10.1002/cbic.201900661
ChemBioChem
FULL PAPER
prior to adding to the reaction mixture, by adding 0.60 mL of 100 mM
copper(II) and 2.40 mL of 100 mM sodium ascorbate, resulting in a yellow
suspension. To a solution of DFOB-N3 (30.0 mg, 51 µmol) and acetylene
(102 µmol) in tert-BuOH (5.40 mL) was added catalyst solution (2.8 mL)
under stirring. The reaction mixture was stirred for 18 h. The pH of the
mixture was then adjusted to pH = 2 (few drops of conc. HCl), and the
solvents removed in vacuo. The mixture was re-suspended in aqueous
TFA (0.05%) and loaded onto a C18 cartridge (2 g). The cartridge was
washed with 5.0 mL each of aqueous TFA (0.05%), 10% acetonitrile in
aqueous TFA (0.05%), and eluted with acetonitrile (80% in aqueous TFA
(0.05%). The eluent was directly used for preparative HPLC (system A) to
purify the product.
of the crude product FOB-N3 (21.8 mg, 34 μmol) used in the general
CuAAC procedure with 1-ethynyladamantane (16.4 mg, 102 μmol). LC
system A: using column B at 20 mL min-1, running 25–45% mobile phase
B in mobile phase A from 0–20 min. The product eluted at 15.9 min. Pooled
fractions were reduced in vacuo to approx. 20% of initial volume, frozen
and freeze-dried to yield 21.7 mg of red-brown compound Fe-25 (80%).
HR-MS (ESI+) observed [M-3H++Fe+H+]+ 800.38726 (100), C37H60N8O8Fe
requires m/zcalc 800.38780.
Synthesis of DFOB-triazole-pentane (26). DFOB-N3 (30.0 mg, 51 µmol),
hept-1-yne (20.1 µL, 14.8 mg, 153 µmol). LC system A: using column A at
5 mL min-1, running 27% mobile phase B in mobile phase A over 30 min.
The product eluted at 13.9 min. Pooled fractions were reduced in vacuo to
approx. 20% of initial volume, frozen and freeze-dried to yield 18.1 mg of
off-white powder 26 (52%). HR-MS (ESI+) observed [M+Na+]+ 683.44406
(100), C32H58N8O8Na requires m/zcalc 683.44504. 1H-NMR (DMSO-d6, 600
MHz) δH 9.66 – 9.61 (3 × s, 3H, 3 × N-OH), 7.81 (s, 1H, H-31), 7.77 (t, J =
5.6 Hz, 2H, H-9 and H-20), 4.26 (t, J = 7.1 Hz, 2H), 3.45 (t, J = 7.0 Hz, 3H),
2.99 (q, J = 6.6 Hz, 4H), 2.57 (m, 6H), 2.26 (t, J = 7.2 Hz, 4H), 1.96 (s, 3H,
H-1), 1.78 (p, J = 7.4 Hz, 2H), 1.57 (p, J = 7.5 Hz, 3H), 1.49 (m, 6H), 1.37
(m, 5H), 1.28 (m, 5H), 1.21 (m, 6H), 0.86 (t, J = 6.9 Hz, 3H). 13C-NMR
(DMSO-d6, 125.7 MHz): δC 172.01, 171.33, 170.16, 146.85, 121.55, 49.06,
47.09, 46.87, 46.80, 38.44, 30.84, 29.91, 29.37, 28.83, 28.70, 27.59, 27.55,
26.04, 25.68, 25.00, 23.51, 22.99, 21.88, 20.37, 13.92.
Synthesis of DFOB-triazole-t-butyl (23). DFOB-N3 (30.0 mg, 51 µmol),
3,3-dimethyl-1-butyne (18.9 µL, 12.6 mg, 153 µmol). LC system A: using
column A at 5 mL min-1, running 25% mobile phase B in mobile phase A
over 30 min. The product eluted at 14.1 min. Pooled fractions were
reduced in vacuo to approx. 20% of initial volume, frozen and freeze-dried
to yield 19.6 mg of off-white powder 23 (57%). HR-MS (ESI+) observed
[M+Na+]+ 691.41127 (100), C31H56N8O8Na requires m/zcalc 691.41133. 1H-
NMR (DMSO-d6, 600 MHz) δH 9.63 (s, 3H), 7.83 (s, 1H), 7.77 (t, J = 5.3
Hz, 2H), 4.25 (t, J = 7.2 Hz, 2H), 3.45 (m, 6H), 2.99 (q, J = 6.3 Hz, 4H),
2.57 (m, 4H), 2.26 (t, J = 7.2 Hz, 4H), 1.96 (s, 3H), 1.80 (p, J = 7.4 Hz, 2H),
1.49 (m, 6H), 1.37 (m, 4H), 1.26 (s, 9H), 1.21 (m, 6H). 13C-NMR (DMSO-
d6, 125.7 MHz): δC 171.99, 171.33, 170.15, 156.13, 151.38, 128.92,
119.60, 49.10, 47.09, 46.88, 46.80, 38.44, 30.38, 30.33, 29.89, 29.39,
29.38, 28.83, 27.54, 26.04, 25.68, 23.51, 23.06, 20.36.
Synthesis of DFOB-triazole-phenyl (27). DFOB-N3 (30.0 mg, 51 µmol),
ethynylbenzene (16.9 µL, 15.7 mg, 153 µmol). LC system A: using column
A at 5 mL min-1, running 15–35% mobile phase B in mobile phase A from
0–30 min. The product eluted at 27.4 min. Pooled fractions were reduced
in vacuo to approx. 20% of initial volume, frozen and freeze-dried to yield
21.9 mg of off-white powder 27 (62%). HR-MS (ESI+) observed [M+Na+]+
Synthesis of DFOB-triazole-cyclohexane (24). DFOB-N3 (30.0 mg, 51
µmol), ethyn-2-ylcyclohexane (20.0 µL, 16.6 mg, 153 µmol). LC system A:
using column A at 5 mL min-1, running 29% mobile phase B in mobile
phase A over 30 min. The product eluted at 13.9 min. Pooled fractions
were reduced in vacuo to approx. 20% of initial volume, frozen and freeze-
dried to yield 18.1 mg of off-white powder 24 (51%). HR-MS (ESI+)
observed [M+Na+]+ 717.42727 (100), C33H58N8O8Na requires m/zcalc
717.42698. 1H-NMR (DMSO-d6, 600 MHz) δH 9.63 (s, 3H), 7.80 (s, 1H),
7.78 (s, 2H), 4.26 (t, J = 7.1 Hz, 2H), 3.45 (t, J = 7.0 Hz, 5H), 2.99 (q, J =
6.7 Hz, 4H), 2.57 (d, J = 6.7 Hz, 3H), 2.26 (t, J = 7.5 Hz, 4H), 1.96 (s, 3H),
1.91 (d, J = 12.2 Hz, 2H), 1.79 (q, J = 7.5 Hz, 2H), 1.72 (s, 2H), 1.66 (d, J
= 13.0 Hz, 1H), 1.50 (d, J = 16.3 Hz, 6H), 1.35 (dq, J = 22.2, 12.3, 9.7 Hz,
8H), 1.20 (s, 7H). 13C-NMR (DMSO-d6, 125.7 MHz): δC 172.04, 171.39,
170.20, 158.49, 152.06, 120.44, 68.13, 49.18, 47.13, 46.83, 40.05, 39.52,
38.47, 34.65, 32.62, 29.94, 29.38, 28.85, 27.59, 26.07, 25.68, 25.64, 23.53,
23.04, 20.38, 13.32.
711.37907 (100),
C
33H52N8O8Na requires m/zcalc 711.38003. 1H-
NMR (DMSO-d6, 600 MHz) δH 9.67–9.62 (3 × s, 3H, 3 × N-OH), 8.56 (s,
1H, H-31), 7.83 (2 × d, J = 7.5 Hz, 2H, H-34), 7.77 (q, J = 5.9 Hz, 1H), 7.44
(t, J = 7.6 Hz, 2H, H-35), 7.32 (t, J = 7.4 Hz, 1H, H-36), 4.37 (t, J = 7.1 Hz,
2H), 2.99 (q, J = 7.0 Hz, 4H), 2.57 (m, 3H), 2.26 (t, J = 7.3 Hz, 2H), 1.96
(s, 3H, H-1), 1.87 (p, J = 7.3 Hz, 1H), 1.55 (q, J = 7.3 Hz, 1H), 1.49 (m,
4H), 1.37 (m, 4H) 1.21 (m, 6H). 13C-NMR (DMSO-d6, 125.7 MHz): δC
173.53, 146.28, 130.87, 128.89, 127.79, 125.11, 121.21, 117.92, 49.46,
40.05, 38.44, 29.87, 29.28, 28.82, 26.02, 23.50, 23.01.
CuAAC reaction with 2 to form 29
To a solution of 2 (2.4 mg, 4 µmol) and biotin-PEG-alkyne (0.5 mg, 1 µmol)
in tert-BuOH (444 µL) was added a freshly prepared catalyst solution (56
uL). Catalyst solution contained 28 µL of 10 mM CuSO4, 13 µL of 100 mM
sodium ascorbate, and 15 µL of 20 mM THPTA. The reaction progress
was monitored by HR-LCMS (LC system C). After 3 h, the reaction had
gone to completion (> 95% of acetylene consumed), to yield DFOB-PEG-
biotin (29), eluting at 14.78 min. HR-MS (ESI+) observed [M+2H+]+
522.79223 (100), [M+H+]+ 1044.57652 (5), C46H83N11O14S requires m/zcalc
522.79208. After Fe(III)-loading, HR-MS at 13.62 min observed [Fe-M-
3H+2H+]+ 549.24841 (100), [Fe-M-3H+H+]+ 1097.48813 (15),
C46H80N11O14SFe requires m/zcalc 549.24781.
Synthesis of DFOB-triazole-adamantane (25). DFOB-N3 (30.0 mg, 51
µmol), 1-ethynyladamantane (16.4 mg, 153 µmol). LC system A: using
column A at 5 mL min-1, running 33% mobile phase B in mobile phase A
over 30 min. The product eluted at 19.3 min. Pooled fractions were
reduced in vacuo to approx. 20% of initial volume, frozen and freeze-dried
to yield 21.1 mg of off-white powder 25 (55%). HR-MS (ESI+) observed
[M+Na+]+ 769.45833 (100), C37H62N8O8Na requires m/zcalc 769.45828. 1H-
NMR (DMSO-d6, 600 MHz) δH 9.63 (s, 3H), 7.78 (s, 3H), 4.25 (t, J = 7.2
Hz, 2H), 3.45 (t, J = 7.2 Hz, 6H), 2.99 (q, J = 6.6 Hz, 4H), 2.57 (m, 4H),
2.26 (t, J = 7.3 Hz, 4H), 2.00 (s, 3H), 1.96 (s, 3H), 1.79 (p, J = 7.4 Hz, 2H),
1.72 (m, 7H), 1.50 (m, 6H), 1.37 (m, 4H), 1.20 (m, 6H). 13C-NMR (DMSO-
d6, 125.7 MHz): δC 171.98, 171.34, 171.30, 158.42, 158.17, 156.45,
119.39, 109.55, 49.13, 47.10, 46.87, 46.80, 42.24, 40.05, 38.45, 36.26,
32.15, 29.91, 29.37, 28.83, 27.90, 27.57, 26.05, 23.52, 23.05, 20.37.
SPAAC reactions with 2 to form 34
Synthesis of BCN-biotin (33). To a solution of BCN (25.0 mg, 77.1 µmol)
and biotin-NHS (78.9 mg, 231.2 µmol) in MeCN:DMSO (9:1, 4.4 mL) was
added TEA (32.2 µL, 231.2 µmol), and the reaction mixture was left stirring
for 24 h. The reaction mixture was acidified (3 drops HCl conc.) and the
solvent reduced to half under reduced pressure at 30 °C. The solution was
directly used for RP-HPLC purification on LC system A (column A). A
gradient of 15–60% mobile phase B in mobile phase A at 4 mL min-1 was
applied to elute the product at 18.7 min. Pooled fractions were reduced to
approx. 20% of initial volume in vacuo, frozen and freeze-dried to yield
Synthesis of Fe(III)-DFOB-triazole-adamantane (Fe-25). To a solution
of DFOB-N3 (20.0 mg, 34 μmol) in aqueous MeCN (65%, 1.5 mL) was
added Fe(III)Cl3.6H2O (10.1 mg, 37 μmol) which was stirred for 1 h to give
a dark red-brown solution. The solution was reduced to approx. 0.5 mL
under reduced pressure and loaded onto a Sep-Pak C18 SPE cartridge (1
g bed volume), washed with water and eluted with 80% MeCN in aqueous
TFA (0.05%). The eluent was reduced to dryness in vacuo, and a sample
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