Combining spontaneous polymerization and click reactions for the synthesis of polymer brushes: A grafting onto approach

Article abstract of DOI:10.1002/chem.201202534

Two novel benzofulvene monomers bearing propargyl or allyl groups have been synthesized by means of readily accessible reactions, and were found to polymerize spontaneously by solvent removal, in the apparent absence of catalysts or initiators, to give the corresponding polybenzofulvene derivatives bearing clickable propargyl or allyl moieties. The clickable propargyl and allyl groups were exploited in appropriate click reactions to develop a powerful and versatile grafting onto synthetic methodology for obtaining tailored polymer brushes. A clean sweep: Two novel benzofulvene monomers bearing either propargyl or allyl groups were found to polymerize spontaneously by solvent removal in the absence of catalysts or initiators to give the corresponding polybenzofulvene derivatives bearing clickable propargyl or allyl moieties. These derivatives were modified using click reactions, thus demonstrating a grafting onto methodology for the synthesis of tailored polymer brushes (see figure). Copyright

Full text of DOI:10.1002/chem.201202534

DOI: 10.1002/chem.201202534
Combining Spontaneous Polymerization and Click Reactions for the
Synthesis of Polymer Brushes: A “Grafting Onto” Approach
Andrea Cappelli,*[a] Giorgio Grisci,[a] Marco Paolino,[a] Federica Castriconi,[a]
Germano Giuliani,[a] Alessandro Donati,[a] Stefania Lamponi,[a] Raniero Mendichi,[b]
Antonella Caterina Boccia,[b] Filippo Samperi,[c] Salvatore Battiato,[c]
Eugenio Paccagnini,[d] Mariangela Gentile,[d] Mariano Licciardi,[e]
Gaetano Giammona,[e] and Salvatore Vomero[a]
Abstract: Two novel benzofulvene monomers bearing propargyl or allyl groups
have been synthesized by means of readily accessible reactions, and were found to
polymerize spontaneously by solvent removal, in the apparent absence of catalysts
or initiators, to give the corresponding polybenzofulvene derivatives bearing click-
able propargyl or allyl moieties. The clickable propargyl and allyl groups were ex-
ploited in appropriate click reactions to develop a powerful and versatile “grafting
onto” synthetic methodology for obtaining tailored polymer brushes.
Keywords: click reactions · molecu-
lar recognition
·
photografting
·
poly
tion
ACHTUNGTNERmNUNG ers · spontaneous polymeriza-
Introduction
ture, pH, and ionic strength.[1] A brushlike architecture can
be synthesized by means of three different approaches: the
polymerization of macromonomers (grafting through, GT),
the addition to a polymeric backbone of separately prepared
side chains (grafting onto, GO), and the polymerization of
Molecular brushes are macromolecules formed by polymeric
side chains densely grafted onto a linear polymeric back-
bone. They show peculiar features, which are regulated by
the grafting density and by the properties of the components
(backbone and side chains), such as the side-chain length.
Moreover, the conformational behavior of brushlike macro-
molecules can be affected by the characteristics of the sur-
rounding environment, such as solvent properties, tempera-
side chains from
a macroinitiator backbone (grafting
from).[1,2]
According to the original definition by Sharpless and co-
workers, click reactions are efficient and versatile chemical
transformations capable of producing chemical substances
rapidly, quantitatively, without byproducts and side reac-
tions, under mild reaction conditions, and with broad toler-
ance to functional groups and applicability.[3] Among them,
the most popular reactions are the CuI-catalyzed 1,3-dipolar
cycloaddition between organic azides and alkynes
(CuAAC)[4] and the addition of thiols to alkenes, which is
commonly called thiol–ene coupling.[5,6]
[a] Prof. A. Cappelli, Dr. G. Grisci, Dr. M. Paolino, Dr. F. Castriconi,
Dr. G. Giuliani, Prof. A. Donati, Dr. S. Lamponi, Prof. S. Vomero
Dipartimento Farmaco Chimico Tecnologico and
European Research Centre for Drug Discovery and Development
Universitꢀ degli Studi di Siena
Via A. Moro, 53100 Siena (Italy)
Fax : (+39)0577-234333
The application of click reactions to macromolecular sci-
ence has allowed the rapid synthesis of a large variety of
polymers.[7–16] Indeed, the presence of clickable groups,
along the backbone, in the side chains, or at chain termini,
can be exploited for postpolymerization modifications (i.e.,
functionalization) leading to polymers bearing functionali-
ties incompatible with the polymerization conditions.[7] An-
other interesting potential of the application of click reac-
tions to macromolecular science is that a large number of
different functionalized polymers can be prepared starting
from a single clickable polymer.[7] A key step in the postpo-
lymerization modification approach is represented by the in-
sertion of the clickable moieties.
E-mail: andrea.cappelli@unisi.it
[b] Dr. R. Mendichi, Dr. A. C. Boccia
Istituto per lo Studio delle Macromolecole (CNR)
Via E. Bassini 15, 20133 Milano (Italy)
[c] Dr. F. Samperi, Dr. S. Battiato
Istituto di Chimica e Tecnologia dei Polimeri (CNR)
Viale A. Doria 6, 95125 Catania (Italy)
[d] Dr. E. Paccagnini, Dr. M. Gentile
Dipartimento di Biologia Evolutiva
Universitꢀ degli Studi di Siena
Via A. Moro, 53100 Siena (Italy)
[e] Dr. M. Licciardi, Prof. G. Giammona
Dipartimento di Scienze e Tecnologie Molecolari e Biomolecolari
(STEMBIO)
Universitꢀ degli Studi di Palermo
Self-initiating spontaneous polymerization has been dem-
onstrated to occur in a number of different vinyl monomers.
In our prototypical benzofulvene derivative BF1
Via Archirafi 32, 90123 Palermo (Italy)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201202534.
9710
ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2013, 19, 9710 – 9721

