Afterwards, a solution of 2-bromoisobutyryl bromide (3.38 g, 15 mmol, 1.5 eq.) in CH2Cl2 (20 mL) was slowly added through a dropping funnel. The ice bath was.
Amphipathic Homopolymers for siRNA Delivery: Probing Impact of Bifunctional Polymer Composition on Transfection Christian Buerkli,a Soo Hyeon Lee,b Elena Moroz,b Mihaiela C. Stuparu,c Jean-Christophe Leroux,b* Anzar Khana* a
Department of Materials, ETH-Zürich, CH-8093, Switzerland
b
c
Institute of Pharmaceutical Sciences, ETH-Zürich, CH-8093, Switzerland
Institute of Organic Chemistry, University of Zürich, Switzerland
1. Polymer Synthesis 1. 1. Materials 2-(N-Ethylanilino)ethanol, 2-bromoisobutyryl bromide, glycidyl methacrylate, 4,4’-dinonyl-2,2’dipyridyl, Cu(I)Br, 2-(Boc-amino)ethanethiol, lithium hydroxide (LiOH-H2O), valeryl chloride, benzoyl chloride,
heptanoyl chloride, propanoyl chloride,
hydrocinnamoyl chloride, hexanoyl
chloride, butyryl chloride, trifluoroacetic acid and 1-amidinopyrazole hydrochloride were purchased from commercial sources and used without further purification. NMR spectra were recorded on Bruker AV300 MHz spectrometers using CDCl3 and DMSO-d6 as solvents. Infrared spectra were measured using an attenuated total reflection (ATR)-Fourier transform infrared (FTIR) spectrometer equipped with an OPUS 6 software (Bruker Optics Alpha system with a built-in diamond ATR). Analytical GPC measurements were carried out using a PL-GPC 220 instrument with a 2 x PL-Gel Mix-B LS column set equipped with refractive index, viscosity and light-scattering (with 15° and 90° angle) detectors (DMF containing 1 g/L LiBr as eluent at 45 °C). The polymers’ molecular weight was determined using polystyrene standards and refractive index detection. 1. 2. Synthetic procedure 1. 2. 1. ATRP-Initiator 1: Et
N
OH
Br
Br
Et
N
Br
O O
O NEt3
1
Triethylamine (5 mL) was added to a solution of 2-(N-ethylanilino)ethanol (1.65 g, 10 mmol, 1 eq.) in CH2Cl2 (20 mL) and cooled in an ice bath. Afterwards, a solution of 2-bromoisobutyryl bromide (3.38 g, 15 mmol, 1.5 eq.) in CH2Cl2 (20 mL) was slowly added through a dropping funnel. The ice bath was removed and the reaction was carried out at room temperature (r.t.) for 12 h. After this time, the reaction mixture was diluted with CH2Cl2, washed with water (3 x 100 mL), dried over MgSO4, and 1
the solvent was removed under reduced pressure. Column chromatography (SiO2, DCM:Heptane 2:1) yielded 1.7 g (54%) of the ATRP initiator 1. 1H-NMR (δ, ppm, 300 MHz, CDCl3): 7.23 (m, 2H, ArCH), 6.73 (m, 3H, Ar-CH), 4.34 (t, J = 6.3 Hz, 2H, NCH2CH2O), 3.61 (t, J = 6.3 Hz, 2H, NCH2CH2O), 3.44 (q, J = 6.9 Hz, 2H, NCH2CH3), 1.92 (s, J = 6.9 Hz, 6H, C(CH3)2Br), 1.19 (t, 3H, CH2CH3); IR (cm-1): 2973, 2870, 1733, 1597, 1505, 1458, 1371, 1350, 1269, 1158, 1107, 1010, 903, 859, 791, 745, 693, 643, 510. 1. 2. 2. General reactive scaffold synthesis: O Et
