Ó 2003 WILEY-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim (Germany) Supporting Information for ChemBioChem Z 556

Developing a strategy for activity-based detection of in a protein microarray Grace Y. J. Chen, Mahesh Uttamchandani, Qing Zhu, Gang Wang, and Shao Q. Yao*

Supporting Materials

All chemicals were purchased from commercial sources, unless indicated otherwise. The 1H NMR spectra were taken on a Bruker 300 MHz NMR spectrometer. Chemical shifts are reported in parts per million referenced with respect to residual solvent (CHCl3 = 7.26 ppm). All proteins except -1 were purchased from Sigma (St Louis, USA). Their types (and Sigma catalog numbers) are the followings: 1. Type I-S Alkaline Phosphatase, from bovine intestinal mucosa (P- 7640); 2. Type VIII Alkaline Phosphatase, from rabbit intestine (P-2265); 3. Type IV Alkaline Phosphatase, from porcine intestinal mucosa (P-4002); 4. , from papaya latex (C- 8526); 5. , from papaya latex (P-4762); 6. a-Chymotrypsin (C-4129); 7. b-Chymotrypsin (C- 4629); 8. g-Chymotrypsin (C-4754); 9. Proteinase K, from tritirachium album (P-6556); 10. Subtilisin, from bacillus licheniformis (P-5380); 11. Lysozyme, from chicken egg white (L-6876); 12. Lipase, from candida rugosa (L-1754). All proteins were prepared as ~1-10 mg/ml solutions in distilled water, desalted with a NAP-5 column (Amersham Pharmacia, USA) according to manufacturer’s protocols, and stored at - 20 oC as working stock solutions until use. 2 1. Probe Synthesis (1) Probe 1, PT-Cy3 The synthesis and full-scale SDS-PAGE experiments of PT-Cy3 will be reported elsewhere.[7b] Experiments related to caspase-1 probe has been reported previously.[7a]

(2) Probe 2, VS-Cy3. O H O N OH O SH

O

O OEt Dimethyl hydroxyla mine 71% P NaH 87% HOBT, DIEA I OEt O H O N O N O S O OEt P 20 OEt 22 O

Peracet ic acid LAH 65% o 86% O o H O N H O O O S O OEt P + OEt 21 23 O

NaH 43%

O O H O N S Boc-Gly-Leu-Le u-OMe O

66% 24 O N N O I - OH Cy3-Gl y-Leu-Leu-OM e Pi peridi ne/DMF 89%

26

O O

44% H2N S

O N N O H N OH I - N N H H O O O 27 25

HOBt /EDC /DIEA 75%

28

50% TFA/DCM 55%

O N N O H H O O N N S I - N N H H O O

2 OH

4-Methyl-thiophenyl-methyl-diethylphosphonate (20). To a dried flask flushed with N2 was added 4-methyl-thiophenol(1.39 g, 5.0 mmol) in 10ml THF and chilled on ice. Sodium hydride (0.24 g, 6.0 mmol) was slowly added leading to gas evolution. The reaction was stirred at room temperature for 30 min and diethyl iodomethyl phosphonate (1.39 g, 5 mmol) was added as a solution in 3ml THF. The reaction was stirred at room temperature for 2 hours and quenched with 10ml 5% potassium hydrogen sulfate. The resulting aqueous phase was extracted with 3 x 10ml ethyl acetate and the combined organic layers were dried (MgSO4) and concentrated under reduced pressure to give an oil, which was purified by flash chromatography (hexane/ethyl acetate, 1:2 v/v) to give the 1 product 20 as a colorless oil (1.20 g; 87% yield): H NMR (300 MHz, CDCl3) d 7.00-7.40 (m, 4H), 4.12 (m, 4H), 2.31 (s, 3H) 3.15 (d, 2H) , 1.30 (t, 6H). ESI mass spectrum: m/z 275.0 [M+H]+.

4-Methyl-thiophenyl-methyl-diethylphosphonate sulfone (21). To a stirred solution of 20 (1.20 g, 4.38 mmol) in 12ml 1,4-dioxane was slowly added peracetic acid (15ml, 32% solution) under ice. The reaction was stirred under room temperature for 6 h and 40 ml of saturated sodium bicarbonate was added. The aqueous phase was extracted with 3 x 25 ml portions of ethyl acetate. The combined organic layers were washed with brine, dried (MgSO4) and concentrated under reduced pressure. Purification of the crude product by flash chromatography (hexane/ethyl acetate, 1:2 v/v) 1 afforded 21 as a yellowish solid (1.15 g; 86% yield): H NMR (300 MHZ, CDCl3) d 7.30-7.90 (m, 4H), 4.12 (m, 4H), 3.70 (d, 2H), 2.42 (s, 3H) 1.37 (t, 6H). ESI mass spectrum: m/z 307.0 [M+H]+. 3

