A Biliary Tract-Specific Near-Infrared Fluorescent Dye for Image-Guided Hepatobiliary

Surgery

Michael P. Luciano, Ph.D., Jung-Man Namgoong, MD, Roger R. Nani, Ph.D., So-Hyun Nam,
MD, Ph.D., Choonghee Lee, Ph.D., Il Hyung Shin, Ph.D., Martin J. Schnermann, Ph.D., and
Jaepyeong Cha, Ph.D.

Supporting Information - Contents

General Materials and Methods

Experimental Procedures
1H and 13C NMR Spectra
General Materials and Methods. Unless stated otherwise, reactions were conducted in oven-
dried glassware under an atmosphere of nitrogen or argon using anhydrous solvents (passed
through activated alumina columns). All commercially obtained reagents were used as received.
N-[(3-(anilinomethylene)-2-chloro-1-cyclohexen-1-yl)methylene]aniline monohydrochloride and
Indocyanine Green were purchased from Sigma-Aldrich (St. Louis, MO), IR-Dye800CW was
purchased from LI-COR (Lincoln, Nebraska). Flash column chromatography was performed using
reversed phase (100 Å, 20-40 micron particle size, RediSep® Rf Gold® Reversed-phase C18 or
C18Aq) and silica on a CombiFlash® Rf 200i (Teledyne Isco, Inc.). High-resolution LC/MS
analyses were conducted on a Thermo-Fisher LTQ-Orbitrap-XL hybrid mass spectrometer system
with an Ion MAX API electrospray ion source in negative ion mode. Analytical LC/MS was
performed using a Shimadzu LCMS-2020 Single Quadrupole utilizing a Kinetex 2.6 μm C18 100
Å (2.1 x 50 mm) column obtained from Phenomenex, Inc. Runs employed a gradient of 0→90%
MeCN/0.1% aqueous formic acid over 4.5 min at a flow rate of 0.2 mL/min. 1H NMR and 13C

NMR spectra were recorded on Bruker spectrometers (at 400 or 500 MHz or at 100 or 125 MHz)
and are reported relative to deuterated solvent signals. Data for 1H NMR spectra are reported as
follows: chemical shift (δ ppm), multiplicity, coupling constant (Hz), and integration. Data for 13C
NMR spectra are reported in terms of chemical shift. Absorption curves for quantum yield
measurements were performed on a Shimadzu UV-2550 spectrophotometer operated by UVProbe
2.32 software. Fluorescence traces were recorded on a PTI QuantaMaster steady-state
spectrofluorimeter operated by FelixGX 4.2.2 software, with 5 nm excitation and emission slit
widths, 0.1 s integration rate, and enabled emission correction. Data analysis and curve fitting were
performed using MS Excel 2011 and GraphPad Prism 8. See JOC Standard Abbreviations and
Acronyms for abbreviation:

(http://pubs.acs.org/userimages/ContentEditor/1218717864819/joceah_abbreviations.pdf).
Experimental Procedures

Compound 1, FNIR-7741 and UL-7662 were prepared as described previously.

S2. S13 (47 mg, 0.072 mmol) was dissolved in dry MeCN (3.6 mL) in a microwave tube equipped
with a magnetic stir bar and sealed. The solution was sparged with argon for 2 minutes. 2-
(methylamino)ethanol (23 μL, 0.29 mmol, 4 equiv) was added under argon and the reaction
mixture was heated to 80 °C in a sand bath. After 10 minutes, the reaction changed color from
green to dark blue and LC-MS indicated consumption of the starting material. The reaction mixture
was concentrated by a gentle N2 stream and purified by flash chromatography (silica, 0 → 15%
MeOH/CH2Cl2). The main blue fractions were combined and the solvent removed to afford S2
(30.0 mg, 60% yield) as a deep blue film. 1H NMR (400 MHz, Methanol-d4) δ 7.77 (d, J = 13.4
Hz, 2H), 7.39 (d, J = 7.4 Hz, 2H), 7.32 (t, J = 7.8 Hz, 2H), 7.17 – 7.10 (m, 4H), 5.94 (d, J = 13.4
Hz, 2H), 4.01 (t, J = 7.2 Hz, 4H), 3.89 (dd, J = 17.4, 5.0 Hz, 4H), 3.47 (s, 3H), 2.54 (t, J = 6.5 Hz,
4H), 2.33 (t, J = 7.0 Hz, 4H), 1.83 (dd, J = 13.9, 6.9 Hz, 6H), 1.77 – 1.72 (m, 4H), 1.65 (s, 12H)
ppm; 13C NMR (125 MHz, Methanol-d4) δ 178.97, 177.50, 170.39, 144.30, 144.07, 141.82,
129.49, 125.22, 124.39, 123.11, 110.61, 96.87, 61.15, 60.50, 45.03, 44.03, 36.08, 29.47, 27.51,
25.64, 24.07, 23.25 ppm; HRMS (ESI) calculated for C43H56N3O5 (M+H)+ 694.4214, observed
694.4206.

1 Nani, R. R.; Shaum, J. B.; Gorka, A. P.; Schnermann, M. J. Electrophile integrating smiles
rearrangement provides previously inaccessible C4-O-alkyl heptamethine cyanine fluorophores.
Org. Lett. 2015, 17 (2), 302.
2
Cha, J.; Nani, R. R.; Luciano, M. P.; Kline, G.; Broch, A.; Kim, K.; Namgoong, J. M.;
Kulkarni, R. A.; Meier, J. L.; Kim, P.; Schnermann, M. J. A chemically stable fluorescent marker
of the ureter. Bioorg. Med. Chem. Lett. 2018, 28, 2741.
3
Wang, L.; Jin, J.; Chen, X.; Fan, H.; Li, B. K. F.; Cheah, K.W.; Ding, N.; Ju, S.; Wong, W.T.,
Li, C. A cyanine based fluorophore emitting both single photon near-infrared fluorescence and
two-photon deep red fluorescence in aqueous solution. Org. Biomol. Chem. 2012, 10, 5366.
2. AcOH (7 μL, 0.116 mmol) and HATU (40.0 mg, 0.105 mmol) were dissolved in dry DMF (1.0
mL) under argon in a 1-dram vial. DIPEA (20 μL, 0.116 mmol) was added under argon and the
light yellow reaction was stirred at ambient temperature for 10 minutes. In a separate 1-dram vial,
S2 (11.0 mg, 0.015 mmol) was dissolved in dry DMF (500 μL) and sparged with argon. The
activated ester solution (150 μL) was added to the solution of S2 under argon and heated to 35 °C
in a sand bath for 1.5 h, during which time the color changed from blue to green and LC-MS
indicated consumption of the starting material. The solvent was removed by centrifugal
evaporation and the crude mixture was dissolved in saturated aqueous sodium bicarbonate (2 mL)
and purified by reversed-phased flash chromatography (C18Aq, 10 → 70% MeCN/H2O). The main
green fractions were combined and lyophilized to afford 2 (10.0 mg, 86% yield) as an iridescent
green solid. 1H NMR (500 MHz, Methanol-d4, compound exists as a mixture of rotamers, major
rotamer is designated using an asterisk (“*”), and minor rotamers are denoted “ §”) δ 8.13 (two
overlapping d, J = 14.1 Hz, 2H*, 2H§), 7.49 (d, J = 7.4 Hz, 2H*, 2H§), 7.43 – 7.38 (m, 2H*, 2H§),
7.32 (dd, J = 8.0, 2.7 Hz, 2H*, 2H§), 7.24 (td, J = 7.5, 2.4 Hz, 2H*, 2H§), 6.19 (d, J = 6.1 Hz, 2H*),
6.17 (d, J = 6.2 Hz, 2H§), 4.21-4.17 (m, 2H*, 2H§), 4.16-4.11 (m, 4H*, 4H§), 4.02-3.96 (m, 2H*,
2H§), 3.3 (s, 3H*), 3.21 (s, 3H§) 3.00 (s, 3H*), 2.86 (s, 3H§), 2.70 – 2.59 (m, 4H*, 4H§), 2.26 –
2.21 (m, 4H*, 4H§), 1.92 (d, J = 6.4 Hz, 2H*, 2H§), 1.87-1.81 (m, 4H*, 4H§), 1.77-1.73 (m, 4H*,
4H§), 1.73 (s, 12H*), 1.70 (s, 12H§) ppm; HRMS (ESI) calculated for C45H58N3O6 (M+H)+
736.4320, observed 736.4311.
S3. IR-783 (250 mg, 0.33 mmol) was dissolved in dry DMF (4.2 mL) in a microwave tube
equipped with a magnetic stirrer bar and sealed. The solution was sparged with argon for 2 min,
followed by the addition of 2-(Methylamino)ethanol (134 μL, 1.7 mmol) under argon. The solution
was heated to 80 °C in a sand bath for 10 min, during which time the color changed from green to
blue and LC-MS indicated consumption of the starting material. The reaction mixture was cooled,
precipitated into diethyl ether and then centrifuged at 4500 rpm for 5 min. The pellet was dissolved
in water (8 mL) and purified by reversed-phase chromatography (C18, 0 → 40% MeCN/H2O). The
product-containing fractions were combined and lyophilized to afford S3 (202 mg, 77% yield) as
a blue solid. 1H NMR (400 MHz, Methanol-d4) δ 7.77 (d, J = 13.3 Hz, 2H), 7.37 (d, J = 7.4 Hz,
2H), 7.32 (t, J = 7.7 Hz, 2H), 7.16 (d, J = 8.0 Hz, 2H), 7.11 (t, J = 7.5 Hz, 2H), 5.96 (d, J = 13.4
Hz, 2H), 4.1 – 4.0 (m, 4H), 3.95 – 3.85 (m, 4H), 3.48 (s, 3H), 2.87 (t, J = 6.8 Hz, 4H), 2.55 (t, J
= 6.6 Hz, 4H), 2.05 – 1.86 (m, 8H), 1.85 (t, J = 6.5 Hz, 2H), 1.65 (s, 12H) ppm; 13C NMR (125
MHz, Methanol-d4) δ 177.63, 170.31, 144.32, 144.08, 141.79, 129.49, 125.34, 124.33, 123.06,
110.66, 96.89, 61.20, 60.52, 52.01, 45.05, 44.01, 29.50, 26.90, 25.61, 23.68, 23.24 ppm; HRMS
(ESI) calculated for C41H56N3O7S2 (M+H)+ 766.3554, observed 766.3545.

3. S3 (94 mg, 0.12 mmol), glutaric anhydride (41 mg, 0.36 mmol) and DMAP (4.4 mg, 0.04 mmol)
were dissolved in dry DMF (2.5 mL) in a 20 mL reaction vial under argon. DIPEA (42 μL, 0.24
mmol) was added under argon and the reaction mixture was heated to 35 °C for 24 h during which
time the color changed from deep blue to green. The reaction mixture was precipitated into diethyl
ether and centrifuged for 5 min at 4500 RPM. The pellet was resuspended in H2O and directly
purified by reversed-phase flash chromatography (C18, 0 → 40% MeCN/H2O). The main green
fractions were pooled and lyophilized to afford 3 in 38% yield (40.0 mg) as an iridescent green
solid. 1H NMR (400 MHz, Methanol-d4, compound exists as a mixture of rotamers, major rotamer
is designated using an asterisk (“*”), and minor rotamers are denoted “§”) δ 8.13 (two overlapping
d, J = 14.3 Hz, 2H*, 2H§), 7.51 (two overlapping d, J = 12.9, 7.5, 1.2 Hz, 2H*, 2H§), 7.40 (t, J =
7.5, 2H*, 2H§), 7.32 (dd, J = 7.9, 2.2 Hz, 2H*, 2H§), 7.24 (td, J = 7.4, 2.4, 2H*, 2H§), 6.22 (d, J =
4.4 Hz, 2H*), 6.18 (d, J = 4.4 Hz, 2H§), 4.21-4.12 (m, 6H*, 6H§), 4.06-3.96 (m, 2H*, 2H§), 3.3 (s,
3H*), 3.2 (s, 3H§) 2.88 (t, J = 7.1, 4H*, 4H§), 2.73 – 2.57 (m, 6H*, 6H§), 2.41 (t, J = 7.0 Hz,
2H*,2H§), 2.35 (t, J = 7.0 Hz, 2H*, 2H§), 2.03 – 1.90 (m, 12H*, 12H§), 1.74 (s, 12H*), 1.71 (s,
12H§) ppm; HRMS (ESI) calculated for C46H62N3O10S2 (M+H)+ 880.3877, observed 880.3850.

BL-760. To a 1-dram vial containing S3 (115 mg, 0.15 mmol) and a magnetic stirrer bar was added
TFA (1.5 mL) under argon. The purple reaction mixture was heated to 60 °C for 5 min and the
TFA was removed by nitrogen stream and the residue was dried under vacuum for 30 min (< 0.1
Torr). NaHCO3 (61 mg, 0.73 mmol) was added to the vial, followed by dry DMF (3.3 mL) and
Ac2O (41 μL, 0.44 mmol) under argon. The reaction mixture was heated to 60 °C for 2 h, during
which time LC-MS indicated formation of the desired product. The reaction mixture was
precipitated into diethyl ether and centrifuged at 4500 rpm for 5 min. The pellet was dissolved in
H2O (12 mL) and purified by reversed-phase chromatography (C18, 0→40% MeCN/H2O). The
product containing fractions were combined and lyophilized to afford BL-760 (104 mg, 86% yield)
as an iridescent green solid. 1H NMR (400 MHz, Methanol-d4, compound exists as a mixture of
rotamers, major rotamer is designated using an asterisk (“*”), and minor rotamers are denoted “ §”)4
δ 8.14 (two overlapping d, J = 14.2 Hz, 2H*, 2H§), 7.49 (d, J = 7.5, 2H*, 2H§), 7.44 – 7.37 (m,
2H*, 2H§), 7.35 – 7.31 (m, 2H*, 2H§), 7.24 (t, J = 7.4 Hz, 2H*, 2H§), 6.22 (d, J = 5.9 Hz, 2H*),
6.19 (d, J = 5.8 Hz, 2H§), 4.25 – 4.08 (m, 6H*, 6H§), 4.07 – 3.87 (m, 2H*, 2H§), 3.29 (s, 3H*),
3.20 (s, 3H§), 2.88 (td, J = 7.2, 1.7 Hz, 4H*, 4H§), 2.69-2.63 (m, 4H*, 4H§), 2.29 (s, 3H*), 2.22 (s,
3H§), 2.03 – 1.89 (m, 10H*, 10H§), 1.74 (s, 12H*), 1.70 (s, 12H§) ppm; HR-MS (ESI) calculated
for C43H59N3O8S2 (M+2H)2+ 404.6866, observed 404.6861.

4 The issue of the tertiary amide rotamers was described in ref 1.

 1H

8.0
178.97 7.79

180
2.00
177.50 7.76
7.39

7.5
170.39 1.98 7.38

170
2.18 7.34
3.94 7.32
7.31

7.0

160
7.15
7.13
7.13

6.5
7.12

150
144.30 7.10
144.07
MPL-264D-31 diCOOH-4-NME 13C.10.fid

141.82 5.96

6.0

140
2.04
5.93
and 13C NMR Spectra

MPL-264D-31 diCOOH-4-NME 13C.11.fid

129.49

130
5.5
125.22
124.39
123.11

120
5.0
PROTON MeOD /home/JJB058/iconusers/MPL264 MPL264 6

C13CPD MeOD /home/JJB058/iconusers/MPL264 MPL264 6

110.61

110
4.5
4.02
4.01
3.99

100
96.87 3.87 3.91

4.0
f1 (ppm)
4.08 3.90
3.87

90
3.86

f1 (ppm)
3.5
3.00 3.47

80
3.0
2.55

70
2.54
3.76 2.52
2.5
61.15 2.34
4.20 2.33

60
60.50
2.32
1.85
2.0

6.19 1.84

50
4.25 1.82
45.03 11.75 1.81
44.03
1.5

1.75

40
1.74
36.08 1.74
29.47 1.74
1.0

30
27.51 1.72
25.64 1.72
24.07
0.5

23.25

20
10
0.0

0
 8.13
7.50

8.5
7.48
7.40
2.17 7.40

8.0
7.40
7.39
MPL-264D-34.10.fid

1.85 7.33
7.32

7.5
1.91
1.80 7.31
2.00 7.24
7.24

7.0
7.24
7.24
6.20

6.5
6.19
1.03 6.17
0.98 6.16

6.0
4.89
4.15
4.15
4.14

5.5
4.13
4.12
PROTON MeOD /home/JJB058/iconusers/MPL264 MPL264 11

4.12

5.0
3.98
3.32
3.31

4.5
3.31
1.74 3.31
3.30

f1 (ppm)
4.05
3.30

4.0
2.01
3.21
3.00
2.86

3.5
2.86
3.88 2.66
2.66

3.0
3.19 2.64
2.29
2.5 4.43 2.25
2.25
4.31 2.24
2.17 2.23
2.23
2.0

3.73
4.00 2.22
11.51 2.22
1.89
1.5

1.86
1.85
1.84
1.0

1.83
1.75
1.75
1.75
0.5

1.73
1.72
1.70
0.0

1.20
1.20
 8.5

180
177.63

8.0
170.31

170
7.78
2.00
7.76
7.39

7.5
1.98

160
7.37
2.26 7.34
1.93 7.32
2.12

150
7.0
7.31
144.32 7.17
144.08 7.15
141.79 7.13

140
6.5
MPL-264B-186 4-NME-BS-Cy7 13C.1.fid

7.11

MPL-264B-186 4-NME-BS-Cy7 13C.2.fid

7.10
129.49

130
125.34 5.97

6.0
1.91
124.33 5.94
123.06

120
5.5
110.66 4.89

110
4.86

5.0
96.89 4.05
52.01 4.03

100
49.63 4.02
49.51

4.5
3.92
49.46 3.91

90
49.34 3.90

f1 (ppm)
f1 (ppm)
49.28 4.13 3.88

4.0
49.26 4.23 3.87
49.17

80
3.86
49.13
49.08 3.00 3.48

3.5
49.00 3.32

70
48.98 3.31
48.95 3.31
3.0
48.94 3.31
4.13

60
48.92 3.30
48.91 2.89
48.91 3.80
2.5

2.88

50
48.91
2.86
48.90
2.64
48.83
2.56
2.0

48.76

40
8.00 2.55
48.75 2.32
48.74 2.53
12.06 2.04
48.73

30
1.5

48.72 1.95
48.70 1.94
48.69 1.94

20
48.66 1.93
1.0

48.49 1.92
44.01 1.91
1.90

10
29.50
0.5

26.90 1.86
25.61 1.85

0
23.68 1.84
23.24 1.65
0.0

1.63
 8.5
8.17
8.17 8.13

8.5
2.17 8.14 7.54
8.10 7.54

8.0
2.04
7.50 7.52

8.0
7.50 7.52
2.06 7.48 7.40

7.5
2.08 7.48 2.07 7.40
MPL-264C-85 col 1.1.fid

2.00 7.43 7.38

7.5
2.06
2.11 7.42 2.02 7.38
7.42 7.33

7.0
2.06
7.40 7.33

7.0
7.40 7.31
7.39 7.26

6.5
7.39 7.24

6.5
2.12 7.38 7.24
7.34 7.24
1.92 7.24
7.34

6.0
MPL-264A-64 4-NME smiles AcOH diso3cy7.1.fid
7.33 6.21

6.0
7.32 6.17
7.32 4.85

5.5
7.26 4.18

5.5
7.26 4.17
7.26 4.15

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PROTON MeOD /home/JJB058/iconusers/MPL264 MPL264 10

5.0
7.26 3.98

5.0
7.25 3.32
7.24 3.31
7.24 3.31

4.5
7.24 3.31

4.5
7.23 3.30
6.13
7.22 3.30

f1 (ppm)
f1 (ppm)
2.29 5.98

4.0
7.22 2.05 3.20

4.0
7.22 2.90
6.23 2.90
6.21 2.89

3.5
3.5
6.19 2.88
3.63 6.18 2.87
3.13
4.20 2.86

3.0
4.19 2.69
3.0
4.04
4.17 2.68
3.83 4.15 4.33 2.68
4.15 6.44 2.66

2.5
1.93
2.5

4.14 2.65
3.25 4.13 2.64
4.02 2.41

2.0
9.83 4.00 1.99
2.0

11.97
3.99 1.99
11.83 12.00
3.98 1.98
2.90 1.97

1.5
1.5

2.90 1.97
2.88 1.96
2.88 1.96

1.0
1.0

2.87 1.95
2.86 1.94
2.67 1.94
2.65 1.93
0.5

0.5
2.29 1.92
1.74 1.92
1.70 1.74
0.0

0.0
1.71