Coumarin-conjugated multiwalled carbon anotubes for potential biological applications: Development and characterization

Article abstract of DOI:10.1166/jnn.2012.4929

This work presents a novel cascade of chemical functionalization of multiwalled carbon nanotubes which allows the conjugation with differently substituted coumarins. Aim of the present work is to synthesize new materials able to rescue cells from the adverse effect of CNT particles since pristine CNTs are practically insoluble and tend to accumulate inside cells, organs and tissues. Moreover, it was reported that single walled CNTs particles show an adverse effect on keratinocytes through an oxidative mechanism, leading to NF-kB activation. The conjugation with coumarins, known superoxide anion scavengers, could switch the cytotoxicity of the new materials. The cascade functionalization of MWCNTs by sequential steps of carboxylation, acylation, amine modification and finally, coumarin conjugation have been performed and the synthesis and the chemical properties of several f-MWCNTs-coumarins have been exploited. Copyright

Full text of DOI:10.1166/jnn.2012.4929

Copyright © 2012 American Scientific Publishers
All rights reserved
Printed in the United States of America
Journal of
Nanoscience and Nanotechnology
Vol. 12, 5030–5038, 2012
Coumarin-Conjugated Multiwalled Carbon
Nanotubes for Potential Biological Applications:
Development and Characterization
D. Iannazzo1ꢀ∗, A. Piperno1, G. Romeo1, R. Romeo1, A. Ferlazzo1,
A. Pistone2, M. Lanza3, and C. Milone2
1Dept. Farmaco-Chimico, University of Messina, I-98168, Messina, Italy
2Dept. of Industrial Chem. Materials Engineering, University of Messina, I-98166 Messina, Italy
3CNR, Institute for Chemical Physical Processes, I-98123 Messina, Italy
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This work presents a novel cascade of chemical functionalization of multiwalled carbon nanotubes
which allows the conjugation with differently substituted coumarins. Aim of the present work is
Rice University, Fondren Library
to synthesize new materials able to rescue cells from the adverse effect of CNT particles since
IP : 93.80.195.225
pristine CNTs are practically insSoluunbl,e0a7ndOtcetn2d0to12ac0c7u:m2u5la:3te0inside cells, organs and tissues.
Moreover, it was reported that single walled CNTs particles show an adverse effect on keratinocytes
through an oxidative mechanism, leading to NF-kB activation. The conjugation with coumarins,
known superoxide anion scavengers, could switch the cytotoxicity of the new materials. The cascade
functionalization of MWCNTs by sequential steps of carboxylation, acylation, amine modification and
finally, coumarin conjugation have been performed and the synthesis and the chemical properties
of several f-MWCNTs-coumarins have been exploited.
Keywords: Multi Walled Carbon Nanotubes, Coumarins, Nanotoxicity, CCVD, Chemical
Functionalization.
1. INTRODUCTION
for more versatile suspension capabilities and realization
of certain applications, the most important being their
molecular interactions with biological systems.12 Moreover
functionalization remarkably reduces the cytotoxic effects
of CNT.13
A great scientific and technological interest on carbon
nanotubes (CNTs) has been recently registered as a result
of their fascinating properties and their ability to serve
as candidate for a variety of therapeutic and diagnostic
applications.1–4 Besides the ability of functionalized car-
bon nanotubes (f -CNT) to act as carriers for the delivery
of a wide range of therapeutic agents, f -CNTs-drug can
be also considered as a new entity with novel therapeu-
tic or diagnostic properties.5–7 For successful applications
in the biomedical field, bioeffects and safety of CNTs to
the environment and human health need to be addressed.
In this context, research into the toxicity of nanomaterials,
generally referred as “nanotoxicity,” is being carried out,
but at a far slower pace than research into how nanomate-
rials can be employed.8–10
Coumarin derivatives exhibit a broad range of biolog-
ical effects including anticoagulation, antibiotic, antifun-
gal, antipsoriasis, cytotoxic, anti-HIV, anti-inflammatory
activities.14–16 In particular, substituted hydroxycoumarins
are potent superoxide anion scavengers, are able to inhibit
in vitro lipid peroxidation and act as NF-kB inhibitors.17–18
In order to synthesize new material able to rescue cells
from the adverse effect of CNT particles, we have focused
our attention on the synthesis of new f -CNTs linked to
natural antioxidants with a coumarin structure. We have
performed the cascade functionalization of MWCNTs by
sequential steps of carboxylation, acylation, amine modi-
fication and finally, coumarin conjugation. The synthesis
and the chemical properties of several f-CNTs-coumarins
has been exploited (Fig. 1).
