Two polymerizable derivatives of 2,2′-azino-bis(3- ethylbenzthiazoline-6-sulfonic acid)

Article abstract of DOI:10.1021/ol0513443

(Chemical Equation Presented) A versatile strategy for the synthesis of polymerizable derivatives of the redox-active indicator dye 2,2′-azino- bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) is reported. Two products are shown to illustrate how the fin

Full text of DOI:10.1021/ol0513443

ORGANIC
LETTERS
2006
Vol. 8, No. 1
3-6
Two Polymerizable Derivatives of
2,2 -Azino-bis(3-ethylbenzthiazoline-6-sulfonic
′
acid)
Jiangfeng Fei,† Amit Basu,‡ Fengtian Xue,‡ and G. Tayhas R. Palmore*,†,§
DiVison of Engineering, Department of Chemistry, and DiVision of Biology and
Medicine, Brown UniVersity, ProVidence, Rhode Island 02912
tayhas_palmore@brown.edu
Received June 8, 2005 (Revised Manuscript Received November 8, 2005)
ABSTRACT
A versatile strategy for the synthesis of polymerizable derivatives of the redox-active indicator dye 2,2
′
-azino-bis(3-ethylbenzthiazoline-6-
sulfonic acid) (ABTS) is reported. Two products are shown to illustrate how the final step in the synthetic strategy can be used to alter the
physical properties of the product. Both products were characterized spectroscopically and electrochemically. The hydrophilic monomer
(sABTS) was polymerized, and the utility of this polymer (polyABTS) is demonstrated in the context of bioelectrocatalysis.
The molecule 2,2′-azino-bis(3-ethylbenzothiazoline-6-sul-
fonate) (ABTS) is an important redox-active compound with
broad chemical, material, and biomedical applications. For
example, ABTS and its derivatives have been used as the
electrochromic component in smart windows,1 as a chromo-
genic substrate in assays for enzymatic activity,2 and as a medi-
ator for electron transfer in bioelectrocatalysis.3 Interest in
the use of ABTS in the bioelectrocatalytic reduction of oxy-
gen to water has increased primarily because its redox poten-
tial is near that of oxygen under mildly acidic conditions.4
Interest in redox-active polymers stems from their ap-
plication in electrochromic devices,5 biofuel cells,6 and
biosensors, all of which require a high concentration of
electron mediators that are nonleachable.7 Herein we describe
the synthesis of two derivatives of ABTS that can be
polymerized or copolymerized with other vinyl monomers
to tune the physical properties of the resulting low potential,
electrochromic, redox-active polymer.
(3) (a) Zhao, J.; Henkens, R. W.; Stonehuerner, J.; O’Daly, J. P.;
Crumbliss, A. L. J. Electroanal. Chem. 1992, 327, 109. (b) Gelo-Pujic,
M.; Kim, H. H., Butlin, N. G., Palmore, G. T. R. Appl. EnViron. Microbiol.
1999, 65, 5515. (c) Tsujimura, S.; Tatsumi, H.; Ogawa, J.; Shimizu, S.;
Kano, K.; Ikeda, T. J. Electroanal. Chem. 2001, 496, 69. (d) Dodor, D. E.;
Hwang, H. M.; Ekunwe, S. I. N. Enzyme Microb. Technol. 2004, 35, 210.
(e) Luo, T.-J. M.; Fei, J.; Lim, K. G.; Palmore, G. T. R. Nanotechnology
and the EnVironment; ACS Symposium Series 890; American Chemical
Society: Washington, DC, 2004; Chapter 10.
(4) Palmore, G. T. R.; Kim, H. H. J. Electroanal. Chem. 1999, 464,
110.
† Divison of Engineering.
‡ Department of Chemistry.
(5) (a) Wrighton, M. S.; Palmore, G. T. R.; Hable, C. T.; Crooks, R. M.
New Aspects of Organic Chemistry; Yoshida, Z., Shiba, T., Ohshiro, Y.,
Eds.; VCH Publishers: New York, 1989; p 277. (b) Christ, Jr., C. S.; Yu,
J.; Zhao, X.; Palmore, G. T. R.; Wrighton, M. S. Inorg. Chem. 1992, 31,
4439. (c) Palmore, G. T. R.; Smith, D. K.; Wrighton, M. S. J. Phys. Chem.
B 1997, 101, 2437.
