Fluorescent, Through-Bond Energy Transfer Cassettes for Labeling

They absorb strongly at 488 nm (Ar-laser emission) and efficiently transfer the ..... Meineke , Mariano L. Bossi , Haisen Ta , Vladimir N. Belov , Ste...
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Supporting Information Fluorescent, Through-bond Energy Transfer Cassettes for Labeling Multiple Biological Molecules In One Experiment Guan-Sheng Jiao, Lars H. Thoresen, and Kevin Burgess* Department of Chemistry, Texas A & M University, Box 30012, College Station, TX 77842-3012

Contents 1

Syntheses of Compounds 1 – 8

2

Absorption Spectra of Compounds 1 – 8

3

Tabulated Spectroscopic Properties of Compounds 1 – 8

4

Demonstration of the Photostabilities of Cassettes 1 – 4 Relative to Fluorescein

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1. Syntheses of Compounds 1 – 8 Scheme S1. Preparation of Cassette 1. TMS I

O

O TMS

O

AcO

O

CO2H

25 oC, 24 h

Pd(PPh3)4, CuI, Et3N THF, 55 oC, 1 h AcO OAc

O

K2CO3 MeOH/THF

OAc

O

HO

9 95 %

Br

CO2tBu 4

O

K2CO3, DMF 25 oC, 24 h HO

O

tBuO C 2

O O O

5 Pd(PPh3)4, CuI

O

O

O

Et3N, DMF 75 oC, 24 h O OH

H2N

11 62 %

12 73 %

H2N+

HO2C O O O

TIS, TFA, CH2Cl2 25 oC, 24 h

O

10 98 %

H2N+

CO2tBu

O

O

O

OH

H2N 1 43 %

3

Scheme S2. Preparation of Cassette 2. I

I

HO

O

K2CO3, DMF 25 oC, 24 h

O

O

CO2Me 4

Br

CO2H

CO2Me

O O

O

HO

13 50 %

TMS

CO2Me O

TMS

O

Pd(PPh3)4, CuI, Et3N DMF, 25 oC, 17 h HO

CO2H LiOH THF/H2O

O

0 oC, 6 h O

O

O

14 85 % Me2N+

O

O

HO

15 69 % HO2C O O

6 Pd(PPh3)4, CuI

O O

O

Et3N, DMF 75 oC, 8 h OH

Me2N 2 61 %

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Scheme S3. Preparation of Cassette 3. HO2C

