Tuning the properties of penetrating peptides by oxime conjugation

$FFRXQWVDQG5DSLG&RPPXQLFDWLRQVLQ&KHPLFDO6\QWKHVLV Thieme Supporting Information for DOI: 10.1055/s-0036-1588689 © Georg Thieme Verlag KG Stuttgart · New York 2017 ! Tuning the Properties of Penetrating Peptides by Oxime Conjugation Marta Pazoª, Héctor Fernández-Caroª, Juan M. Priegueª, Irene Lostalé-Seijo and Javier Montenegroª* Singular Research Centre in Chemical Biology and Molecular Materials, (CIQUS), Organic Chemistry Department, University of Santiago de Compostela (USC), Santiago de Compostela, Spainª Supporting Information Table of Contents 1. Supporting methods 1.1. Materials and methods S2 1.2. Abbreviations S3 1.3. Synthesis S4 1.3.1. General protocol for the solid phase synthesis S4 1.3.2. Synthesis of AcP(Ox)2 S5 1.3.3. Synthesis of the fluorophore labelled peptide S5 1.3.4. Preparation of AcP(TIso)2 and AcP(TGua)2 S6 1.3.5. Preparation of CFP(TAcet)2, CFP(TGly)2 and CFP(TGua)2 S6 1.4. Circular dichroism S7 1.5. General procedures for vesicle experiments S7 1.5.1. Vesicle preparation S7 1.5.2. DNA transport experiments in vesicles S8 1.6. Cell assays S8 1.6.1. Cell culture and live cell-imaging S8 1.6.2. Uptake quantification by fluorometry S9 1.6.3. Cell viability: MTT assay S10 2. Supporting figures S11 S2! 1. Supporting methods 1.1. Materials and methods Briefly, reagents for synthesis were purchased from Fluka, GL Biochem and Aldrich, amino acid derivatives from Iris Biotech and GL Biochem, Trizma buffer (99.9 %) was from Aldrich, salts of the best grade available were from Scharlau, Panreac and Fischer used as received. 8-Hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS) was from Sigma and p-xylene-bis-pyridinium bromide (DPX) was from Invitrogen. The aldehyde tested (Tgua) was synthesized following reported protocols.1a The CFR8 peptide was prepared as reported elsewhere.1b Egg yolk L-α-phosphatidylcholine (EYPC) was from Avanti Polar Lipids. Deoxyribonucleic acid from herring sperm (Herring DNA) was from Aldrich. All reactions were performed under N2 atmosphere. Unless stated otherwise, column chromatography was carried out on silica gel 60 (Fluka, 40-63 µm). Analytical thin layer chromatography (TLC) was performed in silica gel 60 (Merck, 0.2 mm, F254). Ultra-high-performance liquid chromatography coupled with mass spectrometry (UHPLC-MS) analyses were carried out on Agilent Technologies 1260 Infinity II associated with a 6120 Quadrupole LC-MS using an Agilent SB-C18 column with Solvent A: Solvent B gradients between 5:95, 95:5 (Solvent A: H2O with 0.1 % TFA; Solvent B: CH3CN with 0.1 % TFA). High-performance liquid chromatography (HPLC) preparative purification was carried out on Waters 1525 composed by a binary pump with a dual Waters 2489 detector with a Phenomenex Luna C18(2) 100A column. An Agilent 1200 with an Agilent Luna C18(2) 100A column were used for semi-preparative purifications, with gradients between 95:5 and 5:95 (Solvent A: H2O with 0.1 % TFA; Solvent B: CH3CN with 0.1 % TFA). All the HPLC spectra reported show the absorbance at 222 nm in ordinates axis and the time in minutes in abscises axis. Nuclear Magnetic Resonance (NMR) spectra were recorded on a Bruker 500 MHz spectrometer. Chemical shifts are reported in ppm (δ units) referenced to the following solvent signals: D2O δH 4.79. Accurate mass determinations (HR-MS) using ESI-MS were !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! 