Full Paper
4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (100 mg, 125 mmol), 1-
ethynylanthracene (28 mg, 138 mmol) and freshly prepared Sono-
gashira catalyst solution (3.0 mL). Yield: 71 mg (77 mmol, 62%);
orange-red solid; Rf =0.20 (buffer A/B, 1:1); 1H NMR [500 MHz,
CD3OD/D2O (5:2), 258C]: d=1.27 (t, J=7.5 Hz, 18H), 1.90 (s, 6H),
1.97 (s, 6H), 3.15 (q, J=7.5 Hz, 12H), 7.44–7.52 (m, 5H), 7.77 (d, J=
6.5 Hz,1H), 8.01–8.02 (m, 2H), 8.06 (d, J=8.5 Hz, 1H), 8.46 (s, 1H),
8.72 ppm (s, 1H); APT NMR [126 MHz, CD3OD/D2O (5:2), 258C]: d=
9.26 (p), 9.85 (p), 11.59 (p), 12.45 (p), 15.80 (p), 47.77 (s), 92.27 (q),
92.81(q), 111.24 (q), 112.91 (q), 116.63 (t), 116.84 (t), 120.09 (q),
125.06 (t), 125.73 (t), 127.26 (t), 127.49 (t), 127.52 (t), 128.37 (t),
129.05 (t), 129.23 (q), 129.33 (q), 129.42 (q), 131.31 (q), 131.38 (t),
131.45 (q), 131.62 (t), 132.19 (q), 132.75 (q), 133.26 (q), 133.43 (q),
136.21 (q), 142.61 (q), 158.11 (q), 159.37 (q), 161.29 (q), 180.98 ppm
(q); 19F NMR [282 MHz, CD3OD/D2O (5:2), 258C]: d=À143.50 to
À143.17 (m), À113.22 (s), À113.19 ppm (s); HRMS (ESIÀ): m/z calcd
for C35H23BF4N2O6S2 359.0519 [M]2À; found 359.0531.
we could establish that, with sterically demanding dienes such
as 1-AB–2F, the ribozyme accelerates almost exclusively the for-
mation of one of the four possible stereoisomers, namely 1-
DAPB–2D-2F (S,R,S,R). Considering the crystallographic data
and the highly dynamic nature of the ribozyme, this extraordi-
nary selectivity is likely caused by the potential steric clashes
between the diene and the ribozyme for different binding ori-
entations.[6d,e,13] A second important finding of the current
study that could only be obtained by using these light-up
probes is that the DAse has an ordered bi uni reaction mecha-
nism in which the dienophile binds first to the catalyst, fol-
lowed by binding of the diene. A rate constant for the chemi-
cal step of approximately 17 minÀ1 was determined, in good
agreement with earlier data,[5h] whereas the differences in the
KM values reflect both the different physical meaning of the
constants depending of the mechanistic treatment (random bi
uni vs. ordered bi uni) and the different chemical nature of the
substrates used.
1-AB–OCH3: The reaction was set up with di-triethylammonium-
2,6-disulfonate-1,3,5,7-tetramethyl-8-(4’-bromo-2’-methoxyphenyl)-
4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (50 mg, 63 mmol), 1-ethy-
nylanthracene (14 mg, 69 mmol) and freshly prepared Sonogashira
catalyst solution (2.0 mL). Yield: 31 mg (33 mmol, 53%); orange-red
solid; Rf =0.15 (buffer A/B, 1:1); 1H NMR [500 MHz, CD3OD/D2O
(5:2), 258C]: d=1.25 (t, J=7.5 Hz, 18H), 1.83 (s, 6H), 2.79 (s, 6H),
3.10 (q, J=7.5 Hz, 12H), 3.88 (s, 3H), 7.14 (d, J=7.8 Hz, 1H), 7.45–
7.55 (m, 5H), 7.82 (dd, J1 =7.8 Hz, J2 =0.6 Hz, 1H) 8.03–8.12 (m,
3H), 8.52 (s, 1H), 8.89 ppm (s, 1H); APT NMR [126 MHz, CD3OD/
D2O (5:2), 258C]: d=9.38 (p), 9.88 (p), 11.66 (p), 13.02 (p), 15.88 (p),
47.76 (s), 56.85 (p), 89.89 (q), 94.76 (q), 115.50 (t), 121.10 (q), 124.81
(q), 125.29 (t), 125.88 (t), 126.51 (t), 127.21 (t), 127.42 (t), 127.61 (q),
128.27 (t), 129.06 (t), 129.31 (t), 130.70 (t), 130.92 (t), 131.55 (q),
131.74 (q), 131.99 (t), 132.39 (q), 133.27 (q), 133.40 (q), 135.34 (q),
143.21 (q), 143.41 (q), 156.33 (q), 157.70 ppm (q); 19F NMR
[282 MHz, CD3OD/D2O (5:2), 258C]: d=À143.37 to À143.13 ppm
Although we developed the turn-on BODIPY probes to study
the reaction mechanism of a ribozyme, they or their synthetic
precursors might find applications in other fields. Preliminary
data indicate that 1-AB derivatives are reactive (and light up)
not only in Diels–Alder reactions with maleimides, but also in
other cycloadditions, including those with reactive oxygen spe-
cies. Furthermore, the bromo-BODIPY intermediates reported
in Scheme 2a could be easily used for a one-step attachment
to sensors or enzyme substrates other than anthracene to
create light-up probes for a variety of analytes. Thereby, these
compounds can address the limitations of BODIPY, especially
with respect to biological systems, often caused by their poor
water solubility.[9a,c]
(m); HRMS (ESIÀ): m/z calcd for C36H27BF2N2O7S2 356.0666 [M]2À
;
found 356.0704.
1-AB–CH3: The reaction was set up with di-triethylammonium-2,6-
disulfonate-1,3,5,7-tetramethyl-8-(4’-bromo-2’-methylphenyl)-4,4-di-
fluoro-4-bora-3a,4a-diaza-s-indacene (50 mg, 64 mmol), 1-ethynylan-
thracene (14 mg, 69 mmol) and freshly prepared Sonogashira cata-
lyst solution (2.0 mL). Yield: 32 mg (35 mmol, 55%); orange-red
solid; Rf =0.15 (buffer A/B, 1:1); 1H NMR [500 MHz, CD3OD/D2O
(5:2), 258C]: d=1.29 (t, J=7.5 Hz, 18H), 1.76 (s, 6H), 2.26 (s, 3H),
2.80 (s, 6H), 3.18 (q, J=7.5 Hz, 12H), 7.31 (d, J=7.8 Hz, 1H), 7.52–
7.59 (m, 4H), 7.76–7.77 (m, 2H) 7.83 (d, J=7.0 Hz, 1H), 8.09–8.16
(m, 3H), 8.57 (s, 1H), 8.93 ppm (s, 1H); APT NMR (126 MHz, CD3OD/
D2O (5:2), 258C]: d=9.29 (p), 11.60 (p), 12.97 (p), 14.60 (p), 15.82
(p), 19.19 (p), 47.84 (s), 88.40 (q), 93.53 (q), 119.96 (q), 123.47 (q),
124.05 (t), 124.68 (t), 124.80 (q), 125.98 (t), 126.19 (t), 126.98 (t),
126.99 (t), 127.81 (t), 128.10 (t), 128.25 (t), 129.57 (t), 129.62 (t),
130.31 (t), 130.54 (q), 130.62 (t), 131.17 (q), 132.03 (q), 132.17 (q),
133.23 (q), 134.16 (q), 134.51 (q), 135.03 (q), 136.23 (q), 142.21 (q),
155.66 (q), 170.50 (q), 175.29 (q), 175.97 ppm (q); 19F NMR
[282 MHz, CD3OD/D2O (5:2), 258C]: d=À143.39 to À143.06 ppm
Experimental Section
All solvents were purchased from Sigma–Aldrich and were used
without further purification unless noted otherwise. Diels–Alderase
ribozyme (DAse, 49-mer RNA sequence: 5’-GGA GCU CGC CCG
GGC GAG GCC GUG CCA GCU CUU CGG AGC AAU ACU CGG C-3’ -
synthesised on solid phase) was obtained from chemical synthesis
services (CSS). Standard Diels–Alderase (DAse) buffer (300 mm
NaCl, 30 mm Tris-HCl, 80 mm MgCl2, 2 mm trolox, pH 7.4) was pre-
pared as a 5 stock solution and diluted as required. DAse concen-
trations for the kinetic experiments were measured with a Nano-
Drop ND-1000 spectrophotometer (Peqlab Biotechnologie GmbH)
by using the theoretical extinction coefficient of the DAse se-
quence, e260 =453600mÀ1 cmÀ1. To ensure correct folding, a desired
stock solution of DAse in water was refolded by heating for
2.0 min at 758C and controlled cooling in the thermoshaker
(Eppendorf) within 20 min to RT.
1-AB derivatives were synthesised in a manner similar to that previ-
ously described by using appropriate bromo-BODIPY derivatives
(1 equiv.), 1-ethynylanthracene (1.1 equiv.) and Sonogashira catalyst
solution (see the Supporting Information for the preparation) in
a microwave reactor (CEM2, Discover LabMate with a focused
single-mode reaction chamber, 2.45 GHz).[7] The synthesised
compounds were purified according to final purification and ion
exchange steps (see the Supporting Information).
(m); HRMS (ESIÀ): m/z calcd for C36H27BF2N2O6S2 348.0691 [M]2À
;
found 348.0720.
1-AB–CF3: The reaction was set up with di-triethylammonium-2,6-
disulfonate-1,3,5,7-tetramethyl-8-(4’-bromo-2’-trifluoromethylphen-
yl)-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (57 mg, 68 mmol), 1-
ethynylanthracene (15 mg, 75 mmol) and freshly prepared Sonoga-
shira catalyst solution (2.0 mL). Yield: 22 mg (23 mmol, 34%);
orange-red solid; Rf =0.35 (buffer A/B, 1:1); 1H NMR {500 MHz,
[D3]MeCN/D2O (4:1), 258C}: d=1.20 (t, J=7.5 Hz, 18H), 1.71 (s, 6H),
1-AB–2F: The reaction was set up with di-triethylammonium-2,6-
disulfonate-1,3,5,7-tetramethyl-8-(4’-bromo-2’,6’-difluorophenyl)-
Chem. Eur. J. 2015, 21, 5864 – 5871
5870
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