Syntheses and spectral properties of functionalized, water-soluble BODIPY derivatives

Article abstract of DOI:10.1021/jo702463f

(Chemical Equation Presented) The objective of this work was to form water-soluble 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) derivatives. Sulfonation conditions were developed for several BODIPY dyes to give the monosulfonated products 1a-3a and

Full text of DOI:10.1021/jo702463f

Syntheses and Spectral Properties of Functionalized, Water-Soluble
BODIPY Derivatives
Lingling Li, Junyan Han, Binh Nguyen, and Kevin Burgess*
Department of Chemistry, Texas A & M UniVersity, Box 30012, College Station, Texas 77841
burgess@tamu.edu
ReceiVed NoVember 16, 2007
The objective of this work was to form water-soluble 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY)
derivatives. Sulfonation conditions were developed for several BODIPY dyes to give the monosulfonated
products 1a-3a and the disulfonated products 1b-3b. Compounds 1 are functionalized with an aryl
iodide for organometallic couplings. Similarly, 2 has an aromatic bromide but also two chlorine atoms
N
that could be replaced via S Ar reactions. The amine 3 is amenable to couple to biomolecules via acylation
reactions. A diazotization/azide reaction sequence was used to convert the amines 3 into azides 4; the
latter may be functionalized via click reactions as illustrated by conversion of 4b into 5. Compound 5
was designed to have an acid-functional group to facilitate activation and coupling to amines. Spectral
data for these materials indicate they are highly fluorescent probes in aqueous environments.
Introduction
Behind fluorescein, rhodamine, and possibly cyanine deriva-
amino groups on proteins or DNA-derivatives. Further, such
carboxylic acids can be activated using sulfonated succinimide
reagents; this makes the hydrophobic dyes more water-soluble,
9
1-4
tives, 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene, or BODIPY
hereafter abbreviated to BODIPY), dyes are probably the most
enabling them to be dissolved in aqueous media for coupling
to various water-soluble biomolecules. Once hydrophobic BO-
DIPY dyes are conjugated to biomolecules, they tend to embed
into hydrophobic pockets or even create micellular-like environ-
ments via aggregation effects. This is not always disadvanta-
geous; indeed, variations of BODIPY fluorescence with the
(
useful fluorescent probes in biotechnology. This is because they
tend to absorb UV radiation efficiently and emit relatively sharp
fluorescence peaks at useful wavelengths (typically 520-650
nm) with high quantum yields.
The core of BODIPY dyes is hydrophobic and does not
contain any functionality to attach the probes to proteins. Both
of these obstacles can be surmounted via synthetic modifications.
For instance, there are many BODIPY dyes with carboxylic acid
10-15
polarity of their immediate environment can be useful.
(8) Ueno, T.; Urano, Y.; Kojima, H.; Nagano, T. J. Am. Chem. Soc.
2006, 128, 10640-10641.
5-8
(9) Gee, K. R.; Archer, E. A.; Kang, H. C. Tetrahedron Lett. 1999, 40,
functional groups
that can be activated and then linked to
1
471-1474.
(10) Karolin, J.; Johansson, L. B.-A.; Strandberg, L.; Ny, T. J. Am. Chem.
(
1) http://probes.invitrogen.com. In Molecular Probes; Invitrogen
Corp.: Carlsbad, CA, 2006.
2) Haugland, R. P. Handbook of Fluorescent Probes and Research
Chemicals, 6th ed.; Molecular Probes: Eugene, OR, 1996.
Soc. 1994, 116, 7801-7806.
(11) Bergstrom, F.; Hagglof, P.; Karolin, J.; Ny, T.; Johansson, L. B. A.
Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 12477-12481.
(
(12) Bergstroem, F.; Mikhalyov, I.; Haeggloef, P.; Wortmann, R.; Ny,
T.; Johansson, L. B. A. J. Am. Chem. Soc. 2002, 124, 196-204.
(13) Mikhalyov, I.; Gretskaya, N.; Bergstroem, F.; Johansson, L. B. A.
Phys. Chem. Chem. Phys. 2002, 4, 5663-5670.
(14) Marushchak, D.; Kalinin, S.; Mikhalyov, I.; Gretskaya, N.; Johans-
son, L. B. A. Spectrochim. Acta, Part A 2006, 65A, 113-122.
(15) Baruah, M.; Qin, W.; Flors, C.; Hofkens, J.; Vallee, R. A. L.;
Beljonne, D.; Van der Auweraer, M.; De Borggraeve, W. M.; Boens, N. J.
Phys. Chem. A 2006, 110, 5998-6009.
(
3) Loudet, A.; Burgess, K. Chem. ReV. 2007, 107, 4891-4932.
(4) Ziessel, R.; Ulrich, G.; Harriman, A. New J. Chem. 2007, 31, 496-
5
01.
(
(
5) Li, Z.; Mintzer, E.; Bittman, R. J. Org. Chem. 2006, 71, 1718-1721.
6) Li, J.-S.; Wang, H.; Cao, L.-W.; Zhang, H.-S. Talanta 2006, 69,
1
190-1199.
7) Matsui, M.; Funabiki, K.; Nakaya, K.-i. Bull. Chem. Soc. Jpn. 2005,
8, 464-467.
(
7
1
0.1021/jo702463f CCC: $40.75 © 2008 American Chemical Society
Published on Web 02/14/2008
J. Org. Chem. 2008, 73, 1963-1970
1963

Li et al.
However, in other cases, it is definitely advantageous to have
water-soluble BODIPY dyes that can be conjugated easily and
that will tend to exist in the aqueous environment that surrounds
a biomolecule without perturbing it.
