Electronic Supplementary Information. A biologically relevant fluorescent probe to assess the binding of ceramide to the CERT transfer protein

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This journal is The Royal Society of Chemistry 20 Electronic Supplementary Information A biologically relevant fluorescent probe to assess the binding of ceramide to the CERT transfer protein Stéphanie Combemale, Cécile Santos, Frédéric Rodriguez, Virginie Garcia, Chantal Galaup, Céline Frongia, Valérie Lobjois, Thierry Levade, Cécile Baudoin-Dehoux, Stéphanie Ballereau and Yves Génisson Table of contents C NMR spectrum of compound 8 S-2 C NMR spectrum of compound 15 S-15 C NMR spectrum of compound 9 S-3 C NMR spectrum of compound 18 S-16 C NMR spectrum of compound 3 S-4 Superposition of cristallographic pose C NMR spectrum of compound 10 S-5 (2E3O) with docked poses of C16-Cer S-17 C NMR spectrum of compound S-6 Superposition of eleven docked poses of C NMR spectrum of compound 16 S-7 probe 16 S-18 C NMR spectrum of compound 19 S-8 Details of H-bonding calculated using C NMR spectrum of compound 4 S-9 Molegro Virtual Docker S-19 C NMR spectrum of compound 11 S-10 Binding assay of 16, C6-NBD-Cer and C5- C NMR spectrum of compound 14 S-11 DMB-Cer to START domain S-20 C NMR spectrum of compound 17 S-12 Intracellular distribution of the C5-DMB-Cer S-21 C NMR spectrum of compound 5 S- Intracellular distribution of compound 16 S-22 C NMR spectrum of compound 12 S-14 Intracellular distribution of the C5-NBD-Cer S-23 1

This journal is The Royal Society of Chemistry 20 C NMR spectrum (CDCl 3, 75 MHz) of compound 8 8.7 1.6 51.0 33.4 29.2 29.2 29.1 29.0 29.0 28.7 28.5 28.4 26.3 1 2

This journal is The Royal Society of Chemistry 20 C NMR spectrum (CDCl 3, 75 MHz) of compound 9 9.3 114.1 40.6 33.8 30.1 29.6 29.6 29.6 29.5 29.3 29.1 28.9 28.4 26.8 1 3

This journal is The Royal Society of Chemistry 20 C NMR spectrum (CD 3 OD, 75 MHz) of compound 3 8.7 116.5 73.4 61.6 55.8 36.8 32.5 30.3 30.2 30.1 30.0 30.0 29.9 26.5 23.2 14.2 1 4

This journal is The Royal Society of Chemistry 20 C NMR spectrum (CDCl 3, 75 MHz) of compound 10 174.0 155.0 4.1 128.8 79.0 74.5 62.4 54.5 40.6 36.8 32.3 31.9 28.4 22.7 14.1 1 5

This journal is The Royal Society of Chemistry 20 C NMR spectrum (CD 3 OD, 75 MHz) of compound 178.4 4.5 1.1 73.4 62.1 56.5 41.8 37.1 33.2 32.9 23.5 14.2 1 6

This journal is The Royal Society of Chemistry 20 C NMR spectrum (CDCl 3, 75 MHz) of compound 16 174.0 144.3 6.5 4.1 128.9 98.5 74.6 62.5 54.5 44.0 36.8 32.2 31.9 22.7 14.1 1 7

This journal is The Royal Society of Chemistry 20 C NMR spectrum (CDCl 3 +CD 3 OD, 75 MHz) of compound 19 175.8 175.3 175.1 4.6 0.2 73.5 62.8 62.0 61.0 56.5 55.9 47.4 40.9 40.2 37.1 36.5 33.0 32.6 23.3 14.4 1 8

This journal is The Royal Society of Chemistry 20 C NMR spectrum (CDCl 3, 75 MHz) of compound 4 174.1 7.3 116.6 74.6 62.2 54.0 36.7 31.4 25.4 22.4.9 1 9

This journal is The Royal Society of Chemistry 20 C NMR spectrum (CDCl 3, 75 MHz) of compound 11 174.0 156.1 4.0 128.8 79.1 74.4 62.4 54.5 40.6 36.8 32.3 31.9 22.7 14.1 1 10

This journal is The Royal Society of Chemistry 20 C NMR spectrum (CDCl 3 +CD 3 OD, 75 MHz) of compound 14 174.3 3.9 128.7 73.7 61.9 54.5 48.8 36.6 32.2 31.8 22.6 14.0 1 11

