An Activity-Based Near-Infrared Glucuronide Trapping Probe for Imaging

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1 An Activity-Based Near-Infrared Glucuronide Trapping Probe for Imaging -Glucuronidase Expression in Deep Tissues Ta-Chun Cheng 1#, Steve R. Roffler 2#, Shey-Cherng Tzou 3, Kuo-Hsiang Chuang 3, Yu-Cheng Su 2,4, Chih-Hung Chuang 5, Chien-Han Kao 1, Chien-Shu Chen 6, I-Hong Harn 3, Kuan-Yi Liu 8, Tian-Lu Cheng 3,7* and Yu-Ling Leu 8* 1 Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan; 2 Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan; 3 Department of Biomedical Science and Environmental Biology, Kaohsiung Medical University, Kaohsiung, Taiwan; 4 Institute of Microbiology and Immunology, National Yang-Ming University, Taipei, Taiwan, Taipei, Taiwan; 5 Institutes of Basic Medical Sciences, National Cheng Kung University, Tainan, Taiwan; 6 School of Pharmacy, China Medical University, Taichung, Taiwan; 7 Cancer Center, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan; 8 Department of Pharmacy, Chia Nan University of Pharmacy and Science, Tainan, Taiwan # equal contribution *Correspondance: Dr. Tian-Lu Cheng, Department of Biomedical and Environmental Biology, Kaohsiung Medical University, No. 100, Shiquan 1st Road, Kaohsiung City 80708, Taiwan. Phone: , Fax: , tlcheng@kmu.edu.tw. Dr. Yu-Ling Leu, Department of Pharmacy, Chia Nan University of Pharmacy and Science, No. 60, Section 1, Erh-Ren Road, Tainan City 71710, Taiwan. Phone: , Fax: , yulin@mail.chna.edu.tw page S1.

2 Supporting Information General Procedures: 1 H and 13 C nuclear magnetic resonance were recorded on a Bruker DPX-200 spectrometer. FAB mass spectra were recorded on a Finnigan MAT 95S mass spectrometer. Element analyses for C, H and N were carried out on a Heraeus VarioEL-III elemental analyzer. Thin-layer chromatographic analyses were performed using precoated silica gel (60 F 254, Merck) plates, and the spots were examined under UV light. Column chromatography was carried out on Merck silica gel 60 ( mesh). Reverse phase column chromatography was performed on Merck LiChroprep RP-18 (40-63 mm). Fluoroescein isothiocyanate (FITC) was purchased from ACROS. IR-820 was purchased from Sigma-Aldrich. page S2.

3 Preparation of methyl 1,2,3,4-tetra-O-acetyl-β-D-glucopyranuronate (2) A mixture of D (+)-glucurono-3,6-lactone (1) (88 g, 500 mmol) and CH 3 ONa (0.75 g, 13.9 mmol) in methanol (500 ml) was stirred at room temperature for 2 h. The reaction mixture was concentrated under reduced pressure to give rise to a yellow-orange oily residue. The residue was dissolved in acetic anhydride (340 ml). Perchloric acid (1.5 ml) in acetic anhydride (10 ml) was then added to the mixture drop-wise in an ice bath and stirred for 2 h. The resulting precipitate was collected by filtration to give 2 (86 g, 46%). mp 176, 1H NMR (200 MHz, DMSO-d6) δ 1.98 (t, J = 4.6 Hz, 9 H), 2.07 (s, 3 H), 3.62 (s, 3 H), 4.65 (d, J = 9.8 Hz, 1 H), 4.98 (q, J = 8.4 Hz, 2 H), 5.50 (t, J = 9.5 Hz, 1 H), 6.00 (d, J = 8.1 Hz, 1 H); 13 C NMR (50 MHz, DMSO-d 6 ) δ 21.0, 21.1, 21.2, 21.3, 53.5, 69.7, 70.7, 71.7, 72.3, 91.5, 167.8, 169.6, 169.9, 170.2, Anal. Calcd for C 15 H 20 O 11 (%):C47.88, H 5.36; found: C 47.78, H 5.40 Preparation of methyl 1-α-bromo-1-deoxy-2,3,4-tri-O-acetyl-β-D-glucopyranuronate (3) A solution of 2 (3.5 g, 9.3 mmol) and TiBr 4 (3.5 g, 9.54 mmol) in CH 2 Cl 2 (100 ml) was stirred at room temperature for 24 h. The mixture was washed with ice water (100 ml) and saturated aqueous NaHCO 3 solution (100 ml), dried over Na 2 SO 4, and page S3.