(6) (a) Palmore, G. T. R.; Whitesides, G. M. Enzymatic ConVersion of
Biomass for Fuels Production; ACS Symposium Series 566; American
Chemical Society: Washington, DC, 1994; p 271. (b) Palmore, G. T. R.;
Bertschy, H.; Bergens, S. H.; Whitesides, G. M. J. Electroanal. Chem. 1998,
443, 155. (c) Palmore, G. T. R. Trends Biotechnol. 2004, 22, 99.
(7) (a) Foulds, N. C.; Lowe C. R. Anal. Chem. 1988, 60, 2473 (b) Gregg
B. A.; Heller A. Anal. Chem. l990, 62, 258.
§ Division of Biology and Medicine.
(1) (a) Shelepin, I. V.; Ushakov, O. A.; Karpova, N. I.; Barachevskii,
V. A. Elektrokhimiya 1977, 13, 32.(b) Ushakov, O. A.; Shelepin, I. V.;
Vasil’ev, Y. B. Elektrokhimiya 1979, 15, 1893.
(2) (a) Groome, N. P. J. Clin. Chem. Clin. Biochem. 1980, 18, 345. (b)
Matsuda, H.; Tanaka, H.; Blas, B. L.; Nosenas, J. S.; Tokawa, T.; Ohsawa,
S. Jpn. J. Exp. Med. 1984, 54, 131. (c) Majkic-Singh, N.; Said, B. A.; Spasic,
S.; Berkes, I. Ann. Clin. Biochem. 1984, 21, 504. (d) Kreit, J.; Lefebvre,
G.; Elhichami, A.; Germain, P.; Saghi, M. Lipids 1992, 27, 458. (e) Ukeda,
H.; Fujita, Y.; Ohira, M.; Sawamura, M. J. Agric. Food Chem. 1996, 44,
3858. (f) Dekker: R. F. H.; Ling, K. Y.; Barbosa, A. M. Biotechnol. Lett.
2000, 22, 105.
10.1021/ol0513443 CCC: $33.50
© 2006 American Chemical Society
Published on Web 12/07/2005

Scheme 1. Synthetic Route to sABT and sABTS
The synthesis and characterization of two polymerizable
nitrogen to give an aminothiol product. Chromatographic
isolation of the aminothiol product from several side
products, however, proved difficult. Therefore, the hydrolysis
reaction was performed in the presence of oxygen to give
the corresponding disulfide (2) in 53-55% yield. The
disulfide, which is a yellow crystalline solid, is easier to
isolate because its Rf value is higher than that of 1 or the
aminothiol product obtained in the absence of oxygen. Both
the aminothiol product or 2 will react with carbon disulfide
when refluxed in a mixture of ethanol and NaOH to give 3
as a white crystalline solid.11 Choosing the disulfide product
instead of the aminothiol product simplified the isolation
process to give 3 in 82-84% yield. Diethyl ether was the
solvent of choice for removal of 2 from the reaction mixture.
Benzothiazolinone or thiazolinone are known to react with
the corresponding benzothiazolinethione or thiazolinethione
via phosphorus pentasulfide thiation reaction or Lawesson’s
reagents without an intermediate hydrolysis step.12 Com-
pound 1, however, does not react with those reagents using
either method. Moreover, the Rf values of both benzothia-
zolinone and benzothiazolinethione are identical, making it
difficult to isolate the product from the starting material using
liquid chromatography.
derivatives of ABTS are described: N-(3-methyl-3H-ben-
zothiazol-2-ylidene)-N′-[3-(4-vinyl-benzyl)-3H-benzothiazol-
2-ylidene]-hydrazine (sABT) and 3-methyl-2-{[3-(4-vinyl-
benzyl)-3H-benzothiazol-2-ylidene]-hydrazono}-2,3-dihydro-
benzothiazole-6-sulfonic acid (sABTS).
A five-step synthesis affords polymerizable derivatives of
ABTS (Scheme 1). The first four steps result in a common
intermediate (4) that can be used to manipulate the solubility
of the target molecules and the hydrophilicity of their
corresponding polymers. Reaction of 4 with MBTH results
in a hydrophobic product (sABT), whereas reaction of 4 with
MBTHS results in the hydrophilic product (sABTS). Both
products yield redox-active polymers that are hydrophobic,
hydrophilic, or amphiphilic depending on the ratio of sABT
to sABTS. The first step in the synthetic sequence reacts
2-benzothiazolinone with 4-vinylbenzyl chloride under basic
conditions using methods similar to that described by J.
D’Amico et al. to yield the N-styryl-substituted benzothia-
zolinone (1) as a white crystalline solid in 91-93% yield.8
Optimal reaction conditions were found at 60 °C for 1 h in
a DMF solution containing 9 M KOH. Higher temperatures
or longer reaction times led to more side products and a lower
yield.
Sulfonation of 3-methyl-benzothiazolinone-(2)-hydrazone
(MBTH), available from Aldrich Chemical Co., Milwaukee,
WI) yields 3-methyl-6-(M sulfonate)-benzothiazolinone-(2)-
hydrazone (MBTHS) according to the published patent.13
Both MBTH and MBTHS react with 4 to give the respective
target compounds, sABT and sABTS (Scheme 1).
Hydrolysis of benzothiazolinone9 and thiazolinone10 can
be accomplished using different approaches. The hydrolysis
of 1 was accomplished by purging a mixture of methanol
and water containing a high concentration of KOH with
Methylation of 3 was achieved with dimethyl sulfate14 as
all attempts to methylate 3 with methyl iodide were unsuc-
(8) (a) D’Amico, J. J.; Bollinger, F. G. J. Heterocycl. Chem. 1988, 25,
1601. (b) D’Amico, J. J.; Bollinger, F. G. J. Heterocycl. Chem. 1989, 26,
655. (c) D’Amico, J. J.; Bollinger, F. G. J. Heterocycl. Chem. 1989, 26,
1245.
(9) (a) Kiprianov, A. I.; Pazenko, Z. N. Russ. J. Gen. Chem. 1949, 19,
1519. (b) Duro, F.; Scapini, G.; Vittorio, F. Farmaco-Ed Sci. 1975, 30,
208.
(10) (a) Abd, E.; Eatedal, H. M.; Mellor, J. M. J. Chem. Soc., Chem.
Commun. 1986, 7, 576. (b) Abd, E.; Eatedal, H. M.; Ashmawy, M. I.;
Mellor, J. M. J. Chem. Soc., Perkin Trans. 1 1986, 12, 2729. (c) Nagasawa,
H. T.; Elberling, J. A.; Roberts, J. C. J. Med. Chem. 1987, 30, 1373.
(11) Corbin, J. L.; Work, D. E. J. Org. Chem. 1976, 41, 489.
(12) (a) Sohar, P.; Denny, G. H.; Babson, R. D. J. Heterocycl. Chem.
1969, 6, 163. (b) Scheibye, S.; Kristensen, J.; Lawesson, S. O. Tetrahedron
1979, 35, 1339.
(13) (a) Klose, S.; Deneke, U.; Haid, E.; Weimann, G. U. S. Patent
4,101,381, 1978. (b) Douglas, J. S.; Drexler, K. R. U. S. Patent 5,989,845,
1999.
4
Org. Lett., Vol. 8, No. 1, 2006

cessful. The derivatives of hydrazone, MBTH and MBTHS,
were reacted with the methylated product (4) to obtain sABT
(50%) and sABTS (63%), respectively. The isolation of
product (4) was not necessary,15 and thus, the next step in
the reaction scheme was performed in the same reaction
flask. The conversion from 3 and 4 was ∼95% as indicated
by TLC. Both sABT and sABTS are white powders. It should
be noted that the sodium salt of sABTS is difficult to dissolve
in most organic solvents. Therefore, this product was
prepared for chromatographic isolation and polymerization
by washing with 1 M HCl. After chromatographic isolation,
the purified product was neutralized with an organic base
(i.e., tetrabutylammonium hydroxide) to obtain a product that
exhibits good solubility in both organic solvents and water
for subsequent polymerization.
The tetrabutylammonium salt of sABTS was polymerized
in ethanol for 1 day at 65 °C in the absence of oxygen using
2,2′-azo-bis(isobutyronitrile) (AIBN) (50:1) as the radical
initiator. Cooling the reaction to room temperature subse-
quently stopped polymerization. The resulting viscous prod-
uct was dialyzed (MWCO 3,500) against deionized water
for 1 day. Polymerization of sABTS was indicated by peak
broadening between 5 and 8 ppm in the 1H NMR spectrum
of the dialyzed product and the absence of sharp peaks
corresponding to the vinyl protons in sABTS (see Supporting
Information). The aromatic protons of the styryl substituent
are represented by a broad peak at 7 ppm, which is similar
to what is observed in the 1H NMR spectrum of polystyrene
sulfonate (see Supporting information) and other derivatives
of polystyrene.16 After dialysis, a solution containing poly-
ABTS (∼28 µmol of sABTS monomer, as calculated from
the absorption spectrum using the extinction coefficient of
sABTS) was dried on the surface of an electrode (d ) 4
mm) for cyclic voltammetry and bioelectrocatalysis experi-
ments. A second solution containing polyABTS (∼56 µmol
of sABTS monomer) was dried on the surface of an ITO
electrode (1 cm2) for electrochromic experiments.
Figure 1. CVs of commercially available ABTS (closed circles),
sABT (open circles), sABTS (solid line), and film of polyABTS
(open triangles). The concentration of all three monomers was 2.5
mM, and the electrolyte was 0.1 M tetrabutylammonium tetrafluo-
roborate in DMSO. Also shown is the CV of an electrode coated
with polyABTS immersed in an aqueous solution of 0.2 M KCl.
The scan rate was 10 mV s-1 for all CVs.
of a film of polyABTS prepared by drying a drop of a 25
µM solution of polyABTS on a glassy carbon electrode. E1/2
of the polyABTS film is 500 mV in 0.2 M KCl solution,
which is 120 mV negative to that of its monomer in DMSO.
The peak to peak separation is 168 mV, indicating poor self-
exchange kinetics in a pure film of polyABTS.18
The method of Nicholson was used to determine the rate
constant (kh) for heterogeneous electron transfer between a
glassy-carbon electrode and the polymerizable monomers.19
For sABT, kh ) 1.47 × 10-3 cm s-1, and for sABTS kh )
2.18 × 10-3 cm s-1. For comparison, kh ) 2.02 × 10-3 cm
s-1 for ABTS in 0.1 M tetrabutylammonium tetrafluoroborate
DMSO solution or 4.54 × 10-3 cm s-1 in sodium acetate
buffer (pH 4).
The compound N,N′-bis(3-methyl-3H-benzothiazol-2-
ylidene)-hydrazine (mABT) has been used as the electro-
active component in an electrochromic device.1 Both mABT
and sABTS have similar chemical structures; however,
sABTS possesses an N-styryl group to render the monomer
polymerizable and a sulfonate group to make it and its
corresponding polymer water soluble. Shown in Figure 2 are
the UV-vis spectra of an ITO electrode coated with a film
of polyABTS (∼56 µmol sABTS) while immersed in an
aqueous solution of 0.2 M KCl. Spectra correspond to the
film before (solid line) and after (dotted line) poising the
electrode at 600 mV for 10 s. Application of an oxidizing
potential converts polyABTS (transparent in the visible
region of the absorption spectrum) to polyABTS•+, which
is blue-green in color.
Values for E1/2 of sABT, sABTS and ABTS were obtained
from the cyclic voltammograms (CVs) shown in Figure 1.
All potentials are reported vs SCE. For sABT, E1/2 is 613
mV when dissolved in a DMSO solution containing in 0.1
M tetrabutylammonium tetrafluoroborate. Under identical
conditions, E1/2 of sABTS is 620 mV. The peak to peak
separation of 76 mV for sABT and 78 mV for sABTS
indicates reversible electrochemical reactions.17 The diffusion
coefficients of sABT and sABTS are 1.21 × 10-6 and 8.1
× 10-7 cm2 s-1, respectively. For comparison, the value for
E1/2 of ABTS (commercially available) is 587 mV in DMSO
and 440 mV in sodium acetate buffer (pH 4).4 The diffusion
coefficient of ABTS in DMSO is 1.39 × 10-6 cm2 s-1, and
in sodium acetate buffer (pH 4) the value is 3.22 × 10-6
cm2 s-1. Also shown in Figure 1 is the cyclic voltammogram
Shown in the inset of Figure 2 are the UV-vis spectra of
20 µM solutions of sABT, sABTS, and ABTS in DMSO.
(14) Sutoris, V.; Gaplovsky, A.; Sekerka, V. Chem. Pap. 1986, 40, 103.
(15) Fahmy, H. T. Y.; Rostom, S. A. F.; Saudi, M. N.; Zjawiony, J. K.;
Robins, D. J. Arch. Pharm. 2003, 336, 216.
(16) (a) Yang, J. C.; Jablonsky, M. J.; Mays, J. W. Polymer 2002, 43,
5125. (b) Mathew, A.; Deb, P. C. Macromol. Chem. Phys. 1998, 199, 2527.
(17) Bard, A. J.; Faulkner, L. R. Electrochemical Methods; Wiley: New
York, 1980.
(18) (a) Daum, P.; Lenhard, J. R.; Rolison, D.; Murray, R. W. J. Am.
Chem. Soc. 1980, 102, 4649. (b) Anson, F. C.; Saveant, J. M.; Shigehara,
K. J. Phys. Chem. 1983, 87, 214.
(19) Nicholson, R. S. Anal. Chem. 1965, 37, 1351.
Org. Lett., Vol. 8, No. 1, 2006
5

Figure 3. LSVs of a polyABTS/glassy carbon electrode. The
electrode was immersed in 0.2 M KCl purged with either nitrogen
or oxygen (solid line) or immersed in 0.2 M sodium acetate buffer
(pH 4) containing 1 mg/mL laccase and purged with nitrogen (open
circles) or oxygen (closed circles). The scan rate for all LSVs was
Figure 2. Spectroelectrochemistry of polyABTS/ITO electrode
immersed in 0.2 M KCl. The spectra correspond to before (solid
line) and after (dotted line) application of potential. Inset: UV-
vis spectra of sABT (open circles), sABTS (solid line), and ABTS
(closed circles) in DMSO.
1 mV s-1
.
or both, reductive current is not observed. This result
indicates that polyABTS itself does not catalyze the elec-
trochemical reduction of oxygen. When dioxygen and laccase
are both present, however, reductive current is observed. It
should be noted that reductive current is not observed at an
uncoated electrode in the presence of both dioxygen and
laccase. These results confirm that polyABTS facilitates the
transport of electrons from the working electrode to the active
site of laccase in solution and also confirm that polyABTS
can be used in electrochemical sensors that detect dioxygen
or laccase.
In conclusion, a simple synthetic route was developed that
affords two polymerizable derivatives of the redox-active dye
ABTS. The spectroscopic and electrochemical properties of
both derivatives are reported. The polymerization of one of
the derivatives (sABTS) into a water-soluble, redox-active
polymer (polyABTS) is demonstrated and shown to be useful
in applications such as biofuel cells, enzyme assays, bio-
sensors, and electrochromic devices by demonstrating its
ability to transfer electrons between an electrode and the
active site of an oxidoreductase. Studies on copolymers of
these derivatives and their applications will be reported
elsewhere.
Both sABT and sABTS have absorption peaks at ∼255 nm,
which corresponds to electronic transitions in the styrene ring,
and at ∼340 nm, which corresponds to electronic transitions
in the conjugated system that include the four nitrogen atoms.
This absorption band is observed in the absorption spectrum
of commercial ABTS and therefore confirms the presence
of the same chromophore in both sABT and sABTS. The
extinction coefficients of the reduced forms of sABT and
sABTS dissolved in DMSO were determined to be 3.06 ×
104 M-1 cm-1 at 338 nm and 2.96 × 104 M-1 cm-1 at 341
nm, respectively. We previously reported the extinction
coefficient (3.45 × 104 M-1 cm-1) of ABTS at 340 nm in
0.2 M sodium acetate buffer (pH 4).4 Similar to polyABTS,
a color change is observed upon electrochemical oxidation
of solutions containing the monomers sABT, sABTS, and
ABTS (data not shown).
We have used ABTS to facilitate electron transport
between the cathode of a biofuel cell and the active site of
laccase to generate electrical power.4 Shown in Figure 3 are
linear sweep voltammograms (LSVs) that demonstrate that
polyABTS participates in the bioelectrocatalytic reduction
of oxygen to water. The LSVs were collected under four
different conditions: a polyABTS-coated electrode was
immersed in 0.2 M KCl that was purged with either (1)
nitrogen or (2) oxygen, or the polyABTS-coated electrode
was immersed in 0.2 M sodium acetate buffer (pH 4)
containing 1 mg/mL laccase that was purged with either (3)
nitrogen or (4) oxygen. In the absence of oxygen or laccase
Supporting Information Available: Synthetic details.
This material is available free of charge via the Internet at
http://pubs.acs.org.
OL0513443
6
Org. Lett., Vol. 8, No. 1, 2006