1!a)!Priegue, J. M.; Crisan, D. N.; Martínez-Costas, J.; Granja, J. R.; Fernandez-Trillo, F.; Montenegro, J. Angew. Chem. Int. Ed. 2016, 55, 7492–7495. b) Rothbard, J. B.; Kreider, E.; VanDeusen, C. L.; Wright, L.; Wylie, B. L.; Wender, P. A. J. Med. Chem. 2002, 45 (17), 3612-3618.! S3! performed on a Sciex QSTAR Pulsar mass spectrometer and are reported as mass-percharge ratio m/z. Recalculations of concentrations of the labelled peptide (CF extinction coefficient = 81500 M-1 cm-1) were carried out by ultraviolet-visible (UV-VIS) spectra, which were recorded on a Dynamica HALO XB-10 UV-VIS Single Beam Spectrophotometer. Circular Dichroism (CD) measurements were performed with a Jasco J-1100 CD Spectrometer equipped with a Jasco MCB-100 Mini Circulation Bath. Vesicles were prepared with a Mini-Extruder from Avanti Polar Lipids (pore size 100 nm). Peptide/aldehyde incubation for oxime bond formation was performed in 500 µl eppendorf vials in a Thermo-shaker TS-100C Biosan. Fluorescence measurements were performed with a FluoroMax-2 spectrofluorometer (Jobin-Yvon Spex) equipped with a stirrer and a temperature controller. Cell fluorescent images were acquired with an Andor Zyla 4.2 digital camera mounted on a Nikon Eclipse Ti-E fluorescence microscope. Absorbance and fluorescence of cellular extracts were measured using a microplate reader Tecan Infinite F200Pro. [α]D 22 values were recorded on a Jasco P-1030 Polarimeter (reported for concentrations c in g / 100 ml solvent). IR spectra were recorded on a Perkin Elmer Spectrum One FT-IR spectrometer (ATR, Golden Gate, unless stated otherwise) and are reported as wavenumbers ν in cm-1 with band intensities indicated as s (strong), m (medium), w (weak), b (broad). 1.2. Abbreviations Aa: Amino acid; Arg: Arginine; Boc: tert-Butoxycarbonyl; Calcd: Calculated; Cbz: (Benzyloxy)carbonyl; CF: 5(6)-Carboxyfluorescein; DCM: Dichloromethane; DIEA: N,NDiisopropylethylamine; DMF: N,N-Dimethylformamide; DMSO: Dimethylsulfoxide; DPX: p-Xylene-bispyridinium bromide; EYPC: Egg yolk phosphatidylcholine; HFIP: 1,1,1,3,3,3Hexafluoro-2-propanol; HPTS: 8-Hydroxypyrene-1,3,6-trisulfonic acid trisodium salt; HRMS (ESI): High resolution mass spectrometry (electrospray ionization); LUVs: Large unilamellar vesicles; Lys: Lysine; Tris: Tris(hydroxymethyl)aminomethane; Mtt: 4Methyltrytil; N-HATU: N-[(Dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridine-1- S4! ylmethylene]-N-methylmethanaminium-hexafluorophosphate N-oxide; N-HBTU: N-[(1HBenzotriazol-1-yl)-(dimethylamino)methylene]-N-methylmethanaminium hexafluorophosphate N-oxide; Ox: Oxime; Pbf: 2,2,4,6,7-Pentamethyldihydrobenzofuran5-sulfonyl; SPPS: solid phase peptide synthesis; TFE: Trifluoroethanol; TIS: Triisopropylsilane; TNBS: 2,4,6-Trinitrobenzenesulfonic acid. 1.3 Synthesis 1.3.1. General protocol for the solid phase synthesis The peptide was synthesized by manual Fmoc solid-phase peptide synthesis (SPPS)2 using Rink amide solid support (140 mg, load: 0.71 mg/mmol) previously swelled in DMF (peptide synthesis grade) for 30 min and then filtered and washed with DMF (3 x 4 ml, 3 min). Coupling cycle consisted of Fmoc group deprotection with a solution of piperidine in DMF (20%, 2 x 5 ml, 15 min) and DMF wash (3 x 3 ml, 1 min) followed by coupling with α-amino acids (4 equiv), N-HBTU (4 equiv) and DIEA (4 equiv, 0.195 M solution in DMF) for 30 min. Finally the resin was washed with DMF (3 x 3 ml, 1 min). Each