Biofunctionalization of multiwalled carbon nanotubes by irradiation of electropolymerized poly(pyrrole-diazirine) films

Article abstract of DOI:10.1002/chem.201300873

A photoactivatable poly(pyrrole-diazirine) film was synthesized and electropolymerized as a versatile tool for covalent binding of laccase and glucose oxidase on multiwalled carbon nanotube coatings and Pt, respectively. Irradiation of the functionalized nanotubes allowed photochemical grafting of laccase and its subsequent direct electrical wiring, as illustrated by the electrocatalytic reduction of oxygen. Moreover, covalent binding of glucose oxidase as model enzyme, achieved by UV activation of electropolymerized pyrrole-diazirine, allowed a glucose biosensor to be realized. This original method to graft biomolecules combines electrochemical and photochemical techniques. The simplicity of this new method allows it to be extended easily to other biological systems. A photoactivatable polymer that grafts biomolecules: A simple procedure to fabricate a photoactivatable polymer that uses diazirine groups to attach biomolecules covalently under UV irradiation is reported. For this purpose, a compound containing diazirine and pyrrole groups was synthesized, electropolymerized, and then irradiated in the presence of proteins on both carbon nanotubes and platinum electrodes (see figure). Copyright

Full text of DOI:10.1002/chem.201300873

FULL PAPER
DOI: 10.1002/chem.201300873
Biofunctionalization of Multiwalled Carbon Nanotubes by Irradiation of
Electropolymerized Poly(pyrrole–diazirine) Films
Vladislav Papper,[a] Karine Gorgy,[b] Kamal Elouarzaki,[b] Ayrine Sukharaharja,[a]
Serge Cosnier,*[b] and Robert S. Marks[a, c]
Abstract: A photoactivatable poly(pyr-
role–diazirine) film was synthesized
and electropolymerized as a versatile
tool for covalent binding of laccase and
glucose oxidase on multiwalled carbon
nanotube coatings and Pt, respectively.
Irradiation of the functionalized nano-
tubes allowed photochemical grafting
of laccase and its subsequent direct
electrical wiring, as illustrated by the
electrocatalytic reduction of oxygen.
Moreover, covalent binding of glucose
oxidase as model enzyme, achieved by
UV activation of electropolymerized
pyrrole–diazirine, allowed glucose
biosensor to be realized. This original
method to graft biomolecules combines
electrochemical and photochemical
techniques. The simplicity of this new
method allows it to be extended easily
to other biological systems.
a
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
Keywords: electrochemistry
· en-
zymes · immobilization · nanotubes ·
polymers
Introduction
between biomolecules and polymers through irradiation of
electropolymerized photoactivatable films.[7–10] The binding
is stronger than that resulting from affinity interactions, and
the procedure is spatially addressable by light and faster
than those based on conventional covalent binding through
activated ester groups.
Due to their large specific surface area, nanowire morpholo-
gy, and intrinsic conductivity, carbon nanotubes are attract-
ing wide attention for developing biointerfaces devoted to
energy conversion in biosensing applications. In particular,
carbon nanotube coatings were widely used as an electroac-
tive skeleton for creating highly porous three-dimensional
biological networks.[1] Among the different procedures for
biofunctionalization of carbon nanotubes, the electrochemi-
cal polymerization of organic films is an attractive approach
thanks to the electrochemical addressing of polymers. Due
to this property, the film completely and uniformly covers
the walls of the nanotubes.[2] Biomacromolecules were im-
mobilized by an entrapment process during polymer growth
or by a subsequent step based on affinity interactions.[3–6]
However, these approaches have the disadvantage of low
accessibility of the entrapped biomolecules or a possible loss
of biomolecules due to the reversible character of affinity
In this context, we report here the synthesis and electro-
polymerization of a pyrrolic diazirine derivative and the use
of the resulting polypyrrole film for the irreversible photo-
binding of glucose oxidase and laccase on MWCNT coat-
ings. The two enzymes glucose oxidase and laccase were
chosen as model proteins to illustrate the photografting
properties of the polymer, since their immobilization is
easily demonstrated through their catalytic activity. This im-
mobilization method combines the advantages of the elec-
trochemical addressing of the polymer with spatial localiza-
tion of the protein by light. Compared to the first example
of an electropolymerized film, namely, poly(pyrrole–benzo-
phenone), which allowed reagentless covalent grafting of
proteins on irradiation, the diazirine group exhibits better
photostability and photoreactivity than benzophenone.[11–12]
interactions. A new interesting research direction in bio
cule immobilization lies in creating direct covalent binding
ACHTUNGTNERmNUNG ol-
ACHTUNGTRENNUNGeACHTUNGTRENNUNG
À
In particular, diazirine can react not only with C H but also
À
À
with N H and O H bonds and thus widens the field of ap-
plications. Moreover, compared to conventional photoactive
groups used for photografting molecules to solid supports,
such as diazo and diazopyruvate derivatives, azides and
phenyl azides, tetrazoles, and benzophenone, diazirine has
several advantages.[13–18] Diazirine is more easily and more
efficiently activated by UV radiation of wavelength 330–
370 nm, which preserves the native structure and activity of
immobilized proteins (Scheme 1). In contrast to azides, dia-
zirine is also essentially nontoxic and has better photostabil-
ity. In addition, compared with benzophenone, diazirine
does not require long irradiation times, which can cause seri-
ous damage to the protein configuration.
[a] Dr. V. Papper, A. Sukharaharja, Prof. R. S. Marks
NTU CREATE, Research Wing #02-06
Nanyang Technological University, 138602 (Singapore)
[b] Dr. K. Gorgy, Dr. K. Elouarzaki, Dr. S. Cosnier
Dꢀpartement de Chimie Molꢀculaire
UMR-5250, ICMG FR-2607, CNRS
Universitꢀ Joseph Fourier, 38041 Grenoble (France)
Fax : (+33)456520805
E-mail: Serge.Cosnier@ujf-grenoble.fr
[c] Prof. R. S. Marks
The Department of Biotechnology Engineering
The National Institute for Biotechnology in the Negev
The Ilse Kats Institute for Nanoscale Science and Technology
The Ben Gurion University of the Negev, Beer Sheva, 84105 (Israel)
Chem. Eur. J. 2013, 19, 9639 – 9643
ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
9639

Figure 1. Cyclic voltammogram of 1 (2ꢂ10À3 molLÀ1) in CH3CN+TBAP
(0.1 molLÀ1) recorded at a Pt electrode (5 mm diameter). Inset: Cyclic
voltammogram of poly(pyrrole–diazirine) (obtained by potentiostatic
method at 0.90 V, Q=1 mC) and transferred into CH3CN+0.1 molLÀ1
TBAP free of monomer.
Ag/Ag+), which corresponds to the well-known oxidation of
the pyrrole group (Figure 1, inset). Polymerization of 1 (2ꢂ
10À3 molLÀ1) was carried out by a galvanostatic method or
by controlled-potential electrolysis of the monomer in
CH3CN+0.1 molLÀ1 TBAP. The resulting electrode was
transferred with thorough rinsing to a CH3CN solution free
of monomer. The cyclic voltammogram of this electrode ex-
hibits a poorly reversible peak system at 0.40 V reflecting
the electroactivity of the formed polypyrrole (Figure 1). The
apparent surface coverage of poly(pyrrole–diazirine) of
1.25ꢂ10À8 molcmÀ2 was determined from the charge record-
ed under the oxidation wave of the polypyrrole backbone
(electropolymerization yield: 56%).
Scheme 1. Schematic of biomolecule photografting onto films of poly-
ACHTUNGTRENNUNG(pyrrole–diazirine) electrogenerated on a Pt or MWCNT electrode.
Results and Discussion
Synthesis and characterization of pyrrole–diazirine 1: Pyr-
role-functionalized diazirine monomer 1 was prepared by
amide coupling of N-(3-aminopropyl)pyrrole with NHS-dia-
zirine in the presence of 4-dimethylaminopyridine (DMAP)
in dichloromethane (Scheme 2).The pyrrolic diazirine deriv-
ative was purified by silica-gel chromatography and charac-
terized by 1H NMR spectroscopy, MALDI-TOF MS, and
cyclic voltammetry.
Characterization of the biomaterial functionalized by glu-
cose oxidase (GOx): With the aim of demonstrating its pho-
tochemical grafting properties, polyACTHNUTRGNEU(GN pyrrole–diazirine) and a
control polymer based on the same skeleton but without di-
azirine groups were irradiated in the presence of glucose ox-
idase (GOx), an enzyme chosen as protein model. A solu-
tion of GOx (20 mL, 4 mgmLÀ1) was incubated with poly-
ACHUTNGRENU(NG pyrAHCUTNGTRENrNNGU ole–diazirine) and regular polypyrrole films under irra-
diation for 10 min. This enzyme catalyzes the oxidation of
glucose in the presence of oxygen with production of H2O2.
The activity of grafted GOx can thus be monitored in phos-
phate buffer by the electrochemical oxidation of H2O2 at
the underlying electrode surface potentiostated at 0.7 V (vs.
SCE).
Figure 2 shows the anodic current response of the enzyme
electrodes as a function of glucose concentration. The sensi-
tivity of the poly(pyrrole–diazirine)/GOx electrode deter-
mined by the slope of the linear part of the calibration
curve is 0.49 mAmolÀ1 LcmÀ2 with a maximum current den-
sity (Jmax) of around 24 mAcmÀ2. This sensitivity appears to
be similar to or higher than those previously reported for
the formation of a compact enzyme monolayer based on af-
Scheme 2. Synthesis of pyrrole–diazirine 1.
The electrochemical behavior of pyrrolic diazirine 1 (2ꢂ
10À3 molLÀ1) was investigated in CH3CN+0.1 molLÀ1 tetra-
butylammonium perchlorate (TBAP). On oxidative scan-
ning, monomer 1 exhibits an irreversible peak at 1.05 V (vs.
finity interactions between biotinylated polymers and bio
ACHTUNGTRENNUNGtin-
AHCTUNGTRENNUNG
9640
www.chemeurj.org
ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2013, 19, 9639 – 9643

Biofunctionalization of Multiwalled Carbon Nanotubes
FULL PAPER
+
C
its stable cation radical form ABTS at pH 5. This process
was monitored by UV/Vis spectroscopy; absorption bands
at 420 nm characteristic of the one-electron oxidation of
ABTS (associated with a shoulder at 650 nm) appear and in-
crease with time (Figure 3). The presence of an isosbestic
point at 375 nm demonstrates the selectivity of the oxida-
tion.
Figure 2. Calibration curves for glucose a) at an irradiated polypyrrole/
GOx electrode, b) at a poly(pyrrole–diazirine)/GOx electrode, and c) at
an irradiated poly(pyrrole–diazirine)/GOx electrode in 0.1 molLÀ1 phos-
phate buffer solution (pH 7.0). Applied potential: 0.7 V vs. SCE.
Moreover, the maximum current density was higher than
or similar to those reported for these modified bioelectrodes
or for poly(pyrrole–nitrilotriacetic acid)/Cu2+/histidine-
tagged GOx or between biotinylated polymers and b-cyclo-
dextrin-tagged GOx (3.4–23 mA cmÀ2).[21,22] Since the Jmax
value is directly proportional to the enzyme loading, these
results illustrate efficient covalent binding of GOx and good
permeability of the poly(pyrrole–diazirine) for H2O2. More-
over, the apparent KM value (29 mmolLÀ1) obtained for this
enzyme electrode is similar to that reported for GOx in sol-
ution (KM =33 mmolLÀ1), which reflects the absence of de-
naturation due to photochemical grafting.[23]
In contrast, performing the same incubation and irradia-
tion process on regular polypyrrole film led to a modified
electrode with almost no electroenzymatic activity, and thus
the role of diazirine is corroborated (Figure 2).
A control experiment was carried out in which poly(pyr-
role–diazirine) and GOx were exposed to visible light with-
out specific irradiation. Surprisingly, the resulting modified
electrode provided sensitivity and Jmax values of 0.48 mA
molÀ1 LcmÀ2 and 11 mAcmÀ2, respectively. Although the Jmax
value, which is directly proportional to the enzyme loading,
is markedly lower than that observed with irradiation, these
values reflect the ease of diazirine photoactivation.
Figure 3. Activity of laccase immobilized by irradiation on MWCNT elec-
trodes previously modified with poly(pyrrole–diazirine). Enzymatic activ-
ity was evaluated by UV/Vis spectroscopy at l=420 nm in acetate buffer
(0.1 molLÀ1, pH 5) containing ABTS (1.48ꢂ10À4 molLÀ1) for 15 min.
By comparison with spectrophotometric measurements
performed with the free enzyme, the bioelectrode exhibits
an immobilized activity of 3.2 UmgÀ1. This corresponds to
the immobilization of 60.7 mgcmÀ2 of laccase, assuming that
the immobilized enzyme retains its full activity.
Since laccase and avidin have the same molecular weight
(66 kDa), a close-packed single-molecular layer of laccase
should correspond, by analogy, to 3.3ꢂ1012 moleculescmÀ2
or 362 ngcmÀ2. Hence, the photochemical grafting process
results in the immobilization of 5.53ꢂ1014 moleculescmÀ2 of
laccase corresponding to 167 equivalent compact monolay-
ers of laccase and thus indicates a three-dimensional struc-
ture of the conductive composite biocoating.
The performance of the resulting composite bioelectrode
in oxygen reduction was investigated by cyclic voltammetry
in acetate buffer (pH 5). In presence of oxygen, a catalytic
cathodic current appeared at 0.61 V (vs. SCE), close to the
redox potential of laccase (Figure 4A). Chronoamperomet-
ric measurements at 0.3 V demonstrated the presence of a
steady-state catalytic current (303 mAcmÀ2) reflecting the ac-
tivity of the immobilized laccase (Figure 4B). This corrobo-
rates the efficient photografting of laccase and reflects also
the direct electrical connection of this enzyme by laccase/
poly(pyrrole–diazirine)/MWCNT/GC. The latter shows an
electroenzymatic activity of 47 mUcmÀ2 towards oxygen re-
duction and corresponds to 25% of the preceding spectro-
photometric enzyme activity. This difference may be ascri-
bed to the thickness of the polypyrrolic film, which could
Photografting of laccase: To validate this procedure for the
functionalization of carbon nanotube deposits by proteins, a
poly(pyrrole–diazirine) film was electropolymerized by con-
trolled-potential
electrolysis
(Eapplied =0.9 V,
Q=
12.5 mCcmÀ2) onto elec
trodes modified by a MWCNT coat-
ing and irradiated in presence of laccase. This enzyme cata-
lyzes the reduction of oxygen and has been widely exploited
for the elaboration of biocathodes and hence the fabrication
of biofuel cells. The enzyme activity of the resulting nano-
structured bioelectrode was investigated in the presence of
2,2’-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS). In
the presence of oxygen, ABTS is enzymatically oxidized to
Chem. Eur. J. 2013, 19, 9639 – 9643
ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
9641

S. Cosnier et al.
Figure 5. Evolution of the open-circuit potential of a laccase/poly(pyr-
role–diazirine)/MWCNT/GC electrode under oxygen-saturated condi-
tions in acetate buffer (0.1 molLÀ1, pH 5).
immobilization of proteins by formation of strong and stable
chemical bonds. This procedure combines the advantages of
light-dependent immobilization with those related to the
electrochemical addressing of polymers, in particular, the
possibility of precisely electrogenerating a photoreactive
polymer film on nanoobjects such as carbon nanotubes. The
potential of this fast and generally applicable photoelectro-
chemical approach to biomolecule immobilization for bio-
sensor fabrication was illustrated with GOx as enzyme
model. The resulting biosensor characteristics showed the
efficiency of this concept, and nondenaturation of the cata-
lytic properties of GOx under UV irradiation was demon-
strated. As illustrated with laccase, this method is an effi-
cient and fast way for the functionalization of carbon nano-
tube deposits by proteins. Moreover, the photoactivatable
poly(pyrrole–diazirine) allows direct electron transfer be-
tween MWCNT and redox enzymes. It is thus expected that
this new reagentless procedure for immobilization of biolog-
ical macromolecules on three-dimensional carbon nano-
structures will be useful for the development of bioarchitec-
tures exhibiting extremely high biological activities
Figure 4. A) Laccase/poly(pyrrole–diazirine)/MWCNT/GC
(3 mm diameter disk) in 0.1 molLÀ1 acetate solution (pH 5.0) without
(dashed line) and with oxygen (straight line). Scan rate: 10 mVsÀ1
B) Chronoamperometric response of the laccase/poly(pyrrole–diazirine)/
MWCNT/GC electrode potentiostated at 0.3 V (vs. SCE) in 0.1 molLÀ1
acetate buffer (pH 5.0) before and after oxygen saturation.
electrode
.
partly prevent direct electron transfer. Moreover, photo-
chemical grafting of laccase can lead to random orientation
of the electroactive T1 copper center. The location of the
active center opposite to the surface of the electrode can
thereby prevent electron exchange with the electrode sur-
face. In contrast, the freely diffusing ABTS can assure elec-
trical wiring of all immobilized enzymes whatever their posi-
tion on the polymer and thus lead to higher enzyme activity.
Experimental Section
Materials: Acetonitrile (HPLC grade) used for electrochemistry meas-
urements was obtained from Rathburn and used without further modifi-
cation. Tetrabutylammonium perchlorate (TBAP, Fluka) was used as the
supporting electrolyte in organic media. Glucose oxidase (GOx,
168 UmgÀ1), laccase (13.6 UmgÀ1), and glucose were purchased from
Sigma. All reagents and chemicals purchased from Aldrich were of re-
agent-grade quality and used as received unless otherwise mentioned.
Commercial-grade thin multiwalled carbon nanotubes (MWNTs, 9.5 nm
diameter, purity >95%), obtained from Nanocyl, were used as received
without any purification step. NHS-diazirine (succinimidyl 4,4-azipenta-
noate) was purchased from Thermo Scientific.
Moreover, under oxygen-saturated conditions,
a stable
open-circuit potential of 0.61 V was reached for more than
1 h (Figure 5).
Conclusion
We have demonstrated easy and electrochemically control-
led elaboration of a poly(pyrrole–diazirine) film and the
suitability of the polymerized diazirine for photochemical
For laccase experiments, the glassy carbon (GC) disk used as working
electrode was polished, rinsed, and then sonicated with water before
each measurement.
9642
www.chemeurj.org
ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2013, 19, 9639 – 9643

Biofunctionalization of Multiwalled Carbon Nanotubes
FULL PAPER
The MWCNT electrode was fabricated by using the following method:
MWCNTs (5 mg) were added to 1 mL of N-methyl-2-pyrrolidone
(NMP). The suspension was homogenized by an ultrasonic generator for
30 min. For electrochemical investigations, a portion (10 mL) of this sus-
pension was dropped onto a freshly polished GC disk electrode (3 mm
diameter) and the solvent was removed under reduced pressure. The
electroactive surface was estimated as previously reported.[24]
surfaces and transduction” of the scientific structure “Nanobio campus”
for providing facilities and Arielle Le Pellec for technical assistance and
fruitful discussions. The authors thank Huang Ruo Cheng, Tan Chong
Yuan, and Terence Mak of River Valley High School for their active par-
ticipation in this project in the frame of the program for Singapore Sci-
ence & Engineering Fair. This research was funded in part by the Singa-
pore National Research Foundation and the publication is supported
under the Campus for Research Excellence and Technological Enterprise
(CREATE) program.
For cross-linking reactions between enzymes and diazirine, 20 mL of
enzyme solution (5 mgmLÀ1 laccase or 4 mgmLÀ1 GOx) was deposited
on modified electrodes and then irradiated with an Hg/Xe lamp (Oriel
Instruments) through an optical fiber for 10 min. Filters from Oriel were
used to select the wavelength and to block IR rays to prevent overheat-
ing of the optical fiber.
[1] A. Le Goff, M. Holzinger, S. Cosnier, Electrochim. Acta 2011, 56,
3633–3640.
Instruments: Electropolymerization and cyclic voltammetric experiments
were performed with an Autolab Potentiostat 100 (Ecochemie, Utrecht,
the Netherlands). All electrochemical experiments were carried out in a
conventional three-electrode cell. The amperometric measurements were
performed with a Tacussel PRG-DL potentiostat in phosphate buffer sol-
ution (0.1 molLÀ1, pH 7) or with an Autolab Potentiostat 100 in acetate
buffer (0.1 molLÀ1, pH 5). All enzyme electrodes were prepared on bare
platinum electrodes or on electrodes previously modified with an
MWCNT deposit. An aqueous saturated calomel electrode (SCE) was
used as reference electrode, and a Pt wire placed in a separate compart-
ment containing the aqueous electrolyte as counter electrode. An Ag+
/Ag (10À2 molLÀ1) electrode was used as reference in acetonitrile.
[2] A. Le Goff, S. Cosnier, J. Mater. Chem. 2011, 21, 3910–3915.
[3] S. Cosnier, R. Ionescu, M. Holzinger, J. Mater. Chem. 2008, 18,
5129–5133.
[4] R. Haddad, M. Holzinger, A. Maaref, S. Cosnier, Electrochim. Acta
2010, 55, 7800–7803.
[5] J. Baur, A. Le Goff, S. Dementin, M. Holzinger, M. Rousset, S. Cos-
nier, Int. J. Hydrogen Energy 2011, 36, 12096–12101.
[6] A. Le Goff, K. Gorgy, M. Holzinger, R. Haddad, M. Zimmerman, S.
Cosnier, Chem. Eur. J. 2011, 17, 10216–10221.
[7] T. Konry, A. Novoa, S. Cosnier, R. S. Marks, Anal. Chem. 2003, 75,
2633–2639.
[8] T. Konry, B. Hadadc, Y. Shemer-Avni, S. Cosnier, R. S. Marks, Ta-
lanta 2008, 75, 564–571.
[9] G. Herzog, K. Gorgy, T. Gulon, S. Cosnier, Electrochem. Commun.
2005, 7, 808–814.
[10] R. E. Ionescu, S. Cosnier, G. Herzog, K. Gorgy, G. Leshem, S. Her-
mann, R. S. Marks, Enzyme Microb. Technol. 2007, 40, 403–408.
[11] E. W. S. Chan, S. Chattopadhaya, R. C. Panicker, X. Huang, S. Q.
Yao, J. Am. Chem. Soc. 2004, 126, 14435–14446.
[12] E. Baslꢀ, N. Joubert, M. Pucheault, Chem. Biol. 2010, 17, 213–227.
[13] K. H. Hong, N. Liu, G. Sun, Eur. Polym. Mater. 2009, 45, 2443.
[14] A. Wirsꢀn, H. Sun, L. Emilsson, A. C. Albertsson, Biomacromole-
cules 2005, 6, 2281–2289.
Synthesis: The pyrrole amine was prepared by reduction of 1-(2-cyanoe-
thyl)pyrrole with lithium aluminum hydride in diethyl ether. A suspen-
sion of LiAlH4 (35 mL, 1.0 molLÀ1 in diethyl ether) was stirred under a
nitrogen stream. A solution of 1-(2-cyanoethyl)pyrrole (7.2 g) in anhy-
drous diethyl ether (35 mL) was then added dropwise. The resulting solu-
tion was heated to reflux for 18 h under nitrogen. The resultant reaction
mixture was allowed to cool, and excess LiAlH4 was destroyed by succes-
sive addition of water (3.5 mL), 15% NaOH solution (3.5 mL), and
water (10 mL). The organic layer was separated, heated to 408C for 2 h,
and filtered through powdered Celite/anhydrous sodium sulfate (1/1).
The filtrate was washed twice with diethyl ether (20 mL) and the result-
ing solution evaporated in vacuo providing a yellow oil, which was used
in the next reaction step without further purification.
[15] C. Geismann, A. Yaroshchuk, M. Ulbricht, Langmuir 2007, 23, 76–
83.
[16] L. Wang, Y. Yu, L. Liu, W. Yang, J. Appl. Polym. Sci. 2007, 106,
621–629.
[17] G. Li, G. He, Y. Zheng, X. Wang, H. Wang, J. Appl. Polym. Sci.
2012, 123, 1951–1959.
[18] A. Ovsianikov, Z. Li, J. Torgersen, J. Stampfl, R. Liska, Adv. Funct.
Mater. 2012, 22, 3527–3527.
[19] S. Cosnier, B. Galland, C. Gondran, A. Le Pellec, Electroanalysis
1998, 10, 808–813.
A mixture of N-(3-aminopropyl)pyrrole (0.25 g), NHS-diazirine (0.45 g),
and 4-dimethylaminopyridine (0.08 g) in dichloromethane (10 mL) was
stirred in a flask covered with aluminum foil to prevent irradiation of the
product under nitrogen at room temperature for 5 days. The resulting sol-
ution was filtered, and dichloromethane was removed in vacuo. The re-
sulting crude product was purified by column chromatography on silica
gel eluted with dichloromethane to give a colorless oil in 57% overall
yield. 1H NMR (Bruker, 400 MHz, CDCl3): d=1.02 (s, 3H), 1.72–1.97
(m, 6H), 3.25 (d, 2H), 3.96 (d, 2H), 5.29 (s, 1H), 6.18 (d, 2H), 6.68 ppm
(d, 2H); MS (MALDI-TOF): m/z: 234.30.
[20] S. Cosnier, A. Le Pellec, Electrochim. Acta 1999, 44, 1833–1836.
[21] N. Haddour, S. Cosnier, C. Gondran, J. Am. Chem. Soc. 2005, 127,
5752–5753.
[22] M. Holzinger, M. Singh, S. Cosnier, Langmuir 2012, 28, 12569–
12574.
[23] B. E. P. Swoboda, V. Massey, J. Biol. Chem. 1965, 240, 2209–2215.
[24] K. Elouarzaki, A. Le Goff, M. Holzinger, J. Thery, S. Cosnier, J.
Am. Chem. Soc. 2012, 134, 14078–14085.
Acknowledgements
The authors thank the ANR Emergence-2010 EMMA-043-02 for partial
financial support. The authors thank the platform “functionalization of
Received: March 6, 2013
Published online: June 10, 2013
Chem. Eur. J. 2013, 19, 9639 – 9643
ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
9643