Chemical BioIogy – Peptides
O H N OCH 2 N 3 H O HO2C
Prof. Helma Wennemers
Prof. H. Wennemers 1 Oxytocin Enkephalines OH CH3 O O CH3 H H O +H N N N CO - 3 N N 2 O H H N O O O H O NH HN NH2 HO
H N O 2 NH O S O S NH NH 2 H2N O Dendrotoxin O O N O HN NH
Aspartam
NH2
O O OH H N OCH 2 N 3 Lisinopril N N H H O O CO2H HO2C
Prof. H. Wennemers 2 The 20 most common proteinogenic amino acids
OH
CH3
H N CO H H N CO H H N CO H H N CO H H2N CO2H H2N CO2H H2N CO2H 2 2 2 2 2 2 2 2 Glycin Alanin Valin Isoleucin Leucin Phenylalanin Tyrosin (Gly, G) (Ala, A) (Val, V) (Ile, I) (Leu, L) (Phe, F) (Tyr, Y)
H H N NH N NH 2 NH3 CO2 NH CO N 2 2
H2N CO2H H2N CO2H H2N CO2H H2N CO2H H2N CO2H H2N CO2H Tryptophan Histidin Lysin Arginin Aspartat Glutamat (Trp, W) (His, H) (Lys, K) (Arg, R) (Asp, D) (Glu, E)
CONH2 S OH OH CONH2 SH
N CO2H H H2N CO2H H2N CO2H H2N CO2H H2N CO2H H2N CO2H H2N CO2H Serin Threonin Asparagin Glutamin Cystein Methionin Prolin (Ser, S) (Thr, T) (Asn, N) (Gln, Q) (Cys, C) (Met, M) (Pro, P) Large Diversity Prof. H. Wennemers 3 Diversity of Peptides – A Number Game
Random combinations of 20 amino acids
3-mer: 203 = 8000 different peptides
5-mer: 205 = 3.2x106 different peptides
10-mer: 2010 = 1.0x1013 different peptides
50-mer: 2050 = 1.1x1065 different peptides
Weight of 1.1x1065 single 50-mer peptide molecules: 1.3x1039 kg
Weight of the earth: 5.9x1024 kg
Prof. H. Wennemers 4 Syllabus
1. Occurrence and Functions of Peptides in Nature and Every Day Life …hormones, neurotransmitters, therapeutics, artificial sweetener, …
2. Peptide Synthesis a) Aspartam: Properties of amino acids; nomenclature; solution phase synthesis b) Glucagon: Solid phase peptide synthesis c) Fuzeon, an anti HIV drug: Solution and solid phase peptide synthesis
3. Peptide Structures – Nanostructured Materials – Self-assembly a) Fuzeon: α-helices and coiled-coil structures b) Amyloids: β-sheets c) Collagen: PPII helices
4. Applications of Peptides in Chemistry, Biology and Material Sciences a) Peptide-based materials b) Therapeutically active peptides b) Cyclic peptides and cancer imaging c) Asymmetric Catalysis with Peptides – Combinatorial chemistry
Prof. H. Wennemers 5 Syllabus
1. Occurrence and Functions of Peptides in Nature and Every Day Life …hormones, neurotransmitters, therapeutics, artificial sweetener, …
2. Peptide Synthesis a) Aspartam: Properties of amino acids; nomenclature; solution phase synthesis b) Glucagon: Solid phase peptide synthesis c) Fuzeon, an anti HIV drug: Solution and solid phase peptide synthesis
3. Peptide Structures – Nanostructured Materials – Self-assembly a) Fuzeon: α-helices and coiled-coil structures b) Amyloids: β-sheets c) Collagen: PPII helices
4. Applications of Peptides in Chemistry, Biology and Material Sciences a) Peptide-based materials b) Therapeutically active peptides b) Cyclic peptides and cancer imaging c) Asymmetric Catalysis with Peptides – Combinatorial chemistry
Prof. H. Wennemers 6 Die Entdeckung von Aspartam - Serendipity in Science Gastrin =
H-Glp-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2
Hormon, das im Magen durch G-Zellen freigesetzt wird und die Bildung von Magensäure anregt.
