A new triferrocenyl-tris(hydroxymethyl)aminomethane derivative as a highly sensitive electrochemical marker of biomolecules: Application to the labelling of PNA monomers and their electrochemical characterization

Article abstract of DOI:10.1002/chem.200501466

We have designed and synthesised a new organometallic molecule containing three ferrocene groups for use as a highly sensitive electrochemical marker in biological assays. This trisferrocene derivative was conjugated to different PNA monomers, and the ele

Full text of DOI:10.1002/chem.200501466

FULL PAPER
DOI: 10.1002/chem.200501466
A New Triferrocenyl-tris(hydroxymethyl)aminomethane Derivative as a
Highly Sensitive Electrochemical Marker of Biomolecules: Application to the
Labelling of PNA Monomers andTheir Electrochemical Characterization
Clara Baldoli,[a] Clara Rigamonti,[b] Stefano Maiorana,*[b] Emanuela Licandro,[b]
[c]
Luigi Falciola,*[c] andPatrizia Romana Mussini
Abstract: We have designed and syn-
thesised a new organometallic mole-
cule containing three ferrocene groups
for use as a highly sensitive electro-
chemical marker in biological assays.
This trisferrocene derivative was conju-
gated to different PNA monomers, and
the electrochemical activities of the
conjugates were extensively investigat-
ed in organic solvents, in view of their
potential diagnostic applications. The
results showed that the introduction of
a trisferrocene unit on the PNA mono-
mer triples the current signal in com-
parison with the monoferrocene-label-
led one. Despite their greater molecu-
lar complexity, trisferrocene-conjugated
PNA monomers are even more electro-
chemically active than the reference
ferrocene. By using differential pulse
voltammetry (DPV), the detection
limit can reach 10À8 m in acetonitrile
solution. These results are
a good
Keywords: bioorganometallic
premise for the use of the trisferrocene
unit as an effective electrochemical
probe for biomolecules.
chemistry
ferrocene
·
electrochemistry
metallocenes
·
·
·
peptide nucleic acids
Introduction
acid mimics introduced by Nielsen in 1991,[1] in which the
sugar-phosphate backbone is replaced by a neutral achiral
pseudopeptide chain of N-(2-aminoethyl)glycine units, with
the nucleobases linked to the glycine nitrogen through car-
bomethylene bridges. PNA oligomers can be prepared in
relatively large quantities by using a peptide synthesizer,
and are highly stable in biological environments.[2] The nu-
cleobases form highly specific duplex hybrids with comple-
mentary DNA sequences based on the Watson–Crick base-
pairing rules; these products are much more thermally
stable than the analogous DNA–DNA or DNA–RNA du-
plexes because the PNA chain is neutral and therefore does
not give rise to any repulsive electrostatic interstrand inter-
action with the negatively charged DNA sequence.[3] The
stability of a PNA–DNA duplex, measured on the basis of
melting temperature (Tm),[4,5] is related to the ability of
PNA oligomers (15–20 units) to discriminate even a single
mismatched base in a given DNA sequence,[6–8] an ability
that is further increased in PNAs containing the so-called
“chiral box”.[9,10] These characteristics give PNAs great po-
tential as antigene or antisense drugs,[11,12] and as biomolecu-
lar probes in biotechnologies,[2,13] thus making it worth
trying to equip them with sensitive markers capable of de-
tecting the hybridisation event and converting it into an
easily measurable analytical signal.
There is a growing demand for high affinity and specific bio-
molecular probes capable of detecting selected base sequen-
ces in human, viral, bacterial and vegetal nucleic acids.
For human DNA sequences, the objective is to detect the
disease-related punctiform mutations responsible for heredi-
tary and other disorders, and the different individual re-
sponses to drugs. One promising method for detecting DNA
involves the use of peptide nucleic acids (PNAs), nucleic
[a] Dr. C. Baldoli
CNR-Institute of Molecular Science and Technologies
Via C. Golgi 19, 20133 Milano (Italy)
[b] Dr. C. Rigamonti, Prof. S. Maiorana, Prof. E. Licandro
Department of Organic and Industrial Chemistry and
Centre of Excellence CISI, University of Milano
Via Venezian 21, 20133 Milano (Italy)
Fax : (+39)02-5031-4139
E-mail: stefano.maiorana@unimi.it
[c] Dr. L. Falciola, Prof. P. R. Mussini
Department of Physical Chemistry and Electrochemistry
University of Milano, Via Golgi 19
20133 Milano (Italy)
Fax : (+39)02-5031-4300
E-mail: luigi.falciola@unimi.it
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As a part of our research project concerning bioorgano-
metallic chemistry,[14] we have recently been interested in
PNA properties and we have synthesised some metal-conju-
gated PNA monomers[15] with the aim of evaluating their an-
alytical characteristics as a preliminary step to the synthesis
of new metal-conjugated PNA oligomers to be used as
DNA probes. In the search for new DNA detection meth-
ods, the use of an electro-active DNA as analytical probe
has been widely investigated,[16] because electrochemistry
seems to be a very promising alternative to fluorescence in
terms of sensitivity, response times and costs. However, elec-
trochemistry has been much less used for PNA probes.[17] Of
the various organometallic complexes, useful electrochemi-
cally detectable fragments, ferrocene is known to be one of
the most convenient, because it is very stable and undergoes
reversible electron oxidation.[18] Both ourselves[15] and other
authors[19] have synthesised and electrochemically studied a
number of ferrocene-labelled PNA monomers and oligom-
ers; in particular the electrochemical behaviour and efficien-
cy of a monoferrocene end-labelled PNA 10-mer, in a sur-
face immobilized duplex, was recently investigated by Metz-
ler-Nolte.[20]
tron oxidation wave; this means proportionality between
current density and the number of ferrocene groups.
We selected the structure tris(ferrocenemethylenoxymeth-
yl)aminomethane (2, Tris-Fc),[21b] because it has a versatile
and reactive functional amino group, and
the ferrocene units are electronically iso-
lated in the sense that they have no evi-
dent interactions other than unpredicta-
ble “through space” interactions. These
characteristics should produce the elec-
trochemical equivalence of the ferrocene
groups. In this paper we report, the syn-
thesis of compound 2, its use for labelling PNA monomers
and the electrochemical characterisation of the conjugates
obtained.
