Ferrocene derivatives supported on poly(N-vinylpyrrolidin-2-one) (PVP): Synthesis of new water-soluble electrochemically active probes for biomolecules

Article abstract of DOI:10.1016/j.jorganchem.2006.10.058

Carboxy-terminated polyvinylpyrrolidin-2-one (PVP) has been used as a new water-soluble and biocompatible polymeric support for a series of ferrocene labeled amino acid and peptide nucleic acid (PNA) monomer derivatives 4-7. The organometallic polymer-con

Full text of DOI:10.1016/j.jorganchem.2006.10.058

Journal of Organometallic Chemistry 692 (2007) 1363–1371
www.elsevier.com/locate/jorganchem
Ferrocene derivatives supported on
poly(N-vinylpyrrolidin-2-one) (PVP): Synthesis of new
water-soluble electrochemically active probes for biomolecules
a,
b
b
b
Clara Baldoli *, Claudio Oldani , Emanuela Licandro , Prasanna Ramani ,
b
b
c
c
Antonio Valerio , Paolo Ferruti , Luigi Falciola , Patrizia Mussini
a
CNR-Institute of Molecular Science and Technologies, Via C. Golgi 19, I-20133 Milano, Italy
b
Department of Organic and Industrial Chemistry, University of Milan, Via G. Venezian 21, I-20133 Milano, Italy
Department of Physical Chemistry and Electrochemistry; University of Milan, Via C. Golgi 19, I-20133 Milano, Italy
c
Received 13 September 2006; received in revised form 25 October 2006; accepted 25 October 2006
Available online 7 November 2006
Dedicated to Professor Stefano Maiorana on the occasion of his 70th birthday.
Abstract
Carboxy-terminated polyvinylpyrrolidin-2-one (PVP) has been used as a new water-soluble and biocompatible polymeric support for
a series of ferrocene labeled amino acid and peptide nucleic acid (PNA) monomer derivatives 4–7. The organometallic polymer-conju-
gates thus obtained are new and potentially useful as water-soluble electrochemically active probes for biomolecules. In view of such
application, their electrochemical activity has been evaluated and has proved very high notwithstanding the complexity and bulkiness
of the molecule, affording detection limits down to 10ꢀ8 M in the aqueous medium.
ꢀ 2006 Elsevier B.V. All rights reserved.
Keywords: Bioorganometallic chemistry; Ferrocene; Functionalized PVP; Electrochemically active probes; Polymer conjugation
1. Introduction
conjugates have been mainly restricted to poly(glutamic
acid) (PG), N-(2-hydroxypropyl)methacrylamide (HPMA)
copolymers, and polyethylene glycol (PEG). PEG conjuga-
tion [6] of therapeutically relevant proteins is a well-estab-
lished technology and a number of PEG-ylated proteins
have received market approval or are in advanced clinical
trials. Within the class of biocompatible soluble polymers
another well-known polymeric agent, namely poly(N-vinyl-
pyrrolidin-2-one) (PVP) [7–10] 1 (Fig. 1), might be a prom-
ising candidate for polymer therapeutics. In fact, PVP is a
water-soluble, biocompatible, amphiphilic polymer with a
huge number of relevant chemical and biological properties
[11–14]. However, the absence of reactive functional
groups on the polymer chain prevents its extensive use
for covalent drug conjugation, thus making the functional-
ization of PVP a relevant synthetic target in polymer
chemistry.
Soluble polymers are of great importance for industrial
applications and in biological systems [1,2]; for example the
conjugation of biologically active molecules to water-solu-
ble polymers [3] is a promising strategy for improving their
pharmacological and pharmacokinetic profile and thera-
peutic index [4]. A number of soluble polymers having lin-
ear, hyperbranched or dendritic structures have been tested
for the covalent conjugation of low and high molecular
weight bioactive molecules [5] constituting the so-called
polymer therapeutics. Although many efforts are being
made to develop novel polymeric carriers, synthetic
polymers that have been tested in clinically evaluated drug
*
Corresponding author. Tel.: +39 02503 14143; fax: +39 02503 14139.
E-mail address: clara.baldoli@istm.cnr.it (C. Baldoli).
0022-328X/$ - see front matter ꢀ 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.jorganchem.2006.10.058

1364
C. Baldoli et al. / Journal of Organometallic Chemistry 692 (2007) 1363–1371
ative to this end. In fact, we found that the carboxy group
*
*
n
of 3 can be reacted directly with an amino or hydroxy
group of a drug, thereby directly forming a conjugate
[24] (Scheme 1, path a). On the other hand, PVP–COOH
3 can serve as a convenient starting material for preparing
other functionalized polymers and we were able to obtain a
variety of PVP derivatives (Scheme 1, path b) arising from
the condensation of the carboxy group of 3 with the amino
group of different bifunctional reagents [25]. These new
PVP oligomers are very useful to enlarge the number of
the molecules that can be conjugated and the kind of link-
ers connecting them to the support.
