Chemical Biology A Practical Course

by Waldmann, Herbert; Janning, Petra

• ISBN13: 9783527307784
• ISBN10: 3527307788
• Edition: 1st
• Format: Paperback
• Copyright: 2004-08-06
• Publisher: Wiley-VCH

Summary

Take your first steps in combinatorial synthesis or synthesize bioactive molecules such as antibiotics. Search for mutations in DNA using chemical probes or perform a proteome analysis in yeast. The present selection of 12 inspiring experiments is tailored for maximum learning effect at minimal expense of time and equipment. Almost all currently used laboratory techniques in synthesis and analysis of bioactive compounds are represented at least once. Abundant practical hints are complemented by a thorough treatment of the underlying theory and mechanisms.

Author Biography

Herbert Waldmann (b 1957) received his PhD in 1985 from the University of Mainz (Germany) with H. Kunz. After postdoctoral studies (1985-1986, Harvard University, George Whitesides) and habilitation (1991, University of Mainz) he was appointed associate professor at the University of Bonn in 1991. In 1993 he moved to the University of Karlsruhe as Full Professor of Organic Chemistry. In 1999 he was appointed Director at the Max Planck Institute of Molecular Physiology in Dortmund, where he is heading the Department of Chemical Biology. He also holds a Professorship in Biochemistry at the University of Dortmund. Herbert Waldmann has been the recipient of the Friedrich Weygand Award for the advancement of peptide chemistry, of the Carl Duisberg Award of the Gesellschaft Deutscher Chemiker, the Otto-Bayer-Award and the Steinhofer Award of the Steinhofer Foundation.<br> His current research interests include bioorganic chemistry and natural product synthesis as well as biocatalysis, stereoselective synthesis and combinatorial chemistry. <br> <br> Petra Janning (b. 1967) studied chemistry at the University of Mnnster (Germany). She completed her PhD degree at the University of Dortmund with G. T÷lg and M. Linscheid. From 1995 - 1998 she worked as a chemist in the University Hospital in Aachen. From 1999-2001 she worked in the group of G.H. Degen and H.M. Bolt at the Institute of Occupational Health in Dortmund. In 2001 she joined the Max Planck Institute of Molecular Physiology, where she is currently working in the group of Herbert Waldmann. She is responsible for the Bioorganic Practical Course and the analytics in the department.

