Dynamic Control of 3D Cell Culture via a Chemoselective Photo-Actuated Ligand
Nathan P. Westcott, Wei Luo, Jeffrey Goldstein, and Muhammad N. Yousaf*
Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, NC. 27599-3290,
USA. Department of Chemistry and Centre for Research on Biomolecular Interactions, York University,
Toronto, Ontario, M3J 1P3, Canada
chrchem@yorku.ca
Supporting Information:
Experimental Section
Scheme 1. Synthesis of α-methacrylic -ω-acetoacetate poly (ethylene glycol). Reagents and conditions: i)
TEA, methacrylic anhydride, DCM. rt, 12h, 91%; ii) tert-butyl acetoacetate, toluene, reflux, 12h, 95%.
Scheme 2. Synthesis of γ -3-(4,5-Dimethoxy-2-nitrophenyl)-2-butyl-l-aspartate: Reagents and conditions:
i) NaH, MeI, 0 C, rt, THF, 2h, 98% ii) NaBH4, MeOH, 2h, 98%; iii) HNO3, 1:1 H20 : AcOH, rt, 6h,
98%; iv) 6, α -tert-butyl N-BOC-l-aspartic acid , DCC, DMAP, DCM, rt 12h, 35% v) TFA, DCM, 3h,
98%
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Synthesis of α-methacrylic -ω-acetoacetate poly (ethylene glycol) (I, scheme 1):
Poly (ethylene glycol) methacrylate (2). To a stirring solution of PEG 1500 (1, 1 g, 0.67 mmol) in THF
was added methacrylic anhydride (113 mg, 0.73 mmol) and triethylamine (73 mg, 0.73 mmol). The
reaction was stirred overnight, the solvent was removed in vacuo, and the product was precipitated in
ethyl ether (0.6 mmol, 950 mg) with a 90% yield. 1H NMR (400 MHz, CDCl3, δ): 2 (s, 1H; CH), 3.8 (m,
J=16 Hz, ~140H; -OCH2CH3-), 3.95 (t, J=4 Hz, 2H; OCH2), 5.6 (d, J= 2 Hz, 1H; =CH), 5.85 (d, J=2 Hz,
1H; =CH).
α-methacrylic -ω-acetoacetate poly (ethylene glycol) (I). To a stirring solution of poly(ethylene glycol)
methacrylate (2, 0.7 mmol, 1.1 g) in toluene was added tert-butyl acetoacetate (0.7 mmol, 111 mg) and
was refluxed for 12 h. The solvent was removed in vacuo, and the product was precipitated in ethyl ether
with a 98% yield. (1.15 g, 0.69 mmol) 1H NMR (400 MHz, CDCl3, δ): 2 (s, 1H; CH), 2.4 (s, 3H; CH3),
3.7 (s, 1H; CH), 3.8 (m, J=16 Hz, ~140H; -OCH2CH3-), 4.15 (t, J=4 Hz, 2H; OCH2), 5.6 (d, J= 2 Hz, 1H;
=CH), 5.85 (d, J=2 Hz, 1H; =CH).
Synthesis of γ -3-(4,5-Dimethoxy-2-nitrophenyl)-2-butyl-l-aspartate (II, scheme 2):
3-(3,4-Dimethoxyphenyl)butan-2-one (3). 3,4-Dimethoxyphenylace- tone (3.78 cm3, 21.7 mmol, 1 eq)
was added to a NaH stirring suspension (625 mg, 26 mmol, 1.2 eq) in dry THF (50 mL). After 0.5 h at
room temperature, the solution was cooled to 0 °C, and iodomethane (1.49 mL, 23.8 mmol, 1.1 eq) was
added. After 0.5 h at 0 °C, the reaction mixture was slowly allowed to reach room temperature for 1 h,
and was quenched by a saturated aqueous NaHCO3 solution (150 mL). The aqueous layer was extracted
with EtOAc (3 x 150 mL). The combined organic extracts were dried over anhydrous sodium sulfate,
filtered, and concentrated in vacuo to afford 3 (4.46 g, 21.4 mmol) as a yellow liquid in 98 % yield. 1H
NMR (400 MHz, CDCl3, δ): 1.2 (d, J =8 Hz, 3H; CH3), 1.8 (s, 3H; CH3), 3.69 (q, J=8 Hz, 1H; CH), 3.82
(s, 6H; 2OCH3), 6.55 (m, J=4 Hz, 2H; Ar H), 6.65 (d, 1H; Ar H).
