Supplementary Notes. HTS compatible FRET-based conformational sensors clarify membrane receptor activation

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1 Supplementary otes TS compatible FRET-based conformational sensors clarify membrane receptor activation Pauline Scholler 1,2,3,4, David Moreno-Delgado 1,2,3, athalie Guillet-Lecat 1,2,3, Etienne Doumazane 1,2,3, Carine Monnier 1,2,3, Fabienne Charrier-Savournin 4, Ludovic Fabre 1,2,3,4, Cédric Chouvet 4, Stéphanie Soldevila 4, Laurent Lamarque 4, Geoffrey Donsimoni 4, Thomas Roux 4, Jurriaan M. Zwier 4, Eric Trinquet 4, Philippe Rondard 1,2,3,#, Jean-Philippe Pin 1,2,3,#. General Chemicals were obtained from Sigma, Cisbio Bioassays (Codolet, France) or Cisbio US (Bedford, US) and were used without further purification. BC- 2 has been synthetized by AtlanchimPharma using the procedure described in the literature. 1 Solvents were purchased from Carlo Erba and were used without further purification. Reactions were monitored by LC-MS (low resolution mass spectrometry) on Waters alliance 2695 coupled with Micromass ZQ mass spectrometer (ESI+). R-MS was performed with an Applied Biosystems SCIEXQStar Elite using for ionization an (ESI+) mode and a TF for analyses. The analysis was done in triplicate. Melting points were measured using Buchi Melting point B-540. Purifications were carried out by preparative PLC. The analytical PLC (igh-performance liquid chromatography) was performed on a Thermo-scientific Spectra System P1000XR equipped with dioarray detector UV1000. The preparative PLC was performed on a Shimadzu LC-8A equipped with UV/vis detector SPD. Several chromatographic systems were employed for analytical and preparative PLC which are reported on each experimental procedure. 1 ature Chemical Biology: doi: /nchembio.2286

2 Synthesis alotag-lumi4-tb (1) 2 Cl Cl Lumi4-Tb-S DMS DIPEA 55% Tb 3+ Cl alotag-lumi4-tb (1) To Lumi4-Tb-S (Cisbio Bioassays, Codolet or Cisbio US, Bedford) (4.6 µmol) were added a solution of 2-(2-(6-chlorohexyloxy)ethoxy)ethanamine hydrochloride (Promega)(1.46 mg, 5.61 µmol) dissolved in dry DMS (510 µl) then diisopropylethyl amine (Sigma) (23.5 µmol, 4 µl). The mixture was stirred for 1.5 h at room temperature under inert atmosphere. The progress of the reaction was monitored by analytical PLC (System A). After this period, the reaction went to completion. The mixture was diluted with triethylammonium acetate buffer (p 6, 25 mm) (10 ml) and purified by preparative PLC (System B). The fractions collected containing alotag-lumi4-tb (1) were combined and concentrated in vacuo to lead to the desired product identified by MS as 1. (2.55 µmol, 55%). R-MS (ESI+) calculated for C Cl Tb [M+2] 2+, m/z , found System A Column : XBridge (Waters); RP-C 18 ; 3.5 µm ; 4.6 x 100 mm ; λ = nm. Gradient : A / 25 mm triethylammonium acetate buffer (p 6, 25 mm), B / acetonitrile. t = 0 t = 2 min 5 % B ; t = 2 19 min 40 % B ; Flow rate : 1 ml.min -1. Retention time: Lumi4-Tb-S Rt = 11.9 min, alotag-lumi4-tb Rt = 15.4 min. 2 ature Chemical Biology: doi: /nchembio.2286

3 System B Column : XBridge (Waters); RP-C 18 ; 5 µm ; 50 x 150 mm ; λ = 320 nm. Gradient : A / 25 mm triethylammonium acetate buffer (p 6, 25 mm), B / acetonitrile. t = 0 t = 2 min 5 % B ; t = 2 19 min 40 % B ; Flow rate : 100 ml.min -1. Retention time: alotag-lumi4-tb Rt = 16.7 min. CF 3 2 CF 3 i) DCC, S, DMF ii) BC- 2 Me 2 C 2 Et BC- 2 2 BC-MB-TFA (5) BC-MB- 2 (6) Synthetic scheme for compounds 5 & 6 3 ature Chemical Biology: doi: /nchembio.2286

