Supplementary Information: Click-click chemistry on a peptidic scaffold for the easy access to tetrameric DNA structures

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1 Supplementary Information: Click-click chemistry on a peptidic scaffold for the easy access to tetrameric DNA structures Romaric Bonnet, a Pierre Murat, a Nicolas Spinelli, a and Eric Defrancq* a a Département de Chimie Moléculaire UMR CNRS 55, Université Joseph Fourier BP 5, 8 Grenoble cedex 9, France; eric.defrancq@ujf-grenoble.fr General details:...s General procedure for peptide synthesis:...s Oligonucleotides and conjugates synthesis:...s7 Circular Dichroism studies:...s5 NMR Studies:...S Abreviations: CD: Circular Dichroism, CuAAC: Copper Catalyzed Alkyne-Azide Cycloaddition, DCM: Dichloromethane, DIEA: Diisopropylethylamine, DMF: Dimethylformamide, DMT: Dimethoxytrityl, EDTA: Ethylenediaminetetraacetic acid, ESI -MS: Electrospray Ionisation Mass Spectrometry, Fmoc: Fluorenylmethyloxycarbonyl, HEPES: -(-hydroxyethyl)-- piperazineethanesulfonic acid, MALDI: Matrix Assisted Laser Desorption Ionisation, NMR: Nuclear Magnetic Resonance, PyBOP: benzotriazol--yl-oxytripyrrolidinophosphonium hexafluorophosphate, RP-HPLC: Reverse-Phase High Performance Liquid Chromatography, SAX-HPLC: Strong Anion Exchange High Performance Liquid Chromatography, SEC: Size Exclusion chromatography, TFA: Trifluoroacetic Acid, THAP:,,6-trihydroxyacetophenone, THPTA: Tris-(hydroxypropyltriazolylmethyl)amine, TIS: Triisopropylsilane, TNBS:,,6-trinitrobenzenesulfonic acid, UV: Ultra Violet, ToF: Time of Flight. General details: ESI mass spectra were performed on an Esquire spectrometer from Bruker. Peptides were analyzed in positive mode and oligonucleotides and conjugates in negative mode. MALDI ToF mass spectrum of was performed on Voyager DE mass spectrometer (Perseptive Biosystems) equipped with an N laser (7 nm). Analyses were operated in THAP matrix in presence of ammonium citrate buffer after a treatment with DOWEX 5 W S

2 X8 resin (ammonium form). All solvents and reagents used were of highest purity commercially available. S

3 General procedure for peptide synthesis: The course of reactions were monitored on RP-HPLC using a HPLC system on a Nucleosil C8 column ( Å, 5 x mm, 5 µm) with UV monitoring at nm and 5 nm. A ml/min flow linear gradient from 95% solvent A (.% TFA in water) and 5% solvent B (. % TFA in acetonitrile/water: 9/) to % B for minutes was applied. RP-HPLC purifications were performed on a Nucleosil C8 column ( Å, 5 x mm, 7 µm ) with UV monitoring at nm and 5 nm using a ml/min flow linear gradient from 95% solvent A and 5% solvent B to % solvent B for min.. Linear peptide 8 Peptide 8 was synthesized using the Fmoc-tBu protocol using Fmoc-Gly-SASRIN ( g, loading of.8 mmol/g) in a glass reaction vessel fitted with a sintered glass. Fmoc- Lys(biotin)-OH, Fmoc-Gly-OH, Fmoc-Ala-OH Fmoc-Pro-OH and Fmoc-Lys(Boc)-OH were commercially available. Fmoc-azidonorleucine was obtained using the reported protocol. The following protocol was used for each amino acid coupling: Fmoc protecting group was first removed using three washing (for, 5 and 5 min) with % piperidine in DMF ( ml). The resin loading was monitored by quantification of free dibenzofulvene using UV absorbance at 99 nm. Each coupling reaction was operated using the classical protocol with amino acid ( eq) in DMF (ml) with PyBOP ( eq) as activator. ph was adjusted to 8-9 with DIEA. The completion of the coupling reaction was analyzed using TNBS test after washing the resin with DMF ( x ml) and dichloromethane ( E. D. Goddard-Borger, and R. V. Stick, Org. Lett. 7, 9, S

