Steric-Dependent Label-Free and Washing-Free. Enzyme Amplified Protein Detection with

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1 Supporting Information Steric-Dependent Label-Free and Washing-Free Enzyme Amplified Protein Detection with Dual-Functional Synthetic Probes Chia-Wen Wang, Wan-Ting Yu, Hsiu-Ping Lai, Bing-Yuan Lee, Ruo-Cing Gao, Kui-Thong Tan,, * Department of Chemistry, National Tsing Hua University, 101 Sec. 2, Kuang Fu Rd, Hsinchu 30013, Taiwan (ROC) Frontier Research Center on Fundamental and Applied Sciences of Matters, National Tsing Hua University, 101 Sec. 2, Kuang Fu Rd, Hsinchu 30013, Taiwan (ROC) Corresponding author: Kui-Thong Tan, kttan@mx.nthu.edu.tw S1

2 Figure S1. Fluorescence spectra of 1 μμ HCAII, 2 μμ BTSA and 1 µm FLDA in the absence (red line) and presence (black line) of 2 μμ avidin after 60 minutes of enzyme amplification. The fluorescence enhancement was suppresed in the presence of 50 μμ free biotin (blue line). λ ex = 470 nm. S2

3 (a) (b) Figure S2. LOD of BTSA to detect avidin. (a) Fluorescence spectra for the detection of different concentrations of avidin with BTSA. Detection condition: various concentrations of avidin were incubated with 1 μμ HCAII and 2 μμ BTSA for 10 minutes followed by the addition of 1 µm FLDA to initiate signal amplification. The fluorescence spectra were recorded after 60 minutes of signal amplification. (b) The fluorescence response was linear in the range of μm and the LOD was estimated as 140 nm from three times the standard deviation corresponding to the control measurements (N = 15, without avidin). S3

4 (a) (b) Figure S3. Relative fluorescence intensity for the incubation of 4 μμ corresponding probes with 1 μμ HCAII and 2 μμ avidin. F/F 0 was obtained after 60 minutes of HCAII catalysis to hydrolyze 1 µm FLDA. The error bar was calculated from three independent measurements. S4

5 Figure S4. Reaction mechanism for the covalent transfer of the benzyl group to MGMT. S5

6 (a) (b) (c) (d) Figure S5. Inhibition profile and fluorescence response of different concentrations of (a) BTSA, (b) BASA, (c) BGSA-1 and (d) BGSA-2 against 1 µm HCAII and 1 µm FLDA. S6

7 (a) (b) Figure S6. LOD of BGSA-1 to detect MGMT. (a) Time course of the fluorescent response for the detection of different concentrations of MGMT with BGSA-1. Detection condition: different concentrations of MGMT were incubated with 1 μμ HCAII and 2 μμ BGSA-1 for 90 minutes. Fluorescent measurement started immediately upon the addition of 1 µm FLDA. (b) The fluorescence response was linear in the range of μm and the LOD was estimated as 173 nm, from three times the standard deviation corresponding to the control measurement (without MGMT). S7

8 (a) (b) (c) Figure S7. (a) Time course of the fluorescence response for the detection of MGMT with the longer linker probe BGSA-3. Detection condition: 1 μμ HCAII and 2 μμ BGSA-3 were incubated with or without 2 µm MGMT for 90 minutes. (b) Time course of the fluorescence response for the longer linker probe BGSA-3 and SNAP-tag. Detection condition: 1 μμ HCAII and 1 μμ BGSA-3 were incubated with or without 1 μμ SNAP. For (a) and (b), fluorescent measurement started immediately upon the addition of 1 µm FLDA. The error bar was calculated from three independent measurements. (c) Chemical structure of BGSA-3. S8

9 (a) (b) Figure S8. LOD of BGSA-2 to detect SNAP-tag. (a) Time course of the fluorescent response for different concentrations of SNAP-tag with BGSA-2. Detection condition: Different concentrations of SNAP-tag protein were incubated with 1 μμ HCAII and 0.5 μμ BGSA-2 at 37 C for 60 minutes. Fluorescent measurement started immediately after the addition of 1 µm FLDA. (b) The fluorescence response was linear from 10 nm to 60 nm range and the LOD was estimated as 12 nm from three times the standard deviation corresponding to the control measurement (without SNAP-tag). S9

10 (a) (b) Figure S9. Selectivity test of BGSA-1 and BGSA-2 with twelve other non-targeted proteins. (a) 1 μμ HCAII and 2 μμ BGSA-1 were incubated with different proteins at 2 μμ. (b) 1 μμ HCAII with 1 μμ BGSA-2 were tested with different proteins at 1 μμ. For (a) and (b), fluorescent measurement started immediately upon the addition of 1 µm FLDA. F/F 0 ratio was obtained after 60 minutes of HCAII catalysis to hydrolyze FLDA. The error bar was calculated from three independent measurements. S10

11 phenylboronic acid Figure S10. Time course of the fluorescence response for the detection of lactoferrin with BASA in the presence and absence of 50 um free phenylboronic acid. Detection condition: 1 μμ HCAII and 4 μμ BASA were incubated with or without 4 μμ lactoferrin at 37 C for 60 minutes. The blue line shows the presence of 50 μμ free phenylboronic acid in the detection condition. Fluorescent measurement started immediately upon the addition of 1 µm FLDA. The error bar was calculated from three independent measurements. S11

12 (a) (b) Figure S11. (a) Test of BASA with seven different diols. 5 mm Diols (except hyaluronic acids which were tested at the concentration of 0.5 mg/ml) were incubated respectively with 1 μμ HCAII and 4 μμ BASA at 37 C for 60 minutes. Fluorescent measurement started immediately after the addition of 1 µm FLDA. (b) Time course of the fluorescent response for 50 mm concentrations of glucose and fructose with BASA. S12

13 (a) (b Figure S12. LOD of BASA to detect lactoferrin. (a) Time course of the fluorescent response for different concentrations of lactoferrin with BASA. Detection condition: Different concentrations of lactoferrin were incubated with 1 μμ HCAII and 4 μμ BASA at 37 C for 60 minutes. Fluorescent measurement started immediately after the addition of 1 µm FLDA. (b) The fluorescence response was linear from 0.3 µm to 4 µm range and the LOD was estimated as 350 nm from three times the standard deviation corresponding to the control measurement (without lactoferrin). S13

14 (a) (b) Figure S13. LOD of BGSA-1 to detect MGMT in 10% urine. (a) Time course of the fluorescent response for different concentrations of MGMT with 2 µm BGSA-1 and 1 µm HCAII in 10% urine mixed with HEPES buffer. The fluorescence measurement started immediately upon the addition of 1 µm FLDA. (b) The fluorescence response was linear from 0.1 µm to 1 µm range and the LOD was estimated as 1.3 µm from three times the standard deviation corresponding to the control measurement (without MGMT protein). S14

15 (a) (b) (c) (d) (e) (f) Figure S14. Detection of avidin and SNAP-tag in 10% urine. (a) Time course of the fluorescence response for the detection of 2 µm avidin using 4 µm BTSA and 1 µm HCAII in 10% urine mixed with HEPES buffer. (b) Time course of the fluorescence response for the detection of 1 μμ SNAP-tag using 0.5 μμ BGSA-2 and 1 µm HCAII in 10% urine mixed with HEPES buffer. (c) Time course of the fluorescent S15

16 response for different concentrations of avidin with 4 μμ BTSA and 1 μμ HCAII in 10% urine mixed with HEPES buffer. (d) The LOD was estimated as 7000 nm. (e) Time course of the fluorescent response for different concentrations of SNAP-tag with 0.5 μμ BGSA-2 and 1 μμ HCAII in 10% urine mixed with HEPES buffer. (f) The LOD was estimated as 32 nm. The fluorescent measurement started immediately upon the addition of 1 µm FLDA. The LOD was estimated from three times the standard deviation corresponding to the control measurement (without target proteins). S16

17 (a) (b) Figure S15. Fluorescence amplification of denatured SNAP and MGMT proteins after the chemical probes were covalently attached to the proteins. (a) The data shows that similar HCAII-amplified fluorescence intensity could be obtained even the SNAP-BGSA-2 covalent conjugate was denatured. (b) Time course of fluorescence response for different concentrations of denatured MGMT-BGSA-1 covalent conjugate. S17