In view of diagnostic applications of PVP bioconjugates,
the possibility of inserting an analytically detectable probe
on the polymer might represent an important step in devel-
oping this chemistry. But, since only mono-functionalized
PVP can be obtained by chain-transfer radical polymeriza-
tion, it is necessary to bind, at the end of the chain, a tri-
functional molecule able to link the probe, the polymer
and the biomolecule (Scheme 2). Some x-functionalized
amino acids (i.e. tyrosine, lysine and serine) are suitable
candidates to this aim, and in principle, after the linkage
to some fluorescent or electrochemically active molecules,
their conjugation to PVP could provide new water-soluble
analytical probes, a quite interesting feature for application
in the biomedical field [26].
N
O
1
Fig. 1. Poly(N-vinylpyrrolidin-2-one) (PVP).
The synthesis and characterization of some end- [15–19]
and lactam ring- [20] functionalized PVP have been
recently reported. For example, PVP oligomers bearing
hydroxy [15] or carboxy [18] groups at one chain end, such
as 2 (PVP–OH) and 3 (PVP–COOH) (Fig. 2), have been
prepared by the radical polymerization of 1-vinylpyrroli-
din-2-one (VP) in the presence of suitable chain transfer
agents. These polymers have been used in the synthesis of
copolymers [21] and for the modification of bioactive mol-
ecules or macromolecules [22,23].
The study of further applications of functionalized PVP,
as new water-soluble supports for bioactive and organic
molecules, is certainly an important research field and we
considered the PVP–COOH 3 [18] as the most useful deriv-
O
*
O
*
n
n
OH
OH
N
N
O
O
In this paper, we report our results concerning the cova-
lent support on PVP of mono- and multi-ferrocene labeled
amino acid and PNA monomer derivatives, as well as the
investigation of the electrochemical behaviour of the corre-
sponding water-soluble polymers thus obtained.
2
3
Fig. 2. Hydroxy-(PVP–OH) 2 and carboxy-(PVP–COOH) 3 end-func-
tionalized PVP.
O
Drug
Drug
H N
2
N
H
PVP
O
a
b
PVP
OH
O
Linker
R
N
H
PVP
H N
Linker
R
2
O
*
n
N
R= SH, OH, SSR', NH , CO H,
=
N
PVP
2
2
O
O
Scheme 1. Conjugation of molecules to PVP–COOH.
O
O
Biomolecule
Biomolecule
X
Probe
X
Y
1.
2.
X
Y
PVP
PVP
O
Z
Probe
Probe
OH
PVP
Scheme 2. Probe-containing PVP.

C. Baldoli et al. / Journal of Organometallic Chemistry 692 (2007) 1363–1371
1365
2. Results and discussion
In addition, the PVP conjugation of the previously syn-
thesized and electrochemically investigated [27] mono- and
tri-ferrocene labeled tyrosine Peptide Nucleic Acid (PNA)
monomers 6 and 7 (Fig. 4), was investigated.
PNA oligomers [28], are synthetic pseudo-peptide mim-
ics of DNA, have great potential for DNA recognition as
new therapeutics in anti-sense and anti-gene strategies
and are useful for diagnostic applications. However, a
rapid progress in extensive use of PNA is restrained by
its poor water solubility and cell permeability. The possibil-
ity of conjugating PNA to PVP might be a promising
approach to solve this problem, especially in the presence
of a probe allowing detection of its cellular uptake.
Mono-ferrocene labeled tyrosine 4, was prepared as pre-
viously reported [27d], while tri-ferrocene tyrosine 5 was
synthesized as described in Scheme 3: N-Cbz tyrosine
methyl ester 8 was reacted with 2-(Boc-amino)ethyl bro-
mide 9, in the presence of cesium carbonate and KI in
DMF at room temperature, affording compound 10 in
70% yield. After the cleavage of N-Boc protection, deriva-
tive 11 was condensed with tris-ferrocene isocyanate 12
The first molecules of choice are shown in Fig. 3, and are
constituted by mono- and tri-ferrocene labeled tyrosine
derivatives 4 and 5.
NH2
CO2Me
NH2
CO2Me
Fe
O
O
O
O
O
O
Fe
N
H
N
H
Fe
4
5
Fe
Fig. 3. Ferrocene labeled tyrosine.
O
Me
HN
O
Me
HN
O
O
N
O
N
O
O
H2N
N
O
Fe
O
N
H
NHCbz
H2N
N
N
H
NHCbz
O
Fe
Fe
H
N
H
N
O
O
O
Fe
O
6
7
Fig. 4. Ferrocene-labeled PNA monomers.
O
O
O
NHBoc
CbzHN
Br
TFA
OMe
9
CbzHN
CbzHN
OMe
OMe
Cs2CO3, KI
DMF, r.t., 3h
CH2Cl2, r.t
NH3
CF3COO
O
NHBoc
O
OH
8
10, 70%
11, 95%
1. DIPEA, r.t.
DMF
Fc
O
O
O
O
Fc
OCN
12
CbzHN
HCO2NH4
Pd/C, MeOH
OMe
Fc
O
Fc
O
O
H
N
H
N
5, 72%
O
Fc
O
13, 60%
Fc
Scheme 3. Synthesis of tri-ferrocene labeled tyrosine 5.

1366
C. Baldoli et al. / Journal of Organometallic Chemistry 692 (2007) 1363–1371
[27a] to give 13. Tri-ferrocene labeled tyrosine 5 was even-
tually obtained in 72% yield, after the cleavage of Cbz
group in the presence of Pd/C and ammonium formate.
PNA monomers 6 and 7, bearing a 2-aminoethyl amide
group, were obtained in good yields from the correspond-
ing methyl ester monomers 14 and 15 heated at 50 ꢁC in
ethylendiamine solvent (Scheme 4).
The condensation of PVP–COOH with compounds 4–7
was run in DMF using O-(Benzotriazol-1-yl)-N,N,N0,N0-
tetramethyluronium hexafluorophosphate (HBTU) in the
presence of diisopropylethyl amine (DIEA) as a condens-
ing agent and stirring the mixture at room temperature
for 3 days (Scheme 5). The PVP conjugated 16–19 thus
obtained were isolated, after the evaporation of the solvent,
by precipitation from diethyl ether. The polymers were
then dissolved in water (in which they are fully soluble)
and purified by ultrafiltration through a membrane with
a nominal cut-off of 3000. After lyophilization, polymers
16–19 were isolated as a light brown powder and com-
pletely characterized.
It is worth emphasizing that in the case of tyrosine deriv-
atives 16 and 17 the amide bond, formed with the a-nitro-
gen atom, directly connects the amino acid to the polymer,
while a short amino-ethyl linker is present between PVP
O
O
Me
HN
Me
HN
O
O
N
O
O
N
H2N
NH2
O
O
H2N
N
N
N
H
NHCbz
MeO
NHCbz
50 ˚C, 1h
,
O
R
O
R
6, 90%
7, 85%
14, 15
Fe
O
Fe
Fe
14, 6: R=
15, 7: R=
H2CHNCOHN
;
O
Fe
O
Scheme 4. Synthesis of PNA monomers 6 and 7.
O
CO2Me
N
H
PVP
O
R
T
4, 5
6, 7
16, 17
O
HBTU, DIEA
DMF, r.t., 72h
PVP
OH
O
(M =4535)
3,
n
O
H
N
PVP
N
NHCbz
N
H
O
18, 19
O
R
Fe
O
Fe
Fe
O
N
Me
HN
Fe
T=
16, 18: R=
17, 19: R= H2CHNCOHN
;
O
O
O
Scheme 5. PVP conjugation of the ferrocene derivatives 4–7.

C. Baldoli et al. / Journal of Organometallic Chemistry 692 (2007) 1363–1371
1367
and PNA monomers in 18 and 19, keeping the organome-
tallic moiety a little further from the core of the polymer
(Scheme 5).
In the perspective of using such PVP conjugates as
active water-soluble probes it is important to evaluate
how the presence of the polymer affects their electrochem-
ical activity.
In this context, a voltammetric investigation on the
PVP-conjugated 16–19 using differential pulse voltammetry
(DPV) and square wave voltammetry (SWV) has been car-
ried out. Fig. 5 summarizes the DPV curves of the investi-
gated compounds, at 2 · 10ꢀ4 M concentration in water.
The peak potentials against the Me10Fc+|Me10Fc (decam-
ethylferricinium|decamethylferrocene) reference redox cou-
ple are at 0.463, 0.395, 0.468 and 0.439 V for compounds
16, 17, 18, and 19, respectively. The large difference in
the redox potential between compound 17 and the others
might be connected with a different accessibility of the
active site and/or a locally modified solvental environment
for this compound. In order to better clarify this difference,
a complete cyclovoltammetric characterization in different
solvents accompanied by theoretical molecular modeling
studies, is currently in progress. The results show a good
voltammetric response in terms of current intensity and tri-
pling of the current in a single oxidation wave in the case of
the conjugates with three ferrocenes 17 and19. This obser-
vation points to the three ferrocene groups being indepen-
dent [27c]. Moreover, comparing the peak currents of the
PNA monomers 18, 19 vs. tyrosine derivatives 16, 17,
either mono- or tri-functionalized (recorded both in CV
and DPV mode), the smaller tyrosine derivatives show
lower currents with respect to the bulkier PNA ones. At
constant concentration, reaction mechanism and number
of transferred electrons, normally higher currents are due
3.0
2.5
2.0
19
17
i
1.5
1.0
18
0.5
0.0
16
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
E
/ V (Me10Fc)
Fig. 5. Differential pulse voltammograms of PVP conjugated compounds at 2 · 10ꢀ4 M concentration in H2O (16, thin line; 17, thick line; 18, thin plain
line; 19, thick plain line).
0.90
0.80
0.70
0.60
0.50
0.40
0.30
0.20
0.10
0.00
0.15
0.14
0.13
0.12
0.11
0.10
0.09
0.08
0.07
0.06
0.05
10-7M
10-8M
SWV
10-7M
δ
i
DPV
0.1
0
0.2
0.3
0.4
0.5
0.6
0.7
E
/ V (Me10Fc)
Fig. 6. Differential pulse (DPV) and square wave (SWV) voltammograms of compound 19 at 10ꢀ7–10ꢀ8 M concentration in H2O (thin line: background).

1368
C. Baldoli et al. / Journal of Organometallic Chemistry 692 (2007) 1363–1371
to higher diffusion coefficients and/or higher accessibility of
the active site. Here such desirable conditions appear to be
better complied with in the case of the bulkier PNA frag-
ment 18 and 19.
The interesting and promising results have prompted us
to investigate on the aqueous detection limit for compound
19. Actually, 19 shows (Fig. 6) an appreciable response
down to 10ꢀ7 M and even 10ꢀ8 M, using the SWV, not-
withstanding the complexity and bulkiness of the molecule,
and in the absence of any amplification step.
was purified by column chromatography (light petro-
leum/EtOAc, 3:2; Rf = 0.55), obtaining 10 as a white solid
in 66% yield.
1
(L)–10: m.p. 79–81 ꢁC (pentane); H NMR (CDCl3), d:
1.44 (s, 9H, But); 3.09–2.97 (m, 2H, CH2Ph); 3.45–3.53
(m, 2H, CH2NHBoc); 3.71 (s, 3H, OCH3); 3.96 (t, 2H,
CH2O, J = 5.1 Hz); 4.62–4.59 (m, 1H, CH); 4.98–5.29 (m,
4H, CH2-Cbz+NH-Cbz+NH-Boc); 6.78 (d, 2H, PhO,
J = 8.5 Hz); 6.98 (d, 2H, PhO, J = 8.5 Hz); 7.33–7.35 (bs,
5H, Ph). 13C NMR (CDCl3, d): 28.2, 37.0, 39.9, 52.0,
54.8, 66.6–66.8, 79.4, 114.3, 127.9–128.5, 130.1, 136.1,
155.5, 155.8, 157.4, 171.9. IR (CHCl3, m, cmꢀ1) : 1708–
1718 (NHCO). MS (ESI) m/z 495.3 (M + Na)+.
3. Conclusions
We have investigated the application of poly(vinyl-
pyrrolidin-2-one) (PVP), as a new water-soluble and
biocompatible polymeric support in the field of bioorgano-
metallic chemistry [29]. A series of mono and tri-ferrocene
tyrosine and peptide nucleic acid (PNA) monomer deriva-
tives 4–7, have been supported on carboxy-terminated PVP
affording new and potentially useful water-soluble electro-
chemical probes for biomolecules. In view of such applica-
tion their electrochemical activity has proved to be very
high, which is surprising given the complexity and bulki-
ness of the molecule, affording detection limits down to
10ꢀ8 M in aqueous media.
4.3. Synthesis of compound 11
At room temperature TFA (3 ml, 41 mmol) was added
to a solution of 10 (1.15 g, 2.43 mmol) in CH2Cl2 (5 ml)
and the mixture stirred for 4 days. The solvent was evapo-
rated and the residue was purified by column chromatogra-
phy (EtOAc/MeOH, 9:1 Rf = 0.2) to obtain 11
(trifluoroacetate salt) in 95% yield.
1
(L)–11: Pinkish white sticky solid, H NMR (CD3OD),
d: 3.0–3.11 (m, 2H, CH2Ph); 3.19–3.23 (m, 2H,
CH2NHþ3 ); 3.68 (s, 3H, OCH3); 4.17 (t, 2H, CH2O,
J = 5.1 Hz); 4.36–4.44 (m, 1H, CH); 5.02 (s, 2H, CH2-
Cbz); 6.87 (d, 2H, PhO, J = 9 Hz); 7.11 (d, 2H, PhO,
J = 9 Hz); 7.29–7.31 (m, 5H, Ph Cbz). 19F NMR (CD3OD,
d): ꢀ73.47. 13C NMR (CD3OD, d): 37.78, 41.04, 52.67,
57.13, 67.56–67.65, 115.67, 128.65–128.95, 129.42, 130.92,
131.35, 158.82, 173.97. MS (ESI) m/z 373.3 (M + 1);
395.2 (M + Na)+. IR (nujol, m, cmꢀ1): 1687.9 (COOMe).
4. Experimental
4.1. Materials and methods
All the chemicals used (laboratory or analytical grade)
were purchased from Sigma–Aldrich or Fluka unless other-
wise indicated. All the reactions were performed under
nitrogen atmosphere using a standard vacuum line. The
reactions were monitored by TLC, carried out on Kieselgel
60 F254 plates (Fluka), with detection by UV, KMnO4 or
developing in I2 chamber. Merck silica gel 60 (70–230
mesh) was used for column chromatography purification.
1H and 13C NMR spectra were recorded on a Bruker,
AC300 and AMX 300 MHz instruments. IR spectra were
recorded on a Perkin–Elmer 1725X FT-IR. Melting points
4.4. Synthesis of compound 13
A solution of 12 (0.07 mmol) in DMF (1 ml) was
added, at room temperature, to a solution of 11
(0.08 mmol) in DMF (1 ml), followed by the addition of
DIPEA (0.015 mmol). The reaction mixture was stirred
for 2 days, then diluted with CH2Cl2 (10 ml), washed with
H2O (2 · 10 ml) and then with a 0.3 M solution of
KHSO4 (2 · 10 ml). The aqueous phase was then
extracted with CH2Cl2 (2 · 10 ml); the combined organic
layer was dried over Na2SO4, filtered and evaporated.
