Model systems for flavoenzyme activity: Site-isolated redox behavior in flavin-functionalized random polystyrene copolymers

Article abstract of DOI:10.1021/ol0505407

A model system has been developed to study the redox behaviors of flavin derivatives appended onto random polystyrene copolymers through "click" chemistry strategies. The results demonstrate that flavin units attached onto polymers exhibit site-isolated r

Full text of DOI:10.1021/ol0505407

ORGANIC
LETTERS
2005
Vol. 7, No. 13
2551-2554
Model Systems for Flavoenzyme
Activity: Site-Isolated Redox Behavior
in Flavin-Functionalized Random
Polystyrene Copolymers
Joseph B. Carroll,† Brian J. Jordan,† Hao Xu,† Belma Erdogan,† Lisa Lee,†
Lily Cheng,† Christopher Tiernan,† Graeme Cooke,‡ and Vincent M. Rotello*,†
Department of Chemistry, UniVersity of Massachusetts, Amherst, Massachusetts 01003,
and Centre for Biomimetic Design and Synthesis, Chemistry, School of Engineering &
Physical Sciences, Heriot-Watt UniVersity, Edinburgh, UK EH14 4AS
rotello@chem.umass.edu
Received March 11, 2005
ABSTRACT
A model system has been developed to study the redox behaviors of flavin derivatives appended onto random polystyrene copolymers
through “click” chemistry strategies. The results demonstrate that flavin units attached onto polymers exhibit site-isolated redox behaviors,
-
-
yielding new materials with electrochemically tunable associations (Ka(ox)
(DAP) functionality.
) )
450 M 1, Ka(red) 18 200 M 1) to complementary diamidopyridine
The relationship between complex redox processes and
molecular recognition is a central theme in many different
biological systems. For example, enzymes composed of
redox-active core molecules, including flavins,1 quinones,2
nicotinamides,3 and pterins,4 use specific enzyme-cofactor
interactions to control the activity of a given cofactor. The
deployment of different recognition elements, including
hydrogen bonding, π-stacking, and electrostatic interactions,
in enzymatic processes serves to control the oxidation and
protonation states of a cofactor’s activity. Accordingly,
understanding the cooperative recognition processes that
govern complex redox enzyme activity serves as an attractive
target in the development of redox-active molecular devices5
and model systems.6
† University of Massachusetts.
‡ Heriot-Watt University.
(1) (a) Chaiyen, P.; Sucharitakul, J.; Svasti, J.; Entsch, B.; Massey, V.;
Ballou, D. P. Biochemistry 2004, 43, 3933-3943. (b) Chakraborty, S.;
Massey, V. J. Biol. Chem. 2002, 277, 41507-41516.
(2) (a) Reddy, S. Y.; Bruice, T. C. Protein Sci. 2004, 13, 1965-1978.
(b) Anthony, C. Biochem. J. 1996, 320, 697-711.
(3) Sundaresan, V.; Chartron, J.; Yamaguchi, M.; Stout, C. D. J. Mol.
Biol. 2005, 346, 617-629.
The incorporation of molecular recognition in polymers
provides robust materials with applications from sensors to
chromatography.7 The incorporation of flavin functionality
(4) Pey, A. L.; Thorolfsson, M.; Teigen, K.; Ugarte, M.; Martinez, A. J.
Am. Chem. Soc. 2004, 126, 13670-13678.
10.1021/ol0505407 CCC: $30.25
© 2005 American Chemical Society
Published on Web 06/01/2005

in polymers allows for the study of polymer environmental
effects on flavin binding both in neutral and reduced redox
states. Here we report the development of a model system
seeking to understand flavin-binding interactions within
polymeric systems. In this paper, alkyne flavin derivatives
were appended to azido-functionalized polystyrene copoly-
mers via Huisgen 1,3-dipolar, “click” cycloadditions.8 The
resultant flavin-functionalized polymers exhibit site-isolated
electrochemical behaviors and reversible, redox-modulated
recognition with complementary DAP units with a concomi-
tant increase in association constant of ∼40-fold versus
neutral binding.
Scheme 1a
The synthesis of alkyne-functionalized flavin unit 1
(Scheme 1a) began with the substitution of 4,5-dimethyl-
1,2-phenylenediamine with 6-chlorohexyne (for synthesis of
1 and 2, see Supporting Information). The resultant conden-
sation of the monosubstituted alkyne diamine species with
alloxan monohydrate yielded alkyne-functionalized flavin 1.
Further reaction of 1 with methyl iodide provides control
N-methylated flavin 2, which cannot participate in three-
point hydrogen bonding with DAP.
The synthesis of flavin-functionalized copolymers began
with the displacement of chloromethylstyrene copolymer7e
with sodium azide in DMSO overnight, resulting in polymer
3 (Scheme 1b). To solutions of alkyne-functionalized flavin
1 units and polymer 3 in DMSO was added copper(I) iodide,
resulting in flavin-functionalized polymer 4. Control N-
methylated, flavin-functionalized polymer 5 was synthesized
in analogous fashion to polymer 4.
a Key: (a) Alkyne-functionalized flavin 1 and control N-
methylated flavin 2. (b) Synthesis of flavin polymer 4 and control
polymer 5 via “click” procedures. Flavin 1 and polymer 4 exhibit
specific three-point hydrogen bonding interactions with comple-
mentary DAP, where control flavin 2 and polymer 5 demonstrate
no hydrogen bonding interactions with DAP.
Infrared (IR) spectroscopy of the resultant polymer films
was used to monitor transformations before and after
“clicking” functionality onto polymer 3. IR spectra of the
azide-functionalized polymer 3 films reveal a pronounced
azide stretch at 2100 cm-1 (see Supporting Information).
Upon triazole formation with 1, the azide stretch at 2100
cm-1 completely disappears, direct evidence of complete
functionalization of all of the azide sites of polymer 3. IR
spectra of films of control polymer 5 also display a
pronounced carbonyl and aromatic CdN stretch in the
absence of an N-H stretch at ∼3400 cm-1.
To probe the association (Ka(ox)) between 1 and flavin-
functionalized polymer 4 with complementary DAP, binding
interactions were quantified using fluorescence spectroscopy
in CHCl3 by monitoring the fluorescence intensity of flavin
upon increasing additions of DAP guest (Figure 1). Fluo-
rescence titrations of flavin 1 in the presence of increasing
concentrations of DAP show a dramatic quenching of flavin
fluorescence corresponding to a Ka(ox) ≈ 375 M-1. Titrations
with control N-methylated flavin 2, which cannot participate
in hydrogen bonding interactions, show no quenching of
fluorescence (Ka(ox) ≈ 0 M-1) upon addition of an excess
of DAP.
(5) (a) Liu, Y.; Flood, A. H.; Stoddart, J. F. J. Am. Chem. Soc. 2004,
126, 9150-9151. (b) Lehn, J. M. Supramolecular Chemistry: Concepts
and PerspectiVes; VCH: Weinheim, 1995. (c) Kaifer, A. E.; Gomez-Kaifer,
M. Supramolecular Electrochemistry; Wiley-VCH: Weinheim, 1999.
(6) (a) Hasford, J. J.; Rizzo, C. J. J. Am. Chem. Soc. 1998, 120, 2251-
2255. (b) Kajiki, T.; Moriya, H.; Hoshino, K.; Kuroi, T.; Kondo, S.;
Nabeshima, T.; Yano, Y. J. Org. Chem. 1999, 64, 9679-9689. (c) Deans,
R.; Rotello, V. M. J. Org. Chem. 1997, 62, 4528-4529. (d) Greaves, M.
D.; Rotello, V. M. J. Am. Chem. Soc. 1997, 119, 10569-10572. (e) Legrand,
Y. M.; Gray, M.; Cooke, G.; Rotello, V. M. J. Am. Chem. Soc. 2003, 125,
15789-15795. (f) Gray, M.; Goodman, A. J.; Carroll, J. B.; Bardon, K.;
Markey, M.; Cooke, G.; Rotello, V. M. Org. Lett. 2004, 6, 385-388.
(7) (a) Brunsveld, L.; Folmer, B. J. B.; Meijer, E. W.; Sijbesma, R. P.
Chem. ReV. 2001, 101, 4071-4097. (b) Prins, L. J.; Reinhoudt, D. N.;
Timmerman, P. Angew. Chem., Int. Ed. 2001, 40, 2383-2426. (c) McQuade,
D. T.; Pullen, A. E.; Swager, T. M. Chem. ReV. 2000, 100, 2537-2574.
(d) Pollino, J. M.; Stubbs, L. P.; Weck, M. J. Am. Chem. Soc. 2004, 126,
563-567. (e) Ilhan, F.; Gray, M.; Rotello, V. M. Macromolecules 2001,
34, 2597-2601.
Analogous fluorescence titrations of flavin functionalized
polymer 4 in the presence of increasing concentrations of
DAP also show a dramatic quenching of flavin fluorescence
corresponding to a Ka (ox) ≈ 450 M-1 (Figure 1). Control
titrations with N-methylated flavin polymer 5 and DAP
exhibit a slight increase in fluorescence perhaps indicating
cooperative flavin interactions; however, no dynamic quench-
ing of polymer fluorescence was observed (Ka(ox) ≈ 0 M-1).
In addition to the neutral associations determined via
fluorescence spectroscopy, the half-wave reduction potentials
of the free flavin 1 and polymer 4 (E1/2(u)) and the
corresponding potentials for 1 and polymer 4 in the presence
of DAP (E1/2(b)) were determined in CH2Cl2 through cyclic
voltammetry (CV). Voltammetric traces of 1, 2, polymer 4,
(8) (a) Huisgen, R. Pure Appl. Chem. 1989, 61, 613-628. (b) Huisgen,
R.; Szeimies, G.; Mobius, L. Chem. Ber. 1967, 100, 2494-2507. (c)
Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew.
Chem., Int. Ed. 2002, 41, 2596-2599. (d) Kolb, H. C.; Finn, M. G.;
Sharpless, K. B. Angew. Chem., Int. Ed. 2001, 40, 2004.
2552
Org. Lett., Vol. 7, No. 13, 2005

reduced flavin anion. Upon the addition of an excess of DAP,
a significant positive shift in the half-wave reduction potential
(E1/2) of 1 is observed (∆E1/2 ) +100 mV). In addition to
the dramatic positive shift, the electrochemical behavior of
1 bound to DAP becomes fully reversible with the disap-
pearance of the second oxidation wave seen with 1 alone.11
Control CV traces with N-methylated flavin 2 exhibit no
appreciable shift upon the addition of an excess of DAP (see
Supporting Information).
In contrast to flavin 1, polymer 4 exhibits very different
electrochemical behaviors from its monomeric counterpart.
Polymer 4 undergoes a one-electron reduction with only a
single return oxidation wave (see Supporting Information).
This fully reversible electrochemical behavior demonstrates
that the flavin units appended onto the polystyrene polymer
are isolated from each other preventing flavin-flavin proton
transfer.12 This observed site isolation is important when
engineering electrochemically tunable materials that interact
specifically with a given substrate. Upon the addition of an
excess of DAP, a significant positive shift in the E1/2 of
polymer 4 is observed (∆E1/2 ) +95 mV). Control CV traces
with N-methylated, flavin-functionalized polymer 5 exhibit
no appreciable shift after the addition of an excess of DAP
(see Supporting Information).
Figure 1. Inverse Stern-Volmer plot showing a dramatic quench-
ing of flavin 1 (9 Ka(ox) ) 375 M-1) and flavin-functionalized
polymer 4 fluorescence upon addition of DAP ([) corresponding
to a Ka(ox) ) 450 M-1, whereas interactions with control N-
methylated flavin 2 (b) and control polymer 5 (2) show no
fluorescence quenching (100 µM, λex ) 445 nm, λem ) 460-650
nm).
The large differences in peak shape and relatively small
differences in the reduction potential (E1/2) observed between
flavin 1 (E1/2 ) -1291 ( 3 mV) and flavin-functionalized
polymer 4 (E1/2 ) -1285 ( 3 mV) prompted further
investigation into the thermodynamic behavior of free flavin
versus flavins appended onto polymer scaffolds. To probe
the thermodynamic contributions to the electrochemical
behaviors observed in flavin 1 and polymer 4, variable-
temperature CV (VT-CV) experiments were carried out in
dichloroethane (Figure 2). VT-CV traces reveal dramatic
differences in the redox behavior of free flavin versus flavins
-
and polymer 5 were taken in CH2Cl2 with n-Bu4N+ClO4
(100 µM)9 as the supporting electrolyte.10
Flavin 1 exhibits a single reduction wave followed by two
characteristic return oxidation waves (see Supporting Infor-
mation). In previous studies,11 this redox behavior was
determined to be the result of an electrochemical-chemical-
electrochemical (ece) process where the radical anion form
of the flavin rapidly deprotonates other oxidized flavins in
solution. The newly formed protonated flavin radical then
undergoes a further one-electron reduction to form the fully
Figure 2. VT-CV traces of polymer 4 and flavin 1 exhibiting different flavin redox behaviors at different temperatures (55, 35, 5 °C).
Lowering the temperature (5 °C) induces the presence of a second oxidation in polymer 4, while elevated temperatures signal the disappearance
of the second oxidation in flavin 1 (55 °C).
Org. Lett., Vol. 7, No. 13, 2005
2553

appended onto polymers. At 35 °C, flavin 1 undergoes a
one-electron reduction followed by two characteristic oxida-
tion waves as previously described. Polymer 4 at 35 °C,
however, exhibits only one reversible redox couple. At higher
temperatures (55 °C), flavin 1 does not exhibit a second
oxidation, presumably due to the disruption of flavin-flavin,
hydrogen-bonding interactions. Lower temperatures (5 °C)
promote flavin-flavin hydrogen bonding interactions as
witnessed by the appearance of a second oxidation in
polymer 4. Interestingly enough, these dramatic changes in
peak shape with temperature fail to yield large differences
in the reduction potential of the flavin systems (see Sup-
porting Information).
In general, at lower temperatures the reduction potential
of both flavin 1 and polymer 4 shifts to a more positive
potential (E1/2 ≈ -1270 mV at 5 °C), while raising the
temperature shifts the reduction potential to a more negative
potential (E1/2 ≈ -1298 mV at 55 °C). Using the shifts in
the reduction potentials of 1 and polymer 4 (∆E1/2) obtained
from CV (see Table 1) and the association constants (Ka-
be ∼18 500 M-1, resulting in a 50-fold increase in binding
and a ∼2.3 kcal/mol stabilization of the radical anion of 1.
The Ka(red) between DAP and polymer 4 was determined
to be ∼18 200 M-1, resulting in a 40-fold increase in binding
and a ∼2.2 kcal/mol stabilization of the radical anion of
polymer 4.
In summary, we have synthesized flavin-functionalized
random polystyrene copolymers through versatile “click”
chemistry strategies. Interestingly, flavin units on polymer
scaffolds act independently and are site-isolated from each
other, yielding unique thermal and electrochemical behaviors
not observed with free flavin molecules. The resultant
polymer exhibits reversible, redox-modulated hydrogen
bonding interactions with complementary DAP units, result-
ing in a 40-fold increase in binding and a 2.2 kcal/mol
stabilization of the radical anion of the polymer. Our further
efforts will involve investigating these redox active polymers
and their possible applications in electrochemically controlled
molecular sensing and surface modification.
Acknowledgment. This research was supported by the
NIH (GM 59249) and the EPSRC. J.B.C. would like to thank
the Electrochemical Society for the Joseph W. Richards
Fellowship.
Table 1. Half-Wave Potentiala (mV) and Association
Constants (M-1) Associated with Flavin Systems
flavin system
Ka(ox)b
E1/2(u)c
E1/2(b)d
-1191
∆E1/2
Ka(red)
Supporting Information Available: Synthesis, 1H NMR,
and CV of flavins 1 and 2 and polymers 3 and 4. This
material is available free of charge via the Internet at
http://pubs.acs.org.
1
375
0
-1291
-1354
-1285
-1346
100
0
18 500
2
polymer 4
polymer 5
450
0
-1190
95
0
18 200
a Half-wave potentials were taken from square-wave voltammetry
measurements (E1/2 ( 3 mV vs ferrocene). b Neutral association constants
(Ka(ox)) determined via fluorescence spectroscopy. c Reduction potential
of the unbound flavin species. d Reduction potential of the bound flavin
species with DAP guest.
OL0505407
(9) Extreme caution was exercised in preparation of electrolyte solutions
for CV studies: Sax, N. I.; Lewis, R. J. Dangerous Properties of Industrial
Materials, 7th ed.; Van Nostrand Reinhold: New York, 1989; Vol. 3.
(10) CV traces were recorded for a CH2Cl2 blank, then for a solution of
the flavin or flavin polymer (200 µM) and ferrocene (as a reference), and
finally for the same samples containing excess DAP. Differences in half-
wave potentials were determined using square-wave voltammetry. All scans
(ox)) as determined using fluorescence spectroscopy, we can
determine a binding constant between DAP, flavin 1, and
polymer 4 in the radical anionic form of the flavin (Ka(red))
through the following relationship.
were recorded at a sweep rate of 100 mV s-1
(11) Niemz, A.; Imbriglio, J.; Rotello, V. M. J. Am. Chem. Soc. 1997,
119, 887-892.
.
(12) (a) Galow, T. H.; Ilhan, F.; Cooke, G.; Rotello, V. M. J. Am. Chem.
Soc. 2000, 122, 3595-3598. (b) Gorman, C. B. AdV. Mater. 1998, 10, 295-
308. (c) Kaifer, A. E. Acc. Chem. Res. 1999, 32, 62-71. (d) Dandliker, P.
J.; Diederich, F.; Gross, M.; Knobler, C. B.; Louati, A.; Sanford, E. M.
Angew. Chem., Int. Ed. Engl. 1994, 33, 1739-1741. (e) Dandliker, P. J.;
Diederich, F.; Gisselbrecht, J.-P.; Louati, A.; Gross, M. Angew. Chem., Int.
Ed. Engl. 1995, 34, 2725-2728.
1/2(b)-E1/2(u))
Ka(red) ) Ka(ox)e{(nF/RT)(E
}
Using the above equation, the association constant between
DAP and 1 in its reduced state (Ka(red)) was determined to
2554
Org. Lett., Vol. 7, No. 13, 2005