A unique aliphatic tertiary amine chromophore: fluorescence, polymer ...
Supporting information
A unique aliphatic tertiary amine chromophore: fluorescence, polymer structure and application in cell imaging Miao Sun, Chun-Yan Hong, Cai-Yuan Pan * CAS Key Laboratory of Soft Matter Chemistry, Department of Polymer Science and Engineering, University of Science and Technology of China, Hefei, Anhui, 230026, P. R. China.
Experimental section Materials Triethanolamine, thionyl chloride, thiourea, dichloromethane (≥99. 5%), methanol (≥99, 5%), sodium bicarbonate (≥96%), diisopropylamine chloroform (analytical grade) and anhydrous magnesium sulfate with analytical grade were purchased from Sinopharm Chemical Reagent Co. Ltd. and used as received. Glycidyl methacrylate (GMA, ≥98%), 1, 3-propanedithiol (≥98%), tetrabutylammonium bromide (≥98%), N-methyldiethanolamine (≥99%, Aladdin Reagent), mercaptoethanol containing ~10 mol% bis(2-hydroxyethyl) disulfide were used as received. Ethylene glycol (≥98%, Aladdin Reagent) was dried by refluxing in toluene to remove water. Tetrahydrofuran (≥99%), chloroform (≥99%), acetone (≥99.5%) were purchased from Sinopharm Chemical Reagent Co. Ltd, and acryloyl chloride (≥96%, Aladdin Reagent) was purified by distillation under reduced pressure before use. Characterization
S1
Nuclear Magnetic Resonance (NMR). Structures of the synthesized compounds were characterized by 1H and
13
C NMR spectra on a Bruker 400 MHz NMR spectrometer using
CDCl3 , DMSO-d6 or D2O as solvent. Size exclusion chromatography (SEC) measurements. Molecular weight and molecular weight distribution were obtained by triple detection size exclusion chromatography (TD-SEC) detection at 25oC. The instrumentation consists of the following: a Waters 1515 isotratic HPLC pump with 5 µm Waters styragel columns (guard, 0.5 HR, 1 HR, 3 HR, 4 HR and 5 HR, the molecular weight ranges of these columns are 0-1000, 100-5000, 500-30000, 5000-500000 and 50000-4000000 g/mol, respectively); a Waters 717 PLUS autosamples; a waters 2414 differential refractive index (DRI) detector, the wavelength is 880 nm; a multi angle laser light scattering (MALLS) detector (Wyatt mini Dawn TRISTRA light scattering, three detection angles are 45o, 90o and 135o, the wavelength and power are 690 nm and 220 w); a Wyatt Visco Star viscometer detector; a Waters Breeze data manager. The eluent was HPLC grade THF delivered at 1.0 mL / min. The refractive index increment (dn/dc) was determined using a Wyatt Optilab REX (λ=640 nm) interferometric differential refractometer in bath model at 25 ºC. Measurement of fluorescence spectra. The fluorescence spectra of the resultant polymers were
recorded
at
room
temperature
on
a
Perkin-Elmer
LS55
luminescence
spectrophotometer. Fluorescence microscopy. Fluorescence microscopy images was recorded on an Olympus IX70 microscope with three kinds of filter (WU: 330–385, WB: 460–490, and WG: 510–550 nm). S2
Laser confocal scanning microscope. Laser confocal fluorescence microscope images were performed on a Carl Zeiss LSM 510 with three kinds of filter (WU: 330–385, WB: 460–490, and WG: 510–550 nm).. UV-visible spectroscopy. UV-visible spectra were acquired on a Shimadzu UV-2401PC UV-Visible scanning spectrophotometer at room temperature. The lifetime of fluorescence. The measurements of fluorescence lifetime were conducted on a FLS 920 spectrometer (Edinburgh Instruments, UK) under excitation wavelength of 375nm. High-resolution mass spectra. High-resolution mass spectra were recorded on a Micromass GCT TOF mass spectrometer at resolutions of 7000 FWHM (EI) by direct introduction at a nominal electron energy of 70 eV for EI, a source temperature of 180◦C for EI, or recorded on LTQ Orbitrap XL (Thermo-Fisher Scientific) with Nano ESI+, source spray voltage 3kV and capillary temperature 200.00◦C. The quantum yield. The quantum yield of the polymers was calculated according to the following equation: ΦSA=ΦST(SSA/SST) (ηSA/ηST) 2 Where Φ = quantum yield; S = gradient of the curve obtained from the plot of intensity versus absorbance; η = refractive index of the solvent; SA = the sample, and ST = the standard. Anthracene (quantum yield = 0.305 in CHCl3) was used as a standard. The polymers and anthracene were all dispersed in CHCl3. The slit width kept the same for both the standard and the samples. Absorbance was measured on a Shimadzu UV-2401PC spectrophotometer. Viability Assay S3
The cytotoxicity of HypET-AlpGP was evaluated in vitro by MTT assay. Human hepatocellular liver carcinoma HepG2 cells were seeded in 96-well plates at 8000 cells per well in 100 µL of complete DMEM medium and incubated at 37oC in a 5% CO2 atmosphere for 24 h. And then the cells were treated with HypET-AlpGP in the DMEM medium at varying concentrations. After 24h, it was followed by the addition of 25 µL MTT reagent (in 20 µL of PBS, 5 mg/mL) to each well. The cells were further incubated for 3 h at 37 oC. The medium in each well was then removed and replaced by 150 µL of DMSO. The plate was gently agitated for 15 min before the absorbance (A) at 570 nm was recorded by a microplate reader (Bio-Rad). The cell viability is calculated as A490,treated/A490,control×100%, where A490,treated and A490,control are the absorbance values of the cells cultured with and without HypET-AlpGP, respectively. Each experiment was done in triple. The data are shown as the mean value plus a standard deviation. Cell Internalization Cells were seeded in a µ-Dish35 mm, high (ibidi GmbH, Germany) in culture medium for 24 h. After the cell medium was aspirated, HypET-AlpGP in serum-free DMEM was applied at a fixed final concentration (2 or 4 mg/mL) for 9h. Then being washed by PBS two times. Cells were observed using a Zeiss LSM510 Laser Confocal Scanning Microscope imaging system in PBS medium. Synthesis of ethylene glycol diacrylate (EGDA) Ethylene glycol (6.2 g, 0. 1 mol), triethylamine (TEA, 25.3 g, 0.25 mol) and anhydrous dichloromethane (80 mL) were placed into a 250 mL round-bottom flask with a magnetic S4
stirrer. After the homogeneous solution was cooled to 0oC, acryloyl chloride (22.6 g, 0.25 mol) in 20 mL of anhydrous dichloromethane was dropwise added under N2 atmosphere, and a white precipitate of triethylammonium bromide was formed immediately. The addition was lasted for 45 min, and then the reaction mixture was warmed to room temperature in approximately 2 h, the reaction continued overnight while stirring. After the salt of TEA was removed by filtration, the filtrate was washed with aqueous solution of sodium bicarbonate and sodium chloride three times, respectively. The organic layer was dried over anhydrous magnesium sulfate. After filtration, the solvent was removed in vacuo, the pure product in 50% yield was obtained as colorless oil by subsequent distillation. 1H NMR (CDCl3, pp): 6.40 and 6.15 (4H, CHH=CHCO-); 5.87 (2H, CHH=CHCO-); 4.40 (4H, -OCH2CH2O-). Synthesis of tris(2-mercaptoethyl) amine (TMEA).