N+ CO2H O O

HO

7 Pd(PPh3)4, CuI

O O

O

N

OH

Et3N, DMF 75 oC, 8 h O

O

O O

15

3 75 %

Scheme S4. Preparation of Cassette 4. tBuO

N+

CO2tBu

O

HO

O O

O

N

OH

Et3N, DMF 75 oC, 24 h O

O

O O

8 Pd(PPh3)4, CuI

O

2C

11 16 47 %

N+

HO2C O O O

TIS, TFA, CH2Cl2

O

O

N

OH

25 oC, 24 h

4 67 %

5

Scheme S5. Preparation of 4-Bromorosamine 5. Br Br

OH

60 % H2SO4

+ H2N CHO

160 oC, 24 h H2N

N+H2

O 5 8%

Scheme S6. Preparation of 4-Bromorosamine 6. Br Br

OH

60 % H2SO4

+ N CHO

160 oC, 22 h Me2N

N+Me2

O 6 12%

Scheme S7. Preparation of 4-Bromorosamine 7. Br OH

Br 60 % H2SO4

+

90 oC, 18 h

N CHO

N

O 7 35 %

N+

6

Scheme S8. Preparation of 4-Bromorosamine 8. Br Br neat

+ N

160 oC, 18 h

OH CHO

N

O 8 5%

N+

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Experimental Procedures for the Preparation of Compounds 1 – 8. General Procedures. Melting points are uncorrected. High field NMR spectra were recorded on Varian Unity Plus (1 H at 300 MHz, 1 3C at 75 MHz, 3 1P at 121 MHz) or Inova (1 H at 500 MHz, 1 3C at 125 MHz) NMR spectrometers. Chemical shifts are reported in units of ppm relative to solvent (CDCl3 : 7.27 ppm for 1 H and 77.0 ppm for 1 3C; acetone-d6 : 2.04 ppm for 1 H and 29.9 ppm for 1 3C; DMSO-d6 : 2.50 ppm for 1 H and 39.5 ppm for 1 3C; CD3 OD: 3.30 ppm for 1 H and 49.0 ppm for 1 3C). FT-IR spectra were obtained using thin films on NaCl plates and 4021 GALAXY series instrument. Mass spectra were obtained from the Mass Spectrometry Applications Laboratory at Texas A&M University. Thin layer chromatography was performed using silica gel 60 F254 plates. Flash chromatography was performed using silica gel (230-600 mesh). CH2 Cl2 , THF, DMF, and triethylamine were distilled from appropriate drying agents. 5-Iodofluorescein,1 5-iodofluorescein diacetate,1 6-bromohexanoic acid tert-butyl ester,2 6-bromohexanoic acid methyl ester,2 and 7-hydroxy-N-methyl-2,2,4-trimethy-l,2-dihydroquinoline3 were prepared following the literature procedures. Other chemicals were purchased from commercial suppliers and used as received. Preparative HPLC were run on a Beckman System Gold 125P Solvent Module and 166P UV-Vis detector using a Vydac semi-preparative C-18 column (cat # 218TP101522, 22 x 250 mm), gradient elution was used (A = water, B = MeCN, both with 0.1% v/v TFA) with a constant flow rate of 10 mL/min. Preparation of Cassette 1. 5-(2-Trimethylsilylethynyl)fluorescein diacetate 9. 5-Iodofluorescein diacetate1 (0.87 g, 1.60 mmol), trimethylsilylacetylene (0.79 g, 8.02 mmol), tetrakis(triphenylphosphine)palladium (93 mg, 0.08 mmol), copper(I) iodide (15 mg, 0.08 mmol), and triethylamine (0.81 g, 8.02 mmol) were dissolved in 5 mL freshly distilled THF in a Schlenk tube. The solution was freeze-pump-thawed three times, then stirred 1 h at 55 °C under N2 . The solution was concentrated in vacuo then purified by flash chromatography eluting with 30% EtOAc/hexanes to yield 5-(2-trimethylsilyl-ethynyl)fluorescein diacetate as a pale yellow film (0.78 g, 95% yield). Rf 0.55 (35 % EtOAc/hexanes); 1H NMR (CDCl3, 300 MHz) d 0.29 (s , 9H), 2.33 (s, 6H), 6.82 (m, 4H), 7.10 (m, 2H), 7.13 (d, J = 8.1 Hz, 1H), 7.75 (dd, J = 8.1 Hz, 1.2 Hz, 1H), 8.11 (s, 1H);

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C NMR (CDCl3, 75 MHz) d -0.3, 21.1, 81.8, 102.6, 110.5, 116.0, 117.8, 124.1, 125.6, 126.5,

128.5, 128.9, 138.5, 151.5, 152.1, 186.8; IR(film, cm-1) n 1417, 1611, 1770, 2960; MS (ESI) m/z 513 (M+H)+ . 5-Ethynylfluorescein 10. 5-(2-Trimethylsilylethynyl)fluorescein diacetate 9 (656 mg, 1.28 mmol) and potassium carbonate (884 mg, 6.41 mmol) were combined in 10 mL 1:1 MeOH/THF. The bright orange mixture was stirred for 24 h at 25 °C. The reaction mixture was poured into 40 mL water then acidified to pH 2 with concentrated HCl. The resulting precipitate was collected by filtration,

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rinsed with water, then dried in vacuo to give 5-ethynylfluorescein as an orange solid (445 mg, 98 % yield). Mp >400 °C (dec); Rf 0.30 (50% EtOAc/hexanes); 1H NMR (acetone-d6, 500 MHz) d 3.91 (s, 1H), 6.62 (dd, J = 8.5 Hz, 2.5 Hz, 2H), 6.70 (d, J = 8.5 Hz, 2H), 6.75 (d, J = 2.5 Hz, 2H), 7.31 (dd, J = 8 Hz, 1 Hz, 1H), 7.87 (dd, J = 8 Hz, 1.5 Hz, 1H), 8.01 (dd, J = 1.5 Hz, 0.5 Hz, 1H), 9.16 (bs, 2H);

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C

NMR (acetone-d6, 125 MHz) d 68.0, 81.3, 82.5, 103.3, 110.8, 113.4, 124.9, 125.4, 128.4, 128.5, 130.0, 139.2, 153.3, 153.8, 160.6, 168.6; IR(film, cm-1) n 1258, 1606, 1693, 1756, 3071, 3279; MS (ESI) m/z 357 (M+H)+ ; HRMS (ESI) calc for C22H13O5+ (M+H)+ : 357.0463. Found: 357.0751. 5-Ethynylfluorescein-(5-tert-butoxycarbonyl)pentyl ester 11. 5-Ethynylfluorescein 10 (250 mg, 0.70 mmol) and 6-bromohexanoic acid tert-butyl ester2 (212 mg, 0.84 mmol) were dissolved in 15 mL dry DMF. Potassium carbonate (126 mg, 0.91 mmol) was added and the bright orange reaction mixture was stirred at 25 °C under nitrogen for 24 h. The solvent was removed under reduced pressure and the residue was adsorbed onto silica then purified by flash chromatography eluting with 5 to 10 % MeOH/CHCl3 yielding the title compound as a bright orange solid (230 mg, 62%). Rf 0.38 (10 % MeOH/CHCl3); 1H NMR (CD3OD, 500 MHz) d 0.96 – 1.01 (m, 2H), 1.23 – 1.29 (m, 2H), 1.35 – 1.41 (m, 2H), 1.42 (s, 9H), 2.08 (t, J = 7.3 Hz, 2H), 3.83 (s, 1H), 3.94 (t, J = 6.1 Hz, 2H), 6.70 (dd, J = 9.3, 2.0 Hz, 2H), 6.73 (d, J = 2.0 Hz, 2H), 7.01 (d, J = 9.3 Hz, 2H), 7.42 (d, J = 7.8 Hz, 1H), 7.88 (dd, J = 7.8 Hz, 1.5 Hz, 1H), 8.30 (d, J = 1.5 Hz, 1H);

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C NMR (CD3OD, 125 MHz) d 25.7, 26.4, 28.4, 29.1,

36.0, 66.8, 81.3, 81.4, 81.9, 82.5, 104.4, 116.3, 122.8, 125.9, 131.9, 132.1, 132.4, 135.1, 135.5, 136.9, 155.5, 158.9, 166.1, 174.7; MS (ESI) m/z 527 (M+H)+ . Cassette 1 tert-Butyl Ester 12. 5-Ethynylfluorescein-(5-tert-butoxycarbonyl)-pentyl ester 11 (45 mg, 86 mmol), 4-bromorosamine hydroxide 5 (33 mg, 86 mmol), tetrakis(triphenylphosphine)palladium (10 mg, 9 mmol), copper(I) iodide (3 mg, 17 mmol), and triethylamine (0.12 mL, 860 mmol) were dissolved in 2 mL DMF in a Schlenk tube. The reaction mixture was freeze-pump-thawed three times then stirred at 75 °C under nitrogen for 24 h. The dark brown solution was concentrated in vacuo then purified by flash chromatography eluting with 5 to 15% MeOH/CH2 Cl2 to give the desired product as an orange solid (51 mg, 73 % yield). Rf 0.15 (10 % MeOH/CH2 Cl2 ); 1 H NMR (1:1 CD3 OD/CDCl3 , 500 MHz) d 1.04 – 1.07 (m, 2H), 1.3 – 1.40 (m, 2H), 1.41 (s, 9H), 1.42 – 1.49 (m, 2H), 2.11 (t, J = 7.3 Hz, 2H), 3.99 (t, J = 6.3 Hz, 2H), 6.70 (d, J = 9.0 Hz, 2H), 6.75 (s, 2H), 6.83 (d, J = 2.2 Hz, 2H), 6.87 (dd, J = 9.3, 2.2 Hz, 2H), 7.03 (d, J = 9.0 Hz, 2H), 7.29 (d, J = 9.3 Hz, 2H), 7.42 (d, J = 7.8 Hz, 1H), 7.49 (d, J = 8.4 Hz, 2H), 7.87 (d, J = 8.4 Hz, 2H), 7.97 (dd, J = 7.8, 1.5 Hz, 2H), 8.42 (d, J =1.5 Hz, 1H); MS (ESI) m/z 811 (M+ ).

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Cassette 1. Compound 12 (25 mg, 31 mmol) was dissolved in 2 mL CH2 Cl2 . To this solution, triiso-propylsilane (0.2 mL) and trifluoroacetic acid (1 mL) were added. The mixture was stirred at 25 °C under nitrogen for 24 h. The reaction mixture was concentrated in vacuo then purified by reversed phase preparative HPLC (40 to 90% MeCN/H2 O over 30 min) to give the desired product as an orange solid (10 mg, 43 % yield). 1 H NMR (1:1 CD3 OD/CDCl3 , 500 MHz) d 1.05 – 1.11 (m, 2H), 1.33 – 1.38 (m, 2H), 1.44 – 1.50 (m, 2H), 2.18 (t, J = 7.3 Hz, 2H), 3.99 (t, J = 6.3 Hz, 2H), 6.81 (dd, J = 9.3, 2.2 Hz, 2H), 6.89 (d, J = 2.2 Hz, 2H), 6.98 (d, J = 2.0 Hz, 2H), 7.11 (d, J = 9.3 Hz, 2H), 7.14 (dd, J = 9.5, 2.0 Hz, 2H), 7.42 (d, J = 7.8 Hz, 1H), 7.54 (d, J = 8.4 Hz, 2H), 7.55 (d, J = 9.5 Hz, 2H), 7.92 (d, J = 8.4 Hz, 2H), 7.99 (dd, J = 7.8 Hz, 1,7 Hz, 2H), 8.45 (d, J = 1.7 Hz, 1H); MS (ESI) m/z 755 (M+ ). Preparation of Cassette 2. 5-Iodofluorescein-(5-methoxycarbonyl)pentyl Ester 13. 5-Iodofluorescein1 (350 mg, 0.76 mmol) and 6-bromohexanoic acid methyl ester2 (208 mg, 0.99 mmol) were dissolved in 10 mL dry DMF. Potassium carbonate (137 mg, 0.99 mmol) was added and the bright orange reaction mixture stirred at 25 °C under nitrogen for 24 h. The reaction mixture was adsorbed onto silica then purified by flash chromatography eluting with 10 % MeOH/CHCl3 . Crystallization from acetone/hexanes yielded the title compound as a bright orange solid (225 mg, 50%). Mp 110-112 °C; Rf 0.34 (10 % MeOH/CHCl3 ); 1 H NMR (CD3 OD, 500 MHz) d 0.96 (p, J = 7.5 Hz, 2H), 1.24 (p, J = 7.5 Hz, 2H), 1.40 (p, J = 7.5 Hz, 2H), 2.17 (t, J = 7.0 Hz, 2H), 3.63 (s, 3H), 3.93 (t, J = 6.0 Hz, 2H), 6.70 (dd, J = 9.5 Hz, 2.0 Hz, 2H), 6.73 (d, J = 2.0 Hz, 2H), 7.03 (d, J = 9.5 Hz, 2H), 7.18 (d, J = 8.0 Hz, 1H), 8.17 (dd, J = 8.0 Hz, 2.0 Hz, 1H), 8.57 (d, J = 2.0 Hz, 1H); 1 3C NMR (CD3 OD, 125 MHz) d 25.5, 26.4, 29.0, 34.4, 52.0, 66.9, 96.3, 104.6, 116.3, 123.1, 131.9, 133.3, 133.5, 134.4, 141.1, 142.9, 155.2, 159.0, 165.6, 175.6; IR(film, cm-1) n 1582, 1717, 2912, 3584; MS (ESI) m/z 587 (M+H)+; Anal. Calcd for C2 7H2 3O7 I: C, 55.30; H, 3.95. Found: C, 55.02; H, 3.89. 5-(2-Trimethylsilylethynyl)fluorescein(5-methoxycarbonyl)pentyl Ester 1 4 . 5Iodofluorescein(5-methoxycarbonyl)pentyl ester 13 (150 mg, 0.26 mmol), trimethylsilylacetylene (126 mg, 1.28 mmol), tetrakis(triphenylphosphine)palladium (15 mg, 0.01 mmol), copper(I) iodide (5 mg, 0.03 mmol), and triethylamine (78 mg, 0.77 mmol) were dissolved in 5 mL DMF in a Schlenk tube. The solution was freeze-pump-thawed three times, then stirred 17 h at 25 °C under N2 . The solution was concentrated in vacuo then purified by flash chromatography eluting with 5 % MeOH/CHCl3 yielding the title compound as an orange film (121 mg, 85%): 1 H NMR (CDCl3 ), 500 MHz) d 0.31 (s, 9H), 1.07 (p, J = 7.5 Hz, 2H), 1.34 (p, J = 7.5 Hz, 2H), 1.49 (p, J = 7.5 Hz, 2H), 2.21 (t, J = 7.5 Hz, 2H), 3.64 (s, 3H), 3.97 (t, J = 6.5 Hz, 2H), 6.79 (dd, J = 9.5 Hz, 2.0 Hz, 2H), 6.88 (d, J = 2.0 Hz, 2H), 6.95 (d, J = 9.0 Hz, 2H), 7.27 (d, J = 7.5 Hz, 1H), 7.79 (dd, J = 8.0 Hz, 2.0 Hz, 1H), 8.30 (d, J = 2.0 Hz, 1H);

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C NMR

10

(CDCl3 , 125 MHz) d –0.2, 24.3, 25.2, 27.9, 33.6, 51.5, 65.6, 98.0, 102.6, 103.8, 114.6, 122.2, 125.3, 130.3, 130.4, 130.8, 133.7, 134.4, 135.4, 154.8, 157.7, 164.6, 173.8, 175.6; MS (ESI) m/z 557 (M+H)+ . 5-Ethynylfluorescein(5-carboxy)pentyl Ester 15. 5-Ethynylfluorescein-(5-methoxycarbonyl)pentyl ester (50 mg, 90 mmol) was dissolved in 4 mL THF and 1 mL water. The orange solution was cooled to 0 °C. Lithium hydroxide (11 mg, 0.27 mmol) was added dropwise (solution in 1 mL water) and the reaction was stirred at 0 °C for 6 h. The reaction was quenched by addition of a few drops acetic acid then adsorbed onto silica. Flash chromatography eluting with 10 to 100% MeOH/CH2 Cl2 followed by chromatography on C18-silica (0 to 100% MeOH/water) yielded the desired product in pure form (29 mg, 69%) as a bright orange solid. Mp 170 °C (dec); Rf 0.27 (10% MeOH/CH2 Cl2 ); 1 H NMR (CD3 OD, 500 MHz) d 0.97 (m, 2H), 1.24 (p, J = 7.0 Hz, 2H), 1.41 (m, 2H), 2.09 (t, J = 7.5 Hz, 2H), 3.81 (s, 1H), 3.93 (t, J = 6.0 Hz, 2H), 6.65 (dd, J = 9.0 Hz, 2.0 Hz, 2H), 6.67 (d, J = 2.5 Hz, 2H), 6.96 (d, J = 9.0 Hz, 2H), 7.41 (d, J = 8.0 Hz, 1H), 7.87 (dd, J = 7.5 Hz, 2.0 Hz, 1H), 8.28 (d, J = 1.5 Hz, 1H); IR (KBr, cm-1) n 1591, 1722, 2921, 3395; MS (ESI) m/z 471 (M+H)+ . Cassette 2. 5-Ethynylfluorescein(5-carboxy)pentyl ester 15 (22 mg, 47 mmol), 4bromotetramethylrosamine hydroxide 6 (22 mg, 47 mmol), tetrakis(triphenylphosphine) palladium (3 mg, 2 mmol), copper(I) iodide (1 mg, 5 mmol), and triethylamine (24 mg, 234 mmol) were dissolved in 2 mL DMF in a Schlenk tube. The reaction mixture was freeze-pump-thawed three times then stirred at 75 °C under nitrogen for 8 h. The dark violet solution was concentrated in vacuo then purified by flash chromatography eluting with 10 to 100% MeOH/CH2 Cl2 then 10% AcOH/MeOH then further purified by reversed phase preparative HPLC (40 to 90% MeCN/H2 O over 30 min) to give the desired product as maroon solid (23 mg, 61%). Mp 190 °C (dec); Rf 0.35 (15 % MeOH/CH2 Cl2 ); 1 H NMR (1:1 CD3 OD/CDCl3 , 500 MHz) d 1.14 (m, 2H), 1.42 (p, J = 7.5 Hz, 2H), 1.49 (p, J = 7.5 Hz, 2H), 2.20 (t, J = 7.5 Hz, 2H), 3.33 (s, 12H), 4.00 (t, J = 6.5 Hz, 2H), 6.93 (d, J = 2.5 Hz, 2H), 7.06 (m, 4H), 7.18 (d, J = 1.5 Hz, 2H), 7.36 (d, J = 9.0 Hz, 2H), 7.42 (d, J = 9.5 Hz, 2H), 7.47 (d, J = 8.0 Hz, 1H), 7.52 (d, J = 8.0 Hz, 2H), 7.90 (d, J = 8.0 Hz, 2H), 8.04 (dd, J = 8.0 Hz, 1.5 Hz, 2H), 8.49 (d, J = 2.0 Hz, 1H);

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C NMR (1:1

CD3 OD/CDCl3 , 125 MHz) d 25.1, 26.0, 28.7, 34.4, 41.0, 66.7, 89.9, 92.1, 97.4, 103.7, 114.1, 115.3, 116.8, 121.9, 125.5, 126.7, 130.7, 131.3, 131.6, 132.3, 132.7, 133.0, 133.4, 134.8, 135.0, 136.3, 158.0, 158.4, 158.8, 159.6, 165.3, 173.7, 176.8; IR(film, cm-1) n 1601, 1732, 2931, 3420; MS (ESI) m/z 811 (M+H)+ . Preparation of Cassette 3. 5-Ethynylfluorescein(5-carboxy)pentyl ester 15 (22 mg, 47 mmol), 4-bromorosamine-x hydroxide 7 (25 mg, 47 mmol), tetrakis(triphenylphosphine) palladium (3 mg, 2 mmol), copper(I) iodide (1 mg, 5

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mmol), and triethylamine (24 mg, 234 mmol) were dissolved in 2 mL DMF in a Schlenk tube. The reaction mixture was freeze-pump-thawed three times then stirred at 75 °C under nitrogen for 8 h. The dark violet solution was concentrated in vacuo then purified via flash chromatography eluting with 10 to 100% MeOH/CH2 Cl2 to 2:4:1:3 n-BuOH/n-PrOH/EtOAc/H2 O then further purified by reversed phase preparative HPLC (40 to 90% MeCN/H2 O over 30 min) to give the desired product as dark green solid (32 mg, 75%). Mp 225 °C (dec); Rf 0.38 (15 % MeOH/CH2 Cl2 ); 1 H NMR (1:1 CD3 OD/CDCl3 , 500 MHz) d 1.16 (m, 2H), 1.43 (p, J = 7.5 Hz, 2H), 1.50 (p, J = 7.5 Hz, 2H), 1.99 (p, J = 5.0 Hz, 4H), 2.11 (p, J = 5.0 Hz, 4H), 2.20 (t, J = 7.5 Hz, 2H), 2.74 (t, J = 6.0 Hz, 4H), 3.06 (t, J = 6.0 Hz, 4H), 3.51 (t, J = 6.0 Hz, 4H), 3.55 (t, J = 6.0 Hz, 4H), 4.00 (t, J = 6.5 Hz, 2H), 6.85 (s, 2H), 7.10 (d, J = 9.0 Hz, 2H), 7.23 (d, J = 1.0 Hz, 2H), 7.37 (d, J = 9.5 Hz, 2H), 7.41 (d, J = 9.0 Hz, 2H), 7.44 (d, J = 7.5 Hz, 1H), 7.86 (d, J = 8.0 Hz, 2H), 8.02 (dd, J = 8.0 Hz, 1.5 Hz, 1H), 8.48 (d, J = 1.5 Hz, 1H);

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C NMR (1:1 CD3 OD/CDCl3 , 125 MHz) d

20.3, 20.5, 21.2, 24.9, 25.9, 28.2, 28.6, 34.3, 51.0, 51.5, 66.6, 89.4, 92.3, 103.6, 106.1, 113.2, 117.0, 121.7, 124.67, 124.69, 126.8, 127.1, 130.4, 131.1, 131.4, 132.6, 132.8, 133.5, 134.1, 134.9, 136.2, 152.0, 153.0, 154.3, 159.5, 165.1, 173.3, 176.6; IR(film, cm-1) n 1306, 1596, 1688, 2955, 3483; MS (ESI) m/z 915 (M+H)+ . Preparation of Cassette 4. Cassette 4 tert-Butyl Ester 16. 5-Ethynylfluorescein(5-tert-butoxycarbonyl)pentyl ester 11 (45 mg, 86 mmol), 4-bromorosamine hydroxide 8 (49 mg, 86 mmol), tetrakis(triphenylphosphine) palladium (5 mg, 4 mmol), copper(I) iodide (2 mg, 9 mmol), and triethylamine (0.12 mL, 860 mmol) were dissolved in 2 mL DMF in a Schlenk tube. The reaction mixture was freeze-pump-thawed three times then stirred at 75 °C under nitrogen for 24 h. The dark blue solution was concentrated in vacuo then purified by flash chromatography eluting with 2 to 10% MeOH/CH2 Cl2 to give the desired product as a dark blue solid (41 mg, 47 % yield). Rf 0.25 (10 % MeOH/CH2 Cl2 ); 1 H NMR (1:1 CD3 OD/CDCl3 , 500 MHz) d 1.07 – 1.12 (m, 2H), 1.34 – 1.38 (m, 2H), 1.39 (s, 9H), 1.41 – 1.48 (m, 2H), 1.50 (s, 12H), 1.84 (d, J = 1.0 Hz, 6H), 2.11 (t, J = 7.3 Hz, 2H), 3.16 (s, 6H), 3.99 (t, J = 6.3 Hz, 2H), 5.59 (d, J = 1.0 Hz, 2H), 6.70 (s, 2H), 6.79 (dd, J = 9.3, 2.2 Hz, 2H), 6.86 (d, J = 2.2 Hz, 2H), 6.96 (s, 2H), 7.06 (d, J = 9.3 Hz, 2H), 7.39 (d, J = 7.8 Hz, 1H), 7.49 (d, J = 8.0 Hz, 2H), 7.89 (d, J = 8.0 Hz, 2H), 7.96 (dd, J = 7.8 Hz, 1.6 Hz, 1H), 8.44 (d, J = 1.6 Hz, 1H);

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C NMR (1:1 CD3 OD/CDCl3 , 75 MHz) d 18.4, 24.7, 25.4, 28.1, 29.3, 33.1, 35.4, 38.9,

60.2, 66.0, 68.4, 80.8, 90.0, 91.0, 95.6 104.0, 113.7, 115.2, 122.0, 124.0 124.9, 125.1, 125.6, 128.9, 129.7, 130.3, 131.3, 132.5, 132.6, 133.0, 134.3, 134.4, 135.4, 152.9, 153.6, 154.0, 157.5, 158.5, 165.1, 168.3, 173.7; MS (ESI) m/z 999 (M+ ). Cassette 4. Compound 16 (40 mg, 39 mmol) was dissolved in 2 mL CH2Cl2. To this solution, tri-iso-propylsilane (0.2 mL) and trifluoroacetic acid (1 mL) were added. The mixture was stirred at 25

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°C under nitrogen for 24 h. The reaction mixture was concentrated in vacuo then purified by reversed phase preparative HPLC (40 to 90% MeCN/H2O over 30 min) to give the desired product as a dark blue solid (25 mg, 67 % yield). 1H NMR (1:1 CD3OD/CDCl3, 500 MHz) d 1.06 – 1.11 (m, 2H), 1.32 – 1.38 (m, 2H), 1.43 – 1.50 (m, 2H), 1.51 (s, 12H), 1.84 (d, J = 1.0 Hz, 6H), 2.18 (t, J = 7.3 Hz, 2H), 3.17 (s, 6H), 3.99 (t, J = 6.3 Hz, 2H), 5.61 (d, J = 1.0 Hz, 2H), 6.73 (s, 2H), 6.82 (dd, J = 9.3, 2.2 Hz, 2H), 6.89 (d, J = 2.2 Hz, 2H), 6.97 (s, 2H), 7.11 (d, J = 9.3 Hz, 2H), 7.41 (d, J = 7.8 Hz, 1H), 7.50 (d, J = 8.5 Hz, 2H), 7.90 (d, J = 8.5 Hz, 2H), 7.98 (dd, J = 7.8, 1.7 Hz, 1H), 8.45 (d, J = 1.7 Hz, 1H);

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C NMR (1:1

CD3OD/CDCl3, 75 MHz) d 18.6, 24.9, 25.9, 28.5, 29.4, 33.3, 34.2, 60.6, 66.5, 90.1, 91.8, 96.0, 104.0, 114.2, 116.1, 122.1, 122.5, 124.4, 125.4, 126.0, 126.1, 130.3, 131.2, 131.6, 133.0, 133.3, 133.5, 134.1, 134.9, 136.0, 154.1, 154.5, 158.7, 159.0, 165.3, 174.8, 176.6; MS (ESI) m/z 943 (M+ ). Preparation of 4-Bromorosamine 5. 3-Aminophenol (2.00 g, 18.35 mmol) and 4-bromobenzaldehyde (0.1.70 g, 9.17 mmol) were suspended in 20 mL 6:4 sulfuric acid/water. The reaction mixture was refluxed gently for 24 h. The dark brown mixture was neutralized with ca. 52 mL 10 M potassium hydroxide to pH ª 7. 100 mL water and 100 mL 5 % iPrOH/CH2Cl2 were added. After separation, the aqueous layer was further extracted with 100 mL 5 % iPrOH/CH2Cl2 twice.

The combined organic layers were dried with Na2SO4 and

concentrated in vacuo. The dark brown residue was further purified by flash chromatography (5 to 15% MeOH/CH2Cl2) to give the title compound (the faster eluting unoxidized product was discarded) as a red-orange solid (100 mg, 8 % yield). Rf 0.10 (10 % MeOH/CH2 Cl2 ); 1 H NMR (1:1 CD3 OD/CDCl3 , 500 MHz) d 6.79 (d, J = 2.2 Hz, 2H), 6.82 (dd, J = 9.3 Hz, 2.2 Hz, 2H), 7.21 (d, J = 9.3 Hz, 2H), 7.27 (d, J = 8.5 Hz, 2H), 7.76 (d, J = 8.5 Hz, 2H);

13

C NMR (1:1 CD3 OD/CDCl3 , 125 MHz) d 98.4, 113.8, 117.7,

125.3, 131.5, 131.6, 132.6, 132.7, 157.5, 159.0, 160.3; MS (ESI) m/z 365/367 (M+ ). Preparation of 4-Bromorosamine 6. 3-Dimethylaminophenol (1.00 g, 7.29 mmol) and 4-bromobenzaldehyde (0.67 g, 3.64 mmol) were suspended in 10 mL 6:4 sulfuric acid/water. The reaction mixture was refluxed gently for 22 h. The dark violet mixture was neutralized with ca. 26 mL 10 M potassium hydroxide to pH ª 7. The dark violet precipitate was collected by filtration, rinsed with water then brine and again water, and dried in vacuo. The solid was further purified by flash chromatography (5 to 10% MeOH/CH2Cl2) to give the title compound (the faster eluting unoxidized product was discarded) as a dark violet solid (184 mg, 12 % yield). Mp = 135 °C (dec); Rf 0.24 (10 % MeOH/CH2 Cl2 ); 1 H NMR (CDCl3 , 500 MHz) d 3.37 (s, 6H), 6.86 (d, J = 2.5 Hz, 2H), 7.01 (dd, J = 9.5 Hz, 2.0 Hz, 2H), 7.27 (d, J = 8.5 Hz, 2H), 7.32 (d, J = 9.5 Hz,

13

2H), 7.76 (d, J = 8.5 Hz, 2H);

13

C NMR (CDCl3 , 125 MHz) d 41.2, 96.9, 113.2, 114.6, 125.0, 130.6,

131.0, 131.4, 132.3, 156.3, 157.2, 157.6; IR(film, cm-1) n 1194, 1347, 1598, 1650, 2935, 3462; MS (ESI) m/z 421/423 (M+ ). Preparation of 4-Bromorosamine 7. 8-Hydroxyjulolidine (1.00 g, 5.28 mmol) and 4-bromobenzaldehyde (0.49 g, 2.64 mmol) were suspended in 10 mL 6:4 sulfuric acid/water. The reaction mixture was heated to 90 °C at which point everything dissolved. Stirring was continued at 90 °C for 18 h. The dark violet mixture was neutralized with ca. 26 mL 10 M sodium hydroxide to pH ª 7.

The dark violet precipitate was collected by

filtration; rinsed with water, brine, then water; and dried in vacuo. The solid was further purified by flash chromatography eluting with 5 to 10% MeOH/CH2Cl2 (the faster eluting unoxidized product was discarded) to give the title compound as a dark violet solid (483 mg, 35 % yield). Mp 140 °C (dec); Rf 0.18 (10 % MeOH/CH2 Cl2 ); 1 H NMR (CDCl3 , 500 MHz) d 1.99 (p, J = 5.5 Hz, 4H), 2.10 (p, J = 5.5 Hz, 4H), 2.70 (t, J = 6.0 Hz, 4H), 3.02 (t, J = 6.0 Hz, 4H), 3.53 (t. J = 5.5 Hz, 4H), 3.56 (t. J = 5.5 Hz, 4H), 6.73 (s, 2H), 7.20 (d, J = 8.5 Hz, 2H), 7.87 (d, J = 8.5 Hz, 2H);

13

C NMR (CDCl3 , 125 MHz) d 19.5,

19.8, 20.5, 27.5, 50.4, 50.8, 105.4, 112.4, 123.8, 124.0, 126.1, 130.9, 131.5, 132.0, 151.1, 152.0, 152.6; IR(film, cm-1) n 1296, 1495, 1596, 2931, 3391; MS (ESI) m/z 525/527 (M+ ). Preparation of 4-Bromorosamine 8. 7-Hydroxy-N-methyl-2,2,4-trimethy-l,2-dihydroquinoline3 (1.00 g, 4.93 mmol) and 4bromobenzaldehyde (0.41 g, 2.47 mmol) were weighed into a 10 mL round bottom flask. The mixture was heated to 160 o C and the stirring was continued at 160 o C for 18 h. The dark blue slurry mixture was dissolved in 1 mL 1:1 MeOH/CH2Cl2 and purified by flash chromatography eluting with 5 to 10% MeOH/CH2Cl2 (the faster eluting unoxidized product was discarded) to give the title compound as a dark blue solid (90 mg, 5 % yield). Rf 0.20 (10 % MeOH/CH2Cl2); 1H NMR (CDCl3, 500 MHz) d 1.46 (s, 12H), 1.79 (d, J = 1.2 Hz, 6H), 3.17 (s, 6H), 5.49 (d, J = 1.2 Hz, 2H), 6.1 (s, 2H), 6.85 (s, 2H), 7.27 (d, J = 8.3 Hz, 2H), 7.76 (d, J = 8.3 Hz, 2H);

13

C NMR (CDCl3, 125 MHz) d 18.4, 29.3, 33.3, 59.8,

96.2, 113.5, 121.4, 123.6, 124.8, 125.4, 130.9, 131.5, 132.3, 132.7, 152.8, 153.3, 158.3; MS (ESI) m/z 553/555 (M+ ). (1) Jiao, G.-S.; Han, J. W.; Burgess, K., J. Org. Chem. submitted. (2) Dyke, C. A.; Bryson, T. A. Tetrahedron Lett. 2001, 42, 3959-3961. (3) Mao, F.; Leung, W. Y.; Haugland, R. P. 1999, WO Patent, 99/15517.

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2. Absorption Spectra of Compounds 1 – 8 Procedure for Measurement of Absorption Spectra. UV/Visible spectra were recorded in 1 cm path length quartz cuvettes on a HP 8452 Diode-Array UV/Visible spectrophotometer, with absolute ethanol (APER Alcohol and Chemical Co.) as the solvent. Equimolar solutions were prepared by assuming that the extinction coefficient of the cassettes is the sum of that of the donor and the acceptors, ie, ecassette = edonor + eacceptor Stock solutions of compounds 1 – 8 were prepared first. The equimolar solutions of cassettes 1 – 4 were prepared by diluting the stock solutions of compounds 1 – 4 (the solution of cassette 1 was randomly prepared with A1, 512 is between 0.5 to 1.0) until A1, 512/(e15, 512 + e5, 512) = A2, 512/(e15, 512 + e6, 512) = A3, 512/(e15, 512 + e7, 512) = A4, 512/(e15, 512 + e8, 512) = C (equimolar concentration) (Where, e15, 512 and e5, 512 were directly measured; e6, 512, e7, 512, and e8, 512 were deduced as a function of e15, 512). A solution containing acceptor 5 at the same concentration as the cassette 1 solution was prepared by diluting the stock solution of acceptor 5 until A5, 514/e5, 514 = A1, 514/(e15, 514 + e5, 514) The equimolar solutions of acceptor 6 – 8 to corresponding cassettes 2 – 4 were prepared by diluting the stock solutions of acceptors 6 – 8 until equivalent absorbance at the acceptor absorbance maxima to corresponding cassettes 2 – 4, ie Aacceptor = Acassette

15

Figure S1. Absorption Spectra of Equimolar Cassettes 1 – 4 in Absolute Ethanol. 0.70 0.60

absorbance

0.50

1 2 3 4

0.40 0.30 0.20 0.10 0.00 400

450

500

550

wavelength (nm)

600

650

16

Figure S2. Normalized Absorption Spectra of Compounds 5 – 8 (Acceptors) and 15 (Donor fragment) in Absolute Ethanol. 1.1 1 5 6 7 8 15

normalized absorbance

0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 380

430

480

530

580

wavelength (nm)

630

680

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3. Tabulated Spectroscopic Properties of Compounds 1 – 8 Table S1. Spectroscopic Data of compounds 1 – 8 in Absolute Ethanol. compound

a

absorption maximum l abs (nm)

extinction emission energy fluorescence coefficient maximum lem transfer (ET) enhancement e (cm-1M-1) a efficiency Icassette/Iacceptor b (nm)

relative fluorescence intensity c

1

510

89600

538

-d

1.8 (1.3)

1.00 (1.00)

2

512 556

65600 62200

582

> 95 %

4.0 (2.4)

0.55 (0.52)

3

512 582

51800 71000

603

> 95 %

5.0 (3.8)

0.51 (0.52)

4

512 592

51600 66700

616

> 95 %

7.0 (4.6)

0.44 (0.41)

5

514

51100

532

-e

-e

-e

6

554

60400

579

-e

-e

-e

7

580

70300

602

-e

-e

-e

8

594

68400

616

-e

-e

-e

Measured at the absorption maxima. b Calculated as the ratio of the fluorescence intensity of cassettes to that of corresponding acceptors with excitation at 488 nm. The values in parentheses are those with excitation at 514 nm. c Excited at 488 nm. The values in parentheses are those with excitation at 514 nm. d Not determined. e Not applicable.

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4. Demonstration of the Photostabilities of the Cassettes 1 – 4 Relative to Fluorescein Procedure for Measurement of Relative Photostabilities. A 1000 W tungsten lamp (Oriel Corporation) was employed, with a long-pass filter (1 M KNO3 , 10 cm path; l > 340 nm), as the irradiation source. Each sample, dissolved in 7:3 absolute ethanol/aqueous Tris•HCl solution (50 mM, pH = 8.8), was placed in a 1 cm path length quartz fluorescence cuvette and irradiated for several periods of 10 min (1 h in total). After each irradiation, the fluorescence spectrum was measured on a SLM-Aminco 8100 Spectrofluorometer with excitation at 488 nm.

Figure S3. Photostability of Cassettes 1 – 4 Relative to Fluorescein (F* ). 1.1

normalized fluorescence intensity

1 0.9 0.8 0.7 0.6 0.5

1 2 3 4 F*

0.4 0.3 0.2 0.1 0 0

10

20

30 time (min)

40

50

60