Effect of a strong interfacial electric field on the orientation of the dipole moment of thiolated aib-oligopeptides tethered to mercury on either the N- or C-terminus

Article abstract of DOI:10.1021/ja100486y

Four oligopeptides consisting of a sequence of α-aminoisobutyric acid (Aib) residues, thiolated at either the N- or C-terminus by means of a -(CH₂)₂-SH anchor, were self-assembled on mercury, which is a substrate known to impart a high fluidity to self-assembled monolayers (SAMs). The surface dipole potential of these peptide SAMs was estimated in 0.1 M KCl aqueous solution at a negatively charged electrode, where the interfacial electric field is directed toward the metal. To the best of our knowledge, this is the first estimate of the surface dipole potential of peptide SAMs in aqueous solution. The procedure adopted consisted in measuring the charge involved in the gradual expansion of a peptide-coated mercury drop and then combining the resulting information with an estimate of the charge density experienced by diffuse layer ions. The dipole moment of the tethered thiolated peptides was found to be directed toward the metal, independent of whether they were thiolated at the C- or N-terminus. This result was confirmed by the effect of these SAMs on the kinetics and thermodynamics of the Eu(III)/Eu(II) redox couple. The combined outcome of these studies indicates that a strong interfacial electric field orients the dipole moment of peptide SAMs tethered to mercury, even against their natural dipole moment.

Full text of DOI:10.1021/ja100486y

Published on Web 04/14/2010
Effect of a Strong Interfacial Electric Field on the Orientation
of the Dipole Moment of Thiolated Aib-Oligopeptides Tethered
to Mercury on Either the N- or C-Terminus
Lucia Becucci,*,† Ivan Guryanov,‡ Flavio Maran,‡ and Rolando Guidelli†
Department of Chemistry, Florence UniVersity, Via della Lastruccia 3, 50019 Sesto Fiorentino,
Firenze, Italy, and Department of Chemistry, UniVersity of PadoVa,
Via Marzolo 1, 35131 PadoVa, Italy
Received January 19, 2010; E-mail: lucia.becucci@unifi.it
Abstract: Four oligopeptides consisting of a sequence of R-aminoisobutyric acid (Aib) residues, thiolated
at either the N- or C-terminus by means of a -(CH2)2-SH anchor, were self-assembled on mercury, which
is a substrate known to impart a high fluidity to self-assembled monolayers (SAMs). The surface dipole
potential of these peptide SAMs was estimated in 0.1 M KCl aqueous solution at a negatively charged
electrode, where the interfacial electric field is directed toward the metal. To the best of our knowledge,
this is the first estimate of the surface dipole potential of peptide SAMs in aqueous solution. The procedure
adopted consisted in measuring the charge involved in the gradual expansion of a peptide-coated mercury
drop and then combining the resulting information with an estimate of the charge density experienced by
diffuse layer ions. The dipole moment of the tethered thiolated peptides was found to be directed toward
the metal, independent of whether they were thiolated at the C- or N-terminus. This result was confirmed
by the effect of these SAMs on the kinetics and thermodynamics of the Eu(III)/Eu(II) redox couple. The
combined outcome of these studies indicates that a strong interfacial electric field orients the dipole moment
of peptide SAMs tethered to mercury, even against their “natural” dipole moment.
method, either in vacuo4,5 or in atmosphere,6 and by photo-
electron spectroscopy.5,7 These measurements have been nor-
mally carried out on self-assembled monolayers (SAMs) of
simple or functionalized alkanethiols. In what follows, the
surface dipole potential is taken as positive when the dipoles
point their positive pole toward the metal; moreover, a dipole
moment is regarded as positive when directed from the negative
to the positive pole. Evans and Ulman6 found that the dipole
potential of n-alkanethiols anchored to gold varies linearly with
increasing chain length. By assuming that each additional
methylene unit does not contribute any additional net dipole
moment to that already existing in the molecule, they concluded
that the dielectric constant should decrease as the chain length
increases. This assumption implies ignoring the possibility for
the protons resulting from the deprotonation of the sulfhydryl
groups following thiol adsorption to be located at the distal end
of the alkyl chain. By using the Kelvin probe method, Imanishi
et al.4 estimated, under high-vacuum conditions, the change of
the surface dipole potential of gold upon adsorption of helical
peptides. The peptide chains consisted of an alternating sequence
of alanine (Ala) and R-aminoisobutyric acid (Aib) residues. To
form the SAM on gold, a lipoic acid moiety, with its disulfide
group, was covalently linked either to the C- or the N-terminus
1. Introduction
Electron-transfer (ET) reactions in a biological system occur
through a sequence of steps involving redox moieties surrounded
by a polypeptide matrix. It is generally believed that these
polypeptides act as efficient mediators of electron tunneling.1
In addition, the large dipole moment that develops along helical
peptides2 is thought to facilitate ET if properly positioned with
respect to the electron tunneling pathway. To clarify this aspect,
both photoinduced and electrochemically induced ETs along
helical peptides have been extensively investigated.3
Surface dipole potential values of organic layers on metal
surfaces have been extracted from changes of the metal work
function upon formation of these layers by the Kelvin probe
† Florence University.
‡ University of Padova.
(1) (a) Skourtis, S. S.; Beratan, D. N. AdV. Chem. Phys. 1999, 106, 377.
(b) Gray, H. B.; Winkler, J. R. In Electron Transfer in Chemistry;
Balzani, V., Ed.;,Wiley-VCH: Weinheim, 2001; Vol. 1, p 3. (c) Long,
Y. T.; Abu-Irhayem, E.; Kraatz, H.-B. Chem.sEur. J. 2005, 11, 5186–
5194. (d) Cordes, M.; Giese, B. Chem. Soc. ReV. 2009, 38, 892–901.
(2) Shin, Y.-G.; Newton, M. D.; Isied, S. S. J. Am. Chem. Soc. 2003,
125, 3722–3732.
(3) (a) Fox, M. A.; Galoppini, E. J. Am. Chem. Soc. 1997, 119, 5277–
5285. (b) Galoppini, E.; Fox, M. A. J. Am. Chem. Soc. 1996, 118,
2299–2300. (c) Morita, T.; Kimura, S.; Kobayashi, S.; Imanishi, Y.
J. Am. Chem. Soc. 2000, 122, 2850–2859. (d) Yasutomi, S.; Morita,
T.; Imanishi, Y.; Kimura, S. Science 2004, 304, 1944–1947. (e)
Yasutomi, S.; Morita, T.; Kimura, S. J. Am. Chem. Soc. 2005, 127,
14564–14565. (f) Sek, S.; Tolak, A.; Misicka, A.; Palys, B.; Bilewicz,
R. J. Phys. Chem. B 2005, 109, 18433–18438. (g) Sek, S.; Swiatek,
K.; Misicka, A. J. Phys. Chem. B 2005, 109, 23121–23124.
(4) Imanishi, Y.; Miura, Y.; Iwamoto, M.; Kimura, S.; Umemura, J. Proc.
Jpn. Acad. Ser. B 1999, 75, 287–290.
(5) Ray, S. G.; Cohen, H.; Naaman, R.; Liu, H.; Waldeck, D. H. J. Phys.
Chem. B 2005, 109, 14064–14073.
(6) Evans, S. D.; Ulman, A. Chem. Phys. Lett. 1990, 170, 462–466.
(7) Alloway, D. M.; Hofmann, M.; Smith, D. L.; Gruhn, N. E.; Graham,
A. L.; Colorado, R., Jr.; Wysocki, V. H.; Lee, T. R.; Lee, P. A.;
Armstrong, N. R. J. Phys. Chem. B 2003, 107, 11690–11699.
9
6194 J. AM. CHEM. SOC. 2010, 132, 6194–6204
10.1021/ja100486y  2010 American Chemical Society

Effect of a Strong Interfacial Electric Field
A R T I C L E S
of the peptide chain. The surface dipole potential of the peptides
anchored to gold on the N-terminus side was found to be positive
and to increase linearly with the chain length. On the other hand,
the surface dipole potential of the peptides anchored at the
C-terminus was negative, but it did not show such a propor-
tionality. Naaman and co-workers5 compared the dipole mo-
ments of a number of thiolated organic molecules, estimated
from work function measurements on gold-supported mono-
layers, with those obtained from ab initio calculations of the
isolated molecules. They concluded that ET between the metal
substrate and the monolayer is small for adsorbed molecules
pointing their dipole toward the metal surface. Conversely, ET
from the metal to the molecule is appreciable for adsorbed
molecules pointing their dipole away from the metal surface;
with one particular molecule, the charge transfer was even found
to be large enough to invert the direction of the dipole.
