chemical bioiogy – peptides · occurrence and functions of peptides in nature and every day life...
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Chemical BioIogy – Peptides
Prof. Helma Wennemers
H2N NH
OOCH3
OHO2C
1 Prof. H. Wennemers
Oxytocin
NH
OO
HN
CH3CH3
NHO
H2NS S
NH2
O
NH
NH
NO
OO
NH2
O
NH
OHN
H2NO
O
OH
Dendrotoxin
NH
HN
NH
HN CO2
-+H3N
HO
O
O
O
O
Enkephalines
NH
N
O
O OH
NH2
CO2H
Lisinopril H2N NH
OOCH3
OHO2C
Aspartam
2
Prof. H. Wennemers
The 20 most common proteinogenic amino acids
H2N CO2H H2N CO2H
CH3
H2N CO2H H2N CO2H
H2N CO2H
NH3
H2N CO2H
HN
NH2
NH2
H2N CO2H
N
NH
NH
CO2HH2N CO2H
OH
H2N CO2H
OH
H2N CO2H
CO2
H2N CO2H
CO2
H2N CO2H
CONH2
H2N CO2H
CONH2
H2N CO2H
SH
H2N CO2H
HN
H2N CO2H
OH
Large Diversity
Glycin (Gly, G)
Alanin (Ala, A)
Valin (Val, V)
Isoleucin (Ile, I)
Leucin (Leu, L)
Phenylalanin (Phe, F)
Tyrosin (Tyr, Y)
Tryptophan (Trp, W)
Histidin (His, H)
Lysin (Lys, K)
Arginin (Arg, R)
Aspartat (Asp, D)
Glutamat (Glu, E)
Serin (Ser, S)
Threonin (Thr, T)
Asparagin (Asn, N)
Glutamin (Gln, Q)
Cystein (Cys, C)
Methionin (Met, M)
Prolin (Pro, P)
H2N CO2H
S
H2N CO2H H2N CO2H
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
4 Prof. H. Wennemers
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
Syllabus
5 Prof. H. Wennemers
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
Syllabus
6 Prof. H. Wennemers
Gastrin = H-Glp-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2
Die Entdeckung von Aspartam - Serendipity in Science
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 ....und tut etwas, was man nicht tun sollte...... 7 Prof. H. Wennemers
BACHEM Holding AG │ Company Presentation Seite 8 │ September 2012
Solution Phase: Calcitonin (salmon)
CSNLSTCVLGKLSQELHKLQTYPRTNTGSGTP-NH2
1 - 6 7 - 10 11 - 16 17 - 23 24 - 32
slide by Dr. Thomas Früh, CEO Bachem
Calcitonin
From: N. Sewald, H.-D. Jakubke, “Peptides: Chemistry and Biology”, 2nd Ed., Wiley-VCH, Weinheim, 2009 9
„Adversary“ of calcitonin: Parathyroid Hormon (PTH)
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
Syllabus
10 Prof. H. Wennemers
Solid Phase Peptide Synthesis (SPPS) (Nobelprize for Chemistry 1984: Bruce Merrifield)
Peptidsynthesizer 1966
11 Prof. H. Wennemers
Solid Phase Peptide Synthesis (SPPS) – Fmoc/tBu Strategie in the example: Wang Linker
O
ClR1
CO2FmocHN Cs LinkerOFmocHNO
R1
LinkerOH2NO
R1
LinkerONH O
R1OFmocHN
R2
DMF
Piperidine:DMF 1:4 (1x 3 min, 1x 7 min)
R2
CO2HFmocHN3 equiv.
3 eq. PyBOP, 3 eq. iPr2NEt
repeat Fmoc deprotection/AA couplings to desired peptide length
LinkerONH O
R1OHN
R2OH2N
RnOHN
H O
R1´OHN
R2´OH3N
Rn´95% TFA
(scavengers)
side-chain deprotected peptide
Wang Linker
12 Prof. H. Wennemers
Kupplungsreagenzien
N C NR
R
Carbodiimide R = R´ = cyclohexyl: N,N´-Di-Cyclohexyl Carbodiimid (DCC) R = R´ = iso-propyl: N,N´-Di-Isopropyl Carbodiimid (DIC) R = Et, R´= CH2CH2CH2NMe2 : N-Ethyl-N´-(3-Dimethylaminopropyl) Carbodiimid (EDC)
Phosphonium Reagenzien Guanidinium/Uronium Reagenzien
NN
NO P
N
NN
PF6
NN
NO P
NMe2
NMe2NMe2
PF6
NN
N
OO
P OEt
O
OEt
NN
N PF6
NN
ON N
NN PF6
NN
O
NN
N BF4
NN
O
HBTU HATU
TBTU
BOP (Castro`s Reagent)
PyBOP
DEPBT
13 Prof. H. Wennemers
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
OR
O
O
OR
O OMe
O
Ph
R
O
Cl
O
HN
R
OMeO
MeO
NH
OOR
O
NO2
O
O
R
O
HN
O
S NH
O
NH
R
O OO
Wang SASRIN® Chlorotrityl
Rink Amide Hycram
o-Nitrobenzyl Kenner Safety Catch
15 Prof. H. Wennemers
Peptidsynthese an fester Phase (Nobelpreis für Chemie 1984 für Bruce Merrifield)
Peptidsynthesizer 1966 heute
16 Prof. H. Wennemers
BACHEM Holding AG │ Company Presentation Seite 17 │ September 2012
Solid Phase: Glucagon (1-29, human)
HSQGTFTSDYSKYLDSRRAQDFVQWLMNT
slide by Dr. Thomas Früh, CEO Bachem
BACHEM Holding AG │ Company Presentation Seite 18 │ September 2012
Insulin (Insulin Superfamily) inac+ve hexameric form – bound to Zn2+:
Insulin and Glucagon
Glucagon (Secre3n Family)
18
BACHEM Holding AG │ Company Presentation Seite 19 │ September 2012
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
Human Parathyroid Hormone (1-84)
Parathyroid Hormone (PTH)
T. Michels, R. Dölling, U. Haberkorn, W. Mier, Org. Le(., 2012 , 14, 5218-‐5221. R. Balling, R. G. Erben, Nature Medicine, 2000, 6, 860-‐861.
PTH fragment (46-‐84): AGSQRPRKKEDNVLVESHEKSLGEADKADVNVLTKAKSQ-‐NH2
addition of 5% formic acid to the piperidine
20
Aspartimid Formation
D. Housset, C. Habersetzer-‐Rochat, J.-‐P. As3er, 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.
R NH
HN
NH
O
O
O
O
R1
R2
OtBu
O
R NH
NNH
O
O
OO
R1
R2
O
R NH
HN
NH
O
O
O
O
R1
R2
OH
O
R NH
HN
NH
O
O
O
O
R1
R2
NO
HN
NHO
O
O
R1
R2
HN
O
R
OHO
HN
NHO
O
O
R1
R2
HN
O
R
NO
Acid or Base(epimerisation)
Hydrolysis(epimerisation)
20% Piperidine/DMF(epimerisation)
α-PeptidePiperidide of α-Peptide
21
Side-reactions of Met
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.T. Lührs, C. Ritter, M. Adrian, D. Riek-Loher, B. Bohrmann, H. Döbeli, D. Schubert, R. Riek, PNAS 2005, 102, 48, 17342–17347.
Aqueous Solution Structure of the Alzheimer's Disease Aβ Peptide 3D Structure of Alzheimer's Aβ(1-42) fibrils
22