• für physiologische Aktivität nur die vier C-terminalen Aminosäuren notwendig
H-Trp-Met-Asp-Phe-NH2
1965 - James M. Schlatter (G. D. Searle & Company, heute: Monsanto) sucht nach einem Wirkstoff gegen Magengeschwüre und stellt
H-Asp-Phe-OCH3 her Prof. H. Wennemers ....und tut etwas, was man nicht tun sollte...... 7 Solution Phase: Calcitonin (salmon)
CSNLSTCVLGKLSQELHKLQTYPRTNTGSGTP-NH2
1 - 6 7 - 10 11 - 16 17 - 23 24 - 32
BACHEM Holding AG │ Company Presentation slide by Dr. ThomasSeite Früh, 8 │ September CEO 2012 Bachem Calcitonin
„Adversary“ of calcitonin:
Parathyroid Hormon (PTH)
From: N. Sewald, H.-D. Jakubke, “Peptides: Chemistry and Biology”, 2nd Ed., Wiley-VCH, Weinheim, 2009 9 Syllabus
1. Occurrence and Functions of Peptides in Nature and Every Day Life …hormones, neurotransmitters, therapeutics, artificial sweetener, …
2. Peptide Synthesis a) Aspartam: Properties of amino acids; nomenclature; solution phase synthesis b) Glucagon: Solid phase peptide synthesis c) Fuzeon, an anti HIV drug: Solution and solid phase peptide synthesis
3. Peptide Structures – Nanostructured Materials – Self-assembly a) Fuzeon: α-helices and coiled-coil structures b) Amyloids: β-sheets c) Collagen: PPII helices
4. Applications of Peptides in Chemistry, Biology and Material Sciences a) Peptide-based materials b) Therapeutically active peptides b) Cyclic peptides and cancer imaging c) Asymmetric Catalysis with Peptides – Combinatorial chemistry
Prof. H. Wennemers 10 Solid Phase Peptide Synthesis (SPPS) (Nobelprize for Chemistry 1984: Bruce Merrifield) Peptidsynthesizer 1966
Prof. H. Wennemers 11 Solid Phase Peptide Synthesis (SPPS) – Fmoc/tBu Strategie in the example: Wang Linker
R1 1 Cl R FmocHN CO2 Cs O O FmocHN Linker Wang Linker DMF O
R2 R1 3 equiv. Piperidine:DMF 1:4 O FmocHN CO H Linker 2 (1x 3 min, 1x 7 min) H2N O 3 eq. PyBOP, 3 eq. iPr2NEt
O R1 repeat Fmoc deprotection/AA couplings FmocHN O N Linker to desired peptide length H R2 O
n 1 Rn´ O R1´ R O R H H 95% TFA N O N OH H3N N H2N N Linker (scavengers) H 2´ H O R2 O O R O side-chain deprotected peptide Prof. H. Wennemers 12 Kupplungsreagenzien Carbodiimide R R = R´ = cyclohexyl: N,N´-Di-Cyclohexyl Carbodiimid (DCC) N C N R = R´ = iso-propyl: N,N´-Di-Isopropyl Carbodiimid (DIC)
R R = Et, R´= CH2CH2CH2NMe2 : N-Ethyl-N´-(3-Dimethylaminopropyl) Carbodiimid (EDC)
Phosphonium Reagenzien Guanidinium/Uronium Reagenzien
N N N N NMe2 N PF6 PF6 O P NMe O P N N N 2 PF PF N 6 N 6 N NMe2 N N N N N N N N N O O BOP PyBOP HBTU HATU (Castro`s Reagent) N N N N O N BF N P N 4 O OEt OEt DEPBT N TBTU O O Prof. H. Wennemers 13 Commonly used acid sensitive protecting groups for the functional groups of the amino acid side chains