4 evaporated to dryness to give 3 (3.3 g, 90%), which was used directly in the next step without further purification. Preparation of methyl 1-O-(2-formyl-4-nitrophenyl)-2,3,4-tri-O-acetyl-β-D- glucopyranuronate (4) A suspension of 3 (3.3 g, 8.4 mmol), 2-hydroxy-5-nitrobenzaldehyde (1.4 g, 8.4 mmol) and Ag 2 O (1.94 g, 8.4 mmol) in CH 3 CN (150 ml) was stirred at room temperature for 4 h. Ag 2 O was filtered out. The solvent was removed under reduced pressure to give rise to a dark brown crude product, which was washed with methanol to obtain 4 (2.64 g; 65 %). MS : 483 m/z (FAB - ); 1 H NMR (400 MHz, DMSO-d 6 ) δ 2.02 (m, 9 H,), 3.62 (s, 3 H,), 4.82 (d, J = 9.8 Hz, 1 H), 5.12 (t, J = 9.7 Hz, 1 H), 5.28 (t, J = 8.4 Hz, 1 H), 5.53 (m, 1H), 5.98 (d, J = 7.6 Hz, 1 H), 7.50 (d, J = 9.2Hz, 1 H), 8.41 (d, J = 2.76 Hz, 1 H), 8.54 (dd, J = 9.2 Hz, J = 2.76 Hz, 1 H), 10.1 (s, 1 H); 13 C NMR (100 MHz, DMSO-d 6 ) δ 20.4, 20.5, 20.6, 52.9, 68.9, 70.2, 70.5, 71.3, 96.7, 116.6, 123.5, 125.0, 131.1, 142.8, 161.8, 167.1, 169.6, 169.7, Anal. Calcd for C 20 H 21 O 13 N (%):C 49.70, H 4.38, N 2.90; found: C 49.55, H 4.47, N 2.74 page S4.

5 Preparation of methyl 1-O-(2-difluoromethyl-4-nitro)-2,3,4-tri-O-acetyl-β-D- glucopyranuronate (5) Diethylaminosulfur trifluoride (DAST) (0.6 ml, 4.51 mmol) dissolved in dichloromethane was added drop-wise to compound 4 (2 g, 4.1 mmol) in dichloromethane (50 ml) at room temperature. The reaction mixture was stirred at 25 C for 2h, and then the reaction was quenched by aqueous NaHCO 3 solution. The dichloromethane layer was separated, dried over anhydrous MgSO 4, and filtered. The solvent was removed under reduced pressure to obtain crude product, which was washed with ether to obtain 5. (1.9 g, 92%) MS : 505 m/z (FAB - _ 2); 1 H NMR (200 MHz, DMSO-d 6 ) δ (m, 9 H,), 3.63 (s, 3 H,), 4.81 (d, J = 9.7 Hz, 1 H), (m, 2 H), 5.50 (t, J = 9.42 Hz, 1H), 5.90 (d, J = 7.5 Hz, 1 H), 6.98 (t, J = 54.4Hz, 1H), 7.47 (d, J = 9.3Hz, 1 H), 8.3 (d, J = 2.4 Hz, 1 H), 8.46 (dd, J = 9.2Hz, J = 2.4 Hz, 1 H); 13 C NMR (500 MHz, DMSO-d 6 ) δ 20.7, 53.1, 69.1, 70.3, 70.9, 71.6, 97.1, 106.9, 111.6, 116.0, 116.3, 122.8, (t, J= 92 Hz), 129.0, 142.8, 158.9, 167.3, 169.5, 169.8, 169.9; Anal. Calcd for C 20 H 21 NF 2 O 12 (%):C 47.53, H 4.19, N 2.77; found: C 47.63, H 4.15, N 2.75 page S5.