peptide coupling and deprotection was monitored employing the TNBS test.3 Once the lineal peptide was finished the acetylation capping of N-terminal group was performed by standard Fmoc deprotection conditions (20% piperidine in DMF (2 x 5 ml, 15 min)) followed by treatment with a solution of 2,6-lutidine/acetic anhydride (1:1, 3 ml). The resin suspension was mechanically shaken for 40 min and washed with DMF (3 x 3 ml, 1 min) and DCM (3 x 3 ml, 5 min). The Mtt protective group was selectively removed by mechanically stirring the resin in a mixture of DCM/HFIP/TFE/TIS (6.5:2:1:0.5, 2 x 3 ml, 3 h). After Mtt deprotection, the resin was washed with DCM (3 x 3 ml, 1 min) and DMF (3 x 3 ml, 10 min). A solution of (Boc-aminooxy)acetic acid (2.5 equiv per free Lysine) and N-HATU (2.5 equiv per free Lysine) in DMF (1 ml) was added to resin followed by the dropwise addition of a solution of a DIEA (2.5 equiv per free Lysine) in DMF (1 ml). The resin was then mechanically stirred for 45 min and finally washed with DMF (3 x 3 ml, 5 min) and DCM (3 x 3 ml, 5 min). The peptides were deprotected and cleaved from the resin !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! 2 Behrendt, R.; White, P.; Offer, J. J. Pept. Sci. 2016, 22, 4-27. 3 Kay, C.; Lorthioir, O.E.; Parr N.J.; Congreve, M.; McKeown, S.C.; Scicinski, J.J.; Ley, S.V. Biotechnol. Bioeng. 2000, 71, 110-8.! S5! by standard TFA cleavage at room temperature (TFA/DCM/H2O/TIS, 90:5:2.5:2.5, 2 h) as shown in the scheme in fig. S1. Then, the mixture was filtered and the peptides were precipitated in ice-cold Et2O and filtered. The precipitate was centrifuged and washed with Et2O (2 x 10 mL). Crude peptides were dissolved in CH3CN/H2O (1:1, 1 mL) and purified using a C18 reverse-phase HPLC [Nucleosil 100-7 C18, H2O (0.1% TFA)/CH3CN (0.1% TFA) with a binary gradient of Solvent A and Solvent B, the collected fractions were lyophilized and stored at -20 °C. Purity and characterization were confirmed by analytical HPLC, 1H NMR and mass spectrometry. 1.3.2. Synthesis of AcP(Ox)2 Following the general protocol for the solid phase synthesis, the AcP(Ox)2 was obtained after HPLC purification with an overall yield of 12 %. Rt = 16.7 min. RP-HPLC [Nucleosil 100-7 C18, H2O (0.1% TFA)/CH3CN (0.1% TFA) 95:5→5:95 (5→35 min), 0:100 (>35 min)]. [α]D22: -6.3 (c = 0.0525 in H2O/CH3CN, 1:1). IR (neat): 3292 (m), 3250 (m), 2957 (m), 2805 (w), 1542 (m), 1451 (w), 1340 (w), 1224(w), 1200 (w), 1178 (m), 1136 (m), 1096 (w), 1012 (w), 836 (w), 725 (w). 1H-NMR (500 MHz, D2O): 4.4-4.1 (m, 17H), 3.3-3.2 (m, 10H), 2.1 (s, 3H), 1.9-1.5 (m, 40H), 1.5-1.4 (m, 11H), 1.0-0.85 (m, 32H). MS (ESI, H2O): 1709 (10, [M+H]+), 855 (50, [M+2H]²+), 571 (100, [M+3H]³+), 428 (10, [M+4H]4+) (Fig. S2). HR-MS (ESI) Calcd for: C71H135N25O14 1709.1026 found: 1709.1021. 1.3.3. Synthesis of the fluorophore labelled peptide Following the general protocol for the solid phase synthesis, CFP(Ox)2 was obtained after the last Fmoc group deprotection with a solution of piperidine in DMF (20%, 2 x 5 ml, 15 min) and DMF wash (3 x 3 ml, 1 min) followed by coupling with 5(6)- carboxyfluorescein (2 equiv), N-HBTU (2 equiv) and DIEA (0.25 equiv) for 4 hours (Fig. S5). The CFP(Ox)2 was directly reacted with the corresponding aldehydes and the resulting oximes were purified by HPLC. S6! 