Novel Water-Soluble Near-Infrared Cyanine Dyes: Synthesis, Spectral Properties, and Use in the Preparation of Internally Quenched Fluorescent Probes

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1 ovel Water-Soluble ear-infrared Cyanine Dyes: Synthesis, Spectral Properties, and Use in the Preparation of Internally Quenched Fluorescent Probes Cédric Bouteiller,,, Guillaume Clavé,,, Aude Bernardin,, Bertrand Chipon,, Marc Massonneau, Pierre-Yves Renard, *, and Anthony Romieu *, IRCF, Equipe de Chimie Bio-rganique, UMR 614 CRS, ISA de Rouen et Université de Rouen, 1, rue Lucien Tesnières, Mont-Saint-Aignan Cedex, France and QUIDD, Technopôle du Madrillet, 5, rue Ettore Bugatti, 768 Saint-Etienne du Rouvray, France. * pierre-yves.renard@univ-rouen.fr or anthony.romieu@univ-rouen.fr. Phone: (or 24-15). Fax: IRCF, Equipe de Chimie Bio-rganique QUIDD These authors contributed equally to this work SUPPRTIG IFRMATI S1 Experimental section : Synthesis of thiol-reactive IR5.5-1 derivative 24 and fluorogenic IR5.5-1/IR7.-2 hexapeptide 25. S2 MALDI-TF mass spectrum of probe 25. S3 S4 S5 S6 Absorption spectra of probe 25 in PBS and Et. Emission (Ex. λ = 67 or 75 nm) spectra of probe 25 in PBS and Et. Emission (Ex. λ = 78 nm) spectra of probe 19 in PBS and DMS. Fluorescence emission time course of probe 19 with BSA protein in caspase assay buffer.

2 -S1- Experimental section : Synthesis of thiol-reactive IR5.5-1 derivative 24 and fluorogenic IR5.5-1/IR7.-2 hexapeptide 25. PLC separations. Several chromatographic systems were used for the analytical experiments and the purification steps. Each one of these systems was optimised in order to improve separation conditions. System A: RP-PLC (Thermo ypersil GLD C 18 column, 5µm, 4.6 x 15 mm) with C 3 C and.1% aq. trifluoroacetic acid (aq. TFA,.1%, v/v, p 2.) as the eluents, at a flow rate of 1 ml/min, with the following gradients: A1 : [1% TFA (5 min), linear gradient from to 2% (8 min) and 2 to 8 (27 min) of C 3 C]. Dual UV-Visible detection was achieved at 26 and 68 nm. A2 : [1% TFA (5 min), linear gradient from to 4% (2 min) and 4 to 8% (4 min) of C 3 C]. Triple UV-Visible detection was achieved at 26, 68 and 75 nm. System B: RP-PLC (Waters XTerra MS C 18 column, 5µm, 7.8 x 1 mm) with C 3 C and.1% aq. TFA as the eluents, at a flow rate of 2.5 ml/min, with the following gradients: B1 : [85% TFA (5 min), linear gradient from 15 to 6% (45 min) of C 3 C]. UV detection was achieved at 26 nm. B2 : [8% TFA (5 min), linear gradient from 2 to 8% (6 min) of C 3 C]. UV detection was achieved at 26 nm. B3 : [85% TFA (5 min), linear gradient from 15 to 35% (1 min) and 35 to 8% (45 min) of C 3 C]. UV detection was achieved at 26 nm. Preparation of thiol-reactive IR5.5-1 derivative (24). + + S 3 - IR5.5-1 C 2 TSTU, DIEA, MP S 3-22, quant. yield Cl DIEA, DMF, RT + + S 3-24, 95% SIAB, C 3 C, borate buffer, p 8.1 S 3-23, 75% Synthetic reactions used for the preparation of thiol-reactive IR5.5-1 derivative 24. I 2 (a) Preparation of IR5.5-1 carboxylic acid, Succinimidyl Ester 22. Free carboxylic acid dye IR5.5-1 (6.6 mg, 9.6 µmol) was introduced into a Reacti-Vial TM and dissolved in 14 µl of 2