The crude product was then purified by a column chro-
matography (EtOAc/ETP, 4:6) to get 13 as orange yellow
oil in 60% yield.
were measured with a Buchi B-540 apparatus and are
¨
uncorrected. MS data were obtained by using a VG 70
EQ Micromass or a Thermo-Finningam mass spectrome-
ter. PVP–COOH 3 [18] (Mn = 4535) was dried at 60 ꢁC
under vacuum for 5 h before use. Compound 4, 8 [27d]
and 12 [27a] were prepared as previously reported.
(L)–13: 1H NMR (CDCl3) d: 3.5–3.72 (m, 13H,
CH2O + CH2Tyr + CH2NH + COOMe); 3.8–4.3 (m,
35H, OCH2Fc + Fc + CH2O); 4.68–4.70 (m, 1H, CH);
5.1 (s, 2H, CH2-Cbz); 6.79 (d, 2H, PhO, J = 8.0 Hz); 7.01
(d, 2H, PhO, J = 8.0 Hz); 7.26–7.34 (m, 5H, Ph). 13C
NMR (CDCl3, d): 35.7, 41.1, 51.8, 55.4, 60, 65.4, 66.3,
67.4, 67.8, 68.2, 68.8, 68.9, 83, 121.3, 127.5, 127.7, 128.3,
129.7, 135, 138, 150, 170. IR (CHCl3, m, cmꢀ1): 1723
(COOMe). MS (ESI) m/z 1113.7 (M+); 1136.7
(M + Na)+. Elemental analysis calc. (%) for C58H63Fe3-
4.2. Synthesis of compound 10 [27a]
2-(Boc-amino)ethyl bromide
9
(1 mmol), Cs2CO3
(3 mmol) and KI (0.1 mmol) were added to a solution of
(L)-8 (1 mmol) in DMF (8 ml). The reaction mixture was
stirred at room temperature for 3 h, diluted with water
(50 ml) and extracted with EtOAc (4 · 25 ml). The com-
bined organic layers were washed with water (30 ml), dried
over Na2SO4, filtered and evaporated. The crude product

C. Baldoli et al. / Journal of Organometallic Chemistry 692 (2007) 1363–1371
1369
N3O9: C, 62.55; H, 5.70; N, 3.77. Found: C, 62.49; H, 5.72;
N, 3.75%.
J = 8.5 Hz); 7.34–7.36 (m, 5H, Cbz). 13C NMR (CD3OD,
d): 12.2, 33.2, 38.4, 39.2, 44.7, 49.0, 59.0, 63.4, 67.3, 69.2,
69.5, 78.2, 83.6, 114.5, 127.7–128.1, 130.0, 130.3, 142.2,
157.8, 158.9, 168.5. MS (ESI) m/z: 1351.5 (M + 1);
1373.5 (M + Na)+. IR (nujol, m, cmꢀ1): 1665.5 (CONH).
Elemental analysis calc. (%) for C66H73Fe3N7O11: C,
60.61; H, 5.63; N, 7.50. Found: C, 60.92; H, 5.64; N,
7.49%.
4.5. Synthesis of compound 5
Ammonium formate (0.25 mmol) and 10% Pd/C
(0.02 equiv.) were added to a solution of 13 (21 mg,
0.05 mmol) in MeOH:CH2Cl2, 1:1 (3 ml). The reaction
mixture was stirred at room temperature for 24 h and fil-
tered through a thin pad of Celiteꢂ. The solvent was evap-
orated; the residue was dissolved in CH2Cl2 (10 ml) and
washed with brine (2 · 5 ml). The aqueous phase was
extracted with CH2Cl2 (3 · 10 ml) and the combined
organic phases were dried over Na2SO4, filtered and evap-
orated to get an orange–yellow oil in 72% yield.
4.7. Synthesis of PVP conjugates: general procedure
HBTU (0.3 mmol) and DIEA (0.6 mmol) were added to
a solution of PVP–COOH 3 (0.06 mmol) in dry DMF
(2 ml). The mixture was stirred for 5 min at room temper-
ature then a solution of 4 (0.3 mmol) in DMF (1 ml) was
added. The reaction was stirred for 3 days at room temper-
ature shielded from light, then the solvent evaporated. The
residue was taken up in CH2Cl2 (2 ml) and the solution was
dropped, under stirring, to Et2O (100 ml). After decanta-
tion of the solvent, the brown precipitate was dissolved in
water (40 ml), and purified by ultra-filtration through a
membrane with a nominal cut off of 3000 (four times).
Products 14–17 were isolated as slightly brown powders
after lyophilization.
NMR characterization: In general NMR spectra of PVP
derivatives show many overlapping and broad signals
between 1.4 and 4.0 ppm due to CH2 and CH signals of
the polymer atactic main chain and of the pyrrolidone ring.
Compound 16: 1H NMR (CDCl3) d: 1.46–1.73 (m,
CH2); 2.02 (m, CH2 pyr); 2.21–2.34 (m, CH2CO); 3.18
(m, CH2N); 3.67–3.91 (m, CH); 4.14–4.28 (m, Fc); 4.72–
4.73 (m, OCH2Fc); 6.84 (m, PhO); 6.97 (m, PhO). 13C
NMR (CDCl3, d): 18.3, 31.5, 35.0, 42.1, 43.7, 45.0, 66.6–
69.1, 115.4, 130.3, 175.5.
(L)–5: 1H NMR (CD3OD), d: 3.68–2.9 (m, 13H,
CH2O + CH2Tyr + CH2NH + COOMe); 4.43–4.0 (m,
35H, OCH2Fc + Fc + CH2O); 4.68–4.52 (m, 1H, CH);
6.91 (d, 2H, PhO, J = 9.0 Hz); 7.14 (d, 2H, PhO,
J = 9.0 Hz). 13C NMR (CD3OD, d): 35.3, 40.8, 51.5,
54.9, 60.8, 66.8, 67.1, 67.6, 68.5, 68.9, 69.2, 83.5, 121.6,
129.9, 135.6, 150.4, 172.0. MS (ESI) m/z 979.7 (M + 1).
IR (nujol, m, cmꢀ1): 1687.9 (COOMe). Elemental analysis
calc. (%) for C50H57Fe3N3O7: C, 61.31; H, 5.87; N, 4.29.
Found: C, 61.02; H, 5.89; N, 4.31%.
4.6. Synthesis of PNA monomers 6 and 7
A solution of 6 or 7 (0.2 mmol) in CH2Cl2 (1 ml) was
slowly added into ethylendiamine (6 ml) at 50 ꢁC. The reac-
tion, stirred at the same temperature and monitored by
TLC (eluent: ethyl acetate, Rf = 0.03), was complete 1 h
and 30 min. After evaporation of the solvent, the residue
was taken up with CH2Cl2 (10 ml), washed with brine
(2 · 10 ml) and the aqueous phase was extracted with
CH2Cl2 (3 · 10 ml). The combined organic phases were
washed with water (3 · 10 ml), dried over Na2SO4 and
evaporated in vacuum affording compound 6 or 7.
Compound 17: 1H NMR (CDCl3) d: 1.46–1.73 (m,
CH2); 2.07 (m, CH2 pyr); 2.20– 2.40 (m, CH2CO); 3.25
(m, CH2N); 3.62–3.94 (m, CH); 4.12–4.30 (m, Fc); 6.83
(m, PhO); 6.95 (m, PhO). 13C NMR (CDCl3, d): 18.3,
31.5, 34.7, 41.8, 43.7, 44.5–44.9, 68.4, 69.1–69.6, 114.6–
115.0, 130.3, 151.7, 175.3.
6: Yellow powder. Yield = 91%. m.p. (pentane) 188–
1
189 ꢁC. H NMR (CDCl3), d: 1.81 (s, 3H, CH3); 2.79–3.3
(m, 11H, CH + CH2NH2 + CH2NHCO + CH2PhO +
CH2NCO + CH2NHCbz); 4.17– 4.3 (m, 9H, Fc); 4.75 (s,
2H, OCH2Fc); 5.05 (s, 2H, CH2Cbz); 6.86 (m, 3H,
PhO + CH@); 7.06 (d, 2H, PhO, J = 9.0 Hz); 7.25–7.33
(m, 5H, Cbz). 13C NMR (CDCl3, d): 12.2, 32.9, 39.4,
40.4, 45.6, 49.3, 66.6, 68.5, 69.0, 69.2, 82.4, 110.4, 115.1,
127.9, 128.1, 129.1, 129.9–130.0, 141.0, 151.3, 157.8,
164.7, 168.4, 170.4. MS (ESI) m/z 765.2 (M + 1). IR (nujol,
m, cmꢀ1): 1665.5 (CONH). Elemental analysis calc. (%) for
C37H39FeN5O7: C, 61.59; H, 5.45; N, 9.71. Found: C,
61.72; H, 5.44; N, 9.69%.
Compound 18: 1H NMR (CDCl3) d: 1.42–1.69 (m,
CH2); 2.02 (m, CH2 pyr); 2.22–2.35 (m, CH2CO); 3.20
(m, CH2N); 3.62–3.88 (m, CH); 4.18 (m, Fc); 5.11 (m,
CH2Cbz); 6.83 (m, PhO); 6.95 (m, PhO), 7.30 (m, Cbz).
13C NMR (CDCl3) d: 18.2, 31.3, 34.6, 42.0, 43.6, 44.8,
115.1, 128.2– 130.0, 175.3
Compound 19: 1H NMR (CD3OD) d: 1.3–1.8 (m, CH2);
2.15 (m, CH2 pyr); 2.25–2.5 (m, CH2CO); 3.25 (m, CH2N);
3.9–3.6 (m, CH); 4.0–4.3 (m, Fc); 6.8–6.9 (m, PhO); 7.1–7.2
(m, PhO); 7.3–7.4 (m, Cbz). 13C NMR (CD3OD, d): 18.9,
32.5, 33.9, 35.9, 43.4, 45.5, 69.3–69.5, 70.2–70.8,
116.0,129.6, 155.4, 177.8.