Despite the obvious practical value of water-soluble BODIPY
dyes, very few have been reported in the open literature. This
is even more surprising in view of the fact that BODIPY dyes
1
6
were first reported in 1969 and have been investigated with
3,4
increasing vigor since then. Indeed, the sum total of synthetic
procedures to obtain BODIPY dyes includes only the four
1
7,18
sulfonated derivatives A-D
and several closely related
oligo(ethylene glycol)-containing systems, of which E19 is
illustrative (Figure 1a). The fact that water-soluble BODIPYs
are relatively under-represented in the literature is unsurprising
to anyone who has attempted to sulfonate these dyes. Sulfonation
reagents tend to be highly reactive, whereas the BODIPY core
is relatively fragile. Further, many sulfonated products are not
isolated via flash chromatography. Consequently, reaction
conditions for sulfonation of BODIPY dyes must be controlled
carefully, and some optimization is required to isolate pure
materials from these transformations.
This paper describes several procedures for the preparation
of sulfonated, water-soluble BODIPY systems (Figure 1b).
Mono- (a) and disubstituted (b) tetramethyl-BODIPYs 1 have
a 4-iodobenzene substituent at the meso position to enable
further functionalization via organometallic cross-coupling
reactions. The bromo compounds 2 can be similarly derivatized,
but they are also potentially reactive toward nucleophiles in SN-
2
0-22
Ar reactions.
Compounds 3 are valuable since they can be
coupled to active carbonyl groups, the azides 4 are amenable
2
3,24
to copper-mediated cycloadditions to alkynes,
and the
disulfonate 5 can be activated and coupled to amino groups on
biomolecules. Thus, the end products of this work have potential
uses in many different scenarios for labeling biological mol-
ecules.
Results and Discussion
The following sections describe the preparation of the unusual
BODIPY starting materials, the pivotal sulfonation reactions,
and reactions of the sulfonated products to further transform
them into useful probes. Finally, the spectral properties of the
target molecules are discussed.
BODIPY Starting Materials. The lipophilic starting materi-
als used in this project were generally known compounds,2
5-27
(
16) Treibs, A.; Kreuzer, F.-H. Liebigs Ann. Chem. 1968, 718, 208-
23.
17) Shah, M.; Thangaraj, K.; Soong, M. L.; Wolford, L.; Boyer, J. H.;
Politzer, I. R.; Pavlopoulos, T. G. Heteroatom. Chem. 1990, 1, 389-399.
18) Wories, H. J.; Koek, J. H.; Lodder, G.; Lugtenburg, J.; Fokkens,
R.; Driessen, O.; Mohn, G. R. Recl. TraV. Chim. Pays-Bas 1985, 104, 288-
91.
19) Atilgan, S.; Ekmekci, Z.; Dogan, A. L.; Guc, D.; Akkaya Engin,
U. Chem. Commun. 2006, 4398-4400.
20) Rohand, T.; Baruah, M.; Qin, W.; Boens, N.; Dehaen, W. Chem.
Commun. 2006, 266-268.
21) Rohand, T.; Qin, W.; Boens, N.; Dehaen, W. Eur. J. Org. Chem.
006, 4658-4663.
22) Baruah, M.; Qin, W.; Vallee, R. A. L.; Beljonne, D.; Rohand, T.;
Dehaen, W.; Boens, N. Org. Lett. 2005, 7, 4377-4380.
23) Tornoe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002,
7, 3057-3064.
24) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B.
Angew. Chem., Int. Ed. 2002, 41, 2596-2599.
25) Li, M.; Wang, H.; Zhang, X.; Zhang, H.-s. Spectrochim. Acta, Part
A 2004, 60A, 987-993.
2
(
(
2
(
(
(
2
(
(
6
(
FIGURE 1. (a) Previously known water-soluble BODIPY systems;
b) compounds prepared in this work.
(
(
1964 J. Org. Chem., Vol. 73, No. 5, 2008

Water-Soluble BODIPY DeriVatiVes
SCHEME 1
oxidation sequence and a flash chromatographic separation.
Incorporation of the difluoroboron groups was achieved via the
standard procedure, though we found that the products 8 could
be isolated via recrystallizations rather than column chroma-
tography.
Sulfonation of Various BODIPY Derivatives. Scheme 2
shows the key sulfonation reactions featured in this paper. After
considerable experimentation, it was discovered that monosul-
fonations (Scheme 2a) of BODIPYs tend to proceed efficiently
using 1.2 equiv of fresh chlorosulfonic acid in CH2Cl2. The
sulfonating agent in CH2Cl2 was added dropwise over a few
minutes to a solution of the BODIPY starting material at -40
°
C. After the addition was complete, the cooling bath was
removed and the reaction was allowed to warm to 25 °C and
stirred for 20 min. For syntheses of compounds 1a, 2a, and 9a,
the reactions were quenched with NaHCO3(aq), and (after an
extraction procedure) the crude products were purified via flash
chromatography on silica. A critical observation in this work
was the need for the bicarbonate quench; it appears that the
protic forms of these sulfonic acids tend to be unstable (though
this is not always the case, see below).
SCHEME 2
Disulfonations of the same starting materials to give products
1
b, 2b, and 9b are shown in Scheme 2b. Two equivalents of
the chlorosulfonic acid was used to achieve the second sul-
fonation. Separation in this case is relatively easy because the
disulfonic acids precipitate from the dichloromethane solution
after 20 min at room temperature. The products were collected
by filtration, dissolved in a small amount of aqueous NaHCO3,
evaporated to dryness, and then reprecipitated from brine to give
essentially pure products. No chromatography is involved, so
the procedure is convenient and amenable to scale up.
One exception to the preferred sulfonation conditions was
27,30
for the alkyne-functionalized BODIPY,
as shown in reaction
1
. Here, the disulfonate 10 precipitated out of the CH2Cl2
solution in nearly pure form. This is very fortunate because
compound 10 is unstable in aqueous media, undergoing
relatively rapid hydrolysis at the alkyne group. For that reason,
this is not a particularly useful building block.
with the exception of compounds 8 shown in Scheme 1.
