S1 The Effect of Contrast Agent Charge on Visualization of Articular Cartilage Using Computed Tomography: Exploiting Electrostatic Interactions for Improved Sensitivity Neel S. Joshi 1 ; Prashant N. Bansal 1,2 ; Rachel Stewart 1 ; Brian D. Snyder 2 ; and Mark W. Grinstaff 1 Address correspondence to: Mark W. Grinstaff, Departments of Chemistry and Biomedical Engineering, Boston University, 590 Commonwealth Ave. Boston, MA 02215 (mgrin@bu.edu ). Supporting Information Supporting Information for Publication Full citation for reference #5 in main text: Felson, D. T.; Lawrence, R. C.; Dieppe, P. A.; Hirsch, R.; Helmick, C. G.; Jordan, J. M.; Kingto, R. S.; Lane, N. E.; Nevitt, M. C.; Zhang, Y.; Sowers, M.; McAlindon, T.; Spector, T. D.; Poole, A. R.; Yanovski, S. Z.; Ateshian, G.; Sharma, L.; Buckwalter, J. A.; Brandt, K. D.; Fries, J. F. Ann Intern Med 2000, 133, 635-46. General THF was dried using a solvent column prior to use. All chemicals were purchased from Aldrich and Acros and used without further purification. All reactions were performed under a nitrogen atmosphere and at room temperature unless otherwise noted. NMR spectra were determined by Varian INOVA spectrometer at 400 MHz for 1 H and 100 MHz for 13 C. High resolution mass spectra were obtained on a Waters QTOF spectrometer. DMA = dimethylacetamide, THF = tetrahydrofuran, EtOAc = ethyl acetate. Chondroitinase ABC (C3667) and protease inhibitor cocktail (S8820) were purchased from Sigma. Synthesis of Contrast Agents 3-amino-2,4,6-triiodobenzoyl chloride (SI1). The commercially available (Acros 277650050) 3-amino-2,4,6-triiodobenzoic acid (1.0 g, 1.9 mmol) was placed in 4 mL of neat thionyl chloride and the mixture was refluxed for 6 hrs. During this time, the solid dissolved to form a yellow solution. The thionyl chloride was removed with 2 rounds of rotary evaporation, diluting with ethyl acetate (EtOAc) between rounds. The resulting yellow solid was dissolved in 50 mL of EtOAc and washed 3x with 1:1 saturated NaHCO 3 /brine. The organic layer was dried over NaSO 4 and the EtOAc was removed by rotary evaporation to yield a yellow flaky solid, which was taken on to the next step without further purification or characterization. Formation of the acyl chloride could be confirmed by TLC, with complete disappearance of the starting material (baseline spot), and the appearance of a new spot at higher R f (1:2 EtOAc/Hex). 3-aminoacyl-2,4,6-triiodobenzoyl chloride (SI2). Compound SI1 (1.03 g, 1.94 mmol) was dissolved in 4 mL of anhydrous dimethylacetamide (DMA) and the solution was cooled in a brine/ice bath while stirring. After dropwise addition of acetyl chloride (0.421 mL, 5.8 mmol), the solution was allowed to warm to room temperature and left to react for 4 hrs. The formation of the acylated product was monitored by TLC. The desired product showed up at a lower R f than the starting material. The reaction solution was then concentrated down to ~1 mL by application of high vacuum. The product was precipitated form the concentrated mixture by addition of a mixture of EtOAc and hexanes. The product was isolated by filtration as a pale yellow solid and taken on to the next step without any further purification or characterization. (Boc-ethylenediamine)-3-aminoacyl-2,4,6-triiodobenzoyl amide (SI3). Compound SI2 (0.89 g, 1.6 mmol) was dissolved in 5 mL of DMA along with triethylamine (0.65 mL, 4.7 mmol). The solution was stirred as mono-Boc-protected ethylenediamine (0.37 g, 2.3 mmol) dissolved in 1 mL of DMA was added dropwise. The solution was stirred at room temperature for 15 hrs, during which time the formation of the acylated product could be monitored by TLC (the product was a baseline spot even in 9:1 CH 2 Cl 2 /MeOH). The mixture was then concentrated to ~2-3 mL by application of a high vacuum and then the product was SI1 SI2 NH 2 I I I OH O NH I I I Cl O O H 2 N NHBoc NH I I I H N O O NHBoc NH I I I H N O O NH 3 + NH 2 I I I Cl O SI3 CA 1+