Results andDiscussion
In a previous work we have synthesised the tyrosine-de-
rived ferrocene-labelled monomer 1a, which is chiral and
has amino and carboxylic functions available for the oligo-
merization; this could allow the in-
Synthesis: Tris-Fc 2 was synthesised following the sequence
shown in Scheme 1. The amino group of tris(hydroxymethyl)-
aminomethane was Cbz-protected to give 4, a compound
that had been previously reported (30% yield),[22] but we
improved the yield to 88% by changing the organic solvent
from CH2Cl2 to AcOEt. Compound 4 was then reacted with
an excess of ferrocene methanol 5[15] by heating to 608C in
toluene in the presence of a catalytic amount of trifluoro-
acetic acid. After chromatographic purification, the N-Cbz
protected Tris-Fc 6 was isolated in 73% yield as a yellow
solid, together with a small amount (about 10% each) of
the di- and monosubstituted derivatives 7 and 8. The cleav-
age of the Cbz group in 6 by ammonium formate and Pd/C,
in MeOH at room temperature, afforded 2 as a dark yellow
solid in 73% yield.
sertion of more than one ferrocene
unit into a PNA chain and, in prin-
ciple, the enhancement of the elec-
trochemical response. Cyclic vol-
tammetry (CV) studies of 1a led to
good results, producing a reversible
CV curve, and the product was de-
tected in concentrations as low as
10À7 m by using differential pulse voltammetry (DPV).[15,21a]
As improving sensitivity is valuable for analytical purpos-
es, the above results convinced us to seek more efficient sys-
tems, and thus to design and
synthesise a molecule contain-
ing multiple ferrocene groups
that could act as a flexible,
general marker and would
have improved electrochemical
properties and sensitivity.
In principle, any such mole-
cule should:
1) Be easy and inexpensive to
make.
2) Bear a versatile and reac-
tive functional group for
linking the marker to the
substrate.
3) Have
electrochemically
equivalent ferrocene units
in order to obtain a single,
multiple-independent-elec-
Scheme 1. Synthesis of Tris-Fc 2.
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The next step was the study of a suitable protocol to link
the new marker 2 to the tyrosine PNA monomer 9,[15] to do
this the hydroxyl group of 9 was activated through the for-
mation of the bis-pentafluorophenyl carbonate 10, which
easily reacted with Tris-Fc 2 in CH2Cl2 at room temperature,
affording triferrocenyl monomer 11 in 80% yield
(Scheme 2).
The synthesis of monomer 11 allowed us to quickly reach
two objectives, which were firstly, the verification of the
good reactivity of the amino group in Tris-Fc 2 and secondly,
to check the amplification of the electrochemical signal in
monomer 11 (see discussion below) with respect to the mon-
oferrocene-labelled monomer 1a. To be able to construct
PNA oligomers, it was necessary to check the stability of the
carbamate group in 11 under the alkaline hydrolysis condi-
tions of the methyl ester[23] but, unfortunately, even the mild
treatment of monomer 11 with two equivalents of LiOH at
room temperature caused the cleavage of the carbamate
group.
Thus, it was necessary to select a linker group stable in
basic conditions and we focused on the ureido function.
Therefore, we synthesised the monomer 18 bearing an ami-
noethyl linker at the tyrosine phenol group, to which 2 was
connected to give the new trisferrocene-labelled monomers
20 and 21 (Scheme 3).
The synthesis of the tyrosine PNA monomer 18 is depict-
ed in Scheme 3. l-N-Cbz tyrosine methyl ester 13 reacted
with N-Boc-2-amino ethanol 12 in THF, in the presence of
Scheme 2. Synthesis of Tris-Fc-labelled monomer 11.
Scheme 3. Synthesis of Tris-Fc-labelled tyrosine PNA monomer 21.
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S. Maiorana, L. Falciola et al.
PPh3 and DEAD, under Mitsunobu conditions at room tem-
perature to give the tyrosine derivative 14 in 73% yield.
The cleavage of the Cbz group with ammonium formate
and Pd/C in MeOH at room temperature gave 15, which
was then reacted with N-Cbz aminoacetaldehyde under re-
ductive amination conditions (NaCNBH3/ZnCl2) in metha-
nol at room temperature to afford the corresponding N-ami-
noethyl derivative 16 in very good yield (93%). The subse-
quent condensation of 16 with 1-carboxymethylthymine was
conducted in the presence of 3,4-dihydro-3-hydroxy-4-oxo-
1,2,3-benzotriazine (DhBtOH) and diisopropylcarbodiimide
(DIC) to give compound 17 in 70% yield. After cleavage of
the Boc-protecting group with TFA, the new monomer 18
was isolated as the trifluoroacetate salt in 97% yield.
Compound 18 was linked to the Tris-Fc isocyanate deriva-
tive 19, which was obtained by treating 2 with Boc2Oin the
presence of dimethylamino pyridine,[24] and afforded trisfer-
rocene-labelled monomer 20 in good yield (80%). The sub-
sequent methyl ester hydrolysis was successfully conducted
by using Ba(OH)2 and gave acid 21 in 75% yield.
In addition, as a further possibility for labelling a PNA
strand, we investigated the reaction of Tris-Fc 2 with the ter-
minal amino group of an aminoethylglycine (aeg) PNA
monomer 22. Tris-Fc 2 was transformed into the intermedi-
ate pentafluorophenyl carbamate, which was directly reacted
with monomer 22 in the presence of diisopropylamine and
gave 23 in 75% yield (Scheme 4). Monomer 23 was also ob-
tained from the reaction of Tris-Fc isocianate 19 and 22 in
DMF at room temperature, but in lower yield (58%).
At this stage of the research, after the check of the syn-
thetic efficiency of 2,[25] it was important to study the elec-
trochemical behaviour of the monomers labelled with Tris-
Fc, 11, 20, 21 and 23, in comparison with that of mono-Fc
monomers 1a,b.[15] Thus, all compounds were investigated
by cyclic voltammetry (CV) and differential pulse voltam-
metry (DPV), the results of which are discussed below.
Figure 1. A synopsis of the CV patterns obtained at v=0.2 VsÀ1 for the
four ester substrates 1a, 11, 20 and 23 in the three organic solvents
CH2Cl2, MeCN and DMF, with ferrocene as a reference.
cene derivatives 11, 20 and 23) is shown in Figure 1, which
contrasts the voltammetric be-
haviours of the four com-
pounds at v=0.2 VsÀ1.
The effect of a free carbox-
ylic function is shown in Fig-
ures 2 and 5; these Figures
compare the CV features of
the two PNA acid monomers
1b and 21 with their parent
Scheme 4. Synthesis of Tris-Fc-labelled aeg-monomer 23: a) (C6F5CO)2O, DIPEA, DMF; b) 22, DMF, DIPEA.
esters 1a and 20, respectively.
The effects of the solvent and
molecular structure on peak
currents can be seen in
Electrochemical characterization: The voltammetric features
of ferrocene derivatives 2, 6, 7 and 8 have already been re-
ported.[21b] Figures 1, 2, 5 and Table 1 summarise the most
significant CV features of our six ferrocene-labelled PNA
monomers (1a, 1b, 11, 20, 21 and 23) and the reference
compound ferrocene (Fc) obtained at potential scan rate v=
0.2 VsÀ1 on GC at 298 K. The effect of the molecular struc-
ture of the ester monomers (monoferrocene 1a and triferro-
Figure 4 in which the squared slopes of the linear character-
istics (normalised current densities versus v0.5) are plotted as
a function of the reciprocal of solvent viscosity.
In the case of a diffusive electrochemically reversible
peak and a given number of transferred electrons, such
squared slopes are proportional to the substrate diffusion
coefficients,[26] and should verify Stokesꢁ law (i.e. the diffu-
sion coefficients should be inversely proportional to solvent
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Electrochemistry
FULL PAPER
cene because of the bulkier
structure implying lower diffu-
sion coefficients and, as in the
case of ferrocene, their modu-
lation by the solvent is consis-
tent
with
Stokesꢁ
law
(Figure 3).
The corresponding monofer-
rocene-labelled acid 1b (the
comparison of CV patterns of
1a and 1b is reported in
Figure 2) has a slightly wider
spread of peak potentials, with
a significant anticipation in the
case of DMF. Remarkably, in
the case of CH2Cl2, sharp peak
shapes were observed, espe-
cially at low scan rates and in
relation to the return peak.
Figure 2. Comparison of the CV patterns obtained at v=0.2 VsÀ1 in the three organic solvents CH2Cl2, MeCN
and DMF, for the monoferrocene-labelled ester 1a and acid 1b.
Moreover, the return peak
had a much larger area (i.e., a
larger quantity of charge trans-
ferred in the process) than the forward peak, and this asym-
metry increased as the potential scan rate decreased, that is,
with increasing time between the anode and cathode peak.
Furthermore, the return peak markedly increased when the
potential scan was paused for different times immediately
after the oxidation peak, and when the solution was increas-
ingly stirred during the pause. This phenomenon was studied
in detail for the trisferrocene-labelled acid 21 (see Figure 6),
which showed the same behaviour as the monoferrocene-la-
belled acid 1b. Actually, the CV patterns of both 21 and 1b
in CH2Cl2 closely resemble that of stripping voltammetry,[27]
the most sensitive voltammetric technique, in which, be-
cause its concentration is below the detection limits of CV
and DPV (typically, at ppb level), the analyte is preconcen-
trated on the working electrode. In our case, we could
assume that given the presence of the carboxylic group (as
the same does not occur with the parent ester 1a, Figure 2),
the oxidation product is so poorly soluble in the hydropho-
bic CH2Cl2 solvent that it films the glassy carbon electrode
surface despite its being nearly inert to specific adsorption.
This film results in a large-scale increase in the local concen-
tration of the substrate available for the backward reaction,
and appears to be conductive (which is consistent with the
high density of the ferrocene groups). Accordingly, the peak
associated with the stripping-like process is highly intense
and very reproducible.
Figure 3. Squared slopes of the normalised current versus v0.5 linear char-
acteristics, plotted against the reciprocal of solvent viscosity h (verifica-
~
tion of Stokesꢁ law): the reference ferrocene compound ( ); the monofer-
*
*
rocene-labelled ester and acid, 1a ( ) and 1b ( ); and the trisferrocene-
&
^
labelled esters 11 ( ) and 20 ( ).
viscosity). The electrochemical behaviour of the mono- and
trisferrocene-labelled series are analysed in detail below.
Monoferrocene-labelled derivatives 1a and 1b: Figure 1 and
Table 1 show that, in all solvents, the monoferrocene-label-
led ester 1a has an oxidation peak that is chemically and
electrochemically reversible according to all of the criteria
(see Experimental Section) and, in the case of acetonitrile
(MeCN) and CH2Cl2, is located at a slightly more positive
potential than the reference ferrocene because of the ferro-
cene derivatisation with the aryl ether bridge in the b-posi-
tion. On the whole, both the shape and potential of the
peak seem to be almost unaffected by the nature of the
working solvent. The currents are lower than those of ferro-
Trisferrocene-labelled monomers: The CV characteristics of
the trisferrocene-labelled monomers 11, 20, 21 and 23 are
shown in Figures 1 and 5, together with those of the parent
monoferrocene-labelled monomers. On the whole, the triple
functionalisation only slightly affects reduction peak poten-
tials and shapes.
One relevant feature is that the three ferrocene groups do
not seem to be entirely equivalent in the polar MeCN and
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S. Maiorana, L. Falciola et al.
Table 1. Selected cyclovoltammetric features of the investigated PNA monomers and the reference ferrocene compound on a GC electrode at 298 K in
1
1
2
different organic media: the anodic peak potentials Ep,a, anodic half-peak widths, (EpÀEp/2), half-wave potentials E = =(Ep,a +Ep,c)/2, E’ = and aapp parame-
2
ters from the convolutive analysis (all referring to a scan rate v = 0.2 VsÀ1) and the slopes of the normalised current densities versus v0.5 linear character-
istics.
c [moldmÀ3
]
Ep,a
(EpÀEp/2) [V] E =
E’ = [V] (Fc+/Fc) aapp
d
(ip,a/c)/dv0.5
N
1
1
Substrate Solvent Supporting
electrolyte (0.1m)
2
2
[V] (Fc+/Fc)
[V] (Fc+/Fc)
Fc
1a
1b
11
20
MeCN TBAP
DMF TBAP
CH2Cl2 TBAP
MeCN TBAP
0.000770
0.000753
0.000788
0.000730
0.000761
0.000752
0.000738
0.000806
0.000718
0.000748
0.000758
0.000758
0.000087
0.0000927
0.0000851
0.000405
0.000387
0.030
0.031
0.032
0.072
0.035
0.063
0.069
À0.015
0.028
0.056
0.057
0.026
0.050
À0.042
0.027
À0.026
0.008
0.055
0.057
0.058
0.057
0.059
0.062
0.056
0.059
0.059
0.065
0.069
0.053
0.088
0.079
0.066
0.056
0.055
0.001
0.003
À0.001
À0.003
0.001
1.04
1.03
0.97
0.91
1.03
0.91
1.14
1.08
0.98
0.93
0.86
1.09
0.68
0.76
1.01
1.08
0.96
1.32
0.76
1.25
0.77
0.46
0.64
0.83
0.22
0.76
2.10
1.15
1.94
2.04
0.98
1.49
6.39
2.34
À0.001
0.043
0.039
DMF
TBAP
0.005
0.018
0.040
À0.044
À0.025
0.024
À0.005
CH2Cl2 TBAP
MeCN TBAP
0.015
0.034
DMF
TBAP
À0.050
À0.005
0.022
CH2Cl2 TBAP
MeCN TBAP
DMF
TBAP
0.020
0.015
CH2Cl2 TBAP
MeCN TEATFB
À0.002
À0.001
0.011
0.028
DMF
TEATFB
À0.081
À0.012
À0.026
À0.024
À0.089
À0.004
À0.030
À0.021
CH2Cl2 TEATFB
CH2Cl2 TBAP
CH2Cl2 TBAP
21
23
11 than for the monoferro-
cene-labelled ester 1a (a fun-
damental characteristic in view
of our applicative aim), and
consistent with Stokesꢁ law
(Figure 3).
As a result, despite its bulki-
ness, 11 has significantly more
electrochemical activity than
the reference ferrocene com-
pound (Figure 1), affording
DPV detection limits in the
order of 10À8/10À6 mol dm3, de-
pending on the working sol-
vent (Figure 4), even in the ab-
sence of amplification steps.
The very similar trisferro-
cene-labelled ester 20, which
includes a urea-based linker,
has oxidation peak potentials
that are slightly less positive
and have a wider spread than
those of 11 (Table 1 and
Figure 4. DPV patterns obtained by working with very low concentrations (c) of the Tris-ferrocene-labelled
esters 11 in the three organic solvents CH2Cl2, MeCN, DMF and 23 in CH2Cl2 (dotted line) for the evaluation
of the relevant detection limits.
DMF solvents as the oxidation peak is significantly larger
than in the case of the monoferrocene-labelled ester 1a
(Figure 1); in other words, although they are not conjugated,
the three ferrocene groups apparently maintain some inter-
communication in the polar solvents, probably as a result of
conformational effects. The same does not apply for CH2Cl2,
in which the three ferrocene groups seem to be independ-
ent, possibly because of strong solvation or the fact that the
oxidation product forms a tightly bound ionic couple with
the supporting electrolyte anion.[28] However, the effect is
slight and, in all three solvents, we obtained currents that
were three times higher for the trisferrocene-labelled ester
Figure 1). As for the trisferrocene-labelled ester 11, only in
the case of MeCN and DMF (but not CH2Cl2), the CV
peaks somewhat broaden and reveal some communication
among the ferrocene groups. The currents are still very high,
and only slightly lower than in the case of 11, which is con-
sistent with the slightly bulkier structure of 20.
The NH2-Tris-Fc labelled ester 23, (dotted line in
Figure 1) produces slightly higher peak currents, with re-
spect to compound 20 (consistently with the lower molecular
weight), confirming that triple functionalisation results in a
triplication of the electrochemical signal, despite the bulkier
structures of the compounds. Accordingly, compound 23 af-
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Electrochemistry
FULL PAPER
of the oxidation product on the electrode surface. This, to-
gether with the additivity of the three ferrocene groups in
terms of current, leads to an exceptionally high peak current
for the return peak.
Conclusions
We have designed and synthesised a new organometallic
molecule containing three ferrocene groups for use as a
highly sensitive electrochemical marker in biological assays.
This trisferrocene-derivative was conjugated to different
PNA monomers, and the electrochemical activities of the
conjugates were extensively investigated in view of their po-
tential diagnostic applications. All of the synthesised mono-
and trisferrocene-labelled PNA monomers have clear elec-
trochemical features and, even in the case of the trisferro-
cene conjugates, a single, multiple-independent-electron oxi-
dation wave. The increased molecular complexity only im-
plies a limited reduction in current signals in comparison
with the reference ferrocene molecule. However, the intro-
duction of three ferrocene units triples the current signal,
thus giving the conjugated PNA monomer more electro-
chemical activity than the reference ferrocene. The detec-
tion limits for compound 11 and 23 are therefore 10À6–
10À8 m, depending on the nature of the solvent, with the best
results being observed in acetonitrile (10À8 m). The labelling
of PNA oligomers with Tris-Fc units is currently in progress
in our laboratories with the aim of improving sensitivity in
ferrocene-based electrochemical biosensors.
Figure 5. Comparison of the CV patterns obtained at v=0.2 VsÀ1 in
CH2Cl2 for the Tris-ferrocene-labelled ester 20 and acid 21 derivatives.
Experimental Section
Materials andmethods : All chemicals were purchased from Sigma-Al-
drich or Fluka unless otherwise indicated. All reactions were performed
under a nitrogen atmosphere by using a standard vacuum line. THF was
distilled over sodium benzophenone ketyl, other reagent grade solvents
were dried by standard procedures. Column chromatography was per-
formed by using Merck silica gel 60 (70–230 mesh). 1H and 13C NMR
spectra were recorded at 258C on a Bruker AC 300 and AMX at
300 MHz. IR spectra were recorded on a Perkin–Elmer 1725X FT–IR.
MS data were obtained by using a VG 70 EQ Micromass or a Thermo-
Finningam mass spectrometer. Melting points were measured with a
Büchi B-540 apparatus and are uncorrected. Optical rotations were meas-
ured by using a Perkin–Elmer 243 polarimeter.
Figure 6. Stripping voltammetry characteristics obtained at v=0.2 VsÀ1 in
CH2Cl2 with TBAP (0.1m) for the Tris-Fc 21 (0.0008m): A) Time of depo-
sition at 0.8 V (SCE) ranging from 0 to 30 s, without stirring, B) 30 s dep-
osition time at 0.8 V (SCE), stirring at 500 rpm, C) 30 s deposition time
at 0.8 V (SCE), stirring at 750 rpm and D) 30 s deposition time at 0.8 V
(SCE), stirring at 1000 rpm.
Electrochemical experiments: The CV investigations were carried out in
a cell thermostated at 298 K at scan rates ranging from 0.02–20 VsÀ1 in
the following: 1) MeCN, DMF and CH2Cl2 (Merck, HPLC grade), with
tetraethylammonium tetrafluoborate (TEATFB, 0.1m) or tetrabutylam-
monium perchlorate (TBAP, Fluka, electrochemical grade) as supporting
electrolytes, 2) ultrapure water (Millipore Milli Q system) with NaClO4
(0.1m) as the supporting electrolyte. The solutions were carefully deaer-
ated by nitrogen bubbling and analyzed using an Autolab PGSTAT 12 or
Autolab PGSTAT 30 potentiostat/galvanostat (EcoChemie, The Nether-
lands) run by a PC with GPES software; the ohmic drop was corrected
by means of the positive feedback technique[29] with a glassy carbon GC
(Amel, surface 0.071 cm2) as the working electrode, a platinum counter
electrode and an aqueous saturated calomel electrode (SCE) as the oper-
ating reference electrode. The formal redox potentials of the ferricinium/
ferrocene redox couple recommended by IUPAC for the intersolvental
fords DPV detection limits in the order of 10À7 m in CH2Cl2
(Figure 4).
Finally, in comparison with parent ester 20, the trisferro-
cene-labelled acid 21 in CH2Cl2 (Figures 5 and 6) shows the
same slight but significant peak potential anticipation and
the same peculiar shape, which indicates the accumulation
Chem. Eur. J. 2006, 12, 4091 – 4100
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
4097

S. Maiorana, L. Falciola et al.
comparison of potential scales[30] were 0.390, 0.476 and 0.488 V, respec-
tively, when measured against our aqueous SCE in MeCN, DMF and
CH2Cl2. The optimized polishing procedure for the working GC electrode
consisted of surface treatment with diamond powder (Aldrich, diameter
1 mm) on a wet cloth (DP-Nap, Struers).
Tris(ferrocenemethylenoxymethyl)aminomethane (2): Pd/C (10%, 0.3 g)
and HCO2NH4 (0.3 g, 4.7 mmol) were added, at room temperature under
nitrogen, to
a stirred solution of N-Cbz Tris-ferrocene 4 (0.85 g,
1.0 mmol) in MeOH (10 mL) and CH2Cl2 (18 mL). After 90 min, the mix-
ture was filtered through a pad of celite and the solvent evaporated, the
residue was then dissolved in CH2Cl2 (40 mL), washed with brine (2”
40 mL) and the aqueous phase was extracted with CH2Cl2 (3”40 mL).
The combined organic phases were dried over Na2SO4 and evaporated.
The crude product was purified by column chromatography on silica gel
(Rf =0.25, AcOEt) affording 2 as an orange powder (0.650 g, 90%). M.p.
105–1078C (pentane); 1H NMR (300 MHz, CDCl3): d=4.24 (s, 6H;
OCH2Fc), 4.18 (t, Jortho =1.8 Hz, 6H; Fc), 4.13 (t, Jmeta =1.8 Hz, 6H; Fc),
4.10 (m, 15H; Fc), 3.45 (s, 6H; CCH2O), 2.28 ppm (brs, 2H; NH2);
13C NMR (300 MHz, CDCl3): d=83.84, 71.48, 69.45, 69.08, 68.4, 68.2,
56.51 ppm; IR (Nujol): n˜ =3093 cmÀ1 (NH2); MS (EI): m/z: 715 [M]+,
199 (CH2Fc), 121; elemental analysis calcd (%) for C37H41Fe3NO3: C
62.13, H 5.78, N 1.96; found: C 62.76, H 5.75, N 1.95.
Tris-Fc monomer 11: d,l-Tyrosine monomer 9[15b] (0.5 g, 0.93 mmol) was
added at 08C to a solution of bis(pentafluorophenyl)carbonate (0.38 g,
0.94 mmol) in CH2Cl2 (5 mL). Diisopropylamine (DIPEA, 0.16 mL,
0.93 mmol) was then added to the resulting suspension, and the clear sol-
ution was stirred at 08C for 2 h. After this time, the solution was diluted
with CH2Cl2 (15 mL), washed with NaHCO3 (5%, 2”10 mL) and the
aqueous phase was extracted with CH2Cl2 (2”15 mL). The organic
phase, after washing with 20 mL of H2O, was dried over Na2SO4, concen-
trated to 2 mL and added to a solution of 2 (0.5 g, 0.75 mmol) and
DIPEA (0.16 mL, 0.92 mmol) in CH2Cl2 (5 mL) cooled at 08C. The re-
sulting orange solution was stirred at room temperature for 5 h. After
this time, the mixture was washed with NaHCO3 (10%, 2”10 mL) and
the aqueous phase was extracted with CH2Cl2 (3”10 mL). The combined
organic layers were then washed with H2O(15 mL), dried over Na 2SO4
and evaporated. The crude product was purified by column chromatogra-
phy (AcOEt) to give 11 (0.4 g, 80%) as an orange solid. M.p. 98–1038C
(pentane); 1H NMR (300 MHz, CHCl3): d=8.01 (s, 1H; NH T), 7.25–
7.35 (m, 5H; Ph), 7.10 (d, J=8.47 Hz, 2H; PhO), 7.02 (d, J=8.47 Hz,
2H; PhO), 6.58 (s, 1H, CH=), 5.61 (brs, 1H; NH Cbz), 5.46 (brs, 1H;
NH), 5.19 (d, J=12 Hz, 1H; PhCH2O), 4.99 (d, J=12 Hz, 1H; PhCH2O),
4.52 (d, J=16.2 Hz, 1H; CH2CO), 4.24 (s, 6H; CH2Fc), 4.19 (s, 6H;
CHortho Fc), 4.14 (s, 6H; CHmeta Fc), 4.11 (s, 15H; Fc), 3.82–3.86 (m, 2H;
CH+CH2CO), 3.77 (s, 3H; CO2CH3), 3.67 (brs, 6H; CH2OCH2Fc),
3.00–3.10 (m, 4H; CH2), 2.60 (d, 1H, J=10.5 Hz; CH2NCH), 1.84 ppm (s,
3H; CH3); 13C NMR (300 MHz, CHCl3): d=172, 166.95, 164.1, 156, 155,
151.4, 141.0, 136, 129.9, 128.4, 127, 122.14, 110.8, 83, 69.6, 69.2, 68.6, 68.5,
66.9, 63.6, 60, 50.34, 49.5, 48.7, 39.03, 33.50, 12.3 ppm; IR (Nujol): n˜ =
1670 cmÀ1 (CO); UV (CH2Cl2): lmax (e)=261 (20690 molÀ1 dm3 cmÀ1);
HRMS (ESI): m/z: 1279.30186 [M]+.