18 Figure S16. SDS-PAGE shows the purity of HCAII and SNAP-tag protein after His-tag purification. S18

19 Scheme S1. Synthetic scheme for BTSA. Synthesis of compound 1: A solution of di-tert-butyldicarbonate (Boc 2 O, 2.4 g, mmol) in 100 ml DCM was added dropwise to a solution of ethylenediamine (4.4 ml, mmol) in 50 ml DCM at 0 C. The reaction mixture was allowed to stir overnight at room temperature. The solution was concentrated and the oil residue was re-dissolved in 50 ml 2M K 2 CO 3(aq) and extracted with DCM (30 ml 3). The organic layer was washed with brine, dried over anhydrous MgSO 4 and concentrated in vacuum to yield a colorless oil (1.68 g, 10 mmol, 95%). 1 H -NMR (400 MHz, CDCl 3 ): δ 4.86 (s, 1H), 3.14 (dd, J = 11.4, 5.6 Hz, 2H), 2.77 (t, J = 5.9 Hz, 2H), 1.42 (s, 9H), 1.15 (s, 2H) ppm; 13 C-NMR (100 MHz, CDCl 3 ): δ , 78.19, 42.9, 41.32, ppm; HRMS (ESI): m/z calcd. for C 7 H 17 N 2 O 2 + = , found [M+H] +. S19

20 Synthesis of compound 2: To a solution of EDC.HCl (72 mg, 0.5 mmol), HOBt.H 2 O (91 mg, 0.5 mmol), triethylamine (165 μl, 1 mmol) and biotin (58 mg, 0.2 mmol) in 2 ml DMF was added compound 1 (64 mg, 0.4 mmol in 1 ml DMF) at room temperature and the reaction mixture stirred at room temperature for 17 hours. After being concentrated under reduced pressure, the crude product was purified by column chromatography over silica gel using DCM/MeOH to give product 2 as a colorless oil (51 mg, 0.13 mmol, 56%). 1 H-NMR (400 MHz, MeOD ) δ 4.58 (s, 1H), (m, 1H), 4.30 (dd, J = 7.9, 4.5 Hz, 1H), (m, 4H), 3.13 (t, J = 6.0 Hz, 2H), 2.92 (dd, J = 12.7, 5.0 Hz, 1H), 2.70 (d, J = 12.7 Hz, 1H), 2.20 (t, J = 7.6 Hz, 2H), (m, 6H), 1.44 (s, 9H) ppm; 13 C-NMR (100 MHz, MeOD) δ , , , 80.17, 63.34, 61.63, 56.93, 41.02, 40.96, 40.49, 36.81, 29.73, 29.44, 28.76, ppm. Synthesis of BTSA: Compound 2 (24 mg, 0.06 mmol) was dissolved in 20% TFA/DCM at room temperature and stirred for 30 minutes. The solvent and TFA were removed under reduced pressure and product 3 was used for the next step without further purification. A solution of compound 3 in DMF (1 ml) was added to the solution of 4-sulfamoylbenzoic acid (10 mg, 0.05 mmol), EDC.HCl (20 mg, 0.1 mmol), HOBt (16 mg, 0.1 mmol) and Et 3 N (73 μl, 0.52 mmol) in DMF (1 ml) and the mixture was stirred at room temperature for 18 hours. The reaction mixture was purified by preparative HPLC to give product BTSA as a white powder after lyophilization in 68% yield (16 mg, 0.03 mmol). 1 H -NMR (400 MHz, d-dmso) δ 8.66 (t, J = 5.4 Hz, 1H), 7.97 (d, J = 8.3 Hz, 2H), 7.92 (t, J = 5.7 Hz, 1H), 7.88 (d, J = 8.3 Hz, 2H), 7.47 S20

21 (s, 2H), 6.38 (d, J = 23.3 Hz, 2H), (m, 1H), (m, 1H), (m, 2H), (m, 2H), (m, 1H), 2.79 (dd, J = 12.4, 4.9 Hz, 1H), 2.55 (d, J = 12.4 Hz, 1H), 2.05 (t, J = 7.3 Hz, 2H), (m, 4H), (m, 2H) ppm; 13 C-NMR (100 MHz, d-dmso) δ (C), (C), (C), (C), (C), (CH), (CH), (CH), (CH), (CH), (CH 2 ), (CH 2 ), (CH 2 ), (CH 2 ), (CH 2 ), (CH 2 ), (CH 2 ) ppm; HRMS (ESI): m/z calcd. for C 19 H 28 N 5 O 5 S + 2 = , found [M+H] +. Scheme S2. Synthetic scheme for BASA. Synthesis of BASA: To a solution of 3-carboxyphenylboronic acid (18 mg, 0.11 mmol) in 1 ml DMF was added PyBOP (113 mg, 0.22 mmole), 4-(aminomethyl)benzenesulfonamide HCl (37 mg, 0.16 mmol) and DIPEA (95 μl, 0.55 mmol) and the reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was purified by preparative HPLC to give product BTSA as a white powder after lyophilization in 47% yield (17 mg, 0.05 mmol). 1 H-NMR (400 MHz, d-dmso): δ 9.08 (t, J = 6.0 Hz, 1H), 8.28 (s, 1H), 8.18 (s, 2H), 7.92 (d, J = 7.3 Hz, 1H), 7.89 (d, J = 7.8 Hz, 1H), 7.77 (d, J = 8.4 Hz, 2H), 7.48 (d, J = 8.4 Hz, 2H), 7.43 (t, J = 7.5 Hz, 1H), 7.30 (s, 2H), 4.52 (d, J = 5.9 Hz, 2H) ppm; 13 C-NMR (100 MHz, d-dmso): δ , , , , , , , , , , , ppm; HRMS (ESI): S21

22 m/z calcd. for C 14 H 16 BN 2 O 5 S + = , found [M+H] +. N SO 2 NH 2 O OH SO 2 NH 2 THF, LAH OH SO 2 NH 2 5 H 2 N N N N N H DMF t BuOK DMAP H 2 N N O N N N H 4 BGSA-1 Scheme S3. Synthetic Scheme for BGSA-1. Synthesis of Compound 4: LiAlH 4 (212 mg, 5.59 mmol) was suspended in dry THF (10 ml) under nitrogen. The suspension was cooled to 0 C and 4-sulfamoylbenzoic acid (450 mg, 2.23 mmol, as a suspension in 10 ml of dry THF) was added. The resulting mixture was then heated to reflux overnight. The reaction mixture was cooled to 0 C and quenched by the addition of 3N HCl (10 ml). The quenched mixture was extracted with ethylacetate (30 ml x 3). The combined organic layer was washed with brine and dried over anhydrous magnesium sulfate. The solvent was removed under reduced pressure and purified by flash chromatography to give compound 4 (200 mg, 48%). 1 H-NMR (400 MHz, D-Methanol): δ 7.86 (d, J = 8.6 Hz, 2H), 7.51 (d, J = 8.6 Hz, 2H), 4.67 (s, 2H) ppm; 13 C-NMR (100 MHz, D-Methanol): δ , , , , ppm. S22

23 Synthesis of BGSA-1: To a solution of compound 4 in DMF (2 ml) was added t BuOK (95 mg, mmol) at room temperature. After 30 minutes of stirring, benzylguanine salt 5 and DMAP (8 mg, mmol) were added and stirred at room temperature overnight. The solvent was removed under reduced pressure and the crude residue was purified by flash chromatography to give BGSA-1 (8 mg, 12%). 1 H-NMR (400 MHz, d-dmso): δ 7.84 (S, 1H), 7.83 (d, J = 8.0 Hz, 2H), 7.66 (d, J = 8.0 Hz, 2H), 6.26 (s, 2H), 5.56 (s, 2H) ppm; 13 C-NMR (100 MHz, d-dmso): δ , , , , , , , ppm; HRMS (ESI): calcd for C 12 H 12 N 6 O 3 S: , found: [M+H] +. Scheme S4. Synthetic scheme for BGSA-2. Synthesis of BGSA-2: To a solution of 4-sulfamoylbenzoic acid (8 mg, 0.04 mmol) EDC.HCl (9 mg, 0.05 mmol), HOBt (10 mg, mmol) and triethylamine (6.45 μl, 0.05 mmol) in 1 ml DMF was added BG-NH 2 (10 mg, 0.04 mmol). The reaction mixture was stirred at room temperature for 18 hours. The crude product was purified by column chromatography over silica gel using DCM/MeOH to give product BGSA-2 (15 mg, 0.03 mmol, 87%). 1 H NMR (400 MHz, d-dmso): δ 9.25 (t, J = 6.1 Hz, 1H), 8.34 (d, J = 12.1 Hz, 1H), 8.02 (d, J = 8.1 Hz, 2H), 7.89 (d, J = 8.1 Hz, 2H), 7.50 (d, J = 8.8 Hz, 4H), 7.36 (d, J = 7.8 Hz, 2H), 5.51 (s, 2H), 4.49 (d, J = 5.9 Hz, 2H) ppm; S23

24 13 C-NMR (100 MHz, d-dmso): δ , , , , , , , , , , , , , , 67.69, ppm. S24

25 13C NMR DEPT 135 DEPT 90 S25

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