N
Br
O
Et
O
O
O
N
n
O
2
Cu(I)Br dinonylbipyridine ligand Anisole
1
Br
O O
O O
3
ATRP-initiator 1 (110 µL, 0.35 mmol, 1 eq.), glycidylmethacrylate 2 (2.72 g, 20.5 mmol, 60 eq.) and 4,4’-dinonyl-2,2’-bipyridine (130 mg, 0.31 mmol) were dissolved in 5 mL anisole in a schlenk flask and purged with N2 for 40 min. Afterwards, Cu(I)Br (28 mg, 0.19 mmol, 0.5 eq.) was added and N2 purging was continued for another 5 min. The reaction mixture was stirred for 2 h at r.t., precipitated into 100 mL of isopropanol and filtered. The solid was dissolved in a minimal amount of CH2Cl2 and filtered through a short silica plug (elution with CH2Cl2). The white solid was dried to give polymer 3 as a white powder. 1H-NMR (δ, ppm, 300 MHz, CDCl3): 4.32 (br s, 1H, COOCH2), 3.8 (br s, 1H, COOCH2), 3.23 (br s, 1H, CH2CHCH2O), 2.84 (br s, 1H, COOCH2CHCH2O), 2.65 (br s, 1H, COOCH2CHCH2O), 2.05-1.94 (br s, 2H, CH2CCH3), 0.94 (br s, 3H, CCH3); IR (cm-1): 2998, 2934, 1723, 1448, 1387, 1340, 1254, 1146, 990, 904, 843, 757, 538, 451. 3a: Mn (NMR) = 5600, DP (NMR) = 40, GPC (DMF): Mn = 19200, Mw = 21900, PDI (Mw/Mn) = 1.14; 3b: Mn (NMR) = 6300, DP (NMR) = 45, GPC (DMF): Mn = 15600, Mw = 17800, PDI (Mw/Mn) = 1.15; 3c: Mn (NMR) = 4700, DP (NMR) = 33, GPC (DMF): Mn = 18200, Mw =20500, PDI (Mw/Mn) = 1.12; 3d: Mn (NMR) = 5000, DP (NMR) = 36, GPC (DMF): Mn = 27200, Mw = 32900, PDI (Mw/Mn) = 1.21; 3e: Mn (NMR) = 5600, DP (NMR) = 40, GPC (DMF): Mn = 17800, Mw = 21900, PDI (Mw/Mn) = 1.23. 1. 2. 3. General Thiol–epoxy coupling procedure: Et
N
Br
O O
3
Et
n
O
O O
HS
NHBoc
N
O
n O
O
O OH
LiOH, THF
4
S
NHBoc
Polymer 3 was dissolved in THF, 2-(Boc-amino)ethanethiol (4 eq. per repeat unit (r.u.)) was added and the solution was cooled in an ice bath. LiOH-H2O (0.25 eq. per SH) was added slowly, the cooling 2
bath was removed and the reaction mixture was stirred at r.t., for 12 h. After this time, water (10 mL) was added and the polymer was extracted with CH2Cl2 (4 x 50 mL). The combined organic phase was dried over K2CO3, filtered, and precipitated into petroleum ether and dried. 1H-NMR (δ, ppm, 300 MHz, CDCl3): 5.4 (br s, 1H, NH), 4.1-4.0 (br s, 3H, OCH2CH(OH)CH2S), 3.33 (br s, 2H, NHCH2CH2S), 2.72 (br s, 4H, CH(OH)CH2S, NHCH2CH2S), 1.87-1.85 (br s, 2H, CH2CCH3), 1.45 (br s, 9H, C(CH3)3), 1.08-0.95 (br s, 3H, CCH3); IR (cm-1): 3368, 2977, 2930, 1685, 1510, 1449, 1364, 1248, 1153, 993, 949, 864, 751, 544, 462. 