Tuning the Emission of Semiconducting. Polymer Dots from Green to Near-Infrared by. Alternating Donor Monomers and their

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1 Tuning the Emission of Semionduting Polymer Dots from Green to Ner-Infrred y Alternting Donor Monomers nd their Applitions for In Vivo Biologil Imging Sz-Yu Liou, Chi-Shing Ke, Jhih-Hn Chen, Yun-Wen Luo, Shih-Yu Kuo, You-Hong Chen, Chi-Chi Fng, Chng-Yi Wu, Cho-Ming Ching, nd Yng-Hsing Chn * Deprtment of Chemistry, Ntionl Sun Yt-sen University, 70 Lien Hi Rod, Kohsiung, Tiwn Deprtment of Biologil Sienes, Ntionl Sun Yt-sen University, 70 Lien Hi Rod, Kohsiung, Tiwn Both uthors ontriuted eqully to this work E-mil: yhhn@mil.nsysu.edu.tw Supporting Informtion Mterils. All of the hemils were purhsed from Sigm-Aldrih or Alf Aesr, nd used s reeived unless indited elsewhere. All io-relted gents suh s streptvidin, nitiody, or medium re purhsed form Invitrogen (Life Tehnologies). High purity 1

2 wter (18.2 M m) ws used throughout the experiment. All 1 HNMR nd 13 CNMR spetr were reorded on Bruker AV300 spetrometer (300 MHz). Compounds 2,7-CZ nd ompounds were synthesized ording to previously pulished reports 1,2 nd thus their syntheti proedures were not desried herein. Other ompounds were synthesized with modified proedures nd the pproprite litertures were ited for eh ompounds. Sheme S1. Synthesis routes of monomers nd BT-ontining opolymers. A H B 2,7-CZ 2,7-CZ P2,7-CZBT 1 C 2 3,6-CZ 3 4 3,6-CZ P3,6-CZBT CPD PCPDBT D 8 9 CPD + BTZ DTT PDTTBT E 10 DTT 5 F 11 Si-CPD Si-CPD Si-PCPDBT G 14 Si-PF Si-PF Si-PFBT BTZ PF PFBT 2

3 Ourrene (A) (B) Height (nm) Figure S1. (A) Representtive AFM imge of Si-PCPDBT-COOH Pdots. The inset in the upper-right orner shows the enlrged view of ouple Pdot nnoprtiles. (B) Prtile height histogrm of Si-PCPDBT-COOH Pdots s mesured y AFM. The verge Pdot size determined y AFM ws 6.7 ± 2.4 nm. The sle r is 10 m. Synthesis of 5-(romomethyl)undene, g (38.13 mmol) of triphenylphosphine ws dissolved in 15 ml of CH2Cl2 in round-ottomed single-neked flsk nd then ooled to 0 C. 2 ml (38.14 mmol) of romine ws dded dropwise nd reted for 10 min t 0 C. After tht, 11.6 g (62.63 mmol) of 2-utyl-1-otnol were dded slowly nd the mixture ws wrm up to room temperture while kept stirring for 12 h. After tht, the retion mixture ws wshed with rine. The orgni extrt ws seprted nd dried over MgSO4, nd the solvent ws removed under redued pressure. The rude produt ws purified y olumn hromtogrphy on sili gel using hexne s eluent to yield 3.5 g (22 %) of ompound 1 s olorless oil. 1 H NMR (300 MHz, CDCl3): δ = 3.45 (d, J = 6.0 Hz, 2H), (m, 1H), (m, 16H), (m, 6H). MALDI-MS: m/z: (M + ). Synthesis of 9-(2-utylotyl)-9H-rzole, 2. 4 To round-ottomed two-neked flsk ws dded 1 g (5.98 mmol) of 9H-rzole, nd 0.28 g (11.6 mmol) of NH under nitrogen 3

4 tmosphere nd then ooled to 0 C. The mixture ws dissolved in 7 ml of dry DMF nd then dded dropwise with 1.94 g (7.8 mmol) of ompound 1. The mixture ws wrm up to room temperture nd stirred for 12 h. The solvent ws removed y rotry evportor, nd then the residue ws dissolved in hexne to extrt with rine. The orgni extrt ws seprted nd dried over MgSO4, nd the solvent ws removed under redued pressure. The rude produt ws purified y olumn hromtogrphy on sili gel using hexne s eluent to yield 1.64 g (72 %) of ompound 2 s olorless oil. 1 H NMR (300 MHz, CDCl3): δ = 8.1 (d, J = 9.1 Hz, 2H), (m, 4H), 7.21 (d, J = 9.1 Hz, 2H), 4.16 (d, J = 6.4 Hz, 2H), (m, 1H), (m, 16H), 0.84 (t, J = 6.4 Hz, 6H). MALDI-MS: m/z: (M + ). Synthesis of 3,6-diromo-9-(2-utylotyl)-9H-rzole, 3,6-CZ g (10.5 mmol) of ompound 2 ws dissolved in 16 ml of DMF in round-ottomed single-neked flsk nd then ooled to 0 C g (22.05 mmol) of n-romosuinimide (NBS) dissolved in 15 ml of DMF ws dded into the mixture in one portion. After eing stirred in the drk t room temperture for 12 h, the retion mixture ws diluted with hexne nd extrted with rine. The orgni extrt ws seprted nd dried over MgSO4, nd the solvent ws removed under redued pressure. The rude produt ws purified y olumn hromtogrphy on sili gel using hexne s eluent to yield 2.78 g (54 %) of ompound 3,6-CZ s olorless oil. 1 H NMR (300 MHz, CDCl3): δ = 8.14 (s, 2H ), 7.54 (d, J = 3.0 Hz, 2H), 7.25 (d, J = 9.2 Hz, 2H), 4.10 (d, J = 6.6 Hz, 2H), (m, 1H), (m, 16H), (m, 6H). 13 C NMR (75 MHz, CDCl3): δ = , , , , , , 47.99, 37.95, 31.93, 31.82, 31.65, 29.64, 28.84, 26.61, 23.06, 22.67, 14.14, MALDI-MS: m/z: (M + ). Synthesis of 2,2'-ithiophene, 3. To stirring solution of 10 ml (0.16 mol) of 2- romothiophene in 60 ml of dry DMF ws dded g (0.54 mol) of freshly wshed opper powder under nitrogen tmosphere, nd then the retion mixture ws refluxed t 140 ºC for 24 h. After the mixture ws ooled to room temperture, exess opper ws removed vi flsh hromtogrphy using CH2Cl2 s eluent. The filtrte ws diluted with CH2Cl2 nd extrted with rine. The orgni extrt ws seprted nd dried over MgSO4, 4

5 nd the solvent ws removed under redued pressure. The rude produt ws purified y olumn hromtogrphy on sili gel using hexne s eluent to yield g (84 %) of ompound 3 s olorless rystl. 1 H NMR (300 MHz, CDCl3): δ = (m, 2H ), (m, 2H). MALDI-MS: m/z: (M + ). Synthesis of 3,3,5,5 -tetrromo-2,2 -ithiophene, g (6.18 mmol) of ompound 3 ws dissolved in 15 ml of DMF in round-ottomed single-neked flsk nd then 4.4 g (24.72 mmol) of NBS dissolved in 15 ml of DMF ws dded in one portion. After eing stirred in the drk t room temperture for 12 h, the mixture ws refluxed t 70 ºC for 8 h. The retion mixture ws diluted with CH2Cl2 nd extrted with rine. The orgni extrt ws seprted nd dried over MgSO4, nd the solvent ws removed under redued pressure. The produt ws further repreipitted using CH2Cl2/methnol nd then filtered to fford 2.74 g (92 %) of ompound 4 s white solid 1 H NMR (300 MHz, CDCl3): δ = 7.05 (s, 2H ). MALDI-MS: m/z: (M + ). Synthesis of 3,3'-diromo-[2,2'-ithiophene]-5,5'-diyl)is(trimethylsilne), 5. 7 A solution of 16.0 g (33.21 mmol) of ompound 4 in 400 ml of dry ether ws ooled to -78 C under nitrogen tmosphere ml (73.05 mmol) of n-buli (1.6 M in hexne) ws dded dropwise to the solution, nd then the mixture ws wrm up to 0 C nd stirred for 3 h. After tht, 8.48 ml (66.42 mmol) of hlorotrimethylsilne ws dded in one portion nd the mixture ws stirred for 12 h, followed y refluxing for nother 8 h. After the retion, the solution ws wshed with rine. The orgni extrt ws seprted nd dried over MgSO4, nd the solvent ws removed under redued pressure. The rude produt ws purified y olumn hromtogrphy on sili gel using hexne s eluent nd further wshed y ethnol to yield g (66 %) of ompound 5 s white needle-like solid. 1 H NMR (300 MHz, CDCl3): δ = 7.15 (s, 2H ), 0.35 (s, 18H). MALDI-MS: m/z: (M + ). Synthesis of 2,6-is(trimethylsilyl)-4H-ylopent[1,2-:5,4-']dithiophen-4-one, 6. 6 A solution of 2.0 g (4.27 mmol) of ompound 5 in 16.8 ml of dry THF ws ooled to -78 C under nitrogen tmosphere. 3.9 ml (9.8 mmol) of n-buli (2.5 M in hexne) ws dded dropwise over 10 min to the solution nd then the solution ws stirred for 15 min. After 5

6 tht, 0.43 ml (4.78 mmol) of dimethyl rmoylhloride dissolved in 4 ml of dry THF ws dded dropwise. The retion mixture then slowly wrmed to room temperture nd stirred for 12 h. The retion ws quenhed y dding NH4Cl solution nd the orgni lyer ws wshed with H2O, rine nd dried over MgSO4. The solvent ws removed under redued pressure nd the rude produt ws purified y olumn hromtogrphy on sili gel, eluting with 1:1 (v/v) of hexne /CH2Cl2 to yield 1.02 g (71 %) of 6 s drk red solid. 1 H NMR (300 MHz, CDCl3): δ = 7.07 (s, 18H ), 0.31 (s, 2H). MALDI-MS: m/z: (M + ). Synthesis of 4H-ylopent[1,2-:5,4-']dithiophen-4-one, g (6.78 mmol) of ompound 6 ws dissolved in 35 ml of CHCl3 in round-ottomed single-neked flsk nd then 1.74 ml of trifluoroeti id ws dded in one portion. After eing stirred for 1 h, the solution ws diluted with CH2Cl2 nd then extrted with rine. The orgni extrt ws seprted nd dried over MgSO4, nd the solvent ws removed under redued pressure. The rude produt ws purified y olumn hromtogrphy on sili gel, eluting with 1:1 (v/v) of hexne /Ch2Cl2 to yield 1.15 g (88 %) of ompound 7 s purple needle-like solid. 1 H NMR (300 MHz, CDCl3): δ = 7.04 (d, J = 6.1 Hz, 2H ), 7.00 (d, J = 3.0 Hz, 2H). MALDI-MS: m/z: (M + ). Synthesis of 4H-ylopent[1,2-:5,4-']dithiophene, 8. 7 To suspension solution of 0.14 g (6.78 mmol) of ompound 7 in 15 ml of ethylene glyol ws dded 0.14 g (2.5 mmol) of potssium hydroxide under nitrogen tmosphere. The mixture ws stirred t room temperture for 20 min, nd then 8.4 ml hydrzine ws dded dropwise. After eing stirred for 1 h, the solution ws refluxed t 180 ºC for 8 h. The retion mixture ws quenhed with wter nd then diluted with ether nd extrted with rine. The orgni extrt ws seprted nd dried over MgSO4, nd the solvent ws removed under redued pressure. The rude produt ws purified y olumn hromtogrphy on sili gel using hexne s eluent to yield 0.93 g (72 %) of ompound 8 s white rystl. 1 H NMR (300 MHz, CDCl3): δ = 7.18 (d, J = 6.3 Hz, 2H ), 7.09 (d, J = 6.2 Hz, 2H), 3.54 (s, 2H). MALDI-MS: m/z: (M + ). 6

7 Synthesis of 4,4-dihexyl-4H-ylopent[1,2-:5,4-']dithiophene, 9. 8 To suspension solution of 0.12 g (0.67 mmol) of ompound 8, 0.23 g (1.34 mmol) of romohexne, nd 3.1 mg of potssium iodide in 3 ml of DMSO ws dded 0.12 g (2.21 mmol) of potssium hydroxide in one portion t 0 ºC. The retion solution ws stirred t room temperture for 16 h nd then quenhed with wter. After tht, the mixture ws diluted with hexne nd extrted with rine. The orgni extrt ws seprted nd dried over MgSO4, nd the solvent ws removed under redued pressure. The rude produt ws purified y olumn hromtogrphy on sili gel using hexne s eluent to yield 0.20 g (81 %) of ompound 9 s olorless oil. 1 H NMR (300 MHz, CDCl3): δ = 7.14 (d, J = 6.6 Hz, 2H ), 6.93 (d, J = 3.2 Hz, 2H), 1.82 (t, J = 6.0 Hz, 4H), (m, 12H), (m, 4H), 0.81 (t, J = 6.2 Hz, 6H). MALDI-MS: m/z: (M + ). Synthesis of 2,6-diromo-4,4-dihexyl-4H-ylopent[1,2-:5,4-']dithiophene, CPD g (0.57 mmol) of ompound 9 ws dissolved in 5 ml of DMF in round-ottomed singleneked flsk nd then 0.21 g (1.2 mmol) of NBS ws dded in one portion. After eing stirred in the drk t room temperture for 12 h, the retion mixture ws diluted with ether nd extrted with rine. The orgni extrt ws seprted nd dried over MgSO4, nd the solvent ws removed under redued pressure. The rude produt ws purified y olumn hromtogrphy on sili gel using hexne s eluent to yield 0.25 g (86 %) of ompound CPD s yellow oil. 1 H NMR (300 MHz, CDCl3): δ = 6.92 (s, 2H ), 1.75 (t, J = 9.4 Hz, 4H), (m, 12H), (m, 4H), 0.83 (t, J = 6.2 Hz, 6H). 13 C NMR (75 MHz, CDCl3): δ = , , , , 53.92, 37.02, 31.10, 29.45, 24.35, 22.70, 14.11, MALDI-MS: m/z: (M + ). Synthesis of dithieno[3,2-:2',3'-d]thiophene, ml (10 mmol) of n-buli (1.6 M in hexne) ws slowly dded into 1 ml (10 mmol) of 3-romothiophene in 15 ml of dry ether under nitrogen tmosphere t -78 o C nd the mixture ws stirred for 40 min while keeping t -78 o C. This solution ws then wrmed to 0 o C nd C4H9Br ws removed under vuum. (Note: If C4H9Br is not removed, 3-utyl-S-thiophene will e generted s mjor produt fter the ddition of sulfur powder in the next step). After tht, 15 ml of dry ether ws dded nd the solution ws ooled to -78 o C g (10 mmol) of sulfur ws dded, 7