It has been reported that single walled CNTs particles
show an adverse effect on keratinocytes through an oxida-
tive mechanism leading to NFkB activation.11 Chemical
modification of CNTs by functionalization is often required
Coumarins are spectrophotometer detectable biologi-
cally active molecules and their conjugation with CNTs,
beside the improvement of the biocompatibility, could
∗Author to whom correspondence should be addressed.
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1533-4880/2012/12/5030/009
doi:10.1166/jnn.2012.4929

Iannazzo et al.
Coumarin-Conjugated Multiwalled CNTs for Potential Biological Applications
Fig. 1. Coumarin-conjugated MWCNTs.
allow an active recognition site at the surface of the
nanocarrier.
synthesis the silica support was removed by a solution of
KOH (1 M) at 105 C and the resulting material, washed
ꢀ
with distilled water, was treated with a solution of HCl
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(1 M) in order to remove the remaining iron particles
and subsequently washed with distilled water and dried at
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2. EXPERIMENTAL DETAILS
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ꢀ
200 C to obtain the pristine MWCNTs wich were used
2.1. Materials
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in the subsequently functionalizations. The purity of pris-
tine nanotubes was quantified by thermogravimetric anal-
Solvents were used as received from Carlo Erba Reagents;
reagents were purchased from Sigma-Aldrich Co.; silica
gel was purchased from Merck Chemicals. Sonication was
carried out in a batch using Bandeling Sonorex Type
RK52H equipment. Nuclear magnetic resonance spectra
(1H NMR recorded at 300 MHz, 13C NMR recorded at
75 MHz) were obtained on Varian Instruments and are
referenced in ppm relative to TMS or the solvent signal.
Thin-layer chromatographic separations were performed
on Merck silica gel 60-F254 precoated aluminum plates.
Flash chromatography was accomplished on Merck sil-
ica gel (200–400 mesh). Preparative separations were car-
ried out by MPLC Büchi C-601 using Merck silica gel
0.040–0.063 mm and the eluting solvents were delivered
ꢀ
ysis: pristine MWCNTs are stable below 800 C with a
weight loss ≈2%; thus the purity of synthesized CNTs
was extimated >95% . All the diagnostics analyses were
performed on purified samples.
2.2.2. Carboxylation of Purified MWCNTs
500 mg of pristine MWCNTs, sonicated in a water bath
(60 W, 35 kHz), in 100 mL of sulfuric acid/nitric acid
ꢀ
(3:1 v/v, 98% and 69% respectively) were stirred at 60 C
for 6 h. 500 mL of deionized water was then added and the
mixture was decanted for 2 h and separated from the heavy
MWCNT material. Then the supernatant (∼300 mL) was
recovered, filtered under vacuum on a 0.1 ꢀm Millipore
membrane, carefully washed with deionized water until pH
became neutral and dried. Light oxidized MWCNTs were
obtained in a yield of 40%. Heavy oxidated MWCNT were
recovered with a yield of 50%. Carboxylated MWCNTs
were titrated to determine the concentration of acidic sites
present according to literature method.20 Briefly, MWC-
by a pump at the flow-rate of 3.5–7.0 mL min−1
.
The morphological characterization was carried out by
SEM on a JEOL JSM 5600LV instrument, operating at
20 kV. HRTEM analysis was conducted on a 200 kV
JEOL JEM 2010 analytical electron microscope (LaB6
electron gun) equipped with a Gatan 794 Multi-Scan CCD
camera for digital imaging. UV spectra have been per-
formed by Thermo Nicolet mod. Evolution 500 spec-
trophotometer. TGA analysis have been performed with a
TA mod. SDTQ600 instrument. IR spectra were recorded
on a Nicolet FT-IR Impact 400 D spectrometer.
