Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 3112–3116
Functionalization of the 4,4-difluoro-4-bora-3a,
4a-diaza-s-indacene (BODIPY) core
Lingling Li, Binh Nguyen and Kevin Burgess*
Department of Chemistry, Texas A&M University, Box 30012, College Station, TX 77842-3012, USA
Received 10 October 2007; accepted 29 October 2007
Available online 1 November 2007
Abstract—The new BODIPY systems 1 and 2 were prepared and then used as substrates to explore SNAr and F-B displacement
reactions. Chloride was easily displaced from 1 by a piperidine/ester, methylmagnesium bromide selectively displaced fluoride,
and cyanide could attack both sites. System 2 readily added soft nucleophiles to the electrophilic carbon atoms, providing a new
method for bioconjugation of BODIPYs to proteins while also introducing a 19F probe.
Ó 2007 Elsevier Ltd. All rights reserved.
BODIPY dyes are some of the most widely used fluorescent probes in biotechnology.1,2 They were first prepared in 1969,3 but developments relating to their
syntheses and functionalization reactions are far from
mature. In that regard, two of the most striking recent
innovations in the literature are: (i) SNAr reactions of
3-chloro or 3,5-dichloro systems;4,5 and (ii) nucleophilic
displacement of fluoride atoms from boron.6–13 These
studies, reported from other laboratories, typically feature compounds such as A and B. Hard nucleophiles
were not explored for the SNAr substrates A, cyanide
was not studied for either system, and no attempts have
been made to use nucleophilic amino acid side-chains on
proteins for the functionalization reactions.
Work from our group involving BODIPY dyes has exposed different facets of both reaction types mentioned
above. This communication focuses on BODIPYs 1
and 2 to illustrate those new issues. Compound 1 was
used to introduce groups functionalized for bioconjugation, and to study cases in which nucleophilic displacement of F and SNAr reactions can compete.
Compound 2 was used to illustrate how SNAr reactions
in particular can be used to conjugate dyes to proteins
while simultaneously changing the BODIPY core structure, its fluorescent properties, and enhancing its water
solubility.
to enable
functionalization
later
Br
to enhance SN Ar
reactivity
Tol
Tol
CF3
N
3
Cl
B
F2
N
N
5
Cl
Nu
Tol = 4-MeC6H4
OH
Nu = MeOH HO
SH EtO2C
NH
Ph
N
B
A
EtO2C
B
F2
H
N
CO2Et
N
Cl
Nu
Nu = alkyl, aryl, alkynyl
(lithium or Grignard reagents)
MeO-, ROH/AlCl3
B
F2
N
N
Cl
Cl
1
Nu = MeO2C
B
F2
N
Cl
2
NH , MeMgBr
Nu = H2N
TMSCN/Lewis Acid
HS
CO2H
SO3- , avidin
this work
Ph
Dehaen, Boens
NH2
Ziessel, Nagano, Mély, Bonnet
* Corresponding author. Tel.: +1 979 845 4345; fax: +1 979 845
1881; e-mail: burgess@tamu.edu
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.10.103
Scheme 1 shows the synthesis developed for the 3,5dichloroBODIPY 1. Academically, this follows a procedure similar to that already developed for the similar
compound A14 but practically the physical properties
of the materials and/or minor variations of the
L. Li et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3112–3116
neat
cat. TFA
Ar
25 oC, 1 h
NH HN
H
N
(i) NCS
THF, - 78 oC, 1.5 h
then 25 oC, 3 h
Ar
(ii) DDQ
CH2Cl2, 25 oC, 1 h
34 %
Ar
Ar
HN
N
ArCHO +
3113
Cl
B
F2
N
COOCH3
N
conditions A or B
Cl
N
B
F2
N
HN
X
MeO2C
Ar
3
BF3•OEt2, NEt3
N
CH2Cl2, 25 oC, 24 h
Cl
N
Cl
Cl
53 %
B
F2
A: MeCN, 25 °C, 10 min, gives 3a, 99 %, where X = Cl,
B: MeCN, reflux, 48 h, gives 3b, 93 %, where
N
Cl
X=
1 98 %
N
Ar =
CO2Me
Br
Scheme 1. Synthesis of the 3,5-dichloroBODIPY 1.
Scheme 2. Mono- and bis-substitution on BODIPY 1.
procedure made it possible to scale-up the syntheses to
5 g with minimal chromatography.
(reactions of solubilized cyanide ions were less satisfactory). The reaction of 1 in the presence of tin tetrachloride occurred selectively at the carbon to give compound
6a, but boron trifluoride promoted both types of displacement reactions to give the tetracyanide 6b. Cyanide
has been used in SNAr reactions occurring at the meso
(or 8-position) of non-halogenated-BODIPY dyes,15–17
but this is the first time that either mode of reactivity
shown in Scheme 3 has been reported.
Displacement of the first of the two chlorines in 3,5dichloroBODIPY 1 with a piperidine derivative occurs
rapidly (Scheme 2). The second chlorine can be displaced using extended reaction times at elevated temperatures. This particular piperidine derivative was of
interest because hydrolysis of the methyl ester would unmask two carboxylic acids that could be used for conjugation of this material to biomolecules.
The transformations shown in Scheme 2 were predictable for this substrate in combination with a soft nucleophile. However, reactions of 1 with a harder
nucleophile like methylmagnesium bromide could conceivably occur at either of the electrophilic sites. It transpired that displacement of the B-F bonds occurred
most rapidly, allowing relatively clean production of
the B-dimethylated compound 4. This could then be reacted with a piperidine derivative to give the SNAr product 5.
Cyanide is a softer anion than methylmagnesium bromide, but is harder than amines. The reaction of 3,5dichloroBODIPY 1 with this nucleophile reflects this
intermediate character. Displacement was achieved
using Lewis acidic activation of trimethylsilyl cyanide
Ar
Ar
MeMgBr
N
Cl
B
F2
N
THF, 25 oC, 5 min
Cl
Cl
N
N
B
Me Me
The Lewis-acid-mediated conditions described above
were also applied to the aminated products 3. This led
to the corresponding B-dicyanated compounds 7
(Scheme 4).
Table 1 shows photophysical data for molecules 1 and
3–7. Several trends can be observed using the starting
material 1 as a basis for comparison. Monoamination
and diamination cause red-shifts in the fluorescence, reduced quantum yields, and peak broadening. The
Stokes’ shift for 3a is high because the absorption maximum for this material is blue-shifted. The B-dimethylated compound 4 is spectroscopically similar to the parent
1 in all respects, and, just like 1 amination to give 5 blueshifts the absorption maximum. Replacement of the 3,5dichloro-substituents in 1 with cyanides gave the BF2
compound 6a and the B(CN)2-compound 6b. These
changes did not significantly affect the wavelengths for
the absorption or fluorescence maxima, but they did increase the quantum yields by a factor of 4–5. Again,
monoamination and diamination caused red-shifts in
the fluorescence, peak broadening, and reduced quantum yields.
Cl
4 57 %
Ar
Ar
Lewis Acid A or B
Ar
HN
COOCH3
N
N
B
N Me Me
N
MeCN, 25 °C, 6 h
Ar =
Cl
Cl
B
F2
+ TMSCN
N
NC
1
A: SnCl4, gives 6a, 99 %, where X = F
B: BF3•OEt2, gives 6b, 71%, where X = CN
H3COOC
N
CH2Cl2, 25 oC, 2 h
Cl
B
X2
N
CN
6
Ar =
Br
5 50 %
Scheme 3. Syntheses of compound 6 having cyanide substituents.
Br
3114
L. Li et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3112–3116
a
Ar
Ar
TMSCN, SnCl4
N
B
F2
N
N
N
N
B
N NC CN Cl
CH2Cl2, 25 oC
10 min
Cl
CO2Me
CO2Me
3a
b
7a 93 %
Ar
Ar
TMSCN, SnCl4
N
B
F2
N
N
CO2Me
N
N
B
N NC CN N
CH2Cl2, 25 oC
10 min
N
CO2Me
MeO2C
MeO2C
7b 65 %
3b
Scheme 4. B-F displacements using cyanide anion in: (a) mono-; and
(b) diaminated BODIPY dyes.
Table 1. Spectroscopic data for BODIPYs 1 and 3–7
Ar
N
X
Z
B
N
Z
Y
1
3a
3b
4
5
6a
6b
7a
7b
Dye
kabs a
(nm)
ea (M
1
1
3a
3b
4
5
6a
6b
7a
7b
512
482
573
503
483
514
510
485
517
77600
68400
39900
134300
61800
127400
36500
33700
26100
X
Cl
pip
pip
Cl
pip
CN
CN
pip
pip
cm 1)
Y
Cl
Cl
pip
Cl
pip
CN
CN
Cl
pip
Z
F
F
F
Me
Me
F
CN
CN
CN
Ar =
Br
pip =
CO2Me
N
kemiss a
(nm)
fwhma
(nm)
Ua
523
562
614
516
564
526
523
560
613
27
78
52
30
84
25
25
75
61
0.15 ± 0.01b
0.003c
0.01d
0.13 ± 0.01c
0.002c
0.66 ± 0.01b
0.80 ± 0.02b
0.006c
0.008b
a
In MeOH.
Rhodamine 6G was used as a standard (U = 0.94 in EtOH).
c
Fluorescein was used as a standard (U = 0.92 in 0.1 M NaOHaq).
d
Rhodamine 101 was used as a standard (U = 1.00 in EtOH).
more reactive in SNAr reactions, and we anticipated that
these reactions could be used to directly link the probe
to proteins. Second, the probe is small. Third, the trifluoromethyl group might be useful as an NMR marker.
Finally, the trifluoromethyl substituent makes the carbon that is destined to become the meso-carbon more
electrophilic, and this facilitates synthesis of the probe.
In the event, a convenient synthesis was developed, in
which the trifluoromethyl groups stabilized dipyrromethane and dipyrromethene intermediates 8 and 9 so that
these were isolable (Scheme 5).18
Scheme 6 shows how compound 2 was reacted with
avidin (chosen as a model protein). We assume this
gave the product of displacement of one chlorine atom
since excess of the dye was used, and because of the
experiments depicted in Scheme 6 featuring reactions
with simpler substrates. Indeed, it was necessary to
prepare compounds like 10 and 12 to estimate extinction coefficients for the dyes on proteins, and therefore
dye-to-protein ratios.19 Thus the calculated dye-toprotein ratio was 10:1 for 2-avidin, and 3:1 for 11avidin.
The quantum yields measured for the model compounds
10 and 12 were higher than similar derivatives of 1
(Table 2). This may be attributed to removal of a pathway for non-radiative decay when the freely rotating
meso-aryl group is replaced by CF3, and/or change in
oxidative potential of the BODIPY core leading to reduced photoinduced electron transfer. Absorbance and
fluorescence spectra of 2- and 11-avidin are close to
the control compounds 10 and 12 (Fig. 1 and Table
2); this supports the assertion that the substitution pattern is predominantly that shown in Scheme 6. Finally,
CD spectra recorded for avidin and 2-avidin showed
no significant differences in the 190–320 nm region, indicating the labeling event did not cause a radical change
in the protein tertiary structure.
In summary, the work described in this paper highlights
numerous ways in which the BODIPY core may be
modified via nucleophilic substitution reactions. In the
particular case of SNAr reactions on compound 2 provides a route to label proteins that breaks with well-used
b
CF3
The probes containing piperidine were prepared because
the carboxylic acid might be used to conjugate them to
biomolecules. However, this was not pursued further
since the quantum yields for these derivatives were
low. Further, hydrolysis of the esters gave carboxylic
acids that, at least in the protonated form, tended to
destabilize the structure, perhaps via a pH effect. Consequently, an alternative approach was adopted; this involved derivatives of system 2.
The BODIPY dye 2 was targeted because it might have
several attributes as a biochemical probe. First, the electron withdrawing trifluoromethyl group would make it
OH
HCl, THF
+
F 3C
NH reflux, 2 h
OMe
NH HN
8 48 %
(i) NCS, THF
- 78 oC, 1.5 h
then 25 oC, 3 h
(ii) DDQ, CH2Cl2
25 oC, 2 h
CF3
N
HN
Cl
CF3
BF3•OEt2
NEt3
Cl
CH2Cl2
25 oC, 24 h
9 45 %
Scheme 5. Synthesis of CF3-dichloroBODIPY 2.
N
Cl
B
F2
N
2 99 %
Cl
L. Li et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3112–3116
CF3
a
CF3
pH 8.3 buffer
N
Cl
B
F2
+
N
0.9
avidin
20
oC,
N
1h
Cl
B
F2
Cl
1:1 THF/H2O
25 oC, 12 h
2
3115
Model BODIPY 10
2-Avidin
N
Avidin
2-avidin
0.7
0.5
0.3
H2N ( ) CO2H
5
CF3
0.1
NaS
NaHCO3
1:1 THF/H2O
25 oC, 12 h
N
SO3Na
Cl
B
F2
200
-0.1
N
NH ( ) CO2H
5
N
S
B
F2
N
20
oC,
500
600
b
1
Model BODIPY 10
2-Avidin
0.8
CF3
Avidin
pH 8.3 buffer
400
Wavelength (nm)
10 72%
CF3
300
0.6
N
1h
Cl
S
B
F2
N
Avidin
0.4
0.2
SO3Na
SO3Na
11 58%
11-avidin
H2N ( ) CO2H
5
H2O
25 oC, 48 h
CF3
0
500
550
600
Wavelength (nm)
650
700
c
1
N
NaO3S
S
B
F2
N
NH ( ) CO2H
5
12
0.8
Model BODIPY 12
11-Avidin
0.6
Scheme 6. Syntheses of dye avidin conjugates and model compounds.
0.4
0.2
Table 2. Photophysics studies on compounds 2, 10, 11, and 12
Dye
kabs a
(nm)
ea (M
2b
2-Avidin
10
11
12
11-Avidin
548
481
469
569
477
492
89000
—
17100
57000
41800
—
1
cm 1)
kemiss a
(nm)
Ua
554
546
542
584
584
592
1.00 ± 0.02c
—
0.74 ± 0.02d
0.95 ± 0.02e
0.70. ± 0.01d
—
a
In pH 7.4 buffer (0.1 N lithium phosphate).
In CH2Cl2.
c
Rhodamine B was used as a standard (U = 0.73 in EtOH).
d
Fluorescein was used as a standard (U = 0.92 in 0.1 M NaOHaq).
e
Rhodamine 101 was used as a standard (U = 1.00 in EtOH).
0
200
300
400
500
600
Wavelength (nm)
700
800
d
1
Model BODIPY 12
11-Avidin
0.8
0.6
0.4
b
approaches involving activation of pendant carboxylic
acid functionalities on the dye.
0.2
0
500
550
600
Wavelength (nm)
650
700
Figure 1. (a) Absorption and (b) emission spectra for 2-avidin and its
model compound 10; and (c) absorption and (d) emission for
compound 11-avidin and its model compound 12. Throughout, spectra
were recorded in 0.1 M lithium phosphate buffer, pH 7.4, at 10 6 M
conc in protein or 10 5 M dye.
Acknowledgments
We thank the TAMU/LBMS-Applications Laboratory
for assistance with mass spectrometry. Support for this
work was provided by The National Institutes of Health
(HG 01745 and GM 72041) and by The Robert A.
Welch Foundation.
3116
L. Li et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3112–3116
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.
2007.10.103.
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