6 Preparation of methyl 1-O-(2-difluoromethyl-4-amino)-2,3,4-tri-O-acetyl-β-D- glucopyranuronate (6) 10% Palladium on carbon (106 mg, 0.1 mmol) was added to compound 5 (0.5 g, 1.0 mmol) in ethyl acetate/methanol (9:1; 50 ml). The solution was hydrogenated at room temperature for 1 hr. The reaction mixture was filtered through Celite; the filtrate was concentrated under vacuum to a white solid (6, 0.4 g, 85%). MS : 476 m/z (FAB + _ 1); 1 H NMR (400 MHz, DMSO-d 6 ) δ (m, 9 H,), 3.65 (s, 3 H,), 4.64 (d, J = 9.96 Hz, 1 H), (m, 2 H), 5.17 (s, 2H, NH 2 ), 5.36 (d, J = 7.96 Hz, 1H), 5.46 (t, J = 9.6 Hz, 1 H), 6.68 (d, J = 8.8 Hz, 1H), 6.73 (d, J = 2.3 Hz, 1H), 6.80 (t, J = 55.4Hz, 1H), 6.90 (d, J = 8.8Hz, 1 H); 13 C NMR (50 MHz, DMSO-d 6 ) δ 20.7; 53.0; 69.5; 71.0; 71.3; 71.4; 99.5; 110.6; 112.3; 117.5; 118.5; 124.4; 145.1; 145.6; 167.6; 169.6; 169.8; 170.0; Anal. Calcd for C 20 H 23 NF 2 O 10 (%):C 50.53, H 4.88, N 2.95; found: C 50.47, H 4.86, N 2.91 Preparation of methyl 1-O-(2-difluoromethyl-4-amino)-β-D- glucopyranuronate (7) A suspension of 6 (500 mg, 0.99 mmol) and NaOCH 3 (53 mg, 0.99 mmol) in anhydrous MeOH (20 ml) was stirred at room temperature for 1 h. The dichloromethane solvent was evaporated to dryness and the residue was purified by column chromatography on silica gel (EA) to give 7 (80 mg, 28%); 1 H NMR (200 MHz, page S6.

7 DMSO-d 6 ) δ (m, 3H), 3.66 (s, 3H, OCH 3 ), 3.93 (d, J = 9.2 Hz, 1H), 4.74 (d, J = 7.3 Hz, 1H), 5.07 (br s, 2H, NH 2 ), 5.26 (d, J = 4.2 Hz, 1H, OH), 5.42 ( d, J = 5.5 Hz, 1H, OH), 5.55 (d, J = 4.4 Hz, 1H, OH), (m, 2H, ArH), 6.86 (d, J = 8.7 Hz, 1H, ArH), 7.12 (t, J = 55.5 Hz, 1H, CHF 2 ) ; 13 C NMR (50 MHz, DMSO-d 6 ) δ 52.0, 71.4, 73.1, 75.2, 75.3, 102.9, 109.8, 111.7, 117.3, 118.4, (t, CHF 2 ), 144.6, 145.5, Anal. Calcd for C 14 H 17 O 7 NF 2 (%):C 48.14, H 4.91, N 4.01; found: C 48.25, H 4.97, N 3.96 Preparation of N -fluorescein-n -[4-O-(methyl 2,3,4-tri-O-acetyl- β-d-glucopyranuronate)-3-difluoromethylphenyl]thiourea (8) Compound 6 (190 mg, 0.4 mmol) was dissolved in a solution of TEA (2 ml; 14.3 mmol) in DMF (10 ml) and CH 2 Cl 2 (10 ml), then fluorescein isothiocyanate (FITC) (100 mg, mmol) was added. The reaction was stirred at room temperature for 2h. The mixture was concentrated under reduced pressure to remove the DMF, and then purified by column chromatography to give 142 mg of compound 8 (in 64% yield). LCMS : (M + ); (M + +1); 1 H NMR (200 MHz, DMSO-d 6 ) δ 2.01 (s, 9H), 3.64 (s, 3H), 4.75 (d, J = 9.9 Hz, 1H), (m, 2H), 5.49 (t, J = 9.6 Hz, 1H), 5.66 (d, J = 7.8 Hz, 1H), (m, 7H), 6.89 (t, J = 55.4 Hz, 1H), 7.20 (d, J = 8.3 Hz, 2H), 7.61 (s, 1H), (m, 1H), 8.16 (s, 1H), (m, 4H) page S7.

