SUPPLEMENTARY NOTES. Selective In Vivo Metabolic Cell Labeling Mediated Cancer Targeting

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1 SUPPLEMENTARY NOTES Selective In Vivo Metabolic Cell Labeling Mediated Cancer Targeting Hua Wang b,*, Ruibo Wang b,*, Kaimin Cai b,*, Hua He a, Yang Liu b, Jonathan Yen c, Zhiyu Wang b, Ming Xu b, Yiwen Sun b, Xin Zhou d, Qian Yin b, Li Tang b, Iwona T. Dobrucki e, Lawrence W. Dobrucki c, Eric J. Chaney f, Stephen A. Boppart c,f,g,h, Timothy M. Fan i, 1, Stéphane Lezmi j,1, Xuesi Chen, p,1 Lichen Yin a,1, Jianjun Cheng a,b,c,k,l,m,n,o,1 a Jiangsu Key Laboratory for Carbon-Based Functional Materials & Devices, Institute of Functional Nano & Soft Materials (FUNSOM), Soochow University, Suzhou , Jiangsu, China. b Department of Materials Science and Engineering, c Department of Bioengineering, e Molecular Imaging Laboratory, Beckman Institute for Advanced Science and Technology, f Biophotonics Imaging Laboratory, Beckman Institute for Advanced Science and Technology, g Department of Electrical and Computer Engineering, h Department of Internal Medicine, i Department of Veterinary Clinical Medicine, j Department of Pathobiology at College of Veterinary Medicine, k Department of Chemistry, l Beckman Institute for Advanced Science and Technology, m Micro and Nanotechnology Laboratory, n Institute of Genomic Biology, o Materials Research Laboratory, University of Illinois at Urbana Champaign, Urbana, IL, 61801, USA. d Department of Chemistry, University of Science and Technology of China, Hefei, Anhui , China. p Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Changchun , People's Republic of China *These authors contributed equally to this work. 1 To whom correspondence may be addressed. jianjunc@illinois.edu; lcyin@suda.edu.cn; xschen@ciac.ac.cn; slezmi@illinois.edu; t-fan@illinois.edu. S1

2 Table of Contents Supplementary Scheme 1. Synthetic route of Ac 4 ManAz, Ac 3 ManAzEt, and Ac 3 ManAzNB S3 Synthesis of Ac 4 ManAz (1) S3 Synthesis of Ac 3 ManAzEt (2)and Ac 3 ManAzNB S4 Supplementary Scheme 2. Synthetic route of HDAC/CTSL responsive DCL-AAM (4) S5 Synthesis of AcLys(NHS) (10) S5 Synthesis of (4-aminophenyl)(phenyl)methanol (11) S5 Synthesis of AcLys(DPM) (12) S6 Synthesis of AcLys(DPM-CNCl 3 ) (13) S6 Synthesis of Ac 3 ManAzOH (14) S6 Synthesis of DCL-AAM (4) S7 Supplementary Scheme 3. Synthetic route of HDAC/CTSL reporter (Naph-Lys, 5) S7 Synthesis of Naph-NH 2 (15) S8 Synthesis of Naph-Lys (5) S8 Supplementary Scheme 4. Radiolabeling of DCL-AAM S8 Synthesis of DBCO-DOTA (16) S9 Synthesis of DCL-AAM-DOTA (17) S9 Synthesis of 64 Cu-labeled DCL-AAM (18) S10 Supplementary Scheme 5. Synthetic route of DBCO-VC-Dox (6) S10 Synthesis of DBCO-PEG 1k -NHS S10 S2