electrode. The kinetic behavior of this monolayer was compared
with that of a monolayer in which one-half of the above Fc-
free peptide was replaced by a Fc-free peptide with the cysteine
residue at the N-terminus. The negative surface dipole potential
in the first monolayer should slow the ET from Fc to the metal
(i.e., Fc electro-oxidation) and accelerate the ET in the opposite
direction with respect to the second monolayer, in which the
net surface dipole potential is practically zero. Both effects are
expected to shift the formal potential of the Fc/Fc+ redox couple
of the first monolayer in the positive direction. In fact, the formal
potential in the first monolayer was found to be more positive
than that in the second monolayer by ∼50 mV. The authors
provided a different explanation for this behavior. The opposing
effect of the negative surface dipole potential of an alanine-
rich peptide on Fc oxidation and on Fc+ reduction was pointed
out by Sek et al.,3f by calculating an anodic charge-transfer
coefficient lower than the cathodic one. Morita et al.3c verified
the influence of the surface dipole potential of helical peptides
on photoelectrochemically induced electron transfer. A lipoic
acid residue was linked to the C-terminus of an (Ala-Aib)-based
peptide and a chromophore donor to its N-terminus, or vice
versa. The chromophore was photoexcited in the presence of
an acceptor in solution, giving rise to a cathodic photocurrent
whose quantum efficiency was higher for the peptide with
negative surface dipole potential. This approach was used to
fabricate a molecular photodiode composed of two types of
peptides carrying different chromophores and having opposing
dipole moments.3d
To the best of our knowledge, the surface dipole potential of
SAMs of helical peptides in aqueous solution has never been
estimated. Recently, we devised a procedure to estimate the
surface dipole potential of Hg-supported SAMs.10 The procedure
consists in self-assembling the monolayers on a hanging mercury
drop electrode and then measuring the charge involved during
a progressive expansion of the mercury drop. This measurement
is combined with an estimate of the charge density q experienced
by diffuse layer ions, by measuring the diffuse layer capacitance
of the SAM at different electrolyte concentrations by electro-
chemical impedance spectroscopy. We, therefore, found it
interesting to apply this procedure to the estimate of the surface
dipole potential of a number of Aib-based homo-oligopeptides
thiolated at either the C- or N-terminus. As opposed to peptide
systems based on coded R-amino acids, which start to form
helices only for rather long oligomers,11 Aib homopeptides have
the peculiarity of forming stiff 310-helices even with a low
number of monomeric units.12-14 This is due to the marked
steric hindrance of the R-carbon, which results in a restricted
torsional freedom. Moreover, the increase in the number of units
is accompanied by an increase in the number of intramolecular
hydrogen bonds and a concomitant increase in stiffness. Studies
Galoppini and Fox3b were the first to address the issue of
whether the dipole moment of peptides with an alternating Ala-
Aib backbone could affect the photoinduced ET from a neutral
donor D to a neutral acceptor A. To this end, D and A were
appended, in the order, along the peptide chain either in the
direction of the peptide dipole moment or in the opposite
direction. The rate constant for photoinduced electron transfer
was found to be larger with the former arrangement. This is in
agreement with the fact that, for stable helices, ET is facilitated
when electron tunneling occurs in the direction of the dipole
moment, i.e., when the helix stabilizes the resulting charge
separation state. In keeping with this hypothesis, the same
authors observed that unfolding the helical peptide with a protic
solvent could reduce such a difference in rate constants very
significantly.3a An analogous approach was followed by other
groups to assess the influence of the surface dipole potential of
helical peptides on electrochemically induced ET. To this end,
a disulfide8 or sulfhydryl group3f,9 was linked to the C-terminus
of a helical peptide and an electro-oxidazable ferrocene (Fc)
moiety at its N-terminus, or vice versa. The helix content of
the peptides was estimated from circular dichroism (CD) spectra,
whereas the amide I/amide II absorbance ratio of infrared
reflection absorption spectroscopy (IRRAS) spectra provided
information on the tilt of the helix axis relative to the substrate.8c
By using a peptide with a leucine (Leu)-Aib backbone tethered
to gold via a lipoic acid moiety, the Fc electroxidation rate
constant was found to be higher when the surface dipole
potential of the peptide was positive than when it was negative.8a
It was concluded that ET from Fc to gold is favored by a dipole
moment directed toward the metal. A similar system was also
examined by scanning tunneling spectroscopy.8d Conversely,
the same group found that a different Ala-Aib-based peptide
did not show any appreciable difference in rate constant value
by reversing the direction of the peptide dipole potential. This
behavior was justified by ascribing the rate-determining step to
the ET across the lipoic acid linker.8b
(10) Becucci, L.; Schwan, A. L.; Sheepwash, E. E.; Guidelli, R. Langmuir
2009, 25, 1828–1835.
Mandal and Kraatz9a prepared a leucine-rich peptide with a
cysteine residue at the C-terminus and a Fc moiety at the
N-terminus and diluted it with a large amount of the same
peptide with no Fc; the resulting mixture was tethered to a gold
(11) Goodman, M.; Toniolo, C.; Pallai, P. In Forum Peptides; Castro, B.,
Martinez, J., Eds.; Dhor: Nancy, France, 1985; pp 146-174.
(12) (a) Karle, I.; Balaram, P. Biochemistry 1990, 29, 6747–6756. (b)
Toniolo, C.; Crisma, M.; Formaggio, F.; Peggion, C. Biopolymers
(Pept. Sci.) 2001, 60, 396–419. (c) Toniolo, C.; Bonora, G. M.; Barone,
V.; Bavoso, A.; Benedetti, E.; Di Blasio, B.; Grimaldi, P.; Lelj, F.;
Pavone, V.; Pedone, C. Macromolecules 1985, 18, 895–902. (d)
Toniolo, C.; Benedetti, E. Trends Biochem. Sci. 1991, 16, 350–353.
(13) (a) Polo, F.; Antonello, S.; Formaggio, F.; Toniolo, C.; Maran, F. J. Am.
Chem. Soc. 2005, 127, 492–493. (b) Antonello, S.; Formaggio, F.;
Moretto, A.; Toniolo, C.; Maran, F. J. Am. Chem. Soc. 2003, 125,
2874–2875.
(8) (a) Morita, T.; Kimura, S. J. Am. Chem. Soc. 2003, 125, 8732–8733.
(b) Watanabe, J.; Morita, T.; Kimura, S. J. Phys. Chem. B 2005, 109,
14416–14425. (c) Miura, Y.; Kimura, S.; Imanishi, Y.; Umemura, J.
Langmuir 1998, 14, 6935–6940. (d) Kitagawa, K.; Morita, T.; Kimura,
S. Angew. Chem., Int. Ed. 2005, 44, 6330–6333.
(9) (a) Mandal, H. S.; Kraaz, H.-B. Chem. Phys. 2006, 326, 246–251. (b)
Wain, A. J.; Do, H. N. L.; Mandal, H. S.; Kraatz, H.-B.; Zhou, F. J.
Phys. Chem. C 2008, 112, 14513–14519.
(14) Fabris, L.; Antonello, S.; Armelao, L.; Donkers, R. L.; Polo, F.;
Toniolo, C.; Maran, C. J. Am. Chem. Soc. 2006, 128, 326–336.
9
J. AM. CHEM. SOC. VOL. 132, NO. 17, 2010 6195

A R T I C L E S
Becucci et al.
Chart 1. Peptides 3+, 3-, 5+, and 5- Highlighting Either the
Different Polarity (3+ vs 3-) or the Actual 310-Helix Structure (5+,
5-)a
Preliminary results on the experimental estimate of the dipole
moments of a series of analogous Aib homo-oligomers in
solution18 show that the dipole moment increases linearly with
the number of Aib units; accordingly, we estimate dipole
moments of 11-12 and 16-17 D for the couples of peptides 3
and 5, respectively. These data also show that 310-helices are
far less sensitive to environment than R-helices, as also inferred
from specific electrostatics calculations.19 Despite the strong
secondary structure of Aib peptides and the ensuing large values
of the dipole moments, our electrochemical analysis indicates
that a strong interfacial electric field between the mercury
electrode and the electrolytic solution is capable of orienting
the dipole moment of SAMs of Aib-peptides thiolated at the
C-terminus against their “natural” molecular dipole moment.
2. Experimental Section
Chemicals. The water, obtained from an inverted osmosis unit,
was distilled and then further distilled from alkaline permanganate.
Merck Suprapur KCl was baked at 500 °C before use to remove
any organic impurities. Tetramethyammonium chloride (TMACl)
and hydroxide (TMAOH) were purchased from Merck and used
as such. The following solvents, salts, and reagents were com-
mercially available and used as received. Solvents: methanol,
methylene chloride, diethyl ether, petroleum ether, ethyl acetate
(Carlo Erba). Salts: potassium bisulfate, sodium bicarbonate,
anhydrous sodium sulfate (Carlo Erba); N,N-diisopropylethylamine
(DIEA), trifluoroacetic acid, triisopropylsilane, tert-butylamine,
2-aminoethanethiol (Acros Organics); triphenylmethanol, anhydrous
acetonitrile, trimethylacetyl chloride (Sigma-Aldrich); 1-(3-dim-
ethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl),
1-hydroxy-7-aza-1,2,3-benzotriazole (HOAt) (GL Biochem, Shan-
gai). The syntheses of Ph3C-S-(CH2)2-COOH20 and peptide 3+14
have been already described. The other thiolated Aib homopetides
were prepared as follows.
a The intramolecular CdO· · ·H-N hydrogen bonds are denoted by
dashed lines.
based on X-ray diffraction, IR, and 1H NMR show that helical
structures are formed in the solid state as well as in solution.12c,15
The information so far gathered on this kind of peptides in other
physical states indicates that the 310-helix structure is maintained
unaltered also when the peptides are assembled onto gold
surfaces. IRRAS evidence of it was reported for Aib homopep-
tides, thiolated on the N-terminus through a lipoic acid tether,
in self-assembled monolayers on extended gold surfaces.16
Analogously, we obtained IR and 1H NMR spectroscopy results
showing that the 310-helix conformation is maintained unaltered
also when the same peptides decorate 1-2 nm gold clusters;14
in this case, the peptides were thiolated on the N-terminus
through a -(CH2)2-SH group. We also exploited the unique
features of Aib homopeptides to promptly form stiff helices in
order to introduce oriented dipoles into the capping monolayer
of phenylethanethiolate-protected gold clusters.17 Gradual ex-
change of the original ligands by Aib peptides thiolated at the
N-terminus was found to cause a marked positive shift of the
oxidation peaks of the gold core, thus pointing to very strong
dipolar effects influencing the energy of the highest occupied
molecular orbital of gold.
The peptides examined in the present work contain four or
six Aib units. The molecular sequence is such as to allow the
formation of structures characterized by three or five intramo-
lecular CdO· · ·H-N hydrogen bonds, as shown in Chart 1.
The labeling 3+, 3-, 5+, and 5- denotes the number of such
intramolecular hydrogen bonds and whether the N-terminus (+)
or the C-terminus (-) is thiolated. The two couples of peptides
3+/3- and 5+/5- were devised in order to keep identical both
their lengths and the nature of the group (tert-butyl) facing the
solution.
Z-(Aib)6-OH. To a solution of Z-(Aib)6-OtBu (215 mg, 0.30
mmol) in 3 mL of anhydrous CH2Cl2 was added 3 mL of
trifluoroacetic acid under stirring. After 1 h the solvent was
evaporated and the remaining traces of trifluoroacetic acid were
removed by repetitive evaporations with diethyl ether. The product
was recrystallized from diethyl ether to give a colorless solid (yield
98%) having mp ) 205-206 °C. IR (KBr): 3310, 1737,1704, 1659,
1
1530 cm-1. H NMR (400 MHz, CDCl3): δ 1.37 (s, 6H, 2CH3),
1.42 (s, 6H, 2CH3), 1.45 (s, 6H, 2CH3), 1.47 (s, 16H, 4CH3), 1.51
(s, 6H, 2CH3), 5.10 (s, 2H, CH2), 5.32, 6.29, 7.33 (3s, 3H, 3NH),
7.36 (m, 6H, 1NH, Ph), 7.38 (s, 1H, NH), 7.44 (s, 1H, NH).