1.3.4. Preparation of AcP(TIso)2 and AcP(TGua)2 5 µL of AcP(Ox)2 (50 mM) in H2O/DMSO/AcOH (1:1:0.05) were mixed with 2 equivalents of the different aldehyde for each aminooxyacetate of the peptide (e.g., 10 µl of 100 mM hydrophobic aldehyde in DMSO and adjusted to a total volume to 100 µl with H2O/DMSO/AcOH (1:1:0.05). The mixture was stirred at 60°C for 1 hour and the resulting O-alkyloximes (upon corresponding dilution) were then used for DNA activation experiments. Oxime-bond formation was confirmed by HPLC-MS. RP-HPLC [Nucleosil 100-7 C18, H2O (0.1% TFA)/CH3CN (0.1% TFA) 95:5→5:95 (5→35 min), 0:100 (>35 min)]. AcP(TGua)2: Rt: 20.5 min; MS (ESI, H2O/CH3CN): 1194 (90, [M+3TFA+2H]2+), 720 (20, [M+TFA+3H]3+), 512 (100, [M+4H]4+) (Fig. S4). AcP(Tiso)2: Rt: 22.5 min; MS (ESI, H2O): 1846 (65, [M+H]+), 924 (100, [M+2H]2+), 616 (80, [M+3H]3+) (Fig. S3). 1.3.5 Preparation of CFP(TGly)2, CFP(TGua)2 and CFP(TAcet) Reactive peptides of CFP(Ox)2 in H2O (5 mM) were mixed with 2 equivalents of TGly (60 mM), TGua (60 mM) and TAcet (60 mM) and adjusted the total volume to 500 µl with H2O/DMSO/AcOH (1:1:0.05). The mixture was stirred at 60°C for 1 hour and the resulting O-alkyloximes were then directly used, after dilution in HKR buffer, in HeLa cells experiments. Oxime-bond formation was confirmed HPLC-MS. RP-UHPLC [SBC18, H2O (0.1% TFA)/CH3CN (0.1% TFA) 95:5→5:95 (5→15 min), 0:100 (>15 min)]. CFP(TGly)2: Rt: 11.1 min; MS (ESI, H2O/CH3CN): 724 (100, [M+3H]3+) (Fig S6). CFP(TGua)2: Rt: 10.5 min; MS (ESI, H2O/CH3CN): 788 (100, [M+3H]3+)], 592 (15, [M+4H]4+), 473 (10, [M+5H]5+) (Fig S7). CFP(TAcet)2: Rt: 12 min; MS (ESI, H2O/CH3CN): 703 (100, [M+3H]3+)], 528 (10, [M+4H]4+) (Fig. S8). S7! 1.4. Circular Dichroism Circular dichroism measurements were carried out with the following settings: acquisition range: 300-190 nm; band width: 1.0 nm; accumulation: 3 scans; data pitch: 1 nm; CD scale 200 mdeg/1.0 dOD; D.I.T. (Data Integration Time): 1 s; scanning mode: continuous; scanning speed: 200 nm/min. Measurements were done from 10 ºC to 60 ºC (data interval: 10 ºC; temp. gradient 5 ºC/min) in a quartz cell of 0.2 cm path length at a final volume of 0.5 mL (H2O or TFE) with a final peptide concentration of 200 µM. The results are expressed as the mean residue molar ellipticity [θ]MRt with units of degrees·cm2·dmol-1 and calculated using the following equation S1. [Ɵ]MRt =!.!!!!Ɵ !·!!·!!"#$%&!!"!!"#$%&"# (deg · cm2 · dmol -1) Equation S1: Formula to calculate the ellipticity. Ɵ is the ellipticity (mdeg), C is the peptide concentration (M) and l is the cell path length (cm). 1.5. General procedures for vesicle experiments 1.5.1. Vesicle preparation A thin lipid film was prepared by evaporating a solution of EYPC (25 mg) in a mixture of MeOH and CHCl3 (1:1, 1 ml) on a rotary evaporator (room temperature) and then in vacuo overnight. The resulting film was hydrated with 1.0 ml buffer (5 mM HPTS, 16.5 mM DPX, 10 mM Tris, 72 mM NaCl, pH 7.4) for more than 30 min, subjected to freeze-thaw cycles (7 times) and extrusions (15 times) through a polycarbonate membrane (pore size 100 nm). Extravesicular components were removed by gel filtration (Sephadex G-50) with 10 mM Tris, 107 mM NaCl, pH 7.4. Final conditions: ~5 mM EYPC; inside: 5 mM HPTS, 16.5 mM DPX, 10 mM Tris, 72 mM NaCl, pH 7.4; outside: 10 mM Tris, 107 mM NaCl, pH 7.4. (S1) S8! 