Scheme S1. Synthesis of tris(2-mercaptoethyl) amine 3 The tris(2-mercaptoethyl) amine 3 was synthesized according to Scheme S1. The first step is synthesis of tris(2-chloroethyl)amine 1. A solution of triethanolamine (29.8 g, 0.2 mol) in 50 mL of chloroform was dropwise added into a solution of thionyl chloride (52 mL, 0.7 mol) in chloroform (80 mL) in a 250 mL two-necked round-bottom flask, which was fitted with a dropping funnel and a reflux condenser, while stirring. The addition was carried out at ambient temperature for 1h, the reaction continued at room temperature until gas evolution S5
stopped, and then the mixture was heated to refluxed temperature for 4h. After cooling to room temperature, the white solid product was filtered and washed with 3×50 mL of chloroform, and then the tris(2-chloroethyl)amine hydrochloride 1 was obtained in 80% yield after dried in a vacuum oven at room temperature for 24h. The second step is the preparation of tris(ethylisothiouronium)amine chloride 2. A mixture of the compound 1 (36.15 g, 0.15 mol) and thiourea (34.3 g, 0.45 mol) in 100 mL of ethanol was refluxed at 80oC for 5h. After cooled to room temperature, the 2 was obtained in 90% yield by filtration and following vacuum drying. The third step is synthesis of tris(2-mercaptoethyl) amine 3. A solution of the compound 2 (37.3 g, 0.08 mol) in 80 mL of water was bubbled with N2 for 10 min. After sodium hydroxide (20 g, 0.5 mol) was added, the reaction mixture was heated at 100 °C for 3-5 min under N2 atmosphere. The solution was quickly cooled to 20 °C in an ice bath, and then neutralized by slowly adding the diluted hydrochloric acid solution. The solution was extracted with 3×25 mL of chloroform. The extracts were dried over anhydrous magnesium sulfate, after filtration, the solvent was removed in vacuo. The pure product 3 was obtained in 45% yield as colorless oil by subsequent distillation. 1H NMR (CDCl3, 400 MHz, ppm from TMS): 2.83-2.50 (12H, HSCH2CH2-); 1.75 (3H, HSCH2CH2-);
13
C NMR (CDCl3, ppm):
57.08 (CH2CH2-); 23.04 (HSCH2-); HRMS (EI+): m/z calcd for C6H15NS3: 197.388, found: 198.0442 Synthesis of hyperbranched poly(amine-ester)s (HypETs).
S6
Tris(2-mercaptoethyl) amine 3 (0.29 g , 1.5 mmol) , EGDA (0.51 g, 3 mmol) and 1.5 mL chloroform were successively added into a 5 mL glass tube with a magnetic bar, and then the system was degassed by three freeze-pump-thaw cycles. The tube was sealed under vacuum, and then the sealed tube was placed in an oil bath at 50°C. After the polymerization was carried out for 11h, or 15h, or 20h or 24h, respectively, the tube was cooled to room temperature and opened. Into the reaction mixture, the mercaptoethanol (1.13g) was added, and the tube was sealed again after three freeze-pump-thaw cycles. The reaction was carried out for additional 24h at 50°C. After cooling to room temperature, the tube was opened, and the solution was poured into diethyl ether while stirring vigorously, the polymer was precipitated. After filtration and drying in vacuo at room temperature for 24h, the target hyperbranched polymers, which were respectively marked as HypET11 , HypET15 , HypET20 and HypET24 for the polymers obtained from 11h, 15h, 20h and 24h polymerization, were obtained. Synthesis of bis(2-mercaptoethyl) methylamine 6.
S7
Scheme S2. Synthesis of bis(2-mercaptoethyl) methylamine The bis(2-mercaptoethyl) methylamine 6 was synthesized according to Scheme S2, and the synthetic procedure is similar to synthesis of the compound 3. The first step is synthesis of bis(2-chloroethyl)methylamine hydrochloride 4. The N-methyldiethanolamine (23.8 g, 0.2 mol) in 30 mL of chloroform was dropwise added into a solution of thionyl chloride (36.5 mL, 0.5 mol) in 50 mL of chloroform in a 250 mL two-necked round-bottom flask equipped with a dropping funnel and a refluxing condenser at ambient temperature. The addition was completed in 1h, and the reaction continued at room temperature while stirring until gas evolution stopped. After the reaction mixture was refluxed for 2h, and then cooled to room temperature, the white solid product was filtered and washed with 3×30 mL of chloroform, the bis(2-chloroethyl)methylamine hydrochloride 4 was obtained in 78% yield after dried in a vacuum oven at room temperature for 24h. The second step is synthesis of bis(ethylisothiouronium) methylamine 5. A mixture of 4 (28.9 g, 0.15 mol) and thiourea (22.8 g, 0.3 mol) in 100 mL of ethanol was refluxed at 80oC for 5 h. After cooling the mixture to room temperature, the compound 5 was obtained in 65% yield by filtration and subsequent drying in vacuum. The third step is synthesis of bis(2-mercaptoethyl)methylamine 6. A solution of 5 (13.6 g, 0.06 mol) in 60 mL of water was bubbled with N2 for 10 min, into this solution, the sodium hydroxide (14.4 g, 0.36 mol) was added. The reaction mixture was heated at 100 °C under N2 S8
atmosphere for 3-5 min and then quickly cooled to 20 °C in an ice bath. After the mixture was neutralized by slowly adding a solution of diluted hydrochloric acid, and then extracted with 3×30 mL of chloroform. The extracts were dried over anhydrous magnesium sulfate, after filtration, the solvent in filtrate was removed in vacuo. The pure product 6 was obtained in 40% yield as colorless oil by subsequent distillation. 1H NMR (CDCl3, ppm): 2.68-2.55 (8H, HSCH2CH2-); 2.25 (3H, NCH3); 1.77 (2H, HSCH2CH2-);
13
C NMR (CDCl3, ppm):
60.05 (HSCH2CH2-); 41.44 (CH3N); 22.66 (HSCH2-); HRMS (EI+): m/z calcd for C5H13NS2: 151.295, found: 151.0414. Synthesis of the linear polymer, l-poly(EGDA-BMEA).
Bis(2-mercaptoethyl)methylamine 6 (0.23 g, 1. 5 mmol), EGDA (0.26 g, 1.5 mmol) and 1. 5 mL chloroform were successively added into a 5 mL glass tube with a magnetic bar. After three freeze-pump-thaw cycles, the tube was sealed under vacuum, and then the sealed tube was placed in an oil bath at 50°C for 36h. The product was obtained in 76. 3% yield after precipitation in diethyl ether, filtration and drying in vacuo. Synthesis of 1-acryloyl-1-di(isopropyl) amineethyl-2-methacryloyl-glycol 8.