Z-(Aib)6-NHtBu. To an ice-cold solution of Z-(Aib)6-OH (194
mg, 0.29 mmol) in 20 mL of anhydrous acetonitrile was added
EDC ·HCl (58 mg, 0.30 mmol), followed after 1 h by addition of
tert-buthylamine (315 µL, 3.00 mmol). The reaction mixture was
stirred 3 days at room temperature and then evaporated to dryness.
The crude product was purified by column chromatography using
as the eluent petroleum ether/ethyl acetate (v/v 1/1, 1/2, 1/3, 1/4)
and recrystallized from ethyl acetate/petroleum ether to give
Z-(Aib)6-NHtBu as a colorless solid (yield ) 70%) having mp )
219-221 °C. IR (KBr): 3428, 3321, 2985, 2940, 1703, 1663, 1530,
1
1454 cm-1. H NMR (CDCl3): δ 1.31 (s, 6H, 2CH3), 1.39 (s, 9H,
tBu), 1.43 (s, 6H, 2CH3), 1.48 (s, 18H, 6CH3), 1.51 (s, 6H, 2CH3),
5.11 (s, 2H, CH2), 5.89, 6.49, 6.89 (3s, 3H, 3NH), 7.36 (m, 6H,
1NH, Ph), 7.50 (s, 2H, 2NH), 7.56 (s, 1H, NH).
Ph3C-S-(CH2)2-CO-(Aib)6-NHtBu. To an ice-cold solution
of Ph3C-S-(CH2)2-COOH (150 mg, 0.43 mmol) in 10 mL of
anhydrous CH2Cl2 were added HOAt (60 mg, 0.44 mmol) and
(15) (a) Kennedy, D. F.; Crisma, M.; Toniolo, C.; Chapman, D. Biochem-
istry 1991, 30, 6541. (b) Hanson, P.; Millhauser, F.; Formaggio, F.;
Crisma, M.; Toniolo, C. J. Am. Chem. Soc. 1996, 118, 7618.
(16) Wen, X.; Linton, R. W.; Formaggio, F.; Toniolo, C.; Samulski, E. T.
J. Phys. Chem. A 2004, 108, 9673–9681.
(18) Di Noto, V.; Maran, F., work in progress.
(19) Sengupta, D.; Behera, R. N.; Smith, J. C.; Ullmann, G. M. Structure
2005, 13, 849–855.
(17) Holm, A. H.; Ceccato, M.; Donkers, R. L.; Fabris, L.; Pace, G.; Maran,
F. Langmuir 2006, 22, 10584–10589.
(20) Peggion, C.; Formaggio, F.; Toniolo, C.; Becucci, L.; Moncelli, M. R.;
Guidelli, R. Langmuir 2001, 17, 6585–6592.
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Effect of a Strong Interfacial Electric Field
A R T I C L E S
EDC·HCl (85 mg, 0.44 mmol), followed by addition of a solution
of H-(Aib)6-NHtBu (obtained, and used as such, by catalytic
hydrogenation of 200 mg, 0.28 mmol, of Z-(Aib)6-NHtBu in
MeOH) in anhydrous CH2Cl2 containing DIEA (50 µL, 0.28 mmol).
After stirring for 48 h at room temperature, the reaction mixture
was evaporated to dryness. The crude product was purified by
column chromatography using as the eluent petroleum ether/ethyl
acetate (v/v 1/2, 1/3, 1/4, 1/5) and recrystallized from ethyl acetate/
petroleum ether to give Ph3C-S-(CH2)2- CO-(Aib)6-NHtBu as
a colorless solid (yield ) 67%) having mp ) 251-253 °C. IR
followed by addition of a solution of H-(Aib)4-NHtBu (obtained,
and used as such, by catalytic hydrogenation of 300 mg, 0.55 mmol,
of Z-(Aib)4-NHtBu in MeOH) in 20 mL of anhydrous acetonitrile.
After heating at 80 °C for 3 days the reaction was evaporated to
dryness. The crude product was purified by column chromatography
using as the eluent petroleum ether/ethyl acetate (v/v 1/3) and
recrystallized from ethyl acetate/petroleum ether to give a colorless
solid (yield ) 43%) having mp ) 217-218 °C. IR (KBr): 3427,
1
3293, 2985, 2937, 2871, 1744, 1698, 1656, 1531, 1456 cm-1. H
NMR (CDCl3): δ 1.24 (s, 9H, tBu), 1.42-1.58 (m, 45H, 9H tBu,
12CH3), 6.09, 6.15, 7.30, 7.39, 7.52, 7.63 (6s, 6H, 6NH).
(KBr): 3420, 3308, 2985, 2932, 1660, 1533 cm-1
.
1H NMR
(CD3CN): δ 1.28 (s, 6H, 2CH3), 1.32 (s, 9H, tBu), 1.35 (s, 6H,
2CH3), 1.35 (s, 6H, 2CH3), 1.38 (s, 6H, 2CH3), 1.40 (s, 12H, 4CH3),
2.22 (t, 2H, CH2), 2.42 (t, 2H, CH2), 6.82, 6.90, 6.94 (3s, 3H, 3NH),
7.24-7.44 (m, 16H, 1NH, 3Ph), 7.58, 7.62, 7.68 (3s, 3H, 3NH).
HS-(CH2)2-CO-(Aib)6-NHtBu (5+). Ph3C-S-(CH2)2
-CO-(Aib)6-NHtBu (56 mg, 0.06 mmol) was dissolved in 10
mL of a 7/2/1 mixture of CH2Cl2/trifluoroacetic acid/triisopropyl-
silane. After stirring for 1 h at room temperature, the solvent was
evaporated and the remaining traces of trifluoroacetic acid were
removed by repetitive evaporations with diethyl ether. The crude
product was purified by column chromatography using the eluent
CH2Cl2 and then CH2Cl2/EtOH, 10/1. After crystallization from
diethyl ether HS-(CH2)2-CO-(Aib)6-NHtBu was obtained as a
colorless solid (yield 68%) having mp ) 270-271 °C. IR (KBr):
C(CH3)3-CO-(Aib)4-OH. To a solution of C(CH3)3
-CO-(Aib)4-OtBu (1.00 g, 2.01 mmol) in 3 mL of anhydrous
CH2Cl2 was added 3 mL of trifluoroacetic acid under stirring. After
1 h the solvent was evaporated and the remaining traces of
trifluoroacetic acid were removed by repetitive evaporations with
diethyl ether. The product was recrystallized from diethyl ether to
give a colorless solid (yield ) 80%) having mp ) 228-229 °C.
IR (KBr): 3422, 3356, 3327, 3294, 3177, 2986, 2938, 1747, 1673,
1
1646, 1528 cm-1. H NMR (CDCl3): δ 1.13 (s, 9H, tBu), 1.24 (s,
6H, 2CH3), 1.32 (s, 18H, 6CH3), 7.35, 7.38, 7.50, 7.64 (4s, 4H,
4NH).
C(CH3)3-CO-(Aib)6-OH. To
a solution of C(CH3)3
-CO-(Aib)6-OtBu (130 mg, 0.19 mmol) in 3 mL of anhydrous
CH2Cl2 was added 3 mL of trifluoroacetic acid under stirring. After
1 h the solvent was evaporated and the remaining traces of
trifluoroacetic acid were removed by repetitive evaporations with
diethyl ether. The product was recrystallized from diethyl ether to
give a colorless solid (yield ) 80%) having mp ) 285-286 °C.
IR (KBr): 3418, 3293, 2986, 2940, 2872, 1740, 1657, 1532, 1456
cm-1. 1H NMR (CDCl3): δ 1.22 (s, 9H, tBu), 1.42-1.55 (m, 36H,
12CH3), 7.28, 7.32, 7.40, 7.55, 7.58, 7.66 (6s, 6H, 6NH).
1
3424, 3313, 3049, 2985, 2934, 2872, 1662, 1539 1455 cm-1. H
NMR (CDCl3): δ 1.40 (s, 9H, tBu), 1.42-1.58 (m, 36H, 12CH3),
2.64 (t, 2H, CH2-CO), 2.86 (m, 2H, S-CH2), 6.83, 6.89, 7.51, 7.60,
7.66, 7.75, 7.78 (7s, 7H, 7NH). ESI-TOF: [M + H]+ ) 672.9;
calc
[M + H]+ ) 672.3.
exp
C(CH3)3-CO-(Aib)2-OtBu. To a solution of H-(Aib)2-OtBu
(obtained, and used as such, by catalytic hydrogenation of 200 mg,
0.53 mmol, of Z-(Aib)2-OtBu in MeOH) in 20 mL of anhydrous
CH2Cl2 were added C(CH3)3-CO-Cl (100 µL, 0.81 mmol) and
DIEA (140 µL, 0.81 mmol). After stirring for 3 days at room
temperature, the reaction mixture was evaporated to dryness. The
crude product was purified by column chromatography using, as
the eluent, petroleum ether/ethyl acetate (v/v 3/1) and recrystallized
from ethyl acetate/petroleum ether to give C(CH3)3-CO-
(Aib)2-OtBu as a colorless solid (yield ) 90%) having mp )
139-140 °C. IR (KBr): 3392, 3352, 2978, 2964, 2937, 1721, 1675,
(Ph)3C-S-CH2CH2-NH2 ·H2SO4. To an ice-cold solution of
HS-CH2CH2-NH2 ·HCl (0.26 g, 2.29 mmol) and (Ph)3C-OH
(1.00 g, 3.84 mmol) in 40 mL of acetic acid was added dropwise
H2SO4(conc) until the yellow color remained persistent. After 30
min the reaction mixture was concentrated to 10 mL, and 150 mL
of diethyl ether was added. The white precipitate was filtered and
washed several times with diethyl ether. Yield ) 80%. Mp )
194-195 °C. IR (KBr): 3428, 3052, 2928, 1636, 1513, 1488, 1444,
1221, 1180, 1162, 1061, 1017 cm-1. 1H NMR (DMSO-d6): δ 2.43
(m, 2H, CH2), 2.51 (m, 2H, CH2), 7.21-7.41 (m, 15H, 3Ph), 7.66
(s, 2H, NH2).
