Sensitive thin-layer chromatography detection of boronic acids using alizarin

Article abstract of DOI:10.1055/s-0032-1316543

A new method for the selective and sensitive detection of boronic acids on thin-layer chromatography plates is described. The plate is briefly dipped in an alizarin solution, allowed to dry in ambient air, and observed under 366nm light. Alizarin emits a bright yellow fluorescence only in the presence of a boronic acid.

Full text of DOI:10.1055/s-0032-1316543

LETTER
▌1751
lSeteternsitive Thin-Layer Chromatography Detection of Boronic Acids Using
Alizarin
Sensitive Thin-Layer Chromatograp
a
hy Detection of Boronic Acids
a
a,b
Florine Duval, Teris A. van Beek,* Han Zuilhof
a
Laboratory of Organic Chemistry, Wageningen University, Dreijenplein 8, 6703 HB Wageningen, The Netherlands
Fax +31(317)484914; E-mail: teris.vanbeek@wur.nl
b
Department of Chemical and Materials Engineering, King Abdulaziz University, Jeddah, Saudi Arabia
Received: 27.03.2012; Accepted after revision: 14.05.2012
boronic acids on TLC plates. Alizarin has been used in
combination with concentrated H SO for the fluorescent
detection of boric acid on spot plates. Alizarin is also
Abstract: A new method for the selective and sensitive detection of
boronic acids on thin-layer chromatography plates is described. The
plate is briefly dipped in an alizarin solution, allowed to dry in am-
2
4
9
bient air, and observed under 366 nm light. Alizarin emits a bright known as a TLC staining reagent for the detection of cat-
III
yellow fluorescence only in the presence of a boronic acid.
ions (e.g., Al ), which requires subsequent treatment with
1
0
ammonia.
Key words: boronic acid, TLC, staining reagent, dyes/pigments,
fluorescence
OH
B
R2
O
OH
O
O
OH R2 B(OH)2
R1
O
Boronic acids are key intermediates in organic synthesis
that are involved in, for example, Suzuki and Chan-Lam
1
2
R1
coupling reactions. They have also been used as probes
for saccharides and as potential drugs for diverse thera-
O
O
3
peutic applications, implying the synthesis of target com-
non-fluorescent
fluorescent
pounds that contain boronic acid groups. Easy detection
of boronic acids during reactions, however, remains a
challenge.
alizarin: R1 = H
ARS: R1 = SO3Na
Scheme 1 Structures of alizarin and alizarin red S (ARS) and their
fluorescent complexes with boronic acids
The conversion of boronic acids has been monitored in
Suzuki–Miyaura reaction mixtures to which dihydroxy-
coumarins were added. Fluorescent complexes resulting
from binding of these compounds with boronic acids were
For the detection of boronic acids, preliminary tests
looked promising: after brief immersion of a TLC plate in
a saturated methanolic alizarin solution and spontaneous
evaporation of the solvent, spots that contained phenylbo-
ronic acid (PBA) turned yellow, whereas the rest of the
plate turned pink. Under 366 nm light, the spots showed
an intense yellow fluorescence on a dark background,
even though it is mentioned in the article of Barder and
Buchwald that alizarin would require a significantly high-
4
visualized under a hand-held UV lamp (366 nm). TLC,
however, is a more generally applicable and easier way to
analyze a reaction mixture. The detection of boronic acids
on TLC plates would enable the synthetic chemist to mon-
itor not only the conversion of a boronic acid, but also the
integrity of a boronic acid group during a reaction involv-
ing other functional groups. Moreover, it would not com-
plicate the purification of the product because there would
be no sensing molecule in the reaction mixture.
4
er excitation wavelength (ca. 550 nm).
The detection of 2 μL spots of PBA solutions was opti-
The natural anthraquinone alizarin, a non-fluorescent
compound in its free form, becomes fluorescent when
bound to a boronic acid via its 1,2-diol (Scheme 1). This
mized. A 50 mM solution of Na CO in 50% aqueous
2
3
5
methanol has previously been used to measure the affinity
1
1
of alizarin for boronic acids attached to solid supports.
property has been more commonly exploited with the wa-
ter-soluble derivative alizarin red S (ARS; Scheme 1), and
the mechanism of the involved reaction has recently been
Solubility tests, moreover, showed that 1 mM was near
the saturation concentration of alizarin in methanol. The
following solutions were therefore tested for the detection
of PBA spots on a TLC plate: 1 mM alizarin in (1)
Na CO (20 mM in H O–MeOH, 20:80), (2) methanol,
6
studied in detail. In particular, ARS has been used for the
direct visualization of a boronic acid group on a cellulose
7
8
surface and on glass slides by confocal microscopy.
2
3
2
and (3) HCl (20 mM in H O–MeOH, 20:80). The most in-
2
During our synthetic work involving boronic acids, we
were interested to see whether the commercially available
and inexpensive alizarin would be of help in visualizing
tense fluorescence was observed when pure methanol was
used. Ethanol, ethyl acetate, dichloromethane, tetrahydro-
furan and acetone were also tested, and all solvents gave
similar results in terms of sensitivity: 2 μL spots of PBA
solutions at concentrations down to 0.1 mM (24 ng) could
be easily detected (trace fluorescence at 0.01 mM). Ace-
tone was selected as the preferred solvent because it is in-
SYNLETT 2012, 23, 1751–1754
Advanced online publication: 04.07.2012
0
9
3
6
-
5
2
1
4
1
4
3
7
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2
0
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DOI: 10.1055/s-0032-1316543; Art ID: ST-2012-B0280-L
Georg Thieme Verlag Stuttgart · New York
©

1752
F. Duval et al.
LETTER
expensive, relatively safe, and the solubility of alizarin alizarin can be used to distinguish between boronic and
was higher in acetone than in the other solvents. Alizarin borinic esters.
solutions at different concentrations in acetone were sub-
The selectivity of this method towards boronic acids was
sequently tested (10, 1, 0.1, and 0.01 mM). The most in-
tested by spotting compounds with various functional
tense fluorescence was observed with the initial
groups on a TLC plate (Figure 2). Most compounds gave
concentration of 1 mM. Staining the TLC plate using a
some fluorescence, but PBA at a 10-fold lower concentra-
piece of cotton, or dipping it in the alizarin solution for 1,
tion gave a much more intense fluorescence than any of
5
or 10 seconds resulted in the same sensitivity. Spraying
the other compounds.
the TLC plate with the alizarin solution did not result in
any color change. The following method was therefore
chosen for subsequent experiments:
-
Briefly dip the TLC plate in a 1 mM alizarin solution
in acetone;
-
-
Let dry and wait until the TLC plate becomes pink;
Observe under 366 nm light.
The ability of this method to detect diverse boronic acids
and derivatives was tested (Figure 1). All compounds ap-
peared yellow-orange. Electron-deficient boronic acids
produced a slightly more intense color than electron-rich
boronic acids, which is probably due to the pK depen-
a
1
2
dence of the boronic acid–diol complexation. PBA pina-
col ester was revealed with the same fluorescence
intensity as PBA, which can be explained by the higher af-
1
2
finity of PBA for alizarin than for aliphatic diols. Triflu-
oroborates were revealed with the same fluorescence
intensity as the corresponding boronic acids. PBA N-me-
thyliminodiacetate (MIDA) ester could also be detected,
although it gave a much weaker fluorescence than PBA it-
self. The flavonoid reagent diphenylborinic acid 2-amino-
ethyl ester (DPBA) gave a distinct orange fluorescence
upon contact with alizarin. Under neutral conditions the
catechol group of alizarin, like the catechol group of some
flavonoids (e.g., rutin), probably gives a negatively
charged tetrahedral complex with DPBA after loss of eth-
1
3
Figure 2 Selectivity of alizarin towards boronic acids. Selections
from the original photographs of TLC plates spotted with phenylbo-
ronic acid (10 mM in MeOH, 2 μL) and diverse compounds (100 mM
in CH Cl , 2 μL), briefly immersed in 1 mM alizarin solution in ace-
anolamine. The difference in fluorescence suggests that
2
2
tone and placed under 366 nm light.
The applicability of this method to the analysis of reac-
tions involving boronic acids was tested in 3 examples.
Example 1: Monitoring the consumption of a boronic acid
during a classical Suzuki reaction (Scheme 2). The reac-
tion between 4-(trifluoromethyl)phenylboronic acid and
bromobenzene was carried out, based on the procedure
1
4,15
described by Yang et al.
A sample was spotted every
hour on a TLC plate. TLC analysis using 254 nm light did
not give any indication of the progress of the reaction.
TLC analysis using the proposed method, however,
showed a clear decrease in the concentration of boronic
acid over time. After five hours, the boronic acid was no
longer detected, which suggested that full conversion had
been achieved.
Figure 1 Use of alizarin to detect diverse boronic acids and deriva-
tives thereof. Selections from the original photograph of a TLC plate
spotted with diverse boron compounds (10 mM in MeOH*, 2 μL;
Example 2: Checking the completion of a ligand-free
Suzuki reaction (Scheme 3). The reaction between PBA
and 1-bromonaphthalene was carried out using a modified
*
MIDA ester in anhydrous THF), briefly immersed in 1 mM alizarin
1
6,17
procedure of Liu et al.
After four hours of reaction, the
solution in acetone and placed under 366 nm light.
Synlett 2012, 23, 1751–1754
© Georg Thieme Verlag Stuttgart · New York

LETTER
Sensitive Thin-Layer Chromatography Detection of Boronic Acids
1753
lyzed by TLC. TLC analysis using 254 nm light showed
only weak spots that corresponded to starting material and
a new compound (Scheme 4, left). TLC analysis using the
proposed method, however, showed the same spots with
an intense yellow fluorescence. Apart from much clearer
staining, this method also indicated directly that the new
compound contained a boronic acid moiety (Scheme 4,
right).
Scheme 2 Monitoring the consumption of a boronic acid during a
classical Suzuki reaction using alizarin. Reaction scheme and corre-
sponding TLC, selections from original photographs
reaction mixture was partitioned between dichlorometh-
ane and brine, and both layers were analyzed by TLC.
TLC analysis using 254 nm light (Scheme 3, left) was in-
conclusive. TLC analysis using the proposed method,
however, showed that neither the dichloromethane layer
nor the brine layer contained any significant amount of
PBA, and that full conversion had therefore been obtained
(Scheme 3, right).
Scheme 4 Visualizing the transformation of a boronic acid into an-
other boronic acid using alizarin. Reaction scheme and corresponding
TLC, selections from original photographs.
In conclusion, the difficult monitoring of boronic acids
during organic reactions can be simply overcome by brief-
ly dipping a TLC plate in a 1 mM alizarin solution, to re-
veal boronic acids on TLC plates in a selective and
sensitive manner.
Acknowledgment
We thank Pieken in de Delta Oost-Nederland for financial support.
References and Notes
(
1) Alonso, F.; Beletskaya, I. P.; Yus, M. Tetrahedron 2008, 64,
047.
3
(
(
2) Qiao, J. X.; Lam, P. Y. S. Synthesis 2011, 829.
3) Trippier, P. C.; McGuigan, C. Med. Chem. Commun. 2010,
1
, 183.
(
(
4) Barder, T. E.; Buchwald, S. L. Org. Lett. 2007, 9, 137.
5) Kubo, Y.; Kobayashi, A.; Ishida, T.; Misawa, Y.; James,
T. D. Chem. Commun. 2005, 2846.
Scheme 3 Checking the completion of a ligand-free Suzuki reaction
using alizarin. Reaction scheme and corresponding TLC, selections
from original photographs.
(
6) Tomsho, J. W.; Benkovic, S. J. J. Org. Chem. 2012, 77,
2
098.
Example 3: Visualizing the transformation of a boronic
acid into another boronic acid (Scheme 4). The esterifica-
tion of 4-carboxyphenylboronic acid in acidic ethanol was
carried out by using the procedure of Davison et al.
After one hour of reaction, the reaction mixture was ana-
(
7) Zhang, D.; Thompson, K. L.; Pelton, R.; Armes, S. P.
Langmuir 2010, 26, 17237.
(8) Ivanov, A. E.; Kumar, A.; Nilsang, S.; Aguilar, M. R.;
Mikhalovska, L. I.; Savina, I. N.; Nilsson, L.; Scheblykin,
I. G.; Kuzimenkova, M. V.; Galaev, I. Y. Colloids Surf., B
1
8,19
2
010, 75, 510.
©
Georg Thieme Verlag Stuttgart · New York
Synlett 2012, 23, 1751–1754

1754
F. Duval et al.
LETTER
(9) Szebellédy, L.; Tanay, S. Z. Anal. Chem. 1936, 107, 26.
Et O (1 mL) and 0.1 M HCl (2 mL), and the Et O layer was
2
2
(
(
10) Stahl, E. Thin-layer chromatography: a laboratory
handbook; Springer-Verlag: Berlin, 1969.
11) Duggan, P. J.; Offermann, D. A. Aust. J. Chem. 2007, 60,
spotted on a TLC plate.
(15) Yang, C.; Edsall, R. Jr.; Harris, H. A.; Zhang, X.; Manas,
E. S.; Mewshaw, R. E. Bioorg. Med. Chem. 2004, 12, 2553.
(16) Experimental Procedure for Example 2: A mixture of
phenylboronic acid (122 mg, 1 mmol), 1-bromonaphthalene
(0.15 mL, 1.1 mmol), Na CO (212 mg, 2 mmol) and
8
29.
(12) Springsteen, G.; Wang, B. Tetrahedron 2002, 58, 5291.
13) Matteini, P.; Agati, G.; Pinelli, P.; Goti, A. Monatsh. Chem.
(
2
3
2
011, 142, 885.
14) Experimental Procedure for Example 1: A mixture of
-(trifluoromethyl)phenylboronic acid (190 mg, 1 mmol),
bromobenzene (0.15 mL, 1.4 mmol) and Na CO (318 mg, 3
Pd(OAc) (ca. 1 mg) in acetone (3 mL) and H O (3.5 mL)
was stirred at r.t. in air.
2
2
(
4
(17) Liu, L.; Zhang, Y.; Xin, B. J. Org. Chem. 2006, 71, 3994.
(18) Experimental Procedure for Example 3:
4-Carboxyphenylboronic acid (92 mg, 0.55 mmol) was
dissolved in absolute EtOH (2.5 mL). A mixture of absolute
EtOH (2.5 mL) and concd HCl (2 drops) was added, and the
solution was heated to reflux.
2
3
mmol) in DME (6 mL) and H O (1.6 mL) was degassed with
argon for 10 min and heated to reflux. [Pd(PPh ) ] (58 mg,
2
3
4
0.05 mmol) was added and the reaction mixture was stirred
under reflux. Immediately after adding the catalyst, and then
after 1, 2, 3 and 5 h, aliquots (ca. 0.2 mL, except ca. 0.5 mL
after 5 h) of the reaction mixture were partitioned between
(19) Davison, J. Z.; Jones, W. D.; Zarrinmayeh, H.; Zimmerman,
D. M. US Patent 2003/225266 (A1), 2003.
Synlett 2012, 23, 1751–1754
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