Fluorenymethoxycarbonyl(Fmoc)-Tyr(tBu)-dimethyl hydroxyl amide (22). To a dried flask flushed with N2 was added Fmoc-Tyr(tBu)-OH (0.46 g, 1 mmol), hydroxybenzotriazole (0.17 g, 1.1 mmol), dicyclohexylcarbodiimide (0.23 g, 1.1 mmol) and 4 ml DMF. After stirring 30 min at room temperature, a solid formed and was removed by filtration. N,O-dimethyl hydroxyl amine (0.12 g, 1.2 mmol) was added to the filtered reaction mixture along with N,N-Diisopropylethylamine (0.15 g, 1.2 mmol). The reaction was stirred for an additional 12 h and concentrated by rotary evaporation. The crude oil was dissolved in ethyl acetate and washed with saturated sodium bicarbonate, 0.1M hydrochloric acid and brine. The organic layer was dried over magnesium sulfate and concentrated to dryness. Purification of the crude product by flash chromatography (hexane/ethyl acetate, 2:1 v/v) provided the pure product 22 as a white solid ( 0.36 g; 71% yield). 1 H NMR (300 MHz, CDCl3) d 7.20-7.90 (m, 8H), 6.90-7.10 (m, 4H), 6.09 (d, 1H) 5.09(d, 1H), 4.00-4.40 (m, 3H), 3.61 (s, 3H), 3.15 (s, 3H), 2.90 (b, 2H), 1.34 (s, 12H), ESI mass spectrum: m/z 503.2 [M+H]+.

Fmoc-Tyr(tBu)-H (23). To a stirred solution of 22 (320 mg, 0.64 mmol) in THF at 0oC was cautiously added LiAlH4 (26.7 mg, 0.7 mmol). The reaction was stirred for 30 min and then quenched with 5 ml of 5% potassium hydrogen sulfate. 0.1 M hydrochloride acid (20 ml) was added. The mixture was extracted three times with ethyl acetate. The combined organic layers were washed with brine and dry (MgSO4). After concentration the pure product was obtained by flash chromatograph (hexane/ethyl acetate, 2:1 v/v) to give 23 as a white solid (183 mg; 65% yield). 1 H NMR (300 MHz, CDCl3) d 9.60 (s, 1H), 6.90-7.90 (m, 12H), 5.48 (d, 1H), 4.30-4.60 (m, 3H), 4.21 (t, 1H), 3.10 (d, 2H), 1.34 (s, 12H). ESI mass spectrum: m/z 444.0 [M+H]+.

Fmoc-Tyr(tBu)-vinyl sulfone (24). To a chilled solution of 21 (114 mg, 0.37 mmol) in THF (6 ml) was added sodium hydride (16 mg, 0.40 mmol) under N2. 30 min later, 23 (158.3 mg, 0.36 mmol) in 5ml THF was added. The reaction was stirred for 15min and then quenched with water. The mixture was extracted three times with ethyl acetate. The combined organic layers were washed with saturated NaHCO3, 1N HCl, and brine, followed by drying (MgSO4), filtration and concentration in vacuo. Further purification by flash chromatograph (hexane/ethyl acetate, 2:1 v/v) 1 afforded 24 as a white solid (95.3 mg; 43% yield). H NMR (300 MHZ, CDCl3) d 6.80-7.90 (m, 17H), 6.28 (d, 1H), 4.66 (b, 1H), 4.37 (m, 2H), 4.12 (b, 1H), 2.84(b, 2H), 2.39 (s, 3H), 1.30(s, 9H). ESI mass spectrum: m/z 613.3 [M+Na]+.

H2N-Tyr(tBu)-vinyl sulfone (25). 24 (60.0mg, 0.10mmol) was dissolved in 2ml piperidine/DMF (1:4, v/v). The reaction was stirred for 40 min, then concentrated to an oil and purified by flash column to give the pure product 25 as a yellowish oil (33.4 mg; 89% yield). 1H NMR (300 MHz, CDCl3) d 6.80-7.80 (m, 9H), 6.28 (d, 1H), 3.75 (m, 1H), 2.73(ddd, 2H), 2.39 (s, 3H), 1.30(s, 9H). ESI mass spectrum: m/z 373.9 [M+H]+.

