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131.0, 127.5, 126.6, 110.6, 106.3, 96.8, 61.0, 47.0, 32.4, 21.2,
The organic layer was washed with water (340 mL), separated,
concentrated in vacuo, and purified by semipreparative HPLC
(linear gradient elution). For fluorescence measurements, the prod-
uct was further purified by preparative TLC (CH2Cl2/methanol 20:1
v/v) to give 10b as a black solid (8.8 mg, 31%). Rf (CH2Cl2/MeOH
20:1 v/v): 0.5; 1H NMR (500 MHz, CDCl3): d=8.90 (d, J=16.4 Hz,
1H), 7.76 (d, J=16.5 Hz, 1H), 7.71 (d, J=8.9 Hz, 1H), 7.17 (s, 1H),
6.77 (d, J=7.1 Hz, 1H), 6.53 (s, 1H), 6.37 (s, 1H), 4.18 (q, J=7.0 Hz,
4H), 3.83 (t, J=6.7 Hz, 4H), 3.09 (s, 3H), 2.68 (t, J=6.8 Hz, 4H),
1.28 ppm (t, J=7.1 Hz, 6H); 13C NMR (126 MHz, CDCl3): d=185.0,
171.1, 166.6, 164.4, 151.4, 150.5, 146.9, 140.7, 138.3, 134.2, 132.5,
129.1, 126.4, 125.9, 110.9, 107.0, 96.9, 61.1, 47.1, 32.4, 21.2,
14.1 ppm; IR (neat) n˜max =1722, 1609, 1585, 1398, 1362, 1246, 1194,
+
1130, 851, 802 cmÀ1; HRMS (ESI): m/z calcd for C31H31N6O6 [M+H]+
: 583.2300; found: 583.2305.
Dipropyl 3,3’-({1-[4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl]-3-
oxo-3H-phenoxazin-7-yl}azanediyl)dipropionate (8c)
Compound 6c (20 mg, 0.034 mmol, 1.0 equiv),
7
(10 mg,
0.034 mmol, 1.0 equiv), [PdCl2(dppf)] (1.2 mg, 0.002 mmol,
0.05 equiv), and Cs2CO3 (33 mg, 0.102 mmol, 3.0 equiv) were sus-
pended in 1,4-dioxane (10 mL). The suspension was stirred for
30 min at 808C, after which time it was cooled to room tempera-
ture and CH2Cl2 (30 mL) was added. The organic layer was washed
with brine (340 mL), separated, concentrated in vacuo, and puri-
fied by semipreparative HPLC (linear gradient elution) to give 8c
as a red solid (6.3 mg, 30%). Rf (CH2Cl2/MeOH 20:1 v/v): 0.5;
1H NMR (500 MHz, CDCl3): d=8.67 (d, J=8.2 Hz, 2H), 7.80 (d, J=
8.2 Hz, 2H), 7.56 (d, J=9.1 Hz, 1H), 6.91 (d, J=1.9 Hz, 1H), 6.71
(dd, J=9.1, 2.3 Hz, 1H), 6.54 (d, J=2.2 Hz, 1H), 6.39 (d, J=1.8 Hz,
1H), 4.07 (t, J=6.7 Hz, 4H), 3.82 (t, J=6.9 Hz, 4H), 3.12 (s, 3H), 2.68
(t, J=6.9 Hz, 4H), 1.71–1.62 (m, 4H), 0.94 ppm (t, J=7.2 Hz, 6H);
13C NMR (126 MHz, CDCl3): d=184.9, 171.2, 167.3, 163.9, 151.4,
150.5, 146.6, 144.1, 140.8, 139.9, 132.5, 131.8, 131.7, 131.0, 127.5,
126.6, 110.7, 106.3, 96.8, 66.7, 47.1, 32.3, 21.9, 21.2, 10.3 ppm; IR
14.1 ppm; IR (neat) n˜max =1720, 1607, 1580, 1360, 1200, 1130 cmÀ1
;
+
HRMS (ESI): m/z calcd for C27H29N6O6 [M+H]+: 533.2141; found:
533.2149.