1
1654, 1523 cm-1. H NMR (CDCl3) δ 1.20 (s, 9H, tBu), 1.46 (s,
9H, tBu), 1.53 (s, 6H, 2CH3), 1.57 (s, 6H, 2CH3), 6.47 (s, 1H, NH),
7.03 (s, 1H, NH).
C(CH3)3-CO-(Aib)4-NH-(CH2)2-S-C(Ph)3. To an ice-cold
solution of C(CH3)3-CO-(Aib)4-OH (1.50 g, 3.39 mmol) in 20
mL of anhydrous CH2Cl2 was added EDC ·HCl (0.65 g, 3.39 mmol),
followed after 1 h by addition of (Ph)3C-S-CH2CH2-NH2 ·H2SO4
(2.83 g, 6.78 mmol) and DIEA (1.16 mL, 6.78 mmol). The reaction
mixture was stirred for 3 days at room temperature and evaporated
to dryness. The oily residue was dissolved in ethyl acetate, and the
organic solution was washed with 10% KHSO4, H2O, 5% NaHCO3,
and H2O. The organic layer was dried over anhydrous Na2SO4 and
evaporated to dryness. The product was recrystallized from ethyl
acetate/petroleum ether to give C(CH3)3- CO-(Aib)4
-NH-(CH2)2-S-C(Ph)3 as a colorless solid (yield ) 61%) having
mp ) 245-247 °C. IR (KBr): 3433, 3317, 1660, 1530, 741, 699
cm-1. 1H NMR (DMSO-d6): δ 1.13 (s, 9H, tBu), 1.19 (s, 6H, 2CH3),
1.27 (s, 6H, 2CH3), 1.30 (s, 6H, 2CH3), 1.32 (s, 6H, 2CH3), 2.20
(t, 2H, ꢀ-CH2), 2.90 (m, 2H, R-CH2), 7.20-7.31 (m, 17H, 2NH,
3Ph), 7.55 (s, 1H, NH), 7.63 (s, 1H, NH), 7.78 (s, 1H, NH).
C(CH3)3-CO-(Aib)6-NH-(CH2)2-S-C(Ph)3. To an ice-cold
solution of C(CH3)3-CO-(Aib)6-OH (119 mg, 0.19 mmol) in 20
mL of anhydrous acetonitrile was added EDC ·HCl (45 mg, 0.23
mmol), followed after 1 h by addition of (Ph)3C-S-CH2CH2
-NH2 ·H2SO4 (162 mg, 0.39 mmol) and DIEA (67 µL, 0.39 mmol).
The reaction mixture was stirred for 4 days at room temperature
and evaporated to dryness. The crude product was purified by
column chromatography using as the eluent petroleum ether/ethyl
acetate (v/v 1/2, 1/3, 1/4, 1/5) and recrystallized from ethyl acetate/
C(CH3)3-CO-(Aib)4-OtBu. To a solution of H-(Aib)4-OtBu
(obtained, and used as such, by catalytic hydrogenation of 2.00 g,
3.65 mmol, of Z-(Aib)4-OtBu in MeOH) in 20 mL of anhydrous
CH2Cl2 were added C(CH3)3-CO-Cl (0.47 mL, 3.80 mmol) and
DIEA (1.2 mL, 7.20 mmol). After stirring for 3 days at room
temperature the reaction mixture was treated as described above
for Z-(Aib)2-OtBu to give C(CH3)3-CO-(Aib)4-OtBu as a
colorless solid (yield ) 65%) having mp ) 198-199 °C. IR: 3435,
3417, 3346, 2983, 2936, 2872, 1734, 1663, 1619, 1457 (KBr) cm-1
.
1H NMR (CDCl3): δ 1.20 (s, 9H, tBu), 1.39 (s, 6H, 2CH3), 1.40 (s,
9H, tBu), 1.44 (s, 6H, 2CH3), 1.45 (s, 6H, 2CH3), 1.49 (s, 6H,
2CH3), 5.93, 6.12, 7.19, 7.24 (4s, 4H, 4NH).
C(CH3)3-CO-(Aib)2-OH. To
a solution of C(CH3)3
-CO-(Aib)2-OtBu (150 mg, 0.46 mmol) in 3 mL of anhydrous
CH2Cl2 was added 3 mL of trifluoroacetic acid under stirring. After
1 h the solvent was evaporated and the remaining traces of
trifluoroacetic acid were removed by repetitive evaporations with
diethyl ether. The product was recrystallized from diethyl ether to
give a colorless solid (yield ) 90%) having mp ) 189-190 °C.
IR (KBr): 3394, 3375, 3320, 3300, 2989, 2940, 2875, 1725, 1667,
1
1623, 1517 cm-1. H NMR (CDCl3): δ 1.21 (s, 9H, tBu), 1.53 (s,
6H, 2CH3), 1.56 (s, 6H, 2CH3), 6.16 (s, 1H, NH), 7.61 (s, 1H, NH).
C(CH3)3-CO-(Aib)6-OtBu. To an ice-cold solution of
C(CH3)3-CO-(Aib)2-OH (125 mg, 0.46 mmol) in 10 mL of
anhydrous acetonitrile was added EDC ·HCl (105 mg, 0.55 mmol),
9
J. AM. CHEM. SOC. VOL. 132, NO. 17, 2010 6197

A R T I C L E S
Becucci et al.
petroleum ether to give a colorless solid (yield ) 68%) having mp
) 259-260 °C. IR (KBr): 3358, 3057, 2964, 2932, 2244, 1658,
1
1504 cm-1. H NMR (CDCl3): δ 1.24 (s, 9H, tBu), 1.38 (s, 6H,
2CH3), 1.42 (s, 6H, 2CH3), 1.47 (m, 18H, 3CH3), 1.52 (s, 6H,
2CH3), 2.40 (t, 2H, ꢀ-CH2), 3.19 (m, 2H, R-CH2), 6.03 (s, 1H,
NH), 6.13 (s, 1H, NH), 7.08-7.42 (m, 17H, 2NH, 3Ph), 7.50 (s,
1H, NH), 7.55 (s, 1H, NH), 7.66 (s, 1H, NH).
C(CH3)3-CO-(Aib)4-NH-(CH2)2-SH (3-). C(CH3)3-CO-
(Aib)4-NH-(CH2)2-S-Trt (1.50 g, 2.02 mmol) was dissolved in
20 mL of a 7/2/1 mixture of CH2Cl2/trifluoroacetic acid/triisopro-
pylsilane. The solution was stirred for 2 h at room temperature,
and then the solvent was evaporated. The crude product was washed
with diethyl ether to remove the traces of trifluoroacetic acid by
evaporation. After crystallization from diethyl ether
C(CH3)3-CO-(Aib)4-NH-(CH2)2-SH was obtained as a color-
less solid (yield 83%). IR (KBr): 3416, 3313, 2524, 1665, 1642,
1
1536 cm-1. H NMR (CDCl3): δ 1.24 (s, 9H, tBu), 1.42 (s, 6H,
2CH3), 1.47 (s, 6H, 2CH3), 1.49 (s, 6H, 2CH3), 1.54 (s, 6H, 2CH3),
1.78 (t, 1H, SH), 2.71 (t, 2H, ꢀ-CH2), 2.46 (m, 2H, R-CH2), 5.93
(s, 1H, NH), 6.08 (s, 1H, NH), 7.24 (s, 1H, NH), 7.53 (t, 1H, NH),
Figure 1. Curves of the differential capacitance C against the applied
potential E for Hg-supported 3+ and 3- SAMs in aqueous 0.1 M TMACl,
obtained at 75 Hz upon simulating the SAMs by a resistance and a
capacitance in parallel. The dashed curve is the differential capacitance curve
for bare mercury in the same medium. The inset shows C vs E curves for
Hg-supported 3+, 3-, 5+, and 5- SAMs in aqueous 0.1 M KCl over the
potential ranges in which the SAMs were stabilized.
7.63 (s, 1H, NH). ESI-TOF: [M + H]+calc ) 502.7; [M + H]+
)
exp
502.4.
C(CH3)3-CO-(Aib)6-NH-(CH2)2-SH (5-). C(CH3)3 -CO-
Aib6-NH-(CH2)2-S-Trt (60 mg, 0.0656 mmol) was dissolved
in 10 mL of a 7/2/1 mixture of CH2Cl2/trifluoroacetic acid/
triisopropylsilane. The solution was stirred for 1 h at room
temperature, and then the solvent was evaporated. The crude product
was washed with diethyl ether to remove the traces of trifluoroacetic
acid by evaporation and purified by column chromatography using
the eluent CH2Cl2 and then CH2Cl2/EtOH, 10/1. After crystallization
from diethyl ether C(CH3)3-CO-(Aib)6-NH-(CH2)2-SH was
obtained as a colorless solid (yield 85%) having mp ) 262-264
°C. IR (KBr): 3432, 3301, 2984, 2937, 1657, 1531 cm-1. 1H NMR
(CDCl3): δ 1.24 (s, 9H, tBu), 1.42 (s, 6H, 2CH3), 1.48 (s, 24H,
8CH3), 1.56 (s, 6H, 2CH3), 1.81 (t, 1H, SH), 2.70 (m, 2H, CH2),
3.46 (m, 2H, CH2), 6.19 (s, 1H, NH), 6.21 (s, 1H, NH), 7.40 (s,
1H, NH), 7.60 (s, 1H, NH), 7.63 (m, 1H, NH), 7.71 (s, 1H, NH).
ESI-TOF: [M + H]+ ) 672.9; [M + H]+ ) 672.4.
during the drop expansion, while the self-assembled molecules
undergo a progressive tilt without incorporating water molecules.
Denoting by θ the tilt angle of the self-assembled molecules with
respect to the monolayer normal, the monolayer thickness, d(θ), at
a given stage of the expansion is given by d cos θ. Incidentally,
the quantities whose dependence upon θ is not explicitly expressed
refer to the initial unexpanded drop. The expansion of the drop
area, A(θ), does not alter the monolayer volume, A(θ) × d(θ) ) A
× d; consequently, A(θ) is equal to A/cos θ. An accurate
measurement of the volume of the spherical mercury drop progres-
sively extruded from the capillary of the HMDE allows cos θ to
be estimated from the A/A(θ) ratio. The constancy of the number
of SAM molecules during the drop expansion causes their number
density N to decrease according to the relationship N(θ) ) NA/
A(θ) ) N cos θ. The dipole moment normal component, µ, of the
molecules of the unexpanded SAM, if any, undergoes the same tilt
as d during the drop expansion, thus causing it to vary according
to the relationship µ(θ) ) µ cos θ. The above approximate
assumptions exclude the possibility that the drop expansion may
cause the formation of thiol-free and thiol-covered areas, which
would result in an abrupt increase in capacitance. Such an abrupt
increase in capacitance, indicative of monolayer breakdown, was
reported, for instance, with mercury-supported monolayers of
n-alkanethiols with more than 14 carbon atoms.23a,b No such
behavior was observed with the present peptides, as their capaci-
tance increased smoothly with drop expansion.
calc
exp
Electrochemical Apparatus and Methodologies. All measure-
ments were carried out in aqueous solutions of KCl or TMACl.
Use was made of a homemade hanging mercury drop electrode
(HMDE), described elsewhere.21 A homemade glass capillary with
a finely tapered tip, about 1 mm in outer diameter, was employed.
Capillary and mercury reservoir were thermostated at 25 ( 0.1 °C
in a water-jacketed box to avoid any changes in drop area due to
a change in temperature. Chronocoulometric, cyclic voltammetric,
and electrochemical impedance spectroscopy measurements were
carried out with an Autolab PGSTAT 12 instrument (Echo Chemie,
Utrecht, The Netherlands) supplied with an FRA2 module for
impedance measurements, SCAN-GEN scan generator, and GPES
4.9005 Beta software. Potentials were measured vs a Ag|AgCl
electrode immersed in the KCl or TMACl working solution and
are referred to a Ag|AgCl|0.1 M KCl reference electrode.
The self-assembly of the thiolated-peptide monolayers on the
HMDE was carried out by keeping the mercury drop immersed in
ethanol solutions of the thiolated peptides for about 90 min. The
peptide-coated mercury electrode was stabilized by scanning the
applied potential several times over the appropriate potential range
(see further, inset of Figure 1) and by recording the differential
capacitance C, measured at 75 Hz upon simulating the SAM by a
resistance and a capacitance in parallel.
The rationale behind the procedure for measuring the surface
dipole potential of Hg-supported SAMs is described in detail
elsewhere.10 Suffice it here to say that the opposite of the surface
dipole potential produced by the dipole moment of the SAM
molecules, ꢁ ) Nµ/(∇∇0), where ∇0 is the permittivity of free space,
is given by the slope of the quantity
ꢂ(θ) ≡ {q(θ) cos θ/CSAM + ψ[c, q(θ)]}
(1)
against cos2 θ, at constant applied potential, E. Here, q(θ) is the
charge density experienced by the diffuse layer ions, CSAM is the
A SAM-coated mercury drop can often be expanded to a large
extent without causing the SAM to collapse. This situation is
encountered with SAMs of phospholipids,22 n-alkanethiols with
chain lengths less than 14,23 and thiolipids.20,24 In this case, the
amount of SAM material on the drop surface remains constant
(22) Becucci, L.; Moncelli, M. R.; Herrero, R.; Guidelli, R. Langmuir 2000,
16, 7694–7700.
(23) (a) Slowinski, K.; Chamberlain, R. V.; Miller, C. J.; Majda, M. J. Am.
Chem. Soc. 1997, 119, 11910–11919. (b) Bruckner-Lea, C.; Kimmel,
R. J.; Janata, J.; Conroy, J. F. T.; Caldwell, K. Electrochim. Acta 1995,
40, 2897–2904.
(21) Moncelli, M. R.; Becucci, L. J. Electroanal. Chem. 1997, 433, 91–
(24) Becucci, L.; Guidelli, R.; Liu, Q.; Bushby, R. J.; Evans, S. D. J. Phys.
Chem. B 2002, 106, 10410–10416.
96.
9
6198 J. AM. CHEM. SOC. VOL. 132, NO. 17, 2010

Effect of a Strong Interfacial Electric Field
A R T I C L E S
differential capacitance of the unexpanded SAM, and ψ[c,q(θ)] is
the potential difference across the diffuse layer adjacent to the SAM,
regarded as a function of the electrolyte concentration c and of the
charge density q(θ).
along the external circuit during the drop expansion causes
exclusively a change in the diffuse layer charge. The charge density
q(θ) to be used in eq 1 is obtained by adding the charge Q(θ)
following the drop expansion to the charge Aq on the initial
unexpanded drop, as estimated from a Cd vs Cd(q ) 0)-1 plot,
-1
The only thermodynamically significant charge density on a metal
surface is the “total charge density” σM(E), namely, the charge
density to be supplied to the electrode to keep the applied potential
E constant when the electrode surface is increased by unity, and
the composition of the electrified interface maintains its equilibrium
value during such an increase. It is obtained by starting with an
uncoated electrode immersed in a solution of the supporting
electrolyte alone and stepping the applied potential from the
potential of zero charge (pzc) to a final potential Ef negative enough
to exclude adsorption of the adsorbate under investigation, provided
such a potential is experimentally accessible: the charge ac-
companying the potential step pzc f Ef is just the common value,
σM(Ef), of the total charge density both in the presence and in the
absence of the adsorbate. The charge accompanying a further
potential step from any given initial potential E to Ef at a SAM-
coated electrode in the same supporting electrolyte is equal to σM(Ef)
- σM(E), thus allowing the estimate of the total charge density
σM(E) at E.
The total charge density σM coincides with that, q, experienced
by the diffuse layer ions only when the self-assembling surfactant
molecules are neutral and do not generate a charge in direct contact
with the electrode surface during their self-assembly. Usually, this
requirement is not fulfilled by molecules terminated with a
sulfhydryl group. In fact, during the self-assembly, sulfhydryl groups
may undergo deprotonation. In this case a negative charge is formed
in direct contact with the electrode surface, independent of whether
it may be partially or totally transferred to the electrode. The diffuse
layer ions will then experience both σM and this additional negative
charge. In this case, the total charge density σM at a potential E at
which the SAM is stable, measured by stepping the potential from
E to a potential Ef at which it is desorbed, is positive and relatively
high25 to maintain the applied potential E constant in spite of the
negative charge due to the SAM molecules. It is evident that it is
q, rather than σM, which generates the potential drop across the
SAM, once divided by the differential capacitance CSAM of the
monolayer. However, as opposed to σM, q can only be estimated
on the basis of extrathermodynamic assumptions.
and dividing this sum by A(θ):
q(θ) ) [Q(θ) + Aq]/A(θ)
(2)
3. Results
Differential Capacitance of the Peptide SAMs by ac
Voltammetry at 75 Hz. To verify the possible reductive
desorption of the Aib-peptide SAMs at sufficiently negative
potentials, their differential capacitance C was measured over
the potential range from -0.20 to -2.15 V in aqueous TMACl.
In fact, the double-layer region on bare mercury in this
electrolyte extends up to -2.15 V before the onset of hydrogen
evolution.26 Figure 1 shows the C vs E curves of 3+ and 3-
SAMs in 0.1 M TMACl at 75 Hz upon simulating the SAMs
by a resistance and a capacitance in parallel.
The differential capacitance curve of the 3+ SAM merges
with that of bare mercury at potentials negative of -1.60 V,
thus suggesting complete peptide desorption at these potentials.
However, no pseudocapacitance desorption peak is observed.
Conversely, the differential capacitance curve of the 3- SAM
is lower than that of bare mercury at all accessible potentials.
The differential capacitance curves of the 5+ and 5- SAMs
are very close to those of the 3+ and 3- SAMs, respectively.
The mode of desorption of 3+ and 5+ is thus different from
that of 3- and 5-, as will be argued in connection with
chronocoulometric measurements.
The inset of Figure 1 shows plots of the differential
capacitance curves of Hg-supported SAMs of 3- and 5- from
-0.60 to -1.0 V, and of 3+ and 5+ from -0.30 to -0.80 V,
in aqueous 0.1 M KCl. The curves were obtained after
stabilizing the SAMs by means of repeated potential scans over
the corresponding potential ranges. These potential ranges were
chosen because the SAMs were found to be stable in time and
reproducible therein, thus allowing accurate measurements of
the diffuse layer capacitance by electrochemical impedance
spectroscopy (EIS).
A convenient way to estimate q is based on the use of
Gouy-Chapman theory for the diffuse layer. According to this
approximate theory, the differential capacitance, Cd, of the diffuse
layer depends exclusively upon q and the bulk concentration, c, of
the electrolyte. Thus, Cd-1 values, calculated at different electrolyte
concentrations c and at constant q on the basis of the Gouy-Chapman
theory, can be plotted against the corresponding values calculated
Differential Capacitance of the Peptide SAMs by
Impedance Spectroscopy. The diffuse layer capacitance of the
four Hg-supported thiopeptide SAMs in contact with KCl
aqueous solutions of different concentrations was estimated from
their impedance spectra at constant applied potential E. The Aib-
peptide SAMs were found to be particularly stable in time at
potentials corresponding to the flat minimum of the correspond-
ing differential capacitance curves in the inset of Figure 1. Thus,
the 3+, 5+, and 5- SAMs were investigated at -0.75 V,
whereas the 3- SAM was investigated at -0.95 V. The
impedance spectra were fitted to an equivalent circuit consisting
of four RC meshes in series. These meshes simulate four
different dielectric slabs, namely, the sulfide ion bound to the
mercury surface, the peptide chain, the diffuse layer, and the
aqueous solution adjacent to the SAM. Typical values of the
resistance and capacitance of the 5+ and 5- peptide chains in
0.1 M KCl are 1 MΩ cm2 and 10 µF cm-2; those of the 3+
and 3- peptide chains are 0.9 MΩ cm2 and 13 µF cm-2. The
resistance and capacitance values ascribed to the sulfide ion
bound to mercury are on the order of 10 kΩ cm2 and 50 µF
for zero q, Cd(q ) 0)-1, yielding a series of calculated Cd vs
-1
Cd-(q1 ) 0)-1 curves at different q values.10 Plotting experimental
Cd values, measured at different electrolyte concentrations c,
against the Cd(q ) 0)-1 values calculated for the same electrolyte
concentrations and comparing the resulting plot with the series of
calculated Cd vs Cd(q ) 0)-1 plots allows the q value providing
-1
the best fit to be sorted out.
The expansion of a mercury drop coated with a SAM that does
not collapse is accompanied by the flow of a charge Q(θ) that
consists in an increment of q rather than of σM. In fact, the number
of SAM molecules anchored to the electrode is unchanged during
the expansion, and the same is true for any charge that they keep
in contact with the electrode surface. Hence, to maintain the applied
potential constant, the charge Q(θ) that reaches the metal surface
(25) (a) Widrig, C. A.; Chung, C.; Porter, M. D. J. Electroanal. Chem.
1991, 310, 335–359. (b) Schneider, T. W.; Buttry, D. A. J. Am. Chem.
Soc. 1993, 115, 12391–12397. (c) Yang, D. F.; Wilde, C. P.; Morin,
M. Langmuir 1996, 12, 6570–6577; 1997, 13, 243-248. (d) Kakiuchi,
T.; Usui, H.; Hobara, D.; Yamamoto, M. Langmuir 2002, 18, 5231–
5238. (e) Laredo, T.; Leitch, J.; Chen, M.; Burgess, I. J.; Dutcher,
J. R.; Lipkowski, J. Langmuir 2007, 23, 6205–6211.
cm-2
.
(26) Tadini Buoninsegni, F.; Becucci, L.; Moncelli, M. R.; Guidelli, R. J.
Electroanal. Chem. 2001, 500, 395–407.
9
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A R T I C L E S
Becucci et al.
Figure 3. Plots of ꢂ(θ) vs cos2 θ for Hg-supported SAMs of 3- (open
cirles), 3+ (solid circles), 5- (open squares), and 5+ (solid squares) in
aqueous 0.1 M KCl. The plots refer to -0.75 V for the 5+, 5-, and 3+
SAMs and to -0.95 V for the 3- SAM.
Figure 2. Dashed curves are 1/Cd vs 1/Cd(q ) 0) plots calculated from
the Gouy-Chapman theory for the q values reported on each curve. The
solid squares, open squares, solid circles, and open circles are experimental
values of 1/Cd against the corresponding 1/Cd(q ) 0) values for Hg-
supported SAMs of 5+, 5-, 3+, and 3-, respectively, in aqueous KCl of
concentration 0.1, 5 × 10-2, 3.2 × 10-2, 2 × 10-2, 1.3 × 10-2, 8 × 10-3
,
calculated from the Gouy-Chapman theory and makes only a
small contribution to ꢂ(θ).
5 × 10-3, 3.2 × 10-3, and 2 × 10-3 M (from left to right). Measurements
were carried out at -0.75 V for the 5+, 5-, and 3+ SAMs and at -0.95
V for the 3- SAM.
Figure 3 shows ꢂ(θ) vs cos2 θ plots for all Hg-supported
SAMs in 0.1 M KCl. They were obtained by ascribing to q the
values estimated from Figure 2 at the appropriate potentials and
by setting CSAM equal to 11 µF cm-2 for the 3+ and 3- SAMs
and to 8.5 µF cm-2 for the 5+ and 5- SAMs. The slopes of
the four plots are close. Upon considering the limits of the
experimental errors in the estimate of q and CSAM and their
propagation in the estimate of the ꢂ(θ) function, the surface
dipole potential ꢁ for all four SAMs can be estimated at +220
( 20 mV, positive toward the electrode.
The reciprocal of the diffuse layer capacitance Cd, estimated
at different KCl concentrations by the above procedure, is
plotted against the corresponding calculated Cd(q ) 0)-1 value
in Figure 2. Note that the increase in KCl concentration at
constant applied potential compresses the ionic atmosphere, thus
increasing the diffuse layer capacitance, Cd. The experimental
points in Figure 2 correspond to progressively increasing KCl
concentrations as one proceeds from right to left. Consequently,
Cd increases in the same direction and its reciprocal decreases,
in accordance with the Gouy-Chapman theory. The best
agreement between the four experimental Cd-1 vs Cd(q ) 0)-1
plots and the dashed curves calculated for different q values is
obtained with q(-0.75 V) ) -3 µF cm-2 for the 5+ and 5-
SAMs, with q(-0.75 V) ) -4 µF cm-2 for the 3+ SAM, and
with q(-0.95 V) ) -6 µF cm-2 for the 3- SAM. The more
negative q value for the latter SAM is simply due to the more
negative potential at which it was measured. The difference
between the q(-0.95 V) value for the 3- SAM and the q(-0.75
V) value for the 3+ SAM is compatible with the differential
capacitance CSAM of the peptide chains of the 3+ and 3- SAMs,
which was found to be equal to ∼11 µF cm-2 from ac
voltammetric measurements. Analogous measurements on the
5+ and 5- SAMs yield a CSAM value of ∼8.5 µF cm-2.
It should be noted that the slope of the ꢂ(θ) vs cos2 θ plots
measures the deviation from the behavior of a parallel plate
capacitor. If the SAM molecules point the positive pole of their
dipoles toward the electrode (i.e., if µ is positive), a drop
expansion decreases the positive potential difference created by
the dipole moments as a consequence of their tilt. Consequently,
during the drop expansion, a positive charge must flow to the
electrode surface along the external circuit to maintain the
potential difference across the whole interface constant. This
causes the ꢂ(θ) function to move in the positive direction with
increasing tilt (i.e., with decreasing cos2 θ), imparting a negative
slope to the ꢂ(θ) vs cos2 θ plot. The linear increase of ꢂ with
a decrease of cos2 θ excludes the possibility of a breakdown of
the peptide monolayer during the drop expansion.
It has been reported that the Au-S bond can make an
appreciable contribution to the surface dipole potential of
alkanethiol monolayers6 and helical peptides4,8b tethered to gold.
The present procedure, based on the gradual tilt of the tethered
thiol molecules, should not produce an appreciable tilt of the
Hg-S bond. Therefore, the measured surface dipole potential
should not include the contribution from this bond.
Chronocoulometric Charge Density Measurements on the
Hg-Supported SAMs. Chronocoulometric measurements of the
charge density as a function of the applied potential E were
carried out on all four Aib-peptide SAMs immersed in aqueous
0.1 M TMACl. To this end, a series of potential steps was
carried out from an initial potential Ei equal to that chosen for
q measurements (i.e., Ei ) -0.75 V for 3+, 5+, and 5- and
-0.95 V for 3-) toward both positive and negative final
potentials, Ef, and the resulting charge vs time curves were
recorded. Each potential step was preceded by a rest time of
30 s at Ei. The charge transients show an abrupt increase within
Estimate of the Surface Dipole Potential of the Peptide
SAMs. After determining the charge density q on the Hg-
supported Aib-peptide SAMs experienced by the diffuse layer
ions, the final step for the estimate of the surface dipole potential
of these SAMs consists in measuring the change of q with
increasing the drop size. The charge Q(θ) following the
progressive expansion of the SAM-coated mercury drop was
obtained by extruding mercury gradually from the capillary by
manual advancement of the piston of the mercury reservoir and
by recording the charge increment, ∆Q, flowing as a conse-
quence of each piston advancement. Adding the charge incre-
ment involved in each piston advancement to the sum of all
preceding charge increments yields the charge Q(θ). Inserting
Q(θ) in eq 2 to obtain q(θ), and then q(θ) in eq 1 to obtain
ꢂ(θ), allows the latter quantity to be plotted against cos2 θ. The
diffuse layer potential ψ[c,q(θ)] in the expression of ꢂ(θ) was
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Effect of a Strong Interfacial Electric Field
A R T I C L E S
Figure 6. Difference, ∆Q, between the pristine charge transient and the
immediately subsequent one on a Hg-supported 5+ SAM in aqueous 0.1
M TMACl. The charge transients were obtained by stepping from Ei )
-0.75 V to Ef ) -1.60 (open squares), -1.65 (solid squares), -1.70 (solid
circles), and -1.80 V (open circles). The solid curves were calculated from
the model described in the text for n ) 2, NF ) -2.5 µC cm-2 and for
different values of the kinetic parameter kRkN1/2. This parameter is plotted
against the final potential Ef in the inset.
Figure 4. Curves of the total charge density σM against Ef obtained from
serial charge transients on Hg-supported SAMs of 3- (open circles), 3+
(solid circles), 5- (open squares), and 5+ (solid squares) in aqueous 0.1
M TMACl. The solid circles with error bars are charge densities obtained
from pristine charge transients on a 3+ SAM. The dashed curve is the σM
vs Ef plot on bare mercury.
from the five solid circles with error bars at the bottom of the
figure). The charge vs time curve following each pristine step
exhibits an abrupt increase in charge, which attains a value
practically equal to that for the corresponding serial step. This
is followed by a sigmoidal charge vs time curve, as shown by
curve a in the inset of Figure 5, which refers to the 5+ SAM.
All subsequent steps after the pristine one yield a charge vs
time curve (see curve b in Figure 5) practically equal to that
obtained with the corresponding serial step. The height of this
sigmoidal curve is about equal to 20 µC cm-2 at all final
potential values e -1.60 V. This is shown in Figure 6, which
reports the difference, ∆Q, between the pristine and the second
charge transient on each newly formed drop for the 5+ SAM,
at different values of the final potential Ef.
As distinct from the 3+ and 5+ SAMs, the 3- and 5- SAMs
exhibit a pristine charge transient (see curve c in Figure 5) whose
initial abrupt rise is almost equal to that of the corresponding
serial charge transient. However, the background charge flowing
after this abrupt rise increases in time more rapidly than that
for the corresponding serial charge transient and, even more
so, for the charge transient on the 5+ SAM. The second and
subsequent charge transients after the pristine one (curve d in
Figure 5) are practically equal to the corresponding serial charge
transient.
Figure 5. Charge vs time curves obtained by stepping from Ei ) -0.750
to -1.80 V on newly formed Hg-supported SAMs of 5+ (curves a and b)
and of 5- (curves c and d), in aqueous 0.1 M TMACl; a and c were obtained
from pristine potential steps, b and d from the immediately subsequent steps.
The inset shows the initial portion of the four charge transients, to point
out the sigmoidal shape of curve a.
a few milliseconds, followed by a slow increase due to the
background current. The charge measured immediately after the
abrupt increase, once referred to the unit surface, yields σM(Ef)
- σM(Ei) ≡ ∆σM. Figure 4 shows the ∆σM vs Ef curves for the
four SAMs; the curves are shifted along the vertical axis to
provide a fairly accurate plot of the total charge density σM
against Ef, on the basis of the considerations made in the
subsequent Discussion section.
The peptides thiolated at the N-terminus (3+ and 5+) exhibit
an appreciable difference in the desorption behavior with respect
to those thiolated at the C-terminus (3- and 5-). This difference
between the 5+ and 5- SAMs is shown in Figure 5, but an
entirely analogous difference is encountered between the 3+
and 3- SAMs. This difference is observed when we carry out
the “pristine” potential step on a newly formed SAM-coated
mercury drop from Ei to final potentials Ef e -1.60 V, where
the differential capacitance of the 3+ and 5+ SAMs coincides
with that of bare mercury (see Figure 1). With the Aib-peptides
thiolated at the N-terminus, 3+ and 5+, the absolute value of
the negative charge involved in these pristine potential steps is
higher than that involved in the corresponding “serial” steps,
namely, the steps yielding the charges reported in Figure 4 (apart
Similar charge transients were recorded with Hg-supported
5+ SAMs in aqueous 0.1 M TMAOH + 0.1 M TMACl, where
these peptides are present in the anionic, deprotonated form.
The only difference consisted in steeper sigmoidal charge vs
time curves, under otherwise identical conditions.
4. Discussion
The present extrathermodynamic approach indicates that Hg-
supported SAMs of Aib-peptides thiolated at the C-terminus
have the same positive surface dipole potential as those thiolated
at the N-terminus, within the limits of experimental error. This
result is in apparent contrast with the fact that all free peptides
herein investigated have the dipole moment directed from the
C- to the N-terminus. This behavior can be explained if the
strong interfacial electric field created by the negative charge
density q, experienced by the diffuse layer ions and directed
toward the metal, causes an inversion of the dipole moment of
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A R T I C L E S
Becucci et al.
the peptide molecules thiolated at the C-terminus. Since the latter
molecules are tightly bound to the mercury surface via the Hg-S
bond, this reorientation requires the cleavage of the intramo-
lecular H bonds responsible for the 310-helix and the formation
of intermolecular H-bonds with a favorable orientation of the
CdO bond dipole moments. The negative enthalpic contribution
from the electrostatic interaction of the latter dipole moments
with the interfacial electric field, combined with the positive
entropic contribution from the randomized intermolecular H
bonds, should more than compensate for the positive enthalpic
contribution from the breaking of the more ordered intramo-
lecular H bonds. In other words, a reorientation of the overall
dipole moment of tethered peptide molecules thiolated at the
C-terminus should be associated with a net decrease in free
energy. The formation of intermolecular H bonds at the expense
of intramolecular ones is expected to be much less pronounced
with tethered peptides thiolated at the N-terminus, in view of
the favorable orientation of their “natural” dipole moment with
respect to the interfacial electric field.
In addition to the interfacial electric field, Eief, the “endog-
enous” electric field created by the natural surface dipole
potential of the 3- and 5- SAMs may also cooperate in
reversing the dipole moment of these peptides. By natural
surface dipole potential we mean the dipole potential that the
3- and 5- adsorbed molecules would create if they maintained
the dipole moment of the isolated molecules. As a rough
approximation, this surface dipole potential can be regarded as
equal to and opposite that estimated for the 3+ and 5+ SAMs,
i.e., ꢁ ) -0.220 mV. The endogeneous electric field, Eend, is
given by ꢁ/d, where d is the thickness of the monolayer. Noting
that in 310-helices each amino acid unit determines a structure
elongation of ∼2 Å, and using molecular models of such a
helical structure, d can be estimated at ∼14 Å for the 3+ and
3- peptides and at ∼18 Å for the 5+ and 5- peptides. Taking
3- as an example, Eend is equal to 1.57 × 106 V/cm and is
directed toward the metal. This electric field is to be compared
of the differential capacitance of the 5+ and 5- SAMs to that
of the 3+ and 3- SAMs being about equal to 8.5/11 = 3/4:
for an equal value of the dielectric constant for all SAMs, such
a ratio should be equal to 3/5.
When long-chain thiols undergo deprotonation upon chemi-
sorption on noble metals, they generate a negative charge in
contact with the electrode surface. In such a case, they may
transfer their negative charge to the electrode. However, the
long chain of thiols in SAMs has a dual effect: (i) It interposes
a thick slab of high resistance and low capacitance between the
sulfhydryl functional group and the bulk aqueous phase. This
makes any charge movement from the functional group to the
metal surface hardly appreciable with respect to the bulk phase.
(ii) It decreases the screening of the negative charge of the
functional groups by diffuse layer ions down to a negligible
extent.28 Consequently, this negative charge must be compen-
sated for by an almost equal and opposite charge on the electrode
surface to maintain the electroneutrality of the electrified
interface, independent of whether partial or total charge transfer
from the functional group to the electrode takes place or not.10,29
This implies that a high positive charge density σM on the
electrode surface following the self-assembly of a thiol or
disulfide monolayer does not allow us to establish whether total,
partial, or no charge transfer takes place.
The fact that the pristine charge transients for 3+ and 5+
SAMs are higher than the corresponding serial charge transients
can be explained by assuming that, on a newly formed SAM,
the sulfhydryl groups are totally deprotonated at the initial
potential Ei, independent of the extent of partial charge transfer.
During the series of potential steps yielding the charge vs
potential curves in Figure 4, the Hg-S bonds are progressively
broken, with protonation of the resulting sulfide groups, as the
final potential Ef is made sufficiently negative. For these negative
Ef values, the subsequent backward Ef f Ei potential step causes
the readsorption of the protonated peptides; consequently, the
charge density σM(Ei) becomes progressively less positive, to
maintain the electroneutrality of the interface. Ultimately, for
sufficiently negative Ef values, all the initial negative charge
associated with the SAM of deprotonated thiopeptides is lost,
and the negative charge density involved in the forward Ei f
Ef potential step is decreased by the same amount. This explains
why the pristine charge transients following all potential steps
from Ei to Ef values e -1.60 V are more negative than the
corresponding serial charge transients by about the same amount
of -20 µC cm-2. In aqueous 0.1 M TMAOH + 0.1 M TMACl,
where the thiolated Aib-peptides are expected to be deproto-
nated, the role of protons after Hg-S cleavage can also be
played by K+ or Na+ cations, which may reach the electrode
surface through the partially disorganized SAM.
with the interfacial electric field, which is equal to Eief
=
q/(∇∇0). Noting that the differential capacitance of a SAM is
given by CSAM = ∇∇0/d, we also have Eief = q/(dCSAM). For
the 3- SAM at -0.95 V, q equals -6 µC cm-2 and CSAM equals
11 µF cm-2. Hence, Eief is approximately equal to 3.9 × 106
V/cm. Consequently, the interfacial electric field at a negatively
charged electrode and the endogeneous electric field are
comparable in magnitude and are both directed toward the metal.
According to Naaman and co-workers,5 an endogenous electric
field directed toward the metal induces ET from the metal to
the adsorbed molecule, thus decreasing its effective dipole
moment. However, Naaman’s work function measurements were
carried out in vacuo, on SAMs of molecules of low polarizability
and with no intramolecular hydrogen bonds.27 Under these
conditions, the energetically favored route in response to the
endogenous electric field is a charge transfer from the metal.
In our case, the 3- and 5- SAMs respond to the combined
action of the interfacial and the endogenous electric fields
through a reorganization of hydrogen bonds, leading to a reversal
of the dipole moments.
By using the unit cell size of pBrBz-(Aib)10-OtBu or
Z-(Aib)11-OtBu estimated by X-ray analysis,30 the diameter of
helical Aib-peptides can be set equal to 9.7 Å. By assuming
that the peptide helixes are impenetrable cylinders forming a
hexagonal compact lattice, with the helix axis normal to the
electrode surface, the cross sectional area of a helix is estimated
at 31/2d2/2 ) 81.5 Å2. If we ascribe a single electronic charge
The fact that the surface dipole potentials of the 5+ and 5-
SAMs are close to those of the 3+ and 3- SAMs suggests that
the longer peptides are more tilted than the shorter ones on an
unexpanded mercury drop. This is also confirmed by the ratio
(28) Becucci, L.; Guidelli, R. Soft Matter 2009, 5, 2294–2301.
(29) Kunze, J.; Leitch, J.; Schwan, A. L.; Faragher, R. J.; Naumann, R.;
Schiller, S.; Knoll, W.; Dutcher, J. R.; Lipkowski, J. Langmuir 2006,
22, 5509–5519.
(30) (a) Toniolo, C.; Crisma, M.; Bonora, G. M.; Benedetti, E.; Di Blasio,
B.; Pavone, V.; Pedone, C.; Santini, A. Biopolymers 1991, 31, 129–
138. (b) Gessmann, R.; Bruckner, H.; Petratos, K. J. Pept. Sci. 2003,
9, 753–762.
(27) Cahen, D.; Naaman, R.; Vager, Z. AdV. Funct. Mater. 2005, 15, 1571–
1578.
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Effect of a Strong Interfacial Electric Field
A R T I C L E S
to each helix, a charge density of 23.4 µC cm-2 is estimated
for desorption of a tightly packed Aib-peptide SAM. This
supports the view that the excess charge of pristine charge
transients over the corresponding serial ones is due to desorption
of a deprotonated compact SAM of 3+ or 5+ peptide. The fact
that the same excess charge is observed with the 3+ and 5+
SAMs indicates that the two SAMs have practically the same
surface coverage, even though the closeness of their differential
capacities and surface dipole potentials suggests that the
molecules of the 5+ SAM are more tilted than those of the 3+
SAM.
The sigmoidal ∆Q vs time curves in Figure 6 are reminiscent
of a nucleation and growth process. We can envisage such a
process as follows. Since the detachment of the thiolated peptide
molecules from the electrode surface requires Hg-S bond
cleavage and sulfide group reprotonation, we will briefly refer
to these molecules as “bound” or “unbound”, depending on
whether they maintain the Hg-S bond or not. The breaking of
the Hg-S bond of a single thiolated peptide may not be
sufficient to allow its detachment from the electrode surface,
since it is surrounded by molecules H-bonded to it. Clusters of
contiguous unbound thiopeptide molecules can be regarded as
characterized by a critical size, called “nucleus”. Below this
critical size, the molecules will have a higher tendency to form
again Hg-S bonds than to induce other neighboring bound
molecules to break their Hg-S bonds with a resulting increase
in cluster size. Conversely, above this critical size, the unbound
clusters will have a practically irreversible tendency to grow.
