endosomal

degradation

Endosomal releasing

Nuclear trafficking

INTRACELLULAR

4

1.2 Problem Statement

The success of gene therapy largely relies on the ability of gene delivery

vectors to deliver nucleic acid into the targeted cells with minimal toxicity [25, 30].

About 70% of current gene therapies are using the viral method as the main gene-

therapeutic protocols [27], but it is plagued by some serious health issues as well as

inflammatory response [25]. On the other hand, non-viral vectors present the issue

of toxicity [29], low biocompatibility [28], low cellular uptake as well as tend to

aggregate in blood [24].

Porphyrins are interesting alternative to viral vector-mediated gene delivery.

As they are essential compounds in most natural products, such as haemoglobin,

porphyrins probably have higher biocompatibility with human body cells and thereby,

avoiding immune response as induced by viral vectors. Besides, both hydrophilic

and hydrophobic substituents can be directly anchored on the porphyrins macrocycle

to increase their cell membrane penetration, which in turn, increase their cellular

uptake. Whereas, most non-viral vectors require the incorporation of natural lipid

molecules for exogenous DNA transportation into the cell through a biomimetic

mechanism [29]. As highly hydrophobic substituents may cause extensive self-

aggregation in aqueous solution [15], a shorter length of hydrocarbon such as,

butanal and heptanal were employed as hydrophobic substituents in this study.

Besides, porphyrin molecules can strongly fluorescent which favor the

location identification of vectors in the intracellular domain [16]. On the contrary, a

fluorescent probe is required to attach on most other non-viral vectors’ surfaces for

the delivery process tracking. The introduction of the fluorescent probe may

interrupt the physical or chemical properties of the gene vectors, giving an adverse

effect on the biocompatibility and transfection efficiency [28].

In addition, there are abundant of chemical substituents can be covalently

bonded to porphyrin macrocylces for particular nucleic acid or cell-type application

[31]. Polyamidoamine (PAMAM) dendrimers which have shown high level of

5

transfection through a “proton sponge” mechanism can be conjugated with porphyrin

to form a complex that may enhance transfection level with minimal toxicity effect.

In this study, various cationic porphyrin derivatives with different number,

location and distribution of lipophilic and hydrophilic ligands along the peripheral of

the macrocycle were synthesized using condensation method and low cost reagents.

To evaluate their potential as gene carriers with minimal toxicity effect, their

cyctotoxicity and cellular uptake studies were conducted.

1.3 Objectives of the Study

The objectives of this study are:

1. To prepare and characterize basic cationic porphyrins.

2. To synthesis and characterize amphiphilic cationic porphyrin derivatives.

3. To synthesis and characterize cationic PAMAM-porphyrin conjugate.

4. To evaluate the cytotoxicity and cellular uptake of the cationic porphyrins

as potential gene delivering reagents.

1.4 Scope of the Study

Condensation method was mainly employed in this study to synthesize

diverse type of porphyrins. All cationic porphyrins were prepared using both

methyl-p-toluenesulfonate and methyl iodide as the alkylation agents. Basic cationic

porphyrin anchored with four cationic groups was synthesized. Besides, amphiphilic

cationic porphyrins bearing different number of hydrophobic and hydrophilic

substituents along the peripheral were also prepared in order to facilitate the

penetration of gene carriers through the cell membrane. Polyamidoamine (PAMAM)

dendrimer was also conjugated to porphyrins to produce a complex with improved

cellular uptake and transfection level [32].

6

Cytotoxicity of the synthesized compounds towards the cells were conducted

using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.

The assay was based on cleavage of yellow tetrazolium salt MTT to purple formazan

by the metabolic active cells. In addition, cellular uptakes of the synthetic

porphyrins were investigated in Chinese hamster ovary cells (CHO) and the

qualitative results were obtained using inverted fluorescence microscope.

1.5 Significance of the Study

Porphyrins, as gene delivery agents may have a higher biocompability with

human body cells as they are essential compounds in haemoglobin, thereby avoiding

safety issue as triggered by viral vectors. Synthesis of amphiphilic cationic

porphyrins give a better cell membrane permeability [33], which in turn producing a

better cellular uptake. Self-aggregation is prevented when a shorter length of

hydrocarbons were utilized as hydrophobic groups. Cationic PAMAM-porphyrin

conjugate was also synthesized to enhance transfection level with minimal toxicity

effect. In addition, the fluorescence properties of porphyrins contribute to the use of

porphyrin as the marker candidate.

107

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