Research Collection Doctoral Thesis DNA-encoded chemical libraries Author(s): Mannocci, Luca Publication Date: 2009 Permanent Link: https://doi.org/10.3929/ethz-a-005783014 Rights / License: In Copyright - Non-Commercial Use Permitted This page was generated automatically upon download from the ETH Zurich Research Collection. For more information please consult the Terms of use. ETH Library Diss. ETH No. 18153 DNA-Encoded Chemical Libraries Luca Mannocci ical Libraries Diss. ETH No.18153 Luca Mannocci DNA-Encoded Chem DISS. ETH NO. 18153 DNA-Encoded Chemical Libraries A dissertation submitted to the ETH Zurich For the degree of Doctor of Sciences Presented by Luca Mannocci Dott. Chim. Università degli Studi di Pisa Born September 7, 1979 Citizen of Pisa (Italy) Accepted on the recommendation of Prof. Dr. Dario Neri, examiner Prof. Dr. Karl-Heinz Altmann, co-examiner Zurich, 2009 “I believe in intuition and inspiration. Imagination is more important than knowledge. Knowledge is limited. Imagination embraces the entire world, stimulating progress, giving birth to evolution. It is, strictly speaking, a real factor in scientific research.” Albert Einstein Alla mia famiglia TABLE OF CONTENTS 1. SUMMARY ...........................................................................................7 RIASSUNTO .............................................................................................9 List of abbreviations ...............................................................................11 2. INTRODUCTION ..............................................................................14 2.1 DNA-Encoded Chemical Libraries ................................................................16 2.1.1 Libraries of DNA displaying one covalently linked chemical entity........20 2.1.1.1 DNA-encoded “Split-&-Pool” ............................................................20 2.1.1.2 DNA-assisted “Split-&-Pool” .............................................................21 2.1.1.3 DNA-templated synthesis....................................................................24 2.1.1.4 Stepwise coupling of coding DNA fragments to nascent organic molecules .........................................................................................................28 2.1.2 DNA libraries displaying multiple covalently linked chemical entities ESAC libraries.....................................................................................................30 2.2. The decoding of DNA-encoded chemical libraries........................................38 2.2.1 Microarray-based decoding .......................................................................35 2.2.2 Decoding by high throughput sequencing ................................................38 2.2.2.1 “454” technology.................................................................................40 2.2.2.2 Solexa technology ...............................................................................42 2.2.2.3 SOLiD techonlogy...............................................................................44 2.2.2.4 Single Molecule DNA Sequencing – Helicos technology..................48 3. RESULTS ............................................................................................50 3.1 DNA-Encoded Library “DEL4000”...............................................................50 3.1.1 Library design and synthesis .....................................................................51 3.1.2 Model Compounds .....................................................................................53 3.1.3 Oligonucleotides.........................................................................................54 3.1.4 Compounds.................................................................................................55 3.1.5 HPLC Purification.....................................................................................56 3.1.6 Mass Spectrometry .....................................................................................57 3.1.7 Oligonucleotide concentration determination ..........................................58 3.1.8 Polymerase Klenow encoding....................................................................59 3.1.9 Summary ....................................................................................................59 3.2 Selections using the DEL4000 library............................................................61 3.2.1 Streptavidin selection .................................................................................62 3.2.1.1 Identification of streptavidin binding molecules ...............................64 3.2.1.2 Characterization of streptavidin binding molecules..........................65 3.2.2 Polyclonal human IgG selection ...............................................................68 3.2.2.1 Identification of polyclonal IgG binding molecules ..........................68 3.2.2.2 Characterization of polyclonal IgG binding molecules by affinity chromatography resins ...................................................................................70 3.2.3 Matrix metalloproteinase 3 (MMP3) selection .........................................71 2 3.2.3.2 Characterization of MMP3 binding molecules..................................72 3.2.4 Computational simulation of DEL4000 selections...................................73 3.3 General strategies for the stepwise construction of very large DNA encoded chemical libraries...................................................................................................75 3.3.1 Selective deprotection and reaction of di-amine derivatives ....................75 3.3.1.1 Orthogonal protective group and selective deprotection ...................76 3.3.1.2 Core scaffolds design and synthesis strategy .....................................78 3.3.1.3 Model compounds for N-Fmoc, N’-Nvoc di-amino carboxylic acid core scaffold based library..............................................................................80 3.3.2 Stepwise DNA-encoding ............................................................................82 3.3.2 Encoding by ligation ..............................................................................82 3.3.2.1 Encoding by a combination of Klenow polymerase and ligation......83 3.3.2.2 Encoding by Klenow polymerase........................................................84 3.3.3 Summary ....................................................................................................85 4. DISCUSSION ......................................................................................87 5. MATERIAL AND METHODS .........................................................89 5.1 Reagents and general remarks .......................................................................89 5.2 Synthesis of DEL4000 DNA Encoded Library..............................................89 5.2.1 Synthesis of library model compounds oligonucleotide conjugate..........90 5.2.2 Coupling reactions of 20 Fmoc-protected amino acids............................91 5.2.3 Coupling reactions of 200 carboxylic acids. .............................................91 3 5.2.4 Polymerase Klenow encoding of 200 carboxylic acids reactions.............92 5.2.5 Preparation of D-desthiobiotin oligonucleotide-conjugate (positive control) ................................................................................................................92 5.3 Library DEL 4000 selections...........................................................................93 5.3.1 Streptavidin selection. ................................................................................93 5.3.1.1 Identification of binding molecules....................................................93 5.3.1.2 Synthesis of the binding molecules as fluorescein conjugates..........93 5.3.2 Affinity measurements. .............................................................................94 5.3.3 Polyclonal human IgG selection. ..............................................................95 5.3.3.1 Polyclonal human IgG coating of sepharose beads. .........................95 5.3.3.2 Identification of human IgG binding molecules. ..............................95 5.3.3.3 Synthesis of affinity chromatography resin containing the compound 02-40 or 16-40. ................................................................................................96 5.3.3.4 Polyclonal human IgG Cy5 labeling..................................................97 5.3.3.5 Biotinylated polyclonal human IgG. ..................................................97 5.3.3.6 Affinity chromatography of CHO cells supernatant spiked with human IgG Cy5 labeled or biotinylated human IgG on IgG binding resin. 97 5.3.4 Human MMP3 selection............................................................................98 5.3.4.1 Human MMP3 coating of sepharose beads.......................................98 5.3.4.2 Identification of human MMP3 binding molecules. .........................99 5.3.4.3 Synthesis of the MMP3 binding molecules as fluorescein conjugates. ..........................................................................................................................99 5.3.5 Computational simulation .......................................................................100 4 5.4 Stepwise coupling by selective deprotection and reaction of di-amine derivatives.............................................................................................................100 5.4.1 DNA-compatible cleavage