4 ml). Deprotections and coupling reactions were performed until obtaining the supported NH - free peptide 8. The resin was washed with a % TFA in dichloromethane solution ( x ml) for cleaving the peptide from the resin. Each fraction was collected and neutralized with DIEA. The solution was evaporated under vacuum and the peptide was precipitated with ether to obtain a yellow powder. t r =.6 min. ESI MS(+) m/z calcd: 57.8, found: 57.8 [M+H] +.. Cyclic peptide 9 Peptide 8 (.5 mmol, 76 mg) was dissolved in DMF (5 ml) and PyBOP ( mmol, 5 mg) was added. The ph was adjusted to 8-9 using DIEA and the solution was stirred at room temperature until the complete peptide cyclisation (RP-HPLC monitoring). The solvent was evaporated under vacuum then the crude peptide was precipitated with ether to obtain a yellow powder. t r =.6 min. ESI MS(+) m/z calcd: 99.8, found: 99. [M+H] +.. N-ε-free peptide Peptide 9 (.5 mmol, 75 mg) was treated a TFA/DCM/H O/TIS (5/5/.5/.5) solution ( ml) and stirred at room temperature (h). The solvent was evaporated under vacuum and the crude peptide was precipitated with ether to obtain a yellow powder. t r = 9.6 min ESI MS(+) m/z calcd: 99.6, found: 99.8 [M+H] +.. Protected aminooxy peptide a Peptide (. mmol, 6 mg) was dissolved in anhydrous DMF ( ml). N- Hydroxysuccinimidyl -(-ethoxy-ethylidenaminooxy)acetate (.5 mmol, mg) was then added. ph was adjusted to 8-9 with DIEA. The solution was stirred at room temperature and monitored by RP-HPLC until the completion of the reaction (h). The solvent was evaporated and the peptide was precipitated with ether. The product was purified on RP- HPLC to obtain a white powder (8 mmol, mg, yield: %). t r =. min. ESI MS(+) m/z calcd: 585.9, found: 586. [M+H] + S. Foillard, M. O. Rasmussen, J. Razkin, D. Boturyn, and P. Dumy, J. Org. Chem., 8, 7, S

5 (M+) + / (M-Acm) + / Figure S: ESI mass spectrum of compound a (ESI MS(+) m/z calcd: 585.9),8,6,,,,8,6,,, -, Figure S:, RP-HPLC chromatogram of crude compound a (λ abs =nm). solvents a a, Figure S: RP-HPLC chromatogram of compound a (λ abs = nm). 5. Aminooxy peptide b Peptide a (. mmol, 58 mg) was dissolved in a TFA/DCM/H O/TIS (5/5/.5/.5) solution (5 ml) and the reaction was stirred for one hour at room temperature. The peptide was next precipitated with ether and finally, it was purified on RP-HPLC and freeze dried. b was obtained as a white powder. (. mmol, 6 mg, yield: %). t r = 9. min. ESI MS(+) m/z calcd: 5.7, found: 5.9 [M+H] +. S5

6 (M+) + / M + Figure S: ESI mass spectrum of compound b (ESI MS(+) m/z calcd: 5.7),,,8,6,,, -, Figure S5: b RP-HPLC chromatogram of purified compound b (λ abs = nm). S6

7 Oligonucleotides and conjugates synthesis: Oligonucleotides were synthesized by β-cyanoethylphosphoramidite chemistry using a DNA synthesizer at µmol scale. RP-HPLC analysis was performed on a HPLC system using C8 Nucleosil column (Macherey-Nagel, 5 x.6 mm, Å, 5 µm) with ml/min flow linear gradients of solvent A (5 mm triethylammonium acetate buffer with 5% acetonitrile) and solvent B (acetonitrile with 5% water) with UV-monitoring at 6 nm and 8 nm. Gradients start from % solvent A to % B for minutes. RP-HPLC purifications of oligonucleotides were performed on Nucleosil C-8 column (5 mm x mm, Å, 7µm) using ml/min flow linear gradients with solvent A and B with UV-monitoring at 6 nm and 8 nm. Strong Anion Exchange HPLC (SAX-HPLC) analyses and purifications were performed on a Nucleogel anion exchange column (SAX 5 x.6mm, Å, 8 µm) with monitoring at 6 nm and 8 nm. ml/min flow linear gradient starts from % solvent A (Tris buffer 5 mm ph 8 with % methanol) to 6% solvent B (Tris buffer 5 mm,.5 M NaCl with % methanol) for minutes. Desalting of oligonucleotide was performed by SEC on NAP 5 cartridge using the recommended protocol. Quantification of oligonucleotides was performed at 6 nm (ε= 56 L.mol -.cm -, estimated according to the nearest neighbour model).. -diol oligonucleotide Oligonucleotide was obtained from automated synthesis on a -glyceryl CPG resin at µmol scale. After synthesis cyanoethyl protecting groups were removed using % piperidine in acetonitrile. Cleavage from the resin and deprotections was performed in 8% NH OH for 6h at 55 C. The product was purified on RP-HPLC with a gradient from % to 5% solvent B in solvent A for min. 5 -DMT group was then removed using 8% aqueous acetic acid solution for 5 min at room temperature. After freeze-drying, the residue was diluted in water and washed 5 times with diethyl ether. (78 nmol, yield 78%). t r =.7 min. ESI MS (-): m/z calcd: 856., found: 855. [M-H] - S7

