Microwave-assisted functionalization of bromo-fluorescein and bromorhodamine derivatives

Article abstract of DOI:10.1016/j.tetlet.2003.10.063

The overall goal of this project was to develop methodologies that would allow organometallic couplings of fluorescein and rhodamine derivatives. Consequently, borylation, Suzuki, and Sonogashira reactions of fragments derived from compounds 1 and 2 were investigated. Conventional and microwave heating were compared throughout the study.

Full text of DOI:10.1016/j.tetlet.2003.10.063

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 44 (2003) 9359–9362
Microwave-assisted functionalization of bromo-fluorescein
and bromorhodamine derivatives
Jin Wook Han, Juan C. Castro and Kevin Burgess*
Department of Chemistry, Texas A & M University, Box 30012, College Station, TX 77842-3012, USA
Received 18 September 2003; accepted 7 October 2003
Abstract—The overall goal of this project was to develop methodologies that would allow organometallic couplings of fluorescein
and rhodamine derivatives. Consequently, borylation, Suzuki, and Sonogashira reactions of fragments derived from compounds
1 and 2 were investigated. Conventional and microwave heating were compared throughout the study.
© 2003 Elsevier Ltd. All rights reserved.
Methods to form fluorescein and rhodamine dyes typi-
cally feature high temperature condensation reactions
that are not readily adapted to form small libraries of
derivatives.1–4 A project in these laboratories to prepare
fluorescent tags for labeling and observation of several
biomolecules in one system5 led us to consider ways of
linking fluorescein and rhodamine fragments. More
specifically, it was necessary to link them to form
twisted systems that would be conjugated if they
became planar. Consequently, we decided to investigate
organometallic couplings of starting materials that
could be used to prepare a diverse set of dyes. This
Letter reports conventional and microwave heating6 of
palladium-catalyzed reactions featuring the regioiso-
merically pure brominated starting materials 1 and 2.7
bromide more electron deficient than in compound 1.
Electron deficient aryl bromides tend to give more
efficient coupling reactions in catalytic cycles involving
oxidative addition, while the transmetallation compo-
nent is less sensitive to electronic factors.8 Conse-
quently, it was decided to focus on formation of the
boron-containing fragment from the fluorescein deriva-
tive 1.
The most obvious way to couple fragments 1 and 2 is
to form an organometallic species from one of them,
then couple this with the other. Suzuki couplings8
might be preferred because they are high yielding and
experimentally convenient. Use of this method would
require that one component be converted to a boronic
acid, hence it was necessary to decide which. Molecules
like 2 (rhodamines lacking a carboxylic acid functional-
ity) have been called rosamines.3 Experimentally, they
tend to be more difficult to manipulate than the fluores-
cein derivatives 1, because of their charge. We hypothe-
sized that their positive charge also makes the aryl
Initially, attempts were made to borylate 1 using pina-
colborane,9,10 since that reagent is cheaper than the
corresponding diboron reagents. Conventional heating
was investigated first. The 1,1%-diphenylphosphinoferro-
cenyl-based catalyst, PdCl2(dppf) gave poor conversion
and much of the material that was formed was the
unwanted reduction product 4 (Table 1, entry 1; similar
results were obtained using 100°C reaction temperature,
data not shown).
Keywords: fluorescent labels; microwave acceleration of reactions;
borylation; Suzuki coupling; Sonogashira coupling.
* Corresponding author. Tel.: +1-979-845-4345; fax: +1-979-845-8839;
e-mail: burgess@tamu.edu
One of Li’s phosphinite catalysts,11 that has proved
useful for other coupling reactions, was then investi-
gated; it gave some of the desired product, but reduc-
0040-4039/$ - see front matter © 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2003.10.063

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J. W. Han et al. / Tetrahedron Letters 44 (2003) 9359–9362
Table 1. Borylation of 5-bromofluorescein diacetate 1
Entry
Borylating agent
Base
Catalyst
Solvent
Heating method
(temp. °C, time)
Reactant/product
ratioc 1:3:4
1
2
3
4
5
6
7
8
9
H-BO2C6H12
H-BO2C6H12
NEt3
NEt3
NEt3
KOAc
KOAc
KOAc
KOAc
KOAc
KOAc
PdCl2(dppf)
Dioxane
Dioxane
Dioxane
Dioxane
Toluene
Toluene
Toluene
Toluene
Toluene
Conventional (80, 20 h)
Conventional (80, 20 h)
Conventional (80, 20 h)
Conventional (80, 20 h)
90:0.0:10
0.0:15:85
94:3.0:3.0
35:50:15
0.0:80:20
85:15:0.0
61:39:0.0
0.0:>98:0.0
0.0:>98:0.0
PdCl2(PtBu2OH)2
PdCl2(PtBu2OH)2
PdCl2(PtBu2OH)2
PdCl2(PtBu2OH)2
PdCl2(dppf)
H12C6O2B-BO2C6H12
H12C6O2B-BO2C6H12
H12C6O2B-BO2C6H12
H12C6O2B-BO2C6H12
H12C6O2B-BO2C6H12
H12C6O2B-BO2C6H12
H12C6O2B-BO2C6H12
Conventional (100, 20 h)
Microwave, 50 Wa (25–147, 5 min)
Microwave, 100 Wa (25–157, 5 min)
Microwave, 200 Wa (25–238, 15 min)
Microwaveb (150, 5 min)
PdCl2(dppf)
PdCl2(dppf)
PdCl2(dppf)
a Constant power, temperature allowed to vary.
b Variable power, constant temperature.
c Determined by 1H NMR; errors estimated as 5%.
It was convenient to store compound 3 because when it
is hydrolyzed to the corresponding deacylated boronic
acid 5 cyclotrimerization to the corresponding boroxine
ensues. Consequently, the boronate 3 was hydrolyzed
(K2CO3, 1:1 THF/H2O, 3 h; 79%) to 5 then isolated
immediately prior to use in Suzuki coupling reactions.
tion was still a complication (entry 2). It seemed likely
that the hydrogen that gave the reduction product was
dervied from the pinacolborane, hence the borylating
agent was switched to dipinacolatodiboron.12,13 Entries
4 and 5 demonstrate that when the base was also
switched to KOAc then an appreciable conversion to
the desired product was obtained. Still, however, reduc-
tion products were formed. It may be that the adventi-
tious hydrogen that causes formation of these reduction
products is solvent-derived.
Reaction 1 shows three model reactions that were per-
formed to evaluate Suzuki couplings to compound 5. A
water-soluble palladium catalyst gave a superior result
to Pd(PPh3)4 under these conditions, and the best iso-
lated yield of the product 6 was obtained from the
At this stage, it became evident that the optimization
process was too slow due to the reaction times
involved, hence subsequent studies focused on
microwave acceleration of the reactions.14,15 These reac-
tions were performed using sealed tubes in a CEM
Discover instrument that allows either the microwave
power or the reaction temperature to be held constant,
and the temperature in the reaction vessel to be moni-
tored. Control of the reaction temperature would not
be an option if a domestic microwave instrument was
used. Entries 6–8 illustrate that if the power is modu-
lated then the reaction temperature rises abruptly.
These erratic experiments are scientifically unsatisfying
because they would be hard to reproduce, especially on
a different instrument or for different reaction scales.
However, they did illustrate that high temperatures
were tolerated. Finally, entry 9 shows the most favor-
able conditions identified. The diboron reagent with
KOAc as base, microwave heated at a relatively high
temperature for a short time gave approximately 98%
selectivity for the desired product. A 93% yield of the
desired product was isolated when this reaction was
repeated on a 1 mmol scale (microwave, 150°C, 10 min)
and the crude material was purified via crystallization.16
microwave-accelerated transformation in
vessel.
a sealed
Reaction 2 illustrates how these findings were then
applied to the coupling of the rosamine 2 with com-

J. W. Han et al. / Tetrahedron Letters 44 (2003) 9359–9362
9361
pound 5.17 The water soluble catalyst was used to
couple the rosamine fragments, the product was con-
verted to a protic form, then the counterion was
metathesized to the tetra(3,5-trifluoromethylben-
zene)boronate (BARF) anion; this allows convenient
chromatography isolation of the product.
An in depth study of the spectroscopic properties of
these molecules and a full interpretation of those data
will be published elsewhere. However, Figure 1 shows
the UV absorption spectra and fluorescence emission
spectra of these molecules. The most significant features
are that for the molecules 7 and 9 but not 6, emission
in the 520 nm region, that would be attributed to the
fluorescein component of these systems, is greatly sup-
pressed; most of the fluorescence of 7 and 9 occurs at a
longer wavelength characteristic of the rosamine
fragment.
System 7 would be totally conjugated were it not for
the twists imposed by the adjacent aromatic rings. For
comparison, analogs of this structure with alkyne units
inserted between the rings were desired, hence
microwave-assisted Sonogashira couplings18 of the dyes
were investigated.
Alkyne 8 was formed in a two step process that
involved first coupling trimethylsilylethyne with the
protected fluorescein 1 under microwave conditions (5
mol% Pd(PPh3)4, CuI, NEt3, DMF, 120°C, 25 min;
84% isolated). Like the other microwave reactions
reported in this paper, this coupling was performed in a
sealed tube. The procedure for sealing tubes for use in
the microwave instrument by crimping on an aluminum
ring is very easy. Use of such sealed tubes for reactions
such as this where volatile reagents are used is clearly
an advantage. Deprotection of the alkyne was per-
formed using TBAF (2 equiv., CH2Cl2, −78°C, 5 min;
90%).
Reaction 3 illustrates how the alkyne 8 was coupled
with the bromorosamine 2 to give the desired alkyne-
containing product 9 via a microwave accelerated cou-
pling using the water-soluble catalyst. The product
from this step was hydrolyzed directly to remove the
acetate groups, acidified, then the counterion was
exchanged with BARF− to assist chromatographic
isolation.
Figure 1. (a) UV absorption, and (b) fluorescence emission
spectra of compounds 6, 7 and 9, when excited at 488 nm.

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J. W. Han et al. / Tetrahedron Letters 44 (2003) 9359–9362
Organometallic coupling reactions are rarely used to
functionalize fluorescein- and rhodamime-based sys-
tems. Here, halogenated dyes were transformed into
organoboron compounds, biaryls, and alkynes. Fluo-
rescein and rhodamine derivatives tend to be stable to
high temperatures, so they might be expected to be
amenable to microwave assisted steps. Indeed, the cou-
plings described here worked well using bursts of
microwave irradiation applied to give high tempera-
tures for relatively short times.
4. Orndorff, W. R.; Hemmer, A. J. J. Am. Chem. Soc.
1927, 49, 1272–1280.
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Bergstro¨m, F.; Johansson, L. B.-A. Chem. Commun.
2000, 2203–2204.
6. Larhed, M.; Moberg, C.; Hallberg, A. Acc. Chem. Res.
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7. Jiao, G.-S.; Han, J. W.; Burgess, K. J. Org. Chem. 2003,
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8. Miyaura, N.; Suzuki, A. Chem. Rev. 1995, 95, 2457–
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9. Murata, M.; Watanabe, S.; Masuda, Y. J. Org. Chem.
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Acknowledgements
10. Murata, M.; Oyama, T.; Watanabe, S.; Masuda, Y. J.
Org. Chem. 2000, 65, 164–168.
11. Li, G. Y. Angew. Chem., Int. Ed. 2001, 40, 1513–1516.
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lett 2003, 1204–1206.
We thank Dr. Mike Collins and CEM Corporation for
support with microwave technologies, Dr. George Y. Li
for samples of his catalysts, and Drs. Lars Thoresen
and Guan-Sheng Jiao for useful discussions. Dr. Shane
Tichy and the TAMU/LBMS-Applications Laboratory
are acknowledged. Financial support was provided by
The NIH (HG 01745) and by The Robert A. Welch
Foundation.
15. Furstner, A.; Seidel, G. Org. Lett. 2002, 4, 541–543.
16. The phosphine oxide ligands may be just as useful: the
optimization process was discontinued once a satisfac-
tory result had been obtained.
17. Adamczyk, M.; Grote, J. Bioorg. Med. Chem. Lett.
2003, 13, 2327–2330.
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