Electronic Supplementary Material (ESI) for Molecular BioSystems. This journal is The Royal Society of Chemistry 2016 SUPPORTING INFORMATION Photo-crosslinking of clinically relevant kinases using H89- derived photo-affinity probes Sara C. Stolze, a Nora Liu, a Ruud H. Wijdeven, b Adriaan W. Tuin, a Adrianus M. C. H. van den Nieuwendijk, a Bogdan I. Florea, a Mario van der Stelt, a Gijsbert A. van der Marel, a Jacques J. Neefjes b and Herman S. Overkleeft* a a. Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2300 RA Leiden, The Netherlands. E-mail: h.s.overkleeft@chem.leidenuniv.nl. b. Division of Cell Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands. These authors contributed equally to this work. Table of contents Kinomescan activities of probe 3 and 4 Dose dependency curves for probe 3 and 4 1H and 13 C NMR spectra of all novel compounds
Table S1: Activities of probe 3 and 4 against a panel of kinases. 1 Activities are given as % residual kinase activity at 10 μm probe. Kinase 3 4 Kinase 3 4 PKAc-α 1.5 3.4 TAOK1 72 52 AKT1 2.6 18 MEK2 73 72 ROCK2 3 1.7 FAK 74 100 AKT3 3.4 25 VEGFR2 75 85 PRKCH 5 2.8 CLK2 76 72 PRKCE 20 10 JNK1 78 82 PRKCD 23 14 TYK2 78 97 SNARK 23 24 TRKA 79 82 SGK3 31 32 AURKB 80 79 CDK11 35 78 GSK3B 80 74 PRKCI 35 35 PCTK1 80 84 CSNK1D 39 38 JNK3 81 89 MARK3 40 53 FGFR3 82 84 CDK7 43 48 PIK3CG 82 78 DYRK1B 45 74 PLK1 82 83 ALK 50 62 YANK3 82 88 FLT3 50 60 MAPKAPK2 83 95 MLCK 50 76 PIM1 83 81 CSF1R 51 96 ULK2 83 65 TSSK1B 53 56 ZAP70 83 94 AXL 54 74 DYRK1A 84 85 CHEK1 56 72 ERBB4 84 90 SRPK3 57 86 ERN1 85 57 CDK9 58 70 LKB1 85 94 PDGFRB 58 57 PAK4 86 98 PIP5K1A 58 37 ACVR1B 87 100 PDPK1 59 69 p38-beta 87 100 PLK4 59 57 CSNK1G3 89 91 RET 59 76 ERBB2 89 64 FGFR2 60 67 INSR 89 81 KIT 61 70 PAK2 89 96 AMPK-α2 63 83 TGFBR1 89 88 DCAMKL1 63 86 PIM3 90 94 PRKCQ 63 46 AURKA 91 69 EPHA2 65 66 JAK3 91 84 MAP3K4 65 68 PDGFRA 91 98 AKT2 66 91 RAF1 91 78 CDK2 66 92 BMPR2 92 89 CDK3 66 78 PIK3CA 93 94 ABL1-p 67 81 CSNK1G2 94 80 JAK2 67 90 MEK1 95 83 MET 67 55 PAK1 95 95 RSK2 67 72 BTK 96 92 TIE2 68 86 p38-α 96 94 ERK1 69 70 HIPK2 97 95 JNK2 69 86 EGFR 98 100 PLK3 69 73 PIK3C2B 98 94 PIM2 70 62 IKK-beta 99 80 SRC 70 70 MKNK1 99 92 ADCK3 71 96 PIK4CB 99 97 MKNK2 71 71 BRAF 100 93 MST2 71 73 IKK-alpha 100 86 FLT1 72 69 MTOR 100 100 IGF1R 72 63 MYO3A 100 100 MLK1 72 69 RIOK2 100 88 MUSK 72 77 1 M. W. Karaman, S. Herrgard, D. K. Treiber, P. Gallant, C. E. Atteridge, B. T. Campbell, K. W. Chan, P. Ciceri, M. J. Davis, P. T. Edeen, R. Faraoni, M. Floyd, J. P. Hunt, D. J. Lockhart, Z. V. Milanov, M. J. Morrison, G. Pallares, H. K. Patel, S. Pritchard, L. M. Wodicka and P. P. Zarrinkar, Nat. Biotech., 2008, 26, 127-132.
probe 3, PKA probe 4, PKA Fig.S1: Dose-dependent activity of PKA determined by FRET-based assay for probes 3 and 4. probe 3, AKT1 probe 4, AKT1 Fig.S2: Dose-dependent activity of AKT1 determined by FRET-based assay for probes 3 and 4.
Fig.S3: 1 H NMR (400 MHz, CDCl 3, Me 4Si)for (Diethyl (1-cyano-4-(trityloxy)butyl)phosphonate (7). Fig.S4: 13 H NMR (101 MHz, CDCl 3)for (Diethyl (1-cyano-4-(trityloxy)butyl)phosphonate (7).
10 Fig.S5: 1 H NMR (400 MHz, CDCl 3, Me 4Si) for (E/Z)-2-(4-(3-(trifluoromethyl)-3H-diazirin-3-yl)- benzylidene)-5-(trityloxy)pentanenitrile (10). 10 Fig.S6: 13 C NMR (101 MHz, CDCl 3) for (E/Z)-2-(4-(3-(trifluoromethyl)-3H-diazirin-3-yl)-benzylidene)-5- (trityloxy)pentanenitrile (10).