Dipropyl 3,3’-({1-[2-(6-methyl-1,2,4,5-tetrazin-3-yl)vinyl]-3-
oxo-3H-phenoxazin-7-yl}azanediyl)(E)-dipropionate (10c)
Compound 6c (29 mg, 0.049 mmol, 1.0 equiv),
9
(67 mg,
0.296 mmol, 6.0 equiv), [Pd2(dba)3] (4.5 mg, 0.005 mmol, 0.1 equiv),
QPhos (3.5 mg, 0.005 mmol, 0.1 equiv), and KOAc (39 mg,
0.395 mmol, 8.0 equiv) were suspended in 1,4-dioxane (2 mL). The
suspension was stirred for 120 min at 1008C in a sealed vial, after
which time it was cooled to room temperature, the volatile com-
ponents were removed in vacuo, and CH2Cl2 (30 mL) was added.
The organic layer was washed with water (340 mL), separated,
concentrated in vacuo, and purified by semipreparative HPLC
(linear gradient elution). For fluorescence measurements, the prod-
uct was further purified by preparative TLC (CH2Cl2/methanol 20:1
v/v%) to give 10c as a black solid (2.8 mg, 10%). Rf (CH2Cl2/MeOH
20:1 v/v): 0.5; 1H NMR (500 MHz, CDCl3): d=8.91 (d, J=16.4 Hz,
1H), 7.77 (d, J=16.5 Hz, 1H), 7.72 (d, J=9.0 Hz, 1H), 7.18 (s, 2H),
6.78 (d, J=7.3 Hz, 1H), 6.54 (s, 1H), 6.38 (s, 1H), 4.08 (t, J=6.6 Hz,
4H), 3.83 (t, J=6.6 Hz, 4H), 3.09 (s, 3H), 2.69 (t, J=6.7 Hz, 4H),
1.71–1.63 (m, 4H), 0.95 ppm (t, J=7.3 Hz, 6H); 13C NMR (126 MHz,
CDCl3): d=185.0, 171.2, 166.6, 164.4, 151.4, 150.5, 146.9, 140.7,
138.3, 134.2, 132.5, 129.1, 126.4, 125.9, 110.9, 107.0, 96.9, 66.7, 47.1,
32.3, 21.9, 21.2, 10.3 ppm; IR (neat) n˜max =2922, 1726, 1609, 1585,
1395, 1360, 1186, 1153, 1130 cmÀ1; HRMS (ESI): m/z calcd for
(neat) n˜max =1726, 1603, 1402, 1362, 1263, 1186, 1128 cmÀ1; HRMS
+
(ESI): m/z calcd for C33H35N6O6
[M+H]+: 611.2613; found:
611.2618.
Dimethyl 3,3’-({1-[2-(6-methyl-1,2,4,5-tetrazin-3-yl)vinyl]-3-
oxo-3H-phenoxazin-7-yl}azanediyl)(E)-dipropionate (10a)
Compound 6a (45 mg, 0.085 mmol, 1.0 equiv),
9 (129 mg,
0.592 mmol, 7.0 equiv), [Pd2(dba)3] (7.7 mg, 0.008 mmol, 0.1 equiv),
QPhos (6.0 mg, 0.008 mmol, 0.1 equiv), and KOAc (66 mg,
0.592 mmol, 8.0 equiv) were suspended in 1,4-dioxane (2 mL). The
suspension was stirred for 120 min at 1008C in a sealed vial, after
which time it was cooled to room temperature, the volatile com-
ponents were removed in vacuo, and CH2Cl2 (30 mL) was added.
The organic layer was then washed with water (340 mL), separat-
ed, concentrated in vacuo, and purified by semipreparative HPLC
(linear gradient elution). For fluorescence measurements, the prod-
uct was further purified by preparative TLC (CH2Cl2/methanol 20:1
v/v) to give 10a as a black solid (2.1 mg, 14%). Rf (CH2Cl2/MeOH
20:1 v/v): 0.5; 1H NMR (500 MHz, CDCl3): d=8.90 (d, J=16.4 Hz,
1H), 7.76 (d, J=16.5 Hz, 1H), 7.72 (d, J=9.1 Hz, 1H), 7.18 (s, 1H),
6.76 (d, J=6.9 Hz, 1H), 6.52 (s, 1H), 6.37 (s, 1H), 3.83 (t, J=6.9 Hz,
4H), 3.73 (s, 6H), 3.09 (s, 3H), 2.70 ppm (t, J=6.9 Hz, 4H); 13C NMR
(126 MHz, CDCl3): d=185.0, 171.5, 166.6, 164.4, 151.3, 150.5, 146.8,
140.8, 138.3, 134.2, 132.5, 129.1, 126.4, 125.9, 110.8, 107.0, 96.9,
52.0, 47.1, 32.1, 21.2 ppm; IR (neat) n˜max =2922, 1726, 1601, 1578,
+
C29H33N6O6 [M+H]+: 561.2456; found: 561.2462.
Cell culture and transfections
For labeling experiments, HEK293T cells were maintained in Dul-
becco’s modified Eagle’s medium (DMEM; Gibco, high glucose)
supplemented with 10% FBS (Sigma), 1% l-glutamine (Invitrogen,
Palo Alto, USA), 1% sodium pyruvate (Invitrogen), and 1% penicil-
lin–streptomycin (Invitrogen) in a 5% CO2 atmosphere at 378C.
Cells were passaged at 80% confluency every 2–4 days for up to
20 passages. For imaging, cells were seeded on 24-well glass-bot-
tomed plates (Sensoplate, Greiner Bio-One) at least 16 h prior to
transfection. Prior to cell seeding, 24-well plates were coated with
poly-l-lysine hydrobromide (Sigma) for 4–8 h at room temperature.
Transfections with the previously reported plasmids (GFPY39TAG and
mammalian PylRSAF/tRNAPyl expression plasmids) were performed
with JetPrime reagent (PeqLab, Erlangen, Germany), as previously
described.[17,18] TCO*-lysine stock solution was prepared in 0.2m
NaOH containing DMSO (15% v/v) at 100 mm concentration. We
used TCO*-lysine at a final concentration of 250 mm by diluting the
stock solution 1:4 v/v% in 1m HEPES before adding noncanonical
amino acids (ncAAs) to the medium.[18] After 8 h of incubation with
ncAAs, cells were rinsed with fresh medium and kept overnight in
growth medium without ncAAs.
+
1358, 1256, 1165 cmÀ1; HRMS (ESI): m/z calcd for C25H25N6O6
[M+H]+: 505.1831; found: 505.1836.
Diethyl 3,3’-({1-[2-(6-methyl-1,2,4,5-tetrazin-3-yl)vinyl]-3-oxo-
3H-phenoxazin-7-yl}azanediyl)(E)-dipropionate (10b)
Compound 6b (30 mg, 0.054 mmol, 1.0 equiv),
9
(58 mg,
0.268 mmol, 5.0 equiv), [Pd2(dba)3] (4.9 mg, 0.005 mmol, 0.1 equiv),
QPhos (3.8 mg, 0.005 mmol, 0.1 equiv), and KOAc (32 mg,
0.321 mmol, 6.0 equiv) were suspended in 1,4-dioxane (2 mL). The
suspension was stirred for 120 min at 1008C in a sealed vial, after
which time it was cooled to room temperature, the volatile com-
ponents were removed in vacuo, and CH2Cl2 (30 mL) was added.
Chem. Eur. J. 2016, 22, 8972 – 8979
8978
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim