Fast, long-range electron-transfer reactions of a "blue" copper protein coupled non-covalently to an electrode through a stilbenyl thiolate monolayer.

Article abstract of DOI:10.1039/b312936e

A self-assembled monolayer (SAM), formed by the insitu saponification of a stilbenyl thioacetate on a gold electrode, yields fast electron transfer (ET)(the exchange rate at zero driving force exceeds 1600 s-1) with adsorbed molecules of the blue copper p

Full text of DOI:10.1039/b312936e

Fast, long-range electron-transfer reactions of a ‘blue’ copper protein
coupled non-covalently to an electrode through a stilbenyl thiolate
monolayer
Fraser A. Armstrong,a Nicola L. Barlow,a Paul L. Burn,b Kevin R. Hoke,a Lars J. C. Jeuken†,a
Catherine Shentona and Graham R. Websterb
a Inorganic Chemistry Laboratory, Oxford University, South Parks Road, Oxford, England OX1 3QR
b The Dyson Perrins Laboratory, Oxford University, South Parks Road, Oxford University, England OX1
3QY
Received (in Cambridge, UK) 15th October 2003, Accepted 20th November 2003
First published as an Advance Article on the web 7th January 2004
A self-assembled monolayer (SAM), formed by the in situ
saponification of a stilbenyl thioacetate on a gold electrode,
yields fast electron transfer (ET) (the exchange rate at zero
driving force exceeds 1600 s21) with adsorbed molecules of the
‘blue’ copper protein, azurin, over a distance exceeding 15 Å.
There is great interest in the chemical modification of electrodes to
achieve reversible electrochemistry of adsorbed redox enzymes.1–8
Now, in the quest for electrode surfaces of increasing biological
relevance, complexity, and stability, it is becoming highly desirable
to achieve fast and specific electron transfer (ET) over a far greater
distance than can be achieved with SAMs of short-chain alkane
thiols. Recent studies by Creager and Chidsey have identified redox
systems that form SAMs on gold electrode surfaces and exhibit
electrodes were prepared following a recently reported procedure in
remarkably high rates of long-range ET.9–11 Chidsey has studied
which the oxidatively unstable thiol was generated by saponifica-
the distance dependence of ET rates in systems consisting of
tion of the corresponding thioacetate immediately prior to mod-
ferrocenyl-terminated oligomeric phenylenevinylene (OPV) thiols
of varying lengths linked to the electrode and concluded that the
electronic coupling distance decay constant b is close to zero.9 This
contrasts with the results obtained for long-chain aliphatic alkyl
linkers for which rates decrease exponentially with increasing chain
length and b-values are around 1 Å21. Furthermore the b-value for
OPVs is lower than those measured for similar molecules based on
phenyleneethynyl units.11 It was thus important to determine
whether an OPV thiol could provide such excellent electron
coupling when the redox centre is the active site of a non-covalently
bound protein molecule. In this Communication we report the
synthesis and ET properties of a short OPV-type molecule: a
stilbenyl thiol (I) designed to bind non-covalently the ‘blue’ copper
protein azurin and give fast electron exchange with the active site
over a distance greater than 15 Å.
ification of the electrode.12
Fig. 1 shows voltammograms obtained, at a scan rate of 100 V
s21, for azurin adsorbed on a Au electrode modified with SAMs of
I (dark line) and II (light line).¶ The uniform background has been
subtracted to emphasise the faradaic features. At pH 4.0, the redox
reaction of the blue Cu site is not complicated by coupling to a
proton-transfer reaction.8 The smaller peak separation shows that
ET at I is much faster, even though II is shorter. At slow scan rates
(not shown) the half-height widths are about 100 mV; close to
expectations for a one-electron reaction in a homogeneous
population of redox couples. The peaks broaden as the scan rate is
raised, although, even at 500 V s21 they remain compact at
approximately 200 mV in width. Typical electroactive coverages of
azurin on the electrode were in the range 8–12 pmol cm22
.