4a: DP (NMR) = 40, GPC (DMF): Mn = 58900, Mw = 70400, PDI (Mw/Mn) = 1.20; 4b: DP (NMR) = 45, GPC (DMF): Mn = 40100, Mw = 53400, PDI (Mw/Mn) = 1.33; 4c: DP (NMR) = 33, GPC (DMF): Mn = 42100, Mw = 48900, PDI (Mw/Mn) = 1.16; 4d: DP (NMR) = 36, GPC (DMF): Mn = 38600, Mw = 47100, PDI (Mw/Mn) = 1.22; 4e: DP (NMR) = 40, GPC (DMF): Mn = 49800, Mw = 65900, PDI (Mw/Mn) = 1.32. 1. 2. 4. General esterification procedure:
Et
N
O
Et
n O
4
O
O OH
O
N
n O
RCOCl DMAP
5
S
O
S
O O
R O
NHBoc
NHBoc
Polymer 4 was dissolved in dry THF and stirred under inert argon atmosphere in an ice bath for 5 min. Afterwards, NEt3 (3 eq. per r.u.) was added drop wise and the reaction mixture was stirred for 10 min. After this time, acid chloride (4 eq. per r.u.) and a catalytic amount of DMAP in 1 mL of dry THF were added and the reaction mixture was stirred at 50 °C overnight. The reaction mixture was poured into ice-cold acidic water (20 mL), extracted with CH2Cl2, and dried over K2CO3. The solution was filtered through a short silica gel plug (elution with CH2Cl2), precipitated into petroleum ether, and dried. 5a (precursor polymer 4b, R = Propyl), Yield: 76 mg (57%), 1H-NMR (δ, ppm, 300 MHz, CDCl3): 5.30 (br s, 1H, NH), 5.15 (br s, 1H, OCH2CH(OR)CH2S), 4.19-4.04 (br s, 2H, OCH2CH(OR)CH2S), 3.32 (br s, 2H, RNHCH2CH2S), 2.72 (br s, 4H, CH(OR)CH2S, NHCH2CH2S), 2.34 (br s, 2H, RCOOCH2CH2CH3), 1.87-1.83 (br s, 2H, CH2CCH3), 1.68 (br s, 2H, RCOOCH2CH2CH3), 1.44 (br s, 9 H, C(CH3)3), 1.01-0.87 (br s, 6H, RCOOCH2CH2CH3, CH2CRCH3). IR (cm-1): 3369, 2968, 1709, 1509, 1452, 1364, 1247, 1162, 1041, 949, 864, 748, 584, 460. DP (NMR) = 45, GPC (DMF): Mn = 33800, Mw = 42700, PDI (Mw/Mn) = 1.26. 5b, (precursor polymer 4c, R = Butyl), Yield: 440 mg (79%), 1H-NMR (δ, ppm, 300 MHz, CDCl3): 5.32 (br s, 1H, NH), 5.15 (br s, 1H, OCH2CH(OR)CH2S), 4.18-4.01 (br s, 2H, OCH2CH(OR)CH2S), 3.3 (br s, 2H, RNHCH2CH2S), 2.70 (br s, 4H, CH(OR)CH2S, NHCH2CH2S), 2.35 (br s, 2H 3
RCOOCH2CH2CH2CH3), 1.82-1.52 (br s, 4H, CH2CCH3, RCOOCH2CH2CH2CH3), 1.45-1.33 (br s, 11H, C(CH3)3, RCOOCH2CH2CH2CH3), 0.95-0.90 (br s, 6H, CH2CRCH3, RCOOCH2CH2CH2CH3); IR (cm-1): 3379, 2961, 2933, 2873, 2251, 1703, 1509, 1454, 1391, 1366, 1248, 1162, 1107, 1051, 949, 912, 866, 779, 730, 646, 460; DP (NMR) = 33; GPC (DMF): Mn = 31300, Mw = 42600, PDI (Mw/Mn) = 1.36. 5c, (precursor polymer 4b, R = Pentyl), Yield: 67 mg (64%), 1H-NMR (δ, ppm, 300 MHz, CDCl3): 5.30 (br s, 1H, NH), 5.15 (br s, 1H, OCH2CH(OR)CH2S), 4.18-4.00 (br s, 2H, OCH2CH(OR)CH2S), 3.33 (br s, 2H, RNHCH2CH2S), 2.72 (br s, 4H, CH(OR)CH2S, NHCH2CH2S), 2.35 (br s, 2H, RCOOCH2CH2CH2CH2CH3),
1.89-1.85
(br
s,
2H,
CH2CRCH3);
1.64
(br
s,
2
H,
RCOOCH2CH2CH2CH2CH3), 1.45 (br s, 9 H, C(CH3)3), 1.25 (br s, 4H, RCOOCH2CH2CH2CH2CH3), 1.01-0.92 (br s, 6H, RCOOCH2CH2CH2CH2CH3, CH2CRCH3); IR (cm-1): 3369, 2958, 2930, 2872, 1709, 1509, 1452, 1391, 1364, 1247, 1159, 1053, 999, 951, 866, 750, 581, 460; DP (NMR) = 45; GPC (DMF): Mn = 33000, Mw = 43600, PDI (Mw/Mn) = 1.32. 5d, (precursor polymer 4a, R = Hexyl), Yield: 100 mg (62%), 1H-NMR (δ, ppm, 300 MHz, CDCl3): 5.36 (br s, 1H, NH), 5.16 (br s, 1H, OCH2CH(OR)CH2S), 4.15-4.06 (br s, 2H, OCH2CH(OR)CH2S), 3.31 (br s, 2H, RNHCH2CH2S), 2.71 (br s, 4H, CH(OR)CH2S, NHCH2CH2S), 2.35 (br s, 2H, RCOOCH2CH2CH2CH2CH2CH3), 1.83 (br s, 4H, CH2CRCH3, RCOOCH2CH2CH2CH2CH2CH3), 1.61 (br
s,
4H,
RCOOCH2CH2CH2CH2CH2CH3),
1.53-1.38
(br
s,
11H,
C(CH3)3,
RCOOCH2CH2CH2CH2CH2CH3), 1.00-0.89 (br s, 6H, CH2CRCH3, RCOOCH2CH2CH2CH2CH2CH3); IR (cm-1): 3381, 2923, 2852, 1713, 1509, 1455, 1366, 1245, 1160, 1053, 951, 867, 778, 747, 720, 608, 460. DP (NMR) = 40; GPC (DMF): Mn = 55000, Mw = 68000, PDI (Mw/Mn) = 1.23. 5e, (precursor polymer 4d, R = Phenyl), Yield: 290 mg (64%), 1H-NMR (δ, ppm, 300 MHz, CDCl3): 8.17 (br s, 2H, Ar-H), 7.68-7.42 (br s, 3H, Ar-H), 5.29 (br s, 1H, NH), 5.1 (br s, 1H, OCH2CH(OR)CH2S), 4.5-4 (br s, 2H, OCH2CH(OR)CH2S), 3.29 (br s, 2H, RNHCH2CH2S), 2.7 (br s, 4H, CH(OR)CH2S, NHCH2CH2S), 1.87-1.61 (br s, 2H, CH2CCH3,), 1.39 (br s, 9H, C(CH3)3), 0.960.94 (br s, 3H, CH2CRCH3); IR (cm-1): 3368, 2975, 2930, 1788, 1710, 1601, 1509, 1451, 1391, 1364, 1315, 1267, 1160, 1107, 1068, 1024, 949, 864, 750, 710, 616, 544, 513, 460; DP (NMR) = 36; GPC (DMF): Mn = 46700, Mw = 53200, PDI (Mw/Mn) = 1.14. 5f, (precursor polymer 4e, R = CH2CH2Ph), Yield: 500 mg (71 %), 1H-NMR (δ, ppm, 300 MHz, CDCl3): 7.22-6.95 (br s, 5 H, Ar-H), 5.30 (br s, 1H, NH), 5.08 (br s, 1H, OCH2CH(OR)CH2S), 4.163.96 (br s, 2H, OCH2CH(OR)CH2S), 2.9 (br s, 2H, RNHCH2CH2S), 2.61 (br s, 4H, CH(OR)CH2S, NHCH2CH2S), 2.43 (br s, 2H, RCOOCH2CH2Ph), 1.89-1.51 (br s, 2H, CH2CCH3), 1.49 (br s, 9H, C(CH3)3), 1.28 (br s, 2H, RCOOCH2CH2Ph), 0.97-0.91 (br s, 3H, CH2CRCH3); IR (cm-1): 3368, 2926, 1729, 1496, 1454, 1366, 1248, 1142, 1056, 949, 866, 748, 697, 558, 490; DP (NMR) = 40; GPC (DMF): Mn = 58100, Mw = 71000, PDI (Mw/Mn) = 1.22. 4
1. 2. 5. General BOC-deprotection procedure: Polymers 5a-f (50-500 mg) were dissolved in a mixture of DCM (1 mL) and trifluoroacetic acid (1 mL) and stirred at r.t. overnight. The reaction mixture was then precipitated into diethyl ether (20-40 mL), extracted with water, and freeze dried.