7: Dark brown sticky oil. Yield 90%. 1H NMR
(CD3OD), d: 1.89 (m, 3H, CH3); 2.30–3.70 (m, 18H,
CH2N + CH2PhO + CH2NHCbz + CH2NHCO + CH2NH2
+ CH2NHCONH + CH2O); 3.73–4.80 (m, 38H, CH +
CH2CO + CH2O + Fc + CH2Fc); 5.1 (s, 2H, CH2Cbz);
6.80–6.90 (m, 3H, PhO + CH@); 7.1–7.2 (d, 2H, PhO,
4.8. Electrochemical measurements
The preliminary cyclovoltammetric (CV) screening and
the following differential pulse voltammetry (DPV) and

1370
C. Baldoli et al. / Journal of Organometallic Chemistry 692 (2007) 1363–1371
[9] V. Buhler, Kollidon: Polyvinylpyrrolidone for the Pharmaceutical
¨
square wave voltammetry (SWV) characterizations of the
PVP conjugated polymers 16–19 were carried out using
an Autolab PGSTAT 12 potentiostat/galvanostat
(EcoChemie, The Netherlands) run by a PC with GPES
software, with the following set of experimental parame-
ters: 0.002 s modulation time; 0.1 s interval time; 0.005 V
step potential; 0.025 V modulation amplitude, for DPV,
and 1000 Hz frequency; 0.005 V step potential, 0.025 V
amplitude, in the case of SWV.
Industry, 1999, BASF Ed.
[10] S. Vijayasekaran, T.V. Chirila, Y. Hong, S.G. Tahija, P.D. Dalton,
I.J. Constable, I.L. McAllister, J. Biomater. Sci. Polym. Ed. 7
(1996) 685.
[11] W.R. Gombtz, S.C. Pankey, R. Phan, R. Drager, K. Donaldons,
K.P. Antonsen, A.S. Hoffman, H.V. Raff, Pharm. Res. 11 (1994)
624.
[12] V.P. Torchillin, J. Microencapsul. 15 (1998) 1.
[13] G.P. Bettinetti, P. Mura, A. Liguori, G. Bramanti, Il Farmaco, 1988,
p. 43.
The solutions of the four PVP conjugates (2 · 10ꢀ4 M)
were prepared in highly deionized water (Millipore Milli-
Qꢂ system), with 0.1 M sodium perchlorate (NaClO4,
Sigma–Aldrich, 98+%) as supporting electrolyte. The
working electrode was a glassy carbon GC (Amel, surface
0.071 cm2), the counter electrode was a platinum wire,
while the operating reference electrode was an aqueous
saturated calomel electrode (SCE). The data have been
subsequently referred to the Me10Fc+|Me10Fc (decamethyl-
ferricinium|decamethylferrocene) reference redox couple,
currently proposed as an improved alternative [30–32] to
ferrocene, proposed by IUPAC [33,34], and whose formal
redox potentials in water are ꢀ0.126 V and ꢀ0.283 V
against our aqueous SCE reference and against ferrocene,
respectively. The cell was thermostated at 298 K and the
solutions were carefully deareated by nitrogen bubbling
before the scans. The optimized polishing procedure for
the working GC electrode consisted of surface treatment
with diamond powder (Aldrich, diameter 1 lm) on a wet
cloth (DP-Nap, Struers).
[14] M.C. Tros de llarduya, C. Martin, M.M. Goni, M.C. Martinez-
Oharriz, Drug Dev. Ind. Pharm. 24 (1998) 295.
[15] P. Ferruti, E. Ranucci, G. Spagnoli, L. Sartore, F. Bignotti,
Macromol. Chem. Phys. 196 (1995) 763.
[16] L. Sartore, I. Peroni, P. Ferruti, R. Latini, R. Bernasconi, J. Biomat.
Sci. Polym. Ed. 8 (1997) 741.
[17] F.M. Veronese, L. Sartore, P. Caliceti, O. Schiavon, E. Ranucci, P.
Ferruti, J. Bioact. Compat. Polym. 5 (1990) 167.
[18] (a) E. Ranucci, M. Tarabic, M. Gilberti, A.-C. Albertsson, Macro-
mol. Chem. Phys. 201 (2000) 1219;
(b) E. Ranucci, L. Macchi, R. Annunziata, P. Ferruti, F. Chiellini,
Macromol. Biosci. 4 (2004) 706.
[19] (a) E. Ranucci, P. Ferruti, R. Annunziata, I. Gerges, G. Spinelli,
Macromol. Biosci. 6 (2006) 216;
(b) Z. Liu, S. Rimmer, Macromolecules 35 (2002) 1200.
[20] (a) M. Bencini, E. Ranucci, P. Ferruti, C. Oldani, E. Licandro, S.
Maiorana, Macromolecules 38 (2005) 8211;
(b) W. He, K.E. Gonsalves, J.H. Pickett, C. Halberstadt, Biomacro-
molecules 4 (2003) 75;
(c) J.U.A. Egstro¨m, L.J. Lindgreen, B. Helgee, Macromol. Chem.
Phys. 207 (2006) 536–544.
[21] L. Luo, M. Ranger, D.G. Lessare, D. Le Garrec, S. Gori, J.-C.
Leroux, S. Rimmel, D. Smith, Macromolecules 37 (2004) 4008.
[22] (a) P. Caliceti, O. Schiavon, M. Morpurgo, F.M. Veronese, L.
Sartore, E. Ranucci, P. Ferruti, J. Bioact. Compat. Polym. 10 (1995)
103;
Acknowledgements
(b) L. Sartore, E. Ranucci, P. Ferruti, P. Caliceti, O. Schiavon, F.M.
