Supplmntry Mtril (ESI) for hmil ommunitions This journl is () Th Royl Soity of hmistry 2008 Eltroni Supplmntry Informtion Thrmlly Stbl n Photortiv Polyltis by th Trminl onjugtion of Bio-bs ffi Ai Trn Hng Thi, b Mihiy Mtsuski, n Mitsuru Akshi * Dprtmnt of Appli hmistry, Grut Shool of Enginring, sk Univrsity, 2-1 Ym-ok, Suit 565-0871, Jpn. kshi@hm.ng.osk-u..jp b Fulty of Thnology of rgni hmistry, ollg of hmistry, Ministry of Inustry, Tin Kin, Lm Tho, Phu Tho, Vit Nm 1. Mtrils 2. Synthsis of polyl-ltis (PLLAs) 2-1. Synthti mtho 2-2. Anlysis of hmil strutur 3. Synthsis of 3,4-itoxyinnmi i (DAA) 4. Synthsis of 3,4-itoxyinnmoyl hlori (DA) 5. Synthsis of DAA-PLLAs 5-1. Anlysis of hmil strutur 5-2. Solubility of PLLAs n DAA-PLLAs 5-3. Thrml proprtis of PLLAs n DAA-PLLAs 5-3-1. Anlysis of DS 5-3-2. Anlysis of TGA 5-4. rystllinity of PLLAs n DAA-PLLAs 5-5. Photortivity of PLLAs n DAA-PLLAs 5-6. Molulr wight hng of PLLAs n DAA-PLLAs by UV irrition. 5-7. Photo-onjugtion of fluorsnt y onto PLLAs n DAA-PLLA films 5-7-1. Synthsis of DHA onjugt Alx fluor555 5-7-2. Photo-onjugtion mtho of th fluorsnt y onto th films 1
Supplmntry Mtril (ESI) for hmil ommunitions This journl is () Th Royl Soity of hmistry 2008 1. Mtrils L-lti (LLA; Tokyo hmil Inustry (TI)) ws rrystlliz from thyl tt, n thn ri in vuo t room tmprtur for 24 h. L-lti i (Tokyo hmil Inustry (TI)) ws us s riv. Thionyl hlori (Sl 2 ), 3,4-ihyroxyinnmi i (DHA), ti nhyri (A 2 ) n Alx fluor 555 vrin isoium slt (Wko Pur hmil Inustris, Lt.) wr us without purifition. 2. Synthsis of polyl-ltis (PLLAs) 2-1. Synthti mtho H H 3 H + H 3 H 3 Snt 2 160 o -4h H H 3 n H LLA L-lti PLLA n = 14, 36, 50, 73, 112 Shm S1. Th synthsis of PLLA pro vi ring-opning polymriztion (RP) of L-lti using L-lti i s n inititor n tin (II) thyl hxnot (Snt 2 ) s tlyst. PLLA with L-lti/L-lti i t molr rtio of 10/1 mol/mol ws prpr s follows. A totl of 14.4 g of L-lti (100 mmol), 0.9 g of L-lti i (10 mmol) n 43 mg of Snt 2 (0.3 wt% of monomr) ws issolv in 0.43 ml of tolun, n ws mix n mhnilly stirr in vuo t room tmprtur for 1h to rmov ny ir. Tbl S1 Synthti onitions n thrml proprtis of PLLAs Smpl b L-lti L-Lti i M n M w /M n LLA unit Yil T m T (mmol) (mmol) (Mn/72) (wt%) ( o ) ( o ) ( o ) PLLA14 100 50 1020 2.1 14 43 125 91 241 PLLA36 100 20 2620 2.4 36 74 148 91 231 PLLA50 100 10 3570 3 50 78 153 N 333 PLLA73 100 4 5290 2.8 73 85 156 N 248 PLLA112 100 2 8110 1.4 112 79 168 N 254 Th polymriztion of PLLAs ws rri out in th prsn of Snt 2 s tlyst (0.3 wt% of monomr) with mhnil stirring t 160 o for 4 h. b Th numbrs in th smpl nm r th unit numbrs of LLA. Th molulr wights n numbr of LLA units wr stimt by GP with polystyrn stnrs (lunt: THF). Th yils rprsnt rsults ftr purifition. Th T m n T wr msur by DS upon son hting (10 o min -1 ), th "T " mns th rystlliztion tmprtur. f Th 10% wight-loss tmprturs, T 10, wr msur by TGA unr nitrogn (20 o min -1 ). "N" mns not tt. T 10 f 2
