Modulating the pH-dependent redox potential of a flavin analog via incorporation into a self-assembled monolayer on gold

Article abstract of DOI:10.1039/a806315j

We report the first example of modifying the pH-dependent electrochemical properties of a flavin analog on gold by changing the pK(a) value of its N¹ proton.

Full text of DOI:10.1039/a806315j

Modulating the pH-dependent redox potential of a flavin analog via
incorporation into a self-assembled monolayer on gold
Suk-Wah Tam-Chang,* James Mason, Isaac Iverson, Ki-Oh Hwang and Christa Leonard
Department of Chemistry, University of Nevada, Reno, Nevada 89557, USA. E-mail: tchang@chem.unr.edu
Received (in Columbia, MO, USA) 10th August 1998, Accepted 19th November 1998
We report the first example of modifying the pH-dependent
electrochemical properties of a flavin analog on gold by
changing the pKa value of its N1 proton.
Flavoenzymes are important electron shuttles in a wide range of
biological redox reactions.1 The redox potential of flavin (the
redox active moiety of flavoenzymes) varies by at least 600 mV,
as governed by binding in different apoproteins. Non-covalent
interactions (including p-stacking,2 hydrophobic effects, hydro-
gen bonding, and electrostatic interactions)3 between flavin and
apoproteins play important roles; however, it is difficult to
examine the effects of individual interactions in complex
biological systems. A fundamental understanding of environ-
mental effects on the flavin redox potential can be expected to
provide insight into biological mechanisms for tuning the redox
potential of flavoenzymes. In addition, control over its redox
activity may increase selectivity of electrocatalysis in synthetic
devices.4 Monolayers prepared from alkanethiols or disulfides
with electroactive derivatives5 are commonly used for studying
environmental effects on electron-transfer kinetics.6 The pre-
sent work presents novel self-assembled monolayer (SAM)
formation based on an alkanethiolate derivatized flavin analog7
and the modulation of its redox properties via incorporation into
SAMs.
Disulfide 1 was synthesized according to the procedure
outlined in Scheme 1.8 SAMs containing isoalloxazine (the
heterocyclic ring system of flavin) were prepared by immersing
a gold coated (500 Å) silicon wafer in an acetonitrile solution of
1 and were characterized by cyclic voltammetry (Fig. 1). Flavin
and its analogs undergo two reversible one-electron reduction
steps with overlapping potentials in aqueous buffer solutions
(Scheme 2).9 The surface coverage was calculated to be ca. 5 3
10210 mol cm22 by integrating the area under the oxidation or
reduction peak. This surface coverage coincides with a densely
Fig. 1 Cyclic voltammogram (steady state cycle) of (a) (······) a SAM from
1 in an aqueous buffer solution of pH 2.3, (b) (––––) a SAM from 1 in an
aqueous buffer solution of pH 10.13, and (c) (- - - - -) 2 (1 mM) in an
aqueous buffer solution of pH 12.03. A bare gold working electrode is used
in the determination of E0 of 2. Scan rates are 0.1 V s21
.
packed film of the flavin based on modeling the molecular area
expected for a flavin unit. The pH-dependent formal potential
(E0) of the bound isoalloxazine moiety was determined from the
mean value of the cathodic and anodic peak potentials. Upon
changing from acidic to basic conditions, the full-widths at half
maxima of the peaks increase. This change may be indicative of
structural heterogeneity or differences in the rates of electron
transfer for the two electron transfer steps (Fig. 1).
To examine the importance of microenvironment on the one-
electron processes of Scheme 2, we compared the pH dependent
redox potential of flavin analog 2 in solution (shown in Scheme
2) with that of immobilized isoalloxazine. As shown in Fig. 2,
the formal potential of 2 dissolved in solution is linearly related
to pH, with a slope of 20.057 V (pH unit)21 up to pH 6.7. After
this inflection point the redox processes of 2 remain reversible
(Fig. 1); however, the slope changes to 20.027 V (pH unit)21 at
higher pH values;10 The pH at the inflection point is the pKa of
the N1-proton in the reduced form.9–11 A pKa value of 6.7 for the
N1-proton in the reduced form of 2 and the decrease in slope are
consistent with those reported for mononucleotide (FMN) and
flavin adenine dinucleotides (FAD).9,12 In contrast, the slope of
the E0–pH plot for the monolayer of 1 is uniform until pH ≈ 10
Scheme 1
Scheme 2
Chem. Commun., 1999, 65–66
65

modified via incorporation into SAMs, as well as the known
mechanisms such as selective H-bonding and p-stacking.
This research was supported by the National Science
Foundation (CHE-9510344) and the NSF EPSCOR cooperative
agreement OSR-9353227 (WISE Program). C. C. Leonard
acknowledges support from the REU Program, UNR. We
appreciate the helpful suggestions of the reviewers.
Notes and references
1 C. Walsh, Enzymatic Reaction Mechanisms, W. H. Freeman and
Company, New York, 1979; P. Hemmerich, C. Veeger and H. C. S.
Wood, Angew. Chem., Int. Ed. Engl., 1965, 4, 671.
2 Elegant models have been designed to quantify the effects of p-stacking
on flavin redox chemistry in aprotic solvents: A. Niemz, J. Imbriglio and
V. M. Rotello, J. Am. Chem. Soc., 1997, 119, 887; E. C. Breinlinger, A.
Niemz and V. M. Rotello, J. Am. Chem. Soc., 1995, 117, 5379; E. C.
Breinlinger and V. M. Rotello, J. Am. Chem. Soc., 1997, 119, 1165.
3 B. J. Stockman, T. E. Richardson and R. P. Swenson, Biochemistry,
1994, 33, 15298; R. P. Swenson and G. D. Krey, Biochemistry, 1994,
33, 8505; F. C. Chang and R. P. Swenson, Biochemistry, 1997, 36, 9013;
M. Inoue, Y. Okudo, I. Ishida and M. Nakagaki, Arch. Biochem.
Biophys., 1983, 227, 52.
4 S. Fukuzumi, K. Tanii and T. Tanaka, Chem. Commun., 1989, 816;
S. W. Tam, L. Jimenez and F. Diederich, J. Am. Chem. Soc., 1992, 114,
1503; M. Ishikawa, H. Okimoto, M. Masayuki and Y. Matsuda, Chem.
Lett., 1996, 953.
5 C. E. D. Chidsey, C. R. Bertozzi, T. M. Putvinski and A. M. Mujsce,
J. Am. Chem. Soc., 1990, 112, 4301; N. L. Abbott and G. M. Whitesides,
Langmuir, 1994, 10, 1493; D. M. Collard and M. A. Fox, Langmuir,
1991, 71, 1192.
Fig. 2 The pH dependence of redox potential (E0) of isoalloxazine of 1 in
SAM compared with 2 (1 mM) dissolved in aqueous buffer solution.
(Fig. 2). The intersection point of the linear portions yields a pKa
value of 9.7. This dramatic increase in the pKa value indicates
that the microenviroment of the N1-proton in SAM is less polar
than the aqueous solution. These results are in line with the
observations of Whitesides and coworkers who demonstrated
by contact angle measurements increased pKa values of acidic
groups of w-mercaptoalkanecarboxylic and phosphonic acids
upon incorporation into monolayers.13 They proposed that a low
interfacial dielectric constant and/or electrostatic interactions
cause the unfavorable formation of negatively charged species
in a closely packed monolayer compared with that in water.13
We prepared mixed monolayers from unsymmetrical di-
sulfide 6 and dodecyl disulfide or octyl disulfide thereby
reducing the surface coverage of the isoalloxazine moiety to
6 H. C. D. Long and D. A. Buttry, Langmuir, 1992, 8, 2491; S. E. Creager
and G. K. Rowe, Anal. Chim. Acta, 1991, 246, 233; H. O. Finklea and
D. D. Hanshew, J. Am. Chem. Soc., 1992, 114, 3173.
7 For flavin analogs immobilized on gold via other linkers, see: S.
Sakamoto, H. Aoyagi, N. Nakashima and H. Mihara, J. Chem. Soc.,
Perkin Trans. 2, 1996, 2319; B. Mallik and D. Gani, J. Electroanal.
Chem., 1992, 326, 37.
8 Disulfide is used instead of the analogous thiol due to the thiol’s
vulnerability to oxidation by the flavin moiety.
9 B. Janik and P. J. Elving, Chem. Rev., 1968, 68, 295.
10 The theoretical value for the slope of each linear segment of the E0–pH
curve is 0.059 p/n at 25 °C (where p and n are the numbers of protons
and electrons involved respectively) P. J. Elving, Pure Appl. Chem.,
1963, 7, 423.
11 For the methods and theory in determining pKa values from E0–pH
plots, see: W. M. Clark, Oxidation–Reduction Potentials of Organic
Systems, Williams and Wilkin Co., Baltimore, 1960, pp. 118.
12 W. M. Clark, Oxidation–Reduction Potentials of Organic Systems,
Williams and Wilkin Co., Baltimore, 1960, p. 439.
13 C. D. Bain, G. M. Whitesides, Langmuir, 1989, 5, 1370; T. R. Lee,
R. I. Carey, H. A. Biebuyck and G. M. Whitesides, Langmuir, 1994, 10,
741.
about 10% of that in the monolayer from 1.14 This reduction of
charge/charge repulsion at the interface did not lower the pKa
value of the N1-proton. In addition, the change in alkyl chain
length of the mixed SAM in which N1-proton is embedded did
not influence its pKa value. These results suggest that the low
dielectric constant of the underlying alkyl chains may be the
predominant factor influencing the dissociation constant.
In summary, our work demonstrates that the pH-dependent
electrochemical properties of the isoalloxazine moiety can be
14 In addition to the decrease in peak areas, the full-widths at half maxima
and the separation between the anodic and cathodic peaks (ca. 70 mV)
in the mixed SAMs are smaller than those of the SAM from 1.
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Chem. Commun., 1999, 65–66