Cy3-Gly-Leu-Leu-OCH3 (26). Boc-gly-Leu-leu-OCH3 (91.0 mg, 0.22 mmol), prepared by standard solution phase peptide chemistry, was treated with 2 ml TFA/DCM (1/1, v/v) for 1 h at room temperature. The reaction was repeatedly evaporated in vacuo to remove all of the TFA. After taken in 2ml DMF, Cy3 (126.0 mg, 0.22 mmol) was added, together with HOBt (33.7 mg, 0.22 mmol), EDC (44.0 mg, 0.22 mmol), DIEA (57.0 mg, 0.44 mmol), and the reaction was stirred overnight. The reaction was concentrated under reduced pressure to dryness, and further purified by flash chromatography (CH2Cl2/Ethanol, 8:1 v/v) to give 26 as a red solid (149 mg; 66% yield). 1 H NMR (300 MHz, CDCl3) d 8.34 (t, 1H), 7.10-7.50 (m, 10H), 4.30-4.60 (m, 2H), 3.90-4.10 (m, 4H), 3.79 (s,3H), 3.66 (s, 3H), 2.62 (m, 2H), 1.50-2.0 (m, 20H), 0.70-1.00 (m,12H). ESI mass spectrum: m/z 740.5 [M]+. 4 Cy3-Gly-Leu-Leu-OH (27). 26 (87.0 mg, 0.1 mmol) dissolved in 15ml methanol was added K2CO3 (2 g), and the reaction was stirring at room temperature for 12h. The mixture was filtered, concentrated in vacuo. 30 ml dichloromethane was added, and the organic layer was washed with 0.1 M aqueous solution of HCl, brine, dried (MgSO4), followed by concentration under reduced pressure to give 27 (37.3 mg; 44% yield). Further purification by C18 reverse HPLC afforded the 1 pure product as a red solid (18 mg). H NMR (300 MHz, CDCl3) d 8.34 (t, 1H), 7.10-7.50 (m, 8H), 6.48(t, 2H), 4.30-4.60 (m, 2H), 3.90-4.10 (m, 4H), 3.70 (s,3H), 3.64 (s, 3H), 2.43 (t, 2H), 1.0-2.0 (m, 20H), 0.70-1.00 (m,12H). ESI mass spectrum: m/z 726.5 [M]+.

Cy3-Gly-Leu-Leu-Tyr(tBu)-vinyl sulfone (28). To a dried flask flushed with N2 was added 27 (18.0mg, 0.02mmol), hydroxybenzotriazole (3.6 mg, 0.024 mmol), N-ethyl-N’-(3- dimethylaminopropyl) carbodiimide (4.9 mg, 0.026 mmol), DIEA (3.6 mg, 0.028 mmol) and DMF (1ml). The solution was stirred for 30 min before addition of 25 (8.9 mg, 0.024 mmol). After stirring for an additional 12 h, the mixture was evaporated to dryness, and purified by flash chromatography (DCM/Ethanol, 8:1 v/v) to afford 28 (18.5 mg; 75% yield). 1H NMR (300 MHz, CDCl3) d 8.34 (t, 1H), 6.50-7.80 (m, 20H), 3.70-4.40 (m, 7H), 3.64 (s, 3H), 2.5-2.7 (b, 2H), 2.38 (s, 3H), 1.80-2.10 (m,4H), 1.70 (s, 12H), 1.40-1.60 (m,6H), 1.27 (s, 9H), 0.7-1.0 (m, 12H). ESI mass spectrum: m/z 1081.7 [M]+.

Probe 2, VS-Cy3. 28 (18.0 mg, 0.015 mmol) was treated with 1ml TFA/DCM (1/1, v/v) for 1 h at room temperature. Solvents and TFA were repeatedly concentrated in vacuo to remove any residual TFA, followed by purification with C18 reverse HPLC to give 2 as a red solid (9.6 mg; 55% 1 yield). H NMR (300 MHz, CDCl3) d 8.34 (t, 1H), 6.50-7.80 (m, 20H), 3.70-4.40 (m, 7H), 3.49 (s, 3H), 2.5-2.7 (b, 2H), 2.40 (s, 3H), 1.40-2.00 (m, 22H), 0.7-1.0 (m, 12H). ESI mass spectrum: m/z 1025.7 [M]+. 5

(3) Probe 3, FP-Cy3.

O X (Et O) 3, reflux O P O 4. X = OH Yield: 60% TsC l ; Yi eld: 90% 7 5. X = OTs Na I; Yie ld : 75% NaOH, reflux 6. X = I Yield: 90%

O

HO P

O Cy3 = + 8 N N I- CO OH Ru Cl 3/ NaIO 4,RT Yield: 65%

O

H O P CO OH

O 9

DAST, RT + Yield: 57% N N I- O 11 F P CO O H + O 10 H N NH 2

O T B TU/HOB t Yield: 11%

+ O O N N - H I N P F N H O O 3

1-[(p-Toluenesulfonyl)oxy]-10-undecene (5). A solution of 4 (2.0 g, 11.8 mmol, 1.0 equiv) in pyridine (14.0 ml, 177 mmol, 15 equiv) was cooled to 0°C, followed by addition of p- toluenesulfonyl chloride (pTsCl) (4.5 g, 23.6 mmol, 2.0 equiv). The reaction mixture was kept at 0°C for 10 h and then partitioned between ethyl acetate (200 ml) and water (200 ml). The organic layer was washed with 10% aqueous HCl (2 × 200 ml) and saturated aqueous NaCl (200 ml), dried

(Na2SO4), and concentrated under reduced pressure. Chromatography (2% ethyl acetate-hexanes) 1 afforded 5 (3.4 g; 90% yield) as a colorless oil: H NMR (CDCl3, 300 MHz) 7.76 (d, J = 6.5 Hz,