4 BC-MB-TFA (5) CF 3 CF 3 i) DCC, S, DMF ii) BC- 2 C 2 2 BC-MB-TFA (5) To a solution of 4-((2,2,2-trifluoroacetamido)methyl)benzoic acid 2 (400 mg, 1.62 mmol) in dry DMF (14 ml) was added 1,3-dicyclohexylcarbodiimide (400 mg, 1.92 mmol) and -hydroxysuccinimide (221 mg, 1.92 mmol) under inert atmosphere. The mixture was stirred at room temperature for 1 h and the dicyclohexylurea white precipitate was filtered off. To the filtrate was added in one portion aminomethylbenzylcytosine (BC- 2 ) 1 (331 mg, 1.43 mmol) and the mixture was stirred overnight at room temperature under inert atmosphere. The reaction was monitored by PLC using (System D). After this period the reaction went to completion. The solvent was removed in vacuo and the resulting pale yellow solid was triturated with acetonitrile (4 ml), filtered and washed with acetonitrile (4 ml). The resulting white solid (BC-MB-TFA (5)) was pure enough to be used in the next step without further purification. R-MS (ESI+) calculated for C F [M+] +, m/z , found System D Column : Chromolith (Merck); RP-C 18 ; 3.5 µm ; 4.6 x 100 mm ; λ = 280 nm. Gradient : A / 2 0.2% TFA B / acetonitrile. t = 0 t =2 min 5 % B ; t = 2 10 min 15% B ; t = min 50% B; t = min 100% B ; Flow rate : 1 ml.min -1. Retention time: 4-((2,2,2-trifluoroacetamido)methyl)benzoic acid Rt = min, S ester of 4-((2,2,2-trifluoroacetamido)methyl)benzoic acid Rt = min, BC- 2 Rt = 6.82 min, BC-MB-TFA (5) Rt = min. 4 ature Chemical Biology: doi: /nchembio.2286

5 BC-MB- 2 (6) CF 3 2 Me 2 Et BC-MB- 2 (6) To a solution of methylamine, 33% wt. in absolute ethanol (5 ml) was added 5 (264 mg, 0.58 mmol). The resulting white suspension was stirred at room temperature overnight. The reaction was monitored by PLC using System E. After this period the reaction went to completion. The solvent was removed in vacuo and the residue (158 mg) was purified by flash chromatography using Lichroprep RP18 (15-25 µm) with an eluent gradient of triethylammonium acetate buffer p 7, 25 mm / acetonitrile from 100:0 to 84:16 by 3% increment. The fractions collected containing the desired BC- MB- 2 (6) were combined, concentrated in vacuo to give a white solid (61 mg, 29%) identified by MS as 6. Mp 75 C (dec). R-MS (ESI+) calculated for C [M+] +, m/z , found System E Column : Chromolith (Merck); RP-C 18 ; 3.5 µm ; 4.6 x 100 mm ; λ = 280 nm. Gradient : A / 25 mm triethylammonium acetate buffer (p 7, 25 mm), B / acetonitrile. t = 0 t = 2 min 5 % B ; t = 2 10 min 15% B ; t = min 50% B; t = min 100% B ; Flow rate : 1 ml.min -1. Retention time: 5: Rt = 13.1 min, 6: Rt = 4.26 min. 5 ature Chemical Biology: doi: /nchembio.2286

6 CLIP-Lumi4-Tb (2) 2 Lumi4-Tb-S DIPEA DMS 41% Tb CLIP Lumi4-Tb (2) To Lumi4-Tb-S (Cisbio Bioassays, Codolet or Cisbio US, Bedford) (4.53 µmol) was added a solution of 6 (1.8 mg, 4.9 µmol) dissolved in dry DMS (420 µl) then diisopropylethyl amine (Sigma) (59 µmol, 10 µl). The mixture was stirred for 1 h at room temperature under inert atmosphere. The progress of the reaction was monitored by analytical PLC (System A). After this period, the reaction went to completion. The mixture was diluted with triethylammonium acetate buffer (p 6, 25 mm) (9.5 ml) and purified by preparative PLC (System C). The fractions collected containing CLIP-Lumi4-Tb (2) were combined and concentrated in vacuo to lead to the desired product identified by MS as 2. (1.85 µmol, 41%). R-MS (ESI+) calculated for C Cl Tb [M+] +, m/z , found System A Column : XBridge (Waters); RP-C 18 ; 3.5 µm ; 4.6 x 100 mm ; λ = nm. Gradient : A / 25 mm triethylammonium acetate buffer (p 6, 25 mm), B / acetonitrile. t = 0 t = 2 min 5 % B ; t = 2 19 min 40 % B ; Flow rate : 1 ml.min -1. Retention time: Lumi4-Tb-S: Rt = 11.1 min, 2: Rt = min. System C Column : XBridge (Waters); RP-C 18 ; 5 µm ; 19 x 100 mm ; λ = 280 nm. 6 ature Chemical Biology: doi: /nchembio.2286

7 Gradient : A / 25 mm triethylammonium acetate buffer (p 6, 25 mm), B / acetonitrile. t = 0 t = 2 min 5 % B ; t = 2 19 min 40 % B ; Flow rate : 20 ml.min -1. Retention time: 2: Rt = 12 min. CoA-Lumi4-Tb (3) - - Tb S P P Coenzyme A-S P 2 Lumi4-Tb maleimide DMS PIPES 50mM p7 - - Tb 3+ - S P P P 2 CoA Lumi4-Tb (3) To a solution of Coenzyme A-S (Sigma) (5.7 mg ; 7 µmol) in buffer PIPES 50 mm p 6.5 (3 ml) was added a solution of Lumi4-Tb-maleimide (Cisbio Bioassays Codolet or Cis US, Bedford) (7.1 mg, 5 µmol) in dry DMS (1.6 ml). The mixture was stirred for 1 h at room temperature under inert atmosphere. The progress of the reaction was monitored by analytical PLC (System F). After this period, the reaction went to completion. The mixture was diluted with triethylammonium acetate buffer 7 ature Chemical Biology: doi: /nchembio.2286

8 (p 6, 25 mm) (20 ml) and purified by preparative PLC (System G). The fractions collected containing CoA-Lumi4-Tb (3) were combined and concentrated in vacuo to lead to the desired product identified by MS as 3. (1.85 µmol, 38%). R-MS (ESI+) calculated for C P 3 Tb [M+2] 2+, m/z , found System F Column : XBridge (Waters); RP-C 18 ; 3.5 µm ; 4.6 x 100 mm ; λ = 333 nm. Gradient : A / 25 mm triethylammonium acetate buffer (p 6, 25 mm), B / acetonitrile. t = 0 t = 2 min 5 % B ; t = 2 20 min 55 % B ; Flow rate : 1 ml.min -1. Retention time: Lumi4-Tb-maleimide : Rt = 7.4 min, 3 : Rt = 7.9 min. System G Column :XBridge (Waters); RP-C 18 ; 5 µm ; 19 x 100 mm ; λ = 333 nm. Gradient : A / 25 mm triethylammonium acetate buffer (p 6, 25 mm), B / acetonitrile. t = 0 t =2 min 5 % B ; t = 2 19 min 30 % B ; Flow rate : 20 ml.min -1. Retention time: 3 : Rt = min. BG-Atto-465 (4) DIPEA DMS Bg-Atto465 (4) 8 ature Chemical Biology: doi: /nchembio.2286

9 In a 1,5 ml Eppendorf tube a solution of -(4-(((2-amino-9-purin-6- yl)oxy)methyl)benzyl)-4-(aminomethyl)benzamide 2 (BG-MBA- 2, 0.3 mol) dissolved in 100 ml anhydrous DMS), a solution of Atto-465-S (Atto-tec) (0.254 mol dissolved in 100 ml anhydrous DMS) and 3 ml of diisopropylethylamine (DIPEA) were introduced. The reaction mixture was left stirring at ambient temperature for 1.5 hours. The reaction was monitored by PLC on a XbridgeC18, 3.5 mm, 4.6x100 mm column with a gradient of acetonitrile in water containing 0.2% of trifluoroacetic acid. The purification by preparative PLC was carried out on an Xbridge C18, BTM, 19x100 mm column with a gradient of acetonitrile in water containing 0.2% of trifluoroacetic acid. The fraction were collected and concentrated under reduced pressure. A yellow-orange solid (0.205 mol, 80% yield) was obtained corresponding to the desired product, BG-Atto-465 (4); MS (ES+) C [M+ + ] m/z 681, found ature Chemical Biology: doi: /nchembio.2286

10 REFERECES 1. BC- 2 has been prepared according to the procedure described in Gautier, Arnaud; Juillerat, Alexandre; einis, Christian; Correa, Ivan Reis; Kindermann, Maik; Beaufils, Florent; Johnsson, Kai. Chemistry & Biology (2008), 15(2), ((2,2,2-trifluoroacetamido)methyl)benzoic acid and BG-MBA- 2 have been prepared according to the procedure described in Zwier, Jurriaan M.; Bazin, ervé; Lamarque, Laurent; Mathis, Gerard. Inorganic Chemistry (2014), 53(4), ature Chemical Biology: doi: /nchembio.2286