FULL PAPER
synthesized, and induced to polymerize spontaneously by
solvent removal to give the corresponding polybenzofulvene
derivatives bearing clickable propargyl or allyl moieties.
These were exploited in the appropriate click reactions, such
as the CuAAC reaction and thiol–ene coupling.
The work described herein demonstrates the viability of
the GO approach to polybenzofulvene brushes. Moreover,
several features of the spontaneous polymerization of the bi-
functional benzofulvene monomers appear to fit the original
definition of click reactions by Sharpless and co-workers.
Results and Discussion
Synthesis: Benzofulvene monomers 6-PO-BF3k and 6-AO-
BF3k were prepared starting from indenone 1,[23] which was
demethylated with BBr3 and alkylated with propargyl bro-
mide[22] or allyl bromide to obtain compounds 3a[22] or 3b
(Scheme 2). These indenone derivatives were submitted to
the usual methylation/dehydration procedure to afford
Scheme 1. Selected benzofulvene derivatives.
benzoACTHNURGTNEfNUG ulvene monomers 6-PO-BF3k and 6-AO-BF3k, which
polymerized, in the apparent absence of catalysts, upon sol-
vent removal to give the corresponding polymers.
(Scheme 1), self-initiating spontaneous polymerization was
shown to occur by solvent removal in the apparent absence
of catalysts or initiators to give poly-BF1, a hydrophobic
polymer showing vinyl structure stabilized by aromatic
stacking interactions.[17,18] These results required the explo-
ration of the reactivity of benzofulvene derivatives BF3[19]
and the subsequent introduction of monomethyl oligo(ethyl-
With the aim of developing a versatile synthetic method-
ology for the functionalization of the polybenzofulvene
backbone with a wide variety of side chains, poly-6-PO-
BF3k was reacted with a MOEG-9 chain bearing an azide
group (MOEG-9-N3)[22] as a model, in THF in the presence
of CuBr and DIPEA, to afford poly-6-MOEG-9-TM-BF3k-
GO, which could be compared with its analogous polymer
brush prepared previously by means of a “grafting through”
(GT) approach (poly-6-MOEG-9-TM-BF3k-GT).[22] The op-
timization of the conditions of the click CuAAC reaction re-
quired hard work because poly-6-PO-BF3k is liable to depo-
lymerize when heated in the presence of solvents. After sev-
eral attempts, we found that in the very simple conditions
reported above, the CuAAC reaction proceeded even at
room temperature and became virtually exhaustive by mi-
crowave irradiation without any significant depolymeriza-
tion.
ACHTUNGTRENNUNGene glycol) (MOEG) side chains onto the polybenzofulvene
backbone of poly-BF1 and poly-BF3k (Scheme 1), which led
to the development of a new family of polymer brushes in-
cluding poly-2-MOEG-9-BF1,[20] poly-6-MOEG-9-BF3k,[21]
and poly-6-MOEG-9-TM-BF3k-GT (TM=triazolylme-
thoxy).[22]
These macromolecules showed interesting properties and
significantly different behaviors in the interaction with the
water environment, which ranged from the formation of a
compact physical hydrogel (poly-2-MOEG-9-BF1)[20] to the
apparent water solubility of poly-6-MOEG-9-TM-BF3k-
GT.[22] The results obtained suggest that MOEG-9 side
chains and their location in the monomeric unit play an im-
portant role in determining polymer features, such as inter-
molecular interactions and aggregation properties. The
latter component of the family (poly-6-MOEG-9-TM-BF3k-
GT) was considered to be particularly attractive because it
demonstrated that click can be used as an easy access to
On the other hand, thiol–ene functionalization of poly-6-
AO-BF3k with a MOEG-7 chain bearing a thiol group
(MOEG-7-SH) was more difficult to optimize because the
conditions used to generate the sulfenyl radical produced
detectable amounts of the monomer. The best results were
obtained by means of UV excitation in the presence of
photoACTHNURTGNEUiGN nitiator molecules, such as 2,2-dimethoxy-2-phenyl
poly
benzofulvene derivatives tailored for specific applica-
acetophenone (DMPA), but the thiol–ene photoreaction
could not be considered exhaustive (see below).
tions and, more generally, to the enlargement of the accessi-
ble chemical space around polybenzofulvene derivatives.
However, the reaction conditions used in the methylation/
dehydration procedure of the published GT approach may
limit the variety of the functional groups present in the side
chains.
To develop a powerful and versatile GO methodology for
the synthesis of tailored polymer brushes, benzofulvene
monomers bearing propargyl or allyl groups were designed,
Properties of the new polybenzofulvene derivatives: All the
newly synthesized polybenzofulvene derivatives showed an
excellent solubility in the most common organic solvents. In
dichloromethane or chloroform the polymers dissolved rap-
idly, whereas the solubility in water can be considered as a
distinctive feature between the couple of macromolecular
precursors poly-6-PO-BF3k/poly-6-AO-BF3k and function-
Chem. Eur. J. 2013, 19, 9710 – 9721
ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
9711

A. Cappelli et al.
BF3k (poly-6-MOEG-9-TM-BF3k-GT).[22] On the other
hand, poly-6-MOEG-7-SPO-BF3k interacts with water to
produce a pale yellow hydrogel, which is broken rapidly by
ultrasound to provide a milky dispersion. In static condi-
tions, the dispersion generated an opalescent sediment easy
to be newly suspended in water by gentle stirring. Thus, the
behavior shown by poly-6-MOEG-7-SPO-BF3k in the inter-
action with the water environment appears to be similar to
that shown by previously reported poly-6-MOEG-9-BF3k,[21]
and this close analogy is easily explained in terms of struc-
tural similarity between the side chains of these polybenzo-
fulvene brushes.
Characterization of molecular weight distribution: The mo-
lecular characterization of the new polybenzofulvene deriva-
tives was performed by means of an absolute multiangle
laser light scattering (MALS) detector online to a size-ex-
clusion chromatography (SEC) apparatus. A MALS detec-
tor online to a SEC system furnishes for each polymeric
eluting fraction the molecular weight (M) and the size of
the macromolecules, denoted as radius of gyration Rg. Con-
sequently, a SEC-MALS system furnishes the molecular
weight distribution (MWD) and the radius of gyration distri-
bution (RGD) of the polymer. Furthermore, if the MWD of
the polymer is relatively broad the conformation plot, that
is, the scaling law Rg =f(M), is obtained from a single injec-
tion into the chromatographic system. The slope a of the
conformation plot, obtained directly from the SEC-MALS
system, shows the conformation of the macromolecules.
The different solubility properties of the polymers re-
quired the use of two different SEC mobile phases. In par-
ticular, pure THF was used for the couple of macromolecu-
lar precursors poly-6-PO-BF3k/poly-6-AO-BF3k. A solvent
mixture of THF and DMF+0.01m LiBr (80:20) was em-
ployed in the case of functionalized polymers poly-6-
MOEG-9-TM-BF3k-GO and poly-6-MOEG-7-SPO-BF3k.
The most important results of SEC-MALS molecular and
conformational characterization by means of these two
mobile phases are summarized in Table 1. Basically, the data
Scheme 2. Synthesis and click functionalization of poly-6-PO-BF3k and
poly-6-AO-BF3k. Reagents: i) BBr3, CH2Cl2; ii) for compound 3a: prop-
argyl bromide, K2CO3, NaI, DMF; for compound 3b: allyl bromide,
K2CO3, NaI, DMF; iii) Al
neous polymerization) solvent elimination; vi) CH
DIPEA, THF; vii) CH3A(OCH2CH2)7SH, DMPA, CHCl3. PTSA=p-tolu-
enesulfonic acid monohydrate, DIPEA=N,N-diisopropylethylamine,
ACHTUNGTRENNUNG
CTHUNGTRENNUNG
CHTUNGTRENNUNG
ACHTUNGTRENNUNG
show
that
spontaneous
polymerization
of
DMPA=2,2-dimethoxy-2-phenylacetophenone.
6-PO-BF3k and 6-AO-BF3k afforded polymers character-
ized by MWD features very similar to those of parent poly-
mer poly-BF3k. In particular, poly-6-PO-BF3k and poly-6-
AO-BF3k show ultrahigh molecular weight values (Mw =
1794 and 1497kgmolÀ1, respectively) and relatively broad
polydispersity indexes (PDI=Mw/Mn =2.2 and 2.6, respec-
tively), which appear to be significantly different from those
alized polymers poly-6-MOEG-9-TM-BF3k-GO/poly-6-
MOEG-7-SPO-BF3k. The presence of clickable groups in
poly-6-PO-BF3k and poly-6-AO-BF3k is compatible with
the easy dissolution into the organic solvents commonly
used in click reactions, whereas the properties of the func-
tionalized polymers poly-6-MOEG-9-TM-BF3k-GO and
poly-6-MOEG-7-SPO-BF3k are governed by the side
chains, as expected.
For example, poly-6-MOEG-9-TM-BF3k-GO is soluble in
water and the dissolution was complete after a few hours at
room temperature producing a transparent solution. Re-
markably, this behavior is consistent with that observed with
homopolymer poly-6-MOEG-9-TM-BF3k obtained by spon-
taneous polymerization of macromonomer 6-MOEG-9-TM-
shown by poly-6-MO-BF3k (Mw =347kgmolÀ1
;
PDI=
4.3).[21]
When poly-6-PO-BF3k was reacted with MOEG-9-N3 in
THF in the presence of CuBr and DIPEA to afford poly-6-
MOEG-9-TM-BF3k-GO, we observed both a decrease in
the molecular weight (Mw =821kgmolÀ1) and an increase in
PDI (Mw/Mn =8.9) without the production of significant
monomer concentrations. These observations suggest that
the polybenzofulvene backbone is susceptible to breaking
under the conditions used in the CuAAC reaction. Probably,
9712
www.chemeurj.org
ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2013, 19, 9710 – 9721

Synthesis of Polymer Brushes
FULL PAPER
Table 1. Macromolecular features of the new polybenzofulvene derivatives compared with those shown by
previously reported poly-6-MOEG-9-TM-BF3k-GT (obtained by the GT approach), poly-6-MO-BF3k, and
poly-BF3k.
When poly-6-PO-BF3k was
reacted with MOEG-9-N3 in
THF in the presence of CuBr
and DIPEA to afford poly-6-
[a]
[b]
Polymer
Mp [kgmolÀ1
]
Mw [kgmolÀ1
]
PDI[c]
Rg [nm][d]
K [nm][e]
a[e]
poly-6-PO-BF3k
poly-6-AO-BF3k
poly-6-MO-BF3k
poly-BF3k
poly-6-MOEG-9-TM-BF3k-GO
poly-6-MOEG-9-TM-BF3k-GT
2030
1864
312
1900
484
1794
1497
347
1506
821
2.2
2.6
4.3
3.4
8.9
3.9
51.5
47.7
19.4
49.9
33.6
15.5
0.0101
0.0231
0.0058
0.0066
0.0249
0.58
0.52
0.61
0.60
0.50
MOEG-9-TM-BF3k-GO,
we
observed the complete disap-
pearance of the signals attribut-
ed to the propargyl moiety
from the 13C NMR spectra of
the latter polymer (Figure 2).
This result suggests that the
conditions used in the CuAAC
reaction allowed exhaustive
grafting to be obtained.
193
358
[a] Mp: peak molecular weight. [b] Mw: weight-average molecular weight. [c] Polydispersity index PDI=Mw/
Mn, in which Mn denotes the number-average molecular weight. [d] Rg: radius of gyration, that is, the dimen-
sion of the macromolecules. [e]K, a: intercept and slope of conformation plot Rg =KMa.
multiple ruptures occurred randomly along the backbone up
to the production of macromolecules showing a molecular
weight quite similar to that shown by poly-6-MOEG-9-TM-
BF3k-GT obtained by spontaneous polymerization of the
macromonomer.
Moreover, the correspondence between the 13C NMR
spectrum of poly-6-MOEG-9-TM-BF3k-GO obtained by
functionalization of poly-6-PO-BF3k (GO approach) and
the one of the same polymer obtained by spontaneous poly-
merization of macromonomer 6-MOEG-9-TM-BF3k-GT
(GT approach)[22] was considered to be the final evidence
required to close the circle.
Similarly, the results of MWD characterization of poly-6-
MOEG-7-SPO-BF3k performed by SEC-MALS suggest
that the polybenzofulvene backbone is also susceptible to
multiple ruptures under the conditions used in the photo-
grafting reaction. In fact, when poly-6-AO-BF3k was reacted
with MOEG-7-SH by UV excitation in the presence of
DMPA to afford poly-6-MOEG-7-SPO-BF3k, we obtained a
decrease in the molecular weight (Mw around 70kgmolÀ1)
and in the PDI (Mw/Mn =1.7; for details see the Supporting
Information). The comparison of the degree of polymeriza-
tion shown by macromolecular precursor poly-6-AO-BF3k
(around 5000) with the corresponding value shown by poly-
6-MOEG-7-SPO-BF3k (around 100) suggests that a consid-
erable number of ruptures occurred during the photograft-
ing reaction. Moreover, the relatively low polydispersity of
both poly-6-MOEG-7-SPO-BF3k samples (for details see
the Supporting Information) is reminiscent of a living radi-
cal character of the backbone segmentation products.
On the other hand, 13C NMR analysis of poly-6-MOEG-7-
SPO-BF3k revealed the presence of minor signals (marked
Structure of the new polybenzofulvene derivatives
NMR spectroscopy: The 13C NMR spectra of the couple of
macromolecular precursors poly-6-PO-BF3k and poly-6-
AO-BF3k were compared with that of parent macromole-
cule poly-6-MO-BF3k (Figure 1) to ascertain the corre-
spondence between the structure of these polybenzofulvene
derivatives and the presence of intact clickable groups in the
new polymers poly-6-PO-BF3k and poly-6-AO-BF3k. The
comparison of both the low-field portion containing C-1’
and C-3 signals and the upfield region containing C-1 and
C-D (which may be affected by the competing 1,4-polymeri-
zation) confirms the retention of the spontaneous 1,2-poly-
merization mechanism. Moreover, the presence of the addi-
tional signals attributed to propargyl (poly-6-PO-BF3k) or
allyl (poly-6-AO-BF3k) side chains demonstrates that
1,2-polymerization involves selectively the benzofulvene
exocyclic double bond.
Figure 1. 13C NMR spectra (CDCl3) of newly synthesized poly-6-PO-
BF3k and poly-6-AO-BF3k and, for comparison, those of previously re-
ported poly-6-MO-BF3k and 6-MO-BF3k-MUM (monomeric unit
model) representing the model of its monomeric unit.[21,22]
Chem. Eur. J. 2013, 19, 9710 – 9721
ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
9713

A. Cappelli et al.
with asterisks in Figure 3) attributable to the residual vinyl
groups, thus suggesting that a relatively small amount of
these reactive functionalities remained unmodified after
prolonged exposure to the thiol–ene photoreaction condi-
tions.
Depolymerization studies: The thermoreversible polymeriza-
tion behavior, typical of the polybenzofulvene deriva-
tives,[17,19–21] was evaluated in the couple of macromolecular
precursors poly-6-PO-BF3k and poly-6-AO-BF3k as well as
in functionalized polymers poly-6-MOEG-9-TM-BF3k-GO
and poly-6-MOEG-7-SPO-BF3k by using our standard pro-
tocol. Thus, solutions of the polymers (5.0 mg in 0.5 mL of
1
[D5]nitrobenzene) were heated at 1508C and the H NMR
spectra were recorded at regular time intervals.
The experiments performed with poly-6-PO-BF3k and
poly-6-AO-BF3k showed the expected behavior for the mac-
romolecular precursors. The most significant steps (t=0 and
24 h) of the depolymerization processes are summarized in
Figure 4. The presence of intact clickable groups in the
formed monomers suggests that they are not involved in the
thermoreversible polymerization/depolymerization mecha-
nism.
Figure 2. 13C NMR spectrum (CDCl3) of poly-6-MOEG-9-TM-BF3k-GO
obtained by functionalization of poly-6-PO-BF3k (grafting onto) and, for
comparison, that of the same polymer obtained by spontaneous polymer-
ization of macromonomer 6-MOEG-9-TM-BF3k-GT (grafting through).
Interestingly, the thermoreversible polymerization can be
exploited to evaluate the grafting degree obtained by means
of our coupling procedures. The results of the depolymeriza-
tion experiments, summarized in Figure 5, show the usual
depolymerization behavior for poly-6-MOEG-9-TM-BF3k-
GO obtained by the GO strategy. The expected monomer
was obtained in its pure form at significant concentrations
after 2 h at 1508C, and its concentration increased until the
decomposition phenomenon became apparent (96 h). This
behavior is similar to that previously observed with the
same polymer obtained by spontaneous polymerization of
macromonomer 6-MOEG-9-TM-BF3k (GT approach)[22]
and can be interpreted in terms of close structural similarity.
The absence of the signals attributable to monomer 6-PO-
BF3k (e.g., the peaks at 2.89 and 4.87 ppm of the propargyl
moiety, compare Figure 5 with Figure 4) substantiates the
high grafting density assumed for poly-6-MOEG-9-TM-
BF3k-GO on the basis of its 13C NMR spectrum. Finally, the
persistence of the thermoreversible properties after func-
tionalization opens the way to the production of new tail-
ored monomers (e g., bioactive monomers).
On the other hand, the depolymerization experiments
performed with poly-6-MOEG-7-SPO-BF3k (Figure 6)
showed the presence of a relatively small amount of mono-
mer 6-AO-BF3k, thus confirming that the conditions applied
in the thiol–ene photoreaction were inadequate to produce
an exhaustive grafting of poly-6-AO-BF3k with a MOEG-7
chain bearing a thiol group (MOEG-7-SH).
MALDI-TOF mass spectrometry: The structures and the
end groups of the newly prepared polybenzofulvene deriva-
tives were also studied by matrix-assisted laser desorption/
ionization time-of-flight mass spectrometry (MALDI-TOF
MS)[24,25] in positive-ion mode, using different matrices.
Figure 3. 13C NMR spectra (CDCl3) of newly synthesized poly-6-MOEG-
7-SPO-BF3k and poly-6-AO-BF3k and, for comparison, that of previous-
ly reported poly-6-MOEG-9-BF3k.[21] The signals marked with asterisks
were assigned to the residual vinyl groups remained unmodified after the
exposition to the thiol–ene photoreaction conditions.
9714
www.chemeurj.org
ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2013, 19, 9710 – 9721