8 (M-) - / (M-) - / Figure S6: ESI mass spectrum of compound (m/z calcd: 856.),5,,,,, Figure S7: RP-HPLC chromatogram of purified compound (λ abs =6 nm).. -aldehyde oligonucleotide Sodium metaperiodate ( µmol, 86 µg) was added to a solution of oligonucleotide ( nmol) in water ( µl). The mixture was stirred for h at room temperature in dark conditions. Excess of NaIO was then removed by SEC. Crude oligonucleotide was used in the next step without further purification. (7 nmol, yield 86%). t r =.7 min. ESI MS(-) m/z calcd: 8., found: 8. [M-H] -. (M-) - / Figure S8: ESI mass spectrum of compound (ESI MS(-) m/z calcd: 8.) S8

9 ,,,8,6,,, Figure S9: RP-HPLC chromatogram of crude compound (λ abs =6 nm).. 5 -alkyne oligonucleotide Oligonucleotide was obtained from automated synthesis using commercially available 5 - hexynyl (β-cyanoethyl) phosphoramidite at µmol scale. Cleavage from the resin and subsequent deprotection were performed in 8% ammoniac solution for 6h at 55 C. The crude was purified on RP-HPLC with a gradient from % to 5% solvent B in solvent A for min (85 nmol, yield 85%). t r = 5. min. ESI MS(-) m/z calcd: 86., found: 86. [M-H] -. (M-) - / Figure S: ESI mass spectrum of compound (ESI MS(-) m/z calcd: 86.),,,,8,6,,, -, Figure S: RP-HPLC chromatogram of purified compound (λ abs =6nm). S9

10 . Oligonucleotide 7 Oligonucleotide 7 was obtained from automated synthesis at µmol scale. Cleavage from the resin and deprotections were performed in 8% ammoniac solution for 6h at 55 C. The product was purified on RP-HPLC with a gradient from % to 5% solvent B in solvent A for min. 5 -DMT group was removed using 8% aqueous acetic acid solution for 5 min at room temperature. After freeze-drying, the residue was diluted in water and washed 5 times with diethyl ether. The oligonucleotide was desalted using NAP 5 cartridge. (579 nmol, yield 58%). t r =. min. ESI MS(-) m/z calcd: 7., found: 7. [M-H] -. (M-) - / Figure S: ESI mass spectrum of compound 7 (ESI MS(-) m/z calcd: 7.),7 7,6,5,,,,, -, Figure S: RP-HPLC chromatogram of compound 7 (λ abs =6nm). 5. Conjugates synthesis. a. Strategy (oxime ligation following by CuAAC reaction) Oxime ligation (compound 5: Oligonucleotide (7 nmol) was dissolved in. M ammonium acetate buffer (ph.5, µl) and aminooxy peptide b (7 nmol, µg) was added. The solution was stirred at 55 C S