Veronese, J. Bioact. Compat. Polym. 9 (1995) 411.
[23] C.F. Brunius, E. Ranucci, Macromol. Rapid Commun. 22 (2001)
1474.
[24] Unpublished results.
[25] Unpublished results.
[26] (a) E.W. Neuse, J. Inorg. Organomet. Polym. Mater. 15 (2005) 3;
(b) C. Xue, Z. Chen, F.-T. Luo, K. Palaniappan, D.J. Chesney, J.
Liu, J. Chen, H. Liu, Biomacromolecules 6 (2005) 1810;
(c) J.C. Swarts, Macromol. Symp. 186 (2002) 123.
[27] (a) C. Baldoli, C. Rigamonti, S. Maiorana, E. Licandro, L. Falciola,
P. Mussini, Chem. Eur. J. 12 (15) (2006) 4091;
(b) C. Baldoli, C. Rigamonti, S. Maiorana, P. Mussini, PCT Int.
Appl. (2006), WO 2006006196;
This study was financially supported by the National
Research Council (CNR), Ministero dell’Istruzione e della
Ricerca (MIUR) and PRIN Project ‘‘Design, synthesis and
biomolecular properties of peptide nucleic acids (PNAs)
and their analogs for diagnostic and therapeutic
applications’’.
References
[1] T. Minko, Drug Disc. Today 2 (2005) 15.
[2] T.J. Dickerson, N.N. Reed, K.D. Janda, Chem. Rev. 102 (2002)
3325.
(c) C. Baldoli, E. Licandro, S. Maiorana, D. Resemini, C. Rigamonti,
L. Falciola, M. Longhi, P.R. Mussini, J. Electroanal. Chem. 85
(2005) 97;
(d) C. Baldoli, L. Falciola, E. Licandro, S. Maiorana, P. Mussini, P.
Ramani, C. Rigamonti, G. Zinzalla, J. Organomet. Chem. 89 (2004)
4791.
[3] R. Haag, F. Kratz, Angew. Chem. Int. Ed. (2006) 45.
[4] The therapeutic index of a drug is defined as the ratio of the toxic dose
to the therapeutic dose.
[5] (a) R. Duncan, in: G.S. Kwon (Ed.), Polymeric Drug Delivery
Systems, Marcel Dekker, New York, 2005, pp. 1–92;
(b) R. Duncan, Nat. Rev. Drug Discov. 2 (2003) 347.
[6] (a) F.M. Veronese, G. Pasut, Drug Disc. Today 10 (2005) 1451;
(b) J.M. Harris, R.B. Chess, Nat. Rev. Drug Discov. 2 (2003)
214.
[7] E.S. Barabas, N-Vinyl Amide Polymers, in: H.F. Mark, N.M.
Bikales, C.G. Overberger, G. Menges (Eds.), Encyclopedia of
Polymer Science and Engineering’, vol. 17, John Wiley and Sons,
New York, 1989, pp. 198–257.
[8] E.V. Hart, B.H. Waxman, in: H. Mark, D.F. Othmer, C.G.
Overberger, G.T. Seaborg (Eds.), Encyclopedia of Chemical Tech-
nology, vol. 23, Interscience, New York, 1983, p. 967.
[28] (a) Peter E. Nielsen (Ed.), Peptide Nucleic Acids: Protocols and
Applications, second ed., Horizon Bioscience, Wymondham, UK,
2004;
(b) E. Uhlmann, A. Peyman, G. Breipohl, W.W. David, Angew.
Chem. Int. Ed. 37 (1998) 2796;
(c) A. Jacob, O. Brandt, S. Wuertz, A. Stephan, M. Schnolzer, J.D.
Hoheisel, Production of PNA-arrays for Nucleic Acid Detection, in:
Peter E. Nielsen (Ed.), Peptide Nucleic Acids, second ed., Horizon
Bioscience, Wymondham, UK, 2004, pp. 261–279.
[29] (a) G. Jaouen (Ed.), Bioorganometallics, first ed., Wiley-VCH,
Weinheim, 2005;

C. Baldoli et al. / Journal of Organometallic Chemistry 692 (2007) 1363–1371
1371
´
(b) D.R. Van Stavaren, N. Metzler-Nolte, Chem. Rev. 104 (2004)
5931–5985;
[31] J. Ruiz, D. Astruc, Comptes Rendus Acad. Sci., Serie IIc: Chem. 1
(1998) 21.
(c) T.S. Zatsepin, S.Y. Andreev, T. Hianik, T.S. Oretskaya, Russ.
Chem. Rev. 72 (2003) 537.
[30] L. Falciola, A. Gennaro, A.A. Isse, P.R. Mussini, M. Rossi, J.
Electroanal. Chem. 593 (2006) 47.
[32] I. Noviandri, K.N. Brown, D.S. Fleming, P.T. Gulyas, P.A. Lay,
A.F. Masters, L. Phillips, J. Phys. Chem. B 103 (1999) 6713.
[33] G. Gritzner, J. Kuta, Pure Appl. Chem. 56 (1984) 461.
[34] G. Gritzner, Pure Appl. Chem. 62 (1990) 1839.