8 stirred for 30 min, nd then wrmed up to 0 o C for nother 30 min g (13 mmol) of p- toluenesulfonyl hloride ws dded into the solution t 0 o C, stirred for 30 min, nd then wrmed up to 40 o C for nother 4 h. The seond portion of 3-Li-thiophene (1.2 eq.) ws prepred y the ove desried proedures in whih 9 ml (15 mol) of n-buli (1.6 M in hexne), 1.5 ml (15 mol) of 3- romothiophene, nd 20 ml of dry ether were used. After the retion, the seond portion of 3-lithiophene in ether solution ws dded into the first retion mixture t -78 o C, stirred for 1 h, nd then wrmed up to room temperture for nother 8 h. 4.5 ml (27 mmol) of tetrmethylethylene-dimine (TMEDA) nd 17.5 ml (28 mmol) of n-buli (1.6 M in hexne) ws slowly dded into the ove mixture solution t 0 o C nd stirred for 30 min. The mixture ws then refluxed for 1 h nd llowed to ool down to 0 o C. 4 g (30 mmol) of CuCl2 ws quikly dded into this mixture t, stirred for 1 h, nd then wrmed up room temperture for 12 h. After the retion, the preipittes were filtered out nd the orgni ether portion ws olleted. The solvent ws removed under redued pressure nd the rude produt ws purified y hromtogrphy on sili gel using hexne s eluent to yield 0.50 g (24 %) of ompound 10 s light-yellow solid. 1 H NMR (300 MHz, CDCl3): δ = 7.36 (d, J = 6.3 Hz, 2H ), 7.29 (d, J = 6.3 Hz, 2H). MALDI-MS: m/z: (M + ). Note: This ompound is not stle t room temperture nd should e stored t 4 o C. Synthesis of 2,6-diromo-4,4-dihexyl-4H-ylopent[1,2-:5,4-']dithiophene, DTT g (1.17 mmol) of ompound 10 ws dissolved in 6 ml of THF in round-ottomed singleneked flsk nd then ooled to 0 C g (2.5 mmol) of NBS solid ws diretly dded into the mixture. After eing stirred in the drk t room temperture for 12 h, the solvent ws removed y rotry evportor nd then the solid ws dissolved in CH2Cl2 to extrt with rine. The orgni extrt ws seprted, dried over MgSO4, nd the solvent ws removed under redued pressure. The produt ws otined y repreipitting from CH2Cl2/methnol to fford 0.23 g (55 %) of ompound DTT s light-yellow solid. 1 H NMR (300 MHz, CDCl3): δ = 7.28 (s, 18H ). 13 C NMR (75 MHz, CDCl3): δ = , , , MALDI-MS: m/z: (M + ). 8

9 Synthesis of 4,4-diotyl-2,6-is(trimethylsilyl)-4H-silolo[3,2-:4,5-']dithiophene, A solution of 0.85 g (1.81 mmol) of ompound 5 in 10 ml of dry THF ws ooled to -78 C under nitrogen tmosphere. 2.6 ml (4.15 mmol) of n-buli (1.6 M in hexne) ws dded dropwise to the solution. After eing stirred t -78 C for 15min, 0.75 g (2.17 mmol) of dihlorodiotylsilne ws dded dropwise to the solution nd then wrmed up to room temperture for 12 h. After tht, the retion mixture ws diluted with ether nd extrted with rine. The orgni extrt ws seprted nd dried over MgSO4, nd the solvent ws removed under redued pressure. The rude produt ws purified y olumn hromtogrphy on sili gel using hexne s eluent to yield 0.41 g (40 %) of ompound 11 s yellow oil. 1 H NMR (300 MHz, CDCl3): δ = 7.13 (s, 2H ), (m, 4H), (m, 20H), (m, 10H), 0.34 (s, 18H). MALDI-MS: m/z: (M + ). Synthesis of 2,6-diromo-4,4-diotyl-4H-silolo[3,2-:4,5-']dithiophene, Si-CPD g (3.55 mmol) of ompound 11 ws dissolved in 5 ml of THF in round-ottomed singleneked flsk nd then 1.26 g (7.11 mmol) of NBS in 5 ml of THF ws then dded in one portion. After eing stirred in the drk t room temperture for 4 h, the retion mixture ws diluted with ether nd extrted with rine. The orgni extrt ws seprted nd dried over MgSO4, nd the solvent ws removed under redued pressure. The rude produt ws purified y olumn hromtogrphy on sili gel using hexne s eluent to yield 0.19 g (93 %) of ompound Si-CPD s yellow oil. 1 H NMR (300 MHz, CDCl3): δ = 6.99 (s, 2H ), (m, 24H), (m, 10H). 13 C NMR (75 MHz, CDCl3): δ = , , , , 33.39, 31.97, 29.35, 24.33, 22.98, 22.88, 22.67, MALDI- MS: m/z: (M + ). Synthesis of 4,4'-diromo-2,2'-dinitro-1,1'-iphenyl, g (1.78 mmol) of 1,4- diromo-2-nitroenzene in 6 ml of dry DMF ws dded 0.4 g (6.23 mmol) of pre-wshed opper powder under nitrogen tmosphere, nd then the retion mixture ws refluxed t 125 ºC for 4 h. After the mixture ws llowed to ool to room temperture, 200 ml of toluene ws dded nd exess opper ws removed y filtrtion. The filtrte ws diluted with toluene nd extrted with rine. The orgni extrt ws seprted nd dried over MgSO4, nd the solvent ws removed under redued pressure. The rude produt ws 9

