Supporting Information

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1 Electronic Supplementary Material (ESI) for RSC Advances. This journal is The Royal Society of Chemistry 214 Supporting Information Rapid and sensitive detection of acrylic acid using a novel fluorescence assay Zhi Yi Lee, a,b Sarada S. Raghavan, b Farid J. Ghadessy b and Yin ah Teo a, c a Molecular Engineering Laboratory, Biomedical Sciences Institutes, A*STAR, 61 Biopolis Drive, #3-12 Proteos, Singapore b p53 Laboratory, A*STAR, 8A Biomedical Grove, #6-6 Immunos, Singapore c Division of Chemistry and Biological Chemistry, SPMS, anyang Technological University, Singapore Contents: 1. Synthetic details of diaryltetrazoles and characterization of pyrazoline product 2. Fluorescence characterization of reaction between diaryltetrazoles and acrylic acid 3. Limit of detection of compound 4 4. Reaction monitoring of photoactivated cycloaddition using HPLC and fluorescence 5. GC results of extracted LB samples containing with acrylic acid standards 6. Fluorescence assay of acrylic acid standards in LB media and minimal media 7. Fluorescence of 3-butynoic acid using compound 4 8. Experimental details for fluorescence characterization of reaction between compound 4 and acrylic acid in various ph conditions 1. Synthetic details of diaryltetrazoles and characterization of pyrazoline product All chemicals and solvents were purchased from commerical sources and used directly without purification. Flash chromatography was performed using SiliCycle P6 silica gel (4-63 µm, 6Å). 1 H MR spectra were recorded with Bruker Avance III 4, and chemical shifts were reported in ppm using either TMS or deuterated solvents as internal standards (TMS,.; CDCl 3, 7.26; C 6 D 6, 7.15; DMS-d 6, 2.5). Multiplicity was reported as the follows: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, b = broad. 13 C MR spectra were recorded at 75.4 MHz, and chemical shifts were reported in ppm using the deuterated solvents as internal standards (CDCl 3, 77.; DMS-d 6, 39.5; C 6 D 6, 128.). LC-MS analysis was performed using Waters 31 Single Quadrupole LCMS System. Kinetic studies were performed using Phenomenex Kinetex 2.6u XB-C18 column (5x4.6 mm). Flow rate was 1 ml/min and UV detection was set at 254 and 37 nm as indicated. Gas chromatography was carried out on a Shimadzu GCMS QP 21 using a 3. m x.25 mm, inner diameter of.25 µm HP- IWAX column which was programmed from 4 to 25 C at 1 C /min. Fluorescence detection were performed with Tecan Infinite M1. Compound 1 H Methyl 4-formylbenzoate H 2 S H EtH, rt S H Methyl 4-formylbenzoate (.824 g, 5 mmol) was dissolved in ethanol (5 ml), and benzenesulfonohydrazine (.863 g, 5 mmol) was added. The mixture was stirred at room temperature for 1h, then quenched with water (1 ml) and stirred for 15 min at room temperature. The precipitate was filtered and washed with cold ethanol. The precipitate was then dissolved in pyridine (3 ml) for the next reaction. Aniline (.465 g,.46 ml, 5 mmol) was separately dissolved in water: ethanol (1:1, 8 ml) and concentrated HCl (1.3 ml) was added. a 2 (.346 g, 5 mmol) was also separately dissolved in water (2 ml). The aniline solution was cooled in an ice bath for 5 min before addition of a 2 solution to the aniline solution drop wise in an ice bath. The reaction mixture was added dropwise to the cooled product pyridine, rt Cl- 1 S1

2 from the first reaction in an ice bath. The reaction mixture was stirred for 1 h at room temperature. Extraction was then carried out with ethyl acetate (1 ml X 3). 3 M HCl (25 ml) was added to the combined organic layers and stirred vigorously for 1 min. The organic layer was concentrated and the product was precipitated with hexanes. The product was further washed with cold hexanes to obtain a pale orange solid (.538 g, 38%). 1 H MR (4 MHz, Chloroform-d) δ 8.35 (dd, J = 8.2,.6 Hz, 2H), (m, 4H), (m, 2H), (m, 1H), 3.97 (s, 3H). 13 C MR (11 MHz, CDCl 3 ) δ , , , , , 13.24, , , 127.1, , HRMS (ESI) calcd for C 15 H [M + H+], found Compound 2 H Methyl 4-formylbenzoate H 2 S H EtH, rt S H Methyl 4-formylbenzoate (.82 g, 5 mmol) was dissolved in ethanol (5 ml), followed by addition of benzenesulfonohydrazine (.862 g, 5 mmol). The mixture was stirred at room temperature for 1h, then quenched with water (1 ml) and stirred for 15 min at room temperature. Precipitate was filtered, washed with cold ethanol and dissolved in pyridine (3 ml) to form solution A. 4-fluoroaniline was then dissolve (.555 g,.48 ml, 5 mmol) in water: ethanol (1:1, 8 ml) and concentrated HCl (1.3 ml). a 2 was dissolved (.345 g, 5 mmol) in water (2 ml). Both mixtures were cooled in ice bath for 5 min before addition of a 2 solution to 4-fluoroaniline solution drop wise in ice bath to form solution B. Solution B was added to solution A drop wise in ice bath. Mixture was then stirred for 1h at room temperature. Mixture was extracted with Ethyl Acetate (1 ml X 3). 3M HCl (25 ml) was added to combine organic layer and stirred vigorously for 1 min. rganic layer was concentrated and product was precipitated with hexanes. Product was washed with cold hexanes to obtain pale pink solid (.777 g, 52%) 1 H MR (4 MHz, Chloroform-d) δ (m, 2H), (m, 4H), 7.29 (dd, J = 9.1, 7.9 Hz, 2H), 3.97 (s, 3H). 13 C MR (11 MHz, CDCl 3 ) δ , , 164.4, , , 131.7, 13.24, , , , 116.9, , HRMS (ESI) calcd for C 15 H 11 F [M + H+], found F 2 F Compound 3 H Methyl 4-formylbenzoate H 2 S H EtH, rt S H Methyl 4-formylbenzoate (.82 g, 5 mmol) was dissolved in ethanol (5 ml), followed by addition of benzenesulfonohydrazine (.859 g, 5 mmol). The mixture was stirred at room temperature for 1h, then quenched with water (1 ml) and stirred for 15 min at room temperature. Precipitate was filtered, washed with cold ethanol and dissolved in pyridine (3 ml) to form solution A. 2, 4-fluoroaniline was then dissolve (.645 g,.5 ml, 5 mmol) in water: ethanol (1:1, 8 ml) and concentrated HCl (1.3 ml). a 2 was dissolved (.345 g, 5 mmol) in water (2 ml). Both mixtures were cooled in ice bath for 5 min before addition of a 2 solution to 2, 4-fluoroaniline solution drop wise in ice bath to form solution B. Solution B was added to solution A drop wise in ice bath. Mixture was then stirred for 1h at room temperature. Mixture was extracted with Ethyl Acetate (1 ml X 3). 3M HCl (25 ml) was added to combine organic layer and stirred vigorously for 1 min. layer was concentrated and product was precipitated with hexanes. Product was washed with cold hexanes to obtain red solid (.173 g, 11%). 1 H MR (4 MHz, Chloroform-d) δ (m, 2H), (m, 2H), 7.91 (td, J = 8.6, 5.6 Hz, 1H), (m, 2H), 3.97 (s, 3H). 13 C MR (11 MHz, CDCl 3 ) δ 26.88, 129.9, , , , 77.32, 77.2, 77., 76.68, HRMS (ESI) calcd for C 15 H 1 F [M + H+], found F F Cl- Cl- 3 F F Compound 4 S2