8 Preparation of N -fluorescein-n -[4-O-(methyl β-d-glucopyranuronate)- 3-difluoromethylphenyl]-S-methylthiourea (9) Fluorescein isothiocyanate (FITC) (261 mg, 0.67 mmol) was added to compound 7 (200 mg, 0.67 mmol) in a solution of TEA/DMF (1.4 ml/15 ml) with stirring at room temperature for 1h. Methyl iodine was added to the mixture, and stirring was continued for another 1h. The solvent was evaporated and the residue was purified by chromatography (10% MeOH-CH 2 Cl 2 ). The precipitate obtained with ether give rise to 9 as an orange-color solid (360 mg, 72%). 1 H NMR (200 MHz, DMSO-d 6 ) δ 2.44 (s, 3H, SCH 3 ), (m, 3H), 3.67 (s, 3H, OCH 3 ), 3.87 (d, J = 8.7 Hz, 1H), 5.03(d, J = 6.2 Hz, 1H), 5.33 (br s, 1H), 5.48 (br s, 1H), 5.61 (br s, 1H), (m, 7H), 6.86 (m, 1H), (m, 3H), 7.18 (t, J = 55.5 Hz, 1H, CHF 2 ), 7.78 (d, J = 8.8 Hz, 1H), 7.95 (s, 1H), 8.37 (br s, 1H), 8.94 (br s, 1H), 9.18 (br s, 1H); MS (FAB + +1)C 36 H 30 O 12 N 2 SF 2 (753) Preparation of N -fluorescein-n -[4-O-(β-D-glucopyranuronic acid)-3- difluoromethylphenyl]-s-methylthiourea (10, FITC-TrapG) Potassium trimethylsilanolate (140 mg, 1.11 mmol) was added to a solution of compound 9 (275 mg, 0.37 mmol) in dry THF (30 ml). The mixture was stirred at room temperature for 5 min. The solvent was removed under vacuum. The residue was page S8.

9 dissolved in chilled HCl solution (0.125 N, 9 ml) and washed with dichloromethane (10 ml). The water phase was purified by reverse chromatography (LiChroprep RP-18, m, 40% MeOH in R.O. water) to obtain 10 (102 mg, 37%) 1 H NMR (200 MHz, DMSO-d 6 ) δ 2.44 (s, 3H, SCH 3 ), (m, 3H), 3.87 (t, J = 7.6 Hz, 1H), 4.96 (d, J = 6.7 Hz, 1H), 5.24 (br s, 1H), 5.55 (br s, 1H), (m, 7H), (m, 3H), 7.25 (t, J = 55.2 Hz, 1H, CHF 2 ), 7.50 (d, J = 8.5 Hz, 1H), 7.73 (d, J = 8.5 Hz, 1H), 8.19 (s, 1H), (br s, 2H); 13 C NMR (50 MHz, DMSO-d 6 ) δ 14.8, 71.4, 73.0, 75.3, 75.5, 83.0, 102.0, 102.2, 109.9, 110.2, 112.6, , 117.5, 118.0, 125.6, (t, CHF 2 ),128.1, 129.3, 130.0, 129.1, 135.3, 142.2, 146.2, 147.5, 151.3, 151.9, 157.8, 159.4, 168.2, HRMS (FAB) calc. for C 38 H 28 O 12 N 2 SF 2 O(M + ) Preparation of IR-820.SPh.NO 2 (11) A solution of IR-820 (600 mg; 0.7 mmol) and 4-nitrothiophenol (329 mg; 2.1 mmol) in N, N-dimethylformamide (10 ml) under argon atmosphere was stirred for 24 hours at room temperature. The mixture was evaporated under vacuum to give an oil that was precipitated in acetone, collected by filtration, and washed with acetone. The yellow-green solid was obtained with a 95% yield. 1H NMR (200 MHz, DMSO-d 6 ) 8.63 (d, J = 14.1 Hz, 2H, -C=C-); (m, 4H, ArH); (m, 4H, ArH); 7.78 page S9.

10 (d, J = 8.9 Hz, 2H, ArH); (m, 4H, ArH); (m, 2H, ArH); 6.46 (d, J = 14.1 Hz, 2H, -C=C-), (m, 6H, CH 2 ); (m, 4H, CH 2 ); (m, 4H, CH 2 ); (m, 8H, CH 2 ); 1.71 (s, 12H, CH 3 ); 13 C NMR (DMSO-d 6 ) 20.4, 22.5, 25.9, 26.4, 26.7, 44.0, 50.5, 50.7, 101.8, 111.8, 122.3, 124.7, 125.0, 126.5, 127.4, 127.7, 129.9, 130.4, 131.4, 133.2, 133.7, 139.7, 143.3, 145.0, 145.4, 146.7, Preparation of IR-820.SPh.NH 2 (12) A suspension of compound 11 (640 mg, 0.7 mmol) and SnCl 2 2H 2 O (630 mg, 2.8 mmol) in ethyl acetate (20 ml) was stirred at 80 o C under argon for 2 hrs until compound 11 disappeared on TLC. The mixture was cooled to room temperature and then poured on ice. The ph was adjusted to neutral with 2N NaOH and the mixture was extracted three times with 20% MeOH/CH 2 Cl 2. The combined extracts were dried over anhydrous MgSO 4, filtered and the MeOH/CH 2 Cl 2 was evaporated in vacuum to give an oil. The oil was precipitated and washed with acetone. The green solid was obtained in a 585 mg, 95% yield. 1H NMR (200 MHz, DMSO-d 6 ) 8.66 (d, J = 14 Hz, 2H, -C=C-); 8.26 (d, J = 8.5 Hz, 2H, ArH); (m, 4H, ArH); 7.75 (d, J = 8.9 Hz, 2H, ArH); 7.63 (t, J = 7.5 Hz, 2H, ArH); 7.49 (t, J = 7.5 Hz, 2H, ArH); 7.10 (d, J = 8.4 Hz, 2H, ArH); 6.57 (d, J = 8.5 Hz, 2H, ArH); 6.37 (d, J = 14 Hz, 2H, -C=C-); (m, 6H, CH 2 ); page S10.