Fig. 2 shows the resulting ‘trumpet’ plots, in which the peak
positions for oxidation and reduction are plotted as a function of log
(scan rate). This is a useful method of displaying and analysing the
characteristics of protein film ET reactions over a wide time
domain.2 At low scan rates, the oxidation and reduction peak
positions are close, and their average is the reduction potential E0A
of the Cu site. Although the experiments were performed under
identical conditions of pH and temperature, the E0A value is slightly
Azurin is an ET protein containing a single type 1 Cu centre that
is located about 6 Å below the surface in a hydrophobic region. We
and others have shown recently that azurin can be adsorbed at gold
electrodes modified with alkanethiols, the terminal CH3– function-
ality being well suited for interacting with the hydrophobic patch
close to the Cu.6–8 Alkanethiols form ordered SAMs, as observed
by scanning tunneling microscopy.6 The ET rate constant varies
with the length of the alkyl chain, although evidence suggests6,7
that a limiting value ( < 104 s21) is reached either at short chain
length or high driving force (similar rate limitations are found in
studies with cytochrome c4). To establish the magnitude of ET rates
possible with long, low-b linkers, we have synthesised a prototype
OPV-typemolecule,{3,5-diethoxy-4-[(E)-2-(4-ethylphenyl)vinyl]-
phenyl}methanethiol (I), which presents a hydrophobic ethyl
surface group when assembled as a monolayer on the electrode
surface. We have compared the ET kinetics with those obtained for
azurin adsorbed at decanethiol (II) and dodecanethiol (III), the
lengths of which are, respectively, slightly shorter and longer.
I was prepared and characterised as described below.§ The
alkylthiols II and III were obtained from Aldrich. Electrochemical
methods were as described previously7,8 and the stilbenyl-modified
Fig. 1 Voltammograms for azurin adsorbed at Au-SAM electrodes prepared
using I and II. Electrolyte: 0.02 M NaOAc, 0.1 M Na2SO4, pH 4.0, 0 °C.
Scan rate 100 V s21. Cycles initiated from high-potential limit.
† Current address: School of Physics and Astronomy, University of Leeds,
Leeds, England LS2 9JT.
316
C h e m . C o m m u n . , 2 0 0 4 , 3 1 6 – 3 1 7
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4

lmax(CH2Cl2)/nm (log10e/dm3mol21cm21) 276 (4.31) and 323 (3.83);
dH(400 MHz; CDCl3) 1.43 (6 H, t, J 7.0, CH3), 2.88 (1 H, bs, OH), 4.07 (4
H, q, J 7.0, CH2), 4.64 (2 H, s, ArCH2), 6.49 (2 H, s, ArH) and 10.42 (1 H,
s, CHO); dC(100 MHz; CDCl3) 14.6, 64.5, 64.8, 102.2, 113.2, 150.3, 161.6
and 189.5; m/z [CI+] 225.1 (MH+, 100%).
2 was then reacted to form {3,5-diethoxy-4-[(E)-2-(4-ethylphenyl)vinyl]-
phenyl}methanol 3: Dry THF (5.0 cm3) was added to a stirred mixture of
(4-ethylbenzyl)phosphonic acid diethyl ester17 (0.69 g, 2.7 mmol) and 2
(0.50 g, 2.2 mmol), under Ar. Potassium tert-butoxide (0.55 g, 4.9 mmol)
was added and the reaction mixture was stirred for 22 h. 3 (0.40 g, 55%) was
isolated after work up and purification: m.p. 103–104 °C. (Found: C, 77.0;
H, 8.2. C21H26O3 requires C, 77.3; H, 8.0%); nmax(KBr)/cm21 3378 (OH)
and 1574 (CNC); lmax(CH2Cl2)/nm (log10e/dm3mol21cm21) 317 (4.40),
329 (4.42) and 345sh (4.21); dH(400 MHz; CDCl3) 1.27 (3 H, t, J 7.5, CH3),
1.50 (6 H, t, J 7.0, CH3), 1.83 (1 H, bm, OH), 2.68 (2 H, q, J 7.5, ArCH2),
4.12 (4 H, q, J 7.0, CH2), 4.66 (2 H, d, J 5.5, ArCH2), 6.57 (2 H, s, ArH),
1
7.20 (1 H, 2AAABBA, ArH), 7.44–7.48 (3 H, m, ArH and vinyl H) and 7.68
Fig. 2 ‘Trumpet plots’ comparing electrochemical properties of azurin
adsorbed on SAMs composed of I or II. Conditions: 0.02 M NaOAc, 0.1 M
Na2SO4, pH 4.0, 0 °C. Cycles initiated at high-potential limit.
