A new ferrocene conjugate of a tyrosine PNA monomer: Synthesis and electrochemical properties

Article abstract of DOI:10.1016/j.jorganchem.2004.09.049

A new chiral ferrocene-labelled tyrosine PNA monomer 1 has been synthesised in good yield in both racemic and enantiomerically pure forms. It is suitable for insertion in various positions of PNA oligomers, a possibility that has been preliminarily demonstrated by synthesising the dimer 16. Moreover, in view of possible applications to nucleic acid detection, a preliminary voltammetric investigation on the electrochemical activity of monomer 1 and its synthetic precursors has been carried out in DMF. It appears that, despite the bulkiness of the PNA monomer backbone, its insertion on the ferrocene group only moderately lowers the latter's diffusion coefficients and peak currents, thus affording voltammetric detection limits in the order of 10^-6-10 ^-7 M.

Full text of DOI:10.1016/j.jorganchem.2004.09.049

Journal of Organometallic Chemistry 689 (2004) 4791–4802
www.elsevier.com/locate/jorganchem
A new ferrocene conjugate of a tyrosine PNA monomer:
synthesis and electrochemical properties
a,
b
c
c,
*
Clara Baldoli *, Luigi Falciola , Emanuela Licandro , Stefano Maiorana
,
b
Patrizia Mussini , Prasanna Ramani , Clara Rigamonti , Giovanna Zinzalla
c
c
c
a
CNR – Institute of Molecular Science and Technologies, Via C. Golgi 19, 20133 Milano, Italy
Department of Organic and Industrial Chemistry and Centre of Excellence CISI University of Milan, Via C. Golgi 19, I-20133 Milano, Italy
b
c
Department of Physical Chemistry and Electrochemistry, University of Milan, Via Golgi 19, I- 20133 Milano, Italy
Received 4 August 2004; accepted 21 September 2004
Available online 22 October 2004
Dedicated to Dr. Richard Fish on the occasion of his 65th birthday.
Abstract
A new chiral ferrocene-labelled tyrosine PNA monomer 1 has been synthesised in good yield in both racemic and enantiomer-
ically pure forms. It is suitable for insertion in various positions of PNA oligomers, a possibility that has been preliminarily dem-
onstrated by synthesising the dimer 16. Moreover, in view of possible applications to nucleic acid detection, a preliminary
voltammetric investigation on the electrochemical activity of monomer 1 and its synthetic precursors has been carried out in
DMF. It appears that, despite the bulkiness of the PNA monomer backbone, its insertion on the ferrocene group only moderately
lowers the latterÕs diffusion coefficients and peak currents, thus affording voltammetric detection limits in the order of 10ꢀ6–10ꢀ7 M.
ꢀ 2004 Elsevier B.V. All rights reserved.
Keywords: Ferrocene; Mitsunobu; Tyrosine PNA monomer; Bioorganometallic chemistry
1. Introduction
[2]. However, in view of large-scale applications,
improving PNA biosensor sensitivity is already a valua-
ble target. This could be done by conjugating PNA to an
organometallic moiety as a number of metal complexes
have intense and easily identifiable spectroscopic or
electrochemical signals that are different from those of
the organic bulk of biomolecules. Many examples of
the usefulness of metal-complexes in labelling biomole-
cules have so far been reported [3], among which the
extensive and original work of Jaouen concerning the
use of metal carbonyl complexes as molecular probes
detectable by IR spectroscopy deserves particular
mention [4].
Peptide nucleic acids (PNAs) represent a very prom-
ising class of artificial DNA analogues with excellent
DNA and RNA binding properties [1]. The stability of
PNA/DNA complexes makes PNAs powerful tools in
anti-sense and anti-gene applications, and as markers
in DNA screening assays. In particular, the development
of DNA biosensors based on PNA recognition layers is
a new and exciting area in analytical chemistry because
of the higher specificity, greater discrimination for sin-
gle-base mismatches and faster hybridisation of PNAs
We have recently reported the synthesis of PNA mono-
mers labelled with arene Cr(CO)3 [5] or Fischer-type car-
bene complexes [6] which are easily detected by FT-IR
spectroscopy even at very low concentrations (10ꢀ7 M).
*
Corresponding authors. Tel.: +39 02 503 14142; fax: +39 02 503
14139.
E-mail addresses: clara.baldoli@istm.cnr.it (C. Baldoli), stefano.
maiorana@unimi.it (S. Maiorana).
0022-328X/$ - see front matter ꢀ 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.jorganchem.2004.09.049

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C. Baldoli et al. / Journal of Organometallic Chemistry 689 (2004) 4791–4802
Another suitable candidate marker for PNAs is the ferro-
cene group, which undergoes a stable reversible redox
reaction and can be detected as such, or by adding a
bioelectro-catalytic amplification step pivoting on the
glucose-oxidase mediated oxidation of glucose [7]. Ferro-
cene has been widely used as an active probe for DNA
detection [8], but only a few examples of ferrocene-la-
belled PNA have been reported: the reaction of ferrocen-
ecarboxylic acid with the terminal amino group of a PNA
heptamer [9], and the synthesis of a PNA oligomer in
which a ferrocene unit was attached to a nucleobase [10].
The aim of our study was to synthesise ferrocenyl
multi-labelled PNA oligomers, an objective that can be
reached by inserting a number of mono-labelled mono-
mers along the chain or by introducing a single molecule
containing multiple ferrocenes.
We first considered the synthesis of a suitable mono-
labelled monomer. We here describe the preparation of
the chiral ferrocenyl-labelled tyrosine PNA monomer 1
in racemic and enantiomerically pure forms, in which
the organometallic moiety is bound to the phenolic
group of the tyrosine residue (Fig. 1). In addition to
its chirality [11], monomer 1 meets two important requi-
sites: it can be inserted in various oligomer positions
(using either amino or carboxy functions) and the ‘‘la-
bel’’ does not modify any of the hydrogen-bond sites
of the nucleobases.
nyl conjugate 1 was obtained in low yield (28%) after a
very long reaction time (72 h).
