Total enzymatic synthesis of the cholecystokinin octapeptide (CCK_1tn2_1tn6_1tn-_1tn3_1tn3) [Elektronische Ressource] = Vollenzymatische Synthese des Cholecystokininoctapeptids (CCK_1tn2_1tn6_1tn-_1tn3_1tn3) / vorgelegt von Li-Ping Meng

Informations

• Publié par: eberhard_karls_universitat_tubingen
• Publié le: 01 janvier 2006
• Nombre de lectures: 6
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Total Enzymatic Synthesis of the Cholecystokinin
Octapeptide (CCK ) 26-33


Vollenzymatische Synthese
des Cholecystokininoctapeptids
(CCK ) 26-33


DISSERTATION

der Fakultät für Chemie und Pharmazie
der Eberhard-Karls-Universität Tübingen

zur Erlangung des Grades eines Doktors
der Naturwissenschaften


2006

vorgelegt von


Li-Ping Meng



























Tag der mündlichen Prüfung 10. Juli 2006

Dekan: Prof. Dr. Stefan Laufer
1. Berichterstatter: Prof. Dr. Heiner Eckstein
2. Berichterstatter: Prof. Dr. Günter Häfelinger
This scientific research work was carried out from April 2003 to April 2006 at the
Institute of Organic Chemistry, Faculty of Chemistry and Pharmacy,
Eberhard-Karls-Universität, Tuebingen, Germany, under the guidance of Prof. Dr.
Heiner Eckstein.

I would like to express deep and sincere gratitude to my supervisor Prof. Dr. Heiner
Eckstein for providing the opportunity to study in the perfect working conditions.
Heartfelt appreciation is also expressed for his kind assistance, fruitful suggestions,
excellent instructions and limitless patience throughout the entire research project of
my doctoral dissertation.

I am especially grateful to Prof. Dr. Guenter Haefelinger for his precious time to read
and appreciate my written thesis.

Special thanks go to Mr. H. Bartholomä and Mr. R. Müller for the
FABMS-measurements of samples and to Mr. Wu Su (Max Planck Institute for
Biological Cybernetics, Tuebingen, Germany) for the operation of LC-ESIMS. I also
thank Mrs. Margit Wizemann for her assistance during my laboratory work and to
Mr. Rajendra Joshi for helpful discussion and advices.

My acknowledgements are due to the Pharmacy school, Tongji medical college of
Huazhong university of science and technology, China for supporting my study.

Above all, I am indebted to my parents and family members for their permanent love,
constant care, unconditional support and encouragement during the whole tenure of
my research.











To my family
for all of the love


















Contents I
Contents
1 Introduction...............................................................................................................1
1.1 Chemical peptide synthesis...............................................................................3
1.1.1 Principles.................................................................................................3
1.1.2 Classical peptide synthesis......................................................................7
1.1.3 Solid phase peptide synthesis..................................................................7
1.2 Enzymatic peptide synthesis.............................................................................9
1.2.1 Enzymes..................................................................................................9
1.2.2 Methods of enzyme immobilization .....................................................12
1.2.3 Advantages of enzymatic peptide synthesis .........................................14
1.2.4 Methods of enzymatic peptide synthesis ..............................................15
1.2.4.1 Thermodynamically controlled peptide synthesis ......................15
1.2.4.2 Kinetically controlled peptide synthesis .....................................18
2 State of the art and aim of this research ...............................................................20
3 Results and discussion ............................................................................................29
3.1 Enzyme immobilization..................................................................................29
3.1.1 Immobilization of the enzymes.............................................................29
3.1.2 Activity test of the immobilized enzymes ............................................32
3.2 Enzymatic synthesis of the N-terminal tripeptide
(Phac-Asp(OMe)-Tyr-Met-OAl) ..........................................................................38
3.2.1 The first synthetic strategy....................................................................39
3.2.1.1 Enzymatic synthesis of Phac-Asp(OMe)-Tyr-OAl.....................40
3.2.1.2 Enzymatic synthesis of Phac-Asp(OMe)-Tyr-Met-OAl.............41
3.2.2 The second synthetic strategy ...............................................................42
3.2.2.1 Enzymatic synthesis of Phac-Asp(OMe)-Tyr-OH......................43
3.2.2.2 Enzymatic synthesis of Phac-Asp(OMe)-Tyr-Met-OAl.............43
3.2.3 Influences on the enzymatic syntheses .................................................43
3.3 Chemical synthesis of reference substances ...................................................48
3.3.1 Chemical synthesis of Phac-peptides....................................................48 II Contents
3.3.2 Chemical synthesis of the C-terminal pentapeptides ............................49
3.3.2.1 Chemical synthesis of Fmoc-amino acides pentafluorophenyl
esters .......................................................................................................50
3.3.2.2 Chemical synthesis of Fmoc-Gly-Trp-Met-Asp(OMe)-Phe-NH 50 2
3.3.2.3 Deprotection of Fmoc-Gly-Trp-Met-Asp(OMe)-Phe-NH .........50 2
3.4 Enzymatic synthesis of the C-terminal pentapeptides ....................................51
3.4.1 Enzymatic synthesis of Phac-Gly-Trp-Met-Asp(OMe)-OH.................53
3.4.2 Enzymatic synthesis of Phac-Gly-Trp-Met-Asp(OMe)-Phe-NH ........57 2
3.4.3 Deprotection of the Phac-pentapeptide.................................................59
3.4.4 Influences on the enzymatic syntheses .................................................61
3.5 Synthesis of the octapeptide
(Phac-Asp(OMe)-Tyr-Met-Gly-Trp-Met-Asp(OMe)-Phe-NH )..........................73 2
4 Experimental section ..............................................................................................80
4.1 Apparatus ........................................................................................................80
4.2 Materials .........................................................................................................81
4.3 Methods of chromatography...........................................................................83
4.4 Characterization of the products .....................................................................85
4.5 Enzyme immobilization..................................................................................86
4.6 Activity test of immobilized enzymes ............................................................89
4.7 Preparation of amino acid derivatives.............................................................91
4.8 Peptide synthesis.............................................................................................96
4.8.1 Chemical synthesis of reference peptides .............................................96
4.8.1.1 Chemical synthesis of Phac-peptides..........................................96
4.8.1.2 Chemical synthesis of the C-terminal pentapeptides ..................98
4.8.2 Enzymatic synthesis of peptides .........................................................100
5 Summary................................................................................................................118
6 References..............................................................................................................121
7 Appendix................................................................................................................127 Abbreviations III
Abbreviations
Ac Acetyl
ACN Acetonitrile
AcONH Ammonium acetate 4
Boc t-Butyloxycarbonyl
Bz Benzoyl
Bzl Benzyl
C18 Octadecyl
Cam Carboxamidomethyl
Cbo or Z Benzyloxycarbonyl
CCK Cholecystokinin
α-CHY α-Chymotrypsin
DCC Dicyclohexylcarbodiimide
DCU Dicyclohexylurea
DCM Dichloromethane
DMF Dimethylformamide
DNA Deoxyribonucleic acid
EDTA Ethylenediaminetetraacetic acid
EtOAc Ethyl acetate
EtOH Ethanol
ESI Electron spraying ionization
FAB Fast atom bombardment
Fmoc 9-Fluorenylmethyloxycarbonyl
h hour
HPLC High pressure liquid chromatography
HOBt N-hydroxybenzotriazole
Me Methyl
MeOH Methanol IV Abbreviations
m.p. melting point
min minute
MS Mass spectrometry
OAl Allyl ester
OEt Ethyl ester
OMe Methyl ester
OPfP Pentaflurophenyl ester
PA Penicillin amidase
PE Petroleum ether
PGA Penicillin G amidase
Ph Phenyl
Phac Phenyl acetyl
PIM Positive ion mode
R Retention factor f
RP Reversed phas