2H, ArH), 7.32 (d, J = 7.3 Hz, 2H, ArH), 5.95-5.75 (m, 1H, RCH = CH2), 5.03-4.90 (m, 2H, RCH =

CH2), 3.98 (t, J = 6.5 Hz, 2H, CH2OTs), 2.42 (s, 3H, ArCH3), 2.02 (m, 2H, CH2CH = CH2), 1.65 (p, + J = 6.9 Hz, 2H, CH2CH2OTs), 1.50-1.20 (m, 12H); MS (ESI) m/z 347.2 (M+Na ). 1-Iodo-10-undecene (6). A solution of 5 (3.4 g, 10.5 mmol, 1.0 equiv) in acetone (21 ml, 0.5 M) was treated with NaI (3.2 g, 21 mmol, 2.0 equiv), and the reaction mixture was stirred at reflux for 2 h, producing a yellow-orange solution. The reaction mixture was then partitioned between ethyl acetate (200 ml) and water (200 ml). The organic layer was washed sequentially with saturated aqueous Na2S2O3 (100 ml) and saturated aqueous NaCl (100 ml), dried (Na2SO4), and concentrated under reduced pressure. Chromatography (1-2% ethyl acetate-hexanes) afforded 6 (2.2 g; 75% 1 yield) as a colorless oil. H NMR (CDCl3, 300 MHz) 5.95-5.75 (m, 1H, RCH = CH2), 5.03-4.90

(m, 2H, RCH = CH2), 3.16 (t, J = 7.0 Hz, 2H, CH2I), 2.02 (m, 2H, CH2CH = CH2), 1.80 (p,

J = 6.9 Hz, 2H, CH2CH2I), 1.50-1.20 (m, 12H). MS (FAB): m/z 282.1 (M+1) 1-[Bis(ethoxy)phosphinyl]-10-undecene (7). Triethylphosphite (12.2 ml, 71 mmol, 10 equiv) was added to 6 (2.0 g, 7.1 mmol, 1.0 equiv), and the mixture was stirred at reflux for 15 h. The excess triethylphosphite was removed by distillation and the remaining residue submitted to flash 6 chromatography (25-50% ethyl acetate-hexanes gradient elution) to afford 7 (1.20 g; 60% yield) as 1 a colorless oil. H NMR (CDCl3, 300 MHz) 5.95-5.75 (m, 1H, RCH = CH2), 5.03-4.90 (m, 2H,

RCH = CH2), 4.05 (m, 4H, CH3CH2OP), 2.02 (m, 2H, CH2CH = CH2), 1.80-1.20 (m, 20H). MALDI-FTMS (DHB) ESI: m/z 291.2 (M+1). 1-(Ethoxyhydroxyphosphinyl)-10-undecene (8). A solution of compound 7 (1 g, 3.4 mmol, 1.0 equiv) in EtOH/H2O (40 ml, 1:1/v:v) was added NaOH (2.05 g, 51 mmol, 15 equiv). The reaction was refluxed for 18 h, and 10 N HCl was added to adjust pH to 2. The reaction mixture was extracted with ethyl acetate (2 x 100 ml). The organic layer was washed with saturated NaCl

(200 ml), dried (Na2SO4), and concentrated under reduced pressure. Chromatography (12-20% 1 CH3OH-CHCl3 with 1% aqueous NH4OH) afforded 8 (0.80 g; 90% yield). H NMR (CDCl3,

300 MHz) 5.95-5.75 (m, 1H, RCH = CH2), 5.03-4.90 (m, 2H, RCH =CH2), 4.05 (m, 2H, + CH3CH2OP), 2.02 (m, 2H, CH2CH= CH2), 1.80-1.20 (m, 20H). MS-FAB: m/z 285.2 (M+ Na ). 10-(Ethoxyhydroxyphosphinyl)decanoic acid (9). Compound 8 (0.10 g, 0.38 mmol, 1.0 equiv) in a biphasic solution composed of CCl4-CH3CN-H2O (1.0 ml-1.0 ml-1.5 ml; total volume of 3.5 ml, 0.11 M) was treated sequentially with sodium periodate (0.31 g, 1.56 mmol, 4.1 equiv) and ruthenium trichloride hydrate (0.002 g, 0.009 mmol, 0.022 equiv). The reaction mixture was stirred at 25°C for 2 h and then partitioned between CH2Cl2 (50 ml) and 1 N aqueous HCl (50 ml). The organic layer was washed with saturated aqueous NaCl (25 ml), dried (Na2SO4), and concentrated under reduced pressure. The resulting residue was resuspended in 40 ml of diethyl ether, filtered through a Celite pad, and concentrated under reduced pressure to afford 9 (0.07 g; 65% yield) as a 1 colorless semisolid. H NMR (CDCl3, 300 MHz) 4.05 (m, 2H, CH3CH2OP), 2.32 (t, J = 7.5 Hz, + 2H, CH2COOH), 1.80-1.20 (m, 16H). ESI: m/z 279.1 (M-H ). 10-(Fluorehydroxyphosphinyl)decanoic acid (10). A solution of 9 (0.07 g, 0.25 mmol, 1.0 equiv) in