Fmoc: α-NH group
Boc: Lysine, Tryptophan, Histidine
Pbf: Arginine
Trt: Cysteine, Aspartic acid, Glutamic acid
Acm: Cysteine tBu: Serine, Threonine, Hydroxyproline, Tyrosine
Prof. H. Wennemers 14 Linker für die Festphasensynthese
O O OMe Cl R O R O R O O O O Ph Wang SASRIN® Chlorotrityl R
MeO HN O O R O N H O MeO O Rink Amide Hycram
O NO2 O O R O O O H S N R N N O H H O o-Nitrobenzyl Kenner Safety Catch Prof. H. Wennemers 15 Peptidsynthese an fester Phase (Nobelpreis für Chemie 1984 für Bruce Merrifield) Peptidsynthesizer 1966 heute
Prof. H. Wennemers 16 Solid Phase: Glucagon (1-29, human)
HSQGTFTSDYSKYLDSRRAQDFVQWLMNT
BACHEM Holding AG │ Company Presentation slide by Dr. ThomasSeite Früh, 17 │ September CEO 2012 Bachem Insulin and Glucagon
Glucagon (Secre n Family)
Insulin (Insulin Superfamily) inac ve hexameric form – bound to Zn2+:
BACHEM Holding AG │ Company Presentation Seite 18 │ September 2012 18 Comparison: Solution vs. Solid Phase
Calcitonin Glucagon
- Produced since 1986 1996
- Synthetic steps 104 56
- Purification CCD two-dim. HPLC three-dim. HPLC
- Derivatives needed 158 kg 29 kg for the production of 1kg
- Time for completion 2.5 years 0.6 years
BACHEM Holding AG │ Company Presentation Seite 19 │ September 2012 Parathyroid Hormone (PTH)
PTH fragment (46-84): AGSQRPRKKEDNVLVESHEKSLGEADKADVNVLTKAKSQ-NH Human Parathyroid Hormone (1-84) 2
addition of 5% formic acid to the piperidine
T. Michels, R. Dölling, U. Haberkorn, W. Mier, Org. Le ., 2012 , 14, 5218-5221. 20 R. Balling, R. G. Erben, Nature Medicine, 2000, 6, 860-861. Aspartimid Formation
O OtBu O O R1 H N R N N H H O R2 O O O N OH 1 Acid or Base O O R O O R1 H (epimerisation) H N N R N N R N N H H H H O R2 O O R2 O 1 α-Peptide R O O Piperidide of α-Peptide NH O 20% Piperidine/DMF Hydrolysis O N (epimerisation) (epimerisation) R R N 2 H O O R1 H H O R1 R N N H H N R N N H N O O R2 O H O O O R2 O N O OH
D. Housset, C. Habersetzer-Rochat, J.-P. As er, J. C. Fontecilla-Camps, J. Mol. Biol., 1994, 238, 88–103. M. Mergler, F. Dick, B. Sax, P. Weiler, T. Vorherr, J. Pep de Sci. 2003, 9, 36–46. T. Michels, R. Dölling, U. Haberkorn, W. Mier, Org. Le ., 2012 , 14, 5218-5221. 21
Side-reactions of Met
Aqueous Solution Structure of the Alzheimer's Disease Aβ Peptide 3D Structure of Alzheimer's Aβ(1-42) fibrils
Y. S. Kim, J. A. Moss, K. D. Janda, J. Org. Chem. 2004, 69, 7776-7778. D. Picone et al, ChemBioChem 2006, 7, 257 – 267. 22 T. Lührs, C. Ritter, M. Adrian, D. Riek-Loher, B. Bohrmann, H. Döbeli, D. Schubert, R. Riek, PNAS 2005, 102, 48, 17342–17347.