Diazo Compounds for the Customization of Important Biomolecules Inauguraldissertation zur Erlangung der Würde eines Doktors der Philosophie vorgelegt der Philosophisch-Naturwissenschaftlichen Fakultät der Universität Basel von Na Fei Aus Rizhao, China Basel, 2017 Originaldokument gespeichert auf dem Dokumentenserver der Universität Basel edoc.unibas.ch Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultät auf Antrag von Prof. Dr. Dennis Gillingham Prof. Dr. Florian Seebeck Basel, 24. Mai 2016 Prof. Dr. Jörg Schibler Dekan Dedicated to my mom 真者，精誠之至也 《莊子‧雜篇‧漁父第三十一》 Contents Summary ................................................................................................................................................. 1 Chapter 1 Introduction ............................................................................................................................ 3 1.1 Diazo chemistry ............................................................................................................................. 3 1.2 Carbenoid formation based on α-diazocarbonyl compounds ...................................................... 5 1.2.1 Catalyst comparison for carbenoid chemistry ........................................................................ 6 1.2.2 Modifications in chemical biology with α-diazocarbonyl compounds based on metal- carbenoid XHI ................................................................................................................................ 10 1.3 Alkylation with unstabilized diazo compounds in chemical biology ........................................... 13 Chapter 2 Catalytic NHI of nucleic acids by diazo compounds .............................................................. 17 2.1 Cyclic-di-nucleotides as important signaling molecules .............................................................. 17 2.1.1 c-di-GMP as a ubiquitous second messenger ....................................................................... 18 2.1.2 c-di-AMP as a newly discovered CDN draws attention ........................................................ 20 2.1.3 CDN signaling in eukaryotes domain .................................................................................... 21 2.2 Rh catalyzed NHI for customization of CDNs ............................................................................... 24 2.2.1 Previous synthesis and modification of CDNs ...................................................................... 24 2.2.2 Preparative synthesis of exo-amine modified CDNs ............................................................ 25 2.2.3 Photocrosslinking of modified CDN with its receptor protein ............................................. 29 2.2.4 Conclusion ............................................................................................................................ 31 2.3 Copper catalyzed NHI with diazo compound .............................................................................. 31 2.3.1 Copper (I) catalyzed NHI in dsDNA alkylation ....................................................................... 32 2.3.2 Conclusion ............................................................................................................................ 35 2.4 Experimental part ........................................................................................................................ 36 2.4.1 General ................................................................................................................................. 36 2.4.2 General procedure for Rh2(OAc)4-catalyzed CDN modification with diazocarbonyl compounds. ................................................................................................................................... 36 2.4.3 Protein modification ............................................................................................................. 55 2.4.4 General procedure for Cu(I)-catalyzed hairpin oligonucleotide modification using a- diazocarbonyl compounds. ........................................................................................................... 61 2.4.5 General procedure for auto-tandem catalytic CuAAC/NHI .................................................. 62 Chapter 3 Selective alkylation of biological phosphates by unstabilized diazo compounds ................. 65 3.1 phosphate is one of the most important functional groups in chemical biology ........................ 65 3.1.1 Revealing the mysterious of reversible protein phosphorylation ........................................ 68 3.1.2 Limited methods of chemical labelling of phosphopeptides ................................................ 72 3.1.3 Previous phosphate modification ......................................................................................... 73 3.2 Selective modification of phosphate in aqeous buffer ................................................................ 73 3.2.1 Competition alkylation between carboxylic acid and phosphate ........................................ 74 3.2.2 Substrate scope of modification of bioactive phosphate compound ................................... 75 3.2.3 Src (521-533) and its inhibitor 5mer-peptide modification .................................................. 77 3.2.4 Photo-cleavage of modified product .................................................................................... 80 3.3 Conclusion ................................................................................................................................... 81 3.4 Experimental part ........................................................................................................................ 82 3.4.1 General Methods .................................................................................................................. 82 3.4.2 Synthesis of diazo compounds ............................................................................................. 82 3.4.3 CMP methylation .................................................................................................................. 84 3.4.4 Methylation selectivity towards CMP and benzoic acid with TMSCHN2 .............................. 85 3.4.5 Modification of bioactive phosphate compounds ................................................................ 87 3.4.6 Photo cleavage of Tyrosine phosphate 2-nitro benzyl ester ................................................ 92 3.4.7 Alkylation of phosphate monoester vs phosphodiester ....................................................... 93 3.4.8 Modification of phosphopeptide .......................................................................................... 99 Chapter 4 Investigation of substrate tolerance of RNA demethylase FTO .......................................... 117 4.1 Reversible DNA methylation in gene regulation ....................................................................... 117 4.2 The discovery of FTO as m6A RNA demethylase ........................................................................ 118 4.3 Results and discussion ............................................................................................................... 121 4.3.1 Synthesis of convertible RNA monomers ........................................................................... 121 4.3.2 The FTO protein production ............................................................................................... 124 4.4 Conclusion and outlook ............................................................................................................. 125 4.5 Experimental part ...................................................................................................................... 126 4.5.1 General ............................................................................................................................... 126 4.5.2 Convertible RNA synthesis ................................................................................................. 126 4.5.3 FTO protein production ...................................................................................................... 127 List of abbreviations ............................................................................................................................ 129 References ........................................................................................................................................... 131 Acknowledgement .............................................................................................................................. 140 Summary The selective modification of native biomolecules such as nucleic acid, signaling molecules, and peptides offers new tools for investigation biological processes. These chemically modified analogs, radiolabeled analogs, fluorescent or affinity tags can be used to label, modify, or pull-down a molecule of interest to probe its function. Among these chemical modifications, diazo compounds are the most versatile building blocks for the customization of diverse biomolecules. In chapter 1, I discuss briefly the properties of diazo compounds and their application in chemistry and chemical biology. The transition-metal catalyzed XHI reaction of stabilized diazo compounds is used later in chapter 2 and the alkylation reaction of unstabilized diazo compounds with BrØnsted acids is used in chapter 3. In chapter 2, I first give a short introduction of the biological role of CDNs (c-di-nucleotides) which are very