Et
O
N
Et
n O
O
5
S
O
N
TFA/DCM (1:1)
O O
n O
O
O O
R O
R O
S
NH3
NHBoc
Cationic amphipathic polymers C0A, C3A, C4A, C5A, C6A, PheA, and EtPheA: Polymer C3A, (precursor polymer 5a), Yield: 20 mg (54%), 1H-NMR (δ, ppm, 300 MHz, (CD3)2SO): 8.15 (br s, NH3+), 5.12 (br s, 1H, OCH2CH(OR)CH2S), 4.13-3,84 (br s, 2H, OCH2CH(OR)CH2S), 3.02 (br s, 2H, NHCH2CH2S), 2.77 (br s, 4H, NHCH2CH2S,
CH(OR)CH2S), 2.31 (br s, 2H,
RCOOCH2CH2CH3), 1.77 (br s, 2H, CH2CCH3), 1.57 (br s, 2H, RCOOCH2CH2CH3), 1.08-0.77 (br s, 6H, CH2CRCH3, RCOOCH2CH2CH3); IR (cm-1): 2967, 1726, 1670, 1516, 1458, 1384, 1172, 1132, 955, 894, 839, 798, 750, 723, 599, 519, 435, 411. Polymer C4A, (precursor polymer 5b), Yield: 150 mg (93%), 1H-NMR (δ, ppm, 300 MHz, (CD3)2SO): 8.14 (br s, NH3+), 5.12 (br s, 1H, OCH2CH(OR)CH2S), 4.2-3.8 (br s, 2H, OCH2CH(OH)CH2S), 3.03 (br s, 2H, NHCH2CH2S), 2.8 (br s, 4H, CH(OR)CH2S, NHCH2CH2S), 2.4 (br
s,
2H,
RCOOCH2CH2CH2CH3),
1.8
(br
s,
2H,
CH2CCH3),
1.6-1.3
(br
s,
4H,
RCOOCH2CH2CH2CH3), 0.92-0.87 (br s, 6H, CH2CRCH3, RCOOCH2CH2CH2CH3); IR (cm-1): 2958, 2873, 1729, 1673, 1534, 1427, 1240, 1131, 948, 890, 836, 799, 721, 598, 517, 408. Polymer C5A, (precursor polymer 5c), Yield: 23 mg (79%), 1H-NMR (δ, ppm, 300 MHz, (CD3)2SO): 8.16 (br s, NH3+), 5.13 (br s, 1H, OCH2CH(OR)CH2S), 4.15-3.86 (br s, 2H, OCH2CH(OR)CH2S), 3.09 (br s, 2H, NHCH2CH2S), 2.79 (br s, 4H, NHCH2CH2S,
CH(OR)CH2S), 2.34 (br s, 2H,
RCOOCH2CH2CH2CH2CH3), 1.7 (br s, 2H, CH2CCH3), 1.57 (br s, 2H, RCOOCH2CH2CH2CH2CH3), 1.31
(br
s,
4H,
RCOOCH2CH2CH2CH2CH3),
0.89
(br
s,
6H,
CH2CRCH3,
-1
RCOOCH2CH2CH2CH2CH3); IR (cm ): 2934, 1668, 1459, 1386, 1329, 1133, 962, 842, 799, 750, 723, 602, 519, 435. Polymer C6A, (precursor polymer 5d), Yield: 56 mg (80%), 1H-NMR (δ, ppm, 300 MHz, (CD3)2SO): 8.15 (br s, NH3+), 5.12 (br s, 1H, OCH2CH(OR)CH2S), 4.13-3.94 (br s, 2H, OCH2CH(OR)CH2S), 3.00 (br s, 2H, NHCH2CH2S), 2.76 (br s, 4H, CH(OR)CH2S, NHCH2CH2S), 2.21 (br s, 2H, RCOOCH2CH2CH2CH2CH2CH3),
1.76
(br
s,
2H,
CH2CCH3),
1.54
(br
s,
2H,
RCOOCH2CH2CH2CH2CH2CH3), 1.27 (br s, 6H, RCOOCH2CH2CH2CH2CH2CH3), 0.92-0.86 (br s, 5