Synthesis of Polymer Brushes
FULL PAPER
Figure 4. Thermoinduced depolymerization of poly-6-PO-BF3k (left) and of poly-6-AO-BF3k (right) as followed using 1H NMR spectroscopy
(400 MHz). A solution of the polymer (5.0 mg) in [D5]nitrobenzene (0.5 mL) was heated at 1508C, and the 1H NMR spectra were recorded at regular
time intervals. Only the spectra at t=0 and 24 h are displayed. The arrows indicate the nitrobenzene peaks and H2O the water peak. The spectra of the
corresponding monomers in CDCl3 are given at the top for the sake of comparison.
Table 2. Structural assignments of the peaks observed in the MALDI-TOF spectra of poly-6-PO-BF3k, poly-6-AO-BF3k, and poly-6-MOEG-9-TM-
BF3k-GO samples.
Species
Structures[a]
[M+][b] (n)
Poly-6-PO-
Poly-6-AO-
Poly-6-MOEG-9-
993.2 (3)
1323.6 (4)
1654.0 (5)
1984.4 (6)
2314.8 (7)
2645.2 (8)
etc.
999.2 (3)
1331.6 (4)
1664.0 (5)
1996.4 (6)
2328.8 (7)
2661.2 (8)
1569.8 (2)
2353.7 (3)
3137.6 (4)
3921.5 (5)
4705.4 (6)
5489.3 (7)
An
1007.2 (3)
1337.6 (4)
1668.0 (5)
1998.4 (6)
2328.8 (7)
2659.6 (8)
etc.
1013.2 (3)
1344.6 (4)
1678.0 (5)
2010.4 (6)
2342.8 (7)
2675.2 (8)
1583.8 (2)
2367.7 (3)
3151.6 (4)
3935.5 (5)
4719.4 (6)
5503.3 (7)
Bn
1023.2 (3)
1353.6 (4)
1684.0 (5)
2014.4 (6)
2344.8 (7)
2675.2 (8)
etc.
1029.2 (3)
1361.6 (4)
1694.0 (5)
2026.4 (6)
2358.8 (7)
2691.2 (8)
1599.8 (2)
2383.7 (3)
3167.6 (4)
4735.4 (5)
5519.3 (6)
6303.2 (7)
Cn
[a] R=propargyl for poly-6-PO-BF3k; R=allyl for poly-6-AO-BF3k; R=MOEG-9-TM for poly-6-MOEG-9-TM-BF3k-GO. [b] The mass of the repeat
unit of poly-6-PO-BF3k is 330.4 Da, that of poly-6-AO-BF3k is 332.4 Da, and that of poly-6-MOEG-9-TM-BF3k-GO is 783.9 Da. The mass accuracy was
better than (Æ0.5) and (Æ0.2) Da in the mass spectra recorded in linear and reflectron mode, respectively.
Chem. Eur. J. 2013, 19, 9710 – 9721
ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
9715

A. Cappelli et al.
Figure 6. Thermoinduced depolymerization of poly-6-MOEG-7-SPO-
BF3k as followed by 1H NMR spectroscopy (400 MHz). A solution of
poly-6-MOEG-7-SPO-BF3k (5.0 mg) in [D5]nitrobenzene (0.5 mL) was
1
heated at 1508C, and the H NMR spectra were recorded at regular time
intervals. The arrows indicate the solvent peaks and H2O the water peak.
In the inset, the signals marked with asterisks were assigned to 6-AO-
BF3k and those marked with circles to monomer 6-MOEG-7-SPO-BF3k.
Figure 5. Thermoinduced depolymerization of poly-6-MOEG-9-TM-
BF3k-GO (grafting onto) as followed using 1H NMR spectroscopy
(400 MHz). A solution of poly-6-MOEG-9-TM-BF3k-GO (5.0 mg) in
[D5]nitrobenzene (0.5 mL) was heated at 1508C, and the 1H NMR spec-
tra were recorded at regular time intervals. To appreciate the variation in
monomer concentration, the signals attributable to the vinylene group
were integrated, and the integral values were compared with that of the
lowest field signal of nondeuterated nitrobenzene, which was considered
as an internal standard. Integrals are omitted for the sake of clarity. The
arrows indicate the solvent peaks and H2O the water peak.
Figure 7. MALDI-TOF mass spectrum of poly-6-AO-BF3k recorded in
linear mode. Insets show enlarged sections of the mass spectrum record-
ed in linear (a) and reflectron (b) mode.
ular ions (M+) of linear polybenzofulvene oligomers from
trimer (M3) up to 30-mer (M30). Signals due to polybenzoful-
vene macromolecules with molar mass higher than
15000 Da were not observed in the MALDI mass spectrum
because, as often occurs, a discrimination effect has been ob-
served in the MALDI-TOF MS analysis of these polydis-
perse polymers.[24,25] Generally, low-molecular-mass species
are more easily desorbed than high-molecular-mass ones,
and therefore species with molar mass higher than 10000–
15000 Da were not revealed. MALDI-TOF MS gives abso-
Malononitrile (DCTB, see Experimental Section) has been
found to be the best matrix, and the mass spectrum of the
poly-6-AO-BF3k sample, recorded in linear mode, is dis-
played in Figure 7.
The spectrum shows, in the mass range within m/z 1000
and 10000, a series of three peaks separated from each
other of (332.4Æ0.3) Da, which corresponds to the mass of
the repeating unit. The peak series correspond to the molec-
9716
www.chemeurj.org
ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2013, 19, 9710 – 9721

