Chemical Tools To Probe The Proteasome

Transcription

1 Chemical Tools To Probe The Proteasome PROEFSCHRIFT ter verkrijging van de graad van Doctor aan de Universiteit Leiden, op gezag van Rector Magnificus prof.mr. P.F. van der Heijden, volgens besluit van het College voor Promoties te verdedigen op vrijdag 19 december 2008 klokke uur door Martijn Verdoes geboren te Leiden in 1980

2 Promotiecommissie Promotores : Prof. dr. H. S. Overkleeft : Prof. dr. G. A. van der Marel Overige leden : Prof.dr. J. Brouwer Prof.dr. J.J. Neefjes Prof.dr. J. Lugtenburg Dr. B. M. Kessler

3 As long as we re having fun...

4 Table of contents List of Abbreviations Chapter 1 General Introduction 6 9 Chapter 2 A Fluorescent Broad-Spectrum Proteasome Inhibitor for Labeling Proteasomes In Vitro and In Vivo. 31 Chapter 3 Acetylene functionalized BODIPY dyes and their application in the synthesis of activity based proteasome probes. 51 Chapter 4 Azido-BODIPY acid: a new tool in activity-based protein profiling 63 Chapter 5 Mixing of peptides and electrophilic traps gives rise to potent, broadspectrum proteasome inhibitors. 83

5 Chapter 6 Synthesis and evaluation of subunit specific proteasome probes. 101 Chapter 7 Diels-Alder two-step activity-based proteasome labeling 127 Chapter 8 Summary and Future Prospects 139 Samenvatting 153 List of Publications 156 Curriculum Vitae 158 Nawoord 159

6 List of Abbreviations ABP Activity-based probe BOP benzotriazol-1-yl-oxy-tris- ABPP Activity-based protein profiling (dimethylamino)phosphonium Ac acetyl hexafluorophosphonate AcCl acetyl chloride BSA bovine serum albumine Ac 2 O acetic anhydride Bu butyl AcOH acetic acid calcd. calculated Ada 1-adamantyl acetyl cat. catalytic amount Ahx 6-aminohexanoic acid CDI carbodiimidazole AMC 7-acetoxy-4-methyl-coumarin CLSM confocal laser scanning microscopy AMP adenosine monophosphate CtL chymotrypsin-like APT attached proton test chemical shift ATP adenosine triphosphate d doublet aq. aqueous Da Dalton NA naphthylamide DABCO 1,4-diazabicyclo[2.2.2]octane Bax Bio BM Boc Boc 2 O BODIPY Bcl-2-associated X protein biotin biotinylated molecular marker tert-butyloxycarbonyl tert-butyloxycarbonic anhydride boron-dipyrromethene, boradiazaindacene DBU DC DCE DCM dd ddd DIC diazabicyclo[5.4.0]undec-7-ene dual color marker 1,2-dichloroethane dichloromethane double doublet double double doublet differential interference contrast 6

7 DIC N,N -diisopropyl carbodiimide HOBt N-hydroxybenzotriazole DIFO difluorinated cyclooctyne HOSu N-hydroxysuccinimide DiPEA Diisopropylethylamine hr. hour(s) DMAP 4-(dimethylamino)pyridine HRMS high resolution mass spectrometry DMEM dulbecco's modified eagle's medium Hz Hertz DMF N,N-dimethylformamide i.v. intravenous DMSO dimethylsulfoxide IFN- interferon-gamma dt double triplet I B inhibitor of NF- B DTNB 5,5 -dithio-bis(2-nitrobenzoic acid) J coupling constant DTT dithiothreitol K kelvin DUB deubiquitinating enzyme LC/MS liquid chromatography/ mass dq double quartet spectrometry EDC HCl 1-ethyl-3-(3-dimethyl-aminopropyl)- LiHMDS lithium hexamethyldisilazide carbodiimide HCl salt m multiplet EDTA ethylenediaminetetraacetate M molar EL-4 murine lymphoid cell line M molecular marker Et 3 N triethyl amine MBHA p-methylbenzhydryl amine Et 2 O diethyl ether Me methyl EtOH ethanol MeCN acetonitrile EtOAc ethyl acetate MelJuSo metastasizing human melanoma cell equiv. molar equivalent line ES ethyl methyl sulfone MeOD CD 3 OD FDA United States food and drug MeOH methanol administration MES 2-(N-morpholino)ethane sulfonic acid Fmoc (9H-fluoren-9-yl)methoxycarbonyl min. minute(s) GFP green fluorescent protein nbu n-butyl HCTU (2-(6-Chloro-1H-benzotriazole- 1-yl)- NEM N-ethyl maleimide 1,1,3,3-tetramethylaminium MS (ESI) mass spectrometry (electrospray hexafluorophosphate) ionization) HEK293T human embryonic kidney cell line NF- B nuclear factor- B HeLa human cervix carcinoma cell line NIP nitro-iodophenol HEPES 4-(2-hydroxyethyl)-1- MHC I major histocompatibility piperazineethanesulfonic acid complex class I HMB 3-hydroxy-2-methylbenzamide NMM N-methylmorpholine HMPB 4-hydroxymethylphenyl-butyric acid NMP N-methyl-2-pyrrolidone linker NMR nuclear magnetic resonance 7

8 Ntn N-terminal nucleophilic TBS tert-butyldimethylsilyl m/z mass-to-charge ratio tbu tert-butyl o/n overnight tbuono tert-butyl nitrite OAc acetate tbuoh tert-butanol PBS phosphate buffered saline td triple doublet Pd/C palladium on charcoal TEA triethyl amine PEG polyethyleneglycol TFA trifluoroacetic acid PetEt petroleum ether THF tetrahydrofuran PGPH peptidylglutamyl peptide hydrolytic TIS triisopropyl silane Ph phenyl TL trypsin-like ppm parts per million TLC thin layer chromatography pv. pathovar TMR tetramethylrhodamine PyBOP benzotriazol-1-yl-oxy-tripyrrolidino- TMS trimethylsilyl phosphonium Tol. toluene hexafluorophosphonate Tris 2-amino-2-(hydroxymethyl)-1,3- q quartet propanediol quant. quantitative SDS sodium dodecyl sulphate RAW Mouse leukaemic monocyte SPPS solid phase peptide synthesis macrophage cell line Su succinimidyl RPMI roswell park memorial institute Ub ubiquitin medium UPS Ubiquitin Proteasome rt R t s room temperature retention time singlet VE VO(acac) 2 VS System vinyl ethyl ester vanadium(iv)acetyl acetonate vinyl methyl sulfone sacc. Pier Andrea Saccardo Z benzyloxycarbonyl sat. SDS-PAGE sp. Su t saturated sodium dodecyl sulfate-poly acrylamide gel electroforese species succinimidyl triplet Note: the one and three letter codes for the amino acids follow the recommendations of IUPAC. J. Biol. Chem. 1968, 243, and J. Biol. Chem. 1972, 247, t, tert tertiary T TAP temperature transporter associated with antigen presentation TBAI tetrabutylammonium iodide 8