3 Supplementary Scheme 1. Synthetic route of Ac 4 ManAz, Ac 3 ManAzEt, and Ac 3 ManAzNB Synthesis of 2-azidoacetic acid (7). Bromoacetic acid (20 mmol) and sodium azide (40 mmol) were dissolved in deionized water and stirred at room temperature for 24 h. The resulting solution was adjusted to ph = 1 using hydrogen chloride solution, and then extracted with diethyl ether for three times. The organic phase was collected, dried over anhydrous sodium sulfate, and concentrated to yield 2-azidoacetic acid as colorless oil. Synthesis of N-(2-azidoacetyl) succinimide (8). N,N'-Dicyclohexylcarbodiimide (DCC, 10 mmol) and 7 (10 mmol) were dissolved in anhydrous DMF, followed by the addition of N- hydroxysuccinimide (NHS, 10 mmol). The mixture was stirred at room temperature for 24 h. After filtrating off the precipitate, the solvent was removed to yield a yellow solid. The crude product was recrystallized from DCM/hexane to obtain a white solid. 1 H NMR (CDCl 3, 500 MHz): δ 4.25 (s, 2H, N 3 CH 2 ), 2.88 (s, 4H, CH 2 CH 2 ). 13 C NMR (CDCl 3, 500 MHz): 168.7, 164.4, 48.2, ESI MS (m/z): calculated for C 6 H 7 N 4 O 4 [M+H] , found Synthesis of Ac 4 ManAz (1). D-Mannosamine hydrochloride (2.5 mmol) and triethylamine (2.5 mmol) were dissolved in methanol, followed by the addition of 8 (2.75 mmol). The mixture was stirred at room temperature for 24 h. Solvent was removed under reduced pressure and the residue was re-dissolved in pyridine. Acetic anhydride was added and the reaction mixture was S3

4 stirred at room temperature for another 24 h. After removal of the solvent, the crude product was purified by silica gel column chromatography using ethyl acetate/hexane (1/1, v/v) as the eluent to yield a white solid (1/1 α/β isomers). 1 H NMR (CDCl 3, 500 MHz): δ (ppm) 6.66&6.60 (d, J= 9.0 Hz, 1H, C(O)NHCH), 6.04&6.04 (d, 1H, J=1.9 Hz, NHCHCHO), & (dd, J=10.2, 4.2 Hz, 1H, CH 2 CHCHCH), 5.22&5.16 (t, J=9.9 Hz, 1H, CH 2 CHCHCH), & (m, 1H, NHCHCHO), (m, 2H, CH 2 CHCHCH), 4.07 (m, 2H, C(O)CH 2 N 3 ), (m, 1H, CH 2 CHCHCH), (s, 12H, CH 3 C(O)). 13 C NMR (CDCl 3, 500 MHz): δ (ppm) 170.7, 170.4, 170.3, 169.8, 168.6, 168.3, 167.5, 166.9, 91.5, 90.5, 73.6, 71.7, 70.5, 69.1, 65.3, 65.1, 62.0, 61.9, 52.8, 52.6, 49.9, 49.5, 21.1, 21.0, 21.0, 20.9, 20.9, 20.9, ESI MS (m/z): calculated for C 16 H 22 N 4 O 10 Na [M+Na] , found Synthesis of Ac 3 ManAzEt (2) and Ac 3 ManAzNB (3). Ac 4 ManAz (0.1 mmol, see Supplementary Section Experimental ) and ethanol or 2-nitrobenzylalcohol (0.3 mmol) were dissolved in dry dichloromethane. Boron trifluoride etherate (0.5 mmol) was added. The mixture was stirred overnight and then washed with saturated sodium bicarbonate solution and water. The organic phase was collected and concentrated to yield yellow oil. The crude product was purified by silica gel column chromatography using ethyl acetate/hexane (1/1, v/v) as the eluent. Ac 3 ManAzEt (2): 1 H NMR (CDCl 3, 500 MHz): δ (ppm) 6.52 (d, J = 9.1 Hz, 1H, C(O)NHCH), 5.35 (dd, J = 10.2, 4.2 Hz, 1H, CH 2 CHCHCH), 5.13 (t, J = 10.1 Hz, 1H, CH 2 CHCHCH), 4.77 (d, J = 1.7 Hz, 1H, NHCHCHO), 4.58 (ddd, J = 9.3, 4.3, 1.7 Hz, 1H, NHCHCHO), (dd, J = 12.3, 4.5 Hz, 1H, CH 2 CHCHCH), (dd, J = 12.3, 2.4 Hz, 1H, CH 2 CHCHCH), 4.05 (m, 2H, C(O)CH 2 N 3 ), 3.99 (m, 1H, CH 2 CHCHCH), 3.54&3.73 (dq, J = 9.5, 7.2 Hz, 2H, CH 3 CH 2 O), (s, 9H, CH 3 C(O)), 1.25 (t, 3H, CH 3 CH 2 O). 13 C NMR (CDCl 3, 500 MHz): δ (ppm) 170.8, 170.3, 170.0, 166.9, 98.7, 69.5, 68.3, 66.1, 64.3, 62.4, 52.7, 51.1, 21.0, 20.9, 20.9, HR ESI MS (m/z): calculated for C 16 H 23 N 4 O 9 [M-H] , found Ac 3 ManAzNB (3): 1 H NMR (CDCl 3, 500 MHz): δ (ppm) 8.12 (d, J = 8.2 Hz, 1H, Ph, CHCHCHCHNO 2 ), 7.79 (d, J = 7.5 Hz, 1H, Ph, CHCHCHCHNO 2 ), 7.72 (t, J = 7.5 Hz, 1H, Ph, CHCHCHCHNO 2 ), 7.50 (t, J = 8.4, 7.5 Hz, 1H, Ph, CHCHCHCHNO 2 ), 6.57 (d, J = 8.9 Hz, 1H, C(O)NHCH), 5.41 (dd, J = 10.1, 4.2 Hz, 1H, CH 2 CHCHCH), (m, 2H, C(O)NHCHCHO, CH 2 CHCHCH), 4.94 (dd, J = 8.4, 6.6 Hz, 2H, OCH 2 Ph), 4.76 (ddd, J = 9.3, 4.4, 1.8 Hz, 1H, C(O)NHCHCHO), (dd, J = 12.4, 4.9 Hz, 1H, CH 2 CHCHCH), S4