Condensation of 4-substituted benzaldehydes with pyrrole is a
2
8
known reaction for formation of the dipyrromethane F; this
was repeated here, and an analogous procedure was used to
prepare the bromoderivative 6. Chlorination of compounds
similar to 6 has been reported by Boens and co-workers. Slight
modifications of their procedures enabled us to obtain gram
amounts of the dipyrromethenes 7 after a “one-pot” chlorination
Reaction 2 shows a sulfonation of the relatively electron-
poor BODIPY system 8b with varying equivalents of chloro-
sulfonic acid. A mixture of mono- (11a) and disulfonation (11b)
products formed if less than 3.5 equiv of the sulfonating agents
were used, and neither of these materials precipitated from the
solution; it was, however, possible to obtain the yields indicated
via flash chromatography. Clean disulfonation was obtained
when 3.5 equiv of chlorosulfonic acid was used, and under those
conditions, the product 11b precipitated in a relatively pure form
and the sample could be further purified by reprecipitation from
brine as described for compounds 1b and 2b. The water-
29
(
26) Chen, J.; Burghart, A.; Derecskei-Kovacs, A.; Burgess, K. J. Org.
Chem. 2000, 65, 2900-2906.
27) Wan, C.-W.; Burghart, A.; Chen, J.; Bergstroem, F.; Johansson, L.
(
B.-A.; Wolford, M. F.; Kim, T. G.; Topp, M. R.; Hochstrasser, R. M.;
Burgess, K. Chem. Eur. J. 2003, 9, 4430-4441.
(28) Littler, B. J.; Miller, M. A.; Hung, C.-H.; Wagner, R. W.; O’Shea,
D. F.; Boyle, P. D.; Lindsey, J. S. J. Org. Chem. 1999, 64, 1391-1396.
29) Baruah, M.; Qin, W.; Basaric, N.; DeBorggraeve, W. M.; Boens,
N. J. Org. Chem. 2005, 70, 4152-4157.
(
(30) Burghart, A.; Thoresen, L. H.; Chen, J.; Burgess, K.; Bergstr o¨ m,
F.; Johansson, L. B.-A. Chem. Commun. 2000, 2203-2204.
J. Org. Chem, Vol. 73, No. 5, 2008 1965

Li et al.
solubility of the monosulfonate 11a was lower than the similar
tetramethyl-BODIPY monosulfonates 1a, 3a, 4a, and 9a; for
instance, it was impossible to obtain a clear 13C NMR spectrum
of 11a in D2O.
(benzyltriazolylmethyl)amine (TBTA)32 gave very little product,
but addition of this ligand made the reaction viable. The
disulfonate 5 is freely water-soluble and contains an easily
accessible carboxylic acid for activation and conjugation to
biomolecules.
Derivatization of Some BODIPY Sulfonates. Nitro func-
tionalities on BODIPYs have few direct applications, but this
group allows access to more useful derivatives that contain
amine or azido “handles”. Exploratory work to establish
conditions for hydrogenation of the nitro compounds 9 was not
encouraging. Almost no reduction was observed under 1 atm
of hydrogen in aqueous media. In ethanol, the rates were very
slow, and the product amine was contaminated by N-ethyl
impurities (this is a relatively common occurrence).31 Reactions
featuring SnCl2 in HCl(aq) tended to be complicated, probably
by tin complexes that did not elute from silica. Ultimately,
hydrazine and catalytic palladium on carbon proved to be
effective (Scheme 3). To decrease the risk of an explosion
during this reaction, the hydrazine was added dropwise to the
substrate and reagents in refluxing ethanol. This procedure has
been repeated several times on up to 400 mg of the nitro
compound without any mishaps; however, we advocate use of
a blast shield and certainly do not recommend that this reaction
be performed on a much larger scale. The amino derivatives 3
were isolated via flash chromatography.
Spectroscopic Properties of BODIPY Derivatives. Table
summarizes the spectroscopic data collected for the target
1
materials 1-5 and some interesting intermediates 9 and 11. In
general, all of these compounds are sufficiently water-soluble
to allow their UV and fluorescence properties to be recorded in
aqueous media. All of the disulfonates are freely soluble,
whereas the monosulfonates will not form relatively concen-
trated solutions.
25
All of the compounds shown in Table 1 have absorption
maxima in the range 492-518 nm, and their molar absorption
4
-1
-1
coefficients are high ((5.60-14.9) × 10 M cm ), as is
3
characteristic of BODIPY dyes in general; they fluoresce in
A diazotization/azide treatment reaction was used to convert
the amines 3 into the corresponding azides 4. Visually, this
reaction is interesting. The amine starting materials are weakly
green fluorescent; the reaction mixture becomes more fluores-
cent under acidic conditions, but this fluorescence disappears
when sodium nitrite is added, corresponding to formation of
the diazo compound. However, addition of azide gives a solution
that is more fluorescent than the starting amine.
the 507-540 nm range. Throughout, there are small differences
between the emission maxima of the mono- and disulfonated
forms; in fact, the maxima shift bathochromically between 2
and 4 nm when going from the mono- to disulfonated
compound, except for the nitro derivatives where the opposite
trend is observed. Longest wavelength fluorescence emission
maxima in the series are associated with the dichlorinated
compounds 2 and 11; all of the other probes emit between 507
and 513 nm. Sharp emissions are obtained, as seen in small
fwhm (full width at half-maximum height) values (904-1752
Reaction 3 shows how the disulfonate 4b was functionalized
via a copper-mediated azide-alkyne cycloaddition reaction.