1.5.2. DNA transport experiments in vesicles EYPC-LUV stock solutions (5 µl) were diluted with a buffer (10 mM Tris, 107 mM NaCl, pH 7.4) and placed in a thermostated fluorescence cuvette (25 °C) and gently stirred (total volume 2000 µl; final lipid concentration 13 µM). HPTS efflux was monitored at λ 511 nm (λex 413 nm) as a function of time after addition of oxime-peptide [(20 µl in DMSO) at variable concentrations 0.1-5 µM] at t = 25 s, DNA (20 µl stock solution in buffer, at fixed concentration 70 µM) at t = 50 s and aqueous Triton X-100 (1.2%, 40 µl, 370 µM final concentration) at t = 225 s. Fluorescence intensities were normalized to fractional emission intensity I(t) using equation S2. !(!)=!(!"!!₀) (!!!!₀) (S2) Equation S2: Formula to normalize transport kinetics. I0 = It at DNA addition, I∞ = It at saturation after lysis. 1.6. Cell assays 1.6.1. Cell culture and live cell imaging HeLa cells were grown at 37 ºC, 5 % CO2, in Dulbecco’s Modified Eagle’s Medium (4500 mg/l glucose, L-glutamine, sodium pyruvate and sodium bicarbonate; SigmaAldrich), supplemented with 10 % fetal bovine serum (Sigma-Aldrich) and 1 % of Penicillin-Streptomycin-Glutamine Mix (Fischer). For live cell imaging, HeLa cells grown on glass bottom dishes were washed with HEPES-Krebs-Ringer (HKR) buffer (5 mM HEPES, 137 mM NaCl, 2.68 mM KCl, 2.05 mM MgCl2, 1.8 mM CaCl2, pH 7.4) and incubated for 30 min with 1 µM Hoechst 33342 (ThermoFisher) in HKR to stain the nucleus. This solution was removed and cells were incubated for another 30 min with 4 µM of each (CFP(T)2) diluted in HKR buffer, washed twice with HKR and examined on an epifluorescence microscope (Nikon Eclipse Ti-E). For co-localization studies with lysosomes, cells were washed and then incubated with the different peptides in HKR for 30 min. After three HKR washes, pre-warmed complete culture media was added to the cells and they were incubated for 3 h at 37 ºC. For S15! Fig. S5: General synthetic scheme for the reactive peptide labeled with fluorophore. H2N-RLLAKLARKLARL OxBoc OxBoc OO HO CFHN-RLLAKLARKLARL OxBoc OxBoc COOH HOOC FmocHN 1) 20 % piperidine in DMF 2) 4 equiv L-Fmoc-Aa-OH 4 equiv HBTU in DMF 4 equiv 0.195 M DIEA in DMF A: SPPS elongation (1, 2, 1, 2...) 13 cycles FmocHN-RLLAKLARKLARL Mtt Mtt 1) DCM/HFIP/TFE/TIS (6.5:2:1:0.5) 2) 2.5 equiv BocNHOCH2COOH, 2.5 equiv HATU B: Mtt Cleavage and hydroxyl amine coupling FmocHN-RLLAKLARKLARL OxBoc OxBoc 20 % piperidine in DMF TFA/CH2Cl2/H2O/TIS 90:5:2.5:2.5 E: Resin cleavage and protecting groups removal CFHN-RLLAKLARKLARL Ox Ox CONH2 H2O/DMSO/AcOH CFHN-RLLAKLARKLARL O O O O N N R R C: Fmoc Cleavage D: Carboxyfluoresceine coupling 2 equiv F: Oxime bond formation CONH2 1:1:0.05 Tail S16! Fig. S6: A) Contracted structure of CFP(TGly)2; B) RP-UHPLC [SB-C18, H2O (0.1% TFA)/CH3CN (0.1% TFA) 95:5→5:95 (5→15 min), 0:100 (>15 min)]. Rt 11.1 min. The double peak (both with equal mass) could be due to CF regioisomers; C) ESI-MS. [M+3H]3+! A)! C)! B)! S17! H N NH2 NH2 CONH2 CFHN-RLLAKLARKLARL O O O O NH2 NH2 O O O ON H N H O O H N N O N H O N H NH2 NH2 N H NH2 NH2 CONH2 CFHN-RLLAKLARKLARL Exact Mass: 2364,36 Fig. S7: A) Contracted structure of CFP(TGua)2; B) RP-UHPLC [SB-C18, H2O (0.1% TFA)/CH3CN (0.1% TFA) 95:5→5:95 (5→15 min), 0:100 (>15 min)]. Rt 10.5 min. The double peak (both with equal mass) could be due to CF regioisomers; C) ESI-MS. [M+3H]3+! [M+4H]4+! [M+5H]5+! A)! B)! C)! S18! Fig. S8:A) Contracted structure of CFP(TAcet)2; B) RP-UHPLC [SB-C18, H2O (0.1% TFA)/CH3CN (0.1% TFA) 95:5→5:95 (5→15 min), 0:100 (>15 min)]. Rt 12 min; The double peak (both with equal mass) could be due to CF regioisomers; C) ESI-MS.! [M+3H]3+! [M+4H]4+! B)! C)! A)!