This nucleation and growth process can be treated using a
model developed in ref 31 and based on the Avrami formalism.
Let us denote by Φ the fraction of the unit surface area of the
SAM covered by bound thiolated peptides and by Φ0 its initial
value, before the start of nucleation and growth; clearly, Φ0 is
equal to unity. According to the kinetic model of nucleation
and growth of ref 31, the nucleation rate can be written
product is the same. Therefore, this model may account for the
formation both of small dispersed clusters, when the rate
constant of nucleation is higher than that of radial growth, and
of larger aggregates in the opposite case. Figure 6 shows the
experimental ∆Q(t,E) vs t plots at different potentials together
with the corresponding curves calculated for NF ) -2.5 µC
cm-2, n ) 2, and for different values of kRkN1/2. The inset of
the figure shows the linear dependence of the overall rate
1/2
constant kRkN upon the applied potential Ef. Such a linear
dependence is to be related to the electrostatic interaction
between the interfacial electric field and the water dipoles, which
gradually displace the SAM molecules from the direct contact
with the mercury surface. When the water dipoles are fully
aligned with their positive end toward the electrode, this
electrostatic interaction is proportional to the interfacial electric
field, which varies linearly with E.
The above interpretation of the nucleation and growth process
may provide an explanation for the lack of desorption of the
3- and 5- SAMs, even at the most negative experimentally
accessible potentials. The reversal in the natural dipole moment
of these thiolated peptides, as imposed by the interfacial electric
field, causes these molecules to be much more intermolecularly
H-bonded than the 3+ and 5+ molecules. This makes their
complete detachment from the electrode surface much more
difficult. Moreover, the intercalation of water molecules among
the 3- and 5- molecules at far negative potentials, and their
close vicinity to the sulfide groups, may catalyze a modest
hydrogen evolution; this explains the higher slope of the charge
transients of the 5- SAM (curves c and d in Figure 5) with
respect to those of the 5+ SAM (curves a and b in Figure 5),
after the initial abrupt flow of capacitive charge.
In Figure 4, all the ∆σM vs Ef plots obtained from the serial
potential steps are shifted along the vertical axis by an amount
equal to (20 + q) µC cm-2, where q is the negative charge
experienced by the diffuse layer ions at the initial potential Ei,
at which ∆σM is equal to zero. The rationale behind this shift is
as follows. We assume that the charge density associated with
the SAMs of all four deprotonated thiolated peptides, including
the 3- and 5- SAMs, is equal to -20 µC cm-2. Independent
of whether this charge is totally, partially, or not transferred to
the electrode at Ei, a potential step from Ei to a final potential
Ef negative enough to cause complete thiolated peptide desorp-
tion would release this charge. Incidentally, such a potential
step is actually realized for the 3+ and 5+ SAMs, whereas it
is not realizable for the 3- and 5- SAMs. Since the diffuse
layer ions experience a charge density q at Ei, the charge density
involved in such an Ei f Ef potential step is equal to σM(Ef) -
(20 + q) µF cm-2, where σM(Ef) is the total charge density at
Ef on a bare mercury electrode. Therefore, the vertical shift of
the ∆σM vs Ef plots in Figure 3 by (20 + q) µC cm-2 serves to
position them with respect to the vertical axis of the total charge
density σM.
dN/dt ) kNΦn
(3)
Here, N is the number of nuclei per unit surface area, kN is the
nucleation rate constant, and n is the number of the unbound
peptide molecules composing a critical nucleus. Let us assume
for simplicity that the growing supercritical clusters have a
circular shape, of radius r. Moreover, let the rate of radial growth
of these clusters be proportional to the probability, 2πrΦ, of
bound molecules being adjacent to the perimeter of the unbound
cluster. With these assumptions, the growth rate is given by
d
dt
dr
dt
dr
dt
(πr2) ) 2πr ) kR2πrΦ f
) kRΦ
(4)
where kR is the rate constant of radial growth. Starting from
eqs 3 and 4, the kinetic model involves the numerical solution
of five differential equations. Fitting the calculated ∆Q(t) vs t
curves to the corresponding experimental curves (see Figure 6)
at different applied potentials Ef requires two free parameters,
apart from a potential-independent normalizing factor, NF. They
are the number n of unbound peptide molecules composing a
nucleus and the potential-dependent product kRkN1/2, which
summarizes the kinetic features of the whole nucleation and
growth process. Thus, identical results are obtained with kR much
The closeness of the estimated surface dipole potentials of
the present Hg-supported Aib-peptide SAMs thiolated at the
C- and N-termini is in apparent contrast with opposite effects
that Au-supported peptide SAMs thiolated at the opposite
termini exert upon a number of electrochemically8,9 and
photoelectrochemically3c,d induced electron-transfer processes.
However, the endogeneous electric field created on peptides by
a positively charged N-terminus and a negatively charged
C-terminus has often been found to favor peptide conformations
with the dipole directed toward the C-terminus and to destabilize
conformations with the dipole directed toward the N-terminus.
1/2
lower, equal, or much higher than kN, provided that the kRkN
(31) Becucci, L.; Moncelli, M. R.; Guidelli, R. J. Am. Chem. Soc. 2003,
125, 3785–3792.
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The opposite effects that Au-supported SAMs of peptides
thiolated at the C- and N-terminus, respectively, have sometimes
shown to exert on electrochemical and photoelectrochemical
processes cannot be directly compared with the present results
on Hg-supported peptide SAMs. In fact, the direction and
magnitude of the interfacial electric field on Au-supported SAMs
is usually not known. In addition, an intrinsic difference between
Hg-supported and Au-supported peptide SAMs should be
stressed. The liquid nature of mercury imparts a high degree of
lateral mobility and axial rotation to the tethered peptide
molecules during film stabilization. The formation of an Aib
peptide SAM on mercury requires about 90 min of immersion
in its ethanol solution. Conversely, we found that the formation
of SAMs of the same thiolated peptides on gold requires at least
48 h.18 In other words, the fluid nature of mercury is envisaged
as important for allowing conformational modifications of these
peptides upon electrode polarization.
Figure 7. Cyclic voltammogram of 1 × 10-4 M Eu(III) in 0.1 M KCl on
bare mercury (solid curve) and on mercury coated with a 5+ (dashed curve)
and a 5- SAM (dash-point curve). Scan rate: 50 mV/s.
5. Conclusions
This trend has been observed both with oligoprolines,32 where
no hydrogen bonds are involved, and with certain R- and
ꢀ-peptides.32-34
By taking advantage of the liquid nature of mercury, it was
possible to estimate the surface dipole potential of self-
assembled monolayers of four oligopeptides consisting of a
sequence of R-aminoisobutyric acid units, thiolated at either the
N- or C-terminus by means of a -(CH2)2-SH anchor. To this
end, a novel procedure combining mercury drop expansion and
diffuse layer capacitance measurements was adopted. While the
above peptides adopt a robust 310-helix conformation both in
solution and in the solid state, our results indicate that the
situation may be quite different when they are tightly assembled
onto a mercury surface: when the interfacial electric field is
sufficiently strong and the direction of the field contrasts the
natural molecular dipole moment associated with the 310-helix,
the peptides may be forced to change the direction of the dipole,
which implies a very substantial conformational change. The
endogeneous electric field created by Aib-peptides thiolated at
the C-terminus and tethered to mercury with their natural dipole
moment may also cooperate with the interfacial electric field
in inverting the direction of the surface dipole potential.
The strength of this study rests in providing experimental
evidence that significantly enhances our understanding of how
molecules possessing a strong dipole moment may react toward
imposition of an electric field while anchored to a metal surface;
this is particularly relevant in areas such as sensors, molecular
electronics, and nanosystems. Intriguing questions are also raised
about possible peptide conformational changes under severe
conditions, which could be important to better understand the
mechanism of long-range electron transfers occurring in biologi-
cal systems, such as redox proteins on electrodes.
To verify the effect of the present Hg-supported SAMs upon
a simple redox process taking place over the potential range of
maximum stability of the SAMs, the cyclic voltammograms of
Eu(III) in aqueous 0.1 M KCl were recorded on bare mercury
as well as on mercury coated with 5+ and 5- SAMs. The cyclic
voltammogram of the Eu(III)/Eu(II) redox couple in 0.1 M KCl
is quasi-reversible, with a formal potential of -0.657 V. Its
peak potential varies linearly with the square root of the potential
scan rate, denoting mixed control by diffusion and ET. Figure
7 shows that coating the mercury electrode with the Aib-peptide
SAMs does not depress the voltammetric peak currents. Since
the film is compact, as we already commented upon, this
outcome denotes high electron-conduction properties of these
peptides and is in keeping with our previous results in solution,
obtained with donor-peptide-acceptor systems, showing that
Aib chains mediate electron tunneling very efficiently.13 The
5+ and 5- SAMs exert a similar effect on the voltammetric
behavior of the Eu(III)/Eu(II) redox couple. Both coatings make
the oxidation peak flatter and more rounded; moreover, they
cause a negative shift of the reduction peak potential by 35-37
mV and of the midpoint potential by 8-12 mV. These effects
are modest. However, under the reasonable assumption that they
are due to the surface dipole potentials of the 5+ and 5- SAMs,
they confirm that these dipole potentials act in the same
direction. Similar effects on the Eu(III)/Eu(II) couple are exerted
by the 3+ and 3- SAMs on mercury (data not shown).
Acknowledgment. The financial support by Ente Cassa di
Risparmio di Firenze and that by the Italian Ministero dell’Istruzione,
dell’Universita` e della Ricerca (MIUR) through PRIN 20079Y9578,
are gratefully acknowledged.
(32) Kuemin, M.; Schweizer, S.; Ochsenfeld, C.; Wennemers, H. J. Am.
Chem. Soc. 2009, 131, 15474–15482, and references therein.
(33) Fairman, R.; Shoemaker, K. R.; York, E. J.; Stewart, J. M.; Baldwin,
R. L. Proteins 1989, 5, 1–7.
(34) Hart, S. A.; Bahadoor, A. B. F.; Matthews, E. E.; Qiu, X. J.; Schepartz,
A. J. Am. Chem. Soc. 2003, 125, 4022–4023.
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