10 wshed with old methnol to fford 0.29 g (81 %) of ompound 12 s yellow solid. 1 H NMR (300 MHz, CDCl3): δ = 8.09 (d, J = 9.0 Hz, 2H ), 7.56 (d, J = 9.1 Hz, 2H), 7.16 (d, J = 6.3 Hz, 2H). MALDI-MS: m/z: (M + ). Synthesis of 4,4'-diromo-[1,1'-iphenyl]-2,2'-dimine, g (2.49 mmol) of ompound 12 in ml of ethnol ws dded 7.09 ml of 32 % w/w HCl. After tht, 1.18 g (9.85 mmol) of tin powder ws dded slowly (over 10 min) nd the retion mixture ws refluxed t 100 ºC for 2 h. After ooling to room temperture, the mixture ws poured into 250 ml of ie wter, nd then titrted with 20% w/w NOH solution to ph ~9. The retion mixture ws diluted with ether nd extrted with rine. The orgni extrt ws seprted nd dried over MgSO4, nd the solvent ws removed under redued pressure. The rude produt ws wshed with methnol to fford 0.84 g (98 %) of ompound 13 s light rown solid. 1 H NMR (300 MHz, CDCl3): δ = 6.92 (s, 6H ), 3.68 (rs, 6H). MALDI- MS: m/z: (M + ). Synthesis of 4,4'-diromo-2,2'-diiodo-1,1'-iphenyl, To solution of 0.8 g (1.99 mmol) of ompound 13 in 32 ml of wter nd 32 ml of etonitrile ws dded 8 ml of 32 % w/w HCl, nd then the solution ws ooled to 0 C. 0.7 g (10.1 mmol) of NNO2 dissolved in 4 ml of wter ws dded dropwise nd the mixture ws stirred t 0 ºC for 1 h.after tht, 3.55 g (21.39 mmol) of potssium iodide in 8 ml of wter ws dded dropwise nd the mixture solution ws refluxed t 80 ºC for 12 h. Finlly, the retion mixture ws quenhed with sodium thiosulfte sturted solution, nd extrted with CH2Cl2. The orgni extrt ws seprted nd dried over MgSO4, nd the solvent ws removed under redued pressure. The rude produt ws purified y olumn hromtogrphy on sili gel using hexne s eluent to yield 0.61 g (55 %) of ompound 14 s white rystl-like solid. 1 H NMR (300 MHz, CDCl3): δ = 8.09 (d, J = 9.0 Hz, 2H), 7.56 (d, J = 9.2 Hz, 2H ), 7.03 (d, J = 9.2 Hz, 2H). MALDI-MS: m/z: (M + ). Synthesis of 3,7-diromo-5,5-diotyl-5H-dienzo[,d]silole, Si-PF g (2.66 mmol) of ompound 14 in 30 ml of dry THF ws ooled to -90 C under nitrogen tmosphere, nd then 6.65 ml (10.6 mmol) of n-buli (1.6 M in hexne) ws dded dropwise (over 2 h) to 10

11 the solution. After eing stirred t -90 ºC for 1h, the solution ws dded dropwise with 1.84 g (5.13 mmol) of dihlorodiotylsilne nd then stirred for 12 h t room temperture. After tht, the retion mixture ws diluted with ether to extrt with rine. The orgni extrt ws seprted nd dried over MgSO4, nd the solvent ws removed under redued pressure. The rude produt ws purified y olumn hromtogrphy on sili gel using hexne s eluent to fford 0.2 g (15 %) of ompound Si-PF s olorless oil. 1 H NMR (300 MHz, CDCl3): δ = 7.68 (d, J = 3.0 Hz, 2H), 7.63 (d, J = 6.2 Hz, 2H ), 7.53 (d, J = 6.2 Hz, 2H), (m, 24H), (m, 10H). 13 C NMR (75 MHz, CDCl3): δ = , , , , , 122,36, 33.36, 31.95, 29.29, 29.14, 23.85, 22.77, 14.22, MALDI-MS: m/z: (M + ). 4,7-is(4,4,5,5-tetrmethyl-1,3,2-dioxoroln-2-yl)enzo[][1,2,5]thidizole, BTZ. 11 A solution of 1 g (3.4 mmol) of 4,7-diromo-2,1,3-enzothidizole, 2 g (7.88 mmol) of 4,4,4',4',5,5,5',5'-otmethyl-2,2'-i(1,3,2-dioxorolne), 0.51 g (0.7 mmol) of Pd(dppf)Cl2, nd 2 g (20.38 mmol) of potssium ette in 40 ml of dry 1,4-dioxne ws stirred under nitrogen tmosphere t 85 C for 12 h. After quenhed y the ddition of wter, the retion mixture ws diluted with ether nd extrted with rine. The orgni extrt ws seprted nd dried over MgSO4, nd the solvent ws removed under redued pressure. The rude produt ws purified y olumn hromtogrphy on sili gel, eluting with 1:16 (v/v) of ether /CH2Cl2 to yield 20 mg (15 %) of ompound BTZ s yellow solid. 1 H NMR (300 MHz, CDCl3): δ = 8.13 (s, 2H ), 1.44 (s, 24H). 13 C NMR (75 MHz, CDCl3): δ = , , 84.44, MALDI-MS: m/z: (M + ). Generl Proedures of Polymeriztion. In 100 ml flsk, monomer BTZ (0.125 mmol) nd monomer 2,7-CZ or 3,6-CZ or CPD or DTT or Si-CPD or Si-PF or PF (0.125 mmol) were dissolved in 6 ml of THF, nd then 2 ml of N2CO3 (2 M) ws dded. The mixture solution ws purged with nitrogen for 30 min. After tht, the mixture solution ws degssed nd refilled with N2 (repeted 4 times) efore nd fter the ddition of Pd(PPh3)4 (7 mg, mmol). The retnts were stirred t 80 C for 72 h, nd then 25 mg of phenyloroni id dissolved in 1 ml of THF ws dded. After 2 h, 0.25 ml of romoenzene ws dded nd further stirred for 3 h. The mixture ws poured into 120 ml of methnol. The 11

