Supporting information for:
A Novel Fluorescent Sensor for hydrophobic Amines in
Aqueous Solution
Chun Ren, Jae Seung Lee, Timothy E. Glass*
Department of Chemistry, University of Missouri, Columbia, MO 65211
Email: glasst@missouri.edu
Contents:
1. Experimental procedures and copies of 1H and 13C NMR spectra for compounds
1, 3-9.
2. Additional fluorescence titration spectra of sensor 1.
1. EXPERIMENTAL PROCEDURES
General Fluorescence Titration Procedures. Fluorescence spectra were obtained on a
Shimadzu RF-5301 PC Spectrofluorophotometer. Solutions of sensor (5x10-5 ~ 10-6 M) were
prepared in buffer (20 mM HEPES, 100 mM NaCl, adjusted to pH = 7.0). This solution was
placed in a cuvette (1 ml), then titrated with a solution of the hydrophobic amines (3~118 mM in
20 mM HEPES, pH = 7.0 with 5x10-5 ~ 10-6 M sensor). The fluorescence change and
corresponding concentration of guest were recorded. This data was fitted to a single site binding
isotherm using Graphpad Prism 3.03.
General Synthetic Procedures. All reactions were performed in dried glassware under nitrogen
atmosphere unless otherwise noted. Benzene, tetrahydrofuran, and toluene were distilled from
sodium/benzophenone under nitrogen immediately before use. Dichloromethane (DCM) and
triethylamine (TEA) were distilled from calcium hydride under nitrogen immediately before use.
Flash column chromatography was carried out with 32-63 μm silica gels. NMR spectra were run
with a Bruker ARX-250 MHz, DRX-300 MHz, or DRX-500 MHz in CDCl3, CD3OD, or d6DMSO using tetramethylsilane (TMS) as a reference. IR spectra were run with a Thermo
Scientific Auxiliary Equipment Module (AEM) for Nicolet FT-IR spectrometers. Mass spectra
were run with a Bruker Apex-Qe FTMS at the Old Dominion University in Norfolk, VA.
Control compound 3: To a solution of naphthalene dimer (72 mg, 0.14 mmol) in THF (3 ml)
and methanol (3 ml) was dropwise added NaOH solution (1 ml, 6.0 N). The mixture was
vigorously stirred at room temperature for 16 hours, and then quenched with HCl solution (1.0 N,
10 ml) and extracted with DCM/MeOH three times. The organic layers were combined and dried
over sodium sulfate. After the solvents were stripped, the concentrate was triturated with DCM
two times (10 ml x 2) to obtain compound 3 as a light pink solid (53 mg, 83% yield). m.p.: >200
˚C; 1H-NMR (500 MHz, d6-DMSO) δ: 13.02 (s, br, 2H), 8.67 (d, 2H, J = 9.0 Hz), 7.58 (d, 2H, J
= 9.0 Hz), 7.26 (dd, 2H, J = 2.8, 9.0 Hz), 7.19 (d, 2H, J = 2.5 Hz), 7.11 (d, 2H, J = 9.0 Hz), 6.35
(s, 1H), 5.56 (s, 1H), 4.73 (s, 4H), 2.41 (s, 2H); 13C-NMR (125 MHz, CDCl3) δ: 170.6, 154.3,
148.8, 130.6, 127.5, 126.7, 125.5, 119.7, 119.0, 118.5, 108.8, 91.4, 65.0, 26.6, 22.1; IR (KBr, cm1
): 1730, 1242; HRMS for M+Na calcd. For (C27H20O8)Na+ 495.1050, found 495.1043.
Diformylnaphthalene dimer 4: A solution of naphthalene dimer (250 mg, 0.473 mmol) and
dichloromethyl methyl ether (0.168 ml, 1.89 mmol) in DCM (40 ml) was chilled in ice bath for
15 minutes, followed by the dropwise addition of TiCl4 (1.89 ml, 1.0 M in DCM, 1.89 mmol).
The mixture was kept at 0 °C for 1.5 hours, then slowly warmed up to room temperature and
continued to stir 3 hours under nitrogen. The reaction was quenched with saturated NaHCO3 (100
ml) and extracted with DCM four times (30 ml x 4). The organic layers were combined, dried
over anhydrous MgSO4, filtered and concentrated to get crude product (283 mg). The
diformylnaphthalene dimer 4 was purified with flash silica column chromatography (3~4%
Et2O/DCM) and a yellow solid (181 mg, 0.31 mmol, 66% yield) was isolated. m.p.: 172-173˚C;
H-NMR (250 MHz, CDCl3) δ: 10.89 (s, 1H), 9.07 (d, 2H, J = 9.4 Hz), 8.65 (d, 2H, J = 9.5 Hz),
1
7.28 (d, 2H, J = 9.3 Hz), 7.21 (d, 2H, J = 9.5 Hz), 6.27 (s, 1H), 5.21 (s, 1H), 4.83 (s, 4H), 4.28 (q,
4H, J = 7.2 Hz), 2.43 (t, 2H, J = 2.5 Hz), 1.28 (t, 6H, J = 7.1 Hz); 13C-NMR (125 MHz, CDCl3) δ:
191.9, 168.1, 160.0, 149.5, 130.5, 127.6, 126.7, 125.5, 122.0, 118.4, 118.3, 113.5, 91.1, 66.5, 61.6,
26.6, 22.7, 14.0; IR (KBr, cm-1): 2974, 1759, 1671; HRMS for M+Na calcd. For (C33H28O10)Na+
607.1574, found 607.1573.
Bishydroxymethylnaphthalene dimer 5: To a solution of dialdehyde 4 (734 mg, 1.26 mmol) in
THF (20 ml) and ethanol (20 ml) in ice bath was added NaBH4 (118 mg, 3.14 mmol). The
reaction was kept at 0 °C for 30 minutes, and then quenched with hydrochloride acid (1.0 N) at
0 °C until no bubbles formed. The mixture was extracted with DCM three times (50ml x 3), dried
over sodium sulfate, and evaporated to get crude product (759 mg). After purification with flash
silica column chromatography (10% ~ 25% Et2O/DCM), the desired product 5 was isolated as a
white solid (670 mg, 90% yield). m.p.: 144 - 155˚C; 1H-NMR (300 MHz, CDCl3) δ: 8.45 (d, 2H,
J = 9.5 Hz), 7.95 (d, 2H, J = 9.0 Hz), 7.21 ~ 7.18 (m, 4H), 6.26 (s, 1H), 5.25 (s, 1H), 5.09 (q, 4H,
J = 12.0 Hz), 4.77 (dd, 4H, J = 8.5, 16 Hz), 4.20 (q, 4H, J = 7.5 Hz), 2.41 (s, 2H), 1.25 (t, 6H, J =
7.5 Hz); 13C-NMR (125 MHz, CDCl3) δ: 170.1, 152.0, 149.4, 129.4, 127.9, 125.5, 124.6, 124.4,
120.0, 118.9, 115.2, 91.6, 67.4, 61.9, 55.8, 27.2, 23.2, 14.3; IR (KBr, cm-1): 3428, 2968, 1742,
1207, 1107; HRMS for M+Na calcd. For (C33H32O10)Na+ 611.1888, found 611.1882.
Dibromide 6: To a solution of dialcohol 5 (670 mg, 1.14 mmol) in DCM (40 ml) was dropwise
added PBr3 (271 ml, 2.85 mmol). The mixture was vigorously stirred at room temperature under
nitrogen for 12 hours, and then quenched with icy NaHCO3. The mixture was extracted with
DCM three times (70 ml x3), dried over sodium sulfate, and evaporated to dryness in vacuo to get
product 6 as a gray solid (790 mg, 97% yield) which was used without further purification. m.p.:
174-176˚C; 1H-NMR (500 MHz, CDCl3) δ: 8.50 (d, 2H, J = 9.5 Hz), 7.86 (d, 2H, J = 9.5 Hz),
7.30 (d, 2H, J = 9.5 Hz), 7.19 (d, 2H, J = 9.0 Hz), 6.29 (s, 1H), 5.28 (s, 1H), 5.06 (dd, 4H, J =
10.0, 45.0 Hz), 4.81(s, 4H), 4.26 (q, 4H, J = 4.5 Hz), 2.44 (t, 2H, J = 2.5 Hz), 1.29 (t, 6H, J = 4.0
Hz);
C-NMR (125 MHz, CDCl3) δ: 168.9, 151.9, 149.4, 128.5, 127.5, 125.1, 123.6, 121.1,
13
120.1, 118..9, 114.5, 91.3, 67.0, 61.5, 26.8, 24.7, 23.0, 14.2; IR (KBr, cm-1): 2974, 1754, 1599,
1201; HRMS for M+Na calcd. For (C33H30Br2O8)Na+ 735.0200, found 735.0196.
Alkylated carbaozle 8: To a solution of 2-hydroxycarbazole (1 g, 5.3 mmol) in THF was added
K2CO3 (0.84 g, 6.3 mmol), and ethyl bromoacetate (0.65 ml, 5.8 mmol). The reaction was
vigorously stirred and refluxed under nitrogen for 16 hours, and then quenched with dilute
hydrochloric acid (50 ml 1.0 N HCl + 50 ml water). The mixture was extracted with DCM three
times (100 ml x3), dried over sodium sulfate, and evaporated to get crude product. The desired
product 8 was obtained after purification with flash silica column chromatography (DCM) to get
a gray solid (1.14 g, 80% yield). m.p.: 182-185˚C; 1H-NMR (300 MHz, CDCl3) δ: 7.99 ~ 7.93 (m,
3H), 7.38 ~ 7.32 (m, 2H), 7.24 ~ 7.19 (m, 1H), 6.97 (d, 1H, J = 2.1 Hz), 6.88 (dd, 1H, J = 2.4, 8.4
Hz), 4.72 (s, 2H), 4.29 (q, 2H, J = 7.1 Hz), 1.31 (t, 3H, J = 7.1 Hz); 13C-NMR (125 MHz, CDCl3)
δ: 169.2, 157.2, 140.6, 139.6, 124.9, 123.3, 121.2, 119.7, 118.2, 110.4, 108.3, 96.3, 66.2, 61.4,
14.2; IR (KBr, cm-1): 3334, 1740, 1430, 1229, 1180; HRMS for M+Na calcd. For
(C16H15NO3)Na+ 292.0944, found 292.0938.
8, tBuOK
8
Intermediate 9: To a solution of alkyloxycarbazole 8 (17 mg, 0.063 mmol) in DMF (3 ml) was
added tBuOK (32 ul, 20% wt in THF, 0.053 mmol), followed by the addition of dibromide 6 (15
mg, 0.021 mmol). The mixture was stirred under nitrogen for 24 hours, and then quenched with
hydrochloride acid (1.0 N, 1.0 ml) and evaporated to dryness under reduced pressure. The
concentrate was separated between DCM and water, and extracted with DCM three times (25 ml
x 3). The combined organic layers were dried over MgSO4, filtered, and concentrated to get crude
(34 mg). The desired half tube tetraester 9 was isolated as a gray amorphous solid (21 mg, 0.019
mmol, 87% yield) after purification with flash silica column chromatography (DCM ~ 4%
Et2O/DCM). m.p.: >200˚C; 1H-NMR (500 MHz, CDCl3) δ: 8.52 (d, 2H, J = 9.5 Hz), 7.92 (d, 2H,
J = 7.7 Hz), 7.88 (d, 2H, J = 8.5 Hz), 7.64 (d, 2H, J = 9.3 Hz), 7.35 (d, 2H, J = 8.2 Hz), 7.28 ~
7.22 (m, 4H), 7.12 (t, 2H, J = 7.4 Hz), 6.96 (d, 2H, J = 9.3 Hz), 6.87 (d, 2H, J = 1.9 Hz), 6.78 (dd,
2H, J = 2.1, 8.5 Hz), 6.36 (s, 1H), 5.62 (dd, 4H, J = 15.2, 86.2 Hz), 5.22 (s, 1H), 4.64 (s, 4H),
4.54 (s, 4H), 4.21 ~ 4.14 (m, 8H), 2.32 (s, 2H), 1.22 (t, 6H, J = 7.1 Hz), 1.17 (t, 6H, J = 7.1 Hz);
C-NMR (125 MHz, CDCl3) δ: 169.1, 169.0, 157.0, 151.7, 149.0, 141.8, 141.0, 129.6, 127.4,
13
124.6, 123.4, 123.0, 120.8, 120.0, 119.5, 119.3, 119.0, 118.9, 117.7, 114.2, 109.4, 107.8, 95.1,
91.2, 66.8, 65.9, 61.4, 61.2, 38.7, 26.8, 23.0, 14.1, 14.1; IR (KBr, cm-1): 2978, 1752, 1601, 1458,
1201, 1180; HRMS for M+Na calcd. For (C65H58N2O14)Na+ 1113.3780, found 1113.3805.
Sensor 1: To a solution of open tube tetraester in THF (6 ml) and methanol (8 ml) was dropwise
added a solution of NaOH (2 ml, 6 N). The mixture was vigorously stirred at room temperature
for 16 hours. After the solvents were stripped by nitrogen flow, dilute hydrochloric acid (5 ml, 1.6
N) was added to triturate the residue. A trituration with DCM may be needed if grease is
appreciable. The tetracarboxylic acid 1 was isolated as a black solid (5 mg, 93% yield).
m.p.: >200˚C; 1H-NMR (500 MHz, d6-DMSO) δ: 13.07 (s, br, 4H), 8.78 (d, 2H, J = 9.6 Hz), 7.96
(d, 4H, J = 8.6 Hz), 7.61 (d, 2H, J = 9.3 Hz), 7.53 ~ 7.50 (m, 4H), 7.24 (d, 2H, J = 1.7 Hz), 7.17
(t, 2H, J = 7.4 Hz), 7.07 (t, 2H, J = 7.4 Hz), 6.87 (d, 2H, J = 9.3 Hz), 6.77 (dd, 2H, J = 2.0, 8.5
Hz), 6.21 (s, 1H), 5.93 (dd, 4H, J = 15.3, 25.37 Hz), 5.55 (s, 1H), 4.96 (dd, 4H, J = 8.2, 16.5 Hz),
2.26 (s, 2H); 13C-NMR (125 MHz, d6-DMSO) δ: 170.8, 170.6, 157.4, 152.9, 148.5, 141.9, 140.7,
129.1, 127.2, 125.8, 124.7, 123.6, 122.8, 121.2, 119.9, 119.6, 119.3, 119.2, 118.4, 116.7, 114.9,
110.1, 108.0, 95.2, 91.1, 66.3, 65.1, 55.3, 38.5, 25.9, 22.1; IR (KBr, cm-1): 3423, 2918, 1728,
1598, 1462, 1180; HRMS for M+ calcd. For (C57H42N2O14)Na+ 1001.2528, found 1001.2520.
2. Fluorescence titration spectra
100
Binding isotherm
80
0.6
60
1-I/I0
0.4
40
0.2
20
0.0
0.000
0
376
426
476
0.001
0.002
0.003
Concentration (M)
Figure S1 Fluorescence titration of compound 1 with octylamine in buffer (20 mM
HEPES, 100 mM NaCl; pH = 7.0; [1] = 10-5 M; λex = 366 nm): a) Fluorescence emission
spectra as a function of added octylamine, b) Fit of the titration data at em = 386 nm to a
single-site binding isotherm.
600
Binding isotherm
500
0.8
400
0.6
1-I/I0
300
0.4
200
0.2
100
0.0
0.000
0
376
426
476
0.001
0.002
0.003
0.004
0.005
Concentration (M)
Figure S2 Fluorescence titration of compound 1 with hexanediamine in buffer (20 mM
HEPES, 100 mM NaCl; pH = 7.0; [1] = 10-5 M; λex = 366 nm): a) Fluorescence emission
spectra as a function of added hexanediamine, b) Fit of the titration data at em = 386 nm
to a single-site binding isotherm.
450
Binding isotherm
400
0.8
350
300
0.6
1-I/I0
250
200
150
0.4
0.2
100
50
0.0
0.000
0
376
426
476
0.001
0.002
0.003
0.004
0.005
Concentration (M)
Figure S3 Fluorescence titration of compound 1 with octanediamine in buffer (20 mM
HEPES, 100 mM NaCl; pH = 7.0; [1] = 10-5 M; λex = 366 nm): a) Fluorescence emission
spectra as a function of added octanediamine, b) Fit of the titration data at em = 386 nm
to a single-site binding isotherm.
Binding isotherm
400
350
1.0
300
0.8
250
1-I/I0
200
150
100
0.6
0.4
0.2
50
0.0
0.0000
0
376
426
476
0.0002
0.0004
0.0006
0.0008
Concentration (M)
Figure S4 Fluorescence titration of compound 1 with methylhexanamine in buffer (20
mM HEPES, 100 mM NaCl; pH = 7.0; [1] = 10-5 M; λex = 366 nm): a) Fluorescence
emission spectra as a function of added methylhexanamine, b) Fit of the titration data at
em = 386 nm to a single-site binding isotherm.
400
Binding isotherm
350
1.0
300
0.8
250
1-I/I0
200
150
100
0.6
0.4
0.2
50
0.0
0.0000
0
376
426
0.0002
476
0.0004
0.0006
0.0008
Concentration (M)
Figure S5 Fluorescence titration of compound 1 with tuaminoheptane in buffer (20 mM
HEPES, 100 mM NaCl; pH = 7.0; [1] = 10-5 M; λex = 366 nm): a) Fluorescence emission
spectra as a function of added tuaminoheptane, b) Fit of the titration data at em = 386 nm
to a single-site binding isotherm.
350
Binding isotherm
300
0.8
250
0.6
1-I/I0
200
150
100
0.4
0.2
50
0
-50
376
426
476
0.0
0.000
0.002
0.004
0.006
0.008
Concentration (M)
Figure S6 Fluorescence titration of compound 1 with heptaminol in buffer (20 mM
HEPES, 100 mM NaCl; pH = 7.0; [1] = 10-5 M; λex = 366 nm): a) Fluorescence emission
spectra as a function of added heptaminol, b) Fit of the titration data at em = 386 nm to a
single-site binding isotherm.
400
binding isotherm
350
0.8
300
0.6
1-I/I0
250
200
150
0.4
0.2
100
50
0.0
0.000
0
376
426
0.001
0.002
0.003
0.004
0.005
-1
476
Concentration (M )
Figure S7 Fluorescence titration of compound 1 with phenethylamine in buffer (20 mM
HEPES, 100 mM NaCl; pH = 7.0; [1] = 10-5 M; λex = 366 nm): a) Fluorescence emission
spectra as a function of added phenethylamine, b) Fit of the titration data at em = 386 nm
to a single-site binding isotherm.
450
400
350
300
250
200
150
100
50
0
Binding Isotherm
0.8
1-I/I0
0.6
0.4
0.2
0.0
0.000
376
426
476
0.001
0.002
0.003
0.004
Concentration (M)
Figure S8 Fluorescence titration of compound 1 with pseudoephedrine in buffer (20 mM
HEPES, 100 mM NaCl; pH = 7.0; [1] = 10-5 M; λex = 366 nm): a) Fluorescence emission
spectra as a function of added pseudoephedrine, b) Fit of the titration data at em = 386
nm to a single-site binding isotherm.