Supporting Information

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1 Supporting Information A latent reaction in a model GFP chromophore revealed upon confinement: Photohydroxylation of ortho-halo benzylidene-3-methylimidazolidiones via an electrocylization process Shampa R. Samanta, José P. Da Silva #, Anthony Baldridge, Laren M. Tolbert*, and V. Ramamurthy, * Department of Chemistry, University of Miami, Coral Gables, FL School of Chemistry and Biochemistry, 901 Atlantic Drive, Georgia Institute of Technology, Atlanta, GA # Centro de Investigação em Química do Algarve, FCT, Universidade do Algarve, Campus de Gambelas, Faro, Portugal Table of Contents Title General Experimental Section Experimental Procedures Synthesis Characterization of Compounds Encapsulation Methods Monitorization of the photoreaction Page S2 S3-S6 Spectroscopic Data 1 H NMR spectra of host-guest complexes S7-S8 LC-DAD-MS traces of irradiated samples S9-S16 and S21 1 H NMR spectra of irradiated samples S17-S20, S22 and S23 Emission spectra of irradiated samples S25-S28 S1

2 General Experimental Section All reagents were purchased from commercial sources and used without further purification. NMR spectra were recorded using a Varian Mercury spectrometer (300MHz) and processed using MestRe-C 2.3a. Mass spectroscopy data for characterizing the guests (Figure S1) was completed by the Georgia Tech Mass Spectroscopy Center. Gas chromatography coupled to mass spectrometry (GC-MS) experiments were performed using a Agilent 6890N equipped with a 5973 series mass selective detector (E.I. 70 ev). A DB-5MS capillary column with 30 m length, 0.25 mm I.D., and 0.5-µm film thickness (J&W) was used with the following oven temperature program: 70 ºC for 2 min, 10 ºC per min increase until a final temperature of 280 ºC. The injector was set to 280 ºC and the injection volume was 1 µl. Liquid Chromatography coupled to Diode Array and Mass Spectrometry (LC-DAD-MS) experiments were performed using an Agilent Technologies 1200 Series LC, equipped with a diode array detector and coupled to a Bruker Daltonics HCT ultra (ion trap). A Hamilton PRP-1 reversed phase LC column (15.0 cm length, 2.1 mm internal diameter, 5 Lm), stabilized at 25 C, was used for analyses. The eluent system was a gradient mixture of acetonitrile (A) and water (B), both with 0.1 % ammonia. The gradient started with 15 % of A and 85 % of B, during 1 minute, followed by a linear increase of B to 80 % after 10 minutes and then to 100 % after 14 minutes. The system was allowed to stay at 100 % of B for 4 minutes. Finally the system was allowed to recover the initial composition of the mobile phase (15 % of A and 85 % B) in 1 min and then stabilized for additional 4 min before the next run. All compounds were quantified using the LC traces with absorbance at 350 nm. The quantification of guests (2-7) and of OHderivative (8) was made using calibration curves prepared with pure compounds. The quantification of the trans-products was made assuming a detector response similar to that of the cis-parent compounds at 350 nm. O d O O O O O O O a c O HO HO O O OH OH e O O g f O O O O O O O O b h X N N X = F, Cl, Br 2 ortho X R = CH 3 3 meta X R = CH 3 4 para X R = CH 3 R O OH O OH O OH O OH Octa acid (1) 5 ortho X R = CH 2 CH 2 CH 3 6 meta X R = CH 2 CH 2 CH 3 7 para X R = CH 2 CH 2 CH 3 8 ortho OH R = CH 3 Scheme S1. Chemical structure of octa acid (1) and guest molecules studied. Various proton resonances of the host OA are designated as a-h. S2

3 Experimental Procedures Synthesis Syntheses of guests (Scheme S2) were carried out by a [2+3] cycloaddition of the corresponding aromatic Schiff base with the imine ester (Figure S1-C). Schiff bases were prepared by reaction of the aromatic aldehyde with the corresponding primary alkyl amine according to the literature (Figure S1-A). The imine ester was prepared according to the literature procedure (Figure S1- B). (Baldridge, A.; Kowalik, J.; Tolbert, L. M. Efficient synthesis of new 4-arylideneimidazolin- 5-ones related to the GFP chromophore by 2+3 cyclocondensation of arylideneimines with imidate ylides. Synthesis 2010, ). A CHO R 2 NH 2 R 1 stirring, rt 1 hour R 1 N R 2 B H 2 N O O + NH K 2 CO 3 O Ether, H 2 O Mix Me EtO N H CO 2 Me C R 1 N R 2 + Me EtO N H CO 2 Me Mix R1 N O N R 2 Scheme S2: Synthetic scheme of guests. (A) Synthesis of aromatic Schiff base; (B) Synthesis of imine ester; (C) Synthesis of compounds final guest compounds. Characterization of Compounds 1 H NMR Spectra of Chromophores (300 MHz) 4-(2-fluorobenzylidene)-1,2-dimethyl-1H-imidazol-5(4H)-one: 1 H NMR (300 MHz, CDCl 3 ), δ, ppm: 2.37 (s, 3H); 3.18 (s, 3H); 7.04 (s, 1H); 7.08 (AA of a AA XX spin system, 2H); 8.12 (XX of a AA XX spin system, 2H) 13 C NMR (75 MHz, CDCl 3 ), δ, ppm: 15.65, 26.56, (J CF = 21.8 Hz), , , , (J CF = 8.0 Hz), , , (J CF = Hz), (3-fluorobenzylidene)-1,2-dimethyl-1H-imidazol-5(4H)-one: 1 H NMR (300 MHz, CDCl 3 ), δ, ppm: 2.37 (d, J HH = 0.6 Hz, 3 H), 3.17 (s, 3 H), 7.02 (s, 1 H), (m, 1 H), (m, 1 H), (m, 1 H), (m, 1 H) 13 C NMR (75 MHz, CDCl 3 ), δ, ppm: 15.67, 26.58, (J CF = 22.0 Hz), (J CF = 22.0 Hz), , , (J CF = 7.7 Hz), , , (J CF = Hz), 163.4, S3

4 4-(4-fluorobenzylidene)-1,2-dimethyl-1H-imidazol-5(4H)-one: 1 H NMR (300 MHz, CDCl 3 ), δ, ppm: 2.35 (s, 3 H), 3.16 (s, 3 H), 7.03 (s, 1 H), 7.08 (AA of a AA BB X spin system, 2 H), 8.12 (BB of a AA BB X spin system, 2 H) 13 C NMR (75 MHz, CDCl 3 ), δ, ppm: 15.01, 26.53, (J CF = 21.8 Hz), , , (J CF = 9.2 Hz), , (J CF = Hz), (2-chlorobenzylidene)-1,2-dimethyl-1H-imidazol-5(4H)-one: 1 H NMR (300 MHz, CDCl 3 ), δ, ppm: 2.38 (s, 3H); 3.19 (s, 3H); 7.26 (t of d, J HH = 2.1, 7.6 Hz, 1H); 7.33 (t of d, J HH = 1.5, 7.6 Hz, 1H); 7.41 (dd, J HH = 1.5, 7.9 Hz, 1H); 7.56 (s, 1H); 8.74 (dd, J HH = 1.8, 7.6 Hz, 1H) 13 C NMR (75 MHz, CDCl 3 ), δ, ppm: 15.73, 26.61, , , , , , , , , , (3-chlorobenzylidene)-1,2-dimethyl-1H-imidazol-5(4H)-one: 1 H NMR (300 MHz, CDCl 3 ), δ, ppm: 2.37 (s, 3H); 3.16 (s, 3H); 6.98 (s, 1H); (m, 2H); (m, 1H); 8.20 (s, 1H); 13 C NMR (75 MHz, CDCl 3 ), δ, ppm: 15.66, 26.55, , , , , , , , , (2-bromobenzylidene)-1,2-dimethyl-1H-imidazol-5(4H)-one: 1 H NMR (300 MHz, CDCl 3 ), δ, ppm: 2.36 (s, 3H); 3.18 (s, 3H); 7.17 (t, J HH = 7.9 Hz, 1H); 7.36 (t, J HH = 7.9 Hz, 1H); 7.50 (s, 1H); 7.60 (d, J HH = 7.9 Hz, 1H); 8.70 (d, J HH = 7.9 Hz, 1H) 13 C NMR (75 MHz, CDCl 3 ), δ, ppm: 15.71, 26.58, , , , , , , , , , (3-bromobenzylidene)-1,2-dimethyl-1H-imidazol-5(4H)-one: 1 H NMR (300 MHz, CDCl 3 ), δ, ppm: 2.38 (s, 3H); 3.18 (s, 3H); 6.98 (s, 1H); 7.27 (t, J HH = 7.9 Hz, 1H); 7.48 (d, J HH = 7.9 Hz, 1H); 7.98 (d, J HH = 7.9 Hz, 1H); 8.35 (s, 1H); 13 C NMR (75 MHz, CDCl 3 ), δ, ppm: 15.73, 26.62, , , , , , , , , , (4-bromobenzylidene)-1,2-dimethyl-1H-imidazol-5(4H)-one: 1 H NMR (300 MHz, CDCl 3 ), δ, ppm: 2.36 (s, 3H); 3.17 (s, 3H); 6.99 (s, 1H); 7.51 (d, J HH = 8.8 Hz, 2H); 7.98 (d, J HH = 8.5 Hz, 2H) 13 C NMR (75 MHz, CDCl 3 ), δ, ppm: 15.69, 26.58, , , , , , , , (3-fluorobenzylidene)-2-methyl-1-propyl-1H-imidazol-5(4H)-one: 1 H NMR (300 MHz, CDCl 3 ), δ, ppm: 0.96 (t, J HH = 7.3 Hz, 3H); 1.67 (sextet, J HH = 7.3 Hz, 2H); 2.39 (s, 3H); 3.56 (t, J HH = 7.3 Hz, 2H); 7.02 (s, 1H); 7.05 (t of d, J HH = 8.2, 2.6 Hz, 1H); 7.36 (d of t, J HH = 6.1 Hz, 1H); 7.73 (d, J HH = 7.9 Hz, 1H); 8.05 (ddd, J HH = 10.6, 2.6, 1.5 Hz, 1H); 13 C NMR (75 MHz, CDCl 3 ), δ, ppm: 11.16, 15.77, 22.55, 42.18, (J CF = 21.0 Hz), , , , (J CF = 8.8 Hz), , , (J CF = Hz), , (4-fluorobenzylidene)-2-methyl-1-propyl-1H-imidazol-5(4H)-one: 1 H NMR (300 MHz, CDCl 3 ), δ, ppm: 0.96 (t, J HH = 7.3 Hz, 3H); 1.66 (sextet, J HH = 7.3 Hz, 2H); 2.38 (s, 3H); 3.56 (t, J HH = 7.3 Hz, 2H); 7.04 (s, 1H); 7.10 (AA of a AA BB X spin system, 2 H), 8.14 (BB of a AA BB X spin system, 2 H); 13 C NMR (75 MHz, CDCl 3 ), δ, ppm: 11.20, 15.78, 22.61, 42.19, (J CF = 21.0 Hz), , , (J CF = 8.8 Hz), , (J CF = Hz), S4

5 4-(2-chlorobenzylidene)-2-methyl-1-propyl-1H-imidazol-5(4H)-one: 1 H NMR (300 MHz, CDCl 3 ), δ, ppm: 0.97 (t, J HH = 7.6 Hz, 3H); 1.67 (m, 2H); 2.39 (s, 3H); 3.57 (t, J HH = 7.6 Hz, 2H); 7.27 (t of d, J HH = 1.8, 7.6 Hz, 1H); 7.41 (dd, J HH = 1.8, 7.6 Hz, 1H); 7.33 (t of d, J HH = 1.5, 7.3 Hz, 1H); 7.55 (s, 1H); 8.76 (dd, J HH = 1.8, 7.6 Hz, 1H) 13 C NMR (75 MHz, CDCl 3 ), δ, ppm: 11.20, 15.83, 22.57, 42.21, , , , , , , , , (3-chlorobenzylidene)-2-methyl-1-propyl-1H-imidazol-5(4H)-one: 1 H NMR (300 MHz, CDCl 3 ), δ, ppm: 0.96 (t, J HH = 7.6 Hz, 3H); 1.66 (sextet, J HH = 7.3 Hz, 2H); 2.39 (s, 3H); 3.56 (t, J HH = 7.3 Hz, 2H); 6.99 (s, 1H); (m, 2H); (m, 1H); 8.22 (s, 1H); 13 C NMR (75 MHz, CDCl 3 ), δ, ppm: 11.17, 15.79, 22.55, 42.19, , , , , , , , , (2-bromobenzylidene)-2-methyl-1-propyl-1H-imidazol-5(4H)-one: 1 H NMR (300 MHz, CDCl 3 ), δ, ppm: 0.96 (t, J HH = 7.3 Hz, 3H); 1.67 (sextet, J HH = 7.3 Hz, 2H); 2.38 (s, 3H); 3.57 (t, J HH = 7.3 Hz, 2H); 7.18 (t of d, J HH = 7.9, 1.8 Hz, 1H); 7.37 (t of d, J HH = 7.9, 1.5 Hz, 1H); 7.61 (dd, J HH = 8.2, 1.2 Hz, 1H); 7.50 (s, 1H); 8.72 (dd, J HH = 8.2, 1.5 Hz, 1H); 13 C NMR (75 MHz, CDCl 3 ), δ, ppm: 11.22, 15.84, 22.58, 42.23, , , , , , , , , , (3-bromobenzylidene)-2-methyl-1-propyl-1H-imidazol-5(4H)-one: 1 H NMR (300 MHz, CDCl 3 ), δ, ppm: 0.95 (t, J HH = 7.3 Hz, 3H); 1.66 (sextet, J HH = 7.3 Hz, 2H); 2.39 (s, 3H); 3.56 (t, J HH = 7.6 Hz, 2H); 6.97 (s, 1H); 7.27 (t, J HH = 7.9 Hz, 1H); 7.48 (d, J HH = 7.9 Hz, 1H); 7.99 (d, J HH = 7.6 Hz, 1H); 8.35 (s, 1H); 13 C NMR (75 MHz, CDCl 3 ), δ, ppm: 11.17, 15.80, 22.55, 42.19, , , , , , , , , , , (4-bromobenzylidene)-2-methyl-1-propyl-1H-imidazol-5(4H)-one: 1 H NMR (300 MHz, CDCl 3 ), δ, ppm: 0.95 (t, J HH = 7.3 Hz, 3H); 1.66 (sextet, J HH = 7.3 Hz, 2H); 2.37 (s, 3H); 3.55 (t, J HH = 7.3 Hz, 2H); 6.99 (s, 1H); 7.53 (d, J HH = 8.5 Hz, 2H); 8.00 (d, J HH = 8.5 Hz, 2H); 13 C NMR (75 MHz, CDCl 3 ), δ, ppm: 11.22, 15.83, 22.60, 42.22, , , , , , , , Stoichiometry Titration Titration of OA (1 mm OA dissolved in 10 mm borate buffer solution of D 2 O) with standard solution of guests (BMIs, 60 mm in DMSO-d 6 ) was conducted by 1 H NMR spectroscopy by gradual addition of guests (BMIs) to the host solution. Titration suggested that all chromophores formed 2:1 complex (H : G) with OA. The guest resonances were assigned based upon systematically comparing the variation in the structure of guests and also with the aid of 2D- NMR studies (in some cases). Monitoring Photoreactions by Emission Photoirradiation of 10-5 M solution of BMIs in OA (H : G = 2:1, M) and benzene was done using a 450 W medium pressure Hg lamp and a 310 nm filter. Fluorescence measurements were taken after given increments of time to note the intensity change of the chromophore (excitation 370 nm). S5

6 Monitoring Photoreactions by NMR Spectroscopy A 0.5 mm solution of BMIs was prepared in CD 3 CN and complex solution of these compounds were prepared in 1 mm octa acid solution (H:G = 2:1, 10 mm borate buffer solution of D 2 O). These solutions were irradiated using a 450 W medium pressure Hg lamp and a 310 nm filter. Monitoring Photoreactions by GC-MS and LC-DAD-MS Irradiation of free guests was made using 100 µm concentrations in acetonitrile. The formed products in acetonitrile were studied by GC-MS and LC-DAD-MS. Irradiation of guests@oa complexes was made in aqueous solution of Na 2 B 4 O 7 (10 mm) at ph 9 containing 100 µm of guest and 200 µm of host. Degassing was done by the freeze-pump-thaw technique (3 cycles, 10 2 torr). The photoproducts of guests@oa complexes were followed by LC-DAD-MS. All irradiations were performed using a high-pressure xenon lamp setup in conjunction with a water filter to prevent heating of the sample solution. An additional glass filter was inserted to remove UV light below 320 nm. S6

7 Figure S1. 1 H NMR spectra of capsular assembly of halogen substituted BMI@OA 2, (H:G = 2:1), [OA] = 1 mm in 10 mm buffer (D 2 O). S7

8 Figure S2. 1 H NMR spectra of capsular assembly of halogen and hydroxyl substituted BMI@OA 2, (H:G = 2:1). [OA] = 1 mm in 10 mm buffer (D 2 O). S8

9 Figure S3. GC-MS and HPLC-DAD traces at 350 nm of cis-ortho fluoro N-methyl BMI in acetonitrile and cis-ortho fluoro N-methyl 2 (aqueous borate buffer, ph 9), before and after irradiation (20 min, λ > 320 nm). S9

10 Figure S4. GC-MS and HPLC-DAD traces at 350 nm of cis-ortho chloro N-methyl BMI in acetonitrile and cis-ortho chloro N-methyl 2 (aqueous borate buffer, ph 9), before and after irradiation (20 min, λ > 320 nm). S10

11 Figure S5. GC-MS and HPLC-DAD traces at 350 nm of cis-ortho bromo N-methyl BMI in acetonitrile and cis-ortho bromo N-methyl 2 (aqueous borate buffer, ph 9), before and after irradiation (20 min, λ > 320 nm). S11

12 Figure S6. GC-MS and HPLC-DAD traces at 350 nm of cis-para chloro N-methyl BMI in acetonitrile and cis-para chloro N-methyl 2 (aqueous borate buffer, ph 9), before and after irradiation (20 min, λ > 320 nm). S12

13 Figure S7. GC-MS and HPLC-DAD traces at 350 nm of cis-para bromo N-methyl BMI in acetonitrile and cis-para bromo N-methyl 2 (aqueous borate buffer, ph 9), before and after irradiation (20 min, λ > 320 nm). S13

14 Figure S8. GC-MS and HPLC-DAD traces at 350 nm of cis-meta fluoro N-methyl BMI in acetonitrile and cis-meta fluoro N-methyl 2 (aqueous borate buffer, ph 9), before and after irradiation (20 min, λ > 320 nm). S14

15 Figure S9. GC-MS and HPLC-DAD traces at 350 nm of cis-meta chloro N-methyl BMI in acetonitrile and cis-meta chloro N-methyl 2 (aqueous borate buffer, ph 9), before and after irradiation (20 min, λ > 320 nm). S15

16 Figure S10. GC-MS and HPLC-DAD traces at 350 nm cis-meta bromo N-methyl BMI in acetonitrile and cis-meta bromo N-methyl 2 (aqueous borate buffer, ph 9), before and after irradiation (20 min, λ > 320 nm). S16

17 Figure S11. 1 H NMR spectra of cis/trans isomerization of cis-meta bromo N-propyl BMI@OA 2 (i) cis-meta bromo N-methyl BMI@OA 2 ([OA] = 1 mm in 10 mm borate buffer in D 2 O and [guest] = 0.5 mm), (ii) after photolysis of solution (i) for 10 min > 320 nm. S17

18 Figure S12. 1 H NMR spectra of cis/trans isomerization of cis-para bromo N-propyl 2. (i) cis-para bromo N-propyl@OA 2 ([OA] = 1 mm in 10 mm borate buffer in D 2 O and [guest] = 0.5 mm) (ii) after photolysis of solution (i) for 10 min > 320 nm. S18

19 Figure S13. 1H NMR spectra of photochemical reaction (>320 nm) of cis-ortho fluoro N-methyl in aerated solution of borate buffer in D2O (i) cis-ortho fluoro N-methyl (ii) photolysis of (i) for 2 min, (iii) photolysis of (i) for 30 min followed by heating at 50 oc for 60 min, and (iv) cis-ortho hydroxy N-methyl BMI@OA2. ([OA] = 1 mm in 10 mm borate buffer in D2O and [guest] = 0.5 mm). S19

20 Figure S14. 1 H NMR spectra of photochemical reaction (>320 nm) of cis-ortho bromo N-propyl BMI@OA 2 in aerated solution of borate buffer in D 2 O (i) cis-ortho bromo N-propyl BMI@OA 2, (ii) photolysis of solution (i) for 2 min, (iii) photolysis of solution (i) for 5 min, (iv) photolysis of solution (i) for 10 min, (v) heating of solution (iv) at 50 o C for 30 min, and (vi) ) cis-ortho hydroxy N-propyl BMI@OA 2. ([OA] = 1 mm in 10 mm borate buffer in D 2 O and [guest] = 0.5 mm) S20

21 Figure S15. HPLC-DAD traces at 350 nm of cis-ortho chloro N-methyl 2 (aqueous borate buffer, ph 9), irradiated (20 min, λ > 320 nm) under air equilibrated conditions and after vacuum degassing.. S21

22 Figure S16. 1 H NMR spectra of cis/trans isomerization of cis-ortho bromo N-methyl BMI in CD 3 CN. (i) cis-ortho bromo N-methyl BMI (1 mm in CD 3 CN) (ii) after photolysis of solution (i) for 10 min > 320 nm. S22

23 Figure S17. 1 H NMR spectra of cis/trans isomerization of cis-para bromo N-propyl BMI in CD 3 CN. (i) cis-para bromo N-propyl BMI (1 mm in CD 3 CN), (ii) after photolysis of solution (i) for 10 min > 320 nm. S23

24 Figure S18. Product ratio with respect to duration of irradiation: Irradiation of trans ortho chloro BMI in acetonitrile. S24

25 Figure S19. Emission spectra recorded at various time of irradiation of (>320 nm) of cis-ortho fluoro N-methyl 2 in aerated solvent (see inset). [guest] =10-5 M, [OA] = M). λ ex 370 nm. S25

26 Figure S20. Emission spectra recorded at various time of photolysis (>310 nm) of cis-ortho bromo N-methyl 2 in aerated solvent (see inset). [guest] =10-5 M, [OA] = M). λ ex 370 nm. S26

27 Figure S21. Emission spectra recorded at various time of photolysis (>310 nm) of cis-ortho fluoro N-methyl BMI (10-5 M) in aerated benzene (see inset). λ ex 370 nm. S27

28 Figure S22. Emission spectra recorded at various time of photolysis (>310 nm) of cis-o-br/n- Me (10-5 M) in aerated benzene (see inset). λ ex 370 nm. S28