In the second route, (Scheme 4, path a), the Mitsun-
obu reaction was run on the N-Cbz-protected tyrosine
methyl ester 6 under the same conditions, and gave the
corresponding labelled amino acid 7 in good yield
(81%) and in a relatively short reaction time (18 h). Re-
moval of the Cbz group with ammonium formate and
Pd/C afforded ferrocenyl-tyrosine methylester 8 (75%),
which underwent reductive amination with N-Cbz-2-
amino acetaldehyde 9, using NaCNBH3 and ZnCl2, to
give the labelled backbone 10 in 76% yield. Alterna-
tively, compound 10 can be obtained in comparable
yield (Scheme 3, path b) by running the Mitsunobu reac-
tion on the backbone 3, which was easily obtained by
the reductive amination of tyrosine methyl ester 2 with
9. The target PNA monomer 1 was eventually obtained
in 70% yield after coupling 10 with carboxymethyl thy-
mine 11 in DMF in the presence of DCC at room
temperature.
All of the synthetic steps were performed on racemic
tyrosine and both enantiomers. The enantiomeric purity
of
L and D monomer 1 was checked by HPLC equipped
with a chiral OD column and was complete in both
cases.
As mentioned above, the next step in this work
should be the preparation of a multi-labelled PNA olig-
omer by inserting a number of ferrocene-monomers
within the chain. As PNA oligomer synthesis is usually
performed on solid phase [1], and requires selective
hydrolysis of the ester group and the cleavage of N-
Cbz protection, we first investigated the stability and
reactivity of monomer 1 to these synthetic transforma-
tions. Ester hydrolysis was successfully performed using
Ba(OH)2 or LiOH at room temperature and, after acid-
ification with a 1 M solution of KHSO4, the correspond-
ing acid 12 was isolated in respectively 95% and 80%
yield (Scheme 4). The enantiomeric excess of 12 deter-
mined by HPLC was 88% with Ba(OH)2 hydrolysis
and 85% with LiOH hydrolysis.
2. Results and discussion
The synthesis of a tyrosine PNA monomer has previ-
ously been reported [12], and we considered the Mitso-
nobu reaction with ferrocenemethanol 5 a suitable
method for labelling the tyrosine OH group. Looking
at the scheme for obtaining monomer 4 (Scheme 1), in
principle, the ferrocene unit could be introduced on the
starting amino acid 2, on the backbone 3, or directly on 4.
We investigated all three protocols. We first tried the
Mitsunobu reaction between the racemic tyrosine PNA
monomer 4 and ferrocenemethanol 5 in THF at room
temperature (Scheme 2), but the corresponding ferroce-
The carbobenzyloxy group in monomer 1 was cleaved
using ammonium formate and Pd/C or H2, Pd/C and
O
N
Me
NH
O
O
O
N
oligomerization
oligomerization
CbzHN
OMe
O
Fe
1
Fig. 1.

C. Baldoli et al. / Journal of Organometallic Chemistry 689 (2004) 4791–4802
4793
O
Me
NH
O
PgHN
N
O
Me
NH
COOMe
NH2
COOMe
NH
O
N
O
O
H
O
PgHN
O
N
OH
CbzHN
OMe
OH
2
3
4
OH
O
H
OH
Fe
5
Scheme 1.
O
O
N
Me
Me
NH
NH
N
O
O
O
O
O
OH
O
PPh , DEAD,
3
N
N
Fe
CbzHN
OMe
+
CbzHN
OMe
THF , r.t., 72 h
4
5
1
O
28%
OH
Fe
Scheme 2.
AcOH in methanol at room temperature; this did not
produce any ‘‘free amino’’ monomer, but only gave
ketopiperazine 13 as a result of the intramolecular at-
tack of the deprotected amino group on ester carbonyl.
To circumvent this problem we considered the synthe-
sised dimer 15 in which intramolecular cyclisation could
not take place. Racemic dimer 15 was obtained by react-
ing 12 and a classical glycine PNA monomer 14 [13]
(Scheme 5). The condensation was run in DMF at room
temperature using the trifluoroacetate of 14 in the pres-
ence of N,N-diisopropylethylamine (DIPEA) as base
and diisopropylcarbodiimide (DIC) and 3,4-dihydro-3-
hydroxy-4-oxo-1,2,3-benzotriazine (DhBtOH) as con-
densing agents. Compound 15 was isolated in good
yield.
This result is a good premise for the synthesis of ferro-
cene-multilabelled PNA oligomers.
3. Electrochemistry
In the perspective of using PNA oligomers as active
DNA probes, it is important to evaluate how the func-
tionalisation of ferrocene with the PNA moiety affects
its electrochemical activity in terms of both redox poten-
tials and currents. We therefore performed a preliminary
voltammetric investigation of monomer 1, synthetic
intermediates 7, 8 and 10, and ferrocenemethanol 5 as
a reference compound, in DMF + 0.1 M tetraethyl
ammonium perchlorate (TEAP), using cyclic voltamme-
try (CV) on stationary Pt and glassy carbon (GC) elec-
Dimer 15 was then hydrogenated with HCO2NH4
and Pd/C in MeOH at room temperature, and afforded
the corresponding N-deprotected dimer 16 in 62% yield.
trodes (radius = 1 mm) at scan rates of 20–500 mVsꢀ1
and slow-scan voltammetry on Pt and GC rotating disk
,

4794
C. Baldoli et al. / Journal of Organometallic Chemistry 689 (2004) 4791–4802
Path a
NHCbz
CO Me
NH
2
NHCbz
CO Me
OH
CO Me
2
2
Fe
2
5
-
+
HCOO NH , Pd/C
4
PPH , DEAD, THF
3
r.t., 18 h.
MeOH, 26h
O
O
OH
Fe
Fe
7, 81%
8, 75%
6
H
CbzHN
O
9
ZnCl ,NaCNBH ,
2
3
0 ˚C, MeOH, 18 h
76%
CbzHN
Path b
NH
CbzHN
NH
NH
2
CO Me
2
H
OH
CbzHN
CO Me
O
CO Me
2
2
Fe
9
5
ZnCl , NaCNBH ,
2
3
PPh , DEAD,
3
MeOH, r.t., 15 h
O
THF , r.t., 4 d
Fe
OH
OH
75%
10
2
3, 70%
O
O
Me
Me
NH
NH
N
O
N
O
O
CO H
2
11
O
DCC, DMF,
r.t., 18 h
N
CbzHN
OMe
O
Fe
1, 70%
Scheme 3.
electrodes (RDE; radius = 1 mm) at a scan rate of 5
mVsꢀ1, in order to obtain a sound estimate of the diffu-
sion coefficients from the limiting currents, according to
LevichÕs equation [14].
The CV characteristics of the tested compounds in
the case of the 0.02 Vsꢀ1 scan rate are shown in Fig. 2
and the relevant features are summarized in Table 1.
All of the compounds exhibit a single, reversible mono-
electronic wave that is shifted in the positive direction
for all ferrocenyl tyrosine derivatives with respect to fer-
rocenemethanol 5. The observed potential shift remains
almost unchanged with increasing complexity of the tyr-
osine compounds.
On the contrary, the currents appear to decrease
with the increasing bulkiness of the ferrocene-labelled
molecules, in accordance with the decrease in the rel-
evant diffusion coefficients (Fig. 3) obtained by ana-
lysing the limiting current densities on RDE.
However, this decrease is moderate (conveniently for
our applicative aim) and the diffusion coefficients re-
main satisfactorily high (unlike, for example, in the
case of a recent work by Kraatz et al. [15] in which

C. Baldoli et al. / Journal of Organometallic Chemistry 689 (2004) 4791–4802
4795
O
N
O
N
Me
Me
NH
NH
O
O
1. LiOH or Ba(OH)
THF, r.t., 4 h 30 min
2
O
O
O
O
N
N
CbzHN
OH
CbzHN
OMe
2. KHSO
4
O
O
Fe
Fe
1,
12
Ba(OH)2, y: 95%
LiOH, y: 80%
HCO2NH4 or H2
Pd/C, MeOH
r.t., 2 h
O
O
HN
N
N
O
Me
N
H
O
O
Fe
13, 80%
Scheme 4.
a low D range (1–4 · 10ꢀ7 cm2 sꢀ1) was obtained for
some ferrocene-monosaccharide compounds as a con-
sequence of the high number of groups engaged in
hydrogen bonding with the working aqueous
medium).
Accordingly, preliminary investigation of the voltam-
metric detection limit for our monomer 1 showed that
currents are still perceivable in the 10ꢀ6–10ꢀ7 M range.
The best results (Fig. 4) were obtained working in aceto-
nitrile (which is less viscous than DMF, and thus en-
hances diffusion coefficients and peak currents), and
changing simple CV for differential pulse voltammetry
DPV [16].
coefficients and peak currents, thus affording voltam-
metric detection limits in the order of 10ꢀ6–10ꢀ7 M.
The study of PNA monomers labelled with poly-
ferrocene units [17] is currently in progress with the
aim of improving sensitivity in ferrocene-based electro-
chemical biosensors.
5. Experimental
5.1. General data
All reactions were performed under nitrogen atmos-
phere using a standard vacuum line. THF was distilled
over sodium benzophenone ketyl, other reagent grade
solvent were dried by standard procedures. Column
chromatography was performed using Merck silica gel
4. Conclusions
1
A new chiral ferrocene-labelled PNA monomer 15
can be synthesised in good yield and complete enantio-
meric excess when starting from L or D tyrosine. This
60 (70–230 mesh); H NMR spectra were recorded on
a Bruker AC 300 and AMX 300 MHz. IR spectra were
recorded on a Perkin–Elmer 1725X FT-IR. HPLC anal-
yses were run on Agilent 1100 series, using a chiralcel
OD column. Melting points were measured with a Buchi
¨
B-540 apparatus and are uncorrected. Optical rotations
were measured using a Perkin–Elmer 243 polarimeter.
monomer is suitable for insertion in various positions
of PNA oligomers, as has been preliminarily demon-
strated by synthesising dimer 16. In addition in view
of the applications of such labelled monomers to PNA
biosensors, a preliminary voltammetric investigation
on the electrochemical activity of monomer 1 has been
carried out. It appears that, despite the bulkiness of
the PNA monomer backbone, its insertion on the ferro-
cene group only moderately lowers the latterÕs diffusion
5.2. Ferrocenemethanol (5)
Prepared according literature report [18]. m.p. 78–81
1
ꢁC (pentane). H NMR (CDCl3, d): 1.5 (bs, 1H, OH);

4796
C. Baldoli et al. / Journal of Organometallic Chemistry 689 (2004) 4791–4802
O
N
O
N
Me
Me
O
N
NH
NH
Me
NH
O
O
O
O
O
O
O
DIPEA, DIC
O
N
N
O
12
CbzHN
N
H
OCH3
+
N
DhBtOH, DMF
r.t., 24 h
H3N
-
OCH3
CF3CO2
O
14
15, 65%
Fe
HCO2NH4
Pd/C, MeOH
r.t., 25 h
O
O
N
Me
Me
NH
NH
N
O
O
O
O
O
O
N
N
H2N
N
H
OCH3
O
16, 62%
Fe
Scheme 5.
20
)-3, pale yellow oil. ½aꢁ ¼ þ4:27 (c = 4.3 in CHCl3);
4.16–4.17 (m, 5H); 4.22–4.24 (m, 2H); 4.71 (d, 2H,
J = 6.1 Hz).
(
L
D
20
D
(
D
)-3, ½aꢁ ¼ ꢀ4:98 (c = 4.8 in CHCl3); 1H NMR (CDCl3,
d): 2.5–2.6 (m, 1H, CH2NH); 2.7–2.9 (m, 3H,
CH2NH + CH2PhOH); 3.1–3.2 (m, 2H, CH2NHCbz);
3.4–3.5 (m, 1H, CHCO2Me); 3.65 (s, 3H, OCH3); 5.0
(bs, 2H, PhCH2O); 5.1 ( bs, 1H, OH); 5.3 (bs, 1H,
OCONH); 6.7 (d, 2H, PhOH; J = 8.4 Hz); 7.0 (d, 2H,
PhOH; J = 8.4 Hz); 7.3–7.4 (m, 5H, Ph); 13C NMR
(CDCl3, d): 38.5, 40.5, 47.12, 51.9, 62.5, 66.7, 115.5,
127.9, 128.3, 128.5, 130.0, 155.0, 156.7, 174.9. MS, (EI)
m/z 372 (M+). IR (neat, m cmꢀ1): 3354 (OH), 1698 (CO).
5.3. N-(2-Cbz-aminoethyl)tyrosine methyl ester (3)
A mixture of ZnCl2 (4.2 mmol, 0.6 eq) and NaC-
NBH3(8.4 mmol, 1.2 eq) in dry MeOH (7 ml) was
slowly dropped at 0 ꢁC into a solution of N-Cbz-2-
aminoacetaldehyde 9 (7.0 mmol, 1 eq) and tyrosine
methyl ester 2 (7.7 mmol, 1.1 eq.) in MeOH (8 ml).
The mixture was stirred for 18 h at room tempera-
ture. After evaporation of the solvent, the residue
was taken up with CH2Cl2 (50 ml) and washed with
50 ml of NaHCO3 saturated solution, the aqueous
phase was then extracted with CH2Cl2 (3 · 50 ml).
The combined organic phases were washed with
water, dried over Na2SO4 and evaporated. The crude
product was purified by column chromatography on
silica gel (AcOEt; Rf = 0.40) affording compound 3
in 77% yield.
5.4. Synthesis of tyrosine PNA monomer (4)
A mixture of DCC (2.6 mmol), carboxymethyl thy-
mine 11 (2.6 mmol) and DhBtOH (2.6 mmol) in dry
DMF (7 ml) was stirred for 1 h at room temperature.
From the yellow–green solution a precipitate of DCU
was formed. A solution of 3 (1.6 mmol) in dry DMF
(4 ml) was added to the suspension and the reaction
was stirred for 18 h at room temperature. The DCU
was filtered off and, after distillation of the solvent, the
residue was taken up with AcOEt (40 ml) and washed
(
D
,
L
)-3 white powder, m.p. 87–88 ꢁC (pentane). Anal.
Calc. for C20H24N2O5: C, 64.50.; H, 6.50; N, 7.52.
Found: C, 63.96; H, 6.52; N, 7.49%.

C. Baldoli et al. / Journal of Organometallic Chemistry 689 (2004) 4791–4802
4797
0.150
5
0.125
0.100
0.075
0.050
0.025
0.000
-0.025
-0.050
-0.075
-0.100
8
1
7
10
-0.25 -0.2 -0.15 -0.1 -0.05
0
0.05 0.1 0.15 0.2 0.25
E / V(Fc+|Fc)
Fig. 2. A synopsis of the CV characteristics of the tested substrates, obtained on the GC electrode, in DMF + 0.1 M TEAP medium, at a 20 mVsꢀ1
scan rate and at 298 K.
Table 1
Selected CV features obtained for the tested molecules on GC stationary disk electrodes (1 mm radius) in DMF + 0.1 M TEAP medium, at potential
scan rate v = 0.02 Vsꢀ1, at 298 K: anodic peak potentials Ep,a; anodic half-peak widths (EpꢀEp/2)a; distance between anodic and cathodic peaks,
(Ep,aꢀEp,c); half-wave potentials E1/2, calculated (1) as the average between anodic and cathodic peak potentials, or (2) from convolutive analysis of
the CV characteristics; anodic peak current densities, normalized vs. the substrate concentration, (ip,a/c); and anodic to cathodic peak current ratios,
Ip,a/Ip,c
Molecule
MW
(gmolꢀ1
c
Ep,a
(V(Fc+jFc)
(EpꢀEp/2 a
)
(Ep,aꢀEp,c
)
E1/2 (1)
(V(Fc+jFc))
E1/2 (2)
(V(Fc+jFc))
(ip,a/c)
(A cmꢀ2 molꢀ1 dm3)
Ip,a/Ip,c
)
(mol dmꢀ3
)
(V)
(V)
1
5
736.59
216.06
527.39
393.26
570.46
0.000751
0.000756
0.000746
0.000780
0.000748
0.043
ꢀ0.027
0.036
0.061
0.057
0.058
0.059
0.059
0.078
0.069
0.076
0.069
0.070
0.004
ꢀ0.062
ꢀ0.002
0.003
0
0.008
ꢀ0.059
0.001
0.063
0.102
0.062
0.079
0.057
1.3
1.1
1.3
1.2
0.8
7
8
10
0.037
0.035
0.003
ꢀ0.001
All potentials are referred to the ferrocene couple in DMF solvent.
with a saturated solution of NaHCO3 (40 ml). The aque-
ous phase was extracted with AcOEt (3 · 40 ml). The
combined organic phases were washed with water (40
ml), dried over Na2SO4 and evaporated in vacuo. The
crude product was purified by column chromatography
on silica gel (AcOEt; Rf = 0.28) affording the PNA
monomer 4 as white powder in 70% yield.
Jgem = 15.5 Hz); 3.0–3.03 (m, 1H, NHCH2); 3.14–317
(m, 1H, CH2PhOH); 3.25–3.27 (m, 1H, NHCH2);
3.27–3.29 (m, 1H, CH2PhOH); 3.45–3.47 (m, 1H,
CH2N, Jgem = 15.5 Hz); 3.77 (s, 3H, OCH3); 3.82 (d,
1H, CH2Thym, Jgem = 16.2 Hz); 3.84–3.86 (m, 1H,
CH); 4.9 (d, 1H, CH2Thym, Jgem = 16.2 Hz); 5.0 (d,
1H, PhCH2O, Jgem = 12.3 Hz ); 5.18 (d, 1H, PhCH2O,
Jgem = 12.3 Hz); 5.8 (bs, 1H, Cbz-NH); 6.7 (s, 1H,
CH = Thym); 6.76 (d; 2H, PhOH, J = 8.2 Hz); 6.95 (d;
2H, PhOH, J = 8.2 Hz); 7.2–7.4 (m, 5H, Ph); 7.52 (bs,
1H, OH); 10.35 (s, 1H, NH Thym). 13C NMR (CDCl3,
d): 12.3, 33.11, 38.8, 48.7, 49.5, 52.83, 64.15, 66.9, 110.8,
115.5, 127, 128, 130.2, 136, 141, 151.6, 155.6, 156, 164.5
167, 170.9. MS (FAB+) m/z 539 (M+), 540 (M+ + 1).
(
D
,
L
)-4: white powder, m.p. 181–182 ꢁC (Et2O);
Anal. Calc. for C27H30N4O8: C, 60.22; H, 5.61; N,
10.40. Found: C, 58.96; H, 6.26; N, 10.73%.
(
L
20
D
)-4: white powder, m.p. 104–107 ꢁC (pentane);
½aꢁ ¼ ꢀ92:95
(c = 0.47
in
CHCl3);
(D)-4:
20
½aꢁ ¼ þ91:83 (c = 0.4 in CHCl3); 1H NMR (CDCl3,
D
d): 1.78 (s, 3H, CH3Thym); 2.6 (bd, 1H, CH2N,

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C. Baldoli et al. / Journal of Organometallic Chemistry 689 (2004) 4791–4802
10
9
8
7
6
5
4
3
2
1
5
8
7
10
1
100
200
300
400
500
600
700
800
MW / (gmol-1)
Fig. 3. Diffusion coefficients D of all tested substrates in DMF + 0.1 M TEAP at 298 K, obtained by RDE analysis and plotted against molecular
weights MW.
to a solution of ferrocenemethanol (0.7 mmol, 0.73 eq)
in dry THF (3 ml). The mixture was cooled at ꢀ30 ꢁC
and DEAD (0.96 mmol, 1 eq, 0.48 ml of 2 M solution
in THF) was slowly added. The orange solution was stir-
A
red at room temperature for 18 h. After evaporation of
the solvent, the residue was dissolved in 30 ml of CH2Cl2
and washed with brine (30 ml). The aqueous phase was
extracted with of CH2Cl2 (3 · 30 ml). The combined or-
ganic phases were dried over Na2SO4 and evaporated.
The crude product was purified by column chromatog-
raphy on silica gel (Light petroleum/AcOEt, 7:3;
i
B
0.1 mA/cm2
Rf = 0.30) affording 7 in 81% yield.
(
D
,
L
)-7: orange powder; m.p. 112–113 ꢁC (pentane).
Anal. Calc. for C29H29FeNO5: C, 66.04; H, 5.54; N,
2.66. Found: C, 66.10; H, 5.56; N, 2.57%.
(
B'
20
)-7 orange oil; ½aꢁ ¼ þ31:95 (c = 1.9 in CHCl3);
L
D
20
)-7 orange oil; ½aꢁ ¼ ꢀ32:47 (c = 1.73 in CHCl3).
(
D
D
1H NMR (CDCl3, d): 3.1 (bs, 2H, CH2PhOH); 3.7 (s,
3H, OCH3); 4.1–4.2 (m, 7H, CH, Fc); 4.3 (s, 2H, CH,
Fc); 4.63 (m, 1H, CH); 4.75 (s, 2H, FcCH2O); 5.1 (s,
2H, PhCH2O); 5.2 (bs, 1H, NH); 6.8 (d, 2H, Ph,
J = 8.6 Hz); 7.1 (d, 2H, Ph, J = 8.6 Hz); 7.3–7.4 (m,
5H, Ph); 13C NMR (CDCl3), d: 37.12, 52.08, 54.8,
66.4, 66.7, 68.4, 69.0, 82.35, 114.7, 127.6, 127.9, 128.3,
130.0, 136, 153, 157.8, 171.9. MS (ESI) m/z: 527 (M+),
527 (M+ + 1). IR (CHCl3, m cmꢀ1): 1718 (NHCO).
-0.4 -0.3 -0.2 -0.1 0.0
0.1
0.2
0.3
0.4
E / V (Fc Fc+)
|
Fig. 4. Voltammetric characteristics obtained on GC disk electrodes
(radius = 1.5 mm), at 298 K, working with low-concentration solutions
of PNA monomer 1 in ACN + 0.1 M TBAP: A: CV (0.2 Vsꢀ1), 3.7 ·
10ꢀ6 M substrate; B: DPV (0.05 Vsꢀ1) 3.7 · 10ꢀ6 M substrate; B0:
DPV (0.05 Vsꢀ1), 3.7 · 10ꢀ7 M substrate.
5.6. O-Ferrocenyltyrosine methyl ester (8)
At room temperature 30 mg of Pd/C (10%) and
HCO2NH4 (0.7 mmol, 2.3 eq.) were added to a solution
of 7 (0.3 mmol., 1 eq) in 6 ml of a mixture of CH2Cl2/
MeOH, 3:2. The reaction was stirred overnight at room
temperature, then filtered over a pad of celite. The
5.5. O-Ferrocenyl-N-Cbz-tyrosine methyl ester (7)
N-Cbz-tyrosine methyl ester 6 (0.96 mmol, 1 eq) and
PPh3 (0.96 mmol, 1 eq) were added at room temperature

C. Baldoli et al. / Journal of Organometallic Chemistry 689 (2004) 4791–4802
4799
solvent was evaporated, the residue was dissolved in
AcOEt (10 ml) and washed with brine (2 · 10 ml), the
aqueous phase was extracted with AcOEt (3 · 15 ml).
The combined organic layers, after washing with H2O
(10 ml), were dried over Na2SO4 and evaporated. The
crude product was purified by column chromatography
on silica gel (AcOEt; Rf = 0.2) affording compound 8 in
75% yield.
570.1800. Found 570.1802. IR (CHCl3, m, cmꢀ1) 1698-
1718 (CONH).
5.8. Synthesis of 10 via Mitsunobu reaction between 5
and 3
At room temperature 3 (5.4 mmol, 1.2 eq) and PPh3
(5.4 mmol, 1.2 eq) were added to a solution of ferro-
cenemethanol (4.5 mmol, 1 eq) in dry THF (24 ml).
The mixture was cooled at 0 ꢁC and DEAD (5.4
mmol, 1.2 eq., 2.7 ml of 2 M solution in THF) was
slowly added. The orange solution was stirred at room
temperature for four days. After evaporation of the
solvent, the residue was dissolved in CH2Cl2 (150 ml)
and washed with brine (150 ml). The aqueous phase
was extracted with CH2Cl2 (3 · 150 ml). The combined
organic phases were dried over Na2SO4 and evapo-
rated. The crude product was purified by column chro-
matography on silica gel, as reported above, affording
10 in 75% yield.
(
D
,
L
)-8: orange powder, m.p. 88–90 ꢁC (pentane).
Anal. Calc. for C21H23FeNO3 (393,26): C, 64.14; H,
5.90; N, 3.56. Found: C, 63.99; H, 5.92; N, 3.55%.
(
D
20 20
)-8 orange powder; m.p. 107–108 ꢁC (pentane);
½aꢁ ¼ ꢀ12:5 (c = 0.172 in CHCl3);
L
-8 ½aꢁ ¼ þ14:73
D
D
(c = 0.296 in CHCl3). 1H NMR (CDCl3, d): 1.6 (bs,
2H, NH2); 2.7–2.8 (dd, 1H, CH2Ph, Jgem = 13.6 Hz,
Jvic = 5.1 Hz); 3.0–3.1 (dd, 1H, CH2Ph, Jgem = 13.6
Hz, Jvic = 5.1 Hz); 3.68–3.7 (m, 1H, CH); 3.71 (s,
3H, OCH3); 4.17–4.2 (m, 7H, CH, Fc); 4.3–4.32 (t,
2H, CH, Fc, J = 1.84 Hz); 4.76 (s, 2H, OCH2Fc);
6.87 (d, 2H, Ph, J = 8.5 Hz); 7.1 (d, 2H, Ph, J = 8.5
Hz). 13C NMR (CDCl3, d): 40.1, 52.0, 55.9, 66.6,
68.6, 69.14, 82.3, 114.9, 129.1, 130.2, 157.8, 174.8.
MS (EI) m/z: 393 (M+). IR (CHCl3, m cmꢀ1): 3500
(NH2), 1735 (CO).
5.9. Synthesis of ferrocene-labelled monomer 1 by
coupling of 10 with carboxymethyl thymine 11
A mixture of DCC (0.26 mmol), carboxymethyl
thymine 11 (0.26 mmol) and DhBtOH (0.26 mmol)
in dry DMF (1.5 ml) was stirred for 1 h at room tem-
perature. From the yellow–green solution a precipitate
of DCU was formed. A solution of 10 (0.16 mmol) in
dry DMF (1.5 ml) was added to the suspension and
the mixture stirred for 20 h at room temperature.
The DCU was filtered off and, after distillation of
the solvent, the residue was taken up with AcOEt
(10 ml) and washed with a saturated solution of NaH-
CO3 (10 ml). The aqueous phase was extracted with
AcOEt (3 · 10 ml). The combined organic phases were
washed with water (10 ml), dried over Na2SO4 and
evaporated. The crude product was purified by column
chromatography on silica gel with AcOEt/Light petro-
leum = 6/4 affording the PNA monomer 1 as orange
powder in 70% yield.
5.7. Synthesis of 10 via reductive amination reaction
between 8 and 9
A mixture of ZnCl2 (0.165 mmol) and NaCNBH3
(0.33 mmol) in dry MeOH (1 ml) was slowly added at
0 ꢁC into a solution of N-Cbz-2-aminoacetaldehyde (9)
(0.29 mmol) and 8 (0.30 mmol) in dry MeOH (2 ml).
The reaction was complete after stirring 18 h at room
temperature. After evaporation of the solvent, the resi-
due was taken up with CH2Cl2 (10 ml) and washed with
a saturated solution of NaHCO3(10 ml) and the aqueous
phase was extracted with CH2Cl2 (3 · 10 ml). The com-
bined organic phases were washed with water, dried
over Na2SO4 and evaporated in vacuo. The crude prod-
uct was purified by column chromatography on silica gel
(Light petroleum/AcOEt = 3/7; Rf = 0.32) affording
compound 10 as orange oil in 76% yield.
(
(
D
,
L
)-1: Orange powder, m.p. 181–182 ꢁC (pentane);
20
)-10, orange oil, ½aꢁ ¼ ꢀ6:09
D
,
L
)-10: orange oil, (
L
D
Anal. Calc. for C38H40N4O8Fe: C, 61.96; H, 5.47; N,
7.58. Found: C, 60.98; H, 5.50; N, 7.48%.
D
20
(c = 0.95 in CHCl3). (
)-10: ½aꢁ ¼ þ5:62 (c = 0.97 in
D
CHCl3);. 1H NMR (CDCl3, d): 2.54–2.56 (m, 1H,
CH2NH); 2.75–2.76 (m, 1H, CH2NH); 2.80–2.82 (m,
1H, CH2PhO); 2.90–2.91 (m, 1H, CH2PhO); 3.1–3.3
(m, 2H, NHCH2); 3.43 (bt, 1H, CH); 3.66 (s, 3H,
OCH3); 4.18 (s, 5H, CH, Fc); 4.19 (t, 2H, CH, Fc,
J = 1.8 Hz); 4.30 (t, 2H, CH, Fc, J = 1.8 Hz); 4.74 (s,
2H, OCH2Fc); 5.1 (s, 2H, PhCH2); 5.20 (bs, 1H,
NHCbz); 6.86 (d; 2H, PhO, J = 8.6 Hz); 7.07 (d; 2H,
PhO, J = 8.6 Hz); 7.33–7.35 (m, 5H, Ph). 13C NMR
(CDCl3, d): 38.6, 40.5, 47.2, 51.7, 62.5, 66.5, 68.5, 69.1,
82.6, 114.4, 128.0, 128.4, 129, 130.0, 136.7, 157.8,
156.5, 174.8. HRMS calc. for C31H34FeN2O5:
(
L
)-1: orange powder, 145–150 ꢁC (pentane),
20
D
½aꢁ ¼ ꢀ93:3 (c = 1.0 in CHCl3, e.e. >99% (HPLC, chi-
20
ral OD column, hexane/isopropanol:70/30, 0.6 ml flow);
(
D
)-1: ½aꢁ ¼ þ90:7 (c = 0.63 in CHCl3), e.e. >99%
D
(HPLC). 1H NMR (CDCl3, d): 1.85 (s, 3H, CH3 Thym);
2.6 (d, 1H, CH2N, J = 15 Hz,); 3.0–3.4 (m, 5H,
NCH2CH2N + CH2PhO); 3.78 (bs, 4H, OCH3 + CH);
3.93 (d, 1H, J = 16.3 Hz CH2CO); 4.19 (bs, 7H, CH,Fc);
4.30 (bs, 2H, CH, Fc); 4.58 (d, 1H, J = 16.3 Hz,
CH2CO); 4.77 (s, 2H, CH2Fc); 5.01 (d, 1H, J = 12.2
Hz, PhCH2O); 5.19 (d, 1H, J = 12.2 Hz, PhCH2O); 5.6
(bs, 1H, NHCbz); 6.61 (s, 1H, CH@); 6.88 (d, 2H,

4800
C. Baldoli et al. / Journal of Organometallic Chemistry 689 (2004) 4791–4802
PhO, J = 8.4 Hz); 7.05 (d, 2H, PhO, J = 8.4 Hz); 7.34–
7.36 (m, 5H, Ph); 8.71 (s, 1H, NH Thym). 13C NMR
(CDCl3, d): 12.4, 33.20, 39.06, 48.44, 48.97, 52.80,
63.70, 66.5 66.6, 68.4, 68.9, 82.6, 110.4, 115.2, 128.5,
129.3, 130.1, 141, 141.1, 150.8, 155.6, 157.8, 164.1,
166.95, 170.8. MS, (FAB+) m/z: 737 (M+), 738
(M+ + 1), 739 (M+ + 2). IR (nujol, m cmꢀ1) 1680, 1740
(CONH). UV (CH2Cl2): C = 6.79 · 10ꢀ5 M, A = 0.997,
82.61, 111.5, 115.03, 128.0, 128.38, 129.91, 130.1,
136.4, 141.03, 152.19, 156.68, 157.7, 164.04, 166.50,
172.94 (COOH). HRMS (EI): Calc. for C37H38FeN4O8.
722.20. Found: 722.2014. IR (nujol, m, cmꢀ1): 1673
(CONH).
5.12. Synthesis of dimer 15
k
max = 266 nm.
At room temperature 12 (0.54 mmol 1.1eq) was dis-
solved in dry DMF (5 ml). The yellow solution was
cooled to ꢀ5 ꢁC then DIC (0.97 mmol, 2.0 eq) and
DhBtOH (0.97 mmol, 2.0 eq) were added. After stir-
ring for 15 minutes, PNA monomer 14 [13] (0.49
mmol, 1.0 eq) dissolved in dry DMF (3 ml) was slowly
added over 10 minutes while keeping the temperature
around 0 ꢁC, then DIEA(0.78 mmol;1.6 eq) was added
and the mixture warmed to room temperature and
stirred for 24 h. After distillation of DMF the thick
liquid was dissolved in AcOEt (50 ml) and washed
with a saturated solution of NaHCO3(15 ml) and
H2O (15 ml). The organic phase was dried over
Na2SO4, filtered and evaporated under vacuum. The
crude product was purified by column chromatogra-
phy on silica gel (eluent: EtOAc/MeOH 9:1) affording
the Z-protected dimer 15 as yellowish white powder in
65% yield.
5.10. Synthesis of ferrocene-labelled monomer 1 ‘‘via’’
Mitsunobu reaction between monomer 4 and
ferrocenemethanol
At room temperature PPh3 (0.38 mmol, 1.4 eq) was
added to a solution of ferrocenemethanol 5 (0.27 mmol,
1 eq) and monomer 4 (0.38 mmol, 1.4 eq) in dry THF (6
ml). The mixture was cooled at ꢀ30 ꢁC and DEAD (0.38
mmol, 0.2 ml of 2 M solution in THF, 1.4 eq) was
dropped into the solution, that was stirred for 72 h at
room temperature. The solvent was evaporated and
the residue taken-up with CH2Cl2 (30 ml) and washed
with brine (30 ml), the aqueous phase was then extracted
with CH2Cl2 (3 · 20 ml). The combined organic layers
were dried over Na2SO4 and evaporated to give an or-
ange residue that was purified by silica-gel column chro-
matography, (AcOEt). Monomer 1 was isolated in 28%
yield.
(
D
,
L
)-15, m.p.: 181–190 ꢁC (pentane) Anal. Calc. For
C49H53FeN8O12: C, 58.7; H, 5.30; N, 11.20; found: C,
1
57.37; H, 5.35; N, 11.36. H NMR (CDCl3, d): 1.72,
5.11. Ester hydrolysis in monomer 10 to give 12 using
Ba(OH)2
(s, 3H, CH3 Thym); 1.81 (s, 3H, CH3 Thym); 2.80–
3.41 (m, 13H, CH + CH2); 3.6 (s, 3H, OCH3); 3.75 (s,
4H, CH2 Thym); 4.2 (bs, 7H, CH, Fc); 4.31 (bs, 2H,
CH, Fc); 4.7 (s, 2H, CH2Fc); 5.0 (s, 2H PhCH2O); 6.1
(bs, 1H NHCbz); 6.6–6.7 (s, 2H, CH@); 6.8 ( d, 2H,
PhO); 7.0 ( d, 2H, PhO); 7.3–7.6 (m, 5H, Ph); 9.9 (s,
1H, NH Thym); 10.2 (s, 1H, NH Thym). 13C NMR
(CDCl3, d): 12.04, 12.26, 37.29, 39.27, 39.3, 47.99,
48.13, 48.6, 48.81, 52.64, 62; 66.56, 66.76, 68.75, 69.35,
82, 110.64, 110.21, 115.10, 128.01, 128.4, 130.14,
136.41, 141.31, 141.59, 151.18, 151.36, 157.8, 156.64,
164.70, 167.80, 168.59, 170.39, 171.15. HRMS Calc.
for C49H53FeN8O12: 1001.30, Found 1002.32.
At room temperature to a solution of monomer 1
(0.14 mmol, 1.0 eq) in CH2Cl2 (3 ml), a suspension of
Ba(OH)2 (0.21 mmol, 1.5 eq) in MeOH/H2O,1:1 (3 ml)
was added over 5 min. The reaction mixture was stirred
at room temperature for 4 h and 30 minutes then was
acidified to pH 1 with a solution of KHSO4 1 M. The
layers were separated, the aqueous phase extracted with
CH2Cl2(3 · 25 ml). The combined organic layers was
washed with water (20 ml) dried over Na2SO4 and evap-
orated. The crude product was purifie by a column chro-
matography on silica gel (EtOAc/MeOH,9:1, Rf = 0.1)
affording 12 in 90% yield.
5.13. N-Cbz cleavage on 15 to give 16
(
tane); (
D
,
L
)-12: Orange powder, m.p. 179–182 ꢁC (pen-
L
)-12: orange powder, m.p.: 162–164 ꢁC (pen-
At room temperature, HCOONH4 (1.47 mmol, 10
eq) and 10% Pd/C (60 mg) were added to a solution of
dimer 15 (0.147 mmol, 1.0 eq) dissolved in MeOH (3.5
ml). The reaction mixture was stirred for 25 h, then fil-
tered through a celite pad and evaporated under vac-
uum. The crude product was purified by a column
chromatography on silica gel (CH2Cl2/MeOH, 1:1;
Rf = 0.09) affording 16 as yellowish white powder in
20
D
tane), ½aꢁ ¼ ꢀ90:2, 88%; e.e. (HPLC, chiral OD
column, hexane/isopropanol: 50/50 + 0.1% TFA, 1.0
ml/min flow). 1H NMR (CDCl3, d): 1.87 (s, 3H,
CH3,Thym); 2.5–3.9 (m, 8H, CH + CH2); 4.19 (bs, 7H,
Fc); 4.32 (bs, 2H, Fc); 4.60 (bs, 1H, CH2CO); 4.78 (s,
2H, CH2Fc); 5.29 (s, 2H, PhCH2O); 5.58 (bs, 1H,
NHCbz); 6.89 (bd, 3H, PhO J = 8.4 Hz + CH@); 7.04
(d, 2H, PhO, J = 8.4 Hz); 7.29 (m, 5H, Ph); 10.50 (s,
1H, COOH). 13C NMR (CDCl3, d): 12.26, 33.00,
38.52, 48.86, 49.17, 63.67, 66.60, 66.84, 68.74, 69.29,
62% yield.
(
20
D
L
)-15: M.p: 212–215 ꢁC ½aꢁ ¼ ꢀ34:3ꢂ (c = 0.172 in
MeOH); 1H NMR (CDCl3, d): 1.83 (s, 3H, CH3 Thym);

C. Baldoli et al. / Journal of Organometallic Chemistry 689 (2004) 4791–4802
4801
1.88 (s, 3H, CH3 Thym); 2.5–3.4 (m, 13H, CH + CH2);
3.71 (s, 3H, OCH3); 3.76 (bs, 4H, CH2 Thym); 4.19
(bs, 7H, CH, Fc); 4.3 (bs, 2H, CH, Fc); 4.7 (s, 2H,
CH2Fc); 6.8 (bs, 3H, PhO + CH@); 7.1 ( d, 2H, PhO).
13C NMR (CDCl3, d): 11.6, 11.8, 37.22, 39.1, 39.29,
47.2, 47.8, 48.14, 48.4, 48.7, 52.63, 60.57, 66.56, 68.5,
69.0, 82.0, 110.5, 110.2, 115.0, 127.8, 130.3, 141.3,
141.6, 151.3, 151.5, 156.0, 164.80, 167.9, 168.3, 169.7,
170.72 MS, ESI, 869.3 (M+ + 2). IR (nujol, m, cmꢀ1):
1674 (CONH).
calomel electrode (SCE), inserted into a jacket contain-
ing 3 M aqueous KCl and ensuring contact with the
working solution via a glass joint, in order to avoid
precipitation of the tested substrates within the SCE
porous frit.
Acknowledgements
We thank MIUR, Milan University (National Project
‘‘Stereoselezione in Sintesi Organica. Metodologie e
Applicazioni’’), FIRB Project RBNE015J4Z and the
CNR for their financial support. We also thank Prof.
Rosangela Marchelli and her research group for their
helpful discussion.
5.14. N-Cbz cleavage in 1 to give 13
At room temperature Pd/C (10%) 15 mg and
HCO2NH4 (0.35 mmol, 2.6 eq) were added to a solu-
tion of 1 (0.14 mmol, 1 eq) in 12 ml of DMF/MeOH,
1:1. The reaction was stirred for 2 h, then filtered
over a pad of celite and the solvent evaporated.
The residue was dissolved in CH2Cl2 (15 ml) and
washed with brine (10 ml), the organic phase was
dried over Na2SO4 and evaporated. The crude prod-
uct was taken up with CH2Cl2 (3 ml) and the yellow
solid was filtered, washed with CH2Cl2 (3 · 2 ml) and
dried (80%).
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D
,
L
1
)-13: orange powder, m.p. 221–225 ꢁC (pen-
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