6H, CH2CRCH3, RCOOCH2CH2CH2CH2CH2CH3); IR (cm-1): 2931, 2861, 1731, 1673, 1523, 1459, 1380, 1326, 1139, 951, 894, 837, 798, 747, 723, 701, 598, 517, 435, 392. Polymer PheA, (precursor polymer 5e), Yield: 110 mg (59%), 1H-NMR (δ, ppm, 300 MHz, (CD3)2SO): 8.1 (br s, NH3+), 7.92 (2H, Ar-H), 7.57-7.45 (br s, 3H, Ar-H), 5.3 (br s, 1H, OCH2CH(OR)CH2S), 4.2-3.9 (br s, 2H, OCH2CH(OR)CH2S), 3.01 (br s, 2H, NHCH2CH2S), 2.63 (br s, 4H, NHCH2CH2S, CH(OR)CH2S), 1.6 (br s, 2H, CH2CCH3), 0.71 (br s, 3H, CH2CRCH3); IR (cm1
): 2934, 1720, 1672, 1523, 1489, 1452, 1428, 1386, 1350, 1316, 1271, 1175, 1129, 1067, 1026, 952,
897, 836, 798, 748, 711, 598, 517, 438, 408. Polymer EtPheA, (precursor polymer 5f), Yield: 170 mg (73%). 1H-NMR (δ, ppm, 300 MHz, (CD3)2SO): 8.12 (br s, NH3+), 7.20-7.06 (br s, 5H, Ar-H), 5.11 (br s, 1H, OCH2CH(OR)CH2S ), 4.103.96 (br s, 2H, OCH2CH(OR)CH2S), 3.21 (br s, 2H, NHCH2CH2S), 2.98-2.32 (br s, 8H, NHCH2CH2S, CH(OR)CH2S, COOCH2CH2Ph), 1.8 (br s, 2H, CH2CCH3), 1.12-0.88 (br s, 3H, CH2CRCH3); IR (cm-1): 3412, 3028, 2931, 2502, 1727, 1670, 1454, 1373, 1133, 1061, 966, 836, 801, 748, 698, 596, 558, 462. Control polymer C0A (R = OH, Precursor polymer 4), Yield: 80 mg (90%), 1H-NMR (δ, ppm, 300 MHz, (CD3)2SO): 8.12 (br s, NH3+), 3.89 (br s, 3H, OCH2CH(OH)CH2S), 3.4 (br s, 2H, NHCH2CH2S), 2.78-2.69 (br s, 4H, NHCH2CH2S, CH(OH)CH2S), 1.8 (br s, 2H, CH2CCH3), 1.1-0.88 (br s, 3H, CH2CRCH3); IR (cm-1): 2930, 1669, 1519, 1427, 1330, 1244, 1175, 1126, 995, 942, 895, 836, 798, 721, 598, 517, 465, 408. 1. 2. 6. General guanylation procedure: iPr2NEt (5 eq. per r.u.) was added under inert atmosphere to a solution of polymer C4A, PheA, or EtPheA (80 mg) in dry DMF (1 mL) and stirred for 5 min. 1amidinopyrazole hydrochloride (1.4 eq. per r.u.) was added and the reaction mixture was stirred at r.t. overnight. The solution was precipitated into Et2O, extracted with water, and was purified by using Amicon® Ultra-4 10K centrifugal filter device (MILLIPORE) in the following manner. The filter was washed with Millipore water (2 x 3.5 mL, spin at 4000 rpm for 12 min, r.t., discard flow through). 3.5 mL of the polymer solution in water was added to the filter (spin at 4000 rpm for 12 min, r.t., discard flow through) and the filter was washed with Millipore water (2 x 3.5 mL, spin at 4000 rpm for 12 min, r.t., discard flow through). The concentrated polymer solution was recovered by pipetting from the bottom of the filter and freeze dried. Cationic amphipathic polymers C4G, PheG, and EtPheG:
6
Et Et
N
O
n O
O
S
ClH HN
O O
R
O
N
n O
O
NH2
N N S
O
O O
R O
i-Pr2NEt H2N NH3
NH NH2
Polymer C4G, (precursor polymer C4A), Yield 49 mg (53 %), quantitative conversion, 1H-NMR (δ, ppm, 300 MHz, (CD3)2SO): 7.99 (br s, NH2), 5.35 (br s, NH), 5.15 (br s, OCH2CH(OR)CH2S), 4.3-3.8 (br s, 2H, OCH2CH(OR)CH2S), 3.36 (br s, 2H, NHCH2CH2S), 2.73 (br s, 4H, CH(OR)CH2SCH2), 2.38-2.76 (br s, 2H, RCOOCH2CH2CH2CH3), 1.57-1.37 (br s, 6H, RCOOCH2CH2CH2CH3, CH2CCH3), 0.94-0.85 (br s, 6H, CH2CRCH3, RCOOCH2CH2CH2CH3); IR (cm-1): 3326, 3158, 2958, 2873, 1729, 1655, 1456, 1420, 1387, 1238, 1169, 1133, 996, 949, 836, 801, 747, 721, 662, 591. Polymer PheG, (Precursor polymer PheA), Yield: 29 mg (54 %), quantitative conversion, 1H-NMR (δ, ppm, 300 MHz, (CD3)2SO): 7.95-7.92 (br s, 4H, NH2, Ar-H), 7.58-7.30 (br s, 3H, Ar-H), 5.32 (br s, NH), 5.05 (br s, OCH2CH(OR)CH2S), 4.17-3.82 (br s, 2H, OCH2CH(OR)CH2S), 3.5 (br s, 2H, NHCH2CH2S), 2.8 (br s, 4H, CH(OR)CH2SCH2), 1.69-1.37 (br s, 2H, CH2CCH3), 0.9-0.8 (br s, 3H, CH2CRCH3); IR (cm-1): 3324, 3152, 1717, 1659, 1451, 1388, 1356, 1315, 1272, 1175, 1109, 1070, 1026, 999, 937, 836, 801, 747, 711, 674, 595, 517. Polymer EtPheG, (Precursor polymer EtPheA), Yield: 13 mg (23 %), quantitative conversion, 1HNMR (δ, ppm, 300 MHz, (CD3)2SO): 7.95 (br s, NH2), 7.18-7.16 (br s, 5H, Ar-H), 5.11 (br s, 1H, OCH2CH(OR)CH2S), 5.34 (br s, NH), 4.2-3.9 (br s, 2H, OCH2CH(OR)CH2S), 3.2 (br s, 2H, NHCH2CH2S), 2.95-2.26 (br s, 8H, NHCH2CH2S, CH(OR)CH2S, COOCH2CH2Ph), 1.52 (br s, 2H, CH2CCH3), 1.2-0.8 (br s, 3H, CH2CRCH3).
7
signal intensity (a.u.)
1.00
0.75
0.50
0.25
0.00 14
15
16
17
18
19
retention time (min)
Figure S1. Typical GPC profiles for the reactive scaffold 3 (black), after thiol-epoxy coupling reaction (red, polymer 4), and esterification with an alkyl chain (blue, polymer 5c) in DMF.
e
e a
b
c N
Br
O
d
a
O
d N-Ar c b
9.5
9.0
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1
Figure S2. H-NMR of 1 (CDCl3).
8
1.0
0.5
0.0
ppm
a+b
b
a
Br
O
N
n
O
O
O
d c
e
O
*
cc
d
e
e
*
9.5
9.0
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
ppm
0
ppm
1
Figure S3. H-NMR of 3 (CDCl3). a O
N
n O
O
*
e
O OH
f
c
S
d NH
O
b g
O
a * * b+c e+f
d
g
9
8
7
6
5
4
3
2
1
1
Figure S4. H-NMR of 4 (CDCl3).
