Supplementary Material (ESI) for Chemical Communications This journal is The Royal Society of Chemistry 2008

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1 Giat vesicle formatio through self-assembly of Chitooligosaccharide-based graft copolymers Ke-jig Gao a, Guagtao Li a, *, Xipig Lu, Bo-Qig Xu a ad Jurge-Hirich Fuhrhop b a Key Lab of rgaic ptoelectroics & Molecular Egieerig, Departmet of Chemistry, Tsighua Uiversity, Beijig, , Chia, ad b Istitut fuer Chemie der Freie Uiversitaet Berli/rgaische Chemie, Takustrasse 3, D Berli, Germay 1) Chemicals Chitooligosaccharide (M =1800, the degree of deacetylatio is 96%), purchased from Shezhe Bright Way Novel Biomaterials Tech. Co. Ltd. (P. R. Chia), was dried at 60 uder vacuum. ε-caprolactoe (Acros rgaics, 99%)was purified by vacuum distillatio over CaH 2 ad the fractio collected at was used i polymerizatio. Triethylalumium was obtaied from Fluka Compay ad used as a 1.0M solutio i toluee. Chloroform ad pyridie were purified by usual distillatio method. Hexamethyldisilicae(HMDS) ad other regets were used as received without further purificatio. 2) The preparatio of CS-g-PCL I the work, the followig three-step procedure was used to prepare the targeted copolymer, as show i scheme S1. CH 2 H CH 2 SiMe 3 CH 2 SiMe 3 H 2 N (Me 3 Si) 2 NH 12h,80 æ Me 3 SiNH AlEt 3 2h Me 3 SiNH H 24h CH 2 SiMe 3 Me 3 SiNH H CCH 2 CH 2 CH 2 CH 2 CH 2 i-prh/h 2 /HCl H m 4 AlEt 2 6 CH 2 H 5 1 H 2 N 3 2 CCH 2 CH 2 CH 2 CH 2 CH 2 H m Scheme S1. Sythetic route to CS-g-PCL Trimethylsilylatio of Chitooligosaccharide Chitooligosaccharide (5.0g, 30.9mmol pyraose) was placed i a dried glass reactor that was previously flushed with high purity N 2 for 30 mi. ad added 50ml of DMS. Uder vigorous stirrig, the dispersio was heated at 70 for 6h to form a clear solutio ad the allowed to cool to room temperature. Predetermied amout of hexamethyldisilazae (the molar ratio of hexamethyldisilazae to pyraose uit was 2.0) were added, ad after beig heated at 80 for 12h with stirrig, the mixture was poured ito 150ml of acetoe. The precipitate was filtered, washed with water, ad dried i vacuo to give pale ta powder. The degree of substitutio (DS) of CS was calculated from the C/N value of elemetal aalysis to be 1.1. Graft Copolymerizatio ad Deproctectio The typical of procedure was described as follows: 1.0 g trimethylsilylchitooligosaccharide TMSCS with DS=1.1 was dissolved i Chloroform of 50ml ad the a certai amout of triethylalumium i toluee (the molar ratio of Et 3 Al to H cotaied i TMSCS was 1.2:1) was added dropwise via a syrige through a rubber septum. The reactio was kept for two hours ad the a desired amout of theε-caprolactoe moomer was trasferred ito the reactor. Uder N 2, the graft copolymerizatio was allowed to proceed for 24h at ambiet temperature while stirred. Whe the settig reactio time of 24h elapsed, 5ml mixture of isopropyl alcohol/h 2 /HCl was added ad the reactor was stirred for further 4h to remove the TMS group. The resultig polymer was purified by repeated precipitatio (3 times) usig 10ml of chloroform ad 300ml alcohol as a solvet ad precipitat pair. It was dried for 72h at 50 i vacuo to give the graft copolymer g 1 ad g 2. 1

2 3) 1 H NMR determiatio Supplemetary Material (ESI) for Chemical Commuicatios 1 H-NMR spectra for g1 were take i CDCl 3 by a Bruker AV 400 spectrometer operatig at 400(Figure S1). I the spectra of g 1, the sigal assigmet was cocluded as 1.24, 1.46, 2.26, ad 3.49ppm, respectively referrig to the γ-,δ+β,α-,adε-methylee protos ext to the carboyl group of the PCL brach. 4) GPC determiatio Figure S1. 1 H-NMR spectra of the graft copolymer g 1 The molecular weight (M) ad molecular weight distributio (Mw/M) were measured with a Waters 515 GPC istrumet equipped with Styragel colums (101-, 102-, ad 103-m pore sizes) with tetrahydrofura(thf) as the mobile phase. Polystyree ad Waters Milleium 32 were used as calibratio stadards ad data-processig software, respectively. g2 g Elute time(mi) Figure S2 GPC traces of the resultat g 1 ad g 2 Table S1. Structure details of vesicle-formig graft copolymer [a] Sample M [g mol -1 ] PDI [b] DP [c] g g [a] The degree of polymerizatio of CS used for graftig is 11. [b] PDI: the polydispersity idex of CS-g-PCL. [c] DP: the degree of polymerizatio of PCL brach. M ad PDI were determied from GPC with tetrahydrofura (THF) as a elutio solvet ad moodisperse poly(styree) as stadards, ad DP for PCL was calculated from the sigals at 4.06 (-CH 2 -,repeatig uit) ad 3.65 (-CH 2 H, ed group). 2