11 for h then the crude was purified on RP-HPLC with a gradient from % to 5% solvent B in solvent A for min ( nmol, yield: 59%). t r = 9.8 min ESI MS(-) m/z calcd: 56., found: 557. [M-H] -. (M-) - / (M-) - / Figure S: ESI mass spectrum of compound 5 (ESI MS(-) m/z calcd: 56.),,5,,5,,5, Figure S5: RP-HPLC chromatogram of crude conjugate 5 (λ abs =6 nm). 5,,8,6,,, Figure S6: RP-HPLC chromatogram of conjugate 5 (λ abs =6 nm). CuAAC reaction (compound ) To a solution of oligonucleotide (7 nmol) and conjugate 5 (5 nmol) in mm HEPES buffer (ph 7., µl) solution was added CuSO (88 nmol, µg), THPTA (75 nmol, 7 µg) and sodium ascorbate (5 nmol, 7 µg). The reaction was stirred at room temperature for h. Excess of.5 M EDTA (ph 8) solution ( µl) was added and after min the solution was desalted by SEC. was then purified by using SAX purification (6 nmol, yield 5%). t r =.6 min ESI MS(-) m/z calcd: , found: 8788.[M-H] -. S

12 Sodium ascorbate Figure S7: SAX-HPLC chromatogram of crude compound obtained via the strategy 5 (λ abs =6 nm). b. Strategy (CuAAC reaction following by oxime ligation) CuAAC reaction (compound 6) To a solution of oligonucleotide (5 nmol) and protected aminooxy peptide a (75 nmol, 9 µg) in HEPES buffer (ph 7., µl) was added CuSO (88 nmol, 6 µg), THPTA (75 nmol, 5 µg) and sodium ascorbate (75 nmol, 9 µg). The mixture was stirred at room temperature for h..5 M EDTA (ph8) solution (µl) was added and after min the solution was desalted by SEC. 6 was purified on RP-HPLC with a gradient from % to 8% solvent B in solvent A for min (7 nmol, yield 6%). t r =. min. ESI MS(-) m/z calcd: 5.5, found: 5. [M-H] -. (M-) - / (M-) - / Figure S8: ESI mass spectrum of compound 6 (ESI MS(-) m/z calcd: 5.5), 6,5,,5, Figure S9: RP-HPLC chromatogram of crude conjugate 6 (λ abs =6 nm). S

13 ,,5,,5,,5, -, Figure S: RP-HPLC chromatogram of purified conjugate 6 (λ abs =6 nm). 6 Oxime ligation (compound ): Conjugate 6 ( nmol) was dissolved in % TFA aqueous solution ( µl) and the solution was stirred for minutes for oxyamine deprotection. The product was freeze dried. Oligonucleotide (6 nmol) in. M ammonium acetate buffer (ph.5, µl) was next added. The solution was stirred at 55 C for h and purified by SAX-HPLC (5 nmol, yield: 9%). t r =.6 min. ESI MS(-) m/z calcd.: , found: 8788., Deprotected conjugate 6,5, Figure S: RP-HPLC chromatogram of deprotection of conjugate 6 (λ abs =6 nm) Figure S: SAX-HPLC of crude compound obtained via the strategy (λ abs =6 nm). S

14 % Intensity [M-H] [M-H] - / 9.6 [M-ODN-H] Mass (m/z) Figure S: MALDI-ToF mass spectrum of conjugate (m/z calcd: ). Figure S: ESI mass spectrum of compound (m/z calcd: ) Figure S5: SAX-HPLC of purified compound (λ abs =6nm). S

15 Circular Dichroism studies: Compounds was annealed by heating at 9 C for 5 min in PBS buffer ( mm phosphate buffer containing.7 M KCl and. M NaCl, ph adjusted at ph,ph 5, ph 6,pH 7 and ph 8 by HCl or NaCl) and cooling slowly at room temperature. A control was carried with oligonucleotide 7 in the same conditions. Spectra were recorded on a Jasco J-8 circular dichroism spectropolarimeter using. mm or cm length quartz cuvette. Spectra were recorded every 5 C in a range from 5 to 8 C with a wavelength range from to nm (only to nm was shown). For each temperature, the spectrum was an average of three scans with a.5 s response time, a nm data pitch, a nm bandwidth and a nm.min - scanning speed. Blank spectra of buffer were subtracted for each measure. Measures were obtained with a concentration of 7.5 µm for and with a concentration of µm for 7. Melting temperatures were obtained using Boltzmann fit on Origin software. Each curve fit was only accepted with a r value >.99. ph 7 C 57. C 5 C 56 C 6 Nd. 7 C 7 Nd. C Table : Melting temperature of templated system and intermolecular i-motif 7 depending of the ph. S5

