A bioorganometallic approach for the electrochemical detection of proteins: A study on the interaction of ferrocene-peptide conjugates with papain in solution and on Au surfaces

Article abstract of DOI:10.1002/chem.200601878

In this paper, a new bioorganometallic approach for the detection of proteins using surface-bound ferrocene-peptide conjugates is presented. Specifically, a series of peptide conjugates of l′-aminoferrocene-l- carboxylic acid (ferrocene amino acid, Fca) i

Full text of DOI:10.1002/chem.200601878

FULL PAPER
DOI: 10.1002/chem.200601878
A Bioorganometallic Approach for the Electrochemical Detection of
Proteins: A Study on the Interaction of Ferrocene–Peptide Conjugates
with Papain in Solution and on Au Surfaces
Khaled A. Mahmoud and Heinz-Bernhard Kraatz*[a]
Abstract: In this paper, a new bioor-
ganometallic approach for the detec-
tion of proteins using surface-bound
ferrocene–peptide conjugates is pre-
sented. Specifically, a series of peptide
conjugates of 1’-aminoferrocene-1-car-
boxylic acid (ferrocene amino acid,
Fca) is synthesized: Boc-Fca-Gly-Gly-
thiostic acid linked to the N-terminal
side of Fca in order to facilitate forma-
tion of thin films on gold substrates.
Competitive inhibition towards papain
was determined for Thc-Fca-Gly-Gly-
gest binding affinity to papain. Adsorp-
tion/desorption rate constants were
ka =1.75ꢀ0.05”105 mꢁ1 sꢁ1 and kd =
2.90ꢀ0.05”10ꢁ2 sꢁ1. Interactions of
papain with Fca–peptide 10 were inves-
tigated by cyclic voltammetry. The in-
teraction results were also verified by
measuring the electrochemical re-
sponse of the peptide–papain interac-
tion as function of increasing enzyme
Tyr
Gly-Gly-Arg
Thc-Fca-Gly-Gly-Arg-Tyr-OH
A
N
AHCTREUNG
Tyr
Gly-Gly-Tyr
Thc-Fca-Gly-Gly-Tyr
OH (4), Boc-Fca-Gly-Gly-Arg
Tyr-OMe (7), Thc-Fca-Gly-Gly-Arg-
(Mtr)-Tyr-OMe (8), Thc-Fca-Gly-Gly-
Arg(Mtr)-Tyr-OH (9), Thc-Fca-Gly-
A
N
The binding interaction between the
peptide modified substrates and papain
enzyme was studied using real-time
surface plasmon resonance (SPR)
imaging. Peptide 10 showed the stron-
A
ACHTREUNG
A
N
concentration. A linear relationship
E
was observed for papain concentration
of up to 80 nm. Shifting to higher po-
tentials as well as decrease in the over-
all signal intensity was observed. The
detection limit was 4”10ꢁ9 m.
ACHTREUNG
ACHTREUNG
Keywords:
electrochemistry
ferrocene · peptides
biosensor
enzymes
·
·
Gly-Arg-Tyr-OH (10). The peptide is
conjugated to the C-terminal side of
Fca and compounds 4, 7–10 possess a
·
Introduction
The detection of biomolecular interactions by electro-
chemical methods requires in general the presence of a
redox-active probe as part of the detection system
(Scheme 1). The redox probe can be in solution or can be
covalently attached to the molecule capturing the biomole-
cule from solution. In both cases, the interaction of the sur-
face with the biomolecule will alter the electron-transfer
properties of the systems. For solution-based redox probes,
significant differences exist in the electron-transfer proper-
ties or ability of the redox probe and/or counter ions to dif-
fuse into the film (Scheme 1a). These differences can be ex-
ploited for the detection of protein binding to a range of
capture probes. In essence, this approach is “label-free”,
since no chemical modifications of the capture probe or of
the target protein are necessary.[9,10] An interesting variation
on the theme of a solution-based electroactive probe is the
use of a redox-active polymer. Using a ferrocene (Fc)-la-
beled polythiophene which electrostatically binds to duplex
DNA or duplex DNA–PNA), it was possible to detect
thrombin.[10–13] More recently, electrochemical immunoas-
Enzymatic transformations of substrates are often exploited
for the development of amperometric sensors. The electro-
chemical sensors have received a major attention in the bio-
sensing technology, in particular the use of glucose oxidase
for the development of an electrochemical glucose sensitive
detector.[1–7] The resulting devices exploiting this technology
are simple to use, inexpensive and accurate. More impor-
tantly, they are amenable to miniaturization and hence the
design of high-density arrays.[8]
[a] K. A. Mahmoud, Prof. H.-B. Kraatz
Department of Chemistry, University Of Saskatchewan
110 Science Place, Saskatoon, SK S7N5C9 (Canada)
Fax : (+1)306-966-4730
E-mail: kraatz@skyway.usask.ca
Supporting information for this article is available on the WWW
under http://www.chemeurj.org/ or from the author.
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says (EIA) were developed in which an analyte binds to an
immobilized antibody, followed by binding of a redox medi-
ator bound to an antibody which binds to the electrode-
bound analyte (Scheme 1b).[14,15] In cases where the redox
detection where the interaction does not involve the site of
the redox probe,[17] it will have limited utility for monitoring
peptide–protein interactions. In this case, the presence of a
terminal redox probe may significantly interfere with the in-
teraction and may even prevent it.
We are proposing a new approach making use of our ex-
pertise in bioorganometallic chemistry to join a peptide rec-
ognition sequence to a redox probe. The idea is that a sur-
face-bound redox probe is in close proximity to the elec-
trode surface and thus unaffected by diffusive processes. In-
teraction between the recognition sequence and electrode
surface will not be hindered by the interaction with the
target analyte. This in turn may enhance the sensitivity and
may render a potential device less susceptible to environ-
mental influences.
Fc–peptide conjugates are an attractive class of organo-
metallic peptide conjugates that are conveniently obtained
by solution or solid-phase synthetic strategies,[18] that have
the potential to be tailored to target DNA and specific pro-
teins.[19–22] In a recent study, it was demonstrated that 1’-ami-
noferrocene-1-carboxylic acid (ferrocene amino acid, Fca)
induces a turn into a peptide sequence and thereby allows
control over the peptide secondary structure.[23] Recently,
´
Rapic et al. and Heinze et al. independently used solid-
phase peptide synthesis (SPPS) to obtain Fca-oligopeptides
by combining Fmoc and Boc procedures.[24,25]
In this contribution, we outline how Fca–peptide conju-
gates can be used in protein detection and demonstrate our
approach for the detection of papain, which is a cysteine
protease commonly found in the papaya fruit and used com-
mercially as a meat tenderizer. In our approach (see Fig-
ure 1d), a thin film on gold prepared from a conjugate of
Fca with an inhibitory peptide sequence to papain is used
for the detection of papain. We are reporting the results of a
combined synthetic, spectroscopic and electrochemical study
into the properties of these films and their behavior in the
presence of papain.
Scheme 1. Schematic representation of some of the common electro-
chemical biosensor systems (a–c) and the new proposed system making
use of an ferrocene–peptide conjugate (d): a) Redox active probe in solu-
tion and the target protein blocks its interaction with the surface. b) In
the electrochemical immunoassay (EIA) a protein binds to a surface-
bound capture probe followed by binding labeled antibody. c) Conforma-
tional changes take place upon binding of a target DNA to a single
stranded capture DNA. Changes in the distance between the redox label
and the surface alter the electrochemical behavior of the system. d) Bio-
organometallic capture probe: protein binding is detected by monitoring
the electrochemical properties before and after protein binding.
Results and Discussion
Synthesis: In an early report, it was shown that the organo-
metallic peptide conjugate Fc-Gly-Gly-Tyr-Arg interacts
with papain and acts as a competitive inhibitor.[22] Based on
this result, we decided to work with two peptide systems,
making use of conjugation of Fca while exploiting the
papain-binding sequence Gly-Gly-Tyr-Arg and Gly-Gly-
Arg-Tyr. Conjugation of the tetrapeptide to the C-terminal
side of Fca will allow the interaction with the protein, while
the N-terminal side will be exploited to surface binding of
the Fca conjugate. The synthesis of Fca–peptide conjugate 4
was achieved by carbodiimide coupling in solution and is
summarized in Scheme 2. Compound 4 displayed a broad
band in the IR spectrum assigned to the OH stretch and the
expected carbonyl stretch for the acid group at 1741 cmꢁ1.
Unfortunately, it was not possible to deprotect the guanidine
group of peptide conjugate 4 by hydrogenation, presumably
probe is covalently attached to a terminal position of a mol-
ecule such as single stranded DNA, the interaction with the
appropriate target will result in large structural changes of
the capture probe causing changes in the electron transfer
kinetics of the system. Heegerꢁs DNA-hairpin loop is an ex-
ample for this approach (Scheme 1c).[16] In the absence of a
suitable target, an Fc probe attached to the 5’-position in a
single-stranded DNA is in close proximity to the electrode
surface and gives rise to a significant redox response. Upon
hybridization of a target strand to the (Fc)-labeled capture
strand, significant structural changes occur which change the
distance between the Fc group and the surface, resulting in
a decrease of the electrochemical response. Although this
approach is useful in cases of DNA–DNA or DNA–protein
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Electrochemical Detection of Proteins
FULL PAPER
Mtr group by TFA/anisole re-
sulted in the formation of com-
pound 9 and 10, respectively.
Final purification of compound
10 was carried out by HPLC.
The identity of compounds 9
and 10 was confirmed by full
spectroscopic analysis includ-
ing ToF-MS, IR and 1H NMR
spectroscopy. In the IR com-
pounds
amide-A bands at 3306 and
3447cm ꢁ1
respectively and
carbonyl stretch at 1748 and
9
and 10 showed
,
1725 cmꢁ1,
respectively.
Figure 1 shows the 1H NMR
spectra in [D6]DMSO for the
main three peptides 4, 9, and
Figure 1. 1H NMR of peptides 4, 9, and 10. * [D5]DMSO trace at d=2.50 ppm, water trace at d=3.33 ppm.
a result of Pd catalyst poisoning caused by the thiostic acid
(Thc) disulfide group. In order to overcome this problem,
we decided to implement a small positional change in the
sequence involving the Tyr and Arg, which facilitated the
synthesis and did not require protection of the Tyr. The Arg
guanidine group was protected by Mtr, which could be de-
protected with TFA and thioanisole. The synthesis of Fca–
peptide conjugates 9 and 10 is summarized in Scheme 3.
The peptide building block 6 was synthesized from Boc-
10 under investigation. In all three peptides, the amide as
well as guanidine protons were observed in the region d=
8.6–7.3 ppm. The peaks assigned for Mtr protecting group in
peptide 9 disappeared in peptide 10 indicating the successful
deprotection.
Inhibition studies: Before linking the Fca–peptide conju-
gates onto a gold surface, it was important to evaluate the
Fca conjugates for their ability to inhibit papain in solution.
For this purpose, papain was assayed with the chromogenic
substrate Z-Phe-Arg-NHNp in the absence and presence of
the Fca–peptide conjugates 4, 9 and 10. The Michaelis–
Menten constant (Km) for papain as well as the inhibition
constant Ki for the peptides were determined. The Km value
was determined by monitoring the total papain-catalyzed
Gly2-OH and the corresponding dipeptide H-Arg(Mtr)-Tyr-
A
OMe (5). Boc deprotection and conjugation to the carboxyl-
ic acid group of Fca resulted in the desired conjugate 7.
After Boc deprotection of the amino group of Fca under
anaerobic conditions, Thc-conjugate 8 was formed. Stepwise
deprotection of the methyl ester by hydrolysis and of the
Scheme 2. Synthesis of Fca–peptide conjugate 4. i) TFA/CH2Cl2; Boc-Fca-OBt; Et3N, CH2Cl2; ii) TFA/CH2Cl2; ThcOH, EDCI/HOBt; Et3N, CH2Cl2;
iii) NaOH/CH3OH/H2O. EDCI·HCl = 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride.
Chem. Eur. J. 2007, 13, 5885 – 5895
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H.-B. Kraatz and K. A. Mahmoud
Scheme 3. Synthesis of Fca–peptide conjugates 7–10. i) TFA/CH2Cl2; EDC/HOBt; Et3N/CH2Cl2; ii) TFA/CH2Cl2; Boc-Fca-OBt; Et3N/CH2Cl2; iii) TFA/
CH2Cl2; ThcOH/EDC/HOBt; Et3N/CH2Cl2; iv) NaOH/CH3OH; v) TFA/thioanisole/CH2Cl2.
Table 1. Inhibition constant Ki of the Fc–peptide conjugates 4, 9, and 10
and some literature compounds.
hydrolysis of Z-Phe-Arg-NHNp in the absence of the inhibi-
tors and at high initial concentration of the substrate. A Km
value of 980ꢀ26 mm was determined at pH 6.2 by nonlinear
regression fit of the concentration velocity curve using Mi-
Compound
pH
Ki’ [mm]
Ki [mm]
Ref.
4
9
10
6.2
6.2
6.2
6.0
6.0
6.2
2510.6ꢀ8.8
639.1ꢀ1.1
21.1ꢀ0.3
820.9ꢀ2.5
210ꢀ1.4
6.9ꢀ0.2
150
123
9
this work
this work
chaelis–Menten equation (#=#max [S]/(Km+[S]), where # is
G
this work
the velocity and #max is the maximum velocity, and [S] is the
substrate concentration (Supporting Information, Figure
S2). The value compared well with the previously reported
one of 965ꢀ244 mm.[26] A representative kinetic analysis of
papain inhibition by the two Fca–peptide conjugates 9 and
10 is shown in Figure S3 of the Supporting Information. The
Dixon–Webb plots indicate a time-independent inhibition.
The slopes of the progress curves were directly used to de-
termine Ki’ value, from which Ki value was calculated to be
210ꢀ1.4 mm for compound 9 and 6.9ꢀ0.2 mm for compound
10. Our results suggest that these Fca–peptide conjugates
exhibit competitive inhibition behavior.[26] The inhibition
constants for 10 indicates very good competitive inhibition
of papain, comparable to that of the Fc-conjugate Fc-Gly-
[27]
Gly2TyrArg
Gly2Tyr(Bz)Arg
Fc-Gly2TyrArg
[28]
[22]
interesting to note that switching the peptide sequence from
the original sequence employed by Kaiser for purification of
papain by affinity chromatography Gly-Gly-Tyr-Arg[28] to
Gly-Gly-Arg-Tyr, the peptide became a more potent inhibi-
tor. Blumberg[27] and Kaiser[28] used Gly-Gly-Tyr
(Bzl)-Arg
A
for the purification of papain by affinity chromatography. It
was shown that papain inhibition can be achieved by pep-
tides that have Arg and an aromatic amino acid in the P1
and P2 subsites (nomenclature according to Schechter and
Berger,[32] see Supporting Information Figure S4). It was
found that the peptide sequence Z-Arg-Leu-Val-Gly-DAM
gave the most potent inhibitor of papain with apparent
second order rate constants of the same order of magnitude
as those determined for {1-[N-[(l-3-trans-carboxyoxirane-2-
carbonyl)-l-leucyl]amino]-4-guanidinobutane} E-64, the
Gly-Try-Arg-OH, which has
a Ki =9 mm at pH 6.2
(Table 1).[27]
On the other hand, lower affinities were observed for the
Fca–peptide conjugates 4 (Ki =820.9ꢀ2.5 mm) and 9 (Ki =
210ꢀ1.4 mm), which is rationalized by the presence of pro-
tecting groups which may not allow a proper interaction
with Cys25 and His159 in the active site of papain.[29–31] It is
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Electrochemical Detection of Proteins
FULL PAPER
most common inhibitor of cysteine proteases.[33] Moreover, a
strong electron-withdrawing phenyl groups at the C-termi-
nus constructs a good irreversible inhibitor for papain.[34]
Crystal structures of related papain–inhibitor complexes
showed that the inhibitor extends along the Sn (n=1ꢂ2)
subsites of the enzyme and is stabilized in the active-site
groove by a series of hydrogen bonds and hydrophopic in-
teractions which may have higher priority than the P–S in-
teractions.[34–36] Based on X-ray structures showing the inter-
action of inhibitor molecules with papain, it is proposed that
our inhibitor interacts with His158, Gly66, Asp158 and
Gln19 (Figure 2).[34,36,37] Despite the recent results obtained
the evaluation of the interaction by electrochemical meth-
ods. The idea was to exploit the Thc disulfide of conjugates
4, 9, and 10 for binding to a gold surface. The resulting Fca–
peptide film will have the Fca groups firmly embedded in
the film and the inhibitory peptide exposed allowing papain
to bind. SPR imaging was selected as a method of choice be-
cause of its powerful ability to study the bioaffinity interac-
tions thin films on gold.[39–42] The quantitative measurements
of papain interactions with the Fca–peptide modified surface
by SPR imaging allowed to measure the change in the per-
centage reflectivity (D% R) which was directly proportional
to the fractional surface coverage q of the peptide-modified
surface by papain, providing D% R is below 10%.[43] Using
this method, the adsorption and desorption kinetics of
papain were determined. Peptides 4, 9 and 10 were immobi-
lized on spot ready SPR microchips by spotting ethanolic
solutions of the conjugates onto the four gold pads each per
chip and then monitored the selective binding to papain by
real-time SPR imaging, as described in the Experimental
Section (Figure 3). This was achieved by monitoring changes
in percentage reflectivity as a function of papain concentra-
tion (up to 200 nm) under continuous flow. Conjugate 10 ex-
hibited a significantly higher change in D% R compared
with the other two conjugates 4 and 9. This was in agree-
Figure 2. Proposed structure of peptide 10 interacting with the binding
site of papain. The initial enzyme–inhibitor structure was modeled in the
VEGA ZZ 2.0.5 program[38] based on the papain structure 1PE6 studied
by Yamamoto et al.[36] The papain–peptide complex model was construct-
ed by replacing the inhibitor in structure 1PE6 with peptide 10 as the
chosen inhibitor in our case. The initial geometry of peptide 10 and
papain–peptide complex were modeled by energy minimization using
molecular mechanics calculation method (MM) in Spartan. The enzyme–
inhibitor complex distances were in good agreement with X-ray crystallo-
graphic studies.[36]
by Gütschow[26] that papain prefers aromatic residues over
aliphatic ones in the P2 position, the structural basis of this
argument was not clearly addressed. It appears that the
binding pocket is more flexible with regards to molecular
recognition. Peptide 10 possesses Arg in P2 and Tyr in P1
and exhibits good molecular recognition properties. The
new sequence Thc-Fca-Gly-Gly-Arg-Tyr exhibits the stron-
gest inhibition of papain, Ki =6.9ꢀ0.2 mm. Presumably, N-
conjugation of the inhibitory sequence with Thc-Fca resi-
dues enhances the affinity for papain binding site.[22] As was
demonstrated earlier in solution studies on the Fc derivative
(Fc-Gly-Gly-Tyr-Arg),[22] the organometallic Fca group is
most likely encapsulated, which appears to enhance the in-
teraction with the protein.
Figure 3. SPR imaging measurements showing the interaction of papain
(150 nm) with surface-bound Fca–peptide conjugates 4, 9, and 10 and a
decanethiol control. Papain was dissolved in a buffer solution (0.1m
sodium phosphate pH 6.2, 2.5 mm EDTA, 300 mm DTT, and 30% DMSO,
238C. a) SPR difference image resulting from subtraction of the buffer
image from the ones obtained after interaction with papain. b) Line pro-
file, taken across the difference images, shows significant differences in
the D% R for measurements taken in the absence and presence of
papain.
SPR Imaging: The next step in this investigation was to de-
termine the potential of the Fca–peptide conjugates to be
active towards papain binding when linked on a surface.
This will allow us to evaluate the formation of the peptide–
protein complex on the surface and is a useful step towards
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H.-B. Kraatz and K. A. Mahmoud
ment with the solution studies (see above). Figure 4 shows
the increase in D% R due to papain–surface interaction.
The binding curve reaches a steady state when papain ad-
sorption and desorption rates were equal. At this point,
Figure 5. Plot of g versus papain concentration for conjugate 10 obtained
from fitting the adsorption curves to Equation (2). The linear slope cor-
responds to the adsorption rate constant ka =1.75ꢀ0.05”105 mꢁ1 s1 and
the y intercept corresponds to the desorption rate constant kd =2.90ꢀ
0.05”10ꢁ2 sꢁ1
.
Electrochemistry: Fca–peptide conjugates 10 displayed the
lowest inhibition constant in solution and also performed
well when present as a thin film. Next, we decided to test
our major hypothesis of the new label-based electrochemical
detection and exploit the redox activity of the Fca group to
detect the binding of papain to the inhibitory peptide se-
quence in conjugate 10. The electrochemical studies were
carried out using Fca–peptide films prepared on 25 mm gold
microelectrodes. The peptide films were prepared by im-
mersing the microelectrodes in Thc-terminated peptides pre-
pared in 5% (by volume) acetic acid in ethanol at concen-
tration of approximately 1 mm for 36 h. As described in
Scheme 1d the Fca label is located at the base of the peptide
capturing layer allowing direct connection and close proxim-
ity to the gold surface.
Figure 4. Plot of D% R versus time for the interaction of papain with
thin films of Fca–peptide conjugate 10 on gold. The concentration of
papain was increased from 25–200 nm.
papain-free buffer was injected and flowed over the peptide
array. In order to extract values for the association (ka) and
dissociation (kd) rate constants, sequential fitting of the re-
sponse curves over a series of different protein concentra-
tions was required. Assuming a 1:1 interaction model be-
tween papain and the surface-bound Fca–peptide, Equa-
tion (1) was used to determine the rate of desorption.
Under these conditions, the disulfide-containing Thc
D % RðtÞ ¼ DR expðꢁkdtÞ
ð1Þ
ꢁ
group adsorbed to the gold surface involving a stable Au S
linkage.[48,49] The next step involved the dilution of the Fca–
peptide in order to close available pinholes present in the
film and cover any solution accessible gold. The dilution was
carried out by immersing the Fca–peptide-modified gold mi-
croelectrodes in an ethanolic solution of decanethiol
(0.5 mm) for about two minutes. The electrochemical re-
sponse of the films was evaluated by cyclic voltammetry
(CV) measurements in papain activation buffer at pH 6.2
containing NaClO4 (2m) as supporting electrolyte. Measure-
ments were repeated for ten separate microelectrodes in
order to obtain statistically meaningful results. The films ex-
hibited a single fully reversible one-electron redox peak
with a formal potential E0 of 0.459ꢀ0.005 V at a scan rate
of 10 Vsꢁ1 versus Ag/AgCl. Figure 6 shows the change of
the CV as a function of increasing papain concentration.
As the enzyme concentration increased, the formal poten-
tial of the Fca probe was shifting to higher potentials, indi-
cating that oxidation of the Fca group became increasingly
more difficult as papain was binding to the inhibitory pep-
The adsorption curves are determined using a simplified
Langmuir isotherm by:
D % RðtÞ ¼ DR ð1ꢁegtÞ
ð2Þ
where g = kac + kd with c being the protein concentration.
DR can also be defined as equal to the product DRmaxq,
where DRmax is the maximum SPR signal obtained when all
surface binding sites are occupied and q is the fraction of
the total surface coverage. Figure 5 shows a plot of g versus
papain concentration by fitting the adsorption curves shown
in Figure S5, Supporting Information, to Equation (2). ka =
1.75ꢀ0.05”105 mꢁ1 s1 and kd =2.90ꢀ0.05”10ꢁ2 sꢁ1 were ob-
tained from the slope and intercept, respectively. Moreover,
the equilibrium adsorption constant Kads =6.03”106 mꢁ1 was
obtained from the ratio of ka and kd.
The Kads value was within the reported range of binding
constants for the interaction between proteins with immobi-
lized peptides from 1.7”106 mꢁ1 to 1.5”108.[42,44–47]
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Electrochemical Detection of Proteins
FULL PAPER
do not loose the ability to detect the interaction between
the peptide and the protein. In contrast to our observations,
in some cases severe problems can occur that will result in
the loss of signal. In Katayamaꢁs sensor system,[9] a signifi-
cant reduction in current was observed as a function of c-
AMP addition to the system, ultimately leading to loss of
signal at high c-AMP concentrations. It is likely that these
changes are caused by decreased access of the [Fe(CN)6]3ꢁ/4ꢁ
redox probe to the surface. In aptamer-based systems, the
aptamer is highly dynamic in the absence of the target ana-
lyte allowing efficient electron transfer between the redox
tag and the surface. The electron transfer is affected by ana-
lyte binding and in some cases even inhibited.[51–54]
In order to gain further support for the interaction of
papain with films of conjugate 10, measurements were car-
ried out involving a quartz crystal microbalance. For this
purpose, peptide films were immobilized on the gold pad on
the quartz crystals following the identical procedure used
for gold microelectrodes. In the QCM measurements,
changes in the frequency are monitored as a function of
time as depicted in Figure 8. In the presence of the blank ac-
Figure 6. Cyclic voltammogram of 25 mm diameter gold microelectrodes
modified with a mixed film of Fca–peptide 10 and decanethiol in the
presence of increasing concentrations of papain (The arrow direction in-
dicates a signal decrease and shift to higher potential with increasing the
concentration from 0 to 200 nm). Enzyme activation buffer (0.1m sodium
phosphate pH 6.2, 2.5 mm EDTA, 300 mm DTT, and 30% DMSO, 238C)
with 2m NaClO4. Scan rate of 10 Vsꢁ1, Pt counter, Ag/AgCl reference.
tide. In case of the Fc derivative Fc-Gly-Gly-Tyr-Arg, these
changes were caused by slight changes in the chemical envi-
ronment around the Fc group.[22] This change is illustrated in
Figure 7, which shows a plot of E0 as a function of papain
Figure 8. QCM measurement of papain adsorption on films of Fca–pep-
tide conjugate 10 on gold covered AT-cut type crystals. Papain activation
buffer: 0.1m sodium phosphate pH 6.2, 2.5 mm EDTA, 300 mm DTT, and
30% DMSO, 238C with 2.0m NaClO4.
tivation buffer solution only continuous decay of Df was ob-
served, most likely caused by buffer migration. In the pres-
ence of papain, the protein adsorbs to the film of conjugate
10 and after some time reaches steady state. The change in
frequency Df before and after papain addition is 34.1ꢀ
3 Hz, which is converted into a mass change using the Sauer-
brey equation [Eq. (5)]. The average mass of papain ad-
sorbed to the surface was 245ꢀ3 ngcmꢁ2 or 1.62”
10ꢁ12 molcmꢁ2.
Figure 7. Plot of the formal potential (E0) vs papain concentration.
concentration.
A linear relationship was observed for
papain concentration of up to 80 nm, after this concentration
the potential reaches a steady state indicating potentially
the saturation of the surface with papain. The detection
limit was 4”10ꢁ9 m, estimated from 3
(Sb/m), where Sb is the
G
standard deviation of the measurement signal for the blank
and m the slope of the analytical curve in the linear
region.[50]
In addition, a small decrease in the overall signal intensity
was observed, most likely as a result of less efficient pene-
tration of the film by the supporting electrolyte, thereby de-
creasing the ability of the Fca to be oxidized. However, we
Conclusion
In summary, we have synthesized an bioorganometallic con-
jugate of Fca equipped with an inhibitory peptide sequence
Chem. Eur. J. 2007, 13, 5885 – 5895
ꢀ 2007Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
5891

H.-B. Kraatz and K. A. Mahmoud
solved in CH2Cl2 and cooled in ice bath prior to the dropwise addition of
Et3N (0.45 mL). To this was added a solution of Boc-Fca-OBt (2 mmol,
0.92 g). The reaction mixture was then warmed to room temperature and
left to stir overnight. Purification of the crude product was carried out by
column chromatography on silica gel (CHCl3/MeOH 90:10) to give
yellow crystals of 2 (1.41 g, 76%). 1H NMR ([D6]DMSO) d = 7.43 (d,
2H, J=8 Hz, Bz), 7.38 (t, 2H, J=9 Hz, Bz), 7.32 (t, 1H, J=9 Hz, Bzl),
7.15 (d, 2H, J=8 Hz, Tyr-Bz), 6.89 (d, 2H, J=9 Hz, Tyr-Bz), 5.05 (s, 2H,
that targets papain. Competitive inhibitions from solution
assays and adsorption/desorption kinetics from real-time
SPR imaging studies allowed the selection of peptide 10 as
the most efficient capture probe for papain detection. The
electrochemical measurements of surface-bound Fca–pep-
tide 10 showed a significant electrochemical response of the
sensor upon binding to papain. The signal is shifting anodi-
cally as well as decreasing in intensity, presumably due to
the partial shielding of the Fca group by the papain. This
represents proof of concept that our approach using bioor-
ganometallic sensor systems provides an attractive alterna-
tive for the electrochemical detection of non-labeled non-
redox active proteins, which under current detection
schemes remains a significant challenge. Thus, the next step
in our investigation is to optimize the sensor platform and
expand out studies to include multiplexed measurements of
several analytes by varying the recognition sequence on the
Fca redox probe.
Tyr
(m, 1H, Tyr
4.06 (s, 2H, H-3’, H-4’, Fc), 3.71 (s, 2H, Gly1
(aCH)), 3.61 (s, 3H, COOCH3), 3.03 (m, 2H, CH2 of CH2CH2CH2NH),
ACHTREUNG
A
ACHTREUNG
2
A
-
AHCTREUNG
2.89 (m, 1H, CH2 of Tyr), 2.70 (m, 1H, CH2 of Tyr), 1.69 (m, 1H,
CH2CH2CH2NH), 1.54 (m, 1H, CH2CH2CH2NH), 1.45 (m, 2H, CH2 of
CH2CH2CH2NH), 1.43 ppm (s, 9H, Boc
C
(CH3)3); 13C{1H} NMR
A
([D6]DMSO): d = 172.6, 171.8, 170.4, 169.9, 168.9, 157.4, 153.5, 137.7,
130.7, 130.3, 128.9, 128.2, 128.1, 114.8, 89.6, 76.9, 70.7, 69.5, 69.4, 65.9,
61.4, 45.3, 52.4, 52.2, 42.3, 37.2, 31.4, 28.6, 28.5, 22.5, 14.4 ppm; FTIR
(KBr): n˜ = 3301 (m, N-H), 1741 cmꢁ1 (s, C=O); TOF-MS: m/z: calcd for
C43H53N9O11Fe: 927.3214; found: 927.3217 [M]+.
Thc-Fca-Gly-Gly-Tyr(Bz)-Arg(NO2)-OMe (3): After the removal of the
G
Boc group from 2 (1.1 mmol, 1.01 g) using TFA (1.5 mL), the excess acid
was removed in vacuo and Et3N (1.0 mL) in CH2Cl2 (10 mL) was added.
To this was added a solution of Thc-OBt, prepared in situ from thiostic-
OH (1.0 mmol, 0.206 g), HOBt (1.1 mmol, 0.153 g), and EDCI·HCl
(1.1 mmol, 0.191 g) in dry CH2Cl2 (15 mL, 08C). The reaction mixture
was then warmed to room temperature and left to stir overnight. The re-
action mixture was then treated consecutively with aqueous solutions of
NaHCO3 (sat.), citric acid (10%), and water, dried over Na2SO4, and
evaporated to dryness under reduced pressure. The crude product was
purified by flash column chromatography (silica gel, EtOAc/MeOH 95:5)
to give pale yellow crystals of compound 3 (0.69 g, 68%). 1H NMR
([D6]DMSO): d = 8.38 (s, 1H, NH), 8.36 (s, 1H, NH), 8.10 (brs, 2H,
NH), 8.04 (brs, 2H, NH), 7.43 (d, 2H, J=7Hz, Bz), 7.38 (t, 2H, J=
9 Hz, Bz), 7.32 (t, 1H, J=9 Hz, Bz), 7.15 (d, 2H, J=8 Hz, Tyr-Bz), 6.90
Experimental Section
Materials and general procedure: All syntheses were carried out under
dry nitrogen gas unless otherwise indicated. CH2Cl2 (ACS grade) used
for synthesis was dried (CaH2) and distilled prior to use. CDCl3 (Aldrich)
was dried (CaH2), and stored over molecular sieves (8–12 mesh; 4 Š ef-
fective pore size; Fisher) before use. THF was dried by distillation over
benzophenone/sodium, and stored over molecular sieves (8–12 mesh; 4 Š
effective pore size; Fisher) before use. 2-(1H-Benzotriazol-1-yl)-1,1,3,3-
tetramethyluronium (HBTU), hydroxybenzotriazole (HOBt) (Nova),
MgSO4, and NaHCO3 (VWR) were used as received. For column chro-
matography, a column with a width of 2.7cm (ID) and a length of 45 cm
was packed 18–22 cm high with 230–400 mesh silica gel (VWR). For
TLC, aluminum plates coated with silica gel 60 F254 (EM Science) were
used. NMR spectra were recorded on a Bruker Avance-500 spectrometer
(d, 2H, J=8 Hz, Tyr-Bz), 5.06 (s, 2H, Tyr(O-CH2)), 4.78 (s, 2H, H-2, H-
U
5, Fc), 4.58 (s, 2H, H-2’, H-5’, Fc), 4.44 (1H, m, Tyr aH), 4.42 (s, 2H, H-
3, H-4, Fc), 4.26 (m, 1H, Arg aH), 4.05 (s, 2H, H-3’, H-4’, Fc), 3.96 (s,
2H, Gly1aH), 3.66 (s, 2H, Gly2aH), 3.64 (s, 3H, COOCH3), 3.61 (m, 1H,
CH2 of Thc), 3.20 (m, 1H, CH2 of Thc), 3.14 (m, 1H, CH2 of Thc), 3.03
(m, 2H, CH2 of CH2CH2CH2NH), 2.89 (m, 1H, CH2 of Tyr), 2.70 (m,
1H, CH2 of Tyr), 2.49 (m, 1H, CH2 of Thc), 2.30 (m, 2H, Thc (CH2)),
1.90 (m, 1H, Thc (CH)), 1.75 (m, 4H, two CH2 of Thc), 1.69 (m, 1H,
CH2 of CH2CH2CH2NH), 1.54 (m, 1H, CH2 of CH2CH2CH2NH), 1.53
(m, 2H, Thc (CH2)), 1.45 ppm (m, 2H, CH2 of CH2CH2CH2NH); 13C{1H}
NMR ([D6]DMSO): d = 172.6, 171.8, 171.4, 170.6, 169.7, 168.9, 157.4,
137.7, 130.6, 130.2, 128.8, 128.2, 128.1, 114.8, 97.1, 77.0, 71.7, 69.6, 69.6,
69.4, 66.0, 62.2, 65.6, 54.4, 52.4, 52.2, 42.7, 42.3, 38.6, 37.2, 36.2, 34.7, 34.6,
28.8, 28.7, 28.4, 25.3, 24.7 ppm; FTIR (KBr): n˜ = 3297(brm, N-H), 1741,
1653, 1538 cmꢁ1 (s, C=O); TOF-MS; m/z: calcd for C46H57O10N9S2Fe:
1015.3019; found: 1015.3025 [M]+.
using
a
5 mm broadband probe operating at 500.134 MHz (1H) or
125.766 MHz (13C{1H}). Peak positions in the 1H NMR spectra are report-
ed in ppm relative to TMS. All otherwise it is described 13C{1H} spectra
are referenced to the [D6]DMSO signal at d=39.85 ppm. Mass spectrom-
etry was carried out on a VG Analytical 70/20 VSE instrument. Infrared
spectra were recorded on a Perkin-Elmer model 1605 FTIR spectrome-
ter. Analytical RP-HPLC for peptide 10 was performed on a Dionex
HPLC system equipped with a Dionex Acclaim 3Lm C-18 (150”4.6 mm)
column with a flow rate of 1 mLminꢁ1. Semipreparative RP-HPLC was
performed on a Dionex HPLC system equipped with a Phenomenex
Gemini 5Lm C-18 (250”10 mm) column with
a flow rate of
2.0 mLminꢁ1. Mobile phase A was 0.1% TFA in water, mobile phase B
was 0.1% TFA in acetonitrile. The gradiant was t=0 min, B=5%; t=
20 min, B=65%; t=25 min, B=90%; t=27min, B =95%; t=27.1 min,
B=8%; t=35 min, B=5%.
Fca-Gly-Gly-Tyr(Bz)-Arg(NO2)-OH (4): A solution of 1n NaOH in
G
water (2.6 mL) was added while stirring to a solution of compound 3
(0.50 g, 0.49 mmol) in MeOH (2.0 mL). The reaction was stored at room
temperature for 6 h after which 1n HCl (1.5 mL) was added. The MeOH
was then removed in vacuo followed by cooling of the solution in a ice
bath prior to the dropwise addition of 1n HCl (3.0 mL). The solution
was then stored in the fridge for 3 h after which the precipitate was fil-
tered off and washed three times with cold, distilled water (50 mL) and
dried under reduced pressure overnight to give compound 4 as a yellow
Papain, lyophilized powder ꢃ10 units per mg protein (E1%/280) and
thiostic acid (Thc-OH) were purchased from Sigma. DTT (ꢀ-threo-2,3-
dihydroxy-1,4-butanedithiol) was obtained from Fluka. Z-Phe-Arg-
NHNP was purchased from Bachem, Bubendorf (Switzerland). The
amino acid derivatives were purchased from Advanced ChemTech. Spot-
ready SPR chips having sixteen 1 mm gold spots and plane gold SPR
chips were purchased from GWC technologies. The syntheses of Boc-
1
solid (0.69 g, 68%). M.p. 152–1598C; H NMR ([D6]DMSO): d = 9.44 (s,
1H, COOH), 8.63 (s, 1H, NH), 8.29 (s, 1H, NH), 8.22 (s, 1H, NH), 8.12
(brs, 2H, NH), 7.36 (brs, 1H, NH), 7.42 (d, 2H, J=8 Hz, Bz), 7.38 (t,
2H, J=9 Hz, Bz), 7.32 (t, 1H, J=8 Hz, Bz), 7.15 (d, 2H, J=9 Hz, Tyr-
Gly-Gly-Tyr
N
N
carbonylamino)ferrocene-1-carboxylate (Boc-Fca-OBt)[55] were described
elsewhere.
Bz), 6.90 (d, 2H, J=9 Hz, Tyr-Bz), 5.06 (s, 2H, Tyr(O-CH2)), 4.78 (s, 2H,
N
Boc-Fca-Gly-Gly-Tyr(Bz)-Arg
A
H-2, H-5, Fc), 4.58 (s, 2H, H-2’, H-5’, Fc), 4.44 (m, 1H, Tyr aH), 4.42 (s,
2H, H-3, H-4, Fc), 4.26 (m, 1H, Arg aH), 4.05 (s, 2H, H-3’, H-4’, Fc),
3.96 (s, 2H, Gly1aH), 3.66 (s, 2H, Gly2aH), 3.61 (m, 1H, CH2 of Thc),
3.20 (m, 1H, CH2 of Thc), 3.14 (m, 1H, CH2 of Thc), 3.03 (m, 2H, CH2
Arg(NO2)-OMe (1) (2.2 mmol, 1.54 g) was dissolved in CH2Cl2 and treat-
ACHTREUNG
ed with TFA (3 mL) in CH2Cl2 (3 mL) for 30 min. The TFA and CH2Cl2
were subsequently removed in vacuo. The resulting residue was redis-
5892
www.chemeurj.org
ꢀ 2007Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2007, 13, 5885 – 5895

Electrochemical Detection of Proteins
FULL PAPER
of CH2CH2CH2NH), 2.89 (m, 1H, CH2 of Tyr), 2.70 (m, 1H, CH2 of Tyr),
2.49 (m, 1H, CH2 of Thc), 2.30 (m, 2H, Thc CH2), 1.90 (m, 1H, Thc
CH), 1.75 (m, 4H, 2CH2 of Thc), 1.69 (m, 1H, CH2 of CH2CH2CH2NH),
1.54 (m, 1H, CH2 of CH2CH2CH2NH), 1.53 (m, 2H, Thc CH2), 1.45 ppm
(CHCl3/MeOH 90:10) to give compound 7 as a yellow solid (1.47g,
85%). H NMR ([D6]DMSO): d = 9.22 (s, 1H, Bz-OH), 8.42 (1H, s, NH
1
of guanidine group), 8.30 (d, 1H, J=6.3 Hz, aNH Arg), 8.09 (s, 1H, NH
of guanidine group), 8.05 (s, 1H, NH of guanidine group), 7.96 (d, 1H,
J=6 Hz, aNH Tyr), 6.97(d, 2H, J=8 Hz, Tyr-Bz), 6.68 (s, 1H, Ar-H of
Mtr), 6.64 (d, 2H, J=8 Hz, Tyr-Bz), 6.38 (brs, 2H, NH of guanidine
group), 4.72 (s, 2H, H-2, H-5, Fc), 4.60 (m, 1H, Tyr aCH), 4.47(s, 2H,
H-2’, H-5’, Fc), 4.30 (m, 1H, Arg aCH), 4.24 (s, 2H, H-3, H-4, Fc), 3.95
(s, 2H, H-3’, H-4’, Fc), 3.82 (d, 2H, aCH2 Gly aCH2), 3.78 (s, 3H,
(m, 2H, CH2 of CH2CH2CH2NH); 13C{1H} NMR ([D6]DMSO): d
=
188.5, 171.4, 169.6, 159.8, 157.4, 137.7, 130.7, 128.9, 128.2, 114.8, 97.2,
77.1, 71.6, 69.6, 69.5, 65.9, 62.3, 56.6, 42.6, 38.6, 36.2, 43.7, 28.8, 25.4 ppm;
ꢁ1
FTIR (KBr): n˜ = 3297(brm, N-H), 1471, 1654, 1546 cm
(s, C=O);
TOF-MS: m/z: calcd for C45H55O10N9S2Fe: 1001.2863; found: 1001.2865
[M]+.
COOCH3), 3.70 (d, 2H, Gly aCH2), 3.53 (s, 3H, OCH3(Mtr)), 3.01 (m,
G
2H, CH2 of CH2CH2CH2NH), 2.88 (m, 1H, CH2 of Tyr), 2.82 (m, 1H,
CH2 of Tyr), 2.59 (s, 3H, Ar-CH3 of Mtr), 2.49 (s, 3H, Ar-CH3 of Mtr),
2.04 (s, 3H, Ar-CH3 of Mtr), 1.62 (m, 1H, CH2 of CH2CH2CH2NH), 1.43
Boc-Arg
A
U
HOBt (4.52 mmol, 0.75 g), and EDCI·HCl (4.52 mmol, 0.95 g) were
mixed in dry CH2Cl2 (30 mL, 08C) and allowed to stirred for 30 min. To
this, a solution of H-Tyr-OMe, obtained by treatment of H-Tyr-OMe·HCl
(4.52 mmol, 1.14 g) with Et3N (1.5 mL) in dry CH2Cl2 (20 mL) was added
and the stirring continued at room temperature. The reaction mixture
was then treated consecutively with aqueous solutions of saturated
NaHCO3, citric acid (10%), again saturated NaHCO3 and water, dried
over Na2SO4, filtered and evaporated to dryness under reduced pressure.
The crude product was purified by flash column chromatography (silica
gel, EtOAc/hexane 1:2) to give compound 5 as a white crystalline solid
(2.32 g, 85%). 1H NMR (CDCl3): d = 7.48 (s, 1H, Bz-OH), 6.97 (d, 2H,
J=8 Hz, Tyr-Bz), 6.91 (brs, 1H, aNH Arg), 6.78 (d, 2H, J=8 Hz, Tyr-
Bz), 6.55 (s, 1H, Ar-H (Mtr)), 6.20 (brs, 2H, NH of guanidine), 5.98 (brs,
1H, NH of guanidine group), 5.41 (brs, 1H, Boc NH), 4.79 (m, 1H, Arg
aH), 4.00 (m, 1H, Tyr aH), 3.89 (s, 3H, COOCH3), 3.75 (s, 3H, OCH3 of
Mtr), 3.15 (m, 2H, CH2 of CH2CH2CH2NH), 3.09 (m, 1H, CH2 of Tyr),
2.88 (m, 1H, CH2 of Tyr), 2.70 (s, 3H, Ar-CH3 of Mtr), 2.62 (s, 3H, Ar-
CH3 of Mtr), 2.14 (s, 3H, Ar-CH3 of Mtr), 1.54 (m, 2H, CH2 of
(9H, s, Boc C
C
172.7, 172.4, 170.3, 170.0, 169.4, 157.9, 156.6, 153.5, 138.1, 138.1, 136.1,
130.5, 127.3, 124.0, 115.6, 112.2, 100.0, 79.7, 76.8, 71.7, 69.3, 65.8, 56.0, 5.3,
52.2, 42.9, 42.1, 36.5, 28.6, 24.1, 18.5, 12.2 ppm; TOF-MS: m/z: calcd for
C46H60O12N8SFe: 1004.3401; found: 1004.3402 [M]+.
Thc-Fca-Gly-Gly-Arg(Mtr)-Tyr-OMe (8): The Boc group was removed
H
from compound 7 (1.2 mmol, 1.20 g) by addition of TFA (1.5 mL). After
30 min, the excess acid was removed in vacuo and Et3N (0.7mL) in
CH2Cl2 (10 mL) was added. To this was added a solution of Thc-OH
(1.1 mmol, 0.224 g), HOBt (1.2 mmol, 0.184 g), and EDCI·HCl
(1.2 mmol, 0.230 g) in dry CH2Cl2 (15 mL, 08C). The reaction mixture
was left to stir overnight at room temperature and then treated to an
aqueous work up as described above. The crude product was purified by
flash column chromatography (silica gel, EtOAc/MeOH 90:10) to give a
pale yellow solid of compound 8 (0.89 g, 68%). 1H NMR ([D6]DMSO): d
= 9.22 (s, 1H, Bz-OH), 8.42 (s, 1H, NH of guanidine group), 8.30 (d,
1H, J=6 Hz, aNH Arg), 8.09 (s, 1H, NH of guanidine group), 8.0 (s,
1H, NH of guanidine group), 7.96 (d, 1H, J=6 Hz, aNH Tyr), 6.97(d,
2H, J=8 Hz, Tyr-Bz), 6.68 (s, 1H, Ar-H of Mtr), 6.64 (d, 2H, J=8 Hz,
Tyr-Bz), 6.38 (brs, 2H, NH of guanidine group), 4.72 (s, 2H, H-2,m H-5,
Fc), 4.60 (1H, m, Tyr aH), 4.47(s, 2H, H-2 ’, H-5’, Fc), 4.30 (1H, m, Arg
aH), 4.24 (s, 2H, H-3, H-4, Fc), 3.95 (s, 2H, H-3’, H-4’, Fc), 3.82 (d, 2H,
Gly aH2), 3.78 (s, 3H, COOCH3), 3.70 (d, 2H, Gly aH2), 3.61 (m, 1H,
CH2 of Thc), 3.53 (s, 3H, OCH3 of Mtr), 3.20 (m, 1H, CH2 of Thc), 3.14
(m, 1H, CH2 of Thc), 3.01 (m, 2H, CH2 of CH2CH2CH2NH), 2.88 (m,
1H, CH2 of Tyr), 2.82 (m, 1H, CH2 of Tyr), 2.59 (s, 3H, Ar-CH3of Mtr),
2.49 (m, 1H, CH2 of Thc), 2.49 (s, 3H, Ar-CH3 of Mtr), 2.30 (2H, m, Thc
CH2), 1.90 (m, 1H, Thc (CH)), 2.04 (s, 3H, Ar-CH3 of Mtr), 1.75 (m, 4H,
two CH2 of Thc), 1.62 (m, 1H, CH2 of CH2CH2CH2NH), 1.53 (m, 2H,
Thc (CH2)), 1.36 (m, 2H, CH2 of CH2CH2CH2NH), 1.23 ppm (m, 1H,
CH2 of CH2CH2CH2NH); 13C{1H} NMR ([D6]DMSO): d = 188.5, 172.2,
172.1, 171.9, 171.4, 170.6, 169.8, 169.0, 157.9, 156.6, 149.6, 138.1, 136.1,
135.0, 124.0, 122.0, 112.2, 97.1, 79.7, 77.0, 71.7, 69.4, 66.0, 62.2, 56.5, 56.5,
56.0, 52.3, 38.6, 34.7, 34.5, 25.3, 24.6, 24.1, 12.2 ppm; FTIR (KBr): n˜ =
3315, 3090 (brm, N-H), 1722, 1655, 1548 cmꢁ1 (s, C=O); TOF-MS: m/z:
calcd for C49H64O11N8S3Fe: 1092.3206; found: 1092.3208 [M]+.
CH2CH2CH2NH), 1.42 (s, 9H, Boc C(CH3)3), 1.23 ppm (m, 2H, CH2 of
CH2CH2CH2NH); TOF-MS: m/z: calcd for C31H45O9N5S: 663.2938;
found: 663.2940 [M]+.
E
Boc-Gly-Gly-Arg
G
Boc-Arg(Mtr)-Tyr-OMe
G
(3.01 mmol, 2.00 g) was dissolved in CH2Cl2 (3 mL) and treated with
TFA (3 mL) for 30 min. The CH2Cl2 and TFA were subsequently re-
moved in vacuo. The resulting residue was redissolved in CH2Cl2 (10 mL)
and cooled in an ice bath prior to the dropwise addition of Et3N
(1.2 mL). To this was added a solution of Boc-Gly-Gly-OBt, prepared in
situ from Boc-Gly-Gly-OH (2.74 mmol, 0.64 g), HOBt (3.0 mmol, 0.46 g),
and EDCI·HCl (3.0 mmol, 0.58 g) in dry CH2Cl2 (20 mL, 08C). The reac-
tion mixture was then warmed to room temperature and left to stir over-
night. The resulting solution was then treated as per 5. The product was
purified by flash column chromatography (silica gel, EtOAc/hexane 1:2)
and recrystallized from CHCl3 to yield white crystalline compound 6
(1.73 g, 74%). 1H NMR ([D6]DMSO): d = 9.22 (s, 1H, Bz-OH), 8.42 (s,
1H, NH-C(NH)-NH
NH of guanidine group), 8.05 (s, 1H, NH of guanidine group), 7.96 (d,
1H, J=6 Hz, aNH(Tyr), 6.97(d, 2H, J=8 Hz, Tyr-Bz), 6.68 (s, 1H, Ar-H
of Mtr), 6.64 (d, 2H, J=8 Hz, Tyr-Bz), 6.38 (brs, 2H, NH of guanidine
group), 4.60 (m, 1H, Tyr aH), 4.30 (m, 1H, Arg aH), 3.82 (d, 2H, Gly
aH), 3.78 (s, 3H, COOCH3), 3.70 (d, 2H, Gly aH), 3.53 (s, 3H, OCH3 of
Mtr), 3.01 (m, 2H, CH2 of CH2CH2CH2NH), 2.88 (m, 1H, CH2 of Tyr),
2.82 (m, 1H, CH2 of Tyr), 2.59 (3H, s, Ar-CH3 of Mtr), 2.49 (s, 3H, Ar-
CH3 of Mtr), 2.04 (s, 3H, Ar-CH3 of Mtr), 1.62 (m, 1H, CH2 of
(Arg), 8.09 (s, 1H,
Thc-Fca-Gly-Gly-Arg
0.18 mmol) was deprotected as described for compound 4 to give free
acid as
yellow solid (0.16 g, 82%). M.p. 125–1338C; 1H NMR
A
8
(0.20 g,
9
a
([D6]DMSO): d = 9.58 (s, 1H, COOH), 9.05 (s, 1H, Tyr-OH), 8.49 (s,
1H, NH of guanidine group), 8.35 (d, 1H, J=6 Hz, aNH Arg), 8.18 (s,
1H, NH of guanidine group), 8.05 (s, 1H, NH of guanidine group), 7.95
CH2CH2CH2NH), 1.43 (s, 9H, Boc C(CH3)3), 1.36 (m, 2H, CH2 of
E
CH2CH2CH2NH), 1.23 ppm (m, 1H, CH2 of CH2CH2CH2NH); 13C{1H}
NMR ([D6]DMSO): d = 172.3, 171.9, 170.2, 168.9, 1579, 156.6, 156.5,
156.3, 138.1, 136.1, 130.6, 130.5, 127.5, 115.5, 112.2, 78.6, 56.0, 54.5, 52.3,
52.2, 52.1, 43.7, 42.3, 63.4, 28.7, 24.1, 22.5, 18.5, 12.2 ppm; TOF-MS: m/z:
calcd for C35H51O11N7S: 777.3367; found: 777.3370 [M]+.
(d, 1H, J=6 Hz, aNH
N
N
aNH(Gly)), 6.94 (2H, d, J=8 Hz, Tyr-Bz), 6.67(s, 1H, Ar-H of Mtr),
AHCTREUNG
6.57(2H, d, J=8 Hz, Tyr-Bz), 4.75 (d, 2H, J=9 Hz, H-2, H-5, Fc), 4.60
(m, 1H, Arg aH), 4.59 (d, 2H, J=9 Hz, H-2’, H-5’, Fc), 4.24 (s, 2H, H-3,
H-4, Fc), 4.17(m, 1H, Tyr aH), 3.97(s, 2H, H-3 ’, H-4’, Fc), 3.84 (d, 2H,
Gly aCH2), 3.84 (s, 3H, Ar-O-CH3 of Mtr), 3.75 (d, 2H, Gly aCH2), 3.61
Boc-Fca-Gly-Gly-Arg
U
OMe (1.9 mmol, 1.50 g) was dissolved in CH2Cl2 and treated with TFA
(2 mL) in CH2Cl2 (2 mL) for 30 min. The TFA and CH2Cl2 were subse-
quently removed in vacuo. The resulting residue was re-dissolved in
CH2Cl2 and cooled in ice bath prior to the dropwise addition of Et3N
(0.50 mL). To this was added a solution of Boc-Fca-OBt (1.73 mmol,
0.80 g). The reaction mixture was then warmed to room temperature and
left to stir overnight. The residue was purified by chromatography
(m, 1H, CH2 of Thc), 3.53 (s, 3H, OCH3 of Mtr), 3.17(m, 1H, CH of
2
Thc), 3.11 (m, 1H, CH2 of Thc), 2.97(m, 2H, CH of CH2CH2CH2NH),
2
2.88 (m, 1H, CH2 of Tyr), 2.82 (m, 1H, CH2 of Tyr), 2.60 (s, 3H, Ar-CH3
of Mtr), 2.49 (m, 1H, CH2 of Thc), 2.49 (s, 3H, Ar-CH3 of Mtr), 2.30 (m,
2H, Thc CH2), 1.91 (m, 1H, Thc CH), 2.04 (s, 3H, Ar-CH3 of Mtr), 1.74
(m, 4H, two CH2 of Thc), 1.60 (m, 1H, CH2 of CH2CH2CH2NH), 1.53
(m, 2H, Thc CH2), 1.36 (m, 2H, CH2 of CH2CH2CH2NH), 1.22 (m, 1H,
Chem. Eur. J. 2007, 13, 5885 – 5895
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5893

H.-B. Kraatz and K. A. Mahmoud
CH2 of CH2CH2CH2NH), 0.85 ppm (m, 1H, CH2 of CH2CH2CH2NH);
13C{1H} NMR ([D6]DMSO): d = 188.5, 172.2, 172.1, 171.9, 171.4, 170.6,
169.8, 169.0, 157.9, 156.6, 149.6, 138.1, 136.1, 135.0, 124.0, 122.0, 112.2,
97.1, 79.7, 77.0, 71.7, 69.4, 66.0, 62.2, 56.5, 56.5, 56.0, 52.3, 38.6, 34.7, 34.5,
25.3, 24.6, 24.1, 12.2 ppm; FTIR (KBr): n˜ = 3306, 3086 (m, N-H), 1748
(s, C=O), 1656 (amide I), 1550 cmꢁ1 (amide II); TOF-MS: m/z: calcd for
C48H62O11N8S3Fe: 1078.3049; found: 1078.3049 [M]+.
where # is the rate, #0 is the rate in the absence of the inhibitor, [I] is the
inhibitor concentration and Ki’ is the apparent inhibition constant. The
true inhibition constants Ki were calculated by correction of Ki’ according
to Equation (4)[26]
Ki0
ð4Þ
Ki
¼
1 þ ½SŠ=Km
Thc-Fca-Gly-Gly-Arg-Tyr-OH (10): Thc-Fca-Gly-Gly-Arg(Mtr)-Tyr-OH
A
where [S] is the substrate concentration and Km is the Michaelis–Menten
constant.
(9) (60 mg, 0.07mmol) was suspended in CH 2Cl2 (2 mL) and a mixture
(TFA/thioanisole/H2O 96:3:1, 3 mL) was added and the mixture left to
stir for 4 h at RT. The reaction mixture was concentrated in vacuo, the re-
sulting residue was dissolved in TFA and precipitated by a dropwise ad-
dition to ice-cold Et2O. The precipitate was collected by centrifugation,
and purified by semi-preparative HPLC (tR =22.4 min, l=220 nm) to
yield a faint yellow solid of compound 10 (22 mg, 0.03 mmol, 37%). RP-
HPLC (analytical, tR =9.8 min); m.p. 118–1228C; 1H NMR ([D6]DMSO):
d = 9.48 (s, 1H, COOH), 9.23 (s, 1H, Tyr-OH), 8.30 (1H, m, aNH Arg),
8.22 (s, 1H, NH of guanidine group), 8.02 (1H, s, NH of guanidine
Preparation of peptide arrays: Peptide arrays on spot ready SPR chips
were prepared by spotting three different peptides 4, 9 and 10 on three
arrays. The gold chip was placed on a piece of clean parafilm in a humidi-
ty chamber and the freshly prepared peptide solutions were spotted im-
mediately 5 mm in DMSO (100 mm TEA pH 8) for 20 h. As a control ex-
periment, a 5 mm solution of decanethiol was used to immobilize the dec-
anethiol onto the control array. The excess probe solutions were then re-
moved with a pipet prior to rinsing with the few mL of buffer solution
(0.1m sodium phosphate, pH 6.2, 2.5 mm EDTA, 300 mm DTT, and 30%
DMSO, T=23ꢀ38C).
group), 7.66 (m, 1H, aNH
N
N
J=8 Hz, Tyr-Bz), 6.57(2H, d, J=8 Hz, Tyr-Bz), 4.75 (s, 2H, H-2, H-5,
Fc), 4.60 (s, 2H, H-2’, H-5’, Fc), 4.26 (m, 1H, Arg aH), 4.25 (s, 2H, H-3,
H-4, Fc), 4.01 (m, 1H, Tyr aH), 3.98 (s, 2H, H-3’, H-4’, Fc), 3.80 (d, 2H,
Gly aH2), 3.77 (d, 2H, Gly aH2), 3.61 (1H, m, CH2 of Thc), 3.20 (m, 1H,
CH2 of Thc), 3.18 (1H, m, CH2 of Thc),), 3.11 (m, 1H, CH2 of Tyr), 2.98
(2H, m, CH2 of CH2CH2CH2NH), 2.83 (m, 1H, CH2 of Tyr), 2.71 (2H, s,
NH2 of guanidine group), 2.41 (m, 2H, Thc CH2), 2.36 (1H, m, CH2 of
Thc), 2.15 (m, 1H, CH2 of Thc), 1.66 (2H, m, CH2 of CH2CH2CH2NH),
1.55 (m, 2H, Thc CH2), 1.35 (m, 2H, CH2 of CH2CH2CH2NH), 1.23 (m,
1H, CH2 of CH2CH2CH2NH), 0.85 ppm (m, 1H, CH2 of
CH2CH2CH2NH); 13C{1H} NMR ([D6]DMSO): d = 172.1, 172.0, 171.8,
171.4, 170.5, 169.8, 157.8, 156.6, 138.1, 136.1, 135.0, 123.9, 121.9, 112.2,
97.1, 79.7, 76.9, 71.7, 69.4, 65.9, 62.2, 56.5, 56.4, 52.3, 38.5, 34.7, 34.5, 25.3,
24.6, 24.2 ppm; FTIR (KBr): n˜ = 3447(m, N-H), 1725 (s, C =O), 1658
(amide I), 1547cm ꢁ1 (amide II); TOF-MS: m/z: calcd for
C38H50O8N8S2Fe: 866.2542; found: 866.2541 [M]+.
Preparation of films on flat substrates: Each peptide was immobilized on
the gold surface of a plane SPR chip by soaking the chips overnight in a
1 mm solution of disulfide-terminated peptides 4, 9 and 10 in dry ethanol.
Real-time SPR imaging studies: An SPR imaging system (GWC Technol-
ogies) was used for the real-time monitoring of the interaction between
immobilized peptides and papain enzyme. Briefly, a collimated p-polar-
ized light at a fixed angle reflected from the sample/gold/prism assembly
was sent through a narrow band-pass filter and then detected with a
CCD camera. The data were collected using the software package V++
(Digital Optics, NZ). Custom macros were used so that data could be col-
lected with simultaneous processing of several specific user designated
regions of interest (ROIs) on the array surface.[42] Kinetic data for each
ROI were obtained by collecting one data point approximately every 1 s,
which was the average of five camera frames. The difference in percent
reflectivity for each probe area was normalized with respect to the aver-
age change in percent reflectivity measured for the buffer background.
Kinetic data from multiple identical array elements were averaged to
obtain the final SPR response curves. Microsoft Excel and Origin 7.5
were used for all data processing and kinetic model fitting in these ex-
periments.
Papain inhibition studies: For the inhibition studies, the following stock
solutions were prepared:
A 5 mm stock solution of the chromogenic substrate Z-Phe-Arg-NHNp
was prepared in DMSO.
Stock solutions of the Fca–peptide inhibitors were prepared in DMSO.
Surface electrochemistry: Peptide films were immobilized on home made
25 mm diameter microelectrodes by soaking the gold electrodes in 1 mm
solution of disulfide terminated peptides for 36 h. Then, the concentrated
film was diluted by immersing the modified electrodes in 1 mm ethanolic
solution of decanethiol for 2 min. The CVs were recorded in aqueous sol-
utions of NaClO4 (2.0m). Ag/AgCl was used as a pseudo-reference elec-
trode, and Pt wire as a counter electrode. Ten gold microelectrodes with
peptide films were measured for each compound and all experiments
were carried out at room temperature (23ꢀ38C). Blocking studies were
A papain stock solution was prepared in 1 mm HCl. For daily activation,
the papain stock solution was diluted 1:100 in 0.1m sodium phosphate
pH 6.5, containing 2.5 mm EDTA and 15 mm DTT and was incubated at
238C for 1 h. The activated enzyme was kept on ice.
Inhibition of papain enzyme was assayed at 238C with the chromogenic
substrate Z-Phe-Arg-NHNp (200 mm) in the presence of increasing con-
centrations of the inhibitory peptides 4, 9 and 10. The buffer solution
used for the assay was 0.1m sodium phosphate pH 6.2, 2.5 mm EDTA,
300 mm DTT, and 30% DMSO. Rates were determined by duplicate
measurements of seven different concentrations of each single inhibitor.
The concentration of the liberated p-nitroaniline (pNA) was monitored
spectrophotometrically by measuring the absorbance at l=405 nm
against a blank sample containing no enzyme and an extinction coeffi-
cient of e=9.96 cm2 mmolꢁ1. The final volume made up to 1 mL. The reac-
tion was initiated by addition of the enzyme (20 mL); its final concentra-
carried out in a solution containing 0.1 mm [Ru(NH3)6Cl3] and 0.1n
G
HClO4, see Supporting Information, Figure S6.
Studies using the electrochemical quartz crystal microbalance: An
EQCM system interfaced to a PC (CH instruments 440) was used for re-
cording the frequency change during the process of protein adsorption
onto the peptide film. The EQCM electrodes with a fundamental reso-
nant frequency of 8 MHz and 13.7mm diameter were of the AT-cut type,
with optically polished surfaces coated on both sides with 200 nm thick
gold layer, 5 mm diameter, over a thin chromium adhesion mediator film.
Since the EQCM operated in time-resolved mode, the frequency differ-
ence of the working crystal and the reference crystal was measured. The
relationship between the changes in mass per unit area (m) and frequen-
cy (f) are given by Sauerbrey equation [Eq. (5)]:[56]
tion catalyze the conversation of the substrate with
a rate of 1–
2 mmminꢁ1. Progress curves were monitored over 10 min. A control assay
to determine the total hydrolysis of Z-Phe-Arg-NHNp (480 mm) was car-
ried out in the absence of the inhibitors (70 mL DMSO was added to the
cuvette). The papain concentration was 20-fold higher than the inhibition
assays. The resulting activity was considered as 100%. A Lineweaver–
Burk plot was constructed and the value of the Michaelis–Menten con-
stant (Km) was determined. The apparent inhibition constants Ki’ were
determined by fitting equation 3[26] to the experimental data.
ꢁ2Dmnf02
ð5Þ
Df
¼
A
pffiffiffiffiffiffiffiffiffiffi
mqpq
#
0
where A is the surface area of the electrode, mq is shear modulus of
quartz and 1q is the density of quartz.
# ¼
ð3Þ
1 þ ½IŠ=Ki0
5894
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ꢀ 2007Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2007, 13, 5885 – 5895

Electrochemical Detection of Proteins
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Received: December 24, 2006
Published online: April 23, 2007
Chem. Eur. J. 2007, 13, 5885 – 5895
ꢀ 2007Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
5895