3-(3,4-Dimethoxyphenyl)butan-2-ol (4). 3-(3,4-Dimethoxyphenyl)butan-2-one (717 mg, 3.44 mmol)
was dissolved in 25 mL of methanol and NaBH4 (6.88 mmol) was added. The reaction was stirred for 1.5
h and the solvent was removed in vacuo. The mixture was dissolved in 50 mL ethyl acetate and with with
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3 x 100 mL of saturate aqueous NaHCO3. The organic layer was dried over anhydrous magnesium sulfate
concentrated in vacuo to give 4 (701 mg, 3.37 mmol) as a clear oil in 98% yield. 1H NMR (400 MHz,
CDCl3, δ): 1.2 (d, J =8 Hz, 3H; CH3), 1.45 (d, J=8 Hz, 3H; CH3), 3.69 (q, J=8 Hz, 1H; CH), 3.75 (q, J=8
Hz, 1H; CH), 3.82 (s, 6H; 2OCH3), 4.01 (t, J=4 Hz, 1H; OH), 6.67 (m, J=4 Hz, 2H; Ar H), 7.00 (d, 1H;
Ar H).
3-(4,5-Dimethoxy-2-nitrophenyl)butan-2-ol (5). 4 (700 mg, 3.33 mmol) was dissolved in 20 mL of 1:1
acetic acid:water at 0 °C. 3 mL of 70% nitric acid was added slowly and allowed to stir for 4h. The
reaction was diluted with 130 mL of water and extracted with 5x100 mL of DCM. The combined organic
extracts were dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to give 5 (816 mg,
3.2 mmol) as a yellow liquid in 96% yield. 1H NMR (400 MHz, CDCl3, δ): 1.2 (d, J =8 Hz, 3H; CH3), 1.5
(d, J=8 Hz, 3H; CH3), 3.5 (q, J=6 Hz, 1H; CH), 3.85 (q, J=8 Hz, 1H; CH), 3.82 (s, 6H; 2OCH3), 4.01 (t,
J=4 Hz, 1H; OH), 6.9 (s, 1H; Ar H), 7.2 (s, 1H; Ar H).
α-tert-Butyl γ-3-(4,5-dimethoxy-2-nitrophenyl)-2-butyl N-BOC-l- aspartate (6).
3-(4,5-dimethoxy-2-nitrophenyl)butan-2-ol 5 (300 mg, 1.2 mmol) was dissolved in dry CH2Cl2 (30 mL)
under argon. DCC (500 mg, 2.4 mmol), α -tert-butyl N-BOC-l-aspartic acid (740 mg, 1.8 mmol) and
DMAP (5 mg, cat.) were added. After 12 h at room temperature, the reaction mixture was quenched with a
saturated aqueous NaHCO3 solution (100 mL). The aqueous layer was extracted with CH2Cl2 (3 x 150 mL).
The combined organic extracts were dried over anhydrous sodium sulfate, filtered and concentrated in
vacuo. The residue was purified by flash chromatography (hexane/EtOAc 50:50) to give the product (400
mg, 0.62 mmol) as a yellow solid in 35% yield. 1H-NMR (400MHz, CDCl3, δ): 1.27 (2H, m, J=16 Hz;CH2
β), 1.35 (9H, m, J= 12 Hz; -CH3 OtBu), 2.75 (2H, m, CH2 α), 3.89 (3H, s, OCH3 meta), 3.93 (3H, s, OCH3
para), 4.53 (4H, m, J=8 Hz,; CH benz, CH Fmoc9, CH2 Fmoc), 5.19 (1H, m, J=2 Hz; CH), 5.64 (1H, m,
J=5 Hz; CH α), 7.02 (1H, s, CH, arom6), 7.38 (5H, m, J=8 Hz; CH arom3, CH Fmoc2,3,7,6), 7.59 (2H, d,
J=6.9 Hz; CH Fmoc1,8), 7.76 (2H, d, J=8 Hz; CH Fmoc4,5).
γ-3-(4,5-Dimethoxy-2-nitrophenyl)-2-butyl-l-aspartate (II). tert-butyl
g-3-(4,5-dimethoxy-2-nitrophenyl)-2-butyl N-BOC-l-glutamate (400 mg, 0.62 mmol) was dissolved in
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dry CH2Cl2 (20 mL), trifluoroacetic acid (TFA) (12 mL) was added dropwise at room temperature. The
reaction mixture was diluted with H2O (50 mL) after 5 h at room temperature, and the product was
extracted with EtOAc (3 x 100 mL). The combined organic extracts were dried over anhydrous sodium
sulfate, filtered and concentrated in vacuo to give IV (350 mg, 0.6 mmol, 97 %) as a yellow solid.