Synthesis of Polymer Brushes
FULL PAPER
lute determinations of average molar masses (Mw and Mn)
only for polymers with a narrow dispersity (PDI=Mw/Mn <
1.1); it succeeds independently of their structure, but fails
for polymeric materials with a higher polydispersity (PDI>
1.1) as well as for the polymers investigated in the present
work.
The inset of Figure 7 shows that the most intense peaks
correspond to the molecular ions (M+) of linear polybenzo-
fulvene oligomers terminated with hydrogen at both ends
(H/H; species An in Table 2 and Figure 7). The second in-
tense peak series belongs to the molecular ions of linear
chains having an aldehyde moiety (H/CHO; species Bn in
Table 2 and Figure 7), whereas peaks corresponding to the
polybenzofulvene oligomers terminated with one carboxylic
group (H/COOH; species Cn in Table 2 and Figure 7)
appear with weak intensity.
terminated with the carboxyl group (species Cn) appear with
more intensity (compare Figures 7 and 8). The relative in-
tensity of peaks corresponding to poly-6-PO-BF3k chains
terminated with H/H, H/CHO, and H/COOH is about
54:39:7, as calculated from 20 mass spectra recorded in
linear mode. On the other hand, in the MALDI mass spec-
tra of poly-6-MOEG-9-TM-BF3k-GO, the most intense
peaks were assigned to linear chains terminated with
H/CHO groups (species Bn in Figure 8b), whereas those ter-
minated with H/H (species An) and H/COOH (species Cn)
groups appear with similarly lower intensity. Thus, the rela-
tive intensity of peaks belonging to chains terminated with
H/H, H/CHO, and H/COOH groups is 30:42:28 with a var-
iance of Æ2.5. By way of comparison, previously synthesized
poly-6-MOEG-9-TM-BF3k-GT showed a relative intensity
of 70:28:2 determined with a variance of Æ2.5 by using the
intensities of the corresponding peaks present in the mass
range 1000–8000 of 12 mass spectra recorded using different
laser intensities.
High-resolution mass spectra were also recorded for poly-
6-PO-BF3k and poly-6-MOEG-9-TM-BF3k-GO samples in
reflectron mode. The enlarged sections of their mass spectra
are displayed in Figure 8, whereas the mass spectra recorded
in linear mode are displayed in the Supporting Information
(Figures 2S and 3S).
Macromolecular structure of poly-6-MOEG-9-TM-BF3k-
GO: To obtain information about the aggregation state of
poly-6-MOEG-9-TM-BF3k-GO in water, dynamic light scat-
tering (DLS) studies were carried out by using aqueous dis-
persions of the polymer in the concentration range of 0.1–
1.0 mgmLÀ1 and at 25 and 378C. The analysis of DLS data
obtained with clear solutions at 258C suggested the presence
of objects showing average dimensions of 57 nm. These di-
mensions are significantly smaller than those previously ob-
served with the same polymer synthesized by spontaneous
polymerization of macromonomer 6-MOEG-9-TM-BF3k
(poly-6-MOEG-9-TM-BF3k-GT, 245 nm)[22] and agree with
the radius of gyration as measured by SEC-MALS analysis
(ca. 34 nm).
Furthermore, DLS dimensional data showed the predomi-
nant formation of aggregates at 378C and at a minimum
polyACHTNUGRTNEUNG
mer concentration of 0.5 mgmLÀ1. These aggregates
Figure 8. Enlarged sections of MALDI-TOF mass spectra of a) poly-6-
PO-BF3k and b) poly-6-MOEG-9-TM-BF3k-GO, recorded in reflectron
mode.
Table 3. Size distribution and Z-potential values of poly-6-MOEG-9-TM-
BF3k-GO water dispersions (0.5 mgmLÀ1) at 25 and 378C.
Temperature [8C]
dav [nm]
PDI
Z-potential [mV]
25
37
57
290
0.38
0.17
À7.23
À7.50
Similarly to the mass spectrum of poly-6-AO-BF3k, the
mass spectra of both samples recorded in linear mode show
a series of three peaks (in the mass range from m/z 1000 up
to m/z 15000) separated from each other of (330.4Æ0.4) Da
(in the case of the poly-6-PO-BF3k sample) or of (783.9Æ
0.5) Da (for the poly-6-MOEG-9-TM-BF3k-GO sample),
which correspond to the mass of their repeating units. The
most intense peaks in the poly-6-PO-BF3k spectrum were
attributed to polybenzofulvene chains terminated with H at
both ends (species An). The other two peak series were as-
signed to the oligomers terminated with H/CHO and
H/COOH groups (species Bn and Cn, respectively). The rela-
tive intensity of peaks belonging to the families An and Bn is
comparable with that observed in the spectra of the poly-6-
AO-BF3k sample, whereas the peaks belonging to oligomers
Figure 9. Size distribution curves of poly-6-MOEG-9-TM-BF3k-GO re-
corded at 25 (a) and 378C (b) by DLS (sample concentration
0.5 mgmLÀ1).
Chem. Eur. J. 2013, 19, 9710 – 9721
ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
9717

A. Cappelli et al.
showed an average diameter of 290 nm and PDI of 0.17 (see
Table 3 and Figure 9). Actually, this finding is consistent
with the hypothesis that the polymer dispersion, at the ana-
lyzed concentration, is composed of a homogeneous aggre-
gate population at 378C. Zeta (Z)-potential values measured
at 25 and 378C did not vary significantly, which indicated
that any modification of the exposed surface of the polymer
occurred as a consequence of the polymer aggregation in-
duced by temperature variation.
Briefly, the water solutions of newly synthesized poly-6-
MOEG-9-TM-BF3k-GO show the features of thermores-
ponsive colloidal dispersions similar to those observed with
previously published poly-6-MOEG-9-TM-BF3k-GT, with
the difference that the polymer synthesized by the GO strat-
egy appears to be molecularly dissolved at room tempera-
ture, probably because of its higher Z-potential value
(À7.23 mV versus À1.24 mV shown poly-6-MOEG-9-TM-
BF3k-GT).
result was tentatively rationalized in terms of aggregation
properties and dimension of the aggregates in water.[22] On
the basis of the smaller dimension of the objects generated
by poly-6-MOEG-9-TM-BF3k-GO in aqueous medium, the
potential cytotoxicity of the newly synthesized polymer was
evaluated in two different cell lines (i.e., immortalized
mouse fibroblasts NIH3T3 and human neuroblastoma IMR-
32 cells). IC50 values were found to be >77 mm (NIH3T3)
and 13 mm (IMR-32) and confirmed a low cytotoxic potential
for the tested polymer towards both NIH3T3 and IMR-32
cell lines. The low cytotoxicity shown by poly-6-MOEG-9-
TM-BF3k-GO was considered to be in good agreement with
its low propensity to interact and cross the cytoplasmic
membrane. In fact, preliminary fluorescence microscopy
studies showed a weak fluorescence uptake in both the dif-
ferent cell lines used, even after a prolonged incubation
time.
The data obtained by DLS studies agree with the images
obtained by transmission electron microscopy (TEM) of
poly-6-MOEG-9-TM-BF3k-GO water solutions processed at
room temperature. In fact, Figure 10 shows the presence of
Conclusion
The work described herein shows that benzofulvene mono-
mers bearing propargyl or allyl groups can be synthesized
by means of straightforward reactions and induced to poly-
AHCTUNGTREGmNNNU erize spontaneously, in the apparent absence of catalysts
or initiators, by solvent removal to give the corresponding
polybenzofulvene derivatives showing very high molecular
weight values. In particular, the results of SEC-MALS ex-
periments suggest that spontaneous polymerization of mon-
omers 6-PO-BF3k and 6-AO-BF3k produces polybenzoful-
vene derivatives showing MWD features similar to those of
parent polymer poly-BF3k. NMR studies substantiate the
retention of the spontaneous 1,2-polymerization mechanism,
and the presence in the 13C NMR spectra of additional sig-
nals attributed to propargyl or allyl side chains (clickable
groups) suggests that 1,2-polymerization involves the benzo-
fulvene exocyclic double bond selectively. Moreover, depo-
lymerization experiments confirmed that the clickable
groups are not involved in the thermoreversible polymeriza-
tion/depolymerization mechanism. Thus, the presence of
allyl or propargyl groups does not affect the spontaneous
polymerization of benzofulvene monomers in terms of
MWD and incorporation of allyl or propargyl groups into
the polybenzofulvene backbone. We assume that the benzo-
fulvene derivatives are capable of forming columnar aggre-
gates in concentrated solutions. When the intermonomer
distance becomes compatible with the formation of covalent
bonds among the monomers, the spontaneous polymeriza-
tion starts and proceeds rapidly as a domino reaction lead-
ing to macrobiradical species, which undergo termination.
MALDI-TOF experiments substantiate that the main termi-
nation pathway of the macrobiradical species produced by
spontaneous polymerization of 6-PO-BF3k and 6-AO-BF3k
is hydrogen addition at both ends of the polybenzofulvene
backbone. Moreover, the interaction of the macrobiradical
species with molecular oxygen was assumed to produce the
Figure 10. Structure of brush macromolecules and aggregates found by
TEM analysis of poly-6-MOEG-9-TM-BF3k-GO solution in water.
wormlike objects, which can be assumed to be isolated mac-
romolecules, together with relatively abundant globular spe-
cies presumably resulting from intramolecular collapse, and
rare small aggregates confirming the low propensity towards
aggregation shown by poly-6-MOEG-9-TM-BF3k-GO.[21,22]
Cytotoxicity of poly-6-MOEG-9-TM-BF3k-GO: The ex-
haustive grafting observed in poly-6-MOEG-9-TM-BF3k-
GO taken together with its appreciable water solubility and
the low liability to aggregation led us to envision potential
biological or biotechnological applications for this polymer
brush. Previous fluorescence microscopy studies suggested
that poly-6-MOEG-9-TM-BF3k-GT is unable to interact,
cross the membrane, and alter the morphology of HEK293T
cells, thereby suggesting the absence of toxic effects. The
9718
www.chemeurj.org
ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2013, 19, 9710 – 9721

Synthesis of Polymer Brushes
FULL PAPER
oxidation of the terminal methylene group with the forma-
tion of aldehydic or carboxylic groups.
With the aim of developing a powerful and versatile GO
methodology to obtain tailored polymer brushes, the click-
were not observed.[22] Finally, the lack of any toxic effect
and cell viability impairment found with poly-6-MOEG-9-
TM-BF3k-GO demonstrates that our polybenzofulvene de-
rivatives bearing clickable groups can be functionalized by a
suitable methodology to produce nanostructured materials
potentially useful in a wide range of biological and biotech-
nological applications.
ACHTUNGTRENNUNGable propargyl and allyl groups were exploited in appropri-
ate click reactions. In particular, the CuAAC reaction was
used to transform poly-6-PO-BF3k into poly-6-MOEG-9-
TM-BF3k-GO, which showed a 13C NMR spectrum virtually
indistinguishable from that of the homopolymer obtained by
spontaneous polymerization of macromonomer 6-MOEG-9-
TM-BF3k.[22] This high similarity with the spectrum of the
homopolymer suggests that the CuAAC coupling reaction
led to a very high grafting density, substantiated also by the
lack of signals attributable to the propargyl moiety as well
as by the results of the depolymerization studies. On the
contrary, thiol–ene photografting of poly-6-AO-BF3k with a
MOEG-7 chain bearing a thiol group (MOEG-7-SH) was
less exhaustive, as suggested by the presence of minor sig-
nals attributable to the residual vinyl groups in the
13C NMR spectrum of poly-6-MOEG-7-SPO-BF3k. These
preliminary results suggest that, of the two click reactions
investigated in the present work, the CuAAC coupling is su-
perior to the thiol–ene one, and additional work is probably
required to make the thiol–ene coupling as efficient as the
CuAAC reaction in the GO approach to polybenzofulvene
brushes.
SEC-MALS measurements showed that the transforma-
tion of poly-6-PO-BF3k into poly-6-MOEG-9-TM-BF3k-
GO produces both a decrease in the molecular weight and
an increase in the PDI, thus suggesting that the polybenzo-
fulvene backbone is susceptible to breaking under the condi-
tions used in the CuAAC reaction. We assumed that multi-
ple ruptures occurred randomly along the backbone up to
the production of macromolecules showing a molecular
weight quite similar to that shown by poly-6-MOEG-9-TM-
BF3k-GT obtained by spontaneous polymerization of the
macromonomer. Probably, this behavior is the consequence
of the balance between the increasing steric hindrance
around the polybenzofulvene backbone and its relative sta-
bility to covalent bond scission. In other words, because of
the high grafting density, MOEG-9 side chains are assumed
to repel each other and stretch the polybenzofulvene back-
bone up to its scission,[26] which can be facilitated by the rel-
ative stability of the indenyl radical formed by homolytic
cleavage.
Experimental Section
Synthesis: Melting points were determined in open capillaries in a Gal-
lenkamp apparatus and are uncorrected. UV/Vis spectra were recorded
with a Shimadzu 260 spectrophotometer and the emission spectra were
obtained by means of a Perkin–Elmer LS45 instrument. Merck silica
gel 60 (230–400 mesh) was used for column chromatography. Merck TLC
plates, silica gel 60 F254, were used for TLC. NMR spectra were record-
ed with
a Bruker AC200, Varian Mercury-300, Bruker DRX-400
AVANCE, Bruker DRX-500 AVANCE, or Bruker DRX-600 AVANCE
spectrometer in the indicated solvents (TMS as internal standard): the
values of the chemical shifts are expressed in ppm and the coupling con-
stants (J) in hertz. An Agilent 1100 LC/MSD instrument operating with
an electrospray source was used in mass spectrometry experiments.
Ethyl 1-methylene-3-phenyl-6-(prop-2-ynyloxy)-1H-indene-2-carboxylate
(6-PO-BF3k): A mixture of indenol derivative 4a (0.87 g, 2.5 mmol) in
CHCl3 (50 mL) with p-toluenesulfonic acid monohydrate (PTSA; 0.47 g,
2.5 mmol) was heated under reflux for 1 h and cooled to room tempera-
ture. The reaction mixture was washed with a saturated solution of
NaHCO3 and dried over sodium sulfate to afford a stock (about 0.05m)
solution of the monomer 6-PO-BF3k that was stored under an argon at-
mosphere. 1H NMR (400 MHz, [D]CHCl3, 258C, TMS): d=1.04 (t, J=
7.1 Hz, 3H), 2.53 (t, J=2.3 Hz, 1H), 4.11 (q, J=7.1 Hz, 2H), 4.75 (d, J=
2.3 Hz, 2H), 6.34 (s, 1H), 6.62 (s, 1H), 6.90 (dd, J=8.3 Hz, 2.3, 1H), 7.13
(d, J=8.3 Hz, 1H), 7.33 (d, J=2.3 Hz, 1H), 7.41 ppm (m, 5H); MS
(ESI): m/z: 353 [M+Na+].
Ethyl 6-(allyloxy)-1-methylene-3-phenyl-1H-indene-2-carboxylate (6-AO-
BF3k): A mixture of compound 4b (0.48 g, 1.37 mmol) in chloroform
(28 mL) with PTSA (0.26 g, 1.37 mmol) was heated under reflux for 1 h
and cooled to room temperature. The reaction mixture was washed with
a saturated solution of NaHCO3 and dried over sodium sulfate to afford
a stock (about 0.05m) solution of benzofulvene monomer 6-AO-BF3k,
which was stored under an argon atmosphere. 1H NMR (400 MHz,
[D]CHCl3, 258C, TMS): d=1.04 (t, J=7.1 Hz, 3H), 4.11 (q, J=7.1 Hz,
2H), 4.60 (d, J=5.2 Hz, 2H), 5.29 (dd, J=10.5, 1.1 Hz, 1H), 5.43 (dd, J=
17.2, 1.1 Hz, 1H), 6.07 (m, 1H), 6.32 (s, 1H), 6.60 (s, 1H), 6.84 (dd, J=
8.4, 2.3 Hz, 1H), 7.10 (d, J=8.4 Hz, 1H), 7.27 (d, J=2.3 Hz, 1H),
7.41 ppm (m, 5H); MS (ESI): m/z: 333 [M+H+].
Poly[ethyl
1-methylene-3-phenyl-6-(prop-2-ynyloxy)-1H-indene-2-
carboxylate] (poly-6-PO-BF3k): A solution of benzofulvene monomer 6-
AHCTUNGTRENNUNG
PO-BF3k in chloroform was concentrated under reduced pressure to give
a viscous oil, which was dissolved in chloroform and newly evaporated
(the dissolution/evaporation procedure was repeated three times). The
polymer was purified by precipitation with ethanol (bad solvent) from a
solution of the final residue in chloroform (good solvent) and dried
under reduced pressure to obtain poly-6-PO-BF3k (0.61 g, yield 74%) as
DLS analysis suggested for the aqueous solutions of poly-
6-MOEG-9-TM-BF3k-GO that the properties of thermore-
ACHTUNGTRENNUNGsponsive colloidal dispersions are similar to those observed
1
a white solid. H NMR (400 MHz, [D]CHCl3, 258C, TMS): d=0.3–1.1 (br
with previously published poly-6-MOEG-9-TM-BF3k-GT,[22]
with the difference that the polymer synthesized by the GO
strategy appears to be molecularly dissolved at room tem-
perature. This difference can be explained by the presence
of a relatively high number of chains terminated with car-
boxylic acid groups in poly-6-MOEG-9-TM-BF3k-GO,
thereby leading to a Z-potential value (À7.23 mV) higher
than that measured for poly-6-MOEG-9-TM-BF3k-GT
(À1.24 mV), in which chains terminated with COOH groups
m, 3H), 2.3–2.5 (br m, 1H), 2.6–4.0 (br m, 4H), 4.1–4.9 (br m, 2H), 5.8–
7.3 ppm (br m, 8H).
Poly
G
6-ACTHNGUETRNNU(G allyloxy)-1-methylene-3-phenyl-1H-indene-2-carboxylate]
(poly-6-AO-BF3k): A solution of monomer 6-AO-BF3k in chloroform
was concentrated under reduced pressure to give a viscous yellow oil,
which was dissolved in chloroform and newly evaporated (the dissolu-
tion/evaporation procedure was repeated three times). The polymer was
purified by precipitation with ethanol from a solution of the final residue
in chloroform and dried under reduced pressure to obtain poly-6-AO-
BF3k as a white solid (0.36 g, yield 79%).1H NMR (400 MHz, [D]CHCl3,
Chem. Eur. J. 2013, 19, 9710 – 9721
ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
9719