This journal is The Royal Society of Chemistry 20 C NMR spectrum (CDCl 3, 75 MHz) of compound 17 174.0 144.3 143.9 6.5 4.1 128.8 98.5 74.7 62.5 54.5 44.0 36.8 32.2 22.7 14.1 1 12

This journal is The Royal Society of Chemistry 20 C NMR spectrum (CDCl 3, 75 MHz) of compound 5 171.7 7.1 116.4 73.5 61.4 54.4 22.9 1

This journal is The Royal Society of Chemistry 20 C NMR spectrum (CDCl 3, 75 MHz) of compound 12 170.7 156.0 4.1 128.8 79.1 74.7 62.4 54.4 40.6 32.2 30.0 29.5 29.5 29.4 29.2 29.1 29.0 28.4 26.8 23.4 1 14

This journal is The Royal Society of Chemistry 20 C NMR spectrum (CD 3 OD, 75 MHz) of compound 15 173.4 4.7 1.2 73.7 62.2 57.1 42.3 33.4 32.9 30.8 30.8 30.7 30.7 30.6 30.5 30.3 28.0 22.9 1 15

This journal is The Royal Society of Chemistry 20 C NMR spectrum (CDCl 3, 75 MHz) of compound 18 170.8 144.3 144.0 6.5 4.1 128.8 98.5 74.7 62.4 54.4 44.0 32.2 29.5 29.4 29.3 29.1 29.0 29.0 28.4 26.9 23.4 1 16

This journal is The Royal Society of Chemistry 20 Superposition of cristallographic pose (2E3O, magenta) with docked poses (light purple, cyan, salmon) of C16-ceramide. These results show a good agreement in reproducing conformation and hydrogen bond network at polar head level. Calculation was done in 2E3N cavity (light blue) after structural alignments and gives RMSD of 1.19, 1.05, 1. Angstroms respectively. Calculation was done using Molegro templates (H donor, H acceptor, steric) relative to cristallographic ceramide and flexible residues (45 residues around cavity). 17

This journal is The Royal Society of Chemistry 20 Superposition of eleven docked poses of probe 16 in clipped 2E3N cavity (light blue) corresponding to putative conformations with fluorophore found outside cavity. The pose shown in Fig. 3B is highlighted (blue sticks). 18

This journal is The Royal Society of Chemistry 20 Details of H-bonding calculated using Molegro Virtual Docker ID Donor Energy Length Additional Info 0 (TYR 553) OH O1 (Ligand) -2.5 2.6562 Donor rotatable;;[aa-a-d: 8.106 (%)] 1 (Ligand) N1 OE1 (GLU 446) -2.5 2.76944 ;[AA-A-D: 158.639 (%)];[D-H-A: 152.709 (%)] [AA-A-H: 151.152 (%)] 2 (Ligand) O2 OE1 (GLU 446) -2.5 2.8515 Donor rotatable;;[aa-a-d: 127.954 (%)] 3 (GLN 467) NE2 O2 (Ligand) -2.5 2.80439 ;[AA-A-D: 126.524 (%)];[D-H-A: 43.4826 (0%)] [AA-A-H: 0.659 (%)];[D-H-A: 151.047 (%)] [AA-A-H: 116.454 (%)] 4 (Ligand) O3 OD1 (ASN 4) -2.5 3.01814 Donor rotatable;;[aa-a-d: 122.144 (%)] 5 (THR 447) OG1 N3 (Ligand) -1.66026 2.49923 Donor rotatable; 19

This journal is The Royal Society of Chemistry 20 Binding assay of 16, C6-NBD-Cer and C5-DMB-Cer to START domain 75 Unbound Bound 25 0 Buffer + 16 16 Buffer + C6-NBDceramide C6-NBD-ceramide Buffer + C5-DMBceramide START + C5-DMBceramide 20

This journal is The Royal Society of Chemistry 20 Intracellular distribution of the C5-DMB-Cer C5-DMB-Cer T0 C5-DMB-Cer T30 min CHO LYA CHO LYA hcert CHO LYA CHO LYA hcert Filtre GFP Obj 40x 21

This journal is The Royal Society of Chemistry 20 Intracellular distribution of compound 16 Probe 16 T0 Probe 16 T30 min CHO LYA CHO LYA hcert CHO LYA CHO LYA hcert Filtre GFP Obj 40x 22

This journal is The Royal Society of Chemistry 20 Intracellular distribution of the C6-NBD-Cer C6-NBD-Cer T0 C6-NBD-Cer T30 min CHO LYA CHO LYA hcert CHO LYA CHO LYA hcert Filtre GFP Obj 40x 23

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