Supplmntry Mtril (ESI) for hmil ommunitions This journl is () Th Royl Soity of hmistry 2008 Nxt, th mixtur ws rt t 160 o for 4 h in nitrogn tmosphr. Aftr th rtion, th prout ws issolv in hloroform n purifi by r-pripittion ovr mthnol twi. Th yil ws 78 wt%. Th othr PLLAs wr prpr by n nlogous prour (Tbl S1). Th vrg molulr wight of th PLLAs ws trmin by gl-prmtion hromtogrph (GP) (HL8220GP systm with α M n A900P olumns) in ttrhyrofurn (THF), n thn librt with polystyrn stnrs t flow rt of 0.6 ml/min. 2-2. Anlysis of hmil strutur Th hmil strutur of th PLLA ws nlyz by FT-IR (Figur S1) n 1 H-NMR (Figur S2) msurmnts. () ν - (b) ν - 1300 1250 1200 1150 1100 1050 1000 Wvnumbr (m -1 ) Figur S1. Rprsnttiv FT-IR sptr of () PLLA50 n (b) L-lti. Th FT-IR sptr of th smpls wr msur t 25 o on Prkin Elmr Sptrum n sptropolrimtr btwn 4000-400 m -1. Th strthing vibrtion pks of th - groups in L-lti wr obsrv t 1267 n 1241 m -1, but wr qunh in th FT-IR sptr of PLLAs n shift to 1211 n 1182 m -1, thus initing tht th PLLAs wr sussfully polymriz. 3
Supplmntry Mtril (ESI) for hmil ommunitions This journl is () Th Royl Soity of hmistry 2008 b Dl 3 b () (b) Dl 3 ppm Figur S2. Rprsnttiv 1 H-NMR sptr of () PLLA50 n (b) L-lti. Th 1 H-NMR sptr wr msur with NMR sptromtr (JEL FX 400) t 400 MHz. Th 1 H-NMR hmil shifts in prts pr million (ppm) wr ror ownfil from 7.26 ppm using Dl 3 solvnt s n intrnl rfrn. Th protons of th mthyl or -H groups wr shift into iffrnt position btwn L-lti n PLLAs, n th pks in th sptr of PLLAs wr bror thn in th sptrum of L-lti, thus initing tht th PLLAs wr polymriz. 3. Synthsis of 3,4-itoxyinnmi i (DAA) H H + H 3 H 3 5 H 5 N 0 0-2h, 130 o -5h H 3 H H DHA A 2 DAA H 3 Shm S2. Atyltion of DHA. A totl of 25 g of DHA (140 mmol) ws issolv in 50 ml of istill pyriin t 0 o for 30 min. Nxt, 75 ml of ti nhyri ws n stirr t this tmprtur for 1.5 h. Th mixtur ws thn rt t 130 o unr rflux for 5 h. Aftr th rtion, th prout ws rrystlliz in tolun, wsh by 0.1N Hl twi, n ri t 50 o in vuo for 24 h. Th yil ws 83 wt%. Th hmil strutur of DAA ws stimt by 1 H-NMR msurmnt (Figur S3). 4
Supplmntry Mtril (ESI) for hmil ommunitions This journl is () Th Royl Soity of hmistry 2008 + Dl 3 b f b f ppm Figur S3. 1 H-NMR sptrum of DAA. Th 1 H-NMR sptrum ws msur with NMR sptromtr (JEL FX 400) t 400 MHz. Th 1 H-NMR hmil shifts in prts pr million (ppm) wr ror ownfil from 7.26 ppm using Dl 3 solvnt s n intrnl rfrn. Th 1 H-NMR signls show th prsn of mthyl groups (2.3 ppm) in th DAA, n th intgrl vlu init tht ll H groups of DHA wr strifi with ti nhyri. 4. Synthsis of 3,4-itoxyinnmoyl hlori (DA) H 3 H + Sl 2 DMF 60 o, 7h H 3 l H 3 H 3 DAA DA Shm S3. Synthsis of DA. DA ws synthsiz by 26.4 g of DAA (100 mmol) plus 50 ml of thionyl hlori (Sl 2 ) (700 mmol) using 79 mg of DMF s tlyst in 60 ml of istill ihloromthn s solvnt t 60 o unr rflux for 8 h. Aftr this rtion, th rsiul Sl 2 n solvnt wr rmov in vuo. Th hmil strutur of DA ws nlyz by FT-IR msurmnt (Figur S4). 5
Supplmntry Mtril (ESI) for hmil ommunitions This journl is () Th Royl Soity of hmistry 2008 () (b) ν = (toxyl n rbonyl hlori) ν = (toxyl) ν = (rboxyl) 2000 1900 1800 1700 1600 1500 Wvnumbr (m -1 ) Figur S4. FT-IR sptr of () DA n (b) DAA. Th FT-IR sptr of th smpls wr msur t 25 o on Prkin Elmr Sptrum n sptropolrimtr btwn 4000-400 m -1. Th = groups in th rbonyl hlori of DA ppr t ν = 1730m -1, whrs th = groups in th rboxyl ws not shift t 1670 m -1, initing tht ll rboxyl groups in DAA wr onvrt to rbonyl hlori groups. 5. Synthsis of DAA-PLLAs DAA-PLLA49 ws prpr s follows. A totl of 11.2 g of PLLA50 (3.7 mmol) ws issolv in 40 ml of istill ihloromthn n 2 ml of istill pyriin t 0 o for 30 min, n 4 g of DA (14.9 mmol; molr rtio of PLLA50: DA = 1:4) ws thn, stirr t this tmprtur for 1.5 h, n t room tmprtur for 24 h. Aftr th rtion, th prout ws wsh in ph 3 Hl solution, n r-pripitt into thnol by issolution in DM twi. Th yil ws 94 wt%. Th othr DAA-PLLAs wr prpr by n nlogous prour. Th vrg molulr wight of DAA-PLLAs ws trmin in THF by GP (HL8220GP systm with α M n A900P olumns) librt with polystyrn stnrs t flow rt of 0.6 ml min -1. 6
Supplmntry Mtril (ESI) for hmil ommunitions This journl is () Th Royl Soity of hmistry 2008 5-1. Anlysis of hmil strutur Th hmil struturs of DAA-PLLAs wr nlyz by FT-IR (Figur S5) n 1 H-NMR (Figur S6) msurmnts. DAA-PLLA120 ν = ν = ν p=φ δ s H 3 (lti) DAA-PLLA79 DAA-PLLA49 DAA-PLLA38 DAA-PLLA16 2000 1900 1800 1700 1600 1500 1400 Wv numbr (m -1 ) Figur S5. FT-IR sptr of vrious DAA-PLLAs. Th FT-IR sptr of th smpls wr msur t 25 o on Prkin Elmr Sptrum n sptropolrimtr btwn 4000-400 m -1. Th FT-IR sptr of th DAA-PLLAs onfirm th prsn of th vinyln (ν = 1640m -1 ) n phnyl groups (ν p=φ 1504m -1 ) of DAA, n th mthyl group of PLLAs (δ s 1455m -1 ). In ition, th pk intnsity of th vinyl or phnyl group in th DAA-PLLAs vrsus th pk intnsity of th mthyl group in PLLAs rs with rsing omposition of DAA. Ths rsults init tht DA ws sussfully onjugt to th trminl PLLAs. 7
Supplmntry Mtril (ESI) for hmil ommunitions This journl is () Th Royl Soity of hmistry 2008 Dl 3 b b ppm Figur S6. Rprsnttiv 1 H-NMR sptrum of DAA-PLLA49. Th 1 H-NMR sptr of vrious DAA-PLLAs wr msur with NMR sptromtr (JEL FX 400) t 400 MHz. Th 1 H-NMR hmil shifts in prts pr million (ppm) wr ror ownfil from 0.00 ppm using ttrmthylsiln (TMS) s n intrnl rfrn. Th solvnt ws Dl 3. Th 1 H-NMR signls of DAA-PLLAs show th prsn of DAA n PLLA in th opolymrs. Th omposition of PLLA or DAA in th DAA-PLLAs ws lult by th pk r rtio of th vinyln protons () in DAA n th -H protons (b) in PLLAs. 5-2. Solubility of PLLAs n DAA-PLLAs Solubility of PLLAs n obtin DAA-PLLAs ws vlut using vrious solvnts. Th smpls wr issolv in vrious solvnts with 1 mg/ml onntrtion Tbl S2 Solubility of PLLAs n DAA-PLLAs LLA unit in PLLAs LLA unit in DAA-PLLAs Solvnts 14 36 50 73 112 16 38 49 79 120 Wtr Mthnol Ethnol Aton Atonitril DMF Pyriin NMP DMS Tolun Hxn DM THF hloroform Mrks " " n " " mn solubl n insolubl, rsptivly. 8
Supplmntry Mtril (ESI) for hmil ommunitions This journl is () Th Royl Soity of hmistry 2008 for fw hours. As shown in Tbl S2, solubility of DAA-PLLAs ws intil to PLLA of th sm molulr wight. 5-3. Thrml proprtis of PLLAs n DAA-PLLAs Thrml proprtis of PLLAs n DAA-PLLAs wr nlyz by DS msurmnt (EXSTAR6100, Siko Instrumnts In.) n TGA msurmnt (Q500, TA Instrumnt-Dupont). 5-3-1. Anlysis of DS Th mlting tmprtur (T m ) n rystlliztion tmprtur (T ) of PLLAs n DAA-PLLAs wr stimt t hting n ooling rts of 10 o min -1. n smpl msurmnt ws rri out ovr two yls of hting or ooling. Figur S7 shows DS urvs of ll DAA-PLLAs n PLLAs. Th T m of DAA-PLLA ws highr thn tht of PLLA with th sm molulr wight. ) b) 106 o LLA112 DAA-PLLA120 106 o LLA73 168 o DAA-PLLA79 169 o Eno Exo LLA50 LLA36 91 o 156 o 153 o DAA-PLLA49 DAA-PLLA38 107 o 103 o 147 o 168 o 158 o 91 o 148 o DAA-PLLA16 117 o 157 o LLA14 125 o 137 o 30 60 90 120 150 180 30 60 90 120 150 180 Tmp. ( o ) Tmp. ( o ) Figur S7. DS urvs upon son hting of () PLLAs n (b) DAA-PLLAs. 9
Supplmntry Mtril (ESI) for hmil ommunitions This journl is () Th Royl Soity of hmistry 2008 5-3-2. Anlysis of TGA Th thrml grtion bhvior of th smpls ws obsrv from th TG urvs by hting from 80 o to 500 o t rt of 20 o min -1 unr nitrogn tmosphr with flow rt of 200 ml min -1. 5-4. rystllinity of PLLAs n DAA-PLLAs Smpls t onntrtion of 30 mg ml -1 in hloroform wr st onto glss slis. Thy wr thn ri t room tmprtur for 24 h, n msur ftr nnling t 100 o for 1h by WAXD msurmnt (X-ry iffrtomtr (RINT UltrX18), quipp with sintilltion ountr) using ukα rition (40 kv, 200 ma; wvlngth = 1.5418 Å). (Figur S8). Th rystls of DAA-PLLAs wr intil with PLLAs; i.. DAA i not influn th rystl proprtis of PLLA. () (b) 5 10 15 20 25 30 35 2θ (g) Figur S8. Rprsnttiv WAXD pttrns n iffrntil intrfrn mirosopy (DIM) of () DAA-PLLA49 n (b) PLLA50. 5-5. Photortivity of PLLAs n DAA-PLLAs DAA-PLLA16, DAA-PLLA38, DAA-PLLA49, DAA-PLLA79 n DAA-PLLA120 wr issolv in DMF to onntrtions of 0.08 mg ml -1, 0.18 mg ml -1, 0.25 mg ml -1, 0.4 mg ml -1 n 1.8 mg ml -1, rsptivly. A glss-filtr 10
Supplmntry Mtril (ESI) for hmil ommunitions This journl is () Th Royl Soity of hmistry 2008 high-prssur Hg Lmp (λ > 280 nm, 56 mw m -2 ) ws irrit. Th tim ours of th photortion onvrsion ws monitor by UV-visibl bsorption sptrosopy of th opolymr solutions using HITAHI U3010 Sptrophotomtr. Th ylobutn formtion vi UV-irrition ws tt by 1 H-NMR msurmnt (Figur S9). Smll signls ppr in th hmil shift rng of 4.0-4.2 ppm upon UV irrition, supporting th formtion of n liphti group (ylobutn) substitut by th bnzn ring or rbonyls. () ylobutn (b) ppm Figur S9. Rprsnttiv 1 H-NMR sptr of DAA-PLLA49 () ftr UV irrition for 90 min. n (b) bfor UV irrition. 5-6. Molulr wight hng of PLLAs n DAA-PLLAs by UV irrition. Molulr wight hng of smpls ftr UV irrition t λ > 280 nm ws msur by GP nlysis. Figur S10 shows GP profils of DAA-PLLA49 bfor n ftr UV irrition. Th lution tim of th smpl of ftr UV irrition shift to rlir thn tht bfor UV irrition. In othr wors, imriztion of th PLLA moluls h ourr upon th photortion of trminlly-onjugt DAA. 11
Supplmntry Mtril (ESI) for hmil ommunitions This journl is () Th Royl Soity of hmistry 2008 Aftr 90 min UV Mn=5450, Mw/Mn=2.5 Bfor UV Mn=3690, Mw/Mn=2.8 10 12 14 16 18 20 [min] Figur S10. Rprsnttiv GP hrts of DAA-PLLA49 bfor n ftr UV irrition for 90 min. (λ > 280 nm, 56 mw m -2 ). 5-7. Photo-onjugtion of fluorsnt y onto PLLAs n DAA-PLLA films 5-7-1. Synthsis of DHA onjugt Alx fluor555 H 2 N S 3 S 3 + NH 2 H 2 N S 3 S 3 + NH 2 H WS 2 N + + 2 N + r.t.- 24 h H H NH DHA NH (H 2 ) 5 (H 2 ) 5 NH 2 Alx fluor 555 vrin, isoium slt (y) NH H H Shm S4. Rtion of th y n DHA. Dy/DHA A totl of 0.72 mg of DHA (4 μmol) ws issolv in 0.42 ml of PBS buffr (ph = 7.4), n thn 0.76 mg of WS (4 μmol) ws n stirr t room tmprtur for 10 min. Nxt, 1 mg of Alx fluor 555 vrin isoium slt (y) ws, n 12
Supplmntry Mtril (ESI) for hmil ommunitions This journl is () Th Royl Soity of hmistry 2008 th rtion ws rri out t room tmprtur for 24 h. Aftr this rtion, th prout ws ilyz in wtr for 3 ys to purify it, n frz-ri for 2 ys. 5-7-2. Photo-onjugtion mtho of th fluorsnt y onto th films DAA-PLLA38 or PLLA36 films wr prpr by sting 100 μl of th smpls issolv in DM (10 mg ml -1 ) onto glss slis, n thn ri t room tmprtur for 2 ys. Nxt, 0.5 ml of y/dha issolv in PBS buffr (ph = 7.4, 0.006 wt%) ws ropp onto th films, n xpos to UV light t λ > 280 nm for 100 min. Aftr th UV irrition, th films wr wsh with pur wtr to rmov ny unrt y/dha, n obsrv by onfol fluorsn snning mirosopy. 13