16 A) B) CD signal (mdeg) CD signal (86nm) Wavelenght (nm) temperature ( C) Figure S6: CD analyses of at ph from 5 C to 8 C (7.5 µm in PBS buffer): A) superposition of CD spectra. Arrows indicate the sense of the signal decrease. B) CD melting curve at 86 nm ( : experimental results, curve: Boltzmann fit) A) B) CD signal (mdeg) CD signal (86nm) Wavelenght (nm) temperature ( C) Figure S7: CD analyses of at ph 5 from 5 C to 8 C (7.5 µm in PBS buffer): A) superposition of CD spectra. Arrows indicate the sense of the signal decrease. B) CD melting curve at 86 nm ( : experimental results, curve: Boltzmann fit) A) B) CD signal (mdeg) CD signal (86nm) Wavelenght (nm) temperature ( C) Figure S8: CD analyses of at ph 6 from 5 C to 8 C (7.5 µm in PBS buffer): A) superposition of CD spectra. Arrows indicate the sense of the signal decrease. B) CD melting curve at 86 nm ( : experimental results, curve: Boltzmann fit) S6

17 A) B) 5 5,,5 CD signal (mdeg) - -,5 -, Wavelenght (nm) temperature ( C) Figure S9: CD analyses of at ph 7 from 5 C to 8 C (7.5 µm in PBS buffer): A) superposition of CD spectra. Arrows indicate the sense of the signal decrease. B) CD melting curve at 86 nm ( : experimental results, curve: Boltzmann fit). CD signal (86nm),,5,,5, CD signal (mdeg) Wavelenght (nm) Figure S: CD analyses of at ph 8 from 5 C to 8 C (7.5 µm in PBS buffer). Superposition of CD spectra. Arrows indicate the sense of the signal decrease. S7

18 A) B) 5,5, CD signal (mdeg) CD signal (8.5nm),5,,5 -, -, Wavelenght (nm) temperature ( C) Figure S: CD analyses of 7 at ph from 5 C to 8 C ( µm in PBS buffer): A) superposition of CD spectra. Arrows indicate the sense of the signal decrease. B) CD melting curve at 85 nm ( : experimental results, curve: Boltzmann fit). A) B),6, CD signal (mdeg) - CD signal (8.5nm),,,8,6,,,,8 -, Wavelenght (nm) temperature ( C) Figure S: CD analyses of 7 at ph 5 from 5 C to 8 C ( µm in PBS buffer): A) superposition of CD spectra. Arrows indicate the sense of the signal decrease. B) CD melting curve at 85 nm ( : experimental results, curve: Boltzmann fit). S8

19 A) B) CD signal (mdeg) - CD signal (mdeg) Wavelenght (nm) C), - Wavelenght (nm),5 CD signal (mdeg),,5, -,5 Wavelenght (nm) Figure S: CD analyses of 7 at ph 6 (A), ph 7 (B) and ph 8 (C) from 5 C to 8 C ( µm in PBS buffer). This is the superposition of CD spectra. Arrows indicate the sense of the signal decrease. Any melting curves can not be obtained at 85 nm. S9

20 Normalized CD signals A) B),,9,8,7,6,5,,,,, -, -, -, C) wavelenght (nm) D) Normalized CD signals Normalized CD signals E), -,,,,9,8,7,6,5,,,,, -, -, -, -, -,5 wavelenght (nm),,,9,8,7,6,5,,,,, -, -, -, -, -,5 -,6 -,7 -,8 wavelenght (nm),,9,8,7,6,5,,,,, -, -, -, -, Figure S: Comparison of normalized CD spectra at 5 C of (7.5 µm in PBS buffer, in red) and 7 ( µm in PBS buffer, in black): A) ph, B) ph 5, C) ph 6, D) ph 7, E) ph 8. Normalized CD signals Normalized CD signals,,,,9,8,7,6,5,,,,, wavelenght (nm) -, -, -, -, wavelenght (nm) S

21 NMR Studies: A) B) ph. C) D) ph 7. ppm ppm Figure S5: NMR spectra of at ph (A, B) and 7 (C,D): A) zoom of imino region, B) full spectrum, C) zoom of imino region, D) full spectrum. NMR spectra were recorded at 5 MHz in mm PBS buffer containing mm NaCl. S