5 4.15 (dd, J = 12.9, 3.1 Hz, 1H, CH 2 CHCHCH), 4.06 (m, 2H, C(O)CH 2 N 3 ), 4.02 (m, 1H, CH 2 CHCHCH), (s, 9H, CH 3 C(O)). 13 C NMR (CDCl 3, 500 MHz): δ (ppm) 170.8, 170.0, 167.0, 134.4, 128.8, 128.7, 125.2, 98.7, 69.5, 69.0, 66.6, 65.8, 62.3, 52.7, 50.4, 21.0, 20.9, HR ESI MS (m/z): calculated for C 21 H 26 N 5 O 11 [M+H] , found Supplementary Scheme 2. Synthetic route of HDAC/CTSL responsive DCL-AAM Synthesis of AcLys(NHS) (10). AcLys(OH) (2.0 mmol) and triethylamine (2.1 mmol) was dissolved in anhydrous DMF, followed by the addition of DCC (2.1 mmol) and NHS (2.1 mmol). The reaction mixture was stirred at room temperature for 24 h. The precipitate was filtered off, and the solvent was removed under reduced pressure. The crude product was directly used without further purification. Synthesis of (4-aminophenyl)(phenyl)methanol (11). 4-Aminobenzophenone (5.0 mmol) was dissolved in dry methanol, followed by addition of sodium borohydride (10.0 mmol). The mixture was stirred at room temperature for 24 h. After removal of the solvent, the crude product was purified by silica gel column chromatography using hexane/ethyl acetate (6/1 to 3/1) as the eluent. Compound 11 was obtained as a light yellow solid. 1 H NMR (CDCl 3, 500 MHz): δ (ppm) 7.38 (d, 2H, Ph), 7.32 (t, 2H, Ph), 7.25 (t, 1H, Ph), 7.14 (d, 2H, Ph), 6.63 (d, 2H, Ph), 5.75 (s, 1H, CH(OH)), 3.65 (s, 1H, (CH(OH)), 3.45 (s, 2H, NH 2 ). 13 C NMR (CDCl 3, 500 MHz): δ (ppm) 146.1, 144.4, 134.4, 128.6, 128.2, 127.5, 126.6, 115.3, ESI MS (m/z): calculated for C 13 H 14 NO [M+H] , found S5