Table of Contents

Preface	v
Abbreviations	xix
Introduction: Chemical Biology -- A New Science at the Crossroads of Chemistry and Biology	1	(22)
R. Breinbauer
H. Waldmann
The Chemical Biology of Lipidated Proteins belonging to the Ras Superfamily	4	(3)
Chemical Biology of Rab Proteins	7	(4)
Identifying the Natural Biological Target of FK506 and the Design of Protein Dimerizers as a Research Tool in Chemical Biology	11	(2)
Covalent Trapping of Protein--DNA Complexes	13	(2)
Cellular Imaging and In Vivo Labeling of Proteins with Fluorescent Probes	15	(2)
Modulating Cell Surface Architecture using Chemical Tools	17	(2)
Allele-specific Inhibition of Kinases	19	(1)
Conclusions	20	(1)
Bibliography	21	(2)
DNA Synthesis and DNA Hybridization	23	(12)
S. Ficht
W. Hecker
A. Mattes
O. Seitz
Abstract	23	(1)
Learning Targets	23	(1)
Theoretical Background	24	(8)
Basics	24	(1)
Chemical Synthesis of Oligonucleotides	24	(1)
Stability of Nucleic Acids	24	(2)
Protecting Groups	26	(2)
General Strategy for DNA Synthesis	28	(1)
Hybridization with Synthetic Oligonucleotides	29	(1)
Principles	29	(2)
Specificity of Hybridization	31	(1)
Dyeing Methods	31	(1)
Experimental Procedures	32	(2)
Preparations	32	(1)
Synthesis and Purification of an Oligonucleotide (16 bases) and Yield Determination	32	(1)
Determination of the TM Value of a Match (6--8, 7--9) and a Single Mismatch Duplex (6--9, 7--8)	33	(1)
Materials	33	(1)
Preparation of DNA Solutions	33	(1)
Measurement of the Melting Temperature and Concentration of G+C	33	(1)
Detection of dsDNA in the Presence of ssDNA using Ethidium Bromide	33	(1)
Materials	33	(1)
Calibration	34	(4)
Determination of the Concentration of dsDNA and ssDNA	38
Laboratory Report	34	(1)
Bibliography	34	(1)
Doubly-labeled Peptide Nucleic Acids (PNAs) as Probes for the Detection of DNA Point Mutations	35	(10)
O. Kohler
S. Rottger
O. Seitz
Abstract	35	(1)
Learning Targets	35	(1)
Theoretical Background	36	(4)
Basics of PNA	36	(2)
Synthesis of PNA	38	(2)
Experimental Procedures	40	(4)
Preparations	40	(2)
Synthesis of the PNA Beacon	42	(1)
Solid Phase Synthesis of the PNA--DABCYL Conjugate	42	(1)
Fluorescence Labeling of Ac-Lys(DABCYL)-ACCTACAGCC-HN-Lys-NH2	43	(1)
Detection of Single Nucleotide Polymorphism via Fluorescence Spectrometry	43	(1)
Bibliography	44	(1)
Synthesis and Characterization of a Covalent Oligonucleotide--Streptavidin Conjugate and its Application in DNA-directed Immobilization (DDI)	45	(16)
F. Kukolka
M. Lovrinovic
C.M. Niemeyer
R. Wacker
Abstract	45	(1)
Learning Targets	45	(1)
Theoretical Background	45	(5)
(Strept)avidin--Biotin System and DNA Oligonucleotides as Molecular Tools	45	(2)
Covalent DNA--Streptavidin Conjugates	47	(1)
DNA--Directed Immobilization	48	(2)
Experimental Procedures	50	(9)
Conjugate Synthesis	50	(1)
Oligonucleotide Activation	50	(1)
STV Activation	50	(1)
Purification of Activated Oligonucleotide	50	(1)
Purification of Activated STV	50	(1)
Cross-linking of STV and Oligonucleotide, Quenching and Buffer Exchange	51	(1)
Purification of STV--Oligonucleotide Conjugate	51	(1)
Photometry and Conjugate Quantification	52	(1)
Buffers	53	(1)
Characterization of the Conjugates by Non-denaturing PAGE	53	(1)
Preparation of Separation Gel	53	(1)
Preparation of Stacking Gel	53	(1)
Sample Preparation	54	(1)
Electrophoresis	54	(1)
SybrGold Staining	54	(1)
Fixation	54	(1)
Silver Staining	55	(1)
Documentation	55	(1)
Buffers	55	(1)
Solid Phase Hybridization	56	(1)
Coating of Microplates with STV	56	(1)
Immobilization of Capture Oligonucleotides	56	(1)
Sample Preparation and Dilution Series	56	(1)
DNA-directed Immobilization (DDI)	57	(1)
Enzymatic Reaction	57	(1)
Fluorescence Detection	57	(2)
Buffers	59	(1)
Bibliography	59	(2)
Solid Phase Synthesis of Peptides: Bradykinin Analogs and the Evaluation of Calcium Mobilization in PC-12 Cells	61	(14)
C. Katzka
B. Ludolph
P. Janning
C. Schultz
Abstract	61	(1)
Learning Targets	61	(1)
Theoretical Background	61	(8)
Biological Function of Bradykinin	61	(1)
PC-12 Cells	62	(1)
Calcium Measurements	62	(3)
Peptide Synthesis	65	(1)
Overview	65	(1)
Protecting Group Strategies	65	(2)
Choice of Peptide Sequence	67	(1)
Available Building Blocks	68	(1)
Experimental Procedures	69	(5)
