Development of a new cell penetrating peptide [Elektronische Ressource] : design, synthesis and applications = Entwicklung eines neuen zell-penetrierenden Peptids / vorgelegt von Deepti Jha

Djha

Sujets

• Biologie

Informations

• Publié par: eberhard_karls_universitat_tubingen
• Publié le: 01 janvier 2009
• Nombre de lectures: 24
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Development of a New Cell Penetrating Peptide: Design,
Synthesis and Applications
 
Entwicklung eines neuen zell-penetrierenden Peptids:
Design, Synthese und Anwendungen
 
 
DISSERTATION
 
der Fakultät für Chemie und Pharmazie 
der Eberhard Karls Universität Tübingen 
 
 
zur Erlangung des Grades eines Doktors 
der Naturwissenschaften 
 
2009 
 
vorgelegt von 
Deepti Jha
 
  



















Tag der mündlichen Prüfung: 14.09.09


Dekan: Prof. Dr.Lars Wesemann


1. Berichterstatter Prof. Dr. Martin E. Maier

2. Berichterstatter Dr. Karl-Heinz Wiesmüller

II 
 Acknowledgements
This scientific work was carried out from January 2005 to July 2009 at the Max Planck Institute
for Biological Cybernetics (Chemical Biology Laboratory, High Field Magnetic Resonance
Center) under the supervision of Prof. Dr. Kamil Ugurbil and Dr. Jörn Engelmann in
collaboration with the Institute of Organic Chemistry, Eberhard Karls University, Tübingen, and
EMC microcollections GmbH, Tübingen, supervised by Prof. Dr. Martin E. Maier and Prof. Dr.
Karl-Heinz Wiesmüller.
I thank Prof. Dr. Kamil Ugurbil, Max Planck Institute for Biological Cybernetics, High Field
Magnetic Resonance Center, Tuebingen, for providing me the opportunity, the facilities, and the
financial support to complete this work. I am thankful to Prof. Dr. Martin E. Maier for his
guidance and support. My heartfelt thanks go to Prof. Dr. Karl-Heinz Wiesmueller for his
continuous motivation, excellent guidance and constructive feedbacks.
I am grateful to Dr. Josef Pfeuffer and Dr. Anil Mishra for believing in me and supporting me in
the initial stages of my PhD work.
I would like to express my sincere thanks to Dr. Jörn Engelmann for the helpful advice and
supervision. Special appreciation is expressed for the continuous encouragement, generous
discussions and the critical evaluation of the thesis.
I wish to express my gratitude to Dr. Wu Su for sharing his knowledge and experience with me.
Special thanks go to the Cell Biology group: Dr. Jörn Engelmann, Ms. Ritu Mishra and Ms.
Hildegard Schulz for performing all the biological studies, without them this work would not
have been a reality.
I also thank all the group members: Ms. Aneta Brud, Dr. Anurag Mishra, Dr. Goran Angelovski, 
Ms. Hildegard Schulz, Dr. Ilgar Mamedov, Dr. Jörn Engelmann, Dr. Rajendra Joshi, Ms. Ritu
Mishra, Dr. Sven Gottschalk, and Dr. Xiaozhe Zhang for their helpful discussions and
encouragement throughout my PhD tenure. Special thanks go to Ms. Tina Schröder and Mihai
Vintiloiu for their help.
III 
 I am thankful to Dr. Sven Gottschalk and Dr. Rajendra Joshi for their comments and suggestions
on the thesis.
Thanks to Dipl.-Ing. Michael Beyerlein and Dr. Rolf Pohmann for performing MR experiments,
Dr. Murray Coles for performing NMR experiments and CAT GmbH for determining the optical
purity of the peptide.
Special thanks go to good friends of mine: Anurag Mishra, Ritu Mishra, Arshiya Mishra, Jasmin
Joseph, Suryadeep Dash, Vikram Alva, Kirti Dhingra, Aneta Brud, Deepak Suri, and all well
wishers for their help and support throughout my term in Tuebingen.
I would like to express my sincere thanks to my parents for their affection and encouragement. I
would also like to thank Prof. Dr. Sanjay Tignath, Nitin Jha, Anshuman Jha, Dr. Maneesha
Saxeena, Saurabh Saxeena, Vandana Tignath, Dr. Medha Jha, Sumedha Jha, Narayani Tignath,
Ishan Tignath, and Jahanvi Saxeena for their support.













IV 
 Abbreviations
A Adenine
A (Ala) Alanine
Bhoc Benzhydryloxycarbonyl
Boc Butoxycarbonyl
C Cytosine
C (Cys) Cysteine
CA Contrast agent
CF Carboxyfluorescein
CPP Cell Penetrating Peptide
CyLoP-1 Cytosol Localizing Peptide-1
D (Asp) Aspartic acid
D O Deuterium oxide 2
DCM Dichloromethane
DEE Diethyl ether
DIC N,N'-diisopropylcarbodiimide
DIPEA N,N’-diisopropylethylamine
DMF Dimethylformamide
DMSO Dimethylsulfoxide
DOTA (tris-t-Bu-ester) 1,4,7,10-tetraazacyclododecane-1,4,7-tris(acetic acid-tert-butyl
ester)-10-acetic acid
V 
 DTPA Diethylenetriaminepentaacetic acid
ESI-MS Electrospray Ionization Mass Spectrometry
F(Phe) Phenylalanine
FITC Fluorescein isothiocyanate
GdCl .6H O Gadolinium trichloride hexahydrate 3 2
MeOH Methanol
Fmoc 9-fluorenylmethoxycarbonyl
G Guanine
HATU 2-(1-H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate
HOBt 1-hydroxybenzotriazole
I (Ile) Isoleucine
K (Lys) Lysine
M (Met) Methionine
MR Magnetic resonance
MRI Magnetic resonance imaging
MTBE Methyl tert-butyl ether
N (Asn) Asparagine
P (Pro) Proline
Pbf 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl
PNA Peptide nucleic acid
VI 
 Q (Gln) Glutamine
R (Arg) Arginine
S (Ser) Serine
TCP Tritylchloride polystyrene resin
TCTU 1-H-benzotriazolium-1[bis(dimethylamino)methylene]-5-chloro-
tetrafluoroborate(1-),3-oxide
TFA Trifluoroacetic acid
Trt Trityl
V (Val) Valine
W (Trp) Tryptophan













VII 
 
























VIII 
 Table of contents

PART 1. INTRODUCTION 1
1.1 Intracellular delivery- Cell Penetrating Peptides 3
1.2 Mechanism of internalization 5
1.3 Applications 7
1.4 Limitation and solutions 8
1.5 Crotamine 10
1.5.1 Structure 11
1.5.2 Functions 11
1.5.3 Biological Activity 12
1.6 Aim of the study 21

PART 2. IDENTIFICATION AND OPTIMIZATION OF A NEW CYSTEINE RICH
CELL PENETRATING PEPTIDE
2.1 Result 19
2.1.1 General synthesis 19
2.1.2 Resin selection (C-terminal functional group) 20
2.1.3 Racemization 20
2.1.4 Optimization of cleavage cocktail 21
2.1.5 Ether selection 22
2.1.6 Counter ion purity- TFA and acetate content 22
2.1.7 Uptake studies 23
2.1.8 Structure Activity Relationship Studies 23
2.1.8.1 Substitution of cysteine residues 24
2.1.8.2 Deletion of cysteine residues 25
2.1.8.3 Substitution of tryptophan by proline residues 26
IX 
 2.1.8.4 Substitution of tryptophan by proline residues
and cysteine residues by Abu 27
2.1.8.5 Deletions and substitution of cysteine residues 27
2.1.8.6 Deletions 28
2.1.9 Distinct features of CyLoP-1 29
2.1.10 Optimization of CyLoP-1 31
2.1.10.1 Cysteine substitution by serine residue in CyLoP-1 32
2.1.10.2 Tryptophan substitution 32
2.1.10.3 Stereochemical effects 33
2.2 Discussion 37
2.3 Oxidation studies 40
2.3.1 Results 40
2.3.1.1 Synthesis 40
2.3.2 Internalization studies 45
2.3.3 Discussion 46
2.4 Effect of fluorophores on internalization 47
2.4.1 Results 47
2.4.1.1 Fluorophore at N or C terminus 47
2.4.1.2 Position of fluorophore (amino group of N-terminal lysine) 48
2.4.1.3 Nature of fluorophore 49
2.4.1.4 Dual labeling 50
2.4.2 Discussion 50
2.5 Comparison of uptake efficiency of CyLoP-1 with other CPPs 51
2.6 Summary 53

PART 3. APPLICATION OF CyLoP-1 AS A DELIVERY TOOL
3.1 Efficient intracellular delivery of a MR imaging probe by CyLoP-1
X