6 Synthesis of AcLys(DPM) (12). AcLys(NHS) (1.2 mmol) and compound 11 (1.2 mmol) were dissolved in anhydrous DMF, followed by the addition of triethylamine (1.2 mmol). The mixture was stirred at 40 o C for 24 h. After removal of the solvent, the crude product was purified by silica gel column chromatography using ethyl acetate to ethyl acetate/methanol (20/1, v/v) as the eluent to yield a light yellow solid. 1 H NMR (CD 3 OD, 500 MHz): δ (ppm) 7.50 (d, 2H, Ph), 7.34 (d, 2H, Ph), 7.29 (m, 4H, Ph), 7.21 (t, 1H, Ph), 5.74 (s, 1H, CH(OH)), 4.39 (m, 1H, CHCH 2 CH 2 CH 2 CH 2 NHC(O)CH 3 ), 3.15 (t, 2H, CHCH 2 CH 2 CH 2 CH 2 NHC(O)CH 3 ), 2.00 (s, 3H, CHCH 2 CH 2 CH 2 CH 2 NHC(O)CH 3 ), 1.89 (s, 3H, CHNHC(O)CH 3 ), 1.83&1.72 (m, 2H, CHCH 2 CH 2 CH 2 CH 2 NHC(O)CH 3 ), 1.53 (m, 2H, CHCH 2 CH 2 CH 2 CH 2 NHC(O)CH 3 ), 1.41 (m, 2H, CHCH 2 CH 2 CH 2 CH 2 NHC(O)CH 3 ). 13 C NMR (CD 3 OD, 500 MHz): 172.2, 172.0, 171.7, 144.7, 140.9, 137.3, 128.1, 127.0, 126.5, 120.1, 75.3, 54.4, 38.9, 31.7, 28.9, 23.1, 21.4, ESI MS (m/z): calculated for C 23 H 29 N 3 O 4 Na [M+Na] , found Synthesis of AcLys(DPM-CNCCl 3 ) (13). Compound 12 (0.5 mmol) and CNCCl 3 (5.0 mmol) were dissolved in anhydrous THF, followed by the addition of 1,8-diazabicycloundec-7-ene (DBU, 0.5 mmol). The reaction mixture was stirred at room temperature for 4 h. After removal of the solvent, the crude product was purified by silica gel column chromatography to yield a white solid. 1 H NMR (CD 3 OD, 500 MHz) δ (ppm) 7.99 (m, 1H), 7.53 (d, J = 8.4 Hz, 2H), (m, 7H), 5.27 (s, 1H), 4.43 (m, 1H), 3.73 (m, 1H), 3.17 (m, 2H), 2.02 (s, 3H), 1.91 (s, 3H), 1.84&1.73 (m, 2H), 1.55 (m, 2H), 1.44 (m, 2H). 13 C NMR (CD 3 OD, 500 MHz): , 172.2, 172.1, 171.7, 142.4, 138.5, 137.6, 128.2, 127.3, 126.8, 120.2, 101.8, 85.0, 64.4, 54.3, 39.1, 31.7, 28.9, 23.1, 21.4, Synthesis of Ac 3 ManAzOH (14). Ac 4 ManAz (0.5 mmol) and ammonium carbonate (0.75 mmol) were dissolved in THF/methanol (2/1, v/v) and stirred at room temperature. After 12 h, the solvent was removed and the crude product was purified by silica gel column chromatography to yield a white solid. 1 H NMR (CDCl 3, 500 MHz): δ (ppm) 6.56 (d, J = 9.2 Hz, 1H), 5.44 (dd, J = 10.1, 4.2 Hz, 1H), 5.22 (s, 1H), 5.18 (t, 1H), 4.63 (ddd, J = 9.2, 4.2, 1.8 Hz, 1H), (m, 5H), 3.74 (m, 1H), 2.14&2.08&2.05 (s, 9H). 13 C NMR (CDCl 3, 500 MHz): δ (ppm) 171.0, 170.4, 170.1, 167.1, 93.4, 69.2, 68.4, 66.0, 62.5, 52.7, 50.9, 21.1, 21.0, S6