Preparations	69	(1)
Cultivation of PC-12 Cells	69	(2)
Synthesis of the Hexameric Peptide	71	(1)
Peptide Synthesis	71	(1)
First Steps	71	(1)
Determination of Loading Efficiency	72	(1)
Coupling Procedure	72	(1)
Cleavage Procedure	72	(1)
Purification	72	(1)
Characterization of the Peptides	72	(1)
Measurement of Intracellular Ca2+ Levels	72	(1)
Incubation and Measurement Buffer	72	(1)
Fura-2 Loading	72	(1)
Calcium Measurements	72	(2)
Bibliography	74	(1)
In silico Protein Ligand Design	75	(32)
M. A. Koch
L. Arve
L. Kissau
J. Gerdes
Abstract	75	(1)
Learning Targets	75	(1)
Theoretical Background	76	(15)
Overview	76	(1)
Three-Dimensional Protein Structures	77	(1)
Molecular Modeling	78	(1)
Molecular Mechanics (MM)	78	(2)
Quantum Mechanics (QM)	80	(1)
Computer-aided Drug Design	80	(1)
General Aspects	80	(1)
Program for Engineering Peptides (PEP)	81	(2)
Enzyme Catalysis and Inhibition	83	(2)
Non-Covalent Interactions	85	(1)
Electrostatic Interactions	85	(1)
Hydrophobic Interactions	86	(1)
Hydrogen Bonds	86	(2)
Rational Drug Design	88	(1)
Pharmacodynamics	88	(1)
Pharmacokinetics	88	(1)
Drug Optimization	89	(1)
Quantitative Structure--Activity Relationships (QSAR)	89	(1)
Retrosynthesis	90	(1)
Experimental Procedures	91	(9)
General Remarks	91	(1)
The General Modeling Process	92	(1)
Preparations	93	(1)
Preparation of the Seed Molecule	93	(4)
Minimization of the Protein Structure	97	(1)
Definition of the Binding Site	98	(1)
Generation of Peptides with PEP	99	(1)
Evaluation and Discussion of the Results	99	(1)
Retrosynthesis of a Non-Peptidic FTase Inhibitor	100	(1)
Bibliography	100	(1)
General Literature	100	(1)
Special Literature	101	(1)
Appendices	101	(6)
Appendix A	101	(2)
Appendix B	103	(2)
Appendix C	105	(1)
Appendix D	106	(1)
Lipidation of Proteins and Peptides: Farnesylation of the Ras Protein	107	(16)
S. Sommer
T. Voigt
I. Heinemann
B. Popkirova
R. Reents
J. Kuhlmann
Abstract	107	(1)
Learning Targets	107	(1)
Theoretical Background	108	(8)
Lipidation of Proteins	108	(1)
Ras Proteins	109	(1)
Signal Transduction Pathway	109	(2)
Ras Activation	111	(1)
Farnesylation	112	(1)
In-Vitro Farnesylation	113	(1)
Prenyl Selectivity of FTase	114	(1)
Techniques	114	(1)
Lyophilization	114	(1)
31P-NMR	114	(1)
Triton-X114 Extraction	115	(1)
Mass Spectrometry Employing MALDI-TOF	115	(1)
Experimental Procedures	116	(6)
Preparations	116	(1)
Synthesis of Prenyl- and Alkylpyrophosphates	117	(1)
General	117	(1)
Preparation of the Ion Exchange Column	117	(1)
Preparation of Prenyl- and Alkylpyrophosphates	118	(1)
Farnesylation on an Analytical Scale	118	(1)
Farnesylation on a Preparative Scale	119	(1)
Determination of Protein Concentration	120	(1)
SDS-PAGE-Analysis of the Farnesylated Samples	121	(1)
Mass Spectrometry	121	(1)
Bibliography	122	(1)
Textbooks	122	(1)
Special Literature	122	(1)
Insertion of Lipidated Peptides into Model Membranes	123	(14)
D. Gottlieb
A. Watzke
Abstract	123	(1)
Learning Targets	123	(1)
Theoretical Background	124	(6)
Biological Membranes	124	(2)
Lipidation of Proteins	126	(1)
Biophysical Properties of Lipidated Peptides in Model Membranes	126	(4)
Basic Concepts of Fluorescence and Fluorescence Markers	130	(1)
Experimental Procedures	130	(5)
Preparations	136
Determination of the Partition Coefficient Kp	132	(1)
Preparation of the Peptide--Vesicle Solution	132	(1)
Preparation of Solutions of Empty Vesicles of Different Concentrations	132	(1)
Measurements by Fluorescence Spectroscopy	132	(1)
Interpretation of the Measurements	132	(2)
Vesicle Transfer	134	(1)
Synthesis of Vesicles Containing Fluorescence Quencher and Lipidated Peptides	134	(1)
Preparation of Empty Vesicles	134	(1)
Measurements	134	(1)
Interpretation of the Measurements	134	(1)
Determination of the Flip-Flop Exchange	135	(1)
Measurements	135	(1)
Interpretation of the Measurements	135	(1)
Bibliography	135	(2)
Textbooks	135	(1)
Special Literature	135	(2)
Isolation of Potato Phosphorylase and Enzymatic Synthesis of Amylose	137	(8)
M. Scheck
M. Manger
N. Bisek
Abstract	137	(1)
Learning Targets	137	(1)
Theoretical Background	137	(5)
Overview	137	(2)
Principle of Polysaccharide Synthesis	139	(2)
Isolation of the Potato Phosphorylase	141	(1)
Synthesis of Amylose	141	(1)
Principle of the Phosphate Test	142	(1)
Experimental Procedures	142	(2)
Preparations	142	(1)
Isolation of the Potato Phosphorylase	143	(1)
Synthesis of Amylose and Colorimetric Determination	143	(1)
Bibliography	144	(1)
Textbook	144	(1)
Special Literature	144	(1)
Proteome Analysis: Identification of Proteins Isolated from Yeast	145	(16)
H. Prinz
D. Goehrke
P. Janning
K. Reinecke
Abstract	145	(1)
Learning Targets	145	(1)
Theoretical Background	146	(5)
Introduction	146	(1)
MALDI Mass Spectrometry	146	(2)
Electrospray Mass Spectrometry using Ion Trap Analyzers	148	(1)
Principle	148	(1)
Controlled Fragmentation (MS/MS)	149	(1)
MS/MS Fragmentation of Peptides	149	(1)
High Performance Liquid Chromatography (HPLC)	149	(1)
Two-dimensional Gel Electrophoresis	150	(1)
First Dimension: Isoelectric Focusing	150	(1)
Second Dimension: SDS-PAGE	150	(1)
Proteolytic Digestion	151	(1)
Principle	151	(1)
Characterization by Mass Spectrometry	151	(1)
Experimental Procedures	151	(8)
Preparations	151	(1)
Cell Disruption	152	(1)
Principle	152	(1)
Preparation of Cell Lysate	152	(1)
Two-dimensional Gel Electrophoresis	152	(1)
Introduction	152	(1)
First Dimension: Isoelectric Focusing	152	(1)
Second Dimension: SDS-PAGE	153	(1)
In-gel Digestion of Proteins Separated by Polyacrylamide Gel Electrophoresis	154	(1)
MALDI Mass Spectrometry	155	(1)
Alternative: HPLC ESI-MS/MS	156	(1)
Principle	156	(1)
Protein Quantification	156	(1)
Preparation of a Peptide Mix	157	(1)
HPLC/MS	158	(1)
Data Analysis	158	(1)
Bibliography	159	(2)
Lectins: Determination of the Sugar Specificity of Jacalin by a Sugar--Lectin Binding Assay (SLBA)	161	(10)
E. Gourzoulidou
S. Schlummer
Abstract	161	(1)
Learning Targets	161	(1)
Theoretical Background	162	(5)
Lectins	162	(2)
Sugar--Lectin Binding Assay (SLBA)	164	(3)
Experimental Procedures	167	(3)
Preparations	167	(1)
Periodate-treated BSA	167	(1)
Composition of Buffers	167	(1)
Synthesis of Biotin--Galactose (BG) Conjugate	168	(1)
Sugar--Lectin Binding Assay (SLBA)	168	(1)
Calculations	169	(1)
Bibliography	170	(1)
Combinatorial Synthesis and Genetic Algorithm	171	(10)
T. Lessmann
S. Eichhorn
J. Huber
Abstract	171	(1)
Learning Targets	171	(1)
Theoretical Background	172	(5)
Introduction	172	(7)
Multi-component Reactions	179
Genetic Algorithm	173	(1)
Overview	173	(1)
Encoding the Compounds	174	(1)
Recombination	174	(1)
Mutation	175	(1)
Biological Activity	175	(2)
Course of the Experiment	177	(1)
Experimental Procedures	177	(2)
Synthesis of the a-Amino Amides	177	(1)
Thrombin Assay	177	(1)
Reagents	177	(1)
Procedure	178	(1)
Data Analysis	178	(1)
Tasks and Questions	179	(1)
Bibliography	179	(2)
Textbook	179	(1)
Special Literature	179	(2)
Solid Phase Synthesis of an Antibiotic	181	(12)
C. Peters
C. Rosenbaum
F. Stieber
Abstract	181	(1)
Learning Targets	181	(1)
Theoretical Background	181	(6)
Combinatorial Synthesis of Low Molecular Weight Compound Libraries	181	(1)
Linkers for Solid-phase Synthesis	182	(1)
The Hydrazide Linker: An Oxidation Labile Traceless Linker	183	(1)
Acid-functionalized Resins	184	(1)
Solid-phase Synthesis of Antibiotics	185	(2)
Experimental Procedures	187	(6)
Project A: Synthesis of Functionalized Resin	187	(1)
Procedure for the Preparation of Adipic Acid Monomethyl Ester-functionalized Supports	187	(1)
Hydrolysis of Polymer-bound Methylester to Yield Acid-functionalized Polymeric Support 9	187	(1)
Esterification of Acid-functionalized Resin with 2-Methoxy-5-nitrobenzylester 10 (Determination of Loading)	187	(1)
Determination of Loading by Means of UV-spectroscopic Analysis of 2-Methoxy-5-nitrobenzyl Alcohol 12	188	(1)
Project B: Synthesis of an Antibiotic	188	(1)
Synthesis of 4-Iodophenylhydrazine 13	188	(1)
Polystyrene-bound 4-Iodophenylhydrazide 14	189	(1)
Polystyrene-bound Biphenylaldehyde 20	189	(1)
Synthesis of 4'-biphenylmethyl-(4-Thiomorpholin-4-ylmethyl-phenyl)-amine 21a	189	(1)
General Procedure for the Oxidative Cleavage of the Phenylhydrazide Linker Utilizing Cu(OAc)2, Methanol and Pyridine	190	(1)
Synthesis of 4-(4-Nitrobenzyl)thiomorpholine 17a	190	(1)
Synthesis of 4-Thiomorpholinomethylaniline 18a	190	(1)
Bibliography	191	(2)
Appendix	193	(8)
Subject Index	201