A. Cappelli et al.
258C, TMS): d=0.2–1.1 (br m, 3H), 1.7–4.7 (br m, 6H), 4.8–5.5 (br m,
2H), 5.6–7.8 ppm (br m, 9H).
tween m/z 7000 and 15000. On the other hand, spectra recorded in re-
flectron mode showed significantly intense peaks only in the mass range
lower than m/z 6000. Samples for MALDI analysis were prepared by the
dried-droplet method, in which a mixture of matrix and sample was de-
posited onto the target plate and dried at room temperature under inert
atmosphere (N2 flow). Typically, a THF solution (0.5 mL) of the polymer
(10 mgmLÀ1) was mixed with a matrix solution (0.5 or 1.5 mL), and then
spotted on the MALDI sample holder and dried slowly to allow matrix
crystallization. The better spectra were recorded using a (matrix solu-
tion)/(polymer solution) ratio of 3:1 v/v. The mass peaks corresponding
to the macromolecular species were measured with an accuracy of
Poly
G
6-{[1-(2,5,8,11,14,17,20,23,26-nonaoxaoctacosan-28-yl)-1H-
1,2,3-triazol-4-yl]methoxy}-1-methylene-3-phenyl-1H-indene-2-carboxy-
late) (poly-6-MOEG-9-TM-BF3k-GO): In a microwave tube, a mixture
of poly-6-PO-BF3k (0.15 g, 0.45 mmol in monomer base unit) in THF
(5.0 mL) containing 28-azido-2,5,8,11,14,17,20,23,26-nonaoxaoctacosane
(MOEG-9-N3, 0.41 g, 0.90 mmol), CuBr (0.013 g, 0.091 mmol), and
DIPEA (0.016 mL, 0.092 mmol) was stirred at room temperature for 5 h,
exposed to microwave irradiation in a CEM Discover instrument for
40 min (4ꢂ10 min, T=608C, W =150), and finally evaporated under re-
duced pressure. The residue was dissolved with water (15 mL) and 33%
NH4OH (4.0 mL) to obtain a clear solution after overnight stirring at
room temperature. The aqueous solution was extracted with chloroform
and the organic layer was dried over sodium sulfate and concentrated
under reduced pressure. The polymer was purified by precipitation with
n-hexane (50 mL) from a solution of the final residue in dichloromethane
(5 mL) and dried under reduced pressure to obtain poly-6-MOEG-9-TM-
BF3k-GO as a pale-yellow sticky solid (0.27 g, yield 77%). 1H NMR
(300 MHz, [D]CHCl3, 258C, TMS): d=0.1–1.1 (br m, 3H), 3.3 (s, 3H),
3.4–5.5 (br m, 42H), 5.6–8.2 ppm (br m, 9H).
A
Dynamic light scattering studies and Z-potential measurements: DLS
studies and Z-potential measurements were performed at 25 and 378C by
means of a Malvern Zetasizer NanoZS instrument fitted with a 532 nm
laser at a fixed scattering angle of 908. The polymer samples were molec-
ularly dissolved into doubly distilled water (pH ꢀ6) at concentrations
ranging from 0.1 to 1.0 mgmLÀ1 and the intensity-average hydrodynamic
diameter (size in nm) and polydispersity index (PDI) were obtained by
cumulant analysis of the correlation function. The Z-potential (mV) was
calculated from the electrophoretic mobility by using the Smoluchowsky
relationship and assuming Ka@1 (in which K and a are the Debye–
Hꢃckel parameter and particle radius, respectively). Each experiment
was performed in triplicate.
Poly
[ethyl
6-(2,5,8,11,14,17,20-heptaoxa-23-thiahexacosan-26-yloxy)-1-
(poly-6-MOEG-7-SPO-
methylene-3-phenyl-1H-indene-2-carboxylate]
BF3k): In a vial, poly-6-AO-BF3k (0.030 g, 0.090 mmol in monomer base
unit) was dissolved in CHCl3 (1.0 mL), and O-(2-mercaptoethyl)-O’-
methyl-hexa(ethylene glycol) (MOEG-7-SH, 0.16 g, 0.45 mmol) and
DMPA (0.011 g, 0.043 mmol) were added. The vial was sealed with a
screw cap fitted with a PTFE septum and the reaction mixture was
purged with argon for 5–10 min. Irradiation with a UV lamp (365 nm)
was carried out at room temperature for 6 h. The reaction mixture was
then concentrated under reduced pressure and the residue was washed
with n-hexane and dried under reduced pressure to obtain poly-6-
MOEG-7-SPO-BF3k as a pale-yellow sticky solid (0.047 g).
Transmission electron microscopy:
A drop of poly-6-MOEG-9-TM-
BF3k-GO solution in water was placed onto a 300 mesh formvar-coated
copper grid and observed in a Philips CM10 transmission electron micro-
scope at an acceleration voltage of 80 kV.
Cytotoxicity experiments: NIH3T3 and IMR-32 cells were employed for
cytotoxicity experiments. NIH3T3 cells were kept in Dulbeccoꢄs modified
Eagleꢄs medium (DMEM) and IMR-32 cells in Eagleꢄs minimal essential
medium (EMEM) at 378C in a humidified atmosphere containing 5%
CO2. The culture media were supplemented with 10% fetal calf serum,
1% l-glutamine–penicillin–streptomycin solution, and 1% MEM Non-
Essential Amino Acid Solution. Once at confluence, cells were washed
with phosphate-buffered saline (PBS, 0.1m), taken up with trypsin–ethyl-
enediaminetetraacetic acid solution, and then centrifuged at 1000 rpm for
5 min. The pellet was resuspended in medium solution (dilution 1:15).
Cell viability after 24 h of incubation with the polymer was evaluated by
Neutral Red Uptake assay (Sigma–Aldrich, Switzerland) by the proce-
dure reported previously.[27] The data processing included Studentꢄs t test
with p<0.05 taken as significance level.
SEC-MALS: The MWD characterization of the new polybenzofulvene
derivatives was performed by a MALS light scattering photometer online
to a SEC system. The SEC-MALS system and the corresponding experi-
mental conditions were identical to those used in our previous stud-
ies[18,19] and are not reported in detail herein.
Mass spectrometry: MALDI-TOF mass spectra of the new polybenzoful-
venes were recorded in linear mode with a Voyager DE-STR (Perseptive
Biosystem) mass spectrometry instrument equipped with a nitrogen laser
emitting at 337 nm, with a 3 ns pulse width and working in positive
mode. The accelerating voltage was 25kV, and the grid voltage and delay
time were optimized for each sample to achieve better mass resolution,
expressed as the molecular weight of a given ion divided by the full
width at half maximum (fwhm). The irradiance was maintained slightly
above the threshold. External calibration was performed using a home-
made low-molar-mass (Mn =3000 gmolÀ1) hydroxy-ended poly(bispheno-
l A carbonate) (PC-OH). Mass spectra in reflectron mode were also re-
Interaction with live cells: NIH3T3 and IMR-32 cells were plated on
coverslips and allowed to grow overnight. The coverslips were washed
and incubated with poly-6-MOEG-9-TM-BF3k-GO (0.01 mgmLÀ1) in
PBS for 12 h at room temperature. Cells on coverslips were washed with
PBS, fixed with 4% paraformaldehyde, and mounted for observation
with a Nikon epifluorescence microscope (Zeiss).
corded with
a 4800 Proteomic Analyzer (Applied Biosystems), a
MALDI-TOF/TOF instrument equipped with a Nd:YAG laser at a wave-
length of 355 nm with <500 ps pulse and 200 Hz firing rate. External cali-
bration was performed using an Applied Biosystems calibration mixture
consisting of polypeptides with different molecular weight values. The ir-
radiance was maintained slightly above the threshold, to obtain a mass
resolution of about 6000–8000 fwhm; isotopic resolution was observed
throughout the entire mass range detected (from m/z 1000 up to m/z
6000). Mass accuracy was about 50 ppm. All measurements were per-
formed in positive mode; approximately 1500 laser shots were accumulat-
ed for each mass spectrum. For the analysis of each polymer, four differ-
ent matrices were used: a-cyano-4-hydroxycinnamic acid, 1,8,9-anthrace-
netriol (dithranol), 2-(4’-hydroxybenzeneazo)benzoic acid, and trans-2-[3-
Acknowledgements
Thanks are due to Italian MUR (Ministero dell’Universitꢀ e della Ricer-
ca) for financial support. Prof. Stefania D’Agata D’Ottaviꢄs careful read-
ing of the manuscript is also acknowledged.
[1] S. S. Sheiko, B. S. Sumerlin, K. Matyjaszewski, Prog. Polym. Sci.
2008, 33, 759–785.
[2] M. Zhang, A. H. E. Mꢃller, J. Polym. Sci. Part A 2005, 43, 3461–
3481.
(4-tert-butylphenyl)-2-methyl-2-propenylidene]malononitrile
(DCTB),
0.1m in THF, with and without the addition of cationization salts (i.e.,
CF3COONa or CF3COOAg; 10À3 m in THF). The better spectra were re-
corded using DCTB without salts. Mass spectra recorded in linear mode
showed intense mass-resolved peaks with a fwhm of about 800 within
m/z 1000 and 7000, and also poorly resolved peaks in the mass range be-
[3] H. C. Kolb, M. G. Finn, K. B. Sharpless, Angew. Chem. 2001, 113,
2056–2075; Angew. Chem. Int. Ed. 2001, 40, 2004–2021.
[4] M. Meldal, C. W. Tornoe, Chem. Rev. 2008, 108, 2952–3015.
[5] A. Dondoni, Angew. Chem. 2008, 120, 9133–9135; Angew. Chem.
Int. Ed. 2008, 47, 8995–8997.
9720
www.chemeurj.org
ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2013, 19, 9710 – 9721

