Supporting Information

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1 Supporting Information MegaStokes DIPY-Triazoles as Environmentally-Sensitive Turn-on Fluorescent Dyes Jun Cheng Er, Mui Kee Tang, Chee Geng Chia, Huimin Liew, Marc Vendrell*, and Young-Tae Chang* Department of Chemistry, ational University of Singapore, 3 Science Drive 2, Singapore , Singapore Table of Contents A. In vitro screening of DIPY triazoles 2. Supporting Experiments 4 C. Chemical Structures and Characterization Data for DIPY triazoles 9 D. MR Spectra 10 S1

2 A. In vitro screening of DIPY triazoles Table S1. List of bio-molecules selected for unbiased in vitro screening. uffer: HEPES, 10 mm, ph 7.4 Analyte class Individual analyte molecules Concentrations Control HEPES 10mM, ph 7.4 Viscosity Glycerol Volume %: 20%, 10%, 5%, 2.5% ph ph 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 ucleotides and nucleosides ucleic acids Peptides ATP, CTP, GTP, UTP, cyclic AMP, cyclic GMP Single strand DA (ssda) double strand DA (dsda) transfer RA (t-ra) total RA HA tag (YPYDVPDYA) HA12CA5 tag (CYPYDVPDYA) His tag (HHHHHH) HisG tag (HHHHHG) VSV-G tag (YTDIEMRLGK) V5 tag (CGKPIPPLLGLDST) IQ tag (IQSPHFF) AP tag (KKKGPGGLDIFEAQKIEWH) FLAG tag (DYKDDDK) cmyc tag (EQKLISEEDL) S2 100 μm, 50 μm, 25 μm, 12.5μM 1 mg/ml, 0.5 mg/ml, 0.25 mg/ml, 0.12 mg/ml 100 μg/ml, 50 μg/ml, 25 μg/ml, 12.5 μg/ml Protein Histone, Myoglobin 0.5 mg/ml, 0.25 mg/ml, 0.12 mg/ml, 0.06 mg/ml Insulin, Ubiquitin 0.2 mg/ml, 0.1 mg/ml, 0.05 mg/ml, mg/ml Human Immunoglobulin G (human IgG) ovine Immunoglobulin G (bovine IgG) Human serum albumin (HSA) ovine serum albumin (SA) Holo-Transferrin Apo-Transferrin Concanavalin A Lysozyme Catalase Papapin Cytochrome C bovine heart Haemoglobin Fibrinogen 1 mg/ml, 0.5 mg/ml, 0.25 mg/ml, 0.12 mg/ml Metal ions a, K 1 mm, 0.5 mm, 0.25 mm, 0.12mM Mg 2, Zn 2, Fe 2, Ca μm, 50 μm, 25 μm, 12.5 μm xidoreduction related molecules Ascorbic acid DL-dithiothreitol (DTT) L-Glutathione reduced form (GSH) L-Glutathione oxidized form (GSSG) icotinamide adenine dinucleotide (AD) 2 mm, 1 mm, 0.5 mm, 0.25 mm

3 Miscellaneous molecules icotinamide adenine dinucleotide reduced form (ADH) icotinamide adenine dinucleotide phosphate reduced form (ADPH) Sodium hypochlorite (a) Potassium dioxide (K 2 ) Hydrogen peroxide (H 2 2 ) Iron (II) with hydrogen peroxide (Fe 2+ + H 2 2 ) 2,2-azobis(2-methylpropionamidine) dihydrochloride (AAPH) Glycogen Dextran Chrontroitin Heparin Malachite green Sudan I Melamine Glucose, Glucose phosphate Fructose, Fructose phosphate Caffeine Acetylcholine chloride Gamma.aminobutyric acid (GAA) Gamma butyrolactone (GH) L-Glutamine Monosodium glutamate (MSG) Sarcosine, Histamine, Dopamine 1 mg/ml, 0.5 mg/ml, 0.25 mg/ml, 0.12 mg/ml 2 mm, 1 mm, 0.5 mm, 0.25 mm S3

4 . Supporting Experiments i) Solvatochromic effects a) b) Fig. S1. Effect of solvent polarity on the absorbance and fluorescence of DC-9 (100 μm) at rt. (a) ormalized absorption spectra in different ratios of cyclohexane and ethanol. (b) Corresponding emission spectra of DC-9 in various ratios of cyclohexane and ethanol. S4

5 Fig. S2. Effect of solvent viscosity on the fluorescence intensity of DC-9 (100 μm) at rt. Intensity of DC-9 systematically increased in more viscous (and less polar) alcohols exceeding that of cyclohexane when in n-octanol and n-hexanol. H 2 H 2 F2 F2 A R 1 R 1 H 2 H 2 F2 F2 R 1 C D R 1 Fig. S3. Major resonance structures of DC compounds S5

6 ii) Fluorescence and absorbance response of DC-9 to HSA a) b) Fig. S4. Fluorescence and absorbance response of DC-9 (10 μm) upon incubation with 4.0, 1.0, 0.25, 0.063, 0.016, and 0 mg/ml HSA in 10 mm phosphate buffer (ph = 7.3). (a) ormalized emission spectra; λ exc. : 460 nm. (b) Absorbance spectra. Values are represented as means (n = 3). Measurements were taken at rt. S6

7 iii) Limit-of-detection and linear dynamic range of DC-9 Fig. S5. Fluorescence emission response of DC-9 (10 μm) upon incubation with serial dilutions of HSA in 10 mm phosphate buffer (ph = 7.3); λ exc. : 460 nm, λ em.: 575 nm. Values are represented as means and error bars as standard deviations (n = 3). Measurements were taken at rt. LD = 0.3 μg/ml; linear range = 0.37 to 31 μg/ml, R 2 = iv) Job plot analysis of DC-9 with HSA Fig. S6. Job plot analysis. DC-9 was mixed with HSA (fatty acid free) at different ratios in 10 mm phosphate buffer (ph = 7.3) while maintaining total concentration at 20 μm; λ exc. : 460 nm, λ em.: 575 nm. Values are represented as means and error bars as standard deviations (n = 3). Measurements were taken at rt. S7

8 v) Determination of dissociation constant for DC-9 with HSA Fig. S7. Fluorescence emission of serial dilutions of DC-9 (0, 0.47 to 60 μm) upon incubation with HSA (10 μm) in 10 mm phosphate buffer (ph = 7.3); λ exc. : 460 nm, λ em.: 575 nm; values are represented as means and error bars as standard deviations (n = 3); Measurements were taken at rt; K D = 12.7 ± 0.4 μm (one-site specific binding model). S8

9 C. Chemical Structures and Characterization Data for DIPY triazoles Fig. S8. Chemical structures of the DC, DCAC and DCCA library. Fig. S9. Absorbance maximum trend of the DC, DCAC and DCCA compounds. Concentration = 100 μm in DMS. S9

10 Fig. S10. Fluorescence emission trend of the DC, DCAC and DCCA compounds. Concentration = 100 μm in DMS. Fig. S11. Percentage quantum yield trend of the DC, DCAC and DCCA compounds. Concentration = 100 μm in DMS. S10

11 Table S2. Chemical structures and characterization data for the DC library. Concentration = 100 μm in DMS. Code DC-1 Structure H 2 Purity (254 nm) m/z Calculat ed m/z Experim ental λ max Abs. (nm) λ max Em. (nm) φ (%) 95% F 2 DC-2 H 2 95% F 2 DC-3 H 2 98% F 2 DC-4 H 2 95% F 2 DC-5 H 2 99% F 2 DC-6 H 2 86% F 2 S11

12 DC-7 H 2 100% F 2 DC-8 H 2 88% F 2 DC-9 H 2 97% F 2 DC-10 H 2 92% F 2 DC-11 H 2 97% F 2 DC-12 H 2 90% F 2 S12

13 DC-13 H 2 97% F 2 DC-14 H 2 99% F 2 DC-15 H 2 84% F 2 CF 3 DC-16 H 2 95% F 2 F F DC-17 H 2 99% F 2 DC-18 H 2 87% F 2 S13

14 DC-19 H 2 98% F 2 DC-22 H 2 99% F 2 DC-23 H 2 100% F 2 DC-26 H 2 91% F2 F F DC-27 H 2 58% F 2 DC-29 H 2 93% F 2 S14

15 DC-30 H 2 92% F 2 CF 3 F 3 C DC-31 H 2 96% F 2 F DC-36 H 2 98% F 2 DC-37 H 2 96% F 2 DC-42 H 2 92% F 2 DC-43 H 2 95% F 2 S15

16 DC-44 H 2 94% F 2 DC-45 H 2 84% F 2 DC-46 H 2 98% F 2 DC-47 H 2 90% F 2 DC-48 H 2 96% F 2 DC-49 H 2 92% F 2 S16

17 DC-50 H 2 93% F 2 DC-67 H 2 100% F 2 H DC-69 H 2 92% F 2 H DC-70 H 2 100% F 2 H S17

18 Table S3. Chemical structures and characterization data for the DCAC library. Concentration = 100 μm in DMS. Code DCAC-1 Structure H Purity (254 nm) m/z Calculat ed m/z Experi mental λ max Abs. (nm) λ max Em. (nm) φ (%) 95% F 2 DCAC-2 H 98% F 2 DCAC-3 H 97% F 2 DCAC-4 H 97% F 2 DCAC-5 H 99% F 2 S18

19 DCAC-6 H 93% F 2 DCAC-7 H 98% F 2 DCAC-8 H 98% F 2 DCAC-9 H 97% F 2 DCAC-10 H 91% F 2 DCAC-11 H 96% F 2 S19

20 DCAC-12 H 89% F 2 DCAC-13 H 98% F 2 DCAC-14 H 98% F 2 DCAC-15 H 84% F 2 CF 3 DCAC-16 H 93% F 2 F F DCAC-17 H 99% F 2 S20

21 DCAC-18 H 88% F 2 DCAC-19 H 98% F 2 DCAC-22 H 98% F 2 DCAC-23 H 99% F 2 DCAC-26 H 91% F 2 F F DCAC-27 H 89% F 2 S21

22 DCAC-29 H 95% F 2 DCAC-30 H 93% F 2 CF 3 F 3 C DCAC-31 H 97% F 2 F DCAC-36 H 99% F 2 DCAC-37 H 95% F 2 DCAC-42 H 98% F 2 S22

23 DCAC-43 H 95% F 2 DCAC-44 H 95% F 2 DCAC-45 H 87% F 2 DCAC-46 H 99% F 2 DCAC-47 H 81% F 2 DCAC-48 H 95% F 2 S23

24 DCAC-49 H 96% F 2 DCAC-50 H 94% F 2 DCAC-67 H 97% F 2 H DCAC-69 H 92% F 2 H DCAC-70 H 99% F 2 H S24

25 Table S4. Chemical structures and characterization data for the DCCA library. Concentration = 100 μm in DMS. Code DCCA-1 Structure Purity (254 nm) m/z Calculat ed m/z Experim ental λ max Abs. (nm) λ max Em. (nm) φ (%) 93% H F 2 DCCA-2 52% H F 2 DCCA-3 97% H F 2 DCCA-4 92% H F 2 DCCA-5 98% H F 2 S25

26 DCCA-6 80% H F 2 DCCA-7 99% H F 2 DCCA-8 79% H F 2 DCCA-9 97% H F 2 DCCA % H F 2 DCCA % H F 2 S26

27 DCCA % H F 2 DCCA % H F 2 DCCA % H F 2 DCCA % H F 2 CF 3 DCCA % H F 2 F F DCCA % H F 2 S27

28 DCCA % H F 2 DCCA % H F 2 DCCA % H F 2 DCCA % H F 2 DCCA % H F 2 F F DCCA % H F 2 S28

29 DCCA % H F 2 DCCA % H F 2 CF 3 F 3 C DCCA % H F 2 F DCCA % H F 2 DCCA % H F 2 DCCA % H F 2 S29

30 DCCA % H F 2 DCCA % H F 2 DCCA % H F 2 DCCA % H F 2 DCCA % H F 2 DCCA % H F 2 S30

31 DCCA- 49 H 90% F 2 DCCA- 50 H 87% F 2 DCCA- 67 H 87% F 2 H DCCA- 69 H 89% F 2 H DCCA- 70 H 93% F 2 H S31

32 H 2 F 2 a) mau DAD1, Sig=254,16 Ref=off (MVE2\MVE-DCLIRARY \DC-A10.D) Area: mau mau Area: DAD1 C, Sig=365,16 Ref=off (MVE2\MVE-DCLIRARY \DC-A10.D) DAD1 D, Sig=500,16 Ref=off (MVE2\MVE-DCLIRARY \DC-A10.D) min min min b) mau DAD1, (2402 mau, - ) of DC-A10.D nm *MSD1 SPC, time=7.422 of D:\DATA\MVE2\MVE-DCLIRARY c) d) Max: *MSD2 SPC, time=7.402 of D:\DATA\MVE2\MVE-DCLIRARY Max: m /z m /z Fig. S12 HPLC-MS characterization of DC-9 a) chromatograms at 254, 365 and 500 nm; HPLC conditions: A: H 2 (0.1% HCH), : CH 3 C (0.1% HCH); gradient 5% to 95% (10 min), isocratic 95% (2 min). Reverse-phase Phenomenex C 18 Luna column (4.6 x 50 mm 2, 3.5 µm particle size), flow rate: 1 ml/min. b) absorbance spectrum ( nm); c) ESI-MS positive spectrum; d) ESI-MS negative spectrum. S32

33 D. MR Spectra 2 H H 1 S33

34 2 H H 1 S34

35 2 H 2 S35

36 2 H 2 S36

37 2 F2 3 S37

38 2 F2 3 S38

39 2 F2 3 S39

40 H 2 F2 4 S40

41 H 2 F2 4 S41

42 S42

43 H 2 F 2 DC-9 S43

44 H 2 F 2 DC-9 S44

45 S45