3 dry MP. 2 µl of a solution of TSTU reagent in dry MP (2.88 mg, 9.6 µmol) and 11.7 µl of DIEA (28.8 µmol) were added and the resulting reaction mixture was protected from light and stirred at room temperature for 1 h. The reaction was checked for completion by RP- PLC (system A1) and the resulting succinimidyl ester 22 was used without further purification. PLC (system A1): t R = 33.6 min (compared to t R = 32.6 min for IR5.5-1 carboxylic acid). (b) Synthesis of IR5.5-1 amine 23. Ethylenediamine dihydrochloride (15 mg, mmol) was dissolved in a mixture of deionised water (.9 ml) and DMF (1 ml). The crude reaction mixture containing the succinimidyl ester 22 and a 1% solution of DIEA in DMF (.9 ml, 537 µmol) were sequentially added and the resulting reaction mixture was was protected from light and stirred at room temperature for 1 h. The reaction was checked for completion by RP- PLC (system A1) and the mixture was evaporated to dryness. The resulting residue was purified by RP-PLC (system B1, 2 injections). The product-containing fractions were lyophilised to give the IR5.5-1 amine 23 as a blue amorphous powder (5.2 mg, yield 75%). PLC (system A1): t R = 29.8 min, purity > 95%. MS (MALDI-TF, positive mode, CCA matrix): m/z [M+] +, calcd exact mass for C S (c) Preparation of IR5.5-1 SIAB derivative 24. IR5.5-1 amine 23 (9.6 µmol) was dissolved in a mixture of C 3 C (25 µl) and borate buffer (14 µl, 5 mm, p 8.1). A solution of SIAB reagent (4.14 mg, 1.3 µmol) in a mixture of C 3 C (4 µl) and borate buffer (35 µl) was added. The reaction mixture was protected from light and stirred at room temperature for 9 min. The reaction was checked for completion by RP-PLC (system A1). Finally, the reaction mixture was quenched by dilution with aq. TFA.1% and purified by RP-PLC (system B2, 2 injections). The product-containing fractions were lyophilised to give the thiol-reactive IR5.5-1 derivative 24 as a blue amorphous powder (9.8 mg, yield 95%). PLC (system A1): t R = 32.4 min, purity > 95%. MS (MALDI-TF, positive mode, CCA matrix): m/z [M+] +, [M+a] +, calcd exact mass for C I 5 6 S Synthesis of Ac-Cys(IR5.5-1)-Asp-Glu-Val-Asp-Lys(IR7.-2)- 2 (25). Peptide Ac- Cys-Asp-Glu-Val-Asp-Lys(IR7.-2) (3.4 mg, 2.3 µmol) was introduced into a Reacti-Vial TM and dissolved in 6 µl of sodium bicarbonate buffer (.1 M, p 8.5). 4 µl of a solution of iodoacetyl derivative 24 (.8 mg,.77 µmol) in DMF was added. The reaction mixture was protected from light and stirred at room temperature for 2 h. The reaction was checked for completion by RP-PLC (system A2) and purified by RP-PLC (system B3, 3 injections, t R = 3. min). The product-containing fractions were lyophilised to give the peptide Ac-Cys(IR5.5-2)-Asp-Glu-Val-Asp-Lys(IR7.-2) as a blue-green amorphous powder. This fluorogenic caspase-3 substrate was found to be not soluble in aqueous buffers even after conversion into the triethylammonium salt. Consequently, stock solution of 25 was prepared in PLC grade water (containing 1% DMS) and UV-Visible quantification was achieved in Et at λ max of the IR7.-2 by using the ε value 171 M - 1 cm -1 (yield after RP-PLC purification: 24%). PLC (system A2): t R = 41.5 min, purity > 95%. UV/Visible (water, 25 C): λ max = 646 (broad) and 81 nm. UV-Visible (Et, 25 C): λ max = 682 and 793 nm. MS (MALDI-TF, positive mode, CCA matrix): m/z [M+] +, [M+a] +, [M+K] +, calcd exact mass for C S

4 -S2- MALDI-TF mass spectrum of the fluorogenic substrate of caspase-3 protease 25 a, in the positive mode, [M+] + : m/z: calcd C S , found Structures of peptide 26 and thiol IR dye Voyager Spec #1[BP = 656.6, 5336] [M+K] + 7 % Intensity [M+] + [M+a] Mass (m/z) a Loss of thiol IR dye 21 occurred during the ionisation process. S S S 3 - S S 21 + Ac-Cys-Asp-Glu-Val-Asp-Lys

5 -S3- Absorption spectra of probe 25 at 25 C (concentration 2.2 µm). (a) in PBS. (b) in Et. (a) (b) 5

6 -S4- Emission spectra of probe 25 at 25 C (concentration 2.2 µm) (a) Ex. λ = 67 nm (in PBS and Et). (b) Ex. λ = 75 nm (in PBS and Et). 1 8 (a) 87.2, Intensity (a.u.) , in Et in PBS Wavelength (nm) 15 (b) 81., Intensity (a.u.) 1 5 in Et in PBS Wavelength (nm) 6

7 -S5- Emission spectra of probe 19 at 25 C (concentration 2.6 µm). Ex. λ = 78 nm (in PBS and DMS) , Intensity (a.u.) in DMS in PBS Wavelength (nm) 7

8 -S6- Fluorescence emission time course of probe 19 (concentration 1. µm) with BSA protein (2 µg, incubation time 18 min) in caspase assay buffer (1 mm acl, 4 mm EPES, 1 mm DTT, 1 mm EDTA, 1% (w/v) sucrose and.1% (w/v) CAPS, p 7.2, 37 C) at 75 nm (Ex. λ = 67 nm) and 85 nm (Ex. λ = 77 nm). 25 Intensity (a.u.) Em. λ = 75 nm Em. λ = 85 nm Time (s) 8