Accessory Publication

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1 /CH10199_AC CSIRO 2010 Australian Journal of Chemistry, 2010, 63(9), Accessory Publication Self-Assembly of Protoporphyrin IX-TEG Derivatives into Tunable Nanoscaled Spherical Structures Sheshanath V. Bhosale, A,C Sidhanath V. Bhosale, B,C Mohan B. Kalyankar, A,B Steven J. Langford, A and Ceilica H. Lalander A A School of Chemistry, Monash University, Clayton, Victoria, Australia. B Department of Organic Chemistry, North Maharashtra University, Jalgaon ; C Corresponding Authors. sheshanath.bhosale@sci.monash.edu.au; sidhanath2003@yahoo.co.in Contents 1. Material and measurements 2. Synthetic Scheme 3. UV-vis absorption spectroscopy 4. Dynamic Light Scattering Measurements 5. Transmission electron microscopy TEM) 6. Atomic force microscopy (AFM) 7. References 1. Material and measurements Protoporphyrin IX, DMF, (3-(dimethylamino)propyl)ethyl carbodiimide hydrochloride (EDCI), 1-Hydroxybenzotriazole (HOBt), chloroform, methanol and dichloromethane were purchased from Aldrich and used without purification, unless otherwise specified. UV-vis absorption spectra were recorded on a Perkin Elmer Lambda 40p spectrometer. 1 H NMR, 13 C-NMR spectra were recorded on a Bruker spectrometer using deuterated chloroform as solvent and the trimethylsilane residual solvent peak as the reference. The solvents for spectroscopic studies were of spectroscopic grade and used as received. Selfassembled samples were prepared by dissolving proto-teg amphiphilic 1 in CHCl 3 then diluted with cyclohexane or chloroform/methanol. The sample solution was kept at room temperature for few hours before TEM and AFM measurements.

2 2. Synthetic Scheme 3. Dynamic light scattering Dynamic light scattering (DLS) measurements were performed using a Malvern Nano-ZS zetasizer (Malvern Instruments Ltd, Worcestershire, United Kingdom). The Nano-ZS employs non-invasive back scatter (NIBS ) optical technology and measures real time changes in intensity of scattered light as a result of particles undergoing Brownian motion. The sample is illuminated by a 633 nm Helium-Neon laser and a maximum output power of 100 mw was used as light source.measurements were performed at scattered light is measured at an angle of 30 to 90. The size distribution of the vesicles is calculated from the diffusion coefficient of the particles according to Stokes-Einstein equation. The average diameter and the polydispersity index of the samples are calculated by the software using CONTIN analysis. 1 Freshly prepared samples of proto-teg1with

3 a concentration of 1x10-4 M in cyclohexane were used for DLS measurements (Table S1, entry 1-5). Measurements were performed at certain time intervals to probe any possible changes of the vesicles upon standing in solution. The apparent diffusion coefficient was calculated and extrapolated to zero scattering vectors square. Dynamic Light Scattering measurements show the existence of aggregates with rh = 90 nm in coexistence with several larger and smaller sized species. The relative amount of the two species depends on concentration and time but it was not possible to remove the larger particles by filtration or any other common method. In this manuscript we focus on the analysis of the primary rh = 90 nm assemblies.however, due to the presence of the small amounts of the larger aggregates, it is not possible to further characterize the smaller species by static light scattering. Measurements were also performed with a high sample concentration 1x10-3 M and 1x10-2 M (Table S1, entry 6 and 7 respectively). The results were the same as with the 1x10-4 M solution (Table S1, entry 1-5). Table S1. Size and errors of the aggregates of proto-teg1as determined by DLS with different concentration Serial No. Concentration Hydrodynamic radius error [nm] [nm] 1 1x10-4 M x10-4 M x10-4 M x10-4 M x10-4 M x10-3 M x10-2 M Atomic force microscopy (AFM)

4 Figure S1: AFM images of vesicles formed by proto-teg1 in cyclohexane was spincast on silica wafer before taking the AFM images: a) height image, b) height image, c) phase image of b, d) height images, e) phase image of d, (c) high magnification phase image of 1. Figure S2: AFM and TEM images of vesicles formed by proto-teg1 in chloroform/cyclohexane (v/v 1:1). An AFM image of 1 was spin-cast on silica wafer before taking the AFM images: a) height image, b) cross-section analysis magnified region from image a, c) height image with light contrast. d) TEM images of 1 vesicle

5 formed in chloroform/cyclohexane for sample with concentrations of 1x10-4 M,e) high magnification of a vesicle to identify the interlamellar spacing between two adjacent layers. Bar in TEM represents 250 nm. Figure S3. AFM height image of 1 upon spin-cast on silica wafer shows spherical micellear aggregates in CHCl 3 /MeOH (v/v 6:4). UV/vis absorption spectroscopy

6 Figure S4. (a) UV/vis absorption spectra of protoporphyrin-teg 1 (1x10-4 M) in CHCl 3 (black line) and CHCl 3 /MeOH (6:4, v/v; gray line) at 25 o C. 1 H NMR of Proto-TEG1 (CDCl 3 ) Mass spectrum of Proto-TEG 1

7 5. References [1] (a) S. W. Provencher, Biophys. J.,1976, 16, 27; (b) S. W. Provencher, J. Chem. Phys., 1976, 64, 2772.