(1 H, d, J 16.5, vinyl H); dC(100 MHz; CDCl3) 14.9, 15.6, 28.6, 64.3, 65.6,
103.4, 114.3, 119.2, 126.3, 128.0, 132.0, 137.0, 140.8, 143.1 and 158.1; m/z
[CI+] 327.2 (MH+, 100%).
3 was converted into thiolacetic acid {3,5-diethoxy-4-[(E)-2-(4-ethylphe-
nyl)vinyl]benzyl} ester 4: Diethyl azodicarboxylate (2.8 cm3, 18 mmol) in
dry THF (5.0 cm3) was added to triphenylphosphine (4.6 g, 18 mmol)
cooled to 0 °C under argon. The mixture was stirred for 20 minutes at 0 °C
and then 3 (1.2 g, 3.5 mmol) and thiolacetic acid (1.3 cm3, 18 mmol) in dry
THF (10 mL) were added. The reaction mixture was stirred for 16 h at room
temperature. 4 (0.96 g, 71%) was isolated after work up and purification:
m.p. 87–88 °C; (Found: C, 71.6; H, 7.6. C23H28O3 requires C, 71.8; H,
7.3%); nmax(KBr)/cm21 1687 (CNO); lmax(CH2Cl2)/nm (log10e/
dm3mol21cm21) 320 (4.88), 327 (4.89) 331 (4.90) and 346sh (4.69);
dH(400 MHz; CDCl3) 1.26 (3 H, t, J7.5, CH3), 1.46 (6 H, t, J 7.0, CH3), 2.39
lower at the stilbenyl surface (347 mV compared to 356 mV). This
may reflect a difference in the interfacial potential drop or a small
change in the environment of the Cu site, as the stilbenyl surface
may be more permeable to water molecules. As the scan rate
increases, the oxidation and reduction peak potentials separate, the
shape depending on the ET kinetics (the rate constant k0 is
determined from fits to the Butler–Volmer equation) and the
participation of reactions coupled to electron transfer. The value
obtained for I is 1626 s21, which compares with 481 s21 for II, and
60 s21 for III (data not shown, but reported earlier7). Results were
similar regardless of whether the scan was started from the high or
low potential limit. Similar results were also obtained when the film
was formed using the stilbenyl disulfide which can be formed from
the oxidative coupling of two molecules of I.
The much faster ET rate obtained with I compared to that for
aliphatic SAMs is consistent with a lower value of b as expected for
a conjugated bridge. With the ethyl group as the protein binding
functionality, the interfacial interaction should be very similar to
that for the aliphatic SAMs, and in each case the electron has to
transfer across the remaining distance between the protein surface
and the Cu. However, the important point is that the increase in rate
constant is not several orders of magnitude, as observed for the
ferrocenyl-terminated OPVs.9 This result is important as it supports
an emerging model for protein intermolecular and interfacial ET
reactions, in which the optimal rate of ET is limited by the
probability of achieving, by rapid fluctuations, good electronic
coupling through the interfacial assembly of protein and solvent
molecules.4,6,7,13–15 These processes are probably irrelevant for
coupling to a small molecule through a covalent attachment as in
the ferrocenyl-terminated SAMs, but may be crucial for non-
covalent coupling to a protein. Nevertheless, with an electro-
chemical exchange rate constant exceeding 103 s21, the time
domain complies with turnover rates of most enzymes, so that
interfacial ET need not be rate limiting.16 In practical terms, the
results demonstrate the feasibility of creating SAMs to provide
reversible ET across distances equivalent to those encountered in
biological membranes, thereby allowing fast ET to biological
structures of greater complexity than has been possible with shorter
SAMs.
