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Chemical Science
DOI: 10.1039/C6SC00503A
ARTICLE
Journal Name
3
4
13
=1.3 Hz) ppm. C NMR
1
29.4, 130.2, 131.2, 139.2, 141.2, 141.3, 142.6, 143.2, 164.3, m), 8.37 (1H, dd,
68.5, 169.6 ppm.
J
1
= 8.8 Hz,
J
2
1
3
1
(125 MHz, MeOD): δ ppm. C NMR (150.9 MHz, CDCl
): δ 1.83 (3H, s), 28.1, 28.2, 33.7, 40.4, 55.4, 60.1, 61.9, 63.3, 64.6, 69.0, 69.1,
.22 (2H, t, J = 6.9 Hz), 3.40-3.44 (2H, m), 7.75 (1H, d, 70.4, 70.5, 70.6, 70.6, 129.1, 129.3, 130.3, 131.0, 131.2, 131.3,
3
): δ 24.6,
1
Compound 7. H NMR (600 MHz, DMSO-D
6
3
3
3
3
J = 7.2 Hz), 7.86 (1H, d, J = 7.2 Hz), 7.95-8.01 (2H, m), 8.20- 132.1, 133.2, 141.0, 142.5, 143.1, 143.6, 166.4, 173.5 174.1
13
8
2
1
.29 (3H, m) ppm. C NMR (150.9 MHz, DMSO-D
6
): δ 22.6, ppm; IR (film): 747, 1038, 1119, 1179, 1236, 1264, 1455, 1522,
–
9.6, 37.5, 124.0, 128.6, 129.2, 129.6, 131.2, 131.4, 133.6, 1700, 2867, 2925, 3236 cm , HRMS: (ESI+): m/z calculated for
1
+
40.1, 140.6, 140.7, 142.1, 143.8, 165.4, 169.5 ppm.
C
31
H
39
O
8
N
4
S [M+H] 627.2483, found: 627.2477.
): δ 1.82 (3H, s), Rhodamine-labelled phenazine-1-carboxylic acid (10). To a
.30 (2H, t, J = 6.9 Hz), 3.39-3.43 (2H, m), 7.73 (1H, d, solution of rhodamine (1.9 mg, 0.0043 mmol),
1
Compound 8. H NMR (600 MHz, DMSO-D
6
3
3
3
B
3
4
J = 6.6 Hz), 7.77 (1H, dd, J = 9.2 Hz, J = 1.5 Hz), 7.80 (1H, dd, tetraethyleneglycol phenazine-1-carboxylate 11 (1.7 mg,
3
= 8.6 Hz, J
3
3
= 6.6 Hz), 7.99 (1H, s), 8.04 (1H, d, J = 8.6 Hz), 0.0043 mmol) and PyBop (2.7 mg, 0.0052 mmol, 1.2 eq.) in
J
1
2
3 13
.09 (1H, d, J = 9.2 Hz), 8.28-8.33 (1H, m, NH) ppm. C NMR anhydrous dichloromethane (1 mL) was added Hünigs base
8
6
(150.9 MHz, DMSO-D ): δ 22.5, 30.5, 37.6, 121.9, 125.4, 127.0, (2.9 μL, 4 eq.) at 0 °C. The reaction was allowed to warm to
1
27.5, 129.3, 130.3, 130.9, 140.4, 141.1, 142.0, 142.5, 165.5, room temperature and stirred for 24 h. The solution was
#
69.6 169.8 ppm. *Signal partly obscured, signal only concentrated under reduced pressure and the residue was
1
detectable from HMBC data.
subjected to column chromatography over silica gel (gradient
chloroform/MeOH: 100:0 – 95:5, v/v). The crude product was
subjected to semi-preparative HPLC using a Zorbax Eclipse
Synthesis of phenazine-biotine and -rhodamine probes.
Tetraethyleneglycol phenazine-1-carboxylate (11). Phenazine- XDB-C8 (acetontrile-H
-carboxylic acid (10 mg, 0.044 mmol) was dissolved in 20% acetonitrile, 40% in 4 min, 60% in 12.5 min, 100% in
dichloromethane (5 mL) and cooled to 0 °C. Tetraethylene 0.5 min, 100% for 5 min, 20% in 1 min) to give 2.2 mg of a
2
O, 0.1% formic acid; starting conditions:
1
2
1
3
glycol (26 mg, 0.13 mmol, 3 eq.), DMAP (5 mg, 0.04 mmol) and white solid. Yield: 63%. H NMR (CDCl , 600 MHz) δ = 1.26-1.37
EDC (8.4 mg, 0.05 mmol) were added. After 24 h, water was (12H, m), 3.45-3.49 (3H, m), 3.50-3.59 (9H, m), 3.61-3.64 (2H,
added and the aqueous phase extracted with dichloromethane m), 3.64-3.68 (1H, m), 3.69-3.72 (2H, m), 3.73-3.79 (1H, m),
3
3
3
2 × 10 mL). The combined organic fractions were dried over 3.90 (2H, t, J = 4.8 Hz), 4.12 (2H, t, J = 4.7 Hz), 4.63 (2H, t, J =
(
sodium sulfate und concentrated under reduced pressure. 4.8 Hz), 6.78-6.81 (3H,m), 7.02 (1H, s), 7.04 (1H, s), 7.28 (1H,
3
Column chromatography over silica gel (chloroform/methanol: dd, J
4
= 7.6 Hz, J
1
2
= 0.7 Hz), 7.61-7.82 (3H, m), 7.85-7.91 (3H,
4
= 6.9 Hz, J
3
8:2, v/v) gave the product (15.3 mg) as yellow oil. Yield: 86 %. m), 8.25 (1H, dd, J
3
= 1.1 Hz), 8.26 (1H, dd, J
9
1
2
1
= 8.0
1
3
4
H NMR (600 MHz, MeOD): δ 3.47 (2H, t, J = 4.9 Hz), 3.54 (2H, Hz, J
3
= 1.0 Hz), 8.28-8.32 (2H, m), 8.28 (1H, d, J
2
1
= 8.8 Hz)
3
m), 3.58 (2H, t, J = 4.9 Hz), 3.61 (2H, m), 3.66 (2H, m), 3.73 ppm. C NMR (CDCl
13
3
, 600 MHz) δ = 12.6, 12.6, 12.6, 13.7,
3
3
2H, m), 3.93 (2H, t, J = 4.7 Hz), 4.65 (2H, t, J = 4.7 Hz), 7.96- 46.0, 46.0, 46.0, 47.4, 64.7, 64.8, 68.6, 69.2, 70.4, 70.5, 70.6,
(
3
.99 (3H, m), 8.24-8.27 (1H, m), 8.31 (1H, dd, J = 6.8 Hz, 70.7, 96.4, 113.6, 114.0, 119.0, 128.7, 129.0, 129.6, 129.8,
7
4
3
J = 1.3 Hz), 8.33 (1H, m), 8.40 (1H, dd, J = 8.8 Hz, J = 1.3 Hz) 130.2, 130.3, 131.2, 131.3, 131.3, 131.4, 131.4, 131.7, 132.3,
4
13
ppm. C NMR (150.9 MHz, MeOD): δ 62.2, 66.0, 70.2, 71.4, 132.8, 132.9, 133.0, 133.6, 141.1, 141.9, 142.5, 143.8, 153.8,
7
1
1
3
4
1.6, 71.7, 73.6, 79.5, 130.3, 130.9, 131.0, 132.7, 132.8, 132.9, 155.5, 157.8, 158.8, 165.0, 166.3 ppm. IR (film): 1077, 1126,
33.4, 134.0, 142.0, 143.8, 144.5, 144.9, 168.1 ppm; IR (film): 1183, 1342, 1410, 1462, 1590, 1651, 1722, 2353, 2860, 2923,
–
1
+
53 8 4
119, 1214, 1237, 1274, 1350, 1421, 1523, 1727, 2874, 3318 cm , HRMS: (ESI+): m/z calculated for C49H O N [M]
–1
+
018 cm , HRMS: (ESI+): m/z calculated for C21
H
25
O
6
N
2
[M+H]
825.3858, found: 825.3868.
01.1707, found: 401.1710.
Biotin-labelled
phenazine-1-carboxylic
acid
(9). Phenazine-protein binding assays. The biotinylated probe
9
Tetraethylene-glycol
phenazine-1-carboxylate
(7 mg, was directly mixed with the protein (KS-B - 104.7 kDa) in
0.017 mmol) 11 and biotin (8.5 mg, 0.035 mmol, 2 eq.) were 20 mM Tris-HCl (pH 8.0). Final concentrations of the probe and
dissolved in dry dichloromethane (2 mL) and cooled to 0 °C. protein were 0.5 mM and 20 µM respectively. The reaction
DMAP (2 mg, 0.0.17 mmol) and EDC (8 mg, 0.044 mmol, 2.5 mixture was incubated for 24 h under the following conditions:
eq.) were added. Water (5 mL) was added after 20 h and the a) at 254 nm b) at 370 nm or c) at 22 °C in the presence of
aqueous phase extracted with dichloromethane (2 × 5 mL). 0.5 mM azobisisobutyronitrile (AIBN). For purification of the
The combined organic fractions were dried over sodium biotinylated KS-B, the reaction mixture was diluted twice with
sulfate and concentrated under reduced pressure. Column 50 mM Tris, 0.2 M NaCl, 0.1 % SDS (pH 8.0) and incubated with
chromatography over silica gel (chloroform/methanol: 95:5, 1 mL of streptavidin beads (Thermo Scientific) at 22 °C for 1 h
1
v/v) gave 10.5 mg of a yellow solid. Yield: 96 %. H NMR with shaking at 400 rpm. The beads were washed twice with
3
(500 MHz, CDCl ): δ 1.56-1.70 (6H, m), 2.28-2.32 (2H, m), 2.67- 50 mM Tris, 0.2 M NaCl, 2 % SDS (pH 8.0) after incubation at
2
.73 (1H, m), 2.81-2.88 (1H, m), 3.06-3.13 (1H, m), 3.57-3.65 60 °C for 10 min. Elution of the bound protein was achieved by
3
= 5.4 Hz, J
3
10H, m), 3.72 (2H, dd, J
(
1
2
= 4.1 Hz), 3.91 (2H, t, heating the sample thrice at 60 °C for 30 min in 50 mM Tris,
3
J
1
= 4.8 Hz), 4.14-4.18 (2H, m), 4.23-4.28 (1H, m), 4.43-4.48 0.2 M NaCl, 2% SDS, 3 mM D-biotin (pH 8.0).
3
(
1
1H, m), 4.65 (2H, t, J = 4.8 Hz), 7.82-7.86 (3H, m), 8.20-8.23 The rhodamine-based probe 10 was incubated with the
3
1H, m), 8.23 (1H, dd, J
4
= 7.1 Hz, J
(
1
2
=1.3 Hz), 8.27-8.30 (1H, protein (KS-B, carbonic anhydrase III, albumin) in water or in
6
| J. Name., 2012, 00, 1-3
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