Attempts to perform this reaction in the absence of tris-
-
1
cm ); for comparison, it is informative to consider the series
(
31) Filira, F.; Biondi, L.; Gobbo, M.; Rocchi, R. Tetrahedron Lett. 1991,
(32) Chan, T. R.; Hilgraf, R.; Sharpless, K. B.; Fokin, V. V. Org. Lett.
2004, 6, 2853-2855.
3
2, 7463-7464.
1966 J. Org. Chem., Vol. 73, No. 5, 2008

Water-Soluble BODIPY DeriVatiVes
SCHEME 3
TABLE 1. Spectroscopic Properties of the BODIPY Derivatives
λmax(abs)a
(nm)
λmax(em)a Fwhm
a
dye
log(ꢀmax)a,b
(nm)
(cm )
-1
Φa
1
1
2
2
3
3
4
4
5
9
9
ac
b
494
498
509
512
492
496
494
498
498
497
501
514
518
5.18 ( 0.01
5.00 ( 0.01
4.86 ( 0.01
4.89 ( 0.01
4.93 ( 0.01
5.06 ( 0.01
4.84 ( 0.01
4.89 ( 0.01
4.90 ( 0.01
4.76 ( 0.01
4.96 ( 0.01
4.82 ( 0.01
4.76 ( 0.01
507
509
523
524
507
511
507
509
511
513
511
540
538
1021
994
997
904
1050
945
1062
1053
1027
1752
1409
1440
1177
0.47 ( 0.02d
c
d
0.34 ( 0.02
c
0.27 ( 0.01e
a
c
e
b
a
0.41 ( 0.01
f
0.001d
f
d
b
0.001
f
0.34 ( 0.01d
0.15 ( 0.001
a
f
d
b
g
d
0.49 ( 0.01
ac
0.001d
c
d
b
0.002
0.002
h
e
1
1
1a
1b
h
0.008e
a
In H2O. b ꢀmax were estimated by linear fit of absorbance A vs dye
concentration c at four dye concentrations (one dye concentration was at
c
d
zero). Scheme 2. Fluorescein was used as a standard (Φ ) 0.92 in 0.1
4
0
e
M NaOH(aq)).
Rhodamine 6G was used as a standard (Φ ) 0.95 in
Scheme 3. Reaction 3. Reaction 2.
4
1
f
g h
EtOH).
of water-soluble Nile Red derivatives recently reported;33 these
have fwhm values for their fluorescence emission of between
-
1
1
410 and 1680 cm . Figure 2 shows the spectra obtained.
Quantum yields for the target compounds 1, 2, 4, and 5 were
all reasonably high for fluorescent probes (0.15-0.49). Some
BODIPY dyes have quantum yields that are greater than 0.5;
the slightly diminished values for 1, 2, 4, and 5 can be attributed
to nonradiative decay mechanisms associated with free rotation
of the meso substituent.34 Compounds 3 have a 4-aminobenzene
FIGURE 2. (a) UV absorption and (b) fluorescence spectra for the
mono-sulfonated BODIPYs. (c) UV absorption and (d) fluorescence
spectra for the bis-sulfonated BODIPYs. All of these spectra were
-
6
recorded in deionized water at concentrations of approximately 10
(
33) Jose, J.; Burgess, K. J. Org. Chem. 2006, 71, 7835-7839.
-
7
-6
M for the UV spectra and 10 -10
M for the fluorescence, then
(34) Yamada, K.; Toyota, T.; Takakura, K.; Ishimaru, M.; Sugawara, T.
New J. Chem. 2001, 25, 667-669.
normalized.
J. Org. Chem, Vol. 73, No. 5, 2008 1967

Li et al.
meso substituent; this electron-rich aromatic ring probably
quenches the fluorescence of the BODIPY core via photoin-
duced electron transfer (PeT) in which the excited-state of the
BODIPY fluorophore is reduced by electron transfer from the
relatively high-lying HOMO of the electron-rich meso-substitu-
ent quencher (i.e., reductive electron transfer). Such effects have
General Procedure for the Preparation of Monosulfonated
BODIPYs. A solution of chlorosulfonic acid (1.2 equiv) in CH
Cl (dry, 2 mL) was added dropwise to a solution of BODIPY (100
mg) in CH Cl (dry, 25 mL) over 10 min under N
2
-
2
2
2
2
at -40 °C.
Then the resulting solution was slowly warmed to room temperature.
After 20 min, TLC showed all of the starting material was
consumed, and then aqueous NaHCO
added to neutralize the solution, and the products were separated
from the CH Cl into the aqueous layer. The aqueous layer was
3
(1.2 equiv, 20 mL) was
3
5-39
been elegantly described as a-PeT by Nagano et al.
The
“a” denotes that the fluorescent chromophore acts as an acceptor.
2
2
The low quantum yield observed for compounds 3 is not a
concern if the amine group is transformed into an amide in the
bioconjugation process because that will adjust the oxidation
potential of the meso substituent, bringing down its HOMO level
and restoring the fluorescence.
Compounds 9 and 11 are intermediates rather than target
materials. They both have relatively low quantum yields, and
these can be rationalized via PeT effects. For these materials,
the LUMO of the meso-substituent is relatively low-lying due
to the influence of the 4-nitro group. This means that the excited
state of the BODIPY chromophore can act as an electron donor
to the electron-poor meso substituent quencher (i.e., oxidative
electron transfer).
evaporated to dryness. The residue was dry-loaded onto a silica
gel flash column and eluted using 15% MeOH/CH Cl . All of the
2
2
products were isolated as orange powders with an approximate R
) 0.4 (20% MeOH/CH Cl ).
f
2
2
Sodium 2-Sulfonate-1,3,5,7-tetramethyl-8-(4′-iodophenyl)-4,4-
difluoro-4-bora-3a, 4a-diaza-s-indacence (1a). 1,3,5,7-Tetram-
ethyl-8-(4′-iodophenyl)-4,4-difluoro-4-bora-3a,4a-diaza-s-inda-
cence26 (100 mg, 0.22 mmol) and chlorosulfonic acid (18 µL, 0.27
mmol) were reacted according to the general procedure giving an
1
orange powder (74 mg, 60%): H NMR (500 MHz, CD
3
OD) δ
7
2
.95 (d, 2H, J ) 8.3 Hz), 7.14 (d, 2H, J ) 8.3 Hz), 6.19 (s, 1H),
13
.75 (s, 3H), 2.52 (s, 3H), 1.70 (s, 3H), 1.46 (s, 3H); C NMR
OD) δ 159.3, 152.7, 145.7, 142.3, 139.8, 138.8,
34.4, 133.2, 132.7, 130.2, 129.0, 122.8, 94.8, 14.0, 13.6, 13.0,
(125 MHz, CD
3
1
1
-
-
2 2 3
2.2; MS (ESI) calcd for C19H17BF IN O S (M - Na) 529.01,
found 528.88; IR (thin film) 2922, 1717, 1540, 1312, 1193, 1033,
Conclusions
-
1
1006, 678 cm
.
Sulfonation reactions of BODIPY derivatives are hard to
develop into useful synthetic procedures for two reasons: (i)
inappropriate conditions give mixtures of products and (ii)
sulfonic acid derivatives of BODIPYs can be hard to purify.
The sulfonation reactions shown in Scheme 2 tend to give
predominantly one product, and reactions 1 and 2 give es-
sentially binary mixtures that are easily separated by flash
chromatography. Conjugation of the target materials to biomol-
ecules could be achieved via amide bond formation to amines
or acids or “click” chemistry. Further, some of the dyes
presented here can be derivatized via organometallic couplings
to the organic halide functionalities and, in the case of the
chlorinated derivatives 2 and 11, via SNAr reactions.
Sodium 2-Sulfonate-3,5-dichloro-8-(4′-bromophenyl)-4,4-di-
fluoro-4-bora-3a,4a-diaza-s-indacence (2a). Compound 8a (100
mg, 0.24 mmol) and chlorosulfonic acid (19.2 µL, 0.29 mmol) were
reacted according to the general procedure giving an orange powder
(114 mg, 92%): 1H NMR (500 MHz, CD OD) δ 7.78 (d, 2H, J )
3
8.5 Hz), 7.51 (d, 2H, J ) 8.5 Hz), 7.10 (s, 1H), 7.08 (br, 1H), 6.67
13
3
(d, 1H, J ) 4.6 Hz); C NMR (125 MHz, CD OD) δ 148.9, 145.5,
1
1
4
1
41.4, 136.3, 135.7, 134.7, 133.5, 133.2, 132.1, 131.6, 130.0, 127.0,
-
-
21.7; MS (ESI) calcd for C15
7 2 2 2 3
H BBrCl F N O S (M - Na)
92.88, found 492.76; IR (thin film) 1572, 1379, 1259, 1198, 1119,
-
1
055, 667 cm
.
Sodium 2-Sulfonate-1,3,5,7-tetramethyl-8-(4′-nitrophenyl)-
,4-difluoro-4-bora-3a,4a-diaza-s-indacence (9a). 1,3,5,7-Tet-
4
ramethyl-8-(4′-nitrophenyl)-4,4-difluoro-4-bora-3a,4a-diaza-s-in-
dacence25 (100 mg, 0.27 mmol) and chlorosulfonic acid (22 µL,
Experimental Section
0
.33 mmol) were reacted according to the general procedure giving
1
an orange powder (80 mg, 63%): H NMR (500 MHz, CD
3
OD) δ
Determination of Quantum Yields. Fluorescence quantum yield
measurements were performed on a fluorometer and UV/vis
instrument. The slit width was 5 nm for both excitation and
emission. Relative quantum efficiencies were obtained by compar-
ing the areas under the corrected emission spectrum. The following
equation was used to calculate quantum yield.
8
2
(
1
1
.46 (d, 2H, J ) 8.8 Hz), 7.69 (d, 2H, J ) 8.8 Hz), 6.22 (s, 1H),
1
3
.77 (s, 3H), 2.54 (s, 3H), 1.66 (s, 3H), 1.42 (s, 3H); C NMR
OD) δ 161.2, 154.4, 150.0, 146.6, 142.7, 142.1,
40.8, 134.6, 133.4, 131.3, 129.8, 125.7, 124.3, 15.2, 14.9, 14.2,
125 MHz, CD
3
-
-
3.5; MS (ESI) calcd for C19H17BF N O S (M - Na) 448.09,
found 447.98; IR (thin film) 1513, 1343, 1200, 1086, 988, 806 cm .
2 3 5
-1
2
x
2
Sodium 2-Sulfonate-3,5-dichloro-8-(4′-nitrophenyl)-4,4-dif-
luoro-4-bora-3a,4a-diaza-s-indacence (11a). Compound 8b (100
mg, 0.26 mmol) and chlorosulfonic acid (21 µL, 0.31 mmol) were
reacted according to the general procedure giving an orange powder
Φ ) Φ (I /I )(A /A )(η /η )
x
st
x
st
st
x
st
where Φst is the reported quantum yield of the standard, I is the
integrated emission spectrum, A is the absorbance at the excitation
wavelength, and η is the refractive index of the solvents used. The
subscript x denotes unknown and st denotes standard. Fluorescein
115 mg, 90%): 1H NMR (300 MHz, DMSO-d
(
)
6
d
1
6
) δ 8.42 (d, 2H, J
8.7 Hz), 7.93 (d, 2H, J ) 8.8 Hz), 7.09 (d, 1H, J ) 4.5 Hz),
13
.84 (d, 1H, J ) 4.5 Hz), 6.82 (s, 1H); C NMR (75 MHz, DMSO-
(
Φ ) 0.92 in ethanol) and rhodamine 6G (Φ ) 0.95 in ethanol)
6
) δ 149.6, 145.5, 142.6, 140.1, 138.2, 134.1, 133.4, 132.7, 132.3,
were used as standards.
-
7 2 2 3 5
30.8, 130.0, 124.5, 120.8; MS (ESI) calcd for C15H BCl F N O S
-
(
1
M - Na) 459.95, found 459.85; IR (thin film) 2982, 1558, 1390,
(
35) Tanaka, K.; Miura, T.; Umezawa, N.; Urano, Y.; Kikuchi, K.;
Higuchi, T.; Nagano, T. J. Am. Chem. Soc. 2001, 123, 2530-2536.
36) Ueno, T.; Urano, Y.; Setsukinai, K.; Takakusa, H.; Kojima, H.;
Kikuchi, K.; Ohkubo, K.; Fukuzumi, S.; Nagano, T. J. Am. Chem. Soc.
004, 126, 14079-14085.
37) Sunahara, H.; Urano, Y.; Kojima, H.; Nagano, T. J. Am. Chem.
Soc. 2007, 129, 5597-5604.
38) Gabe, Y.; Urano, Y.; Kikuchi, K.; Kojima, H.; Nagano, T. J. Am.
Chem. Soc. 2004, 126, 3357-3367.
39) Lakowicz, J. R. Principles of Fluorescence Spectroscopy; 2nd ed.;
Kluwer Academic/Plenum Publishers: New York, 1999.
-1
348, 1197, 1030, 667 cm
.
General Procedure for the Preparation of Disulfonated
(
BODIPYs. A solution of chlorosulfonic acid (2 equiv) in dry CH
Cl was added dropwise to a solution of BODIPY in dry CH Cl
over 10 min under N
as the solution mixture warmed slowly to room temperature. The
disulfonic acid was isolated by vacuum filtration and treated with
2
-
2
2
2
2
(
2
at -40 °C. An orange precipitate was formed
(
3
water. The aqueous solution was neutralized with NaHCO (2
equiv), concentrated to 5-10 mL, and treated with brine.
(
1968 J. Org. Chem., Vol. 73, No. 5, 2008

Water-Soluble BODIPY DeriVatiVes
The desired product was reprecipitated afterward to afford an orange
solid (85-100% yield).
Disodium 2,6-Disulfonate-1,3,5,7-tetramethyl-8-(4′-iodophe-
nyl)-4,4-difluoro-4-bora-3a,4a-diaza-s-indacence (1b). 1,3,5,7-
Tetramethyl-8-(4′-iodophenyl)-4,4-difluoro-4-bora-3a,4a-diaza-s-
indacence26 (53 mg, 0.118 mmol) and chlorosulfonic acid
10 min. Hydrazine monohydrate (0.05 mL) and 10% Pd/C (7.9 mg,
0.1 equiv) were added. The mixture was refluxed under N for 30
min. Then Pd/C was removed under vacuum filtration. After
evaporation of the solvent, the residue was dry-loaded onto a silica
2
gel flash column and eluted using 15% MeOH/CH
an orange solid (30 mg, 92%): R ) 0.3 (20% MeOH/CH
NMR (500 MHz, CD OD) δ 6.97 (d, 2H, J ) 8.5 Hz), 6.86 (d,
2H, J ) 8.5 Hz), 6.15 (s, 1H), 2.74 (s, 3H), 2.50 (s, 3H), 1.79 (s,
3H), 1.55 (s, 3H); 13C NMR (125 MHz, CD
OD) δ 159.5, 152.7,
150.7, 147.2, 146.5, 141.2, 134.8, 133.7, 131.1, 130.0, 124.2, 123.5,
2 2
Cl to afford
1
f
2 2
Cl ); H
(
16 µL, 0.236 mmol) were reacted according to the general
3
1
procedure giving an orange powder (68 mg, 88%): H NMR
500 MHz, D O) δ 7.84 (d, 2H, J ) 8.0 Hz), 6.97 (d, 2H, J ) 8.0
Hz), 2.57 (s, 6H), 1.49 (s, 6H); C NMR (75 MHz, D
(
2
3
13
2
O) δ 155.5,
45.7, 144.0, 139.2, 133.1, 132.7, 130.6, 129.7, 95.7, 13.7, 13.0;
-
1
2 3 3
116.6, 15.2, 14.8, 14.1, 13.4; MS (ESI) calcd for C19H19BF N O S
-
-
-
MS (ESI) calcd for C19
found 608.98.
H17BF
2
IN O S
2 6 2
(M - 2Na + H) 608.96,
(M - Na) 418.12 found 418.04; IR (thin film) 3414, 2922, 1608,
-
1
1540, 1519, 1196, 1036, 684 cm
.
Sodium 2-Sulfonate-1,3,5,7-tetramethyl-8-(4′-azidophenyl)-
,4-difluoro-4-bora-3a,4a-diaza-s-indacence (4a). A solution of
Disodium 2,6-Disulfonate-3,5-dichloro-8-(4′-bromophenyl)-
,4-difluoro-4-bora-3a,4a-diaza-s-indacence (2b). Compound 8b
4
3
4
(
a (29 mg, 0.07 mmol) in HCl (2 M, 5 mL) and H
(11.3 mg, 0.16mmol) in H
(2 mL) was added slowly, and the mixture was kept at 0 °C with
stirring for 30 min. Then a solution of NaN (22 mg, 0.33 mmol)
2
O (2 mL) was
500 mg, 0.12 mmol) and chlorosulfonic acid (160 µL, 0.24 mmol)
were reacted by the general procedure giving an orange powder
624 mg, 85%): 1H NMR (500 MHz, D
O) δ 7.73 (d, 2H, J ) 8.4
Hz), 7.45 (d, 2H, J ) 8.4 Hz), 7.27 (s, 2H); C NMR (75 MHz,
O) δ 147.6, 141.9, 133.7, 132.6, 132.3, 131.8, 131.5, 130.0,
cooled to 0 °C. A solution of NaNO
2
2
O
(
2
13
3
in H
continued at room temperature for 1 h after completion of the
addition. The resulting mixture was neutralized with NaHCO and
evaporated to dryness. The residue was dry-loaded onto a silica
gel flash column and eluted using 15% MeOH/CH Cl to afford
an orange solid (23 mg, 75%): R ) 0.3 (20% MeOH/CH
NMR (300 MHz, D O) δ 6.92 (d, 2H, J ) 7.2 Hz), 6.76 (d, 2H, J
) 7.2 Hz), 5.81 (s, 1H), 2.65 (s, 3H), 2.26 (s, 3H), 1.52 (s, 3H),
.04 (s, 3H); 13C NMR (125 MHz, D
O) δ 160.0, 152.0, 146.3,
142.7, 141.5, 140.2, 133.0, 131.9, 130.3, 129.3, 129.1, 123.4, 120.1,
2
O (2 mL) was added dropwise to the mixture. Stirring was
D
2
2-
2-
1
2
6
26.7; MS (ESI) calcd for C15
85.91, found 285.84; IR (thin film) 2968, 1572, 1382, 1206, 1033,
50 cm
6
H BBrCl
F
2 2
N
2
O S
6 2
(M - 2Na)
3
-
1
.
Disodium 2,6-Disulfonate-1,3,5,7-tetramethyl-8-(4′-nitrophe-
nyl)-4,4-difluoro-4-bora-3a,4a-diaza-s-indacence (9b). 1,3,5,7-
2
2
1
f
2 2
Cl ); H
Tetramethyl-8-(4′-nitrophenyl)-4,4-difluoro-4-bora-3a,4a-diaza-s-
2
indacence25 (400 mg, 1.08 mmol) and chlorosulfonic acid (144 µL,
1
2
2
.16 mmol) were reacted according to the general procedure
1
giving an orange powder (630 mg, quant yield): H NMR (300
MHz, D
-
1
4.4, 14.3, 13.5, 12.6; MS (ESI) calcd for C19
H17BF
2
N
5
3
O S
(M
2
O) δ 8.49 (d, 2H, J ) 8.5 Hz), 7.70 (d, 2H, J ) 8.5 Hz),
-
.77 (s, 6H), 1.63 (s, 6H); 13C NMR (75 MHz, D
- Na) 444.11, found 444.02; IR (thin film) 2128, 2105, 1541,
2
1
2
O) δ 156.1, 148.8,
-
1
1
304, 1192, 1023, 686 cm
.
44.0, 143.6, 140.5, 132.9, 130.2, 129.6, 125.3, 13.8, 13.0; MS
2
-
2-
Disodium 2,6-Disulfonate-1,3,5,7-tetramethyl-8-(4′-aminophe-
nyl)-4,4-difluoro-4-bora-3a,4a-diaza-s-indacence (3b). A solution
(
ESI) calcd for C19
found 263.45; IR (thin film) 1522, 1347, 1190, 1004, 853, 669
cm
H
16BF
2
N
3
O
8
S
2
(M - 2Na)
263.52
-
1
of 9b (200 mg, 0.35 mmol) in EtOH (10 mL) was purged with N2
.
for 10 min. Hydrazine monohydrate (0.2 mL) and 10% Pd/C (37.1
1
,3,5,7-Tetramethyl-8-(4′-ethynylphenyl)-4,4-difluoro-4-bora-
a,4a-diaza-s-indacence 2,6-disulfonic Acid (10). A solution of
chlorosulfonic acid (19 µL, 0.276 mmol) in CH Cl (2 mL) was
added dropwise to a solution of 1,3,5,7-tetramethyl-8-(4′-ethy-
mg, 0.1 equiv) were added. The mixture was refluxed under N
2
3
for 30 min. Then Pd/C was removed under vacuum filtration. After
evaporation of the solvent, the residue was dry-loaded onto a silica
2
2
4
2
gel flash column and eluted using 30% MeOH/CH
an orange solid (133 mg, 70%): R ) 0.2 (30% MeOH/CH
O) δ 7.02-6.94 (m, 4H), 2.70 (s, 6H),
.70 (s, 6H); 13C NMR (75 MHz, D
O) δ 154.7, 148.2, 144.1, 132.3,
31.2, 130.0, 123.9, 117.1, 117.0, 13.0 (2); MS (ESI) C19
2
Cl
2
to afford
nylphenyl)-4,4-difluoro-4-bora-3a,4a-diaza-s-indacence (48 mg,
.138 mmol) in CH Cl (5 mL) over 10 min at -40 °C. An orange
f
2
Cl );
2
0
2
2
1
H NMR (300 MHz, D
2
precipitate was formed as the solution mixture warmed slowly to
room temperature. The disulfonic acid was isolated by vacuum
filtration giving the disulfonic acid as an orange powder (42 mg,
1
2
1
Na
H
19BF
2
N
3
-
+
+
0%): 1H NMR (300 MHz, D
2
O
S
6 2
(M + H) 544.0572 found 544.0557; IR (thin film) 3346,
6
2
O) δ 7.66 (d, 2H, J ) 8.8 Hz), 7.29
-
1
2
854, 1608, 1519,1197, 1032, 655 cm
Disodium 2,6-Disulfonate-1,3,5,7-tetramethyl-8-(4′-azidophe-
nyl)-4,4-difluoro-4-bora-3a, 4a-diaza-s-indacence (4b). A solution
of 3b (100 mg, 0.18 mmol) in HCl (2 M, 20 mL) and H O (5 mL)
was cooled to 0 °C. A solution of NaNO (32 mg, 0.46 mmol) in
O (3 mL) was added slowly, and the mixture was kept at 0 °C
with stirring for 30 min. Then a solution of NaN (60 mg, 0.92
mmol) in H O (3 mL) was added dropwise to the mixture. Stirring
was continued at room temperature for 1 h after completion of the
addition. The resulting mixture was neutralized with NaHCO and
evaporated to dryness. The residue was dry-loaded onto a silica
gel flash column and eluted using 30% MeOH/CH Cl to afford
an orange solid (88 mg, 77%): R ) 0.2 (30% MeOH/CH
NMR (300 MHz, D O) δ 7.33-7.26 (m, 4H), 2.75 (s, 6H), 1.67
s, 6H); 13C NMR (75 MHz, D
O) δ 155.5, 146.2, 143.9, 142.0,
.
(
(
d, 2H, J ) 8.5 Hz), 3.48 (s, 1H), 2.63 (s, 6H), 1.54 (s, 6H); MS
-
-
ESI) C21
Disodium 2,6-Disulfonate-3,5-dichloro-8-(4′-nitrophenyl)-4,4-
difluoro-4-bora-3a,4a-diaza-s-indacence (11b). Compound 8b
100 mg, 0.26 mmol) and chlorosulfonic acid (61 µL, 0.91 mmol)
H
18BF
2
N
2
O
6
S
2
(M - H) 507.07, found 507.08.
2
2
(
H
2
were reacted according to the general procedure (the only difference
is using 3.5 equiv of chlorosulfonic acid and being neutralized with
3
2
3
.5 equiv of NaHCO
3
) giving an orange powder (151 mg, 97%):
H NMR (300 MHz, D O) δ 8.30 (d, 2H, J ) 7.5 Hz), 7.70 (d,
H, J ) 7.5 Hz), 7.18 (s, 2H); 13C NMR (75 MHz, D
O) δ 149.4,
45.2, 143.1, 136.8, 134.5, 132.0, 131.3, 124.1 (two carbons may
1
2
3
2
1
2
2-
2
2
beco-incidentinthisspectrum);MS(ESI)calcdforC15
H
6
BCl
2
F
2
N
3
O
8
S
2
1
M - 2Na)2 269.45 found 269.38; IR (thin film) 3113, 1519, 1379,
-
f
2 2
Cl ); H
(
348, 1200, 1030, 848, 692, 680, 664 cm-1
2
1
.
(
2
Sodium 2-Sulfonate-1,3,5,7-tetramethyl-8-(4′-aminophenyl)-
,4-difluoro-4-bora-3a,4a-diaza-s-indacence (3a). A solution of
a (35 mg, 0.07 mmol) in EtOH (10 mL) was purged with N for
2
132.7, 130.9, 129.9, 129.5, 120.5, 13.7, 13.0; MS (ESI) calcd for
4
9
-
-
C
19
H
17BF
2
N
5
O S
6 2
(M - 2Na + H) 523.06, found 523.95; IR
-
1
(
thin film) 2130, 1549, 1295, 1038, 667 cm
Compound 5. Cu (4.5 mg, 0.07 mmol), CuSO
.007 mmol), and TBTA (3.7 mg, 0.007 mmol) were added to a
.
4
2
‚5H O (1.8 mg,
(
48.
40) Weber, G.; Teale, F. W. J. Trans. Faraday Soc. 1958, 54, 640-
0
6
solution of 4b (40 mg, 0.07 mmol) and hexynoic acid (15.7 mg,
.14 mmol) in 1:1 THF/H O (5 mL). The reaction mixture was
(
41) Kubin, A. N.; Fletcher, A. N. J. Luminescence 1982, 27, 455-462.
0
2
(42) Wan, C.-W.; Burghart, A.; Chen, J.; Bergstroem, F.; Johansson, L.
B. A.; Wolford, M. F.; Kim, T. G.; Topp, M. R.; Hochstrasser, R. M.;
Burgess, K. Chem. Eur. J. 2003, 9, 4430-4441.
stirred at room temperature for 12 h and evaporated to dryness.
The residue was dry-loaded onto a silica gel flash column and eluted
J. Org. Chem, Vol. 73, No. 5, 2008 1969

Li et al.
using 40% MeOH/CH
2%): R ) 0.1 (30% MeOH/CH
δ 8.30 (s, 1H) 8.00 (d, 2H, J ) 8.3 Hz), 7.49 (d, 2H, J ) 8.3 Hz),
2
Cl
2
to afford an orange solid (20 mg,
Applications Laboratory directed by Dr. Shane Tichy for
assistance with mass spectrometry. Support for this work was
provided by The National Institutes of Health (GM72041) and
by The Robert A. Welch Foundation.
1
4
f
2 2 2
Cl ); H NMR (500 MHz, D O)
2
.75 (br, 8H), 2.33 (t, 2H, J ) 7.2 Hz), 1.97-1.93 (m, 2H), 1.58
s, 6H); 13C NMR (125 MHz, D
O) δ 180.0, 155.8, 145.1, 143.8,
37.7, 134.3, 132.9, 130.6, 129.8, 122.5, 122.0, 121.7, 39.7, 24.6,
(
2
1
2
2
2-
4.2, 13.8, 13.1; MS (ESI) calcd for C25
H24BF N O S
2 5 8 2
(M -
Supporting Information Available: Characterization data for
compounds 1-11. This material is available free of charge via the
Internet at http://pubs.acs.org.
Na)2 317.56, found 317.50.
-
Acknowledgment. We thank Dr. Valery V. Fokin (Scripps)
for a generous gift of the TBTA ligand and the TAMU/LBMS-
JO702463F
1970 J. Org. Chem., Vol. 73, No. 5, 2008