The chemical and electrochemical reversibility of each well-defined CV
peak were checked by means of classical tests,[26] including analyses of
1) half-peak width (EpÀEp/2), 2) Ep versus logv characteristics, 3) the dis-
tance between the anode and cathode peak potential (Ep,aÀEp,c), 4) Ip
versus v0.5 characteristics and 5) the “stationary” step-like waves obtained
by means of convolutive analysis of the original CV characteristics.[31]
N-Cbz-Tris(hydroxymethyl)aminomethane (4): At room temperature
benzylchloroformate (6.0 g, 35 mmol, 5.2 mL) was slowly dropped into a
stirred suspension of NaHCO3 (53 mmol, 4.4 g) and tris(hydroxymethy-
l)aminomethane (5.0 g, 42 mmol) in H2O(20 mL) and AcOEt (40 mL),
keeping the temperature around 208C. After stirring for 5 h at room tem-
perature, the solid was filtered off and the layers separated. The aqueous
phase was extracted with AcOEt (3”40 mL) and the combined organic
layers were washed with H2O(40 mL). After evaporation of the solvent,
the residue was dissolved in diisopropyl ether (20 mL) and cooled in an
ice-bath. The white solid was filtered to give 4 (9.5 g, 88%). M.p. 101–
1038C (lit.[22] 28%, m.p. 102–1048C).
N-Cbz-Tris(ferrocenylmethylenoxymethyl)aminomethane (6): A solution
of ferrocene methanol 5[15] (2.0 g, 9.26 mmol) in toluene (10 mL) and
CH2Cl2 (3 mL) was heated at 608C under N2. At this temperature a por-
tion of 4 (0.12 g, 0.47 mmol) and CF3CO2H (0.2 mL of a 0.65m toluene
solution) was added. The same amount of the two reagents was added
every 30 min until a further four additions had been carried out. After
two hours, four additions of ferrocenemethanol (0.32 g, 3.8 mmol), each
every 4 h, were made, and the mixture was then heated at the same tem-
perature for 8 h. After evaporation of the solvent, the residue was dis-
solved in CH2Cl2 (20 mL) and washed with saturated NaHCO3 solution
(20 mL). The aqueous phase was extracted with CH2Cl2 (2”20 mL) and
the resulting combined organic phases were washed with water (40 mL),
dried over Na2SO4 and evaporated. The crude product was purified by
column chromatography on silica gel (tert-butyl methyl ether/petroleum
ether 2:8) to afford 6 (1.45 g, 73%) as an orange powder. Compounds 7
(0.1 g, 10%), 8 (0.13 g, 13%) and diferrocenylmethylether (1.5 g) were
also recovered.
Compound6 : M.p. 115–1178C (pentane); 1H NMR (300 MHz, CDCl3):
d=7.32 (m, 5H; Ph), 5.2 (brs; NH), 5.02 (s, 2H; CH2Ph), 4.20 (s, 6H;
CH2Fc), 4.16 (t, J=1.7 Hz, 6H; CH Fc), 4.11 (t, J=1.7 Hz, 6H; CH Fc),
4.09 (s, 15H; CH Fc), 3.67 ppm (s, 6H; CH2O); 13C NMR (300 MHz,
CDCl3): d=156, 137, 128.4, 128.0, 127.9, 83.8, 69.5, 69.0, 68.8, 68.4, 68.2,
66.1, 58.8 ppm; IR (Nujol): n˜ =1723 (NHCO), 1055, 1098 cmÀ1 (Fc); MS
(FAB+): m/z: 850 [M]+; elemental analysis calcd (%) for C45H47Fe3NO5:
C 63.63, H 5.58, N 1.65; found: C 63.44, H 5.59, N 1.65.
Tyrosine derivative 14: N-Boc ethanolamine 12 (0.78 mL, 5.0 mmol), fol-
lowed by PPh3 (1.85 g, 7.0 mmol) was added to a solution of l-N-Cbz ty-
rosine methylester 13 (1.98 g, 6 mmol) in anhydrous THF (50 mL). The
mixture was cooled to À308C and DEAD (3.2 mL of 20% toluene solu-
tion) was added. The mixture was allowed to warm at room temperature
and was then stirred for 48 h. After this time, the solvent was evaporated
and the residue dissolved in AcOEt (100 mL) was washed with NaOH
(0.1m, 2”50 mL), followed by brine (2”50 mL). The organic phase was
dried over Na2SO4 and evaporated. The crude product was purified by
column chromatography (Rf =0.47, AcOEt/petroleum ether 1:1) to give
14 (73%) as a white solid. M.p. 79–818C (pentane); [a]2D0 =+38.8 (c=
Compound7 : Orange oil; 1H NMR (300 MHz, CDCl3): d=7.35–7.38 (m,
5H; Ph), 5.4 (brs; NH), 5.04 (s, 2H; CH2Ph), 4.27 (d, J=11.4 Hz, 2H;
OCH2Fc), 4.21 (d, J=11.4 Hz, 2H; OCH2Fc), 4.17 (t, J=1.76 Hz, 4H;
CH Fc), 4.12 (t, J=1.76 Hz, 4H; CH Fc), 4.10 (s, 10H; CH Fc), 3.77 (d,
J=6.6 Hz, 2H; CH2OH), 3.69 (d, J=9.16 Hz, 2H; CH2OCH2Fc), 3.63
(brs; OH), 3.48 ppm (d, J=9.16 Hz, 2H; CH2OCH2Fc); 13C NMR
(300 MHz, CDCl3): d=156, 136, 128.5, 128.07, 128.0, 83.3, 69.71, 69.56,
69.01–69.08, 69.49, 68.38, 66.6, 65.1, 59.1 ppm; IR (neat): n˜ =3414 (OH),
1714 cmÀ1 (CONH); MS (ESI): m/z: 651.3 [M]+, 674.3 [M+Na]+; ele-
mental analysis calcd (%) for C34H37Fe2NO5: C 60.94, H 6.00, N 3.09;
found: C 60.54; H 5.60, N 3.06.
1
0.23 in CHCl3); H NMR (300 MHz, CDCl3): d=7.33 (brs, 5H; Ph Cbz),
Compound8
: Orange solid; m.p. 113–1158C (decomp) (pentane);
1H NMR (300 MHz, CDCl3): d=7.35 (m, 5H; Ph), 5.74 (s; NH), 5.08 (s,
2H; CH2Ph), 4.3 (s, 2H; OCH2Fc), 4.18 (t, J=1.8 Hz, 2H; CH Fc), 4.16
(t, J=1.8 Hz, 2H; CH Fc), 4.09 (s, 5H; CH Fc), 3.72 (d, J=11.6 Hz, 2H;
CH2OH), 3.58 (s, 2H; CH2OCH2Fc), 3.53 ppm (d, J=11.6 Hz, 2H;
CH2OH); 13C NMR (300 MHz, CDCl3): d=156.5, 136.0, 128.5, 128.2,
128.0, 83.0, 70.8, 69.9, 68.7, 67, 64.5, 59.1 ppm; IR (Nujol): n˜ =3300 (OH),
1685 cmÀ1 (CONH); MS (ESI): m/z: 453.5 [M]+, 476.4 ppm [M+Na]+; el-
emental analysis calcd (%) for C23H27FeNO5: C 60.94, H 6.00, N 3.09;
found: C 60.90, H 5.90, N 3.06.
6.98 (d, J=8.5 Hz, 2H; PhO), 6.78 (d, J=8.5 Hz, 2H; PhO), 5.29–4.98
(m, 4H; CH2-Cbz + NH-Cbz + NH-Boc), 4.62–4.59 (m, 1H; CH), 3.96
(t, J=5.1 Hz, 2H; CH2O), 3.71 (s, 3H; OCH3), 3.45–3.53 (m, 2H;
CH2NHBoc), 3.09–2.97 (m, 2H; CH2Ph), 1.44 ppm (s, 9H; tBu);
13C NMR (300 MHz, CDCl3): d=171.9, 157.4, 155.8, 155.5, 136.1, 130.1,
128.5–127.9, 114.3, 79.4, 66.8–66.6, 54.8, 52.02, 39.9, 37.0, 28.2 ppm; IR
(CHCl3): n˜ =1708–1718 cmÀ1 (NHCO); MS (ESI): m/z: 495.3 [M+Na]+;
elemental analysis calcd (%) for C25H32N2O7: C 63.95, H 6.83, N 5.93;
found: C 63.76, H 6.85, N 5.94.
4098
www.chemeurj.org
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2006, 12, 4091 – 4100

Electrochemistry
FULL PAPER
Tyrosine derivative 15: Ammonium formate (0.5 g, 7.9 mmol) and Pd/C
10% (150 mg) were added to a solution of 14 (1.5 g, 3.2 mmol) in MeOH
(30 mL). The mixture was stirred at room temperature for 3 h, then fil-
tered over a pad of celite. The solvent was evaporated and the residue
was dissolved in AcOEt (100 mL) and washed with brine (60 mL). The
organic phase was dried over Na2SO4 and evaporated. The crude product
15 (colourless oil, 75%) was used for the subsequent reaction without
J=8.5 Hz, 2H; PhO), 6.93 (d, J=8.5 Hz, 2H; PhO), 5.05 (d, Jgem =
12.2 Hz, 2H; PhCH2O), 4.65 (d, J=16.4 Hz, 1H; CH2COTym), 4.34 (d, J=
16.4 Hz, 1H; CH2COTym), 4.20–4.10 (m, 3H; CH2O+CH), 3.90–3.76 (brs,
3H; OCH ), 3.50–3.00 (m, 7H; CH2NCH + CH2Ph + CH2NHCbz +
3
CH2NH3+), 2.60 (m, 1H; CH2N), 1.84 ppm (s, 3H; CH3); 13C NMR
(300 MHz, CD3OD); d=172.2, 169.4, 167, 158.4, 152.8, 143.2, 138.2,
132.2, 131.8, 128.5–128.1, 115.8, 110.9, 67.7, 65.3, 65.0, 52.9, 40.4, 40.1,
34.4, 12.2 ppm; IR (Nujol): n˜ =1660–1719 cmÀ1 (NHCO); MS (ESI): m/z:
582.3 [M+Na]+.
1
any further purification. Rf =0.23 (AcOEt); H NMR (300 MHz, CDCl3):
d=7.10 (d, J=8.5 Hz, 2H; Ph), 6.81 (d, J=8.5 Hz, 2H; Ph), 5.00 (brs,
1H; NH-Boc), 3.97 (t, J=5.1 Hz, 2H; CH2O), 3.80–3.71 (m, 4H;
OCH3 +CH), 3.53–3.48 (m, 2H; CH2NH-Boc); 3.12–2.70 (m, 4H;
CH2Ph+NH2), 1.43 ppm (s, 9H; tBu); MS (ESI): m/z: 339 [M+1]+.
Tris(ferrocenemethylenoxymethyl)methylisocyanate (19): 4-Dimethylami-
nopyridine (45 mg, 0.35 mmol) was added to a solution of 2 (0.250 g,
0.35 mmol) in anhydrous THF (15 mL). The mixture was cooled to
À158C and a solution of Boc2O(0.095 g, 0.43 mmol) in THF (3 mL) was
slowly added. The mixture was allowed to warm at room temperature
and was stirred for 20 h, then the solvent was evaporated. The residue
was dissolved in CH2Cl2 and purified by column chromatography
(AcOEt/light petroleum 3:7) to afford isocyanate 19 as an orange oil
(76%). 1H NMR (300 MHz, CDCl3): d=4.25–4.10 (m, 33H; Fc+
OCH2Fc), 3.46 ppm (s, 6H, CH2O); IR (Nujol): n˜ =2294 cmÀ1 (NCO);
MS (ESI): m/z: 741 [M]+; elemental analysis calcd (%) for
C38H39Fe3NO4: C 61.57, H 5.30, N 1.89; found: C 61.60, H 5.28, N 1.88.
Tyrosine backbone 16: A mixture of ZnCl2 (0.18 g, 1.32 mmol) and
NaCNBH3 (0.165 g, 2.65 mmol) in dry MeOH (1 mL) was slowly added
at 08C, to a solution of 15 (0.82 g, 2.42 mmol) and N-Cbz-2-amino acetal-
dehyde (0.425 g, 2.42 mmol) in dry MeOH (4 mL). The mixture was then
stirred at room temperature for 2 h. After evaporation of the solvent, the
residue was dissolved in AcOEt (100 mL), washed with H2O(2”50 mL)
and the aqueous phase was extracted with AcOEt (3”20 mL). The com-
bined organic phases were washed with H2O(50 mL), dried over Na SO4
2
and evaporated. The crude product 16 was recovered as a colourless oil
(93%). Rf =0.52 (AcOEt); [a]2D0 =À6.60 (c=0.076 in CHCl3); 1H NMR
(300 MHz, CDCl3): d=7.30 (d, J=8.5 Hz, 2H; PhO), 6.76 (d, J=8.5 Hz,
2H; PhO), 5.20–4.90 (m, 4H; NHCbz+OCH2Ph, NHBoc), 3.90 (brt, J=
4.85 Hz, 2H; PhOCH2), 3.60 (s, 3H; OCH3), 3.50–3.40 (m, 3H;
CH2NHBoc+CH), 3.20–3.10 (m, 2H; CH2NHCbz), 2.90–2.70 (m, 3H;
CH2PhO+CH2NH), 2.60–2.50 (m, 1H; CH2NH), 1.84 (s, 3H, CH3), 1.80
(brs, 1H; NHCH), 1.43 ppm (s, 9H; tBu); 13C NMR (300 MHz, CDCl3):
d=174.8, 157.4, 156.4, 155.7, 136.6, 130.1, 129.5, 128.4, 127.9, 114.3, 79.4,
67.0, 66.5, 62.5, 51.6, 47.13, 40.6, 40.0, 38.0, 28.3 ppm; IR (neat): n˜ =
1718 cmÀ1 (NHCO); MS (ESI): m/z: 538 [M+Na]+, 516 [M+1]+; elemen-
tal analysis calcd (%) for C27H37N3O7: C 62.90, H 7.23, N 8.15; found: C
63.01, H 7.22, N 8.16.
Tris-Fc tyrosine ester monomer 20: At room temperature DIPEA
(0.25 mL, 1.45 mmol) was added to a solution of Tris-isocyanate 19
(0.535 g, 0.72 mmol) and PNA monomer 18 (0.335 g, 0.48 mmol) in anhy-
drous DMF (6 mL), and the mixture was stirred at room temperature for
24 h. After this time, CH2Cl2 (20 mL) was added and the solution was
washed with H2O(2”15 mL). The aqueous phase was extracted with
CH2Cl2 (2”15 mL) and the combined organic layers were treated with
KHSO4 (0.3m, 2”15 mL). After the organic phase had been washed with
H2O(20 mL), it was dried over Na SO4 and evaporated. The residue was
2
purified by column chromatography on silica gel (AcOEt/light petroleum
4:6 then AcOEt/MeOH 9:1) to afford monomer 20 (84%) as a yellow
solid and unreacted isocyanate 19 (30%). Rf =0.45 (AcOEt); m.p. 178–
1808C (pentane); [a]2D0 =À67.2 (c=0.064 in CHCl3); 1H NMR (300 MHz,
CDCl3): d=8.80 (s, 1H; NHTym), 7.40–7.20 (m, 5H; Ph), 7.03 (d, J=
8.5 Hz, 2H; PhO), 6.81 (d, J=8.5 Hz, 2H; PhO), 6.60 (s, 1H; CH=), 5.5
PNA monomer 17: At room temperature N,N-diisopropyl carbodiimide
(0.48 mL, 3.1 mmol) was added to a solution of 1-carboxy methyl thy-
mine (0.57 g, 3.1 mmol) in dry DMF (4 mL). DhBtOH (0.5 g, 3.1 mmol)
was then added and the precipitation of N,N-diisopropyl urea (DIU) was
observed. After the mixture had been stirred at room temperature for
2 h, a solution of 16 (0.9 g, 1.75 mmol) in dry DMF (7 mL) was added.
The mixture was stirred for a further 3 h at room temperature, and then
the solvent was evaporated. The resulting residue was dissolved in
CH2Cl2 (10 mL), the DIU was filtered off over a pad of celite and the
solvent was evaporated. Finally, the residue was dissolved in AcOEt
(50 mL), washed with saturated NaHCO3 (4”30 mL) and then with
water (50 mL), dried over Na2SO4 and evaporated. The crude product
was purified by column chromatography on silica gel (AcOEt/light petro-
leum 8:2, Rf = 0.23) to afford the PNA monomer 17 (70%) as a white
solid. M.p. 128–1328C (pentane); [a]2D0 =À82.4 (c=0.074 in CHCl3);
1H NMR (300 MHz, CDCl3): d=8.80 (s, 1H; NH), 7.40–7.20 (m, 5H;
Ph), 7.04 (d, J=8.5 Hz, 2H; PhO), 6.82 (d, