Photoresponsive supramolecular soft materials in aqueous media
Chen, Shaoyu
DOI:
10.33612/diss.107818650
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Publication date: 2019
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Chen, S. (2019). Photoresponsive supramolecular soft materials in aqueous media. Rijksuniversiteit Groningen. https://doi.org/10.33612/diss.107818650
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Chapter 7
Biocompatible Macroscopic Strings
of Motor Amphiphiles for Cell
Growth
Manuscript in preparation:
Shaoyu Chen, Liangliang Yang, Franco King-Chi Leung,* Takashi Kajitani, Marc C.
A. Stuart, Takanori Fukushima, Chaoxia Wang, Patrick van Rijn* and Ben L.
Abstract: Macroscopic unidirectionally aligned and stimuli-responsive hierarchical
supramolecular structures have much potential in adaptive materials, such as biomedical components and soft actuating systems. However, to control the macroscopic morphological transformation in isotropic soft materials, i.e., organogels and hydrogels, remains highly challenging. Towards the design of artificial muscles and future soft robotic systems with excellent biocompatibility, we present in this chapter a macroscopic soft material based on the hierarchical supramolecular assembled structure of motor amphiphiles (MA). Detailed investigations on the photoisomerization by UV-vis absorption and NMR spectroscopies, analysis of the microscopic assembled structure by electronic microscopy, degree of alignment by X-ray diffraction, the actuation process, and biocompatibility are presented. Macroscopic motor amphiphile strings prepared from a calcium chloride solution provide a high degree of alignment and fast response actuations in water. In preliminary experiments, the MA strings show no significant cytotoxicity to the cell cultures, which cell growth observed on the MA strings surface, indicating the potential in developing future biocompatible photoresponsive soft robotic systems.
7.1 Introduction
Hierarchical supramolecular polymeric structures are commonly found in living systems to serve vital roles in key biological functions, e.g., cell division and movement.1–5 Taking inspiration from nature’s precisely controlled supramolecular
polymerization processes, numerous synthetic supramolecular polymers have been developed with tunable organic functionalization and biomimetic structural transformations.6–15 By implementation of stimuli-responsive motifs, i.e., molecular
machines, into the hierarchical supramolecular polymers, various artificial responsive functions in soft materials can be achieved ranging from the molecular level to macroscopic length-scales.10,13 Stimuli-responsive amphiphiles, at the
microscopic level, have shown the capability for assembling into one-dimensional supramolecular polymers to allow advanced functions, such as supramolecular structural transformation16–24 and control of cell growth.23,25,26 One-dimensional
supramolecular polymers can be assembled hierarchically, at the macroscopic level, into a randomly entangled three-dimensional network, with an excellent stimuli-responsiveness, allowing macroscopic gel-sol physical state transformations27–34 and
isotropic gel contractions.35,36 Alternatively, three-dimensional unidirectionally
aligned hierarchical supramolecular structures can be obtained from one-dimensional supramolecular polymers by various macroscopic alignment methodologies, e.g., shear-flow, shear force.37,38 These high anisotropic soft
materials show important opportunities towards applications in anisotropic actuators,39–42 electronic and optoelectronic materials,43–45 and biomedical
materials.46–48 We have demonstrated the first photoresponsive unidirectionally
aligned hierarchical supramolecular structures of motor amphiphiles (MA) in aqueous media, allowing a photocontrolled macroscopic muscle-like actuation in water and in air.39,41,42 This hierarchical supramolecular approach provides an
alternative methodology to macroscopic actuators based on stimuli-responsive crystals, polymeric gels, and polymeric liquid crystals. The large anisotropic macroscopic structural transformations of MA strings have shown interesting macroscopic soft robotic functions in cargo transport processes and weight lifting behaviors,39,41,42 which remains as highly challenging tasks in isotropic motions of
randomly entangled three-dimensional supramolecular networks. Although various robotic functions have been successfully demonstrated, the biocompatibility of the macroscopic MA strings remains unexplored.
On the basis of the recent developments of the cell alignments in hierarchical supramolecular structures of amphiphiles, reported by Stupp’s group,46–48
biocompatible amphiphiles have shown challenging applications in regenerative biomedical materials in arrhythmia, spinal cord injury and tissue engineered blood vessels.49,50 Low amounts of amphiphiles allow to trap cells into macroscopic gels
with a high anisotropically aligned structure, providing a mimic for circumferential alignment as seen in native arteries.49 In this chapter, we demonstrate a
biocompatible hierarchical supramolecular soft materials of MA and its potential in soft robotic materials. The MA nanofibers are assembled by a shear-flow method in a calcium chloride solution to afford unidirectionally aligned macroscopic strings. The macroscopic strings of MA are actuated upon photoirradiation, while the MA strings show no significant cytotoxicity to the cell cultures, allowing for cell growth onto the surface of the MA strings.
Figure 7.1 Schematic illustrations of (a) the reversible photoisomerization and
thermal helix inversion of molecular motor amphiphiles (MA) and (b) cell growth on the surface of macroscopic MA strings and their photoactuation process.
7.2 Results and Discussion
7.2.1 Molecular design and synthesis
Our earlier molecular motor amphiphiles,39,41 for forming macroscopic strings, were
designed with a dodecyl chain attached to the upper half of a second-generation molecular motor core and two carboxyl end-groups connected with two alkyl-linkers to the lower half of the motor core, which need the addition of 2 equiv of sodium hydroxide to allow their dissolution in aqueous media. Such alkaline condition (pH ~11) maybe unsuitable for employing them in biological systems. In this regard, the carboxyl end-groups in our earlier amphiphiles were replaced by phosphate and
sulfate end-groups, which were anticipated to enable the dissolution of the corresponding molecular motor amphiphiles (MAP1 and MAS1) in mild aqueous
media (pH ~7), enabling potential applications for cell growth (Figure 7.1). The general synthesis is summarized in Scheme 7.1.
Scheme 2.1 Synthesis of motor amphiphiles MAP1 and MAS1.
Compound 1 and 5 were prepared by our reported procedure.39 The key step in the
synthesis of MAP1 and MAS1 is the formation of the central overcrowded olefin bond
by diazo-thioketone coupling. The precursor 3 was obtained by a Williamson ether formation of thioxanthone 1 with alkyl bromide 2 in the presence of K2CO3 in DMF,
followed by conversion into the thioketone 4 with Lawesson’s reagent in toluene. Hydrazone 5 was in-situ oxidized with diacetoiodobenzene in DMF into the corresponding diazo compound, and subsequent addition of the freshly prepared thioketone 4 provided the corresponding episulfide 6. Desulfurization with triphenylphosphine in toluene gave the overcrowded alkene 7. Hydrolysis of 7 in the presence of NaOH in water and THF gave overcrowded alkene 8. The motor amphiphiles were functionalized via two different synthetic pathways. MAS1 was
obtained by a sulfate ester formation of 8 with sulfur trioxide pyridine complex, while the MAP1 was provided by phosphonylation of 8 with diphenyl phosphite and
subsequent hydrolysis. The detailed procedures of synthesis and the structures of all new compounds, unambiguously determined by 1H, 13C NMR and high-resolution
ESI mass spectrometry, are provided in the section of Experimental Data.
7.2.2 Photoisomerization of motor amphiphiles
The photochemical and thermal isomerization steps of MAP1 and MAS1 were
examined by 1H NMR and UV-vis spectroscopy. Essentially identical 1H NMR
signal shifts are observed in CD2Cl2 solutions of MAP1 and MAS1 (Figure 7.2), and
upon extending irradiation time, photostationary states (PSS) with an unstable/stable isomer ratio of 85:15 are formed in the CD2Cl2 solutions of MAP1 and MAS1. In the
UV-vis absorption spectra of CH3CN solutions of MAP1 and MAS1, an increase in
the absorption around 310 nm with a concomitant decrease of the absorption band from 330 nm to 370 nm is observed upon irradiating with 365 nm light, which is essentially identical to that observed in MAC10,39,41 indicating the isomerization from
stable configuration to unstable configuration (Figure 7.3). Additionally, an isosbestic point at 327 nm over the course of irradiation indicates that a selective photoisomerization process occurs (Figure 7.3). The transformation from unstable isomer to stable isomer can be induced by heating. The thermal helix inversion processes of MAP1 and MAS1 in CH3CN solutions were studied by means of Eyring
analysis (Figure 7.4). The activation parameters and half-life of MAP1 and MAS1 are
presented in Table 7.1. For example, a Gibbs free energy of activation (Δ‡Go) of
MAP1 was 102.49 kJ mol–1, which corresponded to a half-life (t1/2) of 40.1 h at 25 °C.
Figure 7.2 Selected parts of 1H NMR spectra (CD
2Cl2, 25 °C, 500 MHz) of (a) MAP1
and (b) MAS1 in stable state (black) and a photostationary state mixture (red)
containing 85% unstable isomers after irradiation. For the proton assignment, see Figure 7.1.
Figure 7.3 UV-vis absorption spectra of (a) MAP1 and (b) MAS1 in CH3CN solutions (6.5x10–5 M) before 365 nm light irradiation (black), upon irradiated for 1 min to 3 min (yellow) and after irradiation (red).
Figure 7.4 Kinetic studies of the thermal helix inversion step of (a) MAP1 and (b) MAS1
in CH3CN solutions (55 °C, 60 °C, 65 °C, 70 °C and 75 °C) by UV-vis absorption spectral changes at 342 nm.
Table 7.1. Activation parameters and half-lives.
Samples t1/2 at 293.15 K (h) Δ‡G° (kJ/mol) Δ‡H° (kJ/mol) Δ‡S° (J/K/mol) MAP1 ~40.1 102.50.9 48.40.7 -181.52.1 MAS1 ~77.3 103.83.8 59.92.8 -147.28.4
7.2.3 Supramolecular assembly of motor amphiphiles
A freshly prepared aqueous solution of MAP1 was heated at 80 °C for 10 min and
cooled down to room temperature to afford a colorless transparent solution, indicating that the MAP1 was soluble up to a concentration of 180 mM. Notably, MAP1 also showed excellent solubility in tris-buffer (pH 7.4) by the identical
preparation as mentioned. A Nile red fluorescence assay (NRFA), which probes the internal hydrophobicity of the assemblies,51 revealed a decrease in blue shift when
diluting beyond 0.01 mM. The critical aggregation concentration (CAC) of MAP1 is
determined as 0.76 μM (Figure 7.5a). The MAP1 assemblies were imaged using
cryogenic transmission electronic microscopy (cryo-TEM) to capture their solution-state morphologies. MAP1 assembles into fibers from hundreds of nanometers to
micrometers in length at 3.9 mM concentration (Figure 7.6a). An aqueous solution of MAS1 was prepared using the identical procedure to that for the preparation of a
solution of MAP1. The maximum solubility of MAS1 in water and tris-buffer (pH 7.4)
were identical (180 mM). A higher CAC is observed for MAS1 (1.51 M, Figure 7.5b)
than the one observed for MAP1 (Figure 7.5a). MAS1 assembles into vesicles of
20~30 nm in diameter at a concentration of 3.9 mM (Figure 7.6b).
Figure 7.5 Nile Red fluorescence assay for the determination of critical aggregation
concentration of (a) MAP1 and (b) MAS1 (concentration: 5.0 x 10–7 to 2.0 mM).
Figure 7.6 Cryo-TEM images of aqueous solutions of (a) MAP1 (3.9 mM, above CAC)
A macroscopic string of MAP1 was prepared according to our reported
procedure.39,41,42 Typically, an aqueous solution of MA
P1 (50 mM) was manually
drawn into an aqueous solution of CaCl2 (150 mM) from a pipette, and a noodle-like
string with an arbitrary length was formed. Scanning electronic microscopy image (SEM) of the string, prepared from the solution of CaCl2, shows arrays of
unidirectionally aligned nanofiber bundles (Figure 7.7a), which is essentially identical to that of the MAC10 string prepared from a CaCl2 solution. No macroscopic
string was observed using the shear-flow method with an aqueous solution of MAS1
(50 mM), i.e., a direct dissolution of MAS1 was observed. The freshly prepared string
of MAP1 from the CaCl2 solution shows uniform birefringence in the direction of the
string long axis in polarized optical microscopy (POM) images (Figure 7.7b), which is essentially identical to POM images of that of the MAC10 string. To provide the
structural parameters and orientational order, i.e., degree of alignment, of the MAP1
nanofibers in the macroscopic strings, we carried out through-view small-angle X-ray scattering (SAXS) measurements. In the 2D SAXS image of the MAP1 string,
prepared from the CaCl2 solution on a sapphire substrate at 25 °C (Figure 7.7c), a
pair of spot-like scatterings is observed in a smaller-angle region (q = 0.1–0.45 nm– 1) (Figure 7.7c, inset), which is due to scattering from the unidirectionally aligned
nanofiber bundles. The diffraction arcs with d-spacing of 6.15 nm (Figure 7.7d), are attributed to the diffraction from (001) plane of a lamellar structure, which is constructed by the unidirectionally aligned nanofibers of MAP1 with ionic interaction
between Ca2+ and phosphites of MA
P1 as interfibrillar interaction. The layer spacing
of the lamellar structure (c = 6.15 nm) of the MAP1 prepared from CaCl2 solution is
longer than that observed in a MAC10 string prepared from the CaCl2 solution (c =
5.48 nm), possibly due to a loose packing between MAP1 nanofibers and Ca2+. The
angular dependency of the peak intensity of the diffraction from the (001) plane converted from the through-view 2D SAXS image of the MAP1 string prepared from
the CaCl2 solution (Figure 7.7c) shows intensity maxima at 0° and 180°. The peak
intensity of the diffraction from the (001) plane was quantified by full-width half-maximum (fwhm) to obtain an ~100° azimuthal angle, with a smaller azimuthal angle representing a larger degree of unidirectional alignment. Given that MAC10
string prepared from the CaCl2 solution showed an ~65° azimuthal angle, the results
indicated that a lower degree of unidirectional alignment was present in the MAP1
string. With the good degree of alignment in the MAP1 string, a fast response
macroscopic photoactuation is expected, indeed, a MAP1 string prepared in a cuvette
containing CaCl2 solution bent towards light source from an initial position of 0° to
a flexion angle (90°), upon 365 nm irradiation for 60 s, with an actuation speed of 1.5 °/s (Figure 7.8).
Figure 7.7 (a) SEM and (b) POM images of a macroscopic aligned string composed
of MAP1 prepared from solutions of CaCl2 (150 mM) under crossed polarizers. The POM images of the string was tilted at 45°, 135°, 225°, and 315° relative to the transmission axis of the analyzer. Scale bar applied for all panels. (c) 2D SAXS image of a macroscopic aligned string composed of MAP1 (inset: enlarged 2D image for q
= 0.1–0.45 nm–1 at 25 °C. (d) 1D SAXS patterns of a macroscopic aligned string composed of MAP1 of 2D SAXS images in (c), showing the diffraction pattern in the
direction perpendicular to long axis of the string.
Figure 7.8 Photoisomerization step of MAP1 and photoactuation of a MAP1 string
prepared from a CaCl2 solution, after irradiation with 365 nm light source for 60 s. Scale bar, 5.0 mm.
7.2.4 Biocompatibility and cell growth onto the macroscopic strings
To investigate the biocompatibility of the motor amphiphiles (MA), including
MAC10 and MAP1, human bone marrow-derived mesenchymal stem cells (hBM-MSCs) were cultured in solutions with different concentrations of MAs and
analyzed using an XTT assay.52 Freshly prepared aqueous solutions of MA C10 (50
mM) or MAP1 (50 mM) were diluted with growth medium to obtain a range of
concentrations from 0.5 μM (below CAC) to 2200 μM (above CAC). A solution mixture of hBM-MSCs (cell density: 2500 cells per well) and growth medium (100 μL, containing MAs at a range of concentration: 0.5 μM, 5 μM and 2200 μM) was placed in 96-well plates. The mixture was incubated at 37 oC at 5% CO
2 for 24 h,
and subsequently the growth media were replaced by the fresh media (100 μL). Freshly prepared solutions (50 μL) of XTT reagent and activation solution (v/v = 50/1) were added to the well plates and incubated at 37 oC in a humidified
atmosphere at 5% CO2 for 3 h. During the incubation time, an orange color is formed,
the intensity of which can be measured by a microplate reader. The intensity of the dye is proportional to the number of metabolically active cells, i.e., the greater the number of metabolically active cells in the well, the greater the activity of mitochondrial enzymes, and the higher the concentration of the dye formed.52
Therefore, the dye absorbance is proportional to the number of cells in each well. The absorbances are plotted as a function of the concentration of motor amphiphiles (Figure 7.9). Compared to the absorbance of the sample without MAs, no significant differences of absorbances in the samples containing MAC10 or MAP1 at a
concentration lower than 5 μM were found, indicating that both MAC10 and MAP1
showed no obvious cytotoxicity to the hBM-MSCs.
Figure 7.9 Cytotoxicity measurements of MAC10 or MAP1 solutions. The human bone
marrow derived mesenchymal stem cells (hBM-MSCs) were cultured in the solutions with different concentrations of MAs and analyzed using an XTT assay.
To provide a deep insight in the biocompatibility of MAC10 and MAP1, preliminary
studies towards the growth conditions of hBM-MSCs onto the material surface of macroscopic strings of MAC10 and MAP1 were performed by confocal fluorescence
microscopy. Freshly prepared macroscopic strings of MAC10 and MAP1 were placed
in 6-well plates and washed with growth media (0.5 mL) for 3 times. Subsequently, the hBM-MSCs (cell density: 2500 cells per well) were seeded into the medium with
MA strings for cell growth. The samples were incubated at 37 oC and 5% CO 2 for
72 h. The hBM-MSCs were fixed with 3.7% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min at room temperature, and subsequently washed 3 times with PBS. Subsequently, the cell membrane was permeabilized with a 0.5% TritonX-100 solution for 3 min. The obtained hBM-MSCs were stained with 4′,6-diamidino-2-phenylindole (DAPI) and tetramethylrhodamine isothiocyanate(TRITC)–phalloidin for the cell nuclei and F-actin, respectively, and observed using a confocal laser scanning microscopy (CLSM).53 The fluorescence
images are showed in Figure 7.10.
Figure 7.10 Fluorescence images of hBM-MSCs growth in the present of
macroscopic strings of MAC10 and MAP1 for 72 h. (a) Schematic illustration of
fluorescence images taken by changing the focus positions from the bottom of the strings, i.e., the surface of the well plate, to the middle and to the surface of the strings. The images of a MAC10 string by changing the focus positions from (b)
bottom, to (c) middle and (d) surface. The images of a MAP1 string by changing the
focus positions from (e) bottom, to (f) middle and (g) surface. hBM-MSCs are stained using phalloidin for F-actin (red) and DAPI for the nucleus (blue), Scale bar: 100 μm. It is generally accepted, with few exceptions, that a small, round cell shape is typically indicative of a cell entering apoptosis,54,55 whereas a well-spread and
aligned cell shape is most often quantified as being in a viable state.53,56,57 As depicted
in Figure 7.10, cells spread with well-defined actin stress fibers onto the surfaces of well plates in the present of the MA strings (Figure 7.10b,e), indicating that both
macroscopic strings of MAC10 and MAP1 show no cytotoxicity. Furthermore,
well-defined actin stress fibers and nuclei of hBM-MSCs are observed by focusing on the surface of MA strings (Figure 7.10d,g), indicating that the hBM-MSCs also adhere and grow onto the surface of the macroscopic strings of MAC10 and MAP1.
The initial results demonstrated that both MAC10 and MAP1 show no significant
cytotoxicity to cell cultures, allowing for cell growth on the MA strings surface. This might be a first step towards the development of biocompatible soft robotic systems operating in living organisms.
7.3 Conclusions
Motor amphiphiles with different charged end groups at the lower half of the motor moiety were synthesized and probed for their self-assembling properties. Nanofibers of MAP1 and vesicles of MAS1 were observed by cryo-TEM. As shown by NRFA, a
lower CAC was observed in MAP1 solution than that of observed in MAS1. By
applying a shear-flow method, macroscopic strings of MAP1 could be prepared from
a calcium chloride solution, while direct dissolution of MAS1 was observed. A high
degree of alignment and fast photoactuations were found for the MAP1 string. In
preliminary experiments, both MAP1 and MAC10 showed no significant cytotoxicity
in the XTT assays, allowing for cell growth on the surface of MAP1 and MAC10
strings. The current approach demonstrates the potential of developing multiple responsive macroscopic actuators with potential for future biocompatible soft robotic systems.
7.4 Acknowledgements
This work was supported financially by the China Scholarship Council (no. 201706790063 to S.Y.C.), the Croucher Foundation (Croucher Startup Allowance to F.K.C.L), the Netherlands Organization for Scientific Research (NWO-CW), the European Research Council (ERC; advanced grant no. 694345 to B.L.F.), the Ministry of Education, Culture and Science (Gravitation program no. 024.001.035), and a Grant-in-Aid for Scientific Research on Innovative Areas “π-Figuration” (no. 26102008 and no. 15K21721) of The Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. The synchrotron XRD experiments were performed at the BL45XU in the 8 with the approval of the RIKEN SPring-8 Center (proposal no. 20160027).
7.5 Experimental Data
7.5.1 Materials and Methods
For the general materials and methods, please refer to section A, Materials and Methods, in the Appendix.
For the Cryo-TEM measurement, Nile Red Fluorescence Assay, please see chapter 6.
Synchrotron Radiation X-ray Diffraction Experiments.39,41,42 Through view
X-ray diffraction (XRD) image of the strings of molecular amphiphiles (MA) were obtained using the BL45XU beamline at SPring-8 (Hyogo, Japan) equipped with an R-AXIS IV++ (Rigaku) imaging plate area detector or with a Pilatus3X 2M (Dectris) detector. The scattering vector, q = 4πsinθ/λ, and the position of incident X-ray beam on the detector were calibrated using several orders of layer reflections from silver behenate (d = 58.380 Å), where 2θ and λ refer to the scattering angle and wavelength of the X-ray beam (1.0 Å), respectively. The sample-to-detector distances for through-view XRD measurements were 2.02 m, respectively. The obtained diffraction patterns and images were integrated along the Debye-Scherrer ring to afford one-dimensional (1D) intensity data using the FIT2D software. The lattice parameters were refined using the CellCalc ver. 2.10 software.
Scanning Electron Microscopy and Polarized Optical Microscopy Analysis.39,41,42 Polarized optical microscopy (POM) was performed on a Nikon
model Eclipse LV100POL optical polarizing microscope. Scanning electron microscopy (SEM) was performed on a Hitachi S-5500 Field Emission SEM (FE-SEM). Preparation of a MA string on a sapphire substrate: When an aqueous solution of MA (50 mM) was manually drawn into an aqueous solution of calcium chloride (CaCl2) (150 mM) from a pipette, a noodle-like string with an arbitrary length was
formed. After removal of the solution of CaCl2, the string was washed with deionized
water (three times), and the resulting string was used directly for POM and XRD experiments. A motor string for SEM was dried in air for 48 h before measurement.
Standardized Preparation and Actuation of Motor Amphiphile Strings in Water.39,41,42 Actuation experiments have been performed in water with standardized
parameters as follows: diameter of the strings (300 ± 30 μm) and length of the strings in water (8.0 ± 2.0 mm) (Figure 7.8). The photoactuation speed was determined from the bending process of a string with a saturated flexion angle of 90° within a particular time. All photoactuation speeds were averaged from three set of actuation experiments and recorded with a Nikon Coolpix A900 Digital Camera. On the basis of the UV-Vis absorption and 1H NMR studies in solution shown in Figures 7.2 and
with a ratio of 85:15 (unstable-MA:stable-MA). Therefore, we expect a similar extent of switching in the MA string when the saturated flexion angle of the MA string is reached.
Cell Culture.53-54 Human bone marrow-derived mesenchymal stem cells (hBM-MSCs) obtained from Lonza were used for the cell experiments. The growth medium
consisted of Alpha modifed Eagle medium (Gibco), 10% (v/v) fetal bovine serum (Gibco), 0.1% (v/v) ascorbic acid 2-phosphate (Sigma) and 2% penicillin/streptomycin (Gibco). Cells were incubated at 37 oC, 5% CO
2. The cells
were harvested at ~ 80 – 90% confluence from T75 culture flasks by trypsin for 3–5 min at 37 °C for further subcultures.
XTT assay.52 The cells were cultivated in a flat 96-well plate. To each well was
added 100 µL of growth medium (containing MA at a range of concentration: 0.5 µM, 5 µM and 2200 µM) and 50 µL of freshly prepared reaction solution. The reaction solutions consisted of XTT reagent solutions and activation solution (v/v = 1/50), which were defrosted in a 37 oC bath immediately prior to use. All the samples
were stored in an incubator at 37 oC and 5% CO
2 for 24 h. The well plates, gently
shaken to evenly distribute the dye, were subjected to absorbance measurement against a background control as a blank with a spectrophotometer (BMG LABTECH, Offenburg, Germany) at the wavelength of 450 – 500 nm.
Cell growth on the surface of MA string.53,56 Freshly prepared macroscopic strings
of MAC10 and MAP1 were placed in 6-well plates and washed with growth media
(0.5 mL) for 3 time. Subsequently, the hBM-MSCs with a density of 2500 cells per well were seeded onto the macroscopic strings for cell growth. All samples were stored in an incubator at 37 oC and 5% CO
2 for 72 h. The hBM-MSCs were fixed
with 3.7% paraformaldehyde (Sigma-Aldrich) in phosphate-buffered saline (PBS) for 20 min at room temperature, and subsequently washed three times with PBS. Next, the cell membrane was permeabilized with 0.5% TritonX-100 (Sigma-Aldrich) solution for 3 min. In addition, 4′,6-diamidino-2-phenylindole (DAPI) and tetramethylrhodamine isothiocyanate(TRITC)–phalloidin were used to stain the cell nuclei and F-actin, respectively. Cells were observed using a LEICA TCS SP2 confocal laser scanning microscopy (CLSM) equipped with a 40 × NA 0.80 water immersion objective.
7.5.2 Synthesis and Characterization Compound 3:
A DMF suspension (20 mL) of compound 1 (180 mg, 0.74 mmol) was added K2CO3 (407 mg, 2.95 mmol) and alkyl
bromide 2 (516 mg, 1.85 mmol) at 25 °C, and the mixture was stirred at 85 °C for 18 h. The reaction mixture was allowed to cool to 25 °C and then poured into water (20 mL). The residue
was washed successively with water (50 mL) and ethyl acetate (20 mL), and dried under reduced pressure. The residue was subjected to column chromatography on SiO2 (ethyl acetate/ pentane; v/v = 1/5, Rf = 0.5) to allow isolation of compound 3
(268 mg, 0.42 mmol, 57% yield) as a pale yellow powder.
1H NMR (400 MHz, CDCl 3) δ (ppm) 8.18 (d, J = 8.0 Hz, 2H), 7.35 (t, J = 8.0 Hz, 2H), 7.06 (d, J = 8.0 Hz, 2H), 4.12 (t, J = 6.4 Hz, 4H), 4.00 (t, J = 6.8 Hz, 4H), 2.00 (s, 6H), 1.95 – 1.81 (m, 4H), 1.64 – 1.46 (m, 8H), 1.44 – 1.21 (m, 20H). 13C NMR (100 MHz, CDCl 3) δ (ppm) 181.4, 172.3, 155.5, 131.1, 129.5, 127.0, 122.4, 114.2, 70.6, 65.7, 30.7, 30.6, 30.4, 30.2, 29.8, 27.2, 27.1, 22.1.
HRMS (ESI): calcd. for C37H52O7S [M+Na] 663.3326, found 663.3302. Compound 6:
A toluene solution (10 mL) of compound 3 (267 mg, 0.42 mmol) was added Lawesson’s reagent (680 mg, 1.7 mmol) and the mixture was stirred at 110 °C for 1 h. After solvent removal in vacuum, the residue was subjected to column chromatography on SiO2 (ethyl acetate/ pentane; v/v = 1/5, Rf = 0.5) to allow isolation
of the thioketone compound 4 (252 mg, 0.38 mmol, 91% yield) as a green solid. Meanwhile, a DMF solution (3 mL) of
compound 5 (164 mg, 0.384 mmol) at –50 oC, a DMF solution (2 mL) of
(diacetoxyiodo)benzene (123.7 mg, 0.384 mmol) was added dropwise. After complete addition, the obtained pale pink solution was stirred at –50 °C for 60 s followed by addition of the thioketone compound 4 (240.2 mg, 0.366 mmol) in THF (2 mL). The reaction mixture was stirred at 25 oC for 2 h. Subsequently, the solvent
was removed using rotary evaporation and the residue was subjected to column chromatography on SiO2 (ethyl acetate/pentane; v/v = 1/9, Rf = 0.5) to allow isolation
of episulfide compound 6 (191 mg, 0.181 mmol, 50% yield) as a colorless oil.
1H NMR (400 MHz, CDCl 3) δ (ppm) 8.61 (d, J = 8.0 Hz, 1H), 7.67 (d, J = 8.0 Hz, 1H), 7.50 (d, J = 8.0 Hz, 1H), 7.37 – 7.27 (m, 2H), 7.20 (t, J = 8.0 Hz, 1H), 6.86 (d, J = 8.0 Hz, 1H), 6.70 (s, 1H), 6.31 (dd, J = 20.0, 8.0 Hz, 2H), 6.12 (t, J = 8.0 Hz, 1H), 4.23 – 4.12 (m, 1H), 4.04 (m, 5H), 3.95 – 3.77 (m, 4H), 2.65 – 2.52 (m, 2H), 2.11 (m, 1H), 2.04 (s, 6H), 1.99 – 1.69 (m, 6H), 1.68 – 1.23 (m, 46H), 1.18 (d, J = 4.0 Hz, 3H), 0.89 (t, J = 8.0 Hz, 3H).
13C NMR (100 MHz, CDCl 3) δ (ppm) 172.2, 168.3, 155.3, 154.5, 153.6, 135.8, 133.5, 132.4, 132.1, 131.5, 130.6, 127.8, 127.0, 125.8, 125.4, 125.2, 125.1, 124.9, 124.3, 123.5, 122.7, 110.9, 110.7, 107.0, 70.6, 70.4, 70.3, 66.3, 65.6, 62.7, 41.6, 36.5, 32.9, 30.7, 30.7, 30.7, 30.6, 30.6, 30.5, 30.5, 30.4, 30.4, 30.4, 30.3, 30.3, 30.2, 30.0, 29.6, 29.6, 27.0, 27.0, 27.0, 26.9, 26.9, 23.7, 22.0, 15.1.
HRMS (ESI): calcd. for C63H88O7S3 [M+H] 1053.5765, found 1053.5748. Compound 7:
To a toluene solution (10 mL) of episulfide compound 6 (191 mg, 0.181 mmol), triphenyl phosphine (95 mg, 0.36 mmol) was added and the mixture stirred at 135 °C for 16 h. The solvent was removed in vacuum and the residue was subjected to column chromatography on SiO2 (ethyl acetate/pentane; v/v =
1/9, Rf = 0.4) to allow isolation of compound 7 (158 mg, 0.155 mmol, 85% yield) as a colorless oil.
1H NMR (400 MHz, CDCl 3) δ (ppm) 7.42 (dd, J = 16.0, 8.0 Hz, 2H), 7.23 (d, J = 8.0 Hz, 1H), 7.14 (d, J = 8.0 Hz, 1H), 7.01 (t, J = 8.0 Hz, 1H), 6.91 (s, 1H), 6.81 (t, J = 8.0 Hz, 2H), 6.32 (t, J = 8.0 Hz, 1H), 6.24 (d, J = 8.0 Hz, 1H), 6.04 (d, J = 4.0 Hz, 1H), 4.19 – 4.07 (m, 3H), 4.04 (m, 6H), 3.98 – 3.81 (m, 2H), 3.72 (dd, J = 12.0, 8.0 Hz, 1H), 3.06 (dd, J = 12.0, 4.0 Hz, 1H), 2.01 (s, 6H), 1.90 (m, 4H), 1.78 (m, 2H), 1.67 – 1.17 (m, 46H), 0.86 (t, J = 4.0 Hz, 3H), 0.74 (d, J = 4.0 Hz, 3H). 13C NMR (100 MHz, CDCl 3) δ (ppm) 172.2, 156.5, 155.6, 153.8, 139.7, 137.3, 136.7, 133.1, 132.8, 132.6, 128.7, 127.4, 127.2, 127.0, 126.4, 126.0, 125.4, 124.8, 123.8, 122.6, 120.5, 110.3, 110.2, 106.0, 70.0, 69.9, 69.7, 65.6, 37.6, 32.9, 31.5, 30.7, 30.6, 30.6, 30.6, 30.5, 30.5, 30.5, 30.5, 30.4, 30.4, 30.3, 30.3, 30.3, 30.2, 30.2, 30.1, 29.6, 27.1, 27.1, 27.0, 26.9, 23.7, 22.0, 19.3, 15.1.
HRMS (ESI): calcd. for C63H88O7S2 [M+H] 1021.6044, found 1021.6017. Compound 8:
To a THF/methanol solution (v/v = 1/1, 6 mL) of compound 7 (158 mg, 0.155 mmol), an aqueous NaOH solution (4 M, 0.34 mL, 1.4 mmol) was added and the mixture stirred at 90 oC for 1
h. The reaction mixture was allowed to cool to 25 °C. The solvent was removed in vacuum and then the residue was washed successively with water (50 mL) and ethyl acetate (20 mL). The organic layer was separated and the water layer was extracted
with ethyl acetate (20 mL). The combine organic layers were washed with brine and dried over Na2SO4. The solvent was removed in vacuum and the residue was
1/19, Rf = 0.5) to allow isolation of compound 8 (142 mg, 0.151 mmol, 98% yield) as a colorless oil. 1H NMR (400 MHz, CDCl 3) δ (ppm) 7.42 (dd, J = 12.0, 8.0 Hz, 2H), 7.23 (d, J = 8.0 Hz, 1H), 7.14 (d, J = 8.0 Hz, 1H), 7.02 (t, J = 8.0 Hz, 1H), 6.91 (s, 1H), 6.81 (t, J = 8.0 Hz, 2H), 6.32 (t, J = 8.0 Hz, 1H), 6.24 (d, J = 8.0 Hz, 1H), 6.04 (d, J = 8.0 Hz, 1H), 4.12 (m, 3H), 4.03 (m, 2H), 3.98 – 3.89 (m, 1H), 3.89 – 3.80 (m, 1H), 3.72 (dd, J = 12.0, 8.0 Hz, 1H), 3.60 (t, J = 8.0 Hz, 4H), 3.06 (dd, J = 12.0, 4.0 Hz, 1H), 1.91 (m, 4H), 1.78 (m, 2H), 1.68 – 1.17 (m, 48H), 0.86 (t, J = 8.0 Hz, 3H), 0.74 (d, J = 8.0 Hz, 3H). 13C NMR (100 MHz, CDCl 3) δ (ppm) 156.5, 155.6, 153.8, 139.7, 137.3, 136.7, 133.1, 132.8, 132.6, 128.7, 127.4, 127.4, 127.1, 127.0, 126.4, 126.1, 125.4, 124.8, 123.8, 122.6, 120.5, 110.3, 110.3, 106.0, 70.0,69.9, 69.7, 64.0, 37.6, 33.8, 32.9, 31.5, 30.7, 30.6, 30.6, 30.6, 30.6, 30.6, 30.6, 30.5, 30.5, 30.4, 30.4, 30.3, 30.3, 30.1, 30.1, 27.1, 27.1, 27.0, 26.8, 23.7, 19.3, 15.1.
HRMS (ESI): calcd. for C59H84O5S2 [M+H] 937.5833, found 937.5812. Motor amphiphile (MAP1):
To a pyridine solution (3 mL) of compound 8 (110 mg, 0.117 mmol), diphenyl phosphite (192 mg, 0.819 mmol) was added and the mixture stirred at 25 oC for 16 h. Subsequently, a solution
of triethylamine/water (v/v = 1/1, 2 mL) was added to the reaction mixture and stirring continued at 25 °C for 1 h. The solvent was removed in vacuum and then the residue was washed successively with water (50 mL) and dichloromethane (20 mL). The organic layer was separated and the water layer was
extracted with dichloromethane (20 mL). The combine organic layers were washed with brine and dried over Na2SO4. The solvent was removed in vacuum and the
residue was subjected to column chromatography on SiO2 (methanol (with 10%
triethylamine)/dichloromethane; v/v = 1/9, Rf = 0.5) to allow isolation of MAP1 (48
mg, 0.0379 mmol, 32% yield) as a pale yellow oil.
1H NMR (400 MHz, CDCl 3) δ (ppm) 7.59 (s, 1H), 7.42 (t, J = 12.0 Hz, 2H), 7.25 (t, J = 8.0 Hz, 1H), 7.15 (d, J = 8.0 Hz, 1H), 7.03 (t, J = 8.0 Hz, 1H), 6.92 (s, 1H), 6.82 (t, J = 8.0 Hz, 2H), 6.33 (t, J = 8.0 Hz, 1H), 6.25 (d, J = 8.0 Hz, 1H), 6.05 (d, J = 8.0 Hz, 2H), 4.20 – 4.09 (m, 3H), 4.08 – 3.98 (m, 2H), 3.98 – 3.90 (m, 1H), 3.86 (m, 5H), 3.73 (dd, J = 12.0, 8.0 Hz, 1H), 3.14 – 2.92 (m, 13H), 1.91 (m, 4H), 1.79 (m, 2H), 1.68 – 1.19 (m, 66H), 0.87 (t, J = 6.7 Hz, 3H), 0.74 (d, J = 6.7 Hz, 3H). 13C NMR (150 MHz, CDCl 3) δ (ppm) 156.5, 155.5, 153.7, 139.6, 137.2, 136.6, 133.0, 132.8, 132.5, 128.6, 127.3, 127.1, 126.9, 126.3, 126.0, 125.4, 125.4, 124.7, 123.8,
122.5, 120.4, 110.3, 110.2, 105.9, 70.0, 69.9, 69.6, 65.0 65.0, 46.3, 37.6, 32.8, 31.8, 31.7, 31.4, 30.6, 30.6, 30.6, 30.6, 30.5, 30.4, 30.4, 30.4, 30.3, 30.3, 30.3, 30.3, 30.1, 30.1, 27.1, 27.1, 27.0, 26.8, 23.6, 19.3, 15.1, 9.4.
HRMS (ESI): calcd. for C59H86O9P2S2 [M-H] 1063.5105, found 1063.5129. Motor amphiphile (MAS1):
A mixture of compound 8 (36 mg, 0.038 mmol), sulfur trioxide pyridine complex (37 mg, 0.23 mmol) and triethylamine solution (0.1 mL) in dichloromethane (6 mL) was stirred at 25
oC for 18 h. The solvent was removed in vacuum and the
residue was subjected to column chromatography on SiO2
(methanol (with 10% triethylamine)/dichloromethane; v/v = 1/9, Rf = 0.5) to allow isolation of MAS1 (23 mg, 0.018 mmol,
47% yield) as a pale yellow oil.
1H NMR (400 MHz, CDCl 3) δ (ppm) 9.58 (s, 2H), 7.42 (t, J = 8.0 Hz, 2H), 7.26 (d, J = 8.0 Hz, 1H), 7.15 (d, J = 8.0 Hz, 1H), 7.03 (t, J = 8.0 Hz, 1H), 6.92 (s, 1H), 6.82 (d, J = 8.0 Hz, 2H), 6.33 (t, J = 8.0 Hz, 1H), 6.26 (d, J = 8.0 Hz, 1H), 6.04 (d, J = 8.0 Hz, 1H), 4.25 – 4.08 (m, 3H), 4.02 (m, 6H), 3.98 – 3.80 (m, 2H), 3.73 (m, 1H), 3.11 (m, 13H), 1.92 (m, 4H), 1.78 (m, 2H), 1.66 (m, 4H), 1.61 – 1.19 (m, 60H), 0.87 (t, J = 8.0 Hz, 3H), 0.74 (d, J = 8.0 Hz, 3H). 13C NMR (150 MHz, CDCl 3) δ (ppm) 156.5, 155.5, 153.7, 139.7, 137.3, 136.7, 133.0, 132.8, 132.5, 128.7, 127.3, 127.2, 127.0, 126.3, 126.0, 125.4, 125.3, 124.7, 123.8, 122.5, 120.4, 110.3, 110.3, 106.0, 70.0, 69.9, 69.7, 69.0 55.7, 47.5, 37.6, 32.8, 31.5, 30.6, 30.6, 30.6, 30.5, 30.5, 30.5, 30.4, 30.4, 30.4, 30.3, 30.3, 30.3, 30.2, 30.1, 30.1, 27.1, 27.0, 26.9, 26.8, 23.6, 19.3, 15.0, 9.6.
HRMS (ESI): calcd. for C59H84O11S4 [M-H] 1095.4812, found 1095.4813.
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