11 (m, 4H, CH 2 ); (m, 4H, CH 2 ); (m, 20H, CH 2 & CH 3 ); 13 C NMR (DMSO-d 6 ) 20.7, 22.5, 25.9, 26.3, 27.0, 43.8, 50.5, 50.7, 101.2, 111.8, 115.1, 121.0, 122.3, 124.8, 127.5, 127.6, 128.7, 129.9, 130.3, 131.3, 133.3, 133.5, 139.8, 144.7, 147.7, 152.2, Preparation of IR-820.SPh.NCS (13) A solution of 12 (200 mg, 0.23 mmol) in dichloromethane (15 ml) was added slowly to a solution of thiocarbonyl chloride (0.07 ml, 0.89 mmol) in DMF (15 ml) under an argon atmosphere in an ice bath. The mixture was stirred for 30 min before silica gel was added and evaporation was performed under reduced pressure to obtain a powder. The powder was purified on silica gel using dichloromethane /methanol (90:10) as the mobile phase. The dark-green solid (160mg, 84%) was obtained in acetone. 1H NMR (200 MHz, DMSO-d 6 ) 8.66 (d, J=13.8 Hz, 2H, -C=C-); 8.22 (d, J = 7.9 Hz, 2H, ArH), (m, 4H, ArH), 7.77 (d, J = 8.7 Hz, 2H, ArH), (m, 8H, ArH), 6.43 (d, J = 13.9 Hz, 2H, -C=C-); (m, 4H, CH 2 ); (m, 4H, CH 2 ), 2.56 (m, 2H, CH 2 ), (m, 26H, CH 3 & CH 2 ); 13 C NMR (50 MHz, DMSO-d 6 ) 20.5, 22.5, 25.9, 26.4, 26.8, 43.9, 50.5, 50.6, 101.6, 111.8, 122.3, 124.9, 125.9, 127.3, 127.7, 129.8, 130.4, 131.4, 133.2, 133.6, 137.0, 139.7, 143.7, 147.2, page S11.

12 Preparation of N -(p-aminophenyl)thioether of IR-820-N -[methyl 4-O-(β-D-glucopyranuronate)-3-difluoromethylphenyl]-S-methylthiourea (14) Compound 7 (33 mg, mmol) and added to a solution of thioisocyanate 13 (500 mg, 0.06 mmol) in DMF (10 ml) and stirred for 5 hours at room temperature. Methyl iodine (7.2 ml, 0.12 mmol) was added and stirred for 2 h. The reaction mixture was purified on fresh silica gel using dichloromethane/methanol (80:20) as the mobile phase to obtain a green solid (54 mg, 71%). LC-Mass(FAB + ); 1323 (M + +2); 1 H NMR (400 MHz, DMSO-d 6 ) (m, 12H, CH 3 ), (m, 2H, CH 2 ), (m, 4H, CH 2 ), (m, 4H, CH 2 ), (m, 4H, CH 2 ), (m, 3H, sugarh), (m, 2H, NCH 2 ), (m, 8H, N + CH 2, OCH 3, SCH 3 ), (m, 1H, sugarh), (m, 4H, CH 2 SO 3 H), 4.88 (d, J = 6.8 Hz, 1H, sugarh), 6.30 (d, J = 13.2 Hz, 2H, -C=C-), (m, 2H, ArH), (m, 1H, ArH), 7.07 (t, J = 55.6 Hz, 1H, CHF 2 ), 7.22 (d, J = 8.0 Hz, 2H, ArH), (m, 2H, ArH), (m, 4H, ArH), 7.64 (d, J = 8.8 Hz, 2H, ArH), (m, 4H, ArH), 8.12 (d, J = 8.0 Hz, 2H, ArH), 8.72 (d, J = 13.6 Hz, 2H, -C=C-; 13 C NMR (50MHz, DMSO-d 6 ) 20.5, 22.5, 25.9, 26.4, 27.0, 35.8, 43.8, 50.5, 50.7, 51.9, 64.9, 69.8, 71.4, 72.9, 75.0, 75.1, 100.6, 101.4, 110.0, 111.8, 115.0, 116.0, 117.1, 122.3, 123.0, 124.8, 126.8, 127.4, 127.6, 129.9, 130.3, 131.3, 133.3, 133.6, 139.8, 144.3, 149.6, 163.1, 169.2, page S12.