ꢀ
NTs were heated at 100 C to remove carbon dioxide and
water. Further carboxylated MWCNTs were added into
0.01 N sodium hydroxide and stirred for 48 h. The sample
was centrifuged at 10,000 rpm for 15 minutes; unreacted
NaOH was titrated with 0.01 N hydrochloridric acid. The
number of free OH groups was found to be 1.8 mmol/g.
2.2. Preparation of MWCNTs Acetyl Chlorides
2.2.1. Preparation of Pristine MWCNTs
2.2.3. Acylation of Carboxylated MWCNTs
MWCNTs were produced by the catalytic carbon vapor
deposition (CCVD) and were subjected to purification
according to previously reported procedure.19 Briefly, after
Carboxylated light MWCNTs (100 mg) were heated in
30 mL of neat oxalyl chloride at reflux for 48 h; the excess
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Coumarin-Conjugated Multiwalled CNTs for Potential Biological Applications
Iannazzo et al.
of volatile reagent was removed under reduced pressure to
give the corresponding MWCNTs acetyl chloride which
were used without further purification.
When the TLC revealed the absence of the starting materi-
als, the mixture was filtered, the residue was washed with
ethyl acetate and subjected to purification by MPLC using
a mixture of CHCl3/MeOH (98 :2) as eluent. The obtained
N-protected compound was deprotected with TFA fol-
lowing the procedure reported for the synthesis of com-
pound 2. Linker 5 was purified by MPLC using a mixture
CHCl3/MeOH (9 :1).
2.3. Preparation of the Linker 5
2.3.1. Synthesis of 2-(1,3-dioxoisoindolin-2-yl)ethyl
4-(2-aminoethyl)benzoate 2
2-(1,3-dioxoisoindolin-2-yl)ethyl 4-(2-(3-(2-(2-(2-ami-
noethoxy)ethoxy)ethyl)thioureido)ethyl) benzoate 5,
light yellow oil, 74% yield. H NMR (CD3OD, 300 MHz)
ꢁ 2.94 (t, 2H, J = 7ꢂ5 Hz), 3.13 (t, 2H, J = 5ꢂ0 Hz),
3.58–3.75 (m, 12H), 4.06 ( t, 2H, J = 5ꢂ0 Hz), 4.52 (t, 2H,
J = 5ꢂ0 Hz), 7.30 (d, 2H, J = 8 Hz), 7.78–7.86 (m, 6H,
Ar). 13C NMR (CD3OD, 75 MHz) ꢁ 34.9, 36.7, 39.3, 45.5,
45.9, 61.9, 66.4, 69.1, 69.9, 70.5, 110.7, 114.7, 118.5,
122.8, 127.6, 128.7, 129.3, 129.4, 130.1, 130.9, 134.1,
145.7, 158.9, 165.3, 167.5, 168.0 (See Figs. 7 and 8).
To a solution of 4-(2-aminoethyl)benzoic acid in a 1:1 mix-
ture dioxane/water (10 mL) and in the presence of NaOH
1 M, di-tert-butyl dicarbonate was added and the mixture
was left to stir for 1 h at r.t. After purification by flash
chromatography using a mixture CHCl3/MeOH (98/2), the
protected compound was reacted with ꢀcarbonyldiimida-
zole (CDI) in dioxane for 2 h at 100 C; then,1 eq. of
2-(2-hydroxyethyl) isoindoline-1,3-dione was added and
the mixture was stirred under reflux for 12 h. When the
TLC revealed the absence of the starting compound, the
solution was acidified at pH 5 using HCl 1 M. The mixture
was dried under vacuo and the residue was washed with a
1
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2.4. Preparation of Coumarins 8 and 9
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saturated solution of NaHCO3 and extracted with CHCl3.
The organic layer was dried and the residue was purified by
2.4.1. Synthesis of 5-oxyethyl- tert-butyl carbamate,
IP : 93.80.195.225
7-hydroxy coumarin 7
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MPLC using a mixture CHCl3/MeOH 98/2 as eluent. The
obtained compound as white solid (yield 70%) was depro-
tected using trifluoroacetic acid in CH2Cl2 for 12 h at r.t.
The residue was then dried under vacuo and purified using
a mixture CH3Cl/MeOH 9 :1. 2-(1,3-dioxoisoindolin-2-yl)
ethyl4-(2-aminoethyl) benzoate 2, yellow oil, 70% yield.
1H NMR (CD3OD, 300 MHz) ꢁ 3.12 (t, 2H, J = 6ꢂ5 Hz),
3.28 (t, 2H, J = 6ꢂ5 Hz), 4.23 (t, 2H, J = 4ꢂ2 Hz), 4.69
(t, 2H, J = 4ꢂ2 Hz), 7.50–8.07 (m, 8H, Ar).
To a solution of 5,7-dihydroxy-coumarin22 6 in THF
(20 mL), 2 eq of K2CO3 was added and the mixture was
left to stir at r.t for 15 min; then, 1eq of tert-butyl-2-
bromo-ethylcarbamate was added and the mixture stirred
under reflux for 12 h. The solvent was then removed
and the residue was subjected to purification by MPLC
using a mixture of 98/2 CHCl3/MeOH. Tert-butyl 2-(7-
hydroxy-2-oxo-2H-chromen-5-yloxy)ethylcarbamate 7,
yellow oil, 40% yield. 1H NMR (CDCl3, 300 MHz) ꢁ 1.62
(s, 9H), 3.65 (t, 2H, J = 5ꢂ5 Hz), 4.22 (t, 2H, J = 5ꢂ5 Hz),
5. 20 (bs, 1H), 5.80 (bs, 1H), 6.25 ( d, 1H, J = 8ꢂ5 Hz),
6.50 (s, 1H), 6.60 (s, 1H), 8.18 (d, 1H, J = 8ꢂ5)
2.3.2. Synthesis of tert-butyl 2-(2-(2-isothiocyanato-
ethoxy)ethoxy)ethylcarbamate 4
To a solution of diamine 3 (5 eq.) in dioxane (10 mL),
1 eq. of di-tert-butyl dicarbonate was added dropwise
and the mixture was left to stir for 2 h in an ice bath.
Then, water and chloroform were added; the organic layer
was concentrated in vacuo and the residue was sub-
jected to flash chromatography using as eluent a mixture
CHCl3/MeOH (98/2). The obtained monoprotected deriva-
tive (95% yield) was dissolved in ethanol (5 mL) and
reacted with carbon sulfide (10 eq) ꢀin the presence of tri-
ethylamine (1 eq) for 30 min at 0 C. Then, the mixture
was cooled in an ice bath, added with 1 eq of di-tert-butyl
dicarbonate and 2 mol% of DMAP and left under stirring
at r.t for 1 h. The solvent was then removed under vacuo to
obtain compound 4, in quantitative yield which was used
without further purification.
2.4.2. Synthesis of 5-isothiocyanatoethoxy-7-hydroxy
coumarin 8
Compound 7 was deprotected by reaction with 6 eq of tri-
fluoroacetic acid in CH2Cl2 for 12 h at r.t. The solvent was
then removed and the residue was subjected to flash chro-
matography using a 9 :1 mixture of CH3Cl/MeOH. 5-(2-
aminoethoxy)-7-hydroxy-2H-chromen-2-one, yellow oil,
1
80% yield. H NMR (CD3OD, 300 MHz) ꢁ 3.75 (t, 2H,
J = 5ꢂ5 Hz), 4.62 (t, 2H, J = 5ꢂ5 Hz), 6.25 ( d, 1H, J =
8ꢂ5 Hz), 6.60 (s, 1H), 6.62 (s, 1H), 8.45 (d, 1H, J = 8ꢂ5).
The purified deprotected compound was dissolved in
ethanol (10 mL), treated with 10 eq of carbon sulfide in
the presence of 1 eq of triethylamine and the mixture was
ꢀ
stirred for 30 min at 0 C. The mixture was then cooled
on an ice bath and then, 1 eq of di- tert-butyl dicarbon-
ate, dissoved in absolute ethanol (1 mL) and 2 mol% of
DMAP was added. The mixture was left to stir for 1 h at
r.t. Then, the solvent was removed under vacuum and the
residue, represented by isothiocyanatoethoxy coumarin 8
2.3.3. Synthesis of Linker 5
To a solution of compound 2, in ethanol (10 mL) 1 eq.
of compound 4 was added and the mixture was stirred at
r.t. for 12 h, in the presence of 1 eq. of triethylamine.
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Iannazzo et al.
Coumarin-Conjugated Multiwalled CNTs for Potential Biological Applications
and obtained in quantitative yield was used without further
purification.
acid to give (9Z)-5-(2-aminoethoxy)-2-oxo-2H-chromen-
7-yl octadec-9-enoate. H NMR (CD3OD, 500 MHz) ꢁ
1
0.89 (t, 3H, J = 6ꢂ9 Hz), 1.27 (m, 20H), 1.75 (qt, 2H,
J = 7ꢂ2 Hz), 2.05 (m, 4H), 2.60 (t, 2H, J = 7ꢂ2 Hz), 3.55
(t, 2H, J = 4ꢂ2 Hz), 4.20 (t, 2H, J = 4ꢂ2 Hz), 5.35 (m, 2H),
6.34 (d, 1H, J = 10ꢂ0 Hz), 6.77 (s, 1H), 6.81 (s, 1H), 8.32
(d, 1H, J = 10ꢂ0 Hz). 13C NMR (CD3OD, 125 MHz) ꢁ
36.3, 38.1, 40.7, 63.3, 67.9, 70.5, 71.3, 71.4, 124.1, 124.3,
129.2, 130.1, 130.2, 130.8, 130.9, 133.3, 135.4, 135.5,
146.6, 167.8, 169.7 (See Figs. 9 and 10). The reaction with
carbon sulfide, according to the above reported procedure,
gave the corresponding isothiocyanatoethoxy coumarin 8.
2.4.3. Synthesis of 5-isothiocyanatoethoxy-7-oleate
coumarin 9
To a solution of compound 7 in chloroform (20 mL)
1.2 eq. of Oleoyl chloride and 1.2 eq. of triethy-
lamine were added and the mixture was then stirred
for 12 h at r.t. The solvent was, then removed and the
residue was subjected to purification by MPLC using
a 98/2 mixture of CHCl3/MeOH to obtain (9Z)-5-(2-
aminoethoxy)-2-oxo-2H-chromen-7-yl octadec-9-enoate
ꢃN-Protected with tert-butyl carbamate), yellow oil, 70%
yield. 1H NMR (CDCl3, 300 MHz) ꢁ 0.89 (t, 3H,
J = 7ꢂ5 Hz), 1.27 (m, 20H), 1.45 (s, 9H), 1.73 (t, 2H,
J = 7ꢂ2 Hz), 2.01 (m, 4H), 2.18 (t, 2H, J = 7ꢂ2 Hz),
3.56 (t, 2H, J = 4ꢂ2 Hz), 4.10 (t, 2H, J = 4ꢂ2 Hz), 5.01
(bs, 1H), 5.35 (m, 2H), 6.29 (d, 1H, J = 9ꢂ5 Hz), 6.48
(s, 1H), 6.69 (s, 1H), 8.04 (d, 1H, J = 9ꢂ5 Hz). The
obtained compound was deprotected with trifluoroacetic
2.5. Preparation of Coumarin-Conjugated MWCNTs
To a mixture of MWCNTs acetyl chlorides (50 mg) in
THF (20 mL), 10 eq of the linker 5 (calculated by Kaiser
test21 on MWCNTs-NH2) and 10 eq of triethylamine were
added and the mixture was sonicated for 15 min and
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then left to stir at reflux for 48 h. The solvent was then
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removed and the functionalized MWCNTs were filtered
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by using Durapore Membrane Filter 0.1 ꢀm and washed
Sun, 07 Oct 2012 07:25:30
Fig. 2. Representative HRTEM images of a) pristine MWCNTs and b)
Fig. 3. FTIR Spectra of a) pristine MWCNTs and b) oxidized
oxidized MWCNTs.
MWCNTs.
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Coumarin-Conjugated Multiwalled CNTs for Potential Biological Applications
Iannazzo et al.
with MeOH and CHCl3 till disappearance of the starting
material. The so functionalized MWCNTs (50 mg) were
suspended in ethanol (20 mL) and then added with 10
eq of hydrated hydrazine and the mixture was stirred at
reflux for 24 h. MWCNTs-linker-NH2 dispersed in THF
dry (20 mL) added with 10 eq of coumarin 8 or 9 were
sonicated for 30 min and then left to stir at reflux for two
days. The solvent was then removed and the functional-
ized MWCNTs were filtered by using Durapore Membrane
Filter 0.1 ꢀm and washed with MeOH and CHCl3 till dis-
appearance of the starting material.
3. RESULTS AND DISCUSSION
MWCNTs, produced by catalytic carbon vapor deposi-
tion (CCVD), after purification according to a previously
reported procedure (purity >95%),19 exhibit a clear multi-
walled tube structure of 15–20 layers and the graphite
layers do not stay always continuous along the growth ori-
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entation in some regions, which results from point defects
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and faults between graphitic carbon planes . Moreover, the
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HRTEM analysis revealed an average length of 11–20 ꢀm,
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with a diameter close to 15–20 nm.
The pristine MWCNTs were oxidized with a mix-
ture of sulfuric acid/nitric acid (3:1 v/v, 98% and 69%
respectively) at 60 ꢀC for 6 h. Then, deionized water
was added and the supernatant was recovered, filtered
under vacuum on a 0.1 ꢀm Millipore membrane, care-
fully washed with deionized water until pH became neu-
tral and dried. HRTEM images of pristine and oxidized
MWCNTs show, for the latter, the presence of several
damages on the side walls of nanotubes, as a conse-
quence of the acid attack (Fig. 2(b)). Pristine and carboxy-
lated MWCNTs were also characterized by FTIR spectra.
The pristine MWCNTs show the C=C stretching vibra-
tion at 1631 cm−1 while for carboxylated MWCNTs rep-
resentative additional C=O and C–O stretching vibrations
Fig. 5. Representative HRTEM analysis of MWCNTs conjugated with
a) 7-hydroxy coumarin 8 and b) 7-oleate coumarin 9.
Fig. 4. TGA curves of pristineMCNTs, oxydized MWCNTs, MCNTs-
Linker coumarin oleate conjugated MWCNTs and 7-OH-coumarin Con-
jugated MWCNTs.
Fig. 6. UV spectra of MWCNTs conjugated with 7-oleate coumarin
(red), MWCNTs conjugated with 7-hydroxy coumarin (green),
MWCNTs-Linker (black) and 7-oleate coumarin (pink).
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Iannazzo et al.
Coumarin-Conjugated Multiwalled CNTs for Potential Biological Applications
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Fig. 7. 1H NMR spectrum of compound 5.
are observed at 1713 and 1056 cm−1 respectively (Fig. 3).
Moreover the peak at 3443 cm−1 could be ascribed to the
O–H vibration (Fig. 3(b)).
The TGA results of oxidized MWCNTs reveal a weight
loss attributable to the COOH groups, at 650 ꢀC, under N2
atmosphere, of ∼10%. The thermogravimetrical analysis
of pristine and carboxylated MWCNTs was performed at
ꢀ
ꢀ
10 C/min in temperature range from 30 to 650 C. In the
first stage, up to a temperature of 150 ꢀC , a weight loss of
approximatively 1% is detected for the highly hydrophilic
acid-treated MWCNTs, which corresponds to the evapora-
tion of the adsorbed water.
Fig. 8. 13C NMR spectrum of compound 5.
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Coumarin-Conjugated Multiwalled CNTs for Potential Biological Applications
Iannazzo et al.
Scheme 1. Reagents and conditions: (a) HNO3/H2SO4 (1:3), 60 ꢀC, 6 h;
(b) (COCl)2, reflux, 2d.
Scheme 3. Reagents and conditions: (a) tert-butyl 2-bromoethylcar-
bamate, K2CO3, reflux, 12 h; (b) TFA, CH2Cl2, r.t., 12 h, c) CS2, ETA,
EtOH, Boc2O, DMAP, r.t., 1 h; (d) Oleoyl chloride, ETA, CHCl3ꢄ, r.t.,
12 h.
ꢀ
The second stage, from 150ꢀ to 350 C is attributed
to the decarboxylation of the carboxylic groups present
on the MWCNTs walls. Thermal degradation in the range
between 350 ꢀC and 500 ꢀC may be explained by the
elimination of the hydroxyl functionalities. Finally, at the
temperature higher than 500 C, the observed degradation
corresponds to the thermal oxidation of the remaining dis-
procedure reported in Scheme 2. Coumarins 8 and 9,
where the C-5 position is substituted with the isothio-
cyanatoethoxy group, necessary for the reaction with the
amino group of the linker, were synthesized from the
5,7-dihydroxy-coumarin 6.22
ꢀ
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ordered carbon (Fig. 4).
Carboxylated MWCNTs were titrated to determine the
concentration of the present acidic sites, according to liter-
ature method.20 The number of free OH groups was found
to be 1.8 mmol/g.
The purification and the subsequent oxidation of raw
MWCNTs allowed the removal of catalyst impurities and
graphite particulates, so allowing the effective organic
functionalization on the selected carbon nanotubes.
Oxidized MWCNTs were heated in 30 mL of neat oxa-
lyl chloride at reflux for 48 h in order to obtain the cor-
responding MWCNTs acetyl chlorides which were used
without further purification (Scheme 1).
Coumarins were coupled with MWCNTs by means of
the linker 5 which has an amino group able to react with
acyl MWCNTs. The synthesis of the linker starts from
4-(2-aminoethyl)benzoic acid 1 according to the synthetic
IP : 93.80.195.225
Treatment of 6 with tert-butyl 2-bromo-ethylcarbamate
affords the key intermediate 7 which was converted into the
corresponding 5-isothiocyanatoethoxy-7-hydroxy coumarin
8 and 5-isothiocyanatoethoxy-7-oleate coumarin 9 follow-
ing the procedure reported in Scheme 3.
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Finally, MWCNTs acetyl chlorides were conjugated
with coumarins 8 and 9 by means of the linker 5
(Scheme 4).
These two orthogonal protecting groups of the amino
functionalities in compounds 2 and 4, were removed and
employed selectively by using different reaction condi-
tions. The Boc group was cleaved after the coupling of
2 to 4 by treatment with HCl in dioxane. The obtained
compound 5 was reacted with MWCNTs acetyl chlo-
rides and then the Pht protecting group was removed
using a solution of hydrazine in ethanol at room tempera-
ture. The quantity of the free amino groups (1.5 mmol/g)
was measured with a quantitative Kaiser test.21 The cou-
pling of coumarins 8 or 9 with the NH2 moiety pre-
sented on MWCNTs was performed by reaction in DMF
for two days at room temperature. HRTEM analysis of
Scheme 2. Reagents and conditions: (a) Boc2O, dioxane/water, NaOH
1M, r.t. 1 h; (b) CDI, dioxane, 100 ꢀC, 2 h, then 2-hydroxyethylphtalimide,
100 ꢀC, 12 h; (c) TFA, CH2Cl2, r.t., 12 h; (d) Boc2O, CHCl3 dropwise for
2 h, then r.t, 24 h,; (e)CS2, ETA, EtOH, Boc2O, DMAP, r.t., 1 h; (f) EtOH,
ETA, r.t., 12 h; (g) TFA, CH2Cl2, r.t., 12 h.
Scheme 4. Reagents and Conditions: (a) 5, ETA, THF, reflux, 48 h; (b)
hydrazine, EtOH, reflux, 24 h; (c) 8 or 9, DMF, r.t., 2d.
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Iannazzo et al.
Coumarin-Conjugated Multiwalled CNTs for Potential Biological Applications
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Fig. 9. 1H NMR spectrum of compound 7 (step d).
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coumarin-conjugated multiwalled carbon nanotubes show
the presence of an additional material on the surface
of nanotubes (Fig. 5). This new material is reasonably
attributable to the presence of new molecular aggregates
covalently bonded to the nanotubes. This material is more
considerable for MWCNTs conjugated with coumarin 9,
because of the presence of the oleoyl moieties.
The UV spectra confirm the presence of coumarins
covalently bonded to the nanotubes. MWCNTs conju-
gated with 7-oleate coumarin and 7-hydroxy coumarin
show an absorbance at 240–300 nm, while no absorbance
was detected for the MWCNTs functionalized with
the linker; a different spectrum was registered for
isothiocyanatoethoxy-7-oleate coumarin 9 (Fig. 6).
Fig. 10. 13C NMR spectrum of compound 7 (step d).
J. Nanosci. Nanotechnol. 12, 5030–5038, 2012
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4. CONCLUSIONS
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Received: 1 December 2010. Accepted: 1 May 2011.
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