Scheme S3. Synthesis of 1-acryloyl-1-di(isopropyl)amineethyl-2-methacryloyl-glycol 8. S9
The synthesis of 8 includes two steps. The first step is synthesis of N-di(isopropyl) amine-2-hydroxy-propylene methacrylate 7. GMA (11.4 g, 0.08 mol) and diisopropylamine (12.14 g, 0.12 mol) were added in a 50 mL three-necked flask, and then the reaction was carried out at room temperature under N2 atmosphere for 24h while stirring. After the removal of excess diisopropylamine under reduced pressure, the compound 7 was obtained in 90% yield by vacuum distillation. 1H NMR (CDCl3, TMS, ppm): 6.15 (1H, CHH=C(CH3)CO-); 5.60 (1H, CHH=C(CH3)CO-); 4.46 (1H, CHOH); 3.99 (1H, -OCHHCH); 3.25 (1H, -OCHHCH); 2.96-2.80 (3H, (CH3)2CH- and (C3H7)2NCHH-); 2.67 (1H, (C3H7)2NCHH-); 1.94 (3H, CH2=CCH3CO-); 2.85, 2.64 and 1.50-1.05[12H, CH(CH3)2]. The second step is the reaction of the compound 7 with acryloyl chloride. The compound 7 (12.17 g, 0.05 mol), TEA (7.6 g, 0.075 mol) and anhydrous CH2Cl2 (70 mL) in a three-necked round-bottom flask were cooled to 0oC while stirring. Into this solution, acryloyl chloride (6.8 g, 0.075 mol) in 10 mL anhydrous CH2Cl2 was added slowly at 0oC under N2 atmosphere. After the addition was completed at 0oC for 1h, the reaction continued overnight. The solid was removed by filtration, and the filtrate was washed by the solutions of saturated NaHCO3, the saturated NaCl in water three times, respectively. The organic layer was then dried over anhydrous magnesium sulfate. After filtration, the solvent in the filtrate was removed in vacuo, the pure product 8 was obtained as colorless oil in 55% yield by subsequent distillation (bp 40oC/30 mmHg). 1H NMR (CDCl3, TMS, ppm): 6.5(1H, CHH=CHCO-); 6.21-6.11(2H, CHH=CHCO- and CHH=CCH3CO-); 5.6-5.5(2H, CHH=CHCO- and CHH=CCH3CO-); 4.48(1H, CHH=CHCOOCH); 4.10-3.90 (2H, CHH=CCH3COOCHH) and (C3H7)2NCHH-); 3.89-3.67 (1H, (C3H7)2NCHH-); 3.26 (1H, CHH=CCH3COOCHH); 2.86 S10
and 2.67 (2H, (CH3)2CH-); 1.97(3H, CHH=CCH3COO); 1.49-1.03 (12H, (CH3)2CH-);
13
C
NMR (CDCl3, ppm): 166.72, 166.00 (CHH=C(CH3)CO- and CH2=CHCO-); 135.73 (CH2=C(CH3)CO-); 130.73 (CH2=CH-); 125.95 (CH2=CH-); 125.34 (CH2=C(CH3)-); 76.91 (CH2=CHCOOCH);
65.00
(CH2=C(CH3)COOCH2-);
49.19
((CH3)2CHN-);
44.40
((CH3)2CHNCH2-); 20.37 ((CH3)2CHNCH2-); 18.08 (CH2=C(CH3)CO-); MS-ESI: m/z calcd for C16H27NO4: 297.390, found: 298.2012. Synthesis of linear polymer, l-poly(PPT-APAMA).
The compound 8 (0.297 g, 1 mmol), 1,3-propanedithiol (0.108 g, 1 mmol) and tetrabutyl ammonium bromide (2.10 g, 6.5 mmol) were successively added into a 5 mL glass tube with a magnetic bar, and then the system was degassed by three freeze-pump-thaw cycles. The tube was sealed under vacuum, and then the sealed tube was placed in an oil bath at 100°C. After the polymerization was carried out for 24h, the tube was cooled to room temperature and opened. The product in methanol was precipitated from distilled water while stirring vigorously. Then the solid was dissolved in THF and precipitation was performed in petroleum ether. The linear polymer was obtained in 68. 1% yield after drying under vacuum for 1 d at room temperature. Syntheses of tri(5-carboxylate ethylene-3-sulfoethylene)amine (CESEA) 10.
S11
SH
SH
N
O
O O
S
S
N
O
O O
NaO
S
ONa O
S
S
SH
S
N
O O
O
ONa 10
O 9
Tri(2-mercaptoethyl)amine 3 (0.20 g, 1 mmol), methyl acrylate (0.284 g, 3.3 mol) were added into a 5 mL glass tube with a magnetic bar. After three freeze-pump-thaw cycles, the tube was sealed under vacuum, the reaction was carried out at room temperature for 24h. The product, 9 was obtained in a yield of 98.5% after removing unreacted methyl acrylate under reduced pressure.
1
H NMR (D2O, TMS, ppm): 3.7 (9H, CH3O), 2.89-2.45 (24H,
CH2CH2SCH2CH2). The CESEA 10 was prepared by hydrolysis of the compound 9. The synthetic procedure is as follows: 9 (273.4 mg, 0.6 mmol ) was added into a solution of THF/H2O (9/1, v/v) containing NaOH (72 mg, 1.8 mmol), and then the hydrolysis was carried out at room temperature under N2 atmosphere for 24h. After removal of the solvents under reduced pressure, the hydrolyzed product 10 was obtained after dried in vacuo. 1H NMR (D2O, TMS, ppm): 2.8-2.56(18H, CH2CH2SCH2CH2), 2.43(6H, CH2N). Hydrolysis of HypET24. Hydrolysis of the HypET24 (273.4 mg) was carried out in THF/water (9/1,v/v) solution containing 72 mg of NaOH at 30oC under N2 for 24h, and then the resultant product was dried in vacuo. 1H NMR (D2O, TMS, ppm): 2.8-2.56(18H, 3CH2SCH2CH2), 2.43(6H, CH2N). Synthesis of 1,2:3,4-Di-O-isopropylidene-6-O-acryloyl-α-D-galactopyranose (DIAlGP). S12
Anhydrous ZnCl2 (15.6 g, 114.5 mmol) was dissolved in acetone (163 mL) by stirring under N2 atmosphere, and a very small amount of Zn(OH)2 presented was dissolved by dropwise addition of H2SO4 through septum. Finely ground anhydrous galactose (13 g, 0.072 mol) was added into the solution, and then the solution was stirred for 4h at room temperature. A suspension of 26 g Na2CO3 in 46 mL H2O was added to the solution and the mixture was stirred until the liquid layer contained zinc ions and filtrated. The solid on the filter was washed with acetone (3×15 mL) and the combined filtrates were evaporated using rotary evaporator under reduced pressure (30 °C, 200 mmHg). The residue was extracted with diethyl ether (3×35mL) and the ethereal extract was dried over MgSO4. After the solvent was removed, the residue was dried in vacuum overnight to afford a sirup-like raw product (16.3g). The raw product was distilled and the pure product was collected at 131–135 °C/0.3 mmHg to afforded 14.5 g of product 11 as a colorless viscous liquid (77.1% of yield). 1H NMR (CDCl3, TMS, ppm): 5.58 (1H, anomeric CH), 4.62 (1H, sugar moiety, CHH), 4.35-4.28 (3H, sugar moiety CH), 3.90-3.70 (4H, HOCH2 and sugar moiety CH), 1.54-1.35 (12H, CH3). To a stirred suspension of 1,2:3,4-di-O-isopropylidene-α-D-galactopyranose 11 (8.00 g , 3.07 × 10-2 mol) and basic alumina (6.24 g , 14.11 × 10-2 mol) in distilled acetonitrile (80 S13
mL), acryloyl chloride (12.80 mL, 15.82 × 10-2 mol) was added at room temperature. The reaction mixture was stirred for 3 days. The solids were removed by filtration though a micromembrane. Reaction completion was monitored using thin layer chromatography with hexane/ethyl acetate (2:1) as the solvent. The solvent was removed under reduced pressure to yield light yellow oil, which was purified via column chromatography (hexane/ethyl acetate, 2:1). Viscous light yellow oil was obtained in 56% of yield. 1H NMR (CDCl3, TMS, ppm): 6.40 (1H, CHH=CH-), 6.17 (1H, CH2=CH), 5.84 (1H, CHH=CH), 5.53 (1H, anomeric CH), 4.61 (1H, sugar moiety CHH), 4.13 (4H, sugar moiety 1CHH and 3CH), 4.10 (1H, sugar moiety CH), 1.33-1.53 (12H, CH3). Synthesis of hyperbranced polymer, HypET-DIAlpGP. TMEA 3 (0.29 g, 1.5 mmol), EGDA (0.17 g, 1 mmol), DIAlpGP (0.47 g, 1.5 mmol) and chloroform (1.5 mL) were successively added into a 5 mL glass tube with a magnetic bar. After three freeze-pump-thaw cycles, the tube was sealed under vacuum, and then the sealed tube was placed in an oil bath at 50°C for 30h. The product in 35.8% yield was obtained after precipitation in diethyl ether, filtration and drying in vacuo. The product 0.2 g was dissolved in 10 mL THF, into this solution, aqueous solution of HCl (2 mol/L) was slowly added until the pH reached to about 3, and then the reaction solution was stirred at room temperature for 20h. The HypET-AlpGP was obtained after dialysis (molecular weight (Mw) cutoff: 3500 Da) against deionized water for 24h.