5) The stability of the prepared GVs The GVs suspesio was trasferred ito a observatio chamber ad observed with a phase-cotrast microscope (lympus BX-51), equipped

Slow uiform heatig was achieved with a resistive heatig stage costructed i our lab, cosistig of a backside metal coated microscope coverslip ad a curret limitig

The temperature measuremet was time sychroized with the time readigs o the videotape. The results are displayed i Figure S3.

At temperature above 60, the GVs gradually dissolve i 1, 4-dioxae/water mixtures. 23 40 50 60 80 Figure S3 The shape chage of GVs at differet temperature.

a rate of 30 /h with fial GVs cocetratio of 0.5wt% i dioxae/water mixture i the DSC cell (Figure S4). For GVs obtaied from g1, the ethalpy is 0.

3 5) The stability of the prepared GVs The GVs suspesio was trasferred ito a observatio chamber ad observed with a phase-cotrast microscope (lympus BX-51), equipped with a JVC color video camera ad coected to a image-recordig system. To determie the effect of temperature o the stability of GVs. Slow uiform heatig was achieved with a resistive heatig stage costructed i our lab, cosistig of a backside metal coated microscope coverslip ad a curret limitig stadard power supply. Temperature i the droplet close to the vesicle was measured usig a calibrated micro-sized Cu/Ni thermoelemet. The temperature measuremet was time sychroized with the time readigs o the videotape. The results are displayed i Figure S3. It idicates that the GVs i suspesio are stable over the temperature spaig from 25 to 50. At temperature above 60, the GVs gradually dissolve i 1, 4-dioxae/water mixtures Figure S3 The shape chage of GVs at differet temperature. 6) DSC determiatio of GVs suspesio The thermal phase behavior of GVs self-assembled from g1 ad g2 were recorded usig DSC heatig scas (PerkiElmer Thermal Aalysis) at a rate of 30 /h with fial GVs cocetratio of 0.5wt% i dioxae/water mixture i the DSC cell (Figure S4). For GVs obtaied from g1, the ethalpy is 0.14J/g with a maximum heat capacity at T=53.9. For GVs obtaied from g2, the ethalpy is 0.21 with a maximum heat capacity at T=56.9. The results show that the both the meltig poits ad the degree of crystallie of GVs icrease with PCL brach legth. g 1 g T(Degree Celsius) Figure S4. The DSC traces of GVs self-assembled from g 1 ad g 2 i dioxae/water mixture 3

7) Cofocal fluorescece microscopy of GVs The Nile Red solutio i acetoe (1mM, prepared prior to use) was added to the suspesio of GVs i dioxae/water mixture to make a Nile Red cocetratio of 1μM.

, Tokyo, Japa) ad a argo io laser with 550m excitatio wavelegth. Images were recorded with a digital CCD camera iterfaced to a computer, ad operated by the software provided by the camera maufacturer.

wt%). The copper grid was the dried at room temperature prior to measuremet. TEM was performed o a JEL JEM-1200 electro microscope at a acceleratio voltage of 120KV.

4 7) Cofocal fluorescece microscopy of GVs The Nile Red solutio i acetoe (1mM, prepared prior to use) was added to the suspesio of GVs i dioxae/water mixture to make a Nile Red cocetratio of 1μM. Fluorescece images were record usig the microscopy (lympus BX 61, lympus ptical Co., Tokyo, Japa) with a cofocal optical scaer (Yokogawa Ic., Tokyo, Japa) ad a argo io laser with 550m excitatio wavelegth. Images were recorded with a digital CCD camera iterfaced to a computer, ad operated by the software provided by the camera maufacturer. 8) TEM of the formed aggregates A drop of g 1 ad g2 GVs suspesio was deposited, respectively, o a copper grid coated with a carbo film ad staied with a drop of phosphorous tugsteic acid i water (2 wt%). The copper grid was the dried at room temperature prior to measuremet. TEM was performed o a JEL JEM-1200 electro microscope at a acceleratio voltage of 120KV. The TEM image clearly reveals the vesicular structure, with lamella thickess of ca.12.1m for g1 vesicle ad ca.23.0m for g2 vesicle (Figure S5). 12.1m 23.0m g1 vesicle g2 vesicle Figure S5 TEM images of g1 ad g2 vesicle 9) Self-assembly mechaism sectio The CS-g-PCL molecule has a hydrophilic CS backboe ad hydrophobic PCL braches, so the aggregatio of CS-g-PCL molecules drive by the hydrophobic iteractio i water/dioxae mixture is well expected. the other had, there are a lot of hydroxyl ad amio groups alog the backboe of CS, the formatio of hydroge bods is aother potetial iteractio for the self-assembly. To verify this, trimethylsilyl chitooligosaccharide-graft-poly(caprolactoe) (TMSCS-g-PCL) with trimethylsilylatio of amio ad hydroxyl groups were utilized to self-assemble. The results are show i Figure S6. As show i Figure S6, o GVs were obtaied. Figure S6. The aggregates formed from the solutio of TMSCS-g-PCL 4

5 Variable temperature FTIR was used to detect the hydroge bods i the resultig dried vesicles. Variable temperature FTIR was coducted o Nicolet Avatar 360 uder N 2 from 25 to 80, ad the vesicle solutio was coated o the KBr crystal wafer for the measuremet. The samples were carefully dried before the measuremet. The results are provided i Figure S7. It was foud that with icreasig temperature from 25 to 80, the peak of the hydroxyl groups gradually becomes weaker ad shifts to the high wavelegth side, which clearly idicates that a certai amout of hydroxyl groups have formed hydroge bods i the dried vesicles origiated from the selfassembly of CS-g-PCL molecules. Figure S7. Variable temperature FTIR spectra of dried giat vesicles from CS-g-PCL molecules. The temperature of the related curve from the bottom to the top is 25,50,60,70, 80 ad 100, respectively. The formatio of the vesicles i 1,4-dioxae/water mixtures is evideced by their 1 H NMR spectra (Figure S8). Differet from that measured i 1.4-dioxae (Figure S8a), the peaks related to CS become stroger (Figure S8b). It implies that the PCL segmets form the middle layer of the vesicle wall, ad the CS segmets form the outer layers, which remai i a solvated state. (a) CS (b) CS Figure S8. 1 H NMR spectra of copolymer g1 i differet solvet. (a) Spectra of g1 copolymer solutio i dioxae; (b) Spectra of g1 vesicles i 1,4-dioxae/water mixture with 25wt% water cotet. 5

6 10) The effect of crosslikig o the stability of GVs i differet solvets Figure S9. Photographs of the cossliked vesicles i water (A), dioxae (B) ad ethaol (C) 11) Quatitative iformatio about release of the prepared vesicles i the course of addig water ito vesicle suspesio The quatitative iformatio about release of the prepared vesicles i the course of addig water ito vesicle suspesio was evaluated accordig to the commoly used method (A. D. Tirrell: Lagmuir 2000, 16, 122). To achieve this, calcei loaded vesicles were prepared by addig water ito the mixture of 3ml graft copolymer solutio (0.5wt% i dioxae) ad 30μl calcei solutio (25mM i water) to reach 20wt% water cotet. The, appropriate amout of CoCl 2 (50mM i water) was itroduced to quech the fluorescece of uecapsulated calcei molecules. Efflux of calcei was moitored by measurig the icrease of fluorescece emissio at 525 m (excited at 495 m). The maximum fluorescece itesity was give by the complete release of the ecapsulated calcei caused by additio of 3-fold excess water ito the suspesio. We foud that a relatively low release of calcei occurred i the course of addig water ito the vesicle suspesio, suggestig a high stability of the prepared vesicles. 6

Figure S10 The release of calcei from the prepared vesicles i the course of addig water ito vesicle suspesio: (A) g1 vesicles; (B) g2 vesicles 12) Formatio of the giat vesicles i total water medium

7 Figure S10 The release of calcei from the prepared vesicles i the course of addig water ito vesicle suspesio: (A) g1 vesicles; (B) g2 vesicles 12) Formatio of the giat vesicles i total water medium The formatio of the giat vesicles i total water medium was also achieved. The preparatio procedure is as follows: the sythesized copolymer was first dissolved i dioxae, ad 3ml dioxae solutio was evaporated usig a rotary evaporator to get thi polymer film i a smaller vessel. The, the thi film was covered with pure water (10 ml of Milli-Q water) ad ultrasoicated with a tip for 30 mi at room temperature for the formatio of the vesicles. Figure S11 Photograph of the prepared giat vesicles i total water medium 7

Copper-catalyzed Electrophilic Polyhydroamination of Internal Alkynes

Supportig Iformatio (SI) Copper-catalyzed Electrophilic Polyhydroamiatio of Iteral lkyes ezhao He, Yogwei Wu, ju Qi,, ad e Zhog Tag,, Guagdog Iovative Research Team, State Key Laboratory of Lumiescet Materials