12 preipitte ws filtered, wshed with methnol nd etone to remove monomers nd inorgni slts. The rude produt ws dissolved in CH2Cl2 nd then extrted with rine for 3 times. The orgni extrt ws seprted nd dried over MgSO4, nd the solvent ws removed under redued pressure. The rude polymers were repreipitted in CH2Cl2/methnol nd wshed with etone. For the synthesis of Si-PCPDBT-COOH, the roxyl-funtionlized fluorene, PF-COOH, ws first synthesized ording to our previously reported work 13. Then monomer BTZ (0.125 mmol), monomer Si-PCPDBT (0.121 mmol), nd monomer PF-COOH (0.004 mmol) were dissolved in 6 ml of THF for polymeriztion following the ove-mentioned Suzuki retion. Finlly, the produt ws olleted y filtrtion to fford mg of polymer P2,7-CZBT, P3,6-CZBT, PCPDBT, PDTTBT, Si-PCPDBT, Si-PFBT, or PFBT. GPC: P2,7-CZBT Mn: 1776, Mw: 2274, PDI: 1.93, 1 H NMR (300 MHz, CDCl3): δ = (m, 2H), (m, 8H), (m, 2H, CH2), (m, 3H, CH3); P3,6-CZBT Mn: 1176, Mw: 1473, PDI: 1.25, 1 H NMR (300 MHz, CDCl3): δ = (m, 8H), (m, 2H, CH2), (m, 1H, CH), (m, 16H, CH2), (m, 6H, CH3); PCPDBT Mn: 1146, Mw: 1836, PDI: 1.60, 1 H NMR (300 MHz, CDCl3): δ = (m, 2H), (m, 2H), (m, 4H, CH2), (m, 12H, CH2), (m, 6H, CH3); PDTTBT Mn: 1168, Mw: 1473, PDI: 1.26, 1 H NMR (300 MHz, CDCl3): δ = (m, 2H), (m, 2H); Si-PCPDBT Mn: 1528, Mw: 2497, PDI: 1.63, 1 H NMR (300 MHz, CDCl3): δ = (m, 2H), (m, 2H), (m, 24H, CH2), (m, 10H); Si-PFBT Mn: 1503, Mw: 1962, PDI: 1.30, 1 H NMR (300 MHz, CDCl3): δ = (m, 2H), (m, 28H, CH2), (m, 6H, CH3); PFBT Mn: 1670, Mw: 3450, PDI: 2.01, 1 H NMR (300 MHz, CDCl3): δ = (m, 8H), (m, 4H), (m, 24H), (m, 6H, CH3). Preprtion of BT-Bsed Pdots. Typilly, 200 L of opolymer solution (1 mg/ml in THF) nd 0-20 L of PS-PEG-COOH (2 mg/ml in THF) were dded into 5 ml of THF. This mixture solution ws then quikly injeted into 10 ml of wter under vigorous sonition. After tht, THF ws removed y purging with nitrogen on 96 o C hotplte for 60 min. The resulting Pdot solution ws filtered through 0.2 m ellulose memrne filter to remove ny ggregtes formed during Pdot preprtion. It should e noted tht PE-PEG- 12

13 COOH opolymer ws used in order to funtionlize Pdots nd t the sme time mke smller Pdots. For the preprtion of Si-PCPDBT-COOH Pdots, PS-PEG-COOH ws not dded euse the roxyli id groups hve lredy een inorported within the polymer kone. Bioonjugtion nd Chrteriztion of Pdots. Bioonjugtion ws performed y using the EDC-tlyzed retion etween roxylte-funtionlized BT-sed Pdots nd the respetive mine-ontining streptvidin. In typil ioonjugtion retion, 80 L of polyethylene glyol (5% w/v PEG, MW 3350) nd 80 L of onentrted HEPES uffer (1 M) were dded to 4 ml of Pdot solution, resulting in Pdot solution in 20 mm HEPES uffer with ph of 7.3. Then 240 L of streptvidin (1 mg/ml) ws dded to the solution nd mixed well on vortex. After tht, 80 L of freshly-prepred EDC solution (5 mg/ml in MilliQ wter) ws dded to the solution, nd the mixture ws stirred for 4 hours t room temperture. After iononjugtion, 80 L of BSA (10 wt%) ws dded to the Pdot solution nd the retion ws ontinued for nother 20 minutes. A 80- L liquot of Triton X-100 in MilliQ wter (2.5 wt%) ws dded to the Pdot-streptvidin mixture. The mixture ws then trnsferred to entrifugl ultrfiltrtion tue (Amion Ultr-4, MWCO: 100kD), nd then onentrted to 0.5 ml y entrifugtion. Finlly, the Pdot-streptvidin ioonjugtes were purified y gel filtrtion using Sephryl HR-300 gel medi. The verge prtile size ws determined y dynmi light sttering, trnsmission eletron mirosopy, nd tomi fore mirosopy. TEM imges of the synthesized Pdots were quired using JEOL 2100 trnsmission eletron mirosope t n elertion voltge of 200 kv. For TEM, drop of Pdot queous solution ws pled onto ronoted grid nd llowed to evporte t room temperture. The sorption spetr of Pdots were mesured using UV-visile spetrosopy (Dynmi Hlo DB20S, Dynmi Sientifi). The fluoresene spetr were olleted using Hithi F-7000 fluorometer (Hithi, Tokyo, Jpn) under 450 nm exittion. Asolute fluoresene quntum yield of Pdots ws determined y using n integrting sphere unit of Hithi F-7000 fluoresene spetrophotometer. 13

14 Cell Culture nd Leling. The ervil ner ell line HeL nd rest ner ell line MCF-7 were ordered from Food Industry Reserh nd Development Institute (Tiwn). Primry ultured HeL ells were grown in Duleo s Modified Egle Medium (t. no , Invitrogen) supplemented with 10% fetl ovine serum (FBS) nd 1% peniillinstreptomyin solution (5000 units/ml peniillin G, 50 g/ml streptomyin sulfte in 0.85% NCl) t 37 C with 5% CO2 humidified tmosphere. MCF-7 ells were ultured t 37 C, 5% CO2 in RPMI 1640 medium supplemented with 10% FBS nd 1% peniillinstreptomyin solution. The ells were preultured in T-25 flsk nd llowed to grow for 5-7 dys prior to experiments until ~80% onfluene ws rehed. To prepre ell suspensions, the dherent ner ells were quikly rinsed with medi nd then inuted in 0.8 ml of trypsin-ethylenediminetetreti (EDTA) solution (0.25 w/v % trypsin, 0.25 g/l EDTA) t 37 C for 3 min. The ell suspension solution ws then entrifuged t 1000 rpm for 5 min to preipitte the ells to the ottom of the tue. After tking out the upper medi, the ells ws rinsed nd resuspended in 5 ml of ulture medi. Approximtely tens of thousnds ells were split onto glss-ottomed ulture dish nd llowed to grow for 12 h efore Pdot leling. Prior to fluoresene imging, the ells were rinsed with PBS uffer to remove ny nonspeifilly ound Pdots on the ell surfe. For ell leling experiments, BlokAid TM loking uffer ws purhsed from Invitrogen (Eugene, OR, USA). For leling ell-surfe mrkers with IgG, million MCF-7 ells in 100- L leling uffer (1 PBS, 2 mm EDTA, 1% BSA) ws inuted with 0.3 L of 0.5 mg/ml primry iotin nti-humn CD326 EpCAM ntiody (BioLegend, Sn Diego, CA, USA) on rotry shker in the drk nd t room temperture for 30 minutes, followed y wshing step using leling uffer. Then the ells were inuted with 1.5 nm Pdot-streptvidin onjugtes in BlokAid TM uffer for 30 min on shker in the drk nd t room temperture, followed y two wshing steps with leling uffer. Prior to ell inution, Pdot solutions were sonited for 3 min in order to disperse ny potentil ggregtes. Negtive ontrols were otined y inuting ells with Pdotstreptvidin onjugtes in the sene of primry iotinylted-ntiody. Cell fixtion ws performed y dissolving the ell pellet otined y entrifugtion in 500 μl of fixing uffer (1 PBS, 2 mm EDTA, 1% BSA, 1% PFA). 14

15 For mirotuule-leling experiments, tens of thousnds of HeL ells were plted on mm glss overslip (Biogenesis, Tiwn) nd ultured until the density rehed 60-70% onfluene. The ells were fixed with 4% prformldehyde for 15 minutes, permeilized with 0.5% Triton-X 100 in PBS uffer for 15 min, nd then loked in BlokAid TM loking uffer for nother 40 min. The fixed nd loked HeL ells were susequently inuted with 2 g/ml iotinylted monolonl nti-α-tuulin ntiody (BioLegend, Sn Diego, CA, USA) for 60 min, nd then Pdot-streptvidin onjugtes for 40 min. The Pdot leled ells were then ounterstined with Hoehst for 15 min nd imged immeditely on fluoresene onfol mirosope (Nikon D-Elipse C1). Cell Imging. The fluoresene spetr of Pdot-tgged ells were quired with fluoresene onfol mirosope (Nikon D-Elipse C1) under mient onditions (24 ± 2 o C). The onfol fluoresene imges were olleted using diode lser t 488 nm (~15 mw) s the exittion soure nd n integrtion time of 1.6 s/pixel. A CF1 Pln Fluor 40x (N. A. 0.75, W.D mm) ojetive ws utilized for imging nd spetrl dt quisition; the lser ws foused to spot size of ~7 m 2. The lue fluoresene ws olleted y filtering through 450/35 nd-pss ( ex = 408 nm) while the red fluoresene ws olleted y filtering through 570 long-pss ( ex = 488 nm). MTT Assy. The ellulr ytotoxiity of the Pdots ws exmined on HeL ells. The numer of vile ells ws determined using the MTT ssy with 3-(4,5-dimethylthizole- 2-yl)-2,5-phenyltetrzolium romide. HeL ells were first seeded in eh well of 24- well ulture plte nd then inuted with vrious onentrtions of Pdots (100 pm, 200 pm, nd 400 pm) for 6 h, 12 h, nd 24 h. After tht, 20 L (5 mg/ml) of MTT queous solution ws dded to eh well nd the ells were further inuted for 4 h t 37 C to deoxidize MTT. The medium ws then wshed out nd 300 L of DMSO ws dded into eh well to dissolve formzm rystls. Asorne ws mesured y BioTek ELx800 miroplte reder t 570 nm, while the ells ultured with the pure medium (e.g., without Pdots) served s ontrols. 15

16 In vivo imging on zerfish. The trnsgeni zerfish, Tg(kdrl:eGFP) l116 expressing egfp in the endothelil ells, 12 were mintined t 28 C nd red under stndrd onditions with pprovl from Ntionl SunYt-sen University Animl Cre Committee. For ngiogrphy imging, 37 nl Pdots (125 nm) in 20 mm HEPES uffer ws injeted into the sinus venosus of the nesthetized zerfish emryos 3 dys post fertiliztion (dpf) with 5% (v/v) triine (Sigm). The injeted emryos were reovered for 30 minutes, immoilized in 1.5% low melting point grose (invitrogen), nd then imged immeditely using fluoresene onfol mirosope (Nikon D-Elipse C1). The green fluoresene ws olleted y filtering through 515/30 nd-pss ( ex = 488 nm) while the red fluoresene ws olleted y filtering through 570 long-pss ( ex = 488 nm). 16

17 1 H NMR of 2,7-diromo-9-ethyl-9H-rzole (2,7-CZ) e d d e 17

18 d d d 1 H NMR of 5-(Bromomethyl)undene, 1 18

19 g d f e f g d e g f 1 H NMR of 9-(2-Butylotyl)-9H-rzole, 2 19

20 d e f g d e g f 1 H NMR of 3,6-Diromo-9-(2-utylotyl)-9H-rzole, 3,6-CZ 20

21 1 H NMR of 2,2'-Bithiophene, 3 21

22 1 H NMR of 3,3',5,5'-Tetrromo-2,2'-ithiophene, 4 22

23 1 H NMR of 3,3'-Diromo-[2,2'-ithiophene]-5,5'-diyl)is(trimethylsilne), 5 23

24 1 H NMR of 3,3'-Diromo-[2,2'-ithiophene]-5,5'-diyl)is(trimethylsilne), 6 24

25 1 H NMR of 4H-Cylopent[1,2-:5,4-']dithiophen-4-one, 7 25

26 1 H NMR of 4H-Cylopent[1,2-:5,4-']dithiophen-4-one, 8 26

27 1 H NMR of 4,4-Dihexyl-4H-ylopent[1,2-:5,4-']dithiophene, 9 e f d d e f d e f 27

28 d e d e d e 1 H NMR of 2,6-Diromo-4,4-dihexyl-4H-ylopent[1,2-:5,4-']dithiophene, CPD 28

29 1 H NMR of Dithieno[3,2-:2',3'-d]thiophene, 10 29

30 1 H NMR of 2,6-Diromodithieno[3,2-:2',3'-d]thiophene, DTT 30

31 e d d e d e 1 H NMR of 4,4-Diotyl-2,6-is(trimethylsilyl)-4H-silolo[3,2-:4,5-']dithiophene, 11 31

32 1 H NMR of 2,6-Diromo-4,4-diotyl-4H-silolo[3,2-:4,5-']dithiophene, Si-CPD 32

33 1 H NMR of 4,4'-Diromo-2,2'-dinitro-1,1'-iphenyl, 12 33

34 1 H NMR of 4,4'-Diromo-[1,1'-iphenyl]-2,2'-dimine, 13 34

35 1 H NMR of 4,4'-Diromo-2,2'-diiodo-1,1'-iphenyl, 14 35

36 e d e d 1 H NMR of 3,7-Diromo-5,5-diotyl-5H-dienzo[,d]silole, Si-PF 36

37 1 H NMR of Benzo[][1,2,5]thidizole, 14 37

38 1 H NMR of 4,7-Diromoenzo[][1,2,5]thidizole, 15 38

39 1 H NMR of 4,7-is(4,4,5,5-tetrmethyl-1,3,2-dioxoroln-2- yl)enzo[][1,2,5]thidizole, BTZ 39

40 Referenes (1) Wng, L.; Fu, Y.; Zhu, L.; Cui, G.; Ling, F.; Guo, L.; Zhng, X.; Xie, Z.; Su, Z. Polymer 2011, 52, (2) Neto, B. A. D.; Lopes, A. S. A.; Eeling, G.; Gonçlves, R. S.; Cost, V. E. U.; Quin, F. H.; Dupont, J. Tetrhedron 2005, 61, (3) Bergmnn, J.; Löfstedt, C.; Ivnov, V. D.; Frnke, W. Eur. J. Org. Chem. 2001, 2001, (4) Liu, X.; Xu, D.; Lu, R.; Li, B.; Qin, C.; Xue, P.; Zhng, X.; Zhou, H. Chem. Eur. J. 2011, 17, (5) Yo, K.; Chen, Y.; Chen, L.; Kong, H.; Zhou, W.; Li, F.; He, X.; Wei, Y. J. Polym. Si., Prt A: Polym. Chem. 2010, 48, 434. (6) Pozzi, G.; Orlndi, S.; Cvzzini, M.; Minudri, D.; Mor, L.; Otero, L.; Fungo, F. Org. Lett. 2013, 15, (7) Chen, C.-H.; Hsieh, C.-H.; Duos, M.; Cheng, Y.-J.; Hsu, C.-S. Mromoleules 2010, 43, 697. (8) Li, R.; Liu, J.; Ci, N.; Zhng, M.; Wng, P. J. Phys. Chem. B 2010, 114, (9) Kondo, R.; Ysud, T.; Yng, Y. S.; Kim, J. Y.; Adhi, C. J. Mter. Chem. 2012, 22, (10) Lu, G.; Ust, H.; Risko, C.; Wng, L.; Fhetti, A.; Rtner, M. A.; Mrks, T. J. J. Am. Chem. So. 2008, 130, (11) Lim, B.; Jo, J.; Khim, D.; Jeong, H.-G.; Yu, B.-K.; Kim, J.; Kim, D.-Y. Org. Eletron. 2010, 11, (12) Choi, J.; Dong, L.; Ahn, J.; Do, D.; Hmmershmidt, M.; Chen, J.-N. Dev. Biol. 2007, 304, 735. (13) Chen, C.-P; Hung, Y.-C.; Liou, S.-Y.; Wu, P.-J.; Kuo, S.-Y.; Chn, Y.-H. ACS Appl. Mter. Interfes. 2014, 4,