synthesis and bioassay studies of cationic porphyrins...
TRANSCRIPT
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.
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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
(4-pyridyl)porphyrin (43)
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)
3.5.2 Synthesis of 5,10-dipropyl-15,20-bis 64
(N-methyl-4-pyridyl)porphyrin (58)
3.5.3 Synthesis of 5-propyl-10,15,20-tris 65
(N-methyl-4-pyridyl)porphyrin (59)
3.5.4 Synthesis of 5,10,15-trihexyl-20-(N-methyl 65
-4-pyridyl)porphyrin (60)
3.5.5 Synthesis of 5,15-dihexyl-10,20-bis 66
(N-methyl-4-pyridyl)porphyrin (61)
3.5.6 Synthesis of 5,10-dihexyl-15,20-bis 67
(N-methyl-4-pyridyl)porphyrin (62)
3.5.7 Synthesis of 5-hexyl-10,15,20-tris 68
(N-methyl-4-pyridyl)porphyrin (63)
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
4.4.1 Synthesis of 5-propyl-10,15,20-tris 83
porphyrin (39)
x
4.4.2 Synthesis of 5-hexyl-10,15,20-tris 85
(4-pyridyl)porphyrin (43)
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-pyridyl)porphyrin (17)
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
4.7.1 Synthesis of 5,15-dipropyl-10,20-bis 94
(N-methyl-4-pyridyl)porphyrin (57)
4.7.2 Synthesis of 5,10-dipropyl-15,20-bis 94
(N-methyl-4-pyridyl)porphyrin (58)
4.7.3 Synthesis of 5-propyl-10,15,20-tris 95
(N-methyl-4-pyridyl)porphyrin (59)
4.7.4 Synthesis of 5,10,15-trihexyl-20- 96
(N-methyl-4-pyridyl)porphyrin (60)
4.7.5 Synthesis of 5,15-dihexyl-10,20-bis 96
(N-methyl-4-pyridyl)porphyrin (61)
4.7.6 Synthesis of 5,10-dihexyl-15,20-bis 97
(N-methyl-4-pyridyl)porphyrin (62)
4.7.7 Synthesis of 5-hexyl-10,15,20-tris 98
(N-methyl-4-pyridyl)porphyrin (63)
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
(4-pyridyl)porphyrin (48)
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
(4-pyridyl)porphyrin (51)
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
(4-pyridyl)porphyrin (52)
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
(4-pyridyl)porphyrin (55b)
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
(4-pyridyl)porphyrin (55b)
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
(4-pyridyl)porphyrin (56)
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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