7 Synthesis of DCL-AAM (4). Ac 3 ManAzOH (0.2 mmol) and AcLys(DPM-CNCCl 3 ) (0.22 mmol) were dissolved in anhydrous acetonitrile, followed by the addition of boron trifluoride etherate (2.0 mmol). The mixture was stirred at 0 o C for 1 h. After removal of the solvent, the crude product was purified by silica gel column chromatography and HPLC to yield a white solid. 1 H NMR (CDCl 3, 500 MHz): δ (ppm) 7.55 (m, 2H, Ph), (m, 7H, Ph), 6.73 (m, 1H, CH 3 C(O)NHCH), 6.50 (m, 1H, CH 2 C(O)NHCH), 5.95 (m, 1H, CH 3 C(O)NHCH 2 ), 5.65 (s, 1H, PhCH), 5.43 (ddd, J = 10.1, 8.3, 4.2 Hz, 1H, CH 2 CHCHCH), 5.17 (td, J = 10.2, 3.2 Hz, 1H, CH 2 CHCHCH), (m, 1H, NHCHCHO), 4.68 (m, 1H, NHCHCHO), 4.55 (q, J = 7.3 Hz, 1H, CHCH 2 CH 2 CH 2 CH 2 NHC(O)CH 3 ), 4.18&4.05 (m, 2H, CH 2 CHCHCH), 4.02 (m, 2H, COCH 2 N 3 ), 3.94 (m, 1H, CH 2 CHCHCH), 3.22 (m, 2H, CHCH 2 CH 2 CH 2 CH 2 NHC(O)CH 3 ), 2.12&2.06&2.03&1.99&1.96 (15H, CH 3 C(O)O), 1.93&1.74 (m, 2H, CHCH 2 CH 2 CH 2 CH 2 NHC(O)CH 3 ), 1.51 (m, 2H, CHCH 2 CH 2 CH 2 CH 2 NHC(O)CH 3 ), 1.38 (m, 2H, CHCH 2 CH 2 CH 2 CH 2 NHC(O)CH 3 ). 13 C NMR (CDCl 3, 500 MHz): δ(ppm) 171.5, 171.2, 170.7, 170.4, 170.0, 170.0, 166.8, 141.2, 137.8, 137.2, 135.6, 129.1, 128.8, 128.5, 127.5, 126.7, 120.2, 80.0, 77.5, 77.5, 77.3, 77.2, 77.0, 69.9, 69.0, 65.8, 62.3, 53.9, 52.7, 50.5, 38.8, 31.3, 29.1, 23.4, 22.5, 21.1, HR ESI MS (m/z): calculated for C 37 H 48 N 7 O 12 [M+H] , found Supplementary Scheme 3. Synthetic route of HDAC/CTSL fluorescent reporter (Naph-Lys) S7

8 Syntheis of Naph-NH 2 (15). 4-Amino-1,8-naphthalic anhydride (10 mmol) was dissolved in ethanol (100 ml) under nitrogen atmosphere and brought to reflux. 1-butylamine (15 mmol) was then added and the mixture was further refluxed for 8 h. After cooling down, the solvent was removed under reduced pressure, and the crude product was purified by silica gel column chromatography using DCM/ethyl acetate (2/1, v/v) as the eluent. 1 H NMR (CDCl 3, 500 MHz): δ (ppm) 8.60 (d, 1H, C(O)CCHCHCH), 8.42 (d, 1H, C(O)CCHCHCH), 8.10 (d, 1H C(O)CCHCHCH), 7.66 (t, 1H, C(O)CCHCH), 6.89 (d, 1H, C(O)CCHCH), 4.94 (s, 2H, NH 2 ), 4.18 (t, 2H, CH 2 CH 2 CH 2 CH 3 ), 1.71 (m, 2H, CH 2 CH 2 CH 2 CH 3 ), 1.45 (m, 2H, CH 2 CH 2 CH 2 CH 3 ), 0.97 (s, 3H, CH 2 CH 2 CH 2 CH 3 ). ESI MS (m/z): calculated for C 16 H 17 N 2 O 4 [M+H] , found Synthesis of Naph-Lys (5). Naph-NH 2 (1.0 mmol) and AcLys(OH) (1.0 mmol) were dissolved in DMF, followed by the addition of DCC (1.1 mmol), NHS (1.1 mmol), and triethylamine (1.0 mmol). The mixture was stirred at 45 o C for 48 h. After removal of precipitate and solvent, the crude product was purified by silica gel column chromatography using hexane/ethyl acetate as the eluent to yield a yellow solid. 1 H NMR (CD 3 OD, 500 MHz): δ (ppm) 8.57 (dd, 1H, C(O)CCHCHCH), 8.52 (d, 1H C(O)CCHCH), 8.51 (dd, 1H, C(O)CCHCHCH), 8.12 (dd, 1H, C(O)CCHCH), 7.84 (dd, 1H, C(O)CCHCHCH), 4.62 (dd, 1H, CHCH 2 CH 2 CH 2 CH 2 ), 4.14 (t, 2H, CH 2 CH 2 CH 2 CH 3 ), 3.22 (t, 2H, CHCH 2 CH 2 CH 2 CH 2 ), 2.06 (s, 3H, CH 3 C(O)NHCH), 1.98 (m, 2H, CHCH 2 CH 2 CH 2 CH 2 ), 1.92 (s, 3H, CH 3 C(O)NHCH 2 ), 1.69 (m, 2H, CH 2 CH 2 CH 2 CH 3 ), 1.60 (m, 2H, CHCH 2 CH 2 CH 2 CH 2 ), 1.53 (m, 2H, CHCH 2 CH 2 CH 2 CH 2 ), 1.43 (m, 2H, CH 2 CH 2 CH 2 CH 3 ), 0.99 (m, 3H, CH 2 CH 2 CH 2 CH 3 ). ESI MS (m/z): calculated for C 26 H 33 N 4 O 5 [M+H] , found Supplementary Scheme 4. Radiolabeling of DCL-AAM S8

9 Synthesis of DBCO-DOTA (16). P-SCN-Bn-DOTA (0.05 mmol) and DBCO-amine (0.05 mmol) were suspended in anhydrous DMF (1 ml). Triethylamine (0.2 mmol) was added and the mixture was stirred at 40 o C for 12 h. The solvent was removed under reduced pressure and the product was used without further purification. ESI MS (m/z): calculated for C 42 H 50 N 7 O 9 S [M+H] , found Synthesis of DCL-AAM-DOTA (17). DBCO-DOTA (0.01 mmol) and DCL-AAM (0.01 mmol) were dissolved in methanol. The mixture was stirred at room temperature for 1 h, at which time point HPLC showed complete consumption of the starting materials. The product was lyophilized and used directly without further purification. S9

10 Synthesis of 64 Cu-labeled DCL-AAM (18). The 64 Cu chloride (400 μci, 0.1 ng of 64 Cu) in NH 4 OAc buffer solution (0.1 M, ph = 5.5, 1 ml) was mixed with DCL-AAM-DOTA (2 mg) in PBS. The mixture was vigorously stirred at 80 o C for 1 h, at which time point HPLC showed complete consumption of free 64 Cu. The product solution was directly used without further purification. Supplementary Scheme 5. Synthetic route of DBCO-VC-Dox Synthesis of DBCO-PEG 1k -NHS. DBCO-NHS (0.11 mmol) and NH 2 -PEG 1k -COOH (0.10 mmol) were dissolved in anhydrous DMF, followed by the addition of triethylamine (0.11 mmol). The mixture was stirred at 45 o C for 24 h. DCC (0.12 mmol) and NHS (0.12 mmol) in DMF were added. The reaction mixture was further stirred at 45 o C for 24 h. The precipitate was filtered off and the filtrate was ultracentrifuged (repeated twice) with a cut-off molecular weight of 1k. The residual solution was diluted with DCM, collected, and concentrated to yield a light yellow solid (70% yield). Synthesis of DBCO-VC-Dox (6). Doxorubicin hydrochloride (0.05 mmol), Fmoc-Val-Cit-PAB- PNP (0.05 mmol) and triethylamine (0.06 mmol) were dissolved in dimethylformamide and stirred at 40 o C for 12 h, at which point diisopropylethylamine (100 μl) was added. After 12 h, DBCO-PEG 1k -NHS (0.05 mmol, see Supplementary Section Experimental ) was added and the S10

11 mixture was stirred at 40 o C for another 24 h. The reaction solution was precipitated into diethyl ether to yield a red solid, which was purified by dialysis (1k cut-off molecular weight) for 48 h. MALDI MS (m/z) for DBCO-VC-Dox *n, found (n=20), (n=21), (n=22), (n=23), (n=24), (n=25), (n=26), (n=27), (n=28). S11