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H-NMR (400MHz, CDCl3, δ): 1.4 (2H, m, J=16 Hz;CH2 β), 1.8 (2H, m, CH2 α), 3.89 (3H, s, OCH3
meta), 3.93 (3H, s, OCH3 para), 4.53 (4H, m, J=8 Hz,; CH benz, CH Fmoc9, CH2 Fmoc), 5.19 (1H, m,
J=2 Hz; CH), 5.64 (1H, m, J=5 Hz; CH α), 6.8 (1H, s, CH, arom6), 7.2 (5H, m, J=8 Hz; CH arom3, CH
Fmoc2,3,7,6), 7.4 (2H, d, J=6.9 Hz; CH Fmoc1,8), 7.76 (2H, d, J=8 Hz; CH Fmoc4,5) Calculated
[M+Na+] for C31H32N2O10 = 615.21, Actual [M+Na+] = 615.15.
Coverslip Methacrylation. Glass coverslips (24.5 mm #1.5) were cleaned in boiling acetone for 20
minutes. Then, 50 μL of 3-(trimethoxysilyl)-propyl methacrylate was pipetted onto the coverslips. The
coverslips were placed in a vacuum chamber and allowed to react overnight. After the reaction, the excess
3-(trimethoxysilyl)-propyl methacrylate was rinsed off with ethanol, and the coverslips were dried under
nitrogen.
Hydrogel Polymerization. The generated hydrogels were composed of either 1:0, 95:5, or 90:10 PEGDA
(MW 1623) : KPEGMA (MW 1649). The hydrogel components were dissolved in twice their weight of
pH 8.0 buffer (potassium phosphate monobasic : sodium hydroxide 0.05 M in water) to produce a 1:2
wt%. Each gel was made using 100 μL of buffer and 50 mg of precursor. APS and TEMED were used to
initiate polymerization (1 μL of 10% w/w APS in water, 1 μL of TEMED). The precursor solution and
initiator were then immediately pipetted onto a teflon covered glass slide. The solution was then covered
with a methacrylate-presenting coverslip face down. The hydrogel was cured in vacuo for 45 min.
Solid-Phase Peptide Synthesis. Peptide syntheses of RGDS-oxyamine and photoprotected
GRGDS-oxyamine were performed as previously reported.37 Calculated [M+H+] for C37H60N12O15,
913.44 (photoprotected GRGDS-oxyamine). Actual [M+H+], 913.37. Calculated [M+H+] for
C25H45N11O11, 676.33 (GRGDS-oxyamine). Actual [M+H+], 676.29.
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Ligand Conjugation. Post-polymerized hydrogels were placed gel side down on a 50 μL aliquot of 10
mM ligand solutions and left to react overnight.
Hydrogel Patterning. Once the photoprotected peptide (pRGD) was immobilized to the gel, it was
placed under a Blak-Ray Long Wave UV Lamp (UVP), and a photo-mask was placed on top of the
substrate. The lamp was left on for 1 h, and the hydrogel was submerged in water for 2 h before cell
culture. For global deprotection, the hydrogel substrates in cell media were exposed to UV lamp for 10
min and then replaced in a cell culture hood.
Cell Culture, Fibroblast cells were cultured in Dulbecco’s modified Eagle medium with calf bovine
serum (10%) and penicillin/streptomycin (1%). The cells were released from the culture dish using 0.05%
trypsin in 0.53 mM EDTA.
Cell Spin Down. Fifty-mL centrifuge tubes were filled with polydimethylsiloxane (PDMS) from an
elastomer kit to create a flat surface. The gel substrates were placed in the centrifuge tubes face up. The
cells were released from the flask using 0.05% trypsin in 0.53 mM EDTA and then resuspended in 5 mL
of serum-containing media. One-mL of the resuspended cells was placed into a new cell culture flask with
5 mL of fresh serum-containing media. The remaining unsuspended cells were pipetted into the spin
down tubes. The cells were spun down at 2,000 g. The surfaces were then placed in evaporation-proof
petri dishes with serum-containing media and allowed to grow 1-4 d.
Cell Staining. The gel substrates were placed in phosphate buffered saline (PBS) and allowed to clean for
10 min. The substrates were then placed into 3.2% formaldehyde in PBS for 1 h. The substrate was then
rinsed quickly in PBS and placed in phosphate buffered saline with 1% triton-x 100 (PBS-T) for 1 h. The
substrate was then rinsed in PBS and placed face down on the cell staining solution. The cell staining
solution was 475 μL Goat Serum, 20 μL phalloidin-tetramethylrhodamine B isothiocyanate, and 5 μL
DAPI (4’,6-diamidino-2-phenylindole dihydrochloride) and the substrates were stained overnight.
Confocal Microscopy. The fixed and stained cells were imaged with a LeicaSP2 AOBS upright laser
scanning confocal microscope. Images were taken at 1 m step sizes and processed using Volocity image
processing software.
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Synthesis of PEG 1500 diacrylate. The synthesis was performed following the literature procedure with
PEG 1500.[1]
Supporting References
[1] K. Bott, Z. Upton, K. Schrobback, M. Ehrbar, J. A. Hubbell, M. P. Lutolf, S. C. Rizzi, Biomaterials.
2010, 31, 8454.
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