13 Fig.S7: 1 H NMR (400 MHz, CDCl 3, Me 4Si) for tert-butyl(e)-(5-hydroxy-2-(4-(3-(trifluoromethyl)-3hdiazirin-3-yl)benzylidene)pentyl)(2-(isoquinoline-5-sulfonamido)ethyl)-carbamate (13). 13 Fig.S8: 13 C NMR (101 MHz, CDCl 3) for tert-butyl(e)-(5-hydroxy-2-(4-(3-(trifluoromethyl)-3h-diazirin-3- yl)benzylidene)pentyl)(2-(isoquinoline-5-sulfonamido)ethyl)-carbamate (13).
14 Fig.S9: 1 H NMR (400 MHz, CDCl 3, Me 4Si) for tert-butyl(z)-(5-hydroxy-2-(4-(3-(trifluoromethyl)-3hdiazirin-3-yl)benzylidene)pentyl)(2-(isoquinoline-5-sulfonamido)ethyl)-carbamate (14) 14 Fig.S10: 13 C NMR (101 MHz, CDCl 3) for tert-butyl(z)-(5-hydroxy-2-(4-(3-(trifluoromethyl)-3h-diazirin- 3-yl)benzylidene)pentyl)(2-(isoquinoline-5-sulfonamido)ethyl)-carbamate (14).
Tert-butyl(E)-(5-azido-2-(4-(3-(trifluoromethyl)-3H-diazirin-3- yl)benzylidene)pentyl)(2-(isoquinoline-5-sulfonamido)ethyl)- carbamate Fig.S11: 1 H NMR (400 MHz, CDCl 3, Me 4Si) for tert-butyl(e)-(5-azido-2-(4-(3-(trifluoromethyl)-3hdiazirin-3-yl)benzylidene)pentyl)(2-(isoquinoline-5-sulfonamido)ethyl)-carbamate. Tert-butyl(E)-(5-azido-2-(4-(3-(trifluoromethyl)- 3H-diazirin-3-yl)benzylidene)pentyl)(2- (isoquinoline-5-sulfonamido)ethyl)-carbamate Fig.S12: 13 C NMR (101 MHz, CDCl 3) for tert-butyl(e)-(5-azido-2-(4-(3-(trifluoromethyl)-3h-diazirin-3- yl)benzylidene)pentyl)(2-(isoquinoline-5-sulfonamido)ethyl)-carbamate.
3 Fig.S13: 1 H NMR (400 MHz, CDCl 3, Me 4Si) for (E)-N-(2-((5-azido-2-(4-(3-(trifluoromethyl)-3H-diazirin- 3-yl)benzylidene)pentyl)amino)ethyl)isoquinoline-5-sulfonamide (3). 3 Fig.S14: 13 C NMR (101 MHz, CDCl 3) for (E)-N-(2-((5-azido-2-(4-(3-(trifluoromethyl)-3H-diazirin-3- yl)benzylidene)pentyl)amino)ethyl)isoquinoline-5-sulfonamide (3).
Tert-butyl(Z)-(5-azido-2-(4-(3- (trifluoromethyl)-3h-diazirin-3- yl)benzylidene)pentyl)(2-(isoquinoline-5- sulfonamido)ethyl)carbamate Fig.S15: 1 H NMR (400 MHz, CDCl 3, Me 4Si) for tert-butyl(z)-(5-azido-2-(4-(3-(trifluoromethyl)-3hdiazirin-3-yl)benzylidene)pentyl)(2-(isoquinoline-5-sulfonamido)ethyl)-carbamate. Tert-butyl(Z)-(5-azido-2-(4-(3- (trifluoromethyl)-3h-diazirin-3- yl)benzylidene)pentyl)(2-(isoquinoline- 5-sulfonamido)ethyl)carbamate Fig.S16: 13 C NMR (101 MHz, CDCl 3) for tert-butyl(z)-(5-azido-2-(4-(3-(trifluoromethyl)-3h-diazirin-3- yl)benzylidene)pentyl)(2-(isoquinoline-5-sulfonamido)ethyl)-carbamate.
4 Fig.S17: 1 H NMR (400 MHz, CDCl 3, Me 4Si) for (Z)-N-(2-((5-azido-2-(4-(3-(trifluoromethyl)-3H-diazirin- 3-yl)benzylidene)pentyl)amino)ethyl)isoquinoline-5-sulfonamide (4). 4 Fig.S18: 13 C NMR (101 MHz, CDCl 3) for (Z)-N-(2-((5-azido-2-(4-(3-(trifluoromethyl)-3H-diazirin-3- yl)benzylidene)pentyl)amino)ethyl)isoquinoline-5-sulfonamide (4).
12 Fig.S19: 1 H NMR (400 MHz, CDCl 3, Me 4Si) for (N-(2-aminoethyl)isoquinoline-5-sulfonamide (12). 12 Fig.S20: 13 C NMR (101 MHz, CDCl 3) for (N-(2-aminoethyl)isoquinoline-5-sulfonamide (12).
Fig.S21: 1 H NMR (400 MHz, CDCl 3, Me 4Si) for 4-(3-(Trifluoromethyl)-3H-diazirin-3-yl)benzaldehyde (9). Fig.S22: 13 C NMR (101 MHz, CDCl 3) for 4-(3-(Trifluoromethyl)-3H-diazirin-3-yl)benzaldehyde (9).