13 Preparation of N -(p-aminophenyl)thioether of IR-820-N -[4-O- (β-d-glucopyranuronic acid)-3-difluoromethylphenyl]-s-methylthiourea (15, NIR-TrapG) Potassium trimethylsilanolate (15 mg, 0.12 mmol) was added to a solution of methyl ester of 14 (54mg, 0.04 mmol.) in dry THF (10 ml). The mixture was stirred at ice bath for 5 min. The solvent was removed by vacuum, the residues was dissolved with chilled HCl solution (1.0 N, 1 ml) in 6 ml H 2 O, the green solid was collected by filtration. the crude product was purified by reverse phase column chromatography on RP18 silica gel (30% CH 3 CN-H 2 O) to obtain IR820-TrapG (45mg, 85%); LC-Mass(FAB+); 1308 (M + ); 1 H NMR (200 MHz, DMSO-d 6 ) δ (m, 12H, CH 3 ), (m, 4H, CH 2 ), (m, 2H, CH 2 ), (m, 2H, CH 2 ), (m, 4H, CH 2 ), (m, 2H, NCH 2 ), (m, 2H, N + CH 2 ), 3.51 (s, 3H, SCH 3 ), (m, 3H, sugarh), (m, 1H, sugarh), (m, 4H, CH 2 SO 3 H), (m, 1H, sugarh), 6.43 (d, J = 13.2 Hz, 2H, -C=C-), (m, 4H, ArH, CHF 2 ), (m, 2H, ArH), (m, 2H, ArH), (m, 4H, ArH), (m, 2H, ArH), ( (m, 4H, ArH), 8.21 (d, J = 6 Hz, 2H, ArH), 8.73 (d, J = 12.8Hz, 2H, -C=C-), 13 C NMR (50MHz, DMSO-d 6 ) δ 21.1, 22.6, 26.3, 26.7, 27.3, 29.6, 30.8, 44.3, 51.0, 51.1, 52.5, 71.6, 73.0, 75.4, 75.5, 101.6, 112.2, 122.7, 125.4, 127.6, 127.9, 128.3, 130.4, 130.9, 131.8, page S13.

14 133.8, 134.0, 140.2, 144.3, 154.0, 169.6, LC-Mass(FAB+); 1308 (M + ) (C 67 H 73 O 13 N 4 S 4 F 2 ( )) Biodistribution of FITC-TrapG and NIR-TrapG BALB/c nude mice (n 3) bearing CT26 (in left flanks) and CT26/m G (in right flanks) tumors ( mm 3 ) were intravenously injected with 500 µg/mouse of FITC-TrapG or 100 g/mouse of NIR-TrapG in 100 L PBS. Tumor and selected organs were harvested at 24, 48, and 72 hours after probe injection. The fluorescence intensities of individual organs were recorded on an IVIS50. The ex / em were 490nm/525nm and 710nm/835nm for FITC-TrapG and NIR-TrapG, respectively. The acquisition time of both probes was 10 sec. Figure S1. The biodistribution of FITC-TrapG and NIR-TrapG. (A) FITC-TrapG or (B) NIR-TrapG was intravenously injected to BALB/c nude mice bearing CT26/m G and control CT26 tumors. Tumor and selected organs were harvested at 24 (black column), 48 (gray column), and 72 (white column) hours after probe injection. The fluorescence intensities of individual organs were recorded by an optical imaging system. page S14.

An Efficient Total Synthesis and Absolute Configuration. Determination of Varitriol

An Efficient Total Synthesis and Absolute Configuration Determination of Varitriol Ryan T. Clemens and Michael P. Jennings * Department of Chemistry, University of Alabama, 500 Campus Dr. Tuscaloosa, AL