Synthesis of Polymer Brushes
FULL PAPER
[6] M. J. Kade, D. J. Burke, C. J. Hawker, J. Polym. Sci. Part A 2010, 48,
743–750.
[7] T. P. Lodge, Macromolecules 2009, 42, 3827–3829.
[8] H. Gao, K. Matyjaszewski, J. Am. Chem. Soc. 2007, 129, 6633–6639.
[9] X. Zhang, X. Lian, L. Liu, J. Zhang, H. Zhao, Macromolecules 2008,
41, 7863–7869.
[10] L. M. Campos, K. L. Killops, R. Sakai, J. M. J. Paulusse, D. Damiron,
E. Drockenmuller, B. W. Messmore, C. J. Hawker, Macromolecules
2008, 41, 7063–7070.
[11] A. S. Goldmann, A. Walther, L. Nebhani, R. Joso, D. Ernst, K.
Loos, C. Barner-Kowollik, L. Barner, A. H. E. Mꢃller, Macromole-
cules 2009, 42, 3707–3714.
[20] A. Cappelli, S. Galeazzi, G. Giuliani, M. Anzini, M. Grassi, R. Lapa-
sin, G. Grassi, R. Farra, B. Dapas, M. Aggravi, A. Donati, L. Zetta,
A. C. Boccia, F. Bertini, F. Samperi, S. Vomero, Macromolecules
2009, 42, 2368–2378.
[21] a) A. Cappelli, M. Paolino, P. Anzini, G. Giuliani, S. Valenti, M. Ag-
gravi, A. Donati, R. Mendichi, L. Zetta, A. C. Boccia, F. Bertini, F.
Samperi, S. Battiato, E. Paccagnini, S. Vomero, J. Polym. Sci. Part A
2010, 48, 2446–2461; b) M. Licciardi, G. Amato, A. Cappelli, M.
Paolino, G. Giuliani, B. Belmonte, C. Guarnotta, G. Pitarresi, G.
Giammona, Int. J. Pharm. 2012, 438, 279–286.
[22] A. Cappelli, M. Paolino, G. Grisci, G. Giuliani, A. Donati, R. Men-
dichi, A. C. Boccia, F. Samperi, S. Battiato, E. Paccagnini, E. Giaco-
mello, V. Sorrentino, M. Licciardi, G. Giammona, S. Vomero,
Polym. Chem. 2011, 2, 2518–2527.
[12] S. Fleischmann, H. Komber, B. Voit, Macromolecules 2008, 41,
5255–52644.
[13] M. Munteanu, S. W. Choi, H. Ritter, Macromolecules 2008, 41,
9619–9623.
[14] K. Kempe, A. Krieg, R. Becer, U. S. Schubert, Chem. Soc. Rev.
2012, 41, 176–191.
[15] A. E. van der Ende, J. Harrell, V. Sathiyakumar, M. Meschievitz, J.
Katz, K. Adcock, E. Harth, Macromolecules 2010, 43, 5665–5671.
[16] P. L. Golas, K. Matyjaszewski, Chem. Soc. Rev. 2010, 39, 1338–1354.
[17] A. Cappelli, G. Pericot Mohr, M. Anzini, S. Vomero, A. Donati, M.
Casolaro, R. Mendichi, G. Giorgi, F. Makovec, J. Org. Chem. 2003,
68, 9473–9476.
[18] A. Cappelli, M. Anzini, S. Vomero, A. Donati, L. Zetta, R. Mendi-
chi, M. Casolaro, P. Lupetti, P. Salvatici, G. Giorgi, J. Polym. Sci.
Part A 2005, 43, 3289–3304.
[23] J. H. Ahn, M. S. Shin, S. H. Jung, S.k. Kang, K. R. Kim, S. D. Rhee,
W. H. Jung, S. D. Yang, S. J. Kim, J. R. Woo, J. H. Lee, H. G. Cheon,
S. S. Kim, J. Med. Chem. 2006, 49, 4781–4784.
[24] G. Montaudo, M. S. Montaudo, F. Samperi, In Mass Spectrometry of
Polymers; G. Montaudo, R. P. Lattimer, Eds.; CRC Press: Boca
Raton, 2002; Chapters 2 and 10.
[25] G. Montaudo, M. S. Montaudo, F. Samperi, Progr. Polym. Sci. 2006,
31, 277–357.
[26] S. S. Sheiko, F. C. Sun, A. Randall, D. Shirvanyants, M. Rubinstein,
H. Lee, K. Matyjaszewski, Nature 2006, 440, 191–194.
[27] C. Rossi, A. Foletti, A. Magnani, S. Lamponi, Semin. Cancer Biol.
2011, 21, 207–214.
[19] A. Cappelli, S. Galeazzi, G. Giuliani, M. Anzini, A. Donati, L.
Zetta, R. Mendichi, M. Aggravi, G. Giorgi, E. Paccagnini, S.
Vomero, Macromolecules 2007, 40, 3005–3014.
Received: July 16, 2012
Revised: April 17, 2013
Published online: June 12, 2013
Chem. Eur. J. 2013, 19, 9710 – 9721
ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
9721