(3 H, s, COCH3), 2.66 (2 H, q, J 7.5, ArCH2), 4.09 (6 H, m, ArCH2 and
1
CH2), 6.50 (2 H, s, ArH), 7.19 (2 H, AAABBA, ArH), 7.40–7.47 (3 H, m,
2
ArH and vinyl H) and 7.66 (1 H, d, J 16.5, vinyl H); dC(100 MHz; CDCl3)
14.9, 15.6, 28.6, 30.3, 34.1, 64.3, 105.6, 114.2, 119.1, 126.3, 128.0, 132.0,
137.0, 137.3, 143.1, 158.0 and 195.3; m/z [CI+] 385.4 (MH+, 37%).
4 was converted to I as follows: NH4OH (10 mL) was added to an Ar
purged solution of 4 in DMF (500 mL, 0.5 mM). This mixture was again
purged with Ar and left for 10 min before the gold electrodes were
immersed.
¶ Electrodes for azurin voltammetry were cleaned using standard proce-
dures,7,8 then incubated for 6 hours with a 0.5 mM solution of the
appropriate thiol in DMF. Electrodes were rinsed consecutively with
chloroform, ethanol and water, before azurin was adsorbed by incubating
the electrode overnight in a 2.4 mM solution at 20 °C. Na2SO4 was used to
avoid complications caused by the specific adsorption of chloride ions.
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This research was supported by grants from the EPSRC and
BBSRC (B11675). We thank Professor G. Canters, Leiden
University, for providing a sample of azurin.
12 L. Cai, Y. Yao, J. Yang, D. W. Price, Jr. and J. M. Tour, Chem. Mater,
2002, 14, 2905.
Notes and references
§
I was prepared in four steps. First, (4-bromo-3,5-diethoxyphenyl)me-
13 D. Leys, J. Basran, F. Talfournier, M. J. Sutcliffe and N. S. Scrutton,
Nature Struct. Biol, 2003, 10, 219 and references therein.
14 Z. X. Liang, J. M. Nocek, K. Huang, R. T. Hayes, I. V. Kurnikov, D. N.
Beratan and B. M. Hoffmann, J. Am. Chem. Soc., 2001, 124, 6849.
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16 C. C. Page, C. C. Moser, X. X. Chen and P. L. Dutton, Nature, 1999,
402, 47.
thanol 1 was converted to 2,6-diethoxy-4-(hydroxymethyl)benzaldehyde 2:
n-BuLi in hexane (1.6 M, 9.1 cm3, 23 mmol) was added to a stirred solution
of 1 (2.5 g, 9.1 mmol) in dry THF (100 cm3) at 278 °C under Ar. DMF (7.9
cm3, 90 mmol) was added to the reaction after 1 h. The reaction mixture was
then stirred for 30 min. Aq. HCl (3 M, 50 cm3) was added and the mixture
allowed to warm to room temperature. 2 (1.9 g, 57%) was isolated after
work up and purification: m.p. 94–95 °C. (Found: C, 64.3; H, 7.2. C12H16O4
requires C, 64.3; H, 7.2%); nmax(KBr)/cm21 3494 (OH) and 1684 (CNO);
17 G. M. Day, O. T. Howell, M. R. Metzler and P. D. Woodgate, Aust. J.
Chem., 1997, 50, 425.
C h e m . C o m m u n . , 2 0 0 4 , 3 1 6 – 3 1 7
317