S14
Table S1. Characterization and properties of the hyperbranched polymers, HypET11, HypET15, HypET20 and HypET24 No
a
Mwb (g/mol)
Mw/Mnb
DBc
Φd
τ (ns)
HypET11
7660
2.66
0.71
0.11
－
HypET15
14800
2.64
0.79
0.31
2.45
HypET20
20870
3.50
0.80
0.37
2.46
HypET24
55100
3.66
0.82
0.43
2.57
a)
HypET11, HypET15, HypET20 and HypET24 were prepared by polymerization with molar ratio of EGDA/TMEA=2/1 at 50oC for 11, 15, 20 and 24h, respectively. b) Obtained from TD-SEC measurements. c) Calculated based on the 1H NMR data. d) Quantum yield was measured according to Williams’ method.
S15
e O S
O
O
S f
d S S OH c b a N S
O h' h O i O g' S b' S g O i a' OH
O
d
N f e S c e g N i S O g S S a f O S O h O i b OH h O b'c,f,g,g',h,h' O
i
a'
b
d,e
O O
HypET11
a
6
4
2
0
4
2
0
4
2
0
2
0
HypET15 6
HypET20 6
HypET24 6
4
Chemical shift (ppm)
Figure S1.1H NMR spectra of HypET11, HypET15, HypET20 and HypET24 prepared by Michael addition polymerization of EGDA and TMEA with molar ratio of 2/1 at 50oC for 11h, 15h, 20h and 24h.
S16
Mw,triple=20870 Mw/Mn=3.50
Mw,triple=55100
Mw,triple=14800
Mw/Mn=3.66
Mw/Mn=2.64 Mw,triple=7700 Mw/Mn=2.66
16
18
20
22
24
26
28
Elution volumn (mL)
Figure S2. TD-SEC traces of the HypET11 , HypET15 , HypET20 and HypET24 prepared by Michael addition polymerization of EGDA and TMEA with molar ratio of 2/1 at 50oC for 11h , 15h , 20h and 24h.
S17
g
i
H
O O
(S
O
b
b N
a
H O
S
a
e d
CH3 c
h
O O
f
CH3
O
f
)n
O
S
N
SH
a,b,e g
f
h
i
d
7.0
6.5
6.0
5.5
c
5.0
ppm
8
7
6
5
4
3
2
1
0
Chemical shift (ppm) Figure S3. 1H NMR spectrum of the linear polymer with tertiary amine in the backbone obtained from Michael addition polymerization of EGDA and bis(2-mercaptoethyl) methylamine with molar ratio of 1/1 at 50oC for 24h.
S18
Mn=6750 Mw/Mn=1.74
15
18
21
24
27
Volume (mL)
Figure S4. GPC curve of the linear polymer with tertiary amine in the backbone obtained from Michael addition polymerization of EGDA and bis(2-mercaptoethyl)methylamine with molar ratio of 1/1 at 50oC for 24h.
S19
a H Ha
b O O h
f O
e, e'
k
N g
d cH
m H j
S i
H O d
f O
O
S
l H
n
i
O
H p
O
N
g h
i ,g , d h
c,r
k',e' f
7
p m
l
6
5
e
b
a j
k
4
3
2
1
0
Chemical shift (ppm) Figure S5. 1H NMR spectrum of the linear polymer with the tertiary amine as side group obtained from Michael addition polymerization of 1-acryloyl-1-di(isopropyl) amineethyl2-methacryloyl-glycol and 1,3-propanedithiol with molar ratio of 1/1 at 50oC for 24h.
S20
Mn=4300 Mw/Mn=1.34
15
18
21
24
27
Volume (mL)
Figure S6. GPC curve of the linear polymer with tertiary amine as side group obtained from Michael addition polymerization of 1-acryloyl-1-di(isopropyl) amineethyl–2–methacryloyl –glycol and 1,3-propanedithiol with molar ratio of 1/1 at 50oC for 24h.
S21
HypET24
HypET24-O7
HypET24-O20
4.5
4.0
3.5
3.0
2.5
Chemical shift (ppm) Figure S7. 1H NMR spectra of the HypET24, HypET24-O7 and HypET24-O20 in CDCl3. prepared by oxidization of HypET24 in THF under an air atmosphere at around 35oC for 7 days and 24 days, respectively. Solvent: CDCl3.
S22
HypET24
HypET24-O7
HypET24-O20
-1 1460 cm-1 1361 cm 1184 cm-1 1400 1200
1000
1460 1361 1184
4000
3000
2000
1000 -1
Wavenumber (cm ) Figure S8. IR spectra of the HypET24, HypET24-O7 and HypET24-O20. The HypET24 was prepared by Michael addition polymerization of EGDA and TMEA with molar ratio of 2/1 at 50oC for 24h, HypET24-O7 and HypET24-O20 were prepared by oxidization of the HypET24 in THF under an air atmosphere at around 35oC for 7 days and 20 days, respectively.
S23
a d S c N Sf S e
O O
O l hO O Sg O
O S O d N f e HS c e g S Oi gS f N S h O iO O h
O O
c,d,e,f,g,h
mO b nk O O j
O O
a
i,j,k,m
l
8
7
6
b
5
n
4
3 2 1 0 Chemical Shift (ppm)
Figure S9. 1H NMR spectrum of the HypET-DIAlpGP in CDCl3, the HypET-DIAlpGP was prepared by Michael addition polymerization with molar ratio of EGDA/TMEA/DIAlpGP = 1/1.5/1.5 at 50oC for 30h.
S24
S O S d HS
c
N
f e
O
C
O g O i O S C O h i C O
OH
N
O OH O OH C S O e O N f OH h S g C O l f OH e S n m g O k h j b HO HO S
O
C
HO
S
c,d,e,f,g,h,m,b,k
i,j
nl
8
7
6
5
4
3
2
1
0
Chemical shift (ppm) Figure S10. 1H NMR spectrum of HypET-AlpGP in DMSO. HypET-AlpGP was prepared by polymerization with molar ratio of EGDA/TMEA/DIAlpGP=1/1.5/1.5 at 50oC for 30h, and subsequently, deprotection reaction in diluted aqueous solution of hydrochloric acid.
S25
A unique aliphatic tertiary amine chromophore: fluorescence, polymer structure and application in cell imaging Miao Sun, Chun-Yan Hong, Cai-Yuan Pan * CAS Key Laboratory of Soft Matter Chemistry, Department of Polymer Science and Engineering, University of Science and Technology of China, Hefei, Anhui, 230026, P. R. China.
Experimental section Materials Triethanolamine, thionyl chloride, thiourea, dichloromethane (≥99. 5%), methanol (≥99, 5%), sodium bicarbonate (≥96%), diisopropylamine chloroform (analytical grade) and anhydrous magnesium sulfate with analytical grade were purchased from Sinopharm Chemical Reagent Co. Ltd. and used as received. Glycidyl methacrylate (GMA, ≥98%), 1, 3-propanedithiol (≥98%), tetrabutylammonium bromide (≥98%), N-methyldiethanolamine (≥99%, Aladdin Reagent), mercaptoethanol containing ~10 mol% bis(2-hydroxyethyl) disulfide were used as received. Ethylene glycol (≥98%, Aladdin Reagent) was dried by refluxing in toluene to remove water. Tetrahydrofuran (≥99%), chloroform (≥99%), acetone (≥99.5%) were purchased from Sinopharm Chemical Reagent Co. Ltd, and acryloyl chloride (≥96%, Aladdin Reagent) was purified by distillation under reduced pressure before use. Characterization
S1
Nuclear Magnetic Resonance (NMR). Structures of the synthesized compounds were characterized by 1H and
13
C NMR spectra on a Bruker 400 MHz NMR spectrometer using
CDCl3 , DMSO-d6 or D2O as solvent. Size exclusion chromatography (SEC) measurements. Molecular weight and molecular weight distribution were obtained by triple detection size exclusion chromatography (TD-SEC) detection at 25oC. The instrumentation consists of the following: a Waters 1515 isotratic HPLC pump with 5 µm Waters styragel columns (guard, 0.5 HR, 1 HR, 3 HR, 4 HR and 5 HR, the molecular weight ranges of these columns are 0-1000, 100-5000, 500-30000, 5000-500000 and 50000-4000000 g/mol, respectively); a Waters 717 PLUS autosamples; a waters 2414 differential refractive index (DRI) detector, the wavelength is 880 nm; a multi angle laser light scattering (MALLS) detector (Wyatt mini Dawn TRISTRA light scattering, three detection angles are 45o, 90o and 135o, the wavelength and power are 690 nm and 220 w); a Wyatt Visco Star viscometer detector; a Waters Breeze data manager. The eluent was HPLC grade THF delivered at 1.0 mL / min. The refractive index increment (dn/dc) was determined using a Wyatt Optilab REX (λ=640 nm) interferometric differential refractometer in bath model at 25 ºC. Measurement of fluorescence spectra. The fluorescence spectra of the resultant polymers were
recorded
at
room
temperature
on
a
Perkin-Elmer
LS55
luminescence
spectrophotometer. Fluorescence microscopy. Fluorescence microscopy images was recorded on an Olympus IX70 microscope with three kinds of filter (WU: 330–385, WB: 460–490, and WG: 510–550 nm). S2
Laser confocal scanning microscope. Laser confocal fluorescence microscope images were performed on a Carl Zeiss LSM 510 with three kinds of filter (WU: 330–385, WB: 460–490, and WG: 510–550 nm).. UV-visible spectroscopy. UV-visible spectra were acquired on a Shimadzu UV-2401PC UV-Visible scanning spectrophotometer at room temperature. The lifetime of fluorescence. The measurements of fluorescence lifetime were conducted on a FLS 920 spectrometer (Edinburgh Instruments, UK) under excitation wavelength of 375nm. High-resolution mass spectra. High-resolution mass spectra were recorded on a Micromass GCT TOF mass spectrometer at resolutions of 7000 FWHM (EI) by direct introduction at a nominal electron energy of 70 eV for EI, a source temperature of 180◦C for EI, or recorded on LTQ Orbitrap XL (Thermo-Fisher Scientific) with Nano ESI+, source spray voltage 3kV and capillary temperature 200.00◦C. The quantum yield. The quantum yield of the polymers was calculated according to the following equation: ΦSA=ΦST(SSA/SST) (ηSA/ηST) 2 Where Φ = quantum yield; S = gradient of the curve obtained from the plot of intensity versus absorbance; η = refractive index of the solvent; SA = the sample, and ST = the standard. Anthracene (quantum yield = 0.305 in CHCl3) was used as a standard. The polymers and anthracene were all dispersed in CHCl3. The slit width kept the same for both the standard and the samples. Absorbance was measured on a Shimadzu UV-2401PC spectrophotometer. Viability Assay S3
The cytotoxicity of HypET-AlpGP was evaluated in vitro by MTT assay. Human hepatocellular liver carcinoma HepG2 cells were seeded in 96-well plates at 8000 cells per well in 100 µL of complete DMEM medium and incubated at 37oC in a 5% CO2 atmosphere for 24 h. And then the cells were treated with HypET-AlpGP in the DMEM medium at varying concentrations. After 24h, it was followed by the addition of 25 µL MTT reagent (in 20 µL of PBS, 5 mg/mL) to each well. The cells were further incubated for 3 h at 37 oC. The medium in each well was then removed and replaced by 150 µL of DMSO. The plate was gently agitated for 15 min before the absorbance (A) at 570 nm was recorded by a microplate reader (Bio-Rad). The cell viability is calculated as A490,treated/A490,control×100%, where A490,treated and A490,control are the absorbance values of the cells cultured with and without HypET-AlpGP, respectively. Each experiment was done in triple. The data are shown as the mean value plus a standard deviation. Cell Internalization Cells were seeded in a µ-Dish35 mm, high (ibidi GmbH, Germany) in culture medium for 24 h. After the cell medium was aspirated, HypET-AlpGP in serum-free DMEM was applied at a fixed final concentration (2 or 4 mg/mL) for 9h. Then being washed by PBS two times. Cells were observed using a Zeiss LSM510 Laser Confocal Scanning Microscope imaging system in PBS medium. Synthesis of ethylene glycol diacrylate (EGDA) Ethylene glycol (6.2 g, 0. 1 mol), triethylamine (TEA, 25.3 g, 0.25 mol) and anhydrous dichloromethane (80 mL) were placed into a 250 mL round-bottom flask with a magnetic S4
stirrer. After the homogeneous solution was cooled to 0oC, acryloyl chloride (22.6 g, 0.25 mol) in 20 mL of anhydrous dichloromethane was dropwise added under N2 atmosphere, and a white precipitate of triethylammonium bromide was formed immediately. The addition was lasted for 45 min, and then the reaction mixture was warmed to room temperature in approximately 2 h, the reaction continued overnight while stirring. After the salt of TEA was removed by filtration, the filtrate was washed with aqueous solution of sodium bicarbonate and sodium chloride three times, respectively. The organic layer was dried over anhydrous magnesium sulfate. After filtration, the solvent was removed in vacuo, the pure product in 50% yield was obtained as colorless oil by subsequent distillation. 1H NMR (CDCl3, pp): 6.40 and 6.15 (4H, CHH=CHCO-); 5.87 (2H, CHH=CHCO-); 4.40 (4H, -OCH2CH2O-). Synthesis of tris(2-mercaptoethyl) amine (TMEA).
Scheme S1. Synthesis of tris(2-mercaptoethyl) amine 3 The tris(2-mercaptoethyl) amine 3 was synthesized according to Scheme S1. The first step is synthesis of tris(2-chloroethyl)amine 1. A solution of triethanolamine (29.8 g, 0.2 mol) in 50 mL of chloroform was dropwise added into a solution of thionyl chloride (52 mL, 0.7 mol) in chloroform (80 mL) in a 250 mL two-necked round-bottom flask, which was fitted with a dropping funnel and a reflux condenser, while stirring. The addition was carried out at ambient temperature for 1h, the reaction continued at room temperature until gas evolution S5
stopped, and then the mixture was heated to refluxed temperature for 4h. After cooling to room temperature, the white solid product was filtered and washed with 3×50 mL of chloroform, and then the tris(2-chloroethyl)amine hydrochloride 1 was obtained in 80% yield after dried in a vacuum oven at room temperature for 24h. The second step is the preparation of tris(ethylisothiouronium)amine chloride 2. A mixture of the compound 1 (36.15 g, 0.15 mol) and thiourea (34.3 g, 0.45 mol) in 100 mL of ethanol was refluxed at 80oC for 5h. After cooled to room temperature, the 2 was obtained in 90% yield by filtration and following vacuum drying. The third step is synthesis of tris(2-mercaptoethyl) amine 3. A solution of the compound 2 (37.3 g, 0.08 mol) in 80 mL of water was bubbled with N2 for 10 min. After sodium hydroxide (20 g, 0.5 mol) was added, the reaction mixture was heated at 100 °C for 3-5 min under N2 atmosphere. The solution was quickly cooled to 20 °C in an ice bath, and then neutralized by slowly adding the diluted hydrochloric acid solution. The solution was extracted with 3×25 mL of chloroform. The extracts were dried over anhydrous magnesium sulfate, after filtration, the solvent was removed in vacuo. The pure product 3 was obtained in 45% yield as colorless oil by subsequent distillation. 1H NMR (CDCl3, 400 MHz, ppm from TMS): 2.83-2.50 (12H, HSCH2CH2-); 1.75 (3H, HSCH2CH2-);
13
C NMR (CDCl3, ppm):
57.08 (CH2CH2-); 23.04 (HSCH2-); HRMS (EI+): m/z calcd for C6H15NS3: 197.388, found: 198.0442 Synthesis of hyperbranched poly(amine-ester)s (HypETs).
S6
Tris(2-mercaptoethyl) amine 3 (0.29 g , 1.5 mmol) , EGDA (0.51 g, 3 mmol) and 1.5 mL chloroform were successively added into a 5 mL glass tube with a magnetic bar, and then the system was degassed by three freeze-pump-thaw cycles. The tube was sealed under vacuum, and then the sealed tube was placed in an oil bath at 50°C. After the polymerization was carried out for 11h, or 15h, or 20h or 24h, respectively, the tube was cooled to room temperature and opened. Into the reaction mixture, the mercaptoethanol (1.13g) was added, and the tube was sealed again after three freeze-pump-thaw cycles. The reaction was carried out for additional 24h at 50°C. After cooling to room temperature, the tube was opened, and the solution was poured into diethyl ether while stirring vigorously, the polymer was precipitated. After filtration and drying in vacuo at room temperature for 24h, the target hyperbranched polymers, which were respectively marked as HypET11 , HypET15 , HypET20 and HypET24 for the polymers obtained from 11h, 15h, 20h and 24h polymerization, were obtained. Synthesis of bis(2-mercaptoethyl) methylamine 6.
S7
Scheme S2. Synthesis of bis(2-mercaptoethyl) methylamine The bis(2-mercaptoethyl) methylamine 6 was synthesized according to Scheme S2, and the synthetic procedure is similar to synthesis of the compound 3. The first step is synthesis of bis(2-chloroethyl)methylamine hydrochloride 4. The N-methyldiethanolamine (23.8 g, 0.2 mol) in 30 mL of chloroform was dropwise added into a solution of thionyl chloride (36.5 mL, 0.5 mol) in 50 mL of chloroform in a 250 mL two-necked round-bottom flask equipped with a dropping funnel and a refluxing condenser at ambient temperature. The addition was completed in 1h, and the reaction continued at room temperature while stirring until gas evolution stopped. After the reaction mixture was refluxed for 2h, and then cooled to room temperature, the white solid product was filtered and washed with 3×30 mL of chloroform, the bis(2-chloroethyl)methylamine hydrochloride 4 was obtained in 78% yield after dried in a vacuum oven at room temperature for 24h. The second step is synthesis of bis(ethylisothiouronium) methylamine 5. A mixture of 4 (28.9 g, 0.15 mol) and thiourea (22.8 g, 0.3 mol) in 100 mL of ethanol was refluxed at 80oC for 5 h. After cooling the mixture to room temperature, the compound 5 was obtained in 65% yield by filtration and subsequent drying in vacuum. The third step is synthesis of bis(2-mercaptoethyl)methylamine 6. A solution of 5 (13.6 g, 0.06 mol) in 60 mL of water was bubbled with N2 for 10 min, into this solution, the sodium hydroxide (14.4 g, 0.36 mol) was added. The reaction mixture was heated at 100 °C under N2 S8
atmosphere for 3-5 min and then quickly cooled to 20 °C in an ice bath. After the mixture was neutralized by slowly adding a solution of diluted hydrochloric acid, and then extracted with 3×30 mL of chloroform. The extracts were dried over anhydrous magnesium sulfate, after filtration, the solvent in filtrate was removed in vacuo. The pure product 6 was obtained in 40% yield as colorless oil by subsequent distillation. 1H NMR (CDCl3, ppm): 2.68-2.55 (8H, HSCH2CH2-); 2.25 (3H, NCH3); 1.77 (2H, HSCH2CH2-);
13
C NMR (CDCl3, ppm):
60.05 (HSCH2CH2-); 41.44 (CH3N); 22.66 (HSCH2-); HRMS (EI+): m/z calcd for C5H13NS2: 151.295, found: 151.0414. Synthesis of the linear polymer, l-poly(EGDA-BMEA).
Bis(2-mercaptoethyl)methylamine 6 (0.23 g, 1. 5 mmol), EGDA (0.26 g, 1.5 mmol) and 1. 5 mL chloroform were successively added into a 5 mL glass tube with a magnetic bar. After three freeze-pump-thaw cycles, the tube was sealed under vacuum, and then the sealed tube was placed in an oil bath at 50°C for 36h. The product was obtained in 76. 3% yield after precipitation in diethyl ether, filtration and drying in vacuo. Synthesis of 1-acryloyl-1-di(isopropyl) amineethyl-2-methacryloyl-glycol 8.
Scheme S3. Synthesis of 1-acryloyl-1-di(isopropyl)amineethyl-2-methacryloyl-glycol 8. S9
The synthesis of 8 includes two steps. The first step is synthesis of N-di(isopropyl) amine-2-hydroxy-propylene methacrylate 7. GMA (11.4 g, 0.08 mol) and diisopropylamine (12.14 g, 0.12 mol) were added in a 50 mL three-necked flask, and then the reaction was carried out at room temperature under N2 atmosphere for 24h while stirring. After the removal of excess diisopropylamine under reduced pressure, the compound 7 was obtained in 90% yield by vacuum distillation. 1H NMR (CDCl3, TMS, ppm): 6.15 (1H, CHH=C(CH3)CO-); 5.60 (1H, CHH=C(CH3)CO-); 4.46 (1H, CHOH); 3.99 (1H, -OCHHCH); 3.25 (1H, -OCHHCH); 2.96-2.80 (3H, (CH3)2CH- and (C3H7)2NCHH-); 2.67 (1H, (C3H7)2NCHH-); 1.94 (3H, CH2=CCH3CO-); 2.85, 2.64 and 1.50-1.05[12H, CH(CH3)2]. The second step is the reaction of the compound 7 with acryloyl chloride. The compound 7 (12.17 g, 0.05 mol), TEA (7.6 g, 0.075 mol) and anhydrous CH2Cl2 (70 mL) in a three-necked round-bottom flask were cooled to 0oC while stirring. Into this solution, acryloyl chloride (6.8 g, 0.075 mol) in 10 mL anhydrous CH2Cl2 was added slowly at 0oC under N2 atmosphere. After the addition was completed at 0oC for 1h, the reaction continued overnight. The solid was removed by filtration, and the filtrate was washed by the solutions of saturated NaHCO3, the saturated NaCl in water three times, respectively. The organic layer was then dried over anhydrous magnesium sulfate. After filtration, the solvent in the filtrate was removed in vacuo, the pure product 8 was obtained as colorless oil in 55% yield by subsequent distillation (bp 40oC/30 mmHg). 1H NMR (CDCl3, TMS, ppm): 6.5(1H, CHH=CHCO-); 6.21-6.11(2H, CHH=CHCO- and CHH=CCH3CO-); 5.6-5.5(2H, CHH=CHCO- and CHH=CCH3CO-); 4.48(1H, CHH=CHCOOCH); 4.10-3.90 (2H, CHH=CCH3COOCHH) and (C3H7)2NCHH-); 3.89-3.67 (1H, (C3H7)2NCHH-); 3.26 (1H, CHH=CCH3COOCHH); 2.86 S10
and 2.67 (2H, (CH3)2CH-); 1.97(3H, CHH=CCH3COO); 1.49-1.03 (12H, (CH3)2CH-);
13
C
NMR (CDCl3, ppm): 166.72, 166.00 (CHH=C(CH3)CO- and CH2=CHCO-); 135.73 (CH2=C(CH3)CO-); 130.73 (CH2=CH-); 125.95 (CH2=CH-); 125.34 (CH2=C(CH3)-); 76.91 (CH2=CHCOOCH);
65.00
(CH2=C(CH3)COOCH2-);
49.19
((CH3)2CHN-);
44.40
((CH3)2CHNCH2-); 20.37 ((CH3)2CHNCH2-); 18.08 (CH2=C(CH3)CO-); MS-ESI: m/z calcd for C16H27NO4: 297.390, found: 298.2012. Synthesis of linear polymer, l-poly(PPT-APAMA).
The compound 8 (0.297 g, 1 mmol), 1,3-propanedithiol (0.108 g, 1 mmol) and tetrabutyl ammonium bromide (2.10 g, 6.5 mmol) were successively added into a 5 mL glass tube with a magnetic bar, and then the system was degassed by three freeze-pump-thaw cycles. The tube was sealed under vacuum, and then the sealed tube was placed in an oil bath at 100°C. After the polymerization was carried out for 24h, the tube was cooled to room temperature and opened. The product in methanol was precipitated from distilled water while stirring vigorously. Then the solid was dissolved in THF and precipitation was performed in petroleum ether. The linear polymer was obtained in 68. 1% yield after drying under vacuum for 1 d at room temperature. Syntheses of tri(5-carboxylate ethylene-3-sulfoethylene)amine (CESEA) 10.
S11
SH
SH
N
O
O O
S
S
N
O
O O
NaO
S
ONa O
S
S
SH
S
N
O O
O
ONa 10
O 9
Tri(2-mercaptoethyl)amine 3 (0.20 g, 1 mmol), methyl acrylate (0.284 g, 3.3 mol) were added into a 5 mL glass tube with a magnetic bar. After three freeze-pump-thaw cycles, the tube was sealed under vacuum, the reaction was carried out at room temperature for 24h. The product, 9 was obtained in a yield of 98.5% after removing unreacted methyl acrylate under reduced pressure.
1
H NMR (D2O, TMS, ppm): 3.7 (9H, CH3O), 2.89-2.45 (24H,
CH2CH2SCH2CH2). The CESEA 10 was prepared by hydrolysis of the compound 9. The synthetic procedure is as follows: 9 (273.4 mg, 0.6 mmol ) was added into a solution of THF/H2O (9/1, v/v) containing NaOH (72 mg, 1.8 mmol), and then the hydrolysis was carried out at room temperature under N2 atmosphere for 24h. After removal of the solvents under reduced pressure, the hydrolyzed product 10 was obtained after dried in vacuo. 1H NMR (D2O, TMS, ppm): 2.8-2.56(18H, CH2CH2SCH2CH2), 2.43(6H, CH2N). Hydrolysis of HypET24. Hydrolysis of the HypET24 (273.4 mg) was carried out in THF/water (9/1,v/v) solution containing 72 mg of NaOH at 30oC under N2 for 24h, and then the resultant product was dried in vacuo. 1H NMR (D2O, TMS, ppm): 2.8-2.56(18H, 3CH2SCH2CH2), 2.43(6H, CH2N). Synthesis of 1,2:3,4-Di-O-isopropylidene-6-O-acryloyl-α-D-galactopyranose (DIAlGP). S12
Anhydrous ZnCl2 (15.6 g, 114.5 mmol) was dissolved in acetone (163 mL) by stirring under N2 atmosphere, and a very small amount of Zn(OH)2 presented was dissolved by dropwise addition of H2SO4 through septum. Finely ground anhydrous galactose (13 g, 0.072 mol) was added into the solution, and then the solution was stirred for 4h at room temperature. A suspension of 26 g Na2CO3 in 46 mL H2O was added to the solution and the mixture was stirred until the liquid layer contained zinc ions and filtrated. The solid on the filter was washed with acetone (3×15 mL) and the combined filtrates were evaporated using rotary evaporator under reduced pressure (30 °C, 200 mmHg). The residue was extracted with diethyl ether (3×35mL) and the ethereal extract was dried over MgSO4. After the solvent was removed, the residue was dried in vacuum overnight to afford a sirup-like raw product (16.3g). The raw product was distilled and the pure product was collected at 131–135 °C/0.3 mmHg to afforded 14.5 g of product 11 as a colorless viscous liquid (77.1% of yield). 1H NMR (CDCl3, TMS, ppm): 5.58 (1H, anomeric CH), 4.62 (1H, sugar moiety, CHH), 4.35-4.28 (3H, sugar moiety CH), 3.90-3.70 (4H, HOCH2 and sugar moiety CH), 1.54-1.35 (12H, CH3). To a stirred suspension of 1,2:3,4-di-O-isopropylidene-α-D-galactopyranose 11 (8.00 g , 3.07 × 10-2 mol) and basic alumina (6.24 g , 14.11 × 10-2 mol) in distilled acetonitrile (80 S13
mL), acryloyl chloride (12.80 mL, 15.82 × 10-2 mol) was added at room temperature. The reaction mixture was stirred for 3 days. The solids were removed by filtration though a micromembrane. Reaction completion was monitored using thin layer chromatography with hexane/ethyl acetate (2:1) as the solvent. The solvent was removed under reduced pressure to yield light yellow oil, which was purified via column chromatography (hexane/ethyl acetate, 2:1). Viscous light yellow oil was obtained in 56% of yield. 1H NMR (CDCl3, TMS, ppm): 6.40 (1H, CHH=CH-), 6.17 (1H, CH2=CH), 5.84 (1H, CHH=CH), 5.53 (1H, anomeric CH), 4.61 (1H, sugar moiety CHH), 4.13 (4H, sugar moiety 1CHH and 3CH), 4.10 (1H, sugar moiety CH), 1.33-1.53 (12H, CH3). Synthesis of hyperbranced polymer, HypET-DIAlpGP. TMEA 3 (0.29 g, 1.5 mmol), EGDA (0.17 g, 1 mmol), DIAlpGP (0.47 g, 1.5 mmol) and chloroform (1.5 mL) were successively added into a 5 mL glass tube with a magnetic bar. After three freeze-pump-thaw cycles, the tube was sealed under vacuum, and then the sealed tube was placed in an oil bath at 50°C for 30h. The product in 35.8% yield was obtained after precipitation in diethyl ether, filtration and drying in vacuo. The product 0.2 g was dissolved in 10 mL THF, into this solution, aqueous solution of HCl (2 mol/L) was slowly added until the pH reached to about 3, and then the reaction solution was stirred at room temperature for 20h. The HypET-AlpGP was obtained after dialysis (molecular weight (Mw) cutoff: 3500 Da) against deionized water for 24h.
S14
Table S1. Characterization and properties of the hyperbranched polymers, HypET11, HypET15, HypET20 and HypET24 No
a
Mwb (g/mol)
Mw/Mnb
DBc
Φd
τ (ns)
HypET11
7660
2.66
0.71
0.11
－
HypET15
14800
2.64
0.79
0.31
2.45
HypET20
20870
3.50
0.80
0.37
2.46
HypET24
55100
3.66
0.82
0.43
2.57
a)
HypET11, HypET15, HypET20 and HypET24 were prepared by polymerization with molar ratio of EGDA/TMEA=2/1 at 50oC for 11, 15, 20 and 24h, respectively. b) Obtained from TD-SEC measurements. c) Calculated based on the 1H NMR data. d) Quantum yield was measured according to Williams’ method.
S15
e O S
O
O
S f
d S S OH c b a N S
O h' h O i O g' S b' S g O i a' OH
O
d
N f e S c e g N i S O g S S a f O S O h O i b OH h O b'c,f,g,g',h,h' O
i
a'
b
d,e
O O
HypET11
a
6
4
2
0
4
2
0
4
2
0
2
0
HypET15 6
HypET20 6
HypET24 6
4
Chemical shift (ppm)
Figure S1.1H NMR spectra of HypET11, HypET15, HypET20 and HypET24 prepared by Michael addition polymerization of EGDA and TMEA with molar ratio of 2/1 at 50oC for 11h, 15h, 20h and 24h.
S16
Mw,triple=20870 Mw/Mn=3.50
Mw,triple=55100
Mw,triple=14800
Mw/Mn=3.66
Mw/Mn=2.64 Mw,triple=7700 Mw/Mn=2.66
16
18
20
22
24
26
28
Elution volumn (mL)
Figure S2. TD-SEC traces of the HypET11 , HypET15 , HypET20 and HypET24 prepared by Michael addition polymerization of EGDA and TMEA with molar ratio of 2/1 at 50oC for 11h , 15h , 20h and 24h.
S17
g
i
H
O O
(S
O
b
b N
a
H O
S
a
e d
CH3 c
h
O O
f
CH3
O
f
)n
O
S
N
SH
a,b,e g
f
h
i
d
7.0
6.5
6.0
5.5
c
5.0
ppm
8
7
6
5
4
3
2
1
0
Chemical shift (ppm) Figure S3. 1H NMR spectrum of the linear polymer with tertiary amine in the backbone obtained from Michael addition polymerization of EGDA and bis(2-mercaptoethyl) methylamine with molar ratio of 1/1 at 50oC for 24h.
S18
Mn=6750 Mw/Mn=1.74
15
18
21
24
27
Volume (mL)
Figure S4. GPC curve of the linear polymer with tertiary amine in the backbone obtained from Michael addition polymerization of EGDA and bis(2-mercaptoethyl)methylamine with molar ratio of 1/1 at 50oC for 24h.
S19
a H Ha
b O O h
f O
e, e'
k
N g
d cH
m H j
S i
H O d
f O
O
S
l H
n
i
O
H p
O
N
g h
i ,g , d h
c,r
k',e' f
7
p m
l
6
5
e
b
a j
k
4
3
2
1
0
Chemical shift (ppm) Figure S5. 1H NMR spectrum of the linear polymer with the tertiary amine as side group obtained from Michael addition polymerization of 1-acryloyl-1-di(isopropyl) amineethyl2-methacryloyl-glycol and 1,3-propanedithiol with molar ratio of 1/1 at 50oC for 24h.
S20
Mn=4300 Mw/Mn=1.34
15
18
21
24
27
Volume (mL)
Figure S6. GPC curve of the linear polymer with tertiary amine as side group obtained from Michael addition polymerization of 1-acryloyl-1-di(isopropyl) amineethyl–2–methacryloyl –glycol and 1,3-propanedithiol with molar ratio of 1/1 at 50oC for 24h.
S21
HypET24
HypET24-O7
HypET24-O20
4.5
4.0
3.5
3.0
2.5
Chemical shift (ppm) Figure S7. 1H NMR spectra of the HypET24, HypET24-O7 and HypET24-O20 in CDCl3. prepared by oxidization of HypET24 in THF under an air atmosphere at around 35oC for 7 days and 24 days, respectively. Solvent: CDCl3.
S22
HypET24
HypET24-O7
HypET24-O20
-1 1460 cm-1 1361 cm 1184 cm-1 1400 1200
1000
1460 1361 1184
4000
3000
2000
1000 -1
Wavenumber (cm ) Figure S8. IR spectra of the HypET24, HypET24-O7 and HypET24-O20. The HypET24 was prepared by Michael addition polymerization of EGDA and TMEA with molar ratio of 2/1 at 50oC for 24h, HypET24-O7 and HypET24-O20 were prepared by oxidization of the HypET24 in THF under an air atmosphere at around 35oC for 7 days and 20 days, respectively.
S23
a d S c N Sf S e
O O
O l hO O Sg O
O S O d N f e HS c e g S Oi gS f N S h O iO O h
O O
c,d,e,f,g,h
mO b nk O O j
O O
a
i,j,k,m
l
8
7
6
b
5
n
4
3 2 1 0 Chemical Shift (ppm)
Figure S9. 1H NMR spectrum of the HypET-DIAlpGP in CDCl3, the HypET-DIAlpGP was prepared by Michael addition polymerization with molar ratio of EGDA/TMEA/DIAlpGP = 1/1.5/1.5 at 50oC for 30h.
S24
S O S d HS
c
N
f e
O
C
O g O i O S C O h i C O
OH
N
O OH O OH C S O e O N f OH h S g C O l f OH e S n m g O k h j b HO HO S
O
C
HO
S
c,d,e,f,g,h,m,b,k
i,j
nl
8
7
6
5
4
3
2
1
0
Chemical shift (ppm) Figure S10. 1H NMR spectrum of HypET-AlpGP in DMSO. HypET-AlpGP was prepared by polymerization with molar ratio of EGDA/TMEA/DIAlpGP=1/1.5/1.5 at 50oC for 30h, and subsequently, deprotection reaction in diluted aqueous solution of hydrochloric acid.
S25
