This is the Accepted Manuscript of the following article: Méndez-Ardoy, A., Granja, J., & Montenegro, J. (2018). pH-Triggered self-assembly and hydrogelation of cyclic peptide nanotubes confined in water micro-droplets. Nanoscale Horizons. http://dx.doi.org/10.1039/c8nh00009cThe controlled one-dimensional supramolecular polymerization of synthetic building blocks in confined spaces constitutes a key challenge to simplify the understanding of the fundamental physical principles behind the behavior of more complex encapsulated polymer networks. Cyclic peptide nanotubes constitute an optimal scaffold for the fabrication of hierarchical one-dimensional self-assembled architectures. Herein we report the pH-controlled nanotube formation and fibrillation of supramolecular cyclic peptides in confined aqueous droplets. The externally triggered self-assembly of these peptides gave rise to viscoelastic hydrogels in which the one-dimensional molecular arrangement was perfectly preserved from the nano- to the micro-scale. The cyclic peptide building blocks were confined inside water microdroplets and the base-triggered supramolecular polymerization was externally triggered and followed by confocal microscopy showing that the confined fibrillation spanned and affected the shape of the droplet micro containerThis work was partially supported by the Spanish Agencia Estatal de Investigación (AEI) [SAF2017-89890-R, CTQ2014-59646-R and CTQ2016-78423-R], the Xunta de Galicia
(ED431G/09, ED431C 2017/25 and 2016-AD031) and the ERDF. A. M.-A. received a MCIF from the EC (GLYCONANOPEP-750248).
J. M. holds a Ramón y Cajal (RYC-2013-13784), an ERC-Stg (DYNAP-677786) and a Young Investigator Grant from the HFSP (RGY0066/2017)S

Granja Guillán, Juan Ramón

Montenegro García, Javier

Méndez Ardoy, Alejandro

Nanoscale Horizons

English

Repositorio Institucional da Universidade de Santiago de Compostela

S1Supporting information forpH-Triggered Self-assembly and Hydrogelation of CyclicPeptide Nanotubes Confined in Water MicrodropletsAlejandro Méndez-Ardoy,1 Juan R. Granja*1 and Javier Montenegro*1Centro Singular de Investigación en Química Biolóxica e Materiais Moleculares (CIQUS),Departamento de Química Orgánica, Universidade de Santiago de Compostela, 15782 Santiago deCompostela, SpainTable of contents1. Experimental section.....................................................................................................................S22. Supplementary Figures................................................................................................................. S82.1. Synthesis of CP1 and precursors....................................................................................... S82.2. Assembly studies..............................................................................................................S102.3. Assembly in water-in-oil droplets....................................................................................S162.4. Characterization of cyclic peptides..................................................................................S173. References...................................................................................................................................S19S21. Experimental sectionMaterials and methodsReagents were acquired from Fluka, Aldrich, Iris Biotech or TCI. Egg yolk lysophosphatidylcholine (EYPC) was acquired from Avanti Polard Lipids or Aldrich. Mineral heavy oil wasacquired from Sigma. 1H NMR spectra were acquired in a Varian 300 MHz spectrometer.Chemical shifts (δ) are reported in ppm relative to TMS (δ = 0). All spectra were normalizedwith respect the residual solvent signal. Infrared spectra were acquired in a PerkinElmerSpectrum Two ATR. Analytical HPLC was carried out in an Agilent 1260 Infinity II equippedwith an Agilent SB-C18 column and connected to a 6120 Quadrupole LCMS. HR-MS wasacquired in a Bruker Microtof.AbbreviationsCP: Cyclic peptide, 2CTC: 2-Chlorotrityl chloride, DCM: dichloromethane; DIEA:N,N-Diisopropylethylamine, DMF: N,N-Dimethylformamide, Fmoc:9-Fluorenylmethoxycarbonyl; Mtt: 4-Methyltrityl; N-HATU:N-[(Dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide; N-HBTU: N-[(1H-Benzotriazol-1-yl)-(dimethylamino)methylene]-N-methylmethanaminium hexafluorophosphate N-oxide; HFIP:1,1,1,3,3,3-Hexafluoropropan-2-ol; PyAOP: (7-Azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate; TFA: Trifluoroacetic acid; TFE:2,2,2-Trifluoroethanol; TIS: Triisopropylsilane; UV-vis: Ultraviolet-visible.Synthesis of CP1 precursorCyclic peptides were prepared manually in solid phase. 2-Chlorotrytil chloride resin (2CTC,500 mg, 1.6 mmol Cl-/g resin) were soaked in freshly distilled DCM (4 mL) for 30 min. Thesolvent was filtered off, and a solution of N-α-(9-Fluorenylmethyloxycarbonyl)-L-lysine allylester hydrochloride (178 mg, 0.4 mmol) and DIEA (350 μL, 2 mmol) in freshly distilled DCM(4 mL) was added to the resin. After 2 h, the solvent was filtered off and the resin was washedwith DCM (4 mL). A mixture of DCM-MeOH-DIEA (8.5:1:0.5, 4 mL) was added and theresin was shaken for 1 h, then washed with DCM (3 x 4 mL) and diethyl ether (4 mL). TheS3resin was dried under high vacuum and the loading was determined by quantification of theFmoc group.1 For this, a small portion of the resin (ca 10 mg) was treated with a solution ofpiperidine in DMF (1:4, 2 mL) for 30 min. An aliquot of this solution (20 μL) is diluted to 1mL DMF and the absorbance was read between 290-301 nm. The concentration of thedibenzofulvene-piperidine adduct is obtained by using the extinction coefficients tabulated inthe literature.A portion of resin (0.1 mmol) was used for the synthesis of the peptide. The Fmoc groupwas removed by treatment with piperidine/DMF (1:4, 2-3 mL) for 30 min. The resin waswashed with DMF (6 mL) and then treated with a solution of Fmoc-protected amino acid (0.4mmol), N-HBTU (151 mg, 0.4 mmol) and DIEA (0.1 mL, 0.6 mmol) in DMF (3 mL). Theresin was shaken for 45 min and then washed with DMF (3 mL). The procedure was repeatedwith each corresponding aminoacid (7 cycles). After the coupling of the last amino acid, theresin was washed with DCM (3 x 3 mL) and a solution of PPh3 (39 mg, 0.15 mmol),N-methylmorpholine (110 μL, 1 mmol) and phenylsilane (120 μL, 1 mmol, 10 equiv) in DCM(2 mL) was added, and the mixture was bubbled with argon for 10 min. Then, a solution ofPd(OAc)2 (6.7 mg, 0.03 mmol) in DCM (0.5 mL) was added dropwise and the resin wasfurther bubbled with Ar for 2 min and the suspension was stirred overnight. The resin waswashed with DCM (4 mL), DIEA in DMF (2% v/v, 4 x 4 mL), sodium diethyldithiocarbamate(0.5% w/v in DMF, 4 x 4 mL) and DMF (4 x 4 mL). The resin was stirred withpiperidine/DMF (1:4, 2 mL) for 30 min. Finally, it was washed with DIEA in DMF (2% v/v, 4x 2 mL) and DMF (4 x 4 mL).Cyclization was carried out by adding a solution of PyAOP (208 mg, 0.4 mmol) andDIEA (0.1 mL, 0.6 mmol) in DMF (3 mL) and the suspension was stirred for 12 h. Afterwashing with DMF (3 mL), the reaction was repeated twice in the same conditions. For theselective removal of the 4-methyltrityl group, the resin was washed with DCM (3 x 4 mL),then treated with a mixture of DCM/HFIP/TFE/TIS (6.5:2:1:0.5, 3 mL) and stirred for 1 h.The mixture was filtered off and washed with DCM (3 x 3 mL). The process was carried outagain in the same conditions and washed with DCM (3 x 3 mL) and DMF (3 x 3 mL). Then, asolution of ((tert-butyloxycarbonylaminoxy)acetic acid (48 mg, 0.25 mmol) and N-HATU (94mg, 0.25 mmol) in DMF (1 mL) was added to the resin followed by the dropwise addition ofS4DIEA (42 μL, 0.25 mmol) in DMF (1 mL). The resulting mixture was stirred mechanically for45 min. Finally, the resin was washed with DMF (3 x 3 mL). These deprotection and couplingwith (tert-butyloxycarbonylaminoxy)acetic acid steps were repeated once.The peptide was released from the resin by treatment with freshly prepared TFA cocktail(TFA-DCM-H2O-triisopropylsilane, 0.9:0.05:0.025:0.025, 1 mL/40 mg resin) for 2 h and thenfiltered. The resin was washed with TFA (0.5 mL) and the combined fractions wereevaporated to 1-2 mL by bubbling argon. The concentrated solution was added dropwise tocold diethyl ether (10 mL diethyl ether/ml TFA). The resulting precipitate was centrifuged for10 min at 3000 rpm. The supernatant was discarded, then fresh diethyl ether was added andthe suspension was sonicated and centrifuged. The resulting solid was dried under vacuum.The sample was dissolved in milliQ water and purified by semipreparative HPLC using a C18column [gradient of H2O-0.1% TFA-acetonitrile-0.1% TFA 95:5 (0 min) to 70:30 (20 min)] togive 16.5 mg of 1 (18 %); 1H NMR (300 MHz, D2O) δ: 8.62 (s, 2 H), 7.29, 7.28 (2 soverlapped, 2 H), 4.65 (m, 2 H), 4.62 (s, 2 H), 4.43-4.25 (m, 6 H), 3.88-3.70 (m, 4 H),3.40-3.05 (m, 6 H) 2.97 (t, 2 H, JH,H = 9 Hz), 1.9-1.3 (m, 8 H), 1.32-1.25 (2 d, 6 H, JH,H = 6Hz); Rt = 7.06 min, RP-uHPLC (C18, H2O-acetonitrile (dopped with 0.1% TFA) 100:0 (0 min)→ 25:75 (15 min)); FTIR (neat): 3274 (b), 1669 (m), 1621 (m), 1537 (m), 1432 (m), 1182(m), 1141 (w), 838 (w), 800 (w), 723 (w), 627 (w), 520 (w) cm-1; HRMS calculated forC38H62N15O12+: 920.4697, found: 920.4689.Synthesis of CP1 (Scheme S1)The purified peptide 1 (3.6 mg, 3.92 μmol) was dissolved in a DMSO (100 μL) solutioncontaining of pyrenecarboxyaldehyde (11 mg/mL, 1.1 mg, 4.8 μmol, 1.2 equiv.). The solutionwas stirred at 60 ºC until no starting material was observed by HPLC-MS, typically in 1.5-2 hreaction time. Then, the solution was added to cold diethyl ether (2 mL) and vigorouslyshaken until a yellow precipitate was formed. The organic layer was decanted aftercentrifugation and the solid was sonicated for 10 min with fresh cold diethyl ether (2 mL),centrifuged and decanted. Sonication with diethyl ether and decantation was repeated twice.The solid was dried under vacuum to give 3.8 mg of CP1 (86%); 1H NMR (300 MHz,DMSO-d6) δ: 9.35 (s, 1 H), 8.77 (m, 3 H), 8.40-7.89 (m, 14 H), 7.67 (s, 2 H), 7.23 (2 s, 2 H),S55.05 (m, 1 H), 4.67 (s, 2 H), 4.58 (m, 1 H), 4.30 (m, 4 H), 3.60-2.67 (m, 17 H), 1.72-1.00 (m,14 H); Rt = 9.24 min, RP-uHPLC (C18, H2O-0.1% TFA-acetonitrile-0.1% TFA 95:5 (0 min)→ 5:95 (15 min)); FTIR (neat): 3275 (b), 1670 (m), 1626 (m), 1542 (m), 1204 (w), 1129 (w),844 (w), 799 (w), 722 (w), 625 (w) cm-1; HRMS calculated for C55H70N15O12+: 1132.5323,found: 1132.5327Circular dichroism: Circular dichroism spectra were acquired in a Jasco J-1100 CDspectrometer. Data was collected at 20 ºC in a 2 mm light path quartz cuvette after subtractionof the solvent background signal. In pH titration experiments, a concentration of 0.4 mM CP1was fixed. pH was adjusted by adding small aliquots of NaOH (0.1 M) and the pH wasmeasured using a drop pH-meter. Solutions were equilibrated for 10 min prior measurements.Experiments at a fixed pH and variable CP1 concentration were carried out in HEPES (60mM, pH 8.)Measurement of viscoelastic properties of the gel: Gels of CP1 (2% w/w) were measuredin an Anton Paar MCR-302 rheometer in a parallel plate setup with a gap of 0.5 mm. Infrequency sweep experiments, elastic (G') and viscoelastic (G'') modulus were measured atangular frequencies of 0.1-100 rad/s at a constant shear strain of 5%. All measurements werecarried out at a temperature of 20 ºC.Fluorescence measurements: Fluorescence measurements were carried out in a Varian CaryEclipse fluorescence spectrophotometer. Fluorescence spectra were acquired at 20 ºC with anaveraging time of 0.5 s. Samples were prepared at different pH values and measured with adrop pH meter. Emission measurements were carried out by excitation wavelength of 340 nm.Thioflavin FRET experiments were carried out at a concentration of 20 μM of thioflavin.Epifluorescence and LSCM: Epifluorescence measurements of droplets (see preparationbelow) were carried out in an Olympus BX51 at magnifications of 20x, 40x and 100x.Confocal measurements were carried out in a Leica SP5 exciting at 405 nm and operating at amagnification of 63x. Images were analyzed using ImageJ.2,3 Confocal images of the gel wereacquired from cyclic peptide samples (2% w/w) that were alkalinized with NaOH (0.1 M)until the gel was formed (typically pH around 8). All images were acquired at roomtemperatureS6Transmission and scanning transmission electron microscopy: Cu grids (300 mesh, coatedwith holley carbon grid, Electron _Microscopy Sciences) were used to deposit the samples atconcentration of 0.02% w/w. Samples were stained with uranyl acetate (2%). STEM imageswere acquired in a Zeiss Ultra Plus scanning transmission electron microscope operating at anextra high tension of 20 kV. Transmission electron microscopy images were acquired in anJEOL JEM-1011 operating at 100kV.Formation of gels: For gelification assays, a solution of the peptide in milliQ water (2% w/w,150 μL) was prepared by sonication (pH 3-4). This solution is alkalinized with NaOH (0.1 M)(usually up to pH around 8) until no flow was observed after inversion of the vial.AFM: Atomic force microscopy measurements were carried out in a Park Systems NX-10.ACTA tips were used (silicon tips, nominal values: spring constant = 40 N/m, frequency =300 kHz, ROC less than 10 nm). Aqueous samples at pH 8 of CP1 (15-20 μL, 1.8 mM) weredeposited in freshly cleaved mica. After 15-20 min equilibration, the micas were thoroughlyrinsed with milliQ water and dried with a steam of Ar, then measured immediately. Imageanalysis was carried out with Gwyddion.4 Persistence length was roughly estimated fromAFM images presenting isolated fibers with length longer than 500 nm. After loading theAFM height maps in Easyworm software5, the persistence length was fitted using the meansquare end-to-end model implemented in Easyworm.Preparation hydrophobic glass slides: Hydrophobic glass slides were prepared adapting areported procedure.6 Microscope glass slides were immersed in freshly prepared piranhasolution for 40 min (H2SO4-H2O2 3:1; WARNING: piranha solutions react violently incontact with organic compounds and should be handled with care!). Slides were thoroughlyrinsed with milliQ water, dried with a stream of air and further dried in the oven (65 ºC) forseveral hours. Slides were immersed in a solution of (1H,1H,2H,2H-perfluorooctyl)silane (3mM) in chloroform (HPLC quality) for 20 min. The substrates were rinsed thoroughly withdichlorometane, dried with a stream of air and incubated in the oven for 20 min.Preparation of water-in-oil droplets: Preparation of emulsions of aqueous droplets in oilwere carried out by stirring. Typically, EYPC (10 mg) were dissolved in chloroform (HPLCquality, 1 mL) and a thin film was prepared by rotatory evaporation under reduced pressure.S7The film was further dried at high vacuum for several hours. Heavy mineral oil (5 mL) wasadded to give a phospholipid concentration of 2 mg/mL. The film was sonicated for 1 h andthen equilibrated overnight at room temperature. Oil (200 μL) was mixed with an aqueoussolution of CP1 (1-2% w/w, 1 μL), and the suspension was stirred with a magnetic bar for15-20 min at 700 rpm until a milky suspension was achieved. pH was changed by twodifferent methods; before droplets formation: aqueous solutions of CP1 were diluted withHEPES solution (20 mM, pH 8); after droplets formation: a mixture of Aldrich oil andpropanamine (0.5-1% v/v) was added to the suspension. Images acquired immediately afterdroplet preparation. For this, a small chamber was prepared by mounting hydrophobic slideswith imaging spacers (Secure-Seal Imaging Spacers, 0.12 mm depth, Grace Bio-Labs)S82. Supplementary Figures2.1. Synthesis of CP1 and precursorsScheme S1. Preparation of precursor 1: a) N-α-(9-Fluorenylmethyloxycarbonyl)-L-lysine allylester hydrochloride, DIEA, DCM, 2 h; b) i) piperidine/DMF (1:4), 30 min, ii) amino acid,N-HBTU, DIEA, DMF, 45 min, repeat 7 cycles with the corresponding aminoacid; c) i)Pd(OAc)2, PPh3, phenylsilane, 4-methylmorpholine, DCM, overnight, ii) piperidine/DMF(1:4), 30 min, iii) PyAOP, DIEA, DMF, overnight; d) i) DCM-HFIP-TFE-TIS (6.5:2:1:0.5), 1h, twice ii) [(tert-butoxycarbonyl)aminoxy]acetic acid, N-HATU, DIEA, DMF, 45 min; e)TFA-DCM-H2O-TIS (9:0.5:0.25:0.25).S9Scheme S2. Preparation of CP1.S102.2. Assembly studiesHomodimer formation model:For the equilibriumA+A<−−>A·A (1)with association constantKa=[A·A][A][A](2)Mass balance[A]tot=[A]+2[A·A]=[A]+2Ka[A]2(3)were [A]tot is the total concentration of monomer parted as free monomer and monomer in thecomplex. This be rearranged in the quadratic expression:2Ka[A]2+[A]−[A]tot=0 (4)Which can be analytically solved:[A]=−1+ 1+8Ka[A]tot4Ka(5)At a given wavelength, the ellipticity is given by:Θλ=Bλ[A] l+Cλ[A·A] l (6)where Bλ and Cλ are the molar ellipticity constants for monomer and dimer respectivelyand l is the cell path length. Assuming Bλ = 0, the ellipticity response is given by:Θλ=Cλ l [A·A]=Cλ l Ka[A]2=Cλ l Ka(−1+ 1+8Ka[A]tot4Ka)2 (7)S11Figure S1: Evolution of fluorescence emission at 420 nm upon addition of NaOH (0.1 M) toa solution of CP1 (0.2 mM) in milliQ water. λex = 340 nmFigure S2: IR-ATR of a freeze-dried sample of CP1 (2 % w/w) after alkalinization withNaOH (0.1 M). Base was added until a gel was obtained. The dashed blue box indicates thepresence of intense peaks of the amides I and II at 1623 and 1538 cm−1 respectively.S12Figure S3: a) Normalized absorption (solid lines) and emission (dashed lines) spectra forCP1 (0.2 mM) (red lines, λex = 340 and λem = 420 respectively) in aqueous solutions (slightlyacid) and ThT (10 µM) in water (blue lines, λex = 420 and λem = 500, respectively);Fluorescence spectra of a solution of CP1 (0.1 mM) in the presence (blue lines) or absence(red lines) of ThT b) at pH 3.8; c) at pH 8 in HEPES (20 mM).Figure S4: Absorbance changes after addition of small aliquots of NaOH (0.1 M) to a CP1(0.4 mM) in milliQ water.S13Figure S5: a) Circular dichroism of CP1 at different concentrations in HEPES (60 mM) at pH8; b) fitting to a homodimerization model. A Ka of 4 x 106 M-1 and a molar ellipticity constantof 820 deg cm-2 dmol-1 was obtained.Figure S6: AFM images of CP1 (0.2 % w/w) solutions at different pH deposited on micasurface: a) after dissolving in water (pH ~ 4) b) upon alkalinization (pH ~ 8). Scale bar is 4μm.S14Figure S7: AFM images of CP1 (0.2 % w/w) at pH ca 8 obtained by dilution of the gel: a)High density of nanotubes; b) low density; c) single nanotube. Persistence length wasestimated from the later. Scale bars are 1 μm (a), 500 nm (b and c)Figure S8: Micrographs of CP1 (0.02 % w/w) after dilution of the gel (pH ca 8). Sampleswere stained with uranyl acetate. a) STEM images. Scale bar is 100 nm; b) TEM images.Scale bars are 200 (left), 50 (center) and 50 nm (right).S15Figure S9: Reversibility of fluorescence quenching of CP1 (0.2 mM) upon pH changes (acid,3-4) or basic (ca 8).S162.3. Assembly in water-in-oil dropletsFigure S10: Formation of fibers in water-in-oil emulsions of CP1 (2% w/w); a) dissolved inwater and b) diluted in HEPES (30 mM) at pH 8. Scale bar is 10 µm.S172.4. Characterization of cyclic peptidesFigure S11: a) Chromatogram of 1 after purification; b) MS spectra from the main peak.Figure S12: 1H NMR (300 MHz) of 1 in D2O.S18Figure S13: a) Chromatogram of CP1 after purification; b) MS spectra from the main peak.Figure S14 1H NMR (300 MHz) of CP1 in DMSO-d6.S193. References1 J. Meienhofer, M. Waki, E. P. Heimre, T. J. Lambros, R. C. Makofske and C.-D. Chang, Int. J. Pept.Protein Res., 2009, 13, 35–42; A. Romieu, T. Bruckdorfer, G. Clavé, V. Grandclaude, C. Massif andP.-Y. Renard, Org. Biomol. Chem., 2011, 9, 5337–5342.2 C. A. Schneider, W. S. Rasband and K. W. Eliceiri, Nat. Methods, 2012, 9, 671–675.3 J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C.Rueden, S. Saalfeld, B. Schmid and others, Nat. Methods, 2012, 9, 676–682.4 D. Nečas, P. Klapetek, Cent. Eur. J. Phys. 2012, 10, 181–188.5 G. Lamour, J. B. Kirkegaard, H. Li, T. P. Knowles and J. Gsponer, Source Code Biol. Med., 2014, 9,16.6 J. M. Gorham, A. K. Stover and D. H. Fairbrother, J. Phys. Chem. C, 2007, 111, 18663–18671.

pH-Triggered self-assembly and hydrogelation of cyclic peptide nanotubes confined in water micro-droplets

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