CH2Cl2 (0.4 ml, 0.06 M) at 78°C was treated dropwise with (diethylamino)sulfur trifluoride (0.21 ml, 1.0 mmol, 4.0 equiv), brought to 25°C, and stirred for 5 min, followed by partition between CH2Cl2 (50 ml) and 1 N aqueous HCl (50 ml). The organic layer was washed with saturated aqueous NaCl (25 ml), dried (Na2SO4), and concentrated under reduced pressure to afford 1 10 (0.04 g; 57% yield) as a colorless semisolid. H NMR (CDCl3, 300 MHz) 4.05 (m, 2H, + CH3CH2OP), 2.32 (t, J = 7.5 Hz, 2H, CH2COOH), 1.80-1.20 (m, 16H). ESI: m/z 281.1 (M-H ). Cy3-ethylenediamine (11). A solution of Cy3 (60 mg, 0.105 mmol), prepared from published protocols,1 and 1,1’-carbonyldiimidazole (27 mg, 0.169 mmol) in DMF (2 ml) was allowed to stir for 2h. Ethylenediamine (6 uL, 0.541 mmol) was added and the reaction mixture was stirred overnight and then evaporated to dryness. The crude material was purified by column 1 chromatography to give a red solid 11 (0.052 g; 81% yield). H NMR (CDCl3, 300 MHz) 8.34- 8.27 (m, 1H), 7.35-7.29 (m, 4H), 7.22-7.16 (m, 2H), 7.11-7.07 (m, 2H), 6.82-6.85 ( m, 2H), 4.13 (b, 2H), 3.71 (s, 3H), 3.5-3.2 (m, 4H), 2.47 (b, 2H), 1.85 (m, 4H), 1.65 ( s, 6H), 1.64 (s, 6H). ESI: m/z 485.5 (M-I+). Probe 3, FP-Cy3. Compound 10 (0.1 g, 0.35 mmol), TBTU (0.14 g, 0.42 mmol), and HOBt (0.083 g, 0.54 mmol) were combined in a round-bottom flask containing 10 mL of DMF. This mixture was stirred under argon for 20 min at room temperature, and then 4-methyl morpholine (0.11 g, 1.08 mmol) and Cy3-ethylenediamine (11) (0.17 g, 0.27 mmol) were added to the mixture. After stirring for 24 h, the solvents were removed under vacuum, and the residue was purified by column 1 chromatography to give a red solid 3 (0.026 g; 11% yield). H NMR (CDCl3, 300 MHz) 8.34- 8.27 (m, 1H), 7.35-7.29 (m, 4H), 7.22-7.16 (m, 2H), 7.11-7.07 ( m, 2H), 6.82-6.85( m, 2H), 4.13 (b, 2H), 4.05( m, 2H), 3.71 (s, 3H), 3.5-3.2( m, 4H), 2.47( b, 2H), 2.32(t, 2H), 1.95 (m, 4H), 1.88-1.25 ( m, 32H). ESI: m/z 749.5 (M-I+). 7 1. SDS-PAGE experiments Details of SDS-PAGE experiments with the PT-Cy3 probe are reported elsewhere.[7b] All protein labeling experiments were confirmed independently with SDS-PAGE experiments. To ensure parallel and accurate comparision, the pH-, temperature-dependent, as well as DTT experiments, were performed under standard SDS-PAGE conditions.

(1) Protein detection with the probes. Unless otherwise indicated, reactions between proteins and probes were conducted as follows: 2 ml of protein stocks (~1-10 mg/ml) were diluted with 18 ml of Tris buffer (50 mM, pH 8). 0.2 ml of the probe was added and the reaction was incubated at room temperature in dark for 30-60 min, unless otherwise indicated. The reaction was quenched by addition of 1 vol equiv of standard 2 x SDS-PAGE loading buffer, followed by heating the samples at 90 oC x 5 min before analysis on 10% SDS-PAGE. After SDS-PAGE separation, the Cy3 labeled proteins were visualized with a Typhoon 9200 fluorescence gel scanner (Amersham, USA).

a) VS-Cy3 labeling of cysteine

Protein #: 1 2 3 4 5 6 7 8 9 10 11 12

b) FP-Cy3 labeling of serine

Protein #:1 2 3 4 5 6 7 8 9 10 11 12

(2) Temperature-dependent protein labeling. Reactions between proteins and probes were conducted as follows: 2 ml of protein stocks (~1-10 mg/ml) were diluted with 18 ml of Tris buffer (50 mM, pH 8). The mixture was then incubated at 90 0C x 3 min. A control reaction was run without heating. After cooling down to RT, 0.2 ml of the probe was added and the reaction was incubated at room temperature in 8 dark for 30-60 min, unless otherwise indicated. The reaction was quenched by addition of 1 vol equiv of standard 2 x SDS-PAGE loading buffer, followed by heating the samples at 90 oC x 5 min before analysis on 10% SDS-PAGE. Noted papain (protein 5) was efficiently labeled by VS-Cy3 even after heating.

a) VS-Cy3 labeling Protein #: 4 4 5 5 Heat + - + -

b) FP-Cy3 labeling

Protein #: 6 6 7 7 8 8 9 9 10 10 Heat + - + - + - + - + -

(3) pH-dependent protein labeling. Reactions between proteins and probes were conducted as follows: 2 ml of protein stocks (~1-10 mg/ml) were diluted with a) 18 ml of Tris buffer (50 mM, pH 8); b) 18 ml of Tris buffer (50 mM, pH 2); or c) 18 ml of Tris buffer (50 mM, pH 11). 0.2 ml of the probe was added and the reaction was incubated at room temperature in dark for 30-60 min, unless otherwise indicated. The reaction was quenched by addition of 1 vol equiv of standard 2 x SDS/PAGE loading buffer, followed by heating the samples at 90 oC x 5 min before analysis on 10% SDS-PAGE. Noted most proteins were still labeled to some extent at high pH (e.g. pH 11). 9 a) VS-Cy3 labeling at different pH

Protein #: 4 4 4 5 5 5 pH: 8 2 11 8 2 11

b) FP-Cy3 labeling

Protein #: 6 6 6 7 7 7 8 8 8 9 9 9 10 10 10 pH: 8 2 11 8 2 11 8 2 11 8 2 11 8 2 11

(4) Cysteine activation. Cysteine proteases were labeled with VS-Cy3: a) under normal conditions; or b) after preactivation with DTT. a) Normal. 2 ml of protein stocks (~1-10 mg/ml) were diluted with 18 ml of Tris buffer (50 mM, pH 8). 0.2 ml of the probe was added and the reaction was incubated at room temperature in dark for 30-60 min, unless otherwise indicated. The reaction was quenched by addition of 1 vol equiv of standard 2 x SDS-PAGE loading buffer, followied by heating the samples at 90 oC x 5 min before analysis on 10% SDS- PAGE.

b) Preactivation. 2 ml of protein stocks (~1-10 mg/ml) were diluted with 18 ml of a buffer containing 50 mM Tris (pH 8), 2 mM EDTA and 2 mM fresh DTT. After incubation at room temperature for 1 h to activate the cysteine proteases, 0.2 ml of the probe was added and the reaction was incubated at room temperature in dark for 30-60 min. The reaction was quenched by addition of 1 vol equiv of standard 2 x SDS-PAGE loading buffer, followed by heating the samples at 90 oC x 5 min before analysis on 10% SDS-PAGE. 10

Protein #: 4 4 5 5 Activation: - + - +

*Noted from above experiment, proteins without preactivation were still efficiently labeled by VS-Cy3, although it seems preactivation with DTT did increase the level of labeled proteins.

(5) Inhibition studies of FP-Cy3 labeling of Ser hydrolases with Trypsin Inhibitor (TI). 2 ml of protein stocks (~1-10 mg/ml) were added 4 ml of trypsin inhibitor (~1-10 mg/ml; ~2 eq), 14 ml of Tris buffer (50 mM, pH 8). 0.2 ml of the probe was added and the reaction was incubated at room temperature in dark for 30-60 min, unless otherwise indicated. The reaction was quenched by addition of 1 vol equiv of standard 2 x SDS/PAGE loading buffer, followed by heating the samples at 90 oC x 5 min before analysis on 10% SDS-PAGE. Trypsin inhibitor alone labeled with FP-Cy3 under similar conditions did not give rise to any fluorescently labeled protein.

Protein #: 6 6 7 7 8 8 9 9 10 10 Trypsin Inhibitor: - - - - - + + + + +

*Noted that, for the FP-Cy3 labeling reaction, only trypsin-like serine proteases (e.g. a-, b-, g-chymotrypsin, or protein 6, 7, 8, respectively) were inhibited by TI. Other serine proteases such as proteinase K & subtilisin (protein 9, 10) were not inhibited. 11 2. Array-based experiments. All array-based experiments for detection of proteins using different probes were first validated in solution with SDS-PAGE, unless otherwise indicated. With the exposure time set at one second, the slide images obtained were immediately standardized for visual comparison. All slides were calibrated to the same background intensity and were thus normalized. This adjustment did not change the absolute intensity values (reported in the Excel plots) obtained.

(1) Spotting proteins on the array Epoxy-derivatized slides were prepared from plain glass slides (Sigma, USA) as previously described.[9] NHS slides were also used to spot the proteins but consistently gave inferior results. First, glass slides were cleaned in a piranha solution and derivatized with a 1% solution of 3-glycidoxypropyltrimethoxisilane (95 % ethanol, 16 mM acetic acid) for 1 hr 0 and cured at 150 C for 2 hours. The proteins to be spotted were prepared in 0.1 M NaHCO3 buffer (pH 9) and were arrayed on the epoxy slides using an ESI SMATM arrayer (Ontario, Canada), with a spacing of 180 mm between the spots. After a 2-hour incubation the slides were either used immediately, or stored for future use at 4 oC. The slides, if stored were typically used within 48 hours of printing.

(2) Protein detection with 3 probes based on enzymatic activity Unless otherwise indicated, the probe and reactions on slides were performed as follows. Before use, the slides were quenched with phosphate buffer saline (PBS) with 0.5 M glycine on a shaker for 10 min. The slides were then blocked with PBS, 0.5 M glycine and 1% w/v bovine serum albumin (BSA) for 20 min, followed by washing with distilled water and air- dried. The labeled probe was then applied as below:

a. PT-Cy3: A 2 mM mixture of PT-Cy3 was prepared using 0.5 ml of 200 mM PT-Cy3 (100 x) stock in 49 ml of Tris buffer (50 mM, pH 8), and 0.5 ml BSA (1% w/v) added as a blocking agent to prevent non-specific binding. 50 ml of this freshly prepared mixture was applied to each slide, using the coverslip method, and incubated for 30 min in the dark. The excess probe was washed off after incubation with distilled water, and the slides were subsequently washed with PBS with tween (0.2% v/v) for 15 minutes on a shaker. The slides were then washed with distilled water, air-dried and scanned using an ArrayWorxTM microarray scanner (Applied Precision, USA), under 548/595 nm. b. VS-Cy3: A 2 mM mixture of VS-Cy3 was prepared using 0.5 ml of 200 mM VS-Cy3 (100 x) stock in 49 ml of Tris buffer (50 mM, pH 8), and 0.5 ml BSA (1% w/v) added as a blocking agent to prevent non-specific binding. 50 ml of this freshly prepared mixture was applied to each slide and incubated for 30 min in the dark. The excess probe was washed off after incubation with distilled water, and the slides were subsequently washed with PBS with tween (0.2% v/v) for 15 minutes on a shaker. The slides were then washed with distilled water, air-dried and scanned using an ArrayWorxTM microarray scanner (Applied Precision, USA), under 548/595 nm. c. FP-Cy3: A 2 mM mixture of FP-Cy3 was prepared using 0.5 ml of 200 mM FP-Cy3 stock in 49 ml of Tris buffer (50 mM, pH 8), and 0.5 ml BSA (1% w/v) added as a blocking agent to prevent non-specific binding. 50 ml of this freshly prepared mixture was applied to each slide and incubated for 30 min in the dark. The excess probe was washed off after incubation with distilled water, and the slides were subsequently washed with PBS with tween (0.2% v/v) for 15 minutes on a shaker. The slides were then washed with distilled water, air-dried and scanned using an ArrayWorxTM microarray scanner (Applied Precision, USA), under 548/595 nm. 12

(3) Inhibition experiments with Trypsin Inhibitor (TI) The protocol used was identical to the one explained in step 3 (1) and 3 (2) except for the following considerations. Four separate array slides were incubated with varying concentrations of Trypsin inhibitor (TI) (of original concentration ~ 5 mg/ml), in equivolumetric mixtures comprising uniform amount of FP-Cy3.

The probe mixtures were thus comprised of the followings:

Final Inhibitor concentration:

0.5 mg/ml - 44 ml of Tris buffer (50 mM, pH 8), 5 ml TI, 0.5 ml BSA, 0.5 ml FP-Cy3

0.1 mg/ml - 48 ml of Tris buffer (50 mM, pH 8), 1 ml TI, 0.5 ml BSA, 0.5 ml FP-Cy3

0.05 mg/ml - 48.5 ml of Tris buffer (50 mM, pH 8), 0.5 ml TI, 0.5 ml BSA, 0.5 ml FP-Cy3

0.02 mg/ml - 48.8 ml of Tris buffer (50 mM, pH 8), 0.2 ml TI, 0.5 ml BSA, 0.5 ml FP- Cy3

After thirty minutes of incubation, the slides were washed and scanned, as per protocol 3 (2) above.

(4) Gradient experiments The protocol used was identical to the one explained in step 3 (1) and 3 (2) above except for the following considerations. The slides were printed in a grid of varying concentration of a positive hit protein (in triplicate for each protein concentration). The range of concentrations used spanned from 1-10 mg/ml to 1x10-9 mg/ml. The protein used for each -based inhibitor is indicated in parenthesis next to the respective fluorophore. The lowest detection limit is determined by the lowest protein concentration that produces visible spots under the conditions employed. Of course this need not be the absolute detection limit, just one that we observe within our experimental conditions. By varying the scanning time, or increasing the incubation time with the , one can be certain that this detection limit can be further enhanced. Additionally, this experiment was performed on just one single protease for each of the 3 probes. We can almost be certain that other proteins used will produce different sets of data, and may have consequently differing sensitivity limits. Nonetheless, within our set conditions, we achieved detection of up to 10-6 mg/ml concentrations of spotted protein (example quoted à FP-Cy3), which, if assuming 0.1 nl/spot on the microarray for a protein with MW ~10,000-100,000, gives a detection limit of ~ 10-21 mol per spot. a. FP-Cy3 (with Proteinase K) 13

Conc/ 1 0.5 0.1 5x10-2 1x10-2 5x10-3 1x10-3 5x10-4 1x10-4 1x10-5 1x10-6 mg/ml

Lowest detection limit – 1x10-6 mg/ml

Intensity Plot FP-Cy3

3000 2500 2000 1500 1000 500 0 Relative Spot Intensity 0 2 4 6 8 Log of Sample Concentration

b. PT-Cy3 (with Type VIII Alkaline Phosphatase)

Conc/ 1 0.5 0.1 5x10-2 1x10-2 5x10-3 1x10-3 5x10-4 1x10-5 mg/ml

Lowest detection limit – 1x10-5 mg/ml

Intensity Plot - PT-Cy3

20000 15000 10000 5000 0 0 2 4 6 8 Relative Spot Intensity -5000 Log of Sample Concentration 14

c. VS-Cy3 (with Papain)

Conc/ 0.5 0.1 5x10-2 1x10-2 5x10-3 1x10-3 1x10-4 mg/ml

Lowest detection limit – 1x10-4 mg/ml

Intensity Plot - VS-Cy3 2000

1500

1000

500

Relative Spot Intensity 0 0 2 4 6 8 Log of Sample Concentration 15

(5) Detecting labeled proteins after urea denaturation Proteins I-S Alkaline phosphatase and a-chymotrypsin were chosen as representative targets for PT-Cy3 and FP-Cy3 probes respectively. The proteins were spotted in triplicate and were labeled as described in sections 3.(1) and 3.(2). The slides were scanned and therafter incubated separately for 2 hours in solutions of a) distilled water; b) 6M urea; and c) 1x PBST (PBS containing 1% Tween) on a shaker. Each slide was then washed according to the aforementioned protocol and scanned at the original intensity.

a. PT-Cy3, Results with I-S Alkaline phosphatase

Images of the same grid before (left) and after (right) a 2-hour wash with the respective solutions

Washed with: a) Water b) 6 M Urea c) PBST

Graph of Normalised Intensities

1 0.9 0.8 0.7 0.6 0.5 After 2hr Wash 0.4 Before 2hr Wash 0.3 0.2 0.1 0 Water Urea PBST

Before 2hrAfter Wash 2hr Wash 16

b. FP-Cy3, Results with a -chymotrypsin

Images of the same grid before (left) and after (right) a 2-hour wash with the respective solutions

Washed with: a) Water b) 6 M Urea c) PBST

Graph of Normalised Intensities

1 0.9 0.8 0.7 0.6 0.5 0.4 After 2hr Wash 0.3 Before 2hr Wash 0.2 0.1 0 Water Urea PBST

Before 2hrAfter Wash 2hr Wash

*There was an observed drop in overall intensity detected after the 2 hours wash for all spots. What is significant, however, is that even under strongly denaturing conditions (e.g. 6 M urea for 2 hours), where the protein is expected to be completely unfolded, and devoid of any noncovalent association, the probe is still able to specifically maintain its association to its target protein, indicating the covalent nature of the probe upon binding to the proteins. *The relative drop in intensities obtained after the urea wash is within 15% of the levels obtained with either water or PBST, presumably due to the deteriorating effect of urea towards the integrity of the fluorophore on the probe. The probe is thus efficacious in detecting the proteins even after the protein-probe complex has been exposed to denaturing conditions. This confirms that the probe is covalently binding to the proteins, presumably at their active sites.

(6) Assessing interaction of probes with enzymes with inactivated active sites The serine residue of target proteins to FP-Cy3 were covalently modified using a known small-molecule inhibitor of serine protease/, phenylmethylsulfonyl fluoride (PMSF) (Roche, USA). The modification is site-specific and not expected to perturb the active site of the protein to a great extent. Therefore the PMSF-treated, inactivated enzyme 17 is expected to still bind to its natural substrates efficiently, but exerting no catalytic activity. Each slide was first incubated with 50 ml of 1 mM freshly prepared PMSF (in 50 mM Tris, pH 8) for 30 minutes, rinsed extensively with distilled water and labeled as described in 3.(2). It was found that the probe was unable to label the serine hydrolases after reaction with PMSF, demonstrating the active-site Ser residue, and the catalytic activity of the enzymes are imperative for probe labeling. This, together with other experiments, unambiguously confirmed that our probes are activity-based probes, capable of detecting proteins by their enzymatic activities.

Covalent modification of active-site Ser residue in serine hydrolase by PMSF

OH OR no labeling F

O S O

O S O R = PMSF