9
a lm O
N
n O
O
e
f S
d NH
O
j
h
O O
c
k
i
O
b g
O
a k+m
d
b+c i j
h *
9
8
g
7
6
e
f
5
l
4
3
2
1
0
ppm
1
Figure S5. H-NMR of 5b (CDCl3). * lm N
O
n O
O
e a
O O
f
c
S
d
j
h i
O
k+m
k
b NH3 g d
b + c
l+i+j h
g
9
8
f
a
7
6
5
e
4
*
3
2
1
1
Figure S6. H-NMR of polymer C4A (DMSO-d6). 2. Transfection of siRNA Complexes
10
0
ppm
100
% RLU/protein
80
60
40
20
0
Figure S7. Target luciferase silencing of siRNA complexed with various polymers at N/P ratio of 9 in the absence of serum. Results from polymers C0A, C4G, and PheG are not reported due to severe cytotoxicity under these conditions. Values are represented as a mean ± S.D. (n=3).
160
A
120
140
100
80
100 80
60
60
40
% protein amount
% RLU/protein
120
40 20
20 0
0
siRNA conc. : control
77 nM
control
serum-free for 5h
B
77 nM
154 nM
10% serum for 5h
120
10% serum 25% serum % RLU/protein
100
80
60
40
control
5h
10 h
Incubation time with complex
Figure S8. (A) Comparison of the target luciferase silencing (bar, left y-axis) and total protein amount (filled dot, right y-axis) after the transfection with siRNA/polymer C5A (N/P = 4.5) in the absence or 11
presence of fetal bovine serum. (B) Incubation time-dependent silencing efficiencies with 154 nM of siRNA/polymer C5A (N/P = 4.5) in 10% and 25% serum containing medium. After 5 h and 10 h incubation, the medium was changed with a fresh one and the cells were incubated for 43 h and 38 h, respectively, before analyzing the luciferease expression. Values are represented as a mean ± S.D. (n=3).
*
A
120
**
*
control C5A B-PEI L-PEI
% RLU/protein
100
80
60
40
20
0
Luc-siRNA
mm-siRNA
120
B Cell Viability (%)
100
80
60
40
20
0
control
Luc-siRNA /C5A
Luc-siRNA /B-PEI
Luc-siRNA /L-PEI
Figure S9. Comparison of silencing efficiency (A) and cell viability (B) after the transfection with various carriers in the presence of 25% fetal bovine serum (Luc-siRNA = luciferase targeting siRNA, mm-siRNA = control mismatch siRNA). Values are represented as a mean ± S.D. (n=3). (*) P < 0.001 and (**) P < 0.05 between two groups (one way ANOVA analysis with Turkey test).
3. Polymer Titration The buffering capacities of polymer C5A and B-PEI were determined by acid-base titration. Polymer C5A and B-PEI containing 1 µmol of protonable amine groups were dissolved in 0.5 mL of deionized water and the pH was adjusted to 3 by addition of 0.1 M HCl solution. Polymer solutions were titrated with 0.01 M NaOH solution. It was assumed that all of the NaOH added deprotonate amine groups in the polymer. The buffering capacity was calculated as a percentage of deprotonated amine groups to 12
total protonable amine groups in polymer solution as pH increases from 5.5 to 7.4 (see the following equation). Buffering capacity % =
∆V!"#$ × 0.01 M × 100 (%) 1 µμmol
Wherein ∆VNaOH is the volume of 0.01 M NaOH solution required to change the pH of polymer solution from 5.5 to 7.4.1
10
C5A B-PEI
pH
8
6
Buffering capacity (%)
4
C5A
38.4 ± 1.5
B-PEI
22.8 ± 0.9
*
2 0
50
100
150
V (NaOH), µL
Figure S10. Comparison of buffering capacity of protonable amine groups in polymer C5A and BPEI. (*) P < 0.001 between two values (Student’s t-test).
Reference 1. Zhong, Z.; Feijen, J.; Lok, M. C.; Hennink, W. E.; Christensen, L. V.; Yockman, J. W.; Kim, Y.-H.; Kim, S. W., Low Molecular Weight Linear Polyethylenimine-b-poly(ethylene glycol)-bpolyethylenimine Triblock Copolymers: Synthesis, Characterization, and in Vitro Gene Transfer Properties. Biomacromolecules 2005, 6, (6), 3440-3448.
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