Fluorescent, Through-Bond Energy Transfer Cassettes for Labeling Multiple Biological Molecules in One Experiment

Article abstract of DOI:10.1021/ja037193l

Through-bond energy transfer cassettes based on a fluorescein donor component electronically conjugated to rhodamine-like acceptors have been designed and synthesized. They absorb strongly at 488 nm (Ar-laser emission) and efficiently transfer the energy

Full text of DOI:10.1021/ja037193l

1
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
2
3
4
Syntheses of Compounds 1 – 8
Absorption Spectra of Compounds 1 – 8
Tabulated Spectroscopic Properties of Compounds 1 – 8
Demonstration of the Photostabilities of Cassettes 1 – 4 Relative to Fluorescein

2
1. Syntheses of Compounds 1 – 8
Scheme S1. Preparation of Cassette 1.
TMS
I
O
O
K CO
2
MeOH/THF
3
TMS
CO H
2
O
O
o
Pd(PPh ) , CuI, Et N
3
25 C, 24 h
3 4
THF, 55 C, 1 h
o
AcO
O
OAc
AcO
O
OAc
HO
O
O
9 95 %
10 98 %
t
BuO C
2
+
H N
2
O
t
CO Bu
2
O
O
t
5
Pd(PPh ) , CuI
CO Bu
2
O
Br
3 4
4
O
O
O
K CO , DMF
o
Et N, DMF
3
75 C, 24 h
2
25 C, 24 h
3
o
O
O
HO
OH
H N
2
11 62 %
12 73 %
HO C
2
+
H N
2
O
O
O
TIS, TFA, CH Cl
2
2
O
O
o
25 C, 24 h
OH
H N
2
1 43 %

3
Scheme S2. Preparation of Cassette 2.
I
I
CO Me
2
O
CO Me
2
Br
CO H
2
O
4
K CO , DMF
o
2
25 C, 24 h
3
O
O
HO
HO
O
O
13 50 %
TMS
CO Me
CO H
2
2
LiOH
O
O
O
THF/H O
TMS
Pd(PPh ) , CuI, Et N
2
O
o
0 C, 6 h
3 4
3
o
DMF, 25 C, 17 h
O
O
O
O
HO
HO
14 85 %
15 69 %
HO C
2
+
Me N
2
O
O
O
6
Pd(PPh ) , CuI
3 4
O
O
Et N, DMF
o
3
75 C, 8 h
OH
Me N
2
2 61 %

4
Scheme S3. Preparation of Cassette 3.
HO C
2
+
N
O
O
CO H
2
O
7
Pd(PPh ) , CuI
3 4
O
O
N
O
O
Et N, DMF
o
3
75 C, 8 h
O
O
HO
OH
15
3 75 %
Scheme S4. Preparation of Cassette 4.
t
BuO C
2
+
N
O
t
CO Bu
O
2
O
8
Pd(PPh ) , CuI
O
3 4
O
N
O
O
Et N, DMF
3
75 C, 24 h
o
O
O
HO
OH
11
16 47 %
HO C
2
+
N
O
O
O
TIS, TFA, CH Cl
2
2
O
N
O
o
25 C, 24 h
OH
4 67 %

5
Scheme S5. Preparation of 4-Bromorosamine 5.
Br
Br
OH
60 % H SO
2
+
4
H N
2
o
160 C, 24 h
CHO
+
H N
2
O
N H
2
5 8 %
Scheme S6. Preparation of 4-Bromorosamine 6.
Br
Br
OH
60 % H SO
+
2
4
N
o
160 C, 22 h
CHO
+
Me N
O
N Me
2
2
6 12%
Scheme S7. Preparation of 4-Bromorosamine 7.
Br
OH
Br
60 % H SO
2
4
+
o
N
90 C, 18 h
+
N
O
N
CHO
7 35 %

6
Scheme S8. Preparation of 4-Bromorosamine 8.
Br
Br
neat
+
o
N
OH
160 C, 18 h
+
N
O
N
CHO
8 5 %

7
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 (1H at 300 MHz, 13C at 75 MHz, 31P at 121 MHz) or Inova (1H at 500 MHz, 13C at
125 MHz) NMR spectrometers. Chemical shifts are reported in units of ppm relative to solvent (CDCl3:
7.27 ppm for 1H and 77.0 ppm for 13C; acetone-d6: 2.04 ppm for 1H and 29.9 ppm for 13C; DMSO-d6:
2.50 ppm for 1H and 39.5 ppm for 13C; CD3OD: 3.30 ppm for 1H and 49.0 ppm for 13C). 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). CH2Cl2, 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
1
film (0.78 g, 95% yield). Rf 0.55 (35 % EtOAc/hexanes); H 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); 13C 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,

8
rinsed with water, then dried in vacuo to give 5-ethynylfluorescein as an orange solid (445 mg, 98 %
1
yield). Mp >400 °C (dec); Rf 0.30 (50% EtOAc/hexanes); H 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); 13C
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 %
1
MeOH/CHCl3); H 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); 13C 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/CH2Cl2 to give the desired product as an orange solid (51
1
mg, 73 % yield). Rf 0.15 (10 % MeOH/CH2Cl2); H NMR (1:1 CD3OD/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+).

9
Cassette 1. Compound 12 (25 mg, 31 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 °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 an orange solid (10
1
mg, 43 % yield). H NMR (1:1 CD3OD/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
1
compound as a bright orange solid (225 mg, 50%). Mp 110-112 °C; Rf 0.34 (10 % MeOH/CHCl3); H
NMR (CD3OD, 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); 13C NMR (CD3OD, 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 C27H23O7I: C, 55.30; H, 3.95. Found: C,
55.02; H, 3.89.
5-(2-Trimethylsilylethynyl)fluorescein(5-methoxycarbonyl)pentyl Ester 1 4 .
5-
Iodofluorescein(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
1
title compound as an orange film (121 mg, 85%): 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,
13
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); 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/CH2Cl2 followed by
chromatography on C18-silica (0 to 100% MeOH/water) yielded the desired product in pure form (29 mg,
1
69%) as a bright orange solid. Mp 170 °C (dec); Rf 0.27 (10% MeOH/CH2Cl2); H NMR (CD3OD,
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), 4-
bromotetramethylrosamine 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/CH2Cl2 then 10% AcOH/MeOH then further purified
by reversed phase preparative HPLC (40 to 90% MeCN/H2O over 30 min) to give the desired product as
1
maroon solid (23 mg, 61%). Mp 190 °C (dec); Rf 0.35 (15 % MeOH/CH2Cl2); H NMR (1:1
CD3OD/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); 13C NMR (1:1
CD3OD/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

11
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/CH2Cl2 to 2:4:1:3 n-BuOH/n-PrOH/EtOAc/H2O then further purified by reversed phase
preparative HPLC (40 to 90% MeCN/H2O over 30 min) to give the desired product as dark green solid (32
1
mg, 75%). Mp 225 °C (dec); Rf 0.38 (15 % MeOH/CH2Cl2); H NMR (1:1 CD3OD/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); 13C NMR (1:1 CD3OD/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/CH2Cl2 to give the desired product as a dark blue solid (41
1
mg, 47 % yield). Rf 0.25 (10 % MeOH/CH2Cl2); H NMR (1:1 CD3OD/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); 13C NMR (1:1 CD3OD/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

12
°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
1
solid (25 mg, 67 % yield). H 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); 13C 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
1
red-orange solid (100 mg, 8 % yield). Rf 0.10 (10 % MeOH/CH2Cl2); H NMR (1:1 CD3OD/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); 13C NMR (1:1 CD3OD/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 %
1
yield). Mp = 135 °C (dec); Rf 0.24 (10 % MeOH/CH2Cl2); 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); 13C 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
1
0.18 (10 % MeOH/CH2Cl2); 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); 13C 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 4-
bromobenzaldehyde (0.41 g, 2.47 mmol) were weighed into a 10 mL round bottom flask. The mixture was
heated to 160 oC and the stirring was continued at 160 oC 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); 13C 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.

14
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
1
2
3
4
0.50
0.40
0.30
0.20
0.10
0.00
400
450
500
550
600
650
wavelength (nm)

16
Figure S2. Normalized Absorption Spectra of Compounds 5 – 8 (Acceptors) and 15 (Donor fragment) in
Absolute Ethanol.
1.1
1
5
0.9
6
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
7
8
15
380
430
480
530
580
630
680
wavelength (nm)

17
3. Tabulated Spectroscopic Properties of Compounds 1 – 8
Table S1. Spectroscopic Data of compounds 1 – 8 in Absolute Ethanol.
absorption
maximum
labs (nm)
extinction
coefficient maximum lem transfer (ET) enhancement fluorescence
emission
energy
fluorescence
relative
compound
b
e (cm-1M-1) a
efficiency Icassette/Iacceptor
intensity c
(nm)
538
d
1
2
510
89600
-
1.8 (1.3)
4.0 (2.4)
1.00 (1.00)
512
556
65600
62200
582
603
> 95 %
0.55 (0.52)
0.51 (0.52)
0.44 (0.41)
512
582
51800
71000
3
> 95 %
> 95 %
5.0 (3.8)
7.0 (4.6)
512
592
51600
66700
4
5
616
532
e
e
e
514
51100
-
-
-
e
e
e
6
7
8
554
580
594
60400
70300
68400
579
602
616
-
-
-
e
e
e
-
-
-
e
e
e
-
-
-
b
a Measured at the absorption maxima. 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
c
excitation at 514 nm. Excited at 488 nm. The values in parentheses are those with excitation at 514 nm.
e
d Not determined. Not applicable.

18
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
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
1
2
3
4
F*
0
10
20
30
40
50
60
time (min)