3 H H 4-formylbenzoic acid H 2 S H EtH, rt H S H 4 4-formylbenzoic acid (1. g, 6 mmol) was dissolved in ethanol (1 ml), followed by addition of benzenesulfonohydrazine (1.16 g, 6 mmol). The mixture was stirred at room temperature for 1h, then quenched with water (1 ml) and stirred for 15 min at room temperature. Precipitate was filtered, washed with cold ethanol and dissolved in pyridine (3 ml) to form solution A. Aniline was then dissolved (.587 g,.6 ml, 5 mmol) in water: ethanol (1:1, 8 ml) and concentrated HCl (1.3 ml). a 2 was dissolved (.455 g) in water (2 ml). Both mixtures were cooled in ice bath for 5 min before addition of a 2 solution to aniline solution drop wise in ice bath to form solution B. Solution B was added to solution A drop wise in ice bath. Mixture was then stirred for 1h at room temperature. Mixture was extracted with Ethyl Acetate (1 ml X 3). 3M HCl (25 ml) was added to combine organic layer and stirred vigorously for 1 min. Solvent were removed and then dissolved in dichloromethane. Product was precipitated with hexanes. Product was washed with cold hexanes to obtain red solid (.82 g, 45%). 1 H MR (4 MHz, Methanol-d4) δ (m, 2H), (m, 4H), (m, 2H), 7.63 (d, J = 7.3 Hz, 1H). 13 C MR (11 MHz, MeD) δ 147.1, 141.5, , , 129.6, , , , , , HRMS (ESI) calcd for C 14 H [M + H+], found H Compound 5 H p-tolualdehyde H 2 S H EtH, rt S H p-tolualdehyde (1. g, 8 mmol) was dissolved in ethanol (6 ml), followed by addition of benzenesulfonohydrazine (1.433 g, 8 mmol). The mixture was stirred at room temperature for 1h, then quenched with water (1 ml) and stirred for 15 min at room temperature. Precipitate was filtered, washed with cold ethanol and dissolved in pyridine (3 ml) to form solution A. 1, 4-phenylenediamine was then dissolved(.95 g, 8 mmol) in water: ethanol (1:1, 1 ml) and concentrated HCl (1.3 ml). a 2 was dissolved (.583 g, 8 mmol) in water (2 ml). Both mixtures were cooled in ice bath for 5 min before addition of a 2 solution to 1, 4-phenylenediamine solution drop wise in ice bath to form solution B. Solution B was added to solution A drop wise in ice bath. Mixture was then stirred for 1h at room temperature. Mixture was extracted with Ethyl Acetate (1 ml X 3). 3M HCl (25 ml) was added to combine organic layer and stirred vigorously for 1 min. rganic layer was concentrated to obtain a red solid. Crude product was purified with column chromatography (Hex:EA 1:1) to collect as yellow solid (1.149 g, 54.9%). 1 H MR (4 MHz, Chloroform-d) δ (m, 1H), (m, 2H), 7.76 (s, 1H), 7.54 (d, J = 7.2 Hz, 1H), (m, 2H), (m, 2H), 7.12 (d, J = 7.9 Hz, 2H), 2.32 (s, 3H). 13 C MR (11 MHz, CDCl 3 ) δ , 14.92, , , 13.26, , , , , 29.62, HRMS (ESI) calcd for C 14 H [M + H+], found H 2 5 H 2 Compound 6 H p-tolualdehyde H 2 S H EtH, rt S H p-tolualdehyde (1. g, 8 mmol) was dissolved in ethanol (6 ml), followed by addition of benzenesulfonohydrazine (1.433 g, 8 mmol The mixture was stirred at room temperature for 1h, then quenched with water (1 ml) and stirred for 15 min at room temperature. Precipitate was filtered, washed Cl- Cl- Cl- 6 S3

4 with cold ethanol and dissolved in pyridine (3 ml) to form solution A. 4-methoxyaniline was then dissolved (1.67 g, 8 mmol) in water: ethanol (1:1, 1 ml) and concentrated HCl (1.3 ml). a 2 was dissolved (.583 g, 8 mmol) in water (2 ml). Both mixtures were cooled in ice bath for 5 min before addition of a 2 solution to 4-methoxyaniline solution drop wise in ice bath to form solution B. Solution B was added to solution A drop wise in ice bath. Mixture was then stirred for 1h at room temperature. Mixture was extracted with Ethyl Acetate (1 ml X 3). 3M HCl (25 ml) was added to combine organic layer and stirred vigorously for 1 min. rganic layer was concentrated to obtain a red solid. Crude product was purified with column chromatography (Hex:EA 5:1) to obtain a orange red solid (.8921 g, 41.6%). 1 H MR (4 MHz, Chloroform-d) δ 8.11 (d, J = 8.2 Hz, 2H), (m, 2H), (m, 2H), (m, 2H), 3.86 (s, 3H), 2.41 (s, 3H). 13 C MR (11 MHz, CDCl 3 ) δ 165.5, 16.46, 14.62, , 126.9, , , 55.73, HRMS (ESI) calcd for C 15 H [M + H+], found Compound 7 H Benzaldehyde H 2 S H EtH, rt S H Benzaldehyde (1. g, 8 mmol) was dissolved in ethanol (6 ml), followed by addition of benzenesulfonohydrazine (1.623 g, 8 mmol). The mixture was stirred at room temperature for 1h, then quenched with water (1 ml) and stirred for 15 min at room temperature. Precipitate was filtered, washed with cold ethanol and dissolved in pyridine (3 ml) to form solution A. 4-methoxyaniline was then dissolved (1.67 g, 8 mmol) in water: ethanol (1:1, 1 ml) and concentrated HCl (1.3 ml). a 2 was dissolved (.583 g, 8 mmol) in water (2 ml). Both mixtures were cooled in ice bath for 5 min before addition of a 2 solution to 4-methoxyaniline solution drop wise in ice bath to form solution B. Solution B was added to solution A drop wise in ice bath. Mixture was then stirred for 1h at room temperature. Mixture was extracted with Ethyl Acetate (1 ml X 3). 3M HCl (25 ml) was added to combine organic layer and stirred vigorously for 1 min. rganic layer was concentrated to obtain a red solid. Crude product was purified with column chromatography (DCM: MeH 9:1) to obtain a red solid (1.42 g, 59.7%). 1 H MR (4 MHz, Chloroform-d) δ 8.23 (dd, J = 7.9, 1.7 Hz, 2H), 8.9 (d, J = 9.1 Hz, 2H), (m, 3H), 7.4 (d, J = 9.1 Hz, 2H), 3.87 (s, 3H). 13 C MR (11 MHz, CDCl 3 ) δ , 159.2, 129.1, 127.6, , 12.9, , HRMS (ESI) calcd for C 14 H [M + H+], found Cl- 7 Compound 1P A 4 ml ethyl acetate solution of 1 (2 mg,.713 mmol) and acrylic acid (24.5 µl, 5 equiv molar) were irradiated with 32nm UV lamp for 3h. The excess solvent and reagent were removed by reduced pressure to produce crude product which was subsequently purified by silica gel column chromatography. (Hex: EA, 1:1) Product 1P was collected as yellow solid. 1 H MR (4 MHz, CDCl 3 ) δ 8.94 (s, 1H), 8.5 (d, J = 8.5 Hz, 2H), 7.88 (d, J = 8.3 Hz, 2H), 7.49 (d, J = 7.5 Hz, 2H), (m, 3H), 6.52 (dd, J = 16.8, 1.8 Hz, 1H), 6.3 (t, J = 13.9 Hz, 1H), 5.72 (dd, J = 1.3, 1.8 Hz, 1H), 3.94 (s, 3H). 13 C MR (11 MHz, CDCl 3 ) δ 166.9, , 141.9, 14.99, , , , , , , , 12.3, , 6.19, 52.38, HRMS (ESI) calcd for C 18 H [M + H+], found S4

5 Compound 2P A 4 ml ethyl acetate solution of 2 (2 mg,.671 mmol) and acrylic acid (23 µl, 5 equiv molar) were irradiated with 32nm UV lamp for 3h. The excess solvent and reagent were removed by reduced pressure to produce crude product which was subsequently purified by silica gel column chromatography. (Hex: EA, 1:1) Product 2P was collected as yellow solid. 1 H MR (4 MHz, CDCl 3 ) δ 8.56 (s, 1H), 8.11 (d, J = 8.4 Hz, 2H), 7.89 (d, J = 8.5 Hz, 1H), (m, 1H), 7.12 (dd, J = 9., 8.1 Hz, 2H), 6.52 (dd, J = 16.8, 1.7 Hz, 1H), 6.26 (t, 1H), 5.74 (d, J = 1.8 Hz, 1H), 3.95 (s, 3H). 13 C MR (11 MHz, CDCl 3 ) δ , , 16.2, 15.68, 145.3, , , , , , 13.29, 129.4, , 127.3, , , , , , 59.7, 51.39, HRMS (ESI) calcd for C 18 H 15 F [M + H+], found Compound 3P A 4 ml ethyl acetate solution of 3 (2 mg,.633 mmol) and acrylic acid (21.7 µl, 5 equiv molar) were irradiated with 32nm UV lamp for 3h. The excess solvent and reagent were removed by reduced pressure to produce crude product which was subsequently purified by silica gel column chromatography. (Hex: EA, 1:1) Product 3P was collected as yellow solid. 1 H MR (4 MHz, CDCl 3 ) δ 8.3 (d, J = 7.8 Hz, 2H), 7.86 (d, J = 8.2 Hz, 2H), 7.79 (d, J = 8.6 Hz, 1H), 6.95 (d, J = 8.1 Hz, 2H), 6.54 (dd, J = 16.8, 1.6 Hz, 1H), 6.12 (d, J = 16.9 Hz, 1H), 5.77 (d, J = 1.4 Hz, 1H), 3.94 (s, 3H). 13 C MR (11 MHz, CDCl 3 ) δ , 16.41, , 133.1, , 13.11, 129.3, 127.7, , 123.6, 122.8, 11.13, 19.9, 13.5, 12.79, 12.55, 58.21, 5.26, HRMS (ESI) calcd for C 18 H 14 F [M + H+], found Compound 4P A 4 ml dichloromethane and methanol solution of 4 (2 mg,.751 mmol) and acrylic acid (25.8 µl, 5 equiv molar) were irradiated with 32nm UV lamp for 3h. The excess solvent and reagent were removed by reduced pressure to produce crude product which was subsequently purified by silica gel column chromatography. (MeH: DCM, 3:17) Product 4P was collected as yellow solid. 1 H MR (4 MHz, CDCl 3 ) δ 7.73 (d, J = 8.1 Hz, 2H), 7.68 (d, J = 8.1 Hz, 2H), (m, 2H), 7.45 (dt, J = 13.4, 7.2 Hz, 3H), 6.58 (d, J = 16.8 Hz, 1H), 6.27 (dd, J = 16.7, 1.4 Hz, 1H), 5.78 (d, J = 1.5 Hz, 1H). 13 C MR (11 MHz, CDCl 3 ) δ , , , , , 14.65, , , 131.4, , 129.7, 128.9, , , , , 65.83, 53.38, HRMS (ESI) calcd for C 17 H [M + H+], found Compound 6P S5

6 A 4 ml ethyl acetate solution of 6 (2 mg,.751 mmol) and acrylic acid (25.7 µl, 5 equiv molar) were irradiated with 32nm UV lamp for 3h. The excess solvent and reagent were removed by reduced pressure to produce crude product which was subsequently purified by silica gel column chromatography. (MeH: DCM, 1:2) Product 6P was collected as yellow solid. 1 H MR (4 MHz, CDCl 3 ) δ 7.64 (d, J = 8.1 Hz, 2H), 7.35 (d, J = 8.9 Hz, 2H), 7.1 (d, J = 7.7 Hz, 2H), 6.83 (d, J = 8.9 Hz, 2H), 6.4 (dd, J = 16.8, 1.9 Hz, 1H), 6.18 (s, 1H), (m, 1H), 3.74 (s, 3H), 2.29 (s, 3H 13 C MR (11 MHz, CDCl 3 ) δ , , , , 133.6, 133.6, 13., , 128.2, , , 59.25, 54.48, 28.65, HRMS (ESI) calcd for C 18 H [M + H+], found Compound 7P A 4 ml ethyl acetate solution of 7 (2 mg,.793 mmol) and acrylic acid (27.2 µl, 5 equiv molar) were irradiated with 32nm UV lamp for 3h. The excess solvent and reagent were removed by reduced pressure to produce crude product which was subsequently purified by silica gel column chromatography. (MeH: DCM, 1:2) Product 7P was collected as yellow solid. 1 H MR (4 MHz, CDCl 3 ) δ (m, 2H), (m, 4H), 7.3 (d, J = 6.9 Hz, 2H), 6.83 (d, J = 9. Hz, 2H), 6.42 (dd, J = 16.8, 1.9 Hz, 1H), 6.18 (s, 1H), (m, 1H), 3.74 (s, 3H). 13 C MR (11 MHz, CDCl 3 ) δ , 158.6, , , 13.91, 128.5, 127.6, , , , 113.5, 59.22, 54.49, HRMS (ESI) calcd for C 17 H [M + H+], found S6

7 2. Fluorescence characterization of reaction between diaryltetrazoles and acrylic acid A solution containing each diaryltetrazole (.22 mmol) and acrylic acid ( mmol) was irradiated with a hand-held 32-nm UV lamp for 1 minute. Experiments were performed in a black 96-well plate with a total reaction volume of 1 µl The maximum excitation wavelengths indicated in Table 1 were used for each of the scans. Bandwidth was set at 5nm. Fluorescence Intensity (a.u.) Fluorescence Intensity (a.u.) Fluorescence Intensity (a.u.) Fluorescence Emission of Compound 1 (1 μm) with 1 mm of Acrylic Acid Wavelength (nm) Wavelength (nm) PBS + DMS 1 µm 1 1 mm Acrylic Acid 1 mm Acrylic Acid + 1 µm 1 1 µm 1 without UV Fluorescence Emission of Compound 2 (1 μm) with 1 mm of Acrylic Acid 14 PBS + DMS 12 1 µm mm Acrylic Acid 8 1 mm Acrylic Acid + 1 µm µm 2 without UV 4 Fluorescence Emission of Compound 3 (1 μm) with 1 mm of Acrylic Acid 12 PBS + DMS 1 1 µm mm Acrylic Acid 6 1 mm Acrylic Acid + 1 µm 3 1 µm 3 without UV Wavelength (nm) S7

8 Fluorescence Intensity (a. u.) 4 Fluorescence Emission of Compound 4 (1 μm) with 1 mm of Acrylic Acid Wavelength, nm PBS + DMS 1 µm 4 1 mm Acrylic Acid 1 mm Acrylic Acid + 1 µm 4 1 µm 4 w/o UV Fluorescence Intensity (a.u.) Fluorescence Emission of Compound 6 (1 μm) with 1 mm of Acrylic Acid 18 PBS + DMS µm mm Acrylic Acid 1 1mM Acrylic Acid + 1µM µm 6 without UV Wavelength (nm) Fluorescence Intensity (a.u.) Fluorescence Emission of Compound 7 (1 μm) with 1 mm of Acrylic Acid 18 PBS + DMS µm mm Acrylic Acid 1 1 mm Acrylic Acid + 1 µm µm 7 without UV Wavelength (nm) Figure S1. Fluorescence emission curves of each of the diaryltetrazoles with acrylic acid. S8

9 3. Limit of detection of Compound 4 Conc. of 4 1 µm 1 µm 1 µm 1 µm Conc. of acrylic acid 1 µm 1 µm 1 µm 5 nm Fold increase in fluorescence Emission Spectra of 1 µm 4 with various concentrations of acrylic acid DMS + PBS Fluorescence Intensity (a.u.) µM 4 1 µm Acrylic Acid 5 nm Acrylic Acid 2 nm Acrylic Acid 1 nm Acrylic Acid 1 µm µm Acrylic Acid 1 µm nm Acrylic Acid 1 µm nm Acrylic Acid 1 µm nm Acrylic Acid 1 µm 4 without UV Wavelength (nm) Figure S2. Detection limit of acrylic acid was 5 nm (36 ppb) as determined by measuring the emission spectrum of the reaction between 4 and various concentrations of acrylic acid. Fluorescence Intensity (a. u.) Wavelength (nm) PBS + DMS 1 µm 4 1 µm Acrylic Acid 1 µm Acrylic Acid + 1 µm 4 1 µm 4 without UV Fig. S3. Emission spectra of 4 (1 µm) with acrylic acid (1 µm) in PBS buffer. S9

10 Figure S4. Fold increase in fluorescence upon the addition of acrylic acid at the indicated concentrations to 1 µm of the compound 4 in minimal media (n = 2 ± SD). A 1-fold excess of lactic acid (1 mm) shows negligible fluorescence signal compared to acrylic acid indicating the specificity of 4. S1

11 4. Reaction monitoring of photoactivated cycloaddition using HPLC and fluorescence Experimental details for reaction monitoring with HPLC 1 mm ZY1_69 was dissolved in dichloromethane and methanol (1:1). A separate solution of 1 mm of acrylic acid was dissolved in methanol. 1 µl of 1 mm ZY1_69 and 1 µl of 1 mm AA were then dissolved in 8 µl of methanol. After vigorous stirring, the mixtures were irradiated with 32nm UV lamp for sec, 15 sec, 3 sec, 45 sec, 6 sec, 9 sec and 12 sec, respectively. An aliquot (1 µl) of reaction solution from each sample was withdrawn and immediately injected in the HPLC column. 4 (A) and 4P (B) in each sample were monitored by UV absorbance at 254 nm and 37 nm. A linear gradient of 5-9% MeH was applied after 3 min for 1 min, then kept constant for 1 min before a 7 min linear declining gradient of 9-5% MeH. Compound 4 and compound 4P were eluted at about 13.4 min and 12.4 min respectively. Experimental details for reaction monitoring with fluorescence All samples were prepared with the same method: 1 µl of 1 mm compound 4 and 1 µl of 1 mm acrylic acid were dissolved in 98 µl of methanol. Distances between all sample vials and UV lamp during photoactivation were equal. Fluorescence turn on of reaction mixture after 15 seconds. From left to right: o UV activation, 15s, 3s, 45s, 6s, 9 and 12s of UV activation. S11

12 5. GC results of extracted LB samples containing with acrylic acid standards Experimental details for GCMS detection of acrylic acid Acrylic acid (6.8 µl, 99 µmol) was dissolved in LB medium (993.2 µl) and stirred vigorously for 1 seconds before being diluted to their respective concentration with LB. 1 µl of samples of each concentration were transfer to a 2 ml Eppendorf tube and acidified with 3-5 µl of 5M HCl. Each sample was stirred vigorously for about 1 sec and left to stand for 3-5 min. ph of each sample were tested to ensure the ph 2. Ether (1 µl 2) was then added to the acidified samples for extraction. Combined ether layers were concentrated to about 6 µl for GCMS. Extracted samples were not allowed to evaporate completely as it will affect the GCMS result. Acrylic acid was detected at a retention time of around 17.4 min. Concentration of acrylic acid Peak Intensity 1 mm 22,, 1 mm 22,, 1 mm 5,7, 75 µm 2,6, 5 µm 2,6, 25 µm 1,35, 1 µm 65, 1 µm 35, µm 7, a. Extracted 1 mm acrylic acid in LB Event#1:Scan Ret.Time : [ > 17.17] Scan# : [465 -> 4656] % S12

13 b. Extracted 1 mm acrylic acid in LB c. Extracted 1 mm acrylic acid in LB S13

14 d. Extracted 75 µm acrylic acid in LB e. Extracted 5 µm acrylic acid in LB S14

15 f. Extracted 25 µm acrylic acid in LB g. Extracted 1 µm acrylic acid in LB S15

16 h. Extracted 1 µm acrylic acid in LB i. Extracted LB Sample (no acrylic acid added) S16

17 6. Fluorescence assay of acrylic acid standards in LB media and minimal media Fluorescence Intensity (a. u.) LB + Ethanol DMS + Ether + Ethanol Extracted AA Standards in LB media with 1µM of 4, 1min, Em 1µM AA only 1µM AA + 1µM AA 1µM AA 25µM AA 5µM AA 75µM AA 1mM AA 1mM AA 1mM AA 1µM 4 - Control Concentration Extracted AA Standards in minimal media with 1 µm 4, 1min, Em Fluorescence Intensity (a. u.) MM nly DMS + Ether + Ethanol 1µM AA + Ethanol 1µM AA + 1µM 4- Control 1µM AA+ 1µM AA 25mM AA 5uM AA 75uM AA 1mM AA 1mM AA 1mM AA 1µM 4 Concentration Figure S6. Fluorescence assay of acrylic acid standards in LB media and minimal media (MM), after extraction for GCMS analysis. Control samples are performed in ethanol solvent. S17

18 7. Fluorescence of 3-butynoic acid using compound 4 Figure S7. Fluorescence signals of indicated range of 3-butynoic acid concentrations treated with 5 µm compound 4 (n = 2 +/- SD). 8. Experimental details for fluorescence characterization of reaction between compound 4 and acrylic acid at various ph conditions A solution containing compound 4 (1 µmol) and acrylic acid (1 µmol) was irradiated with a hand-held 32-nm UV lamp for 1 minute. Experiments were performed in Corning flat black 96-well plate with a total reaction volume of 1 µl with buffers of different ph. Excitation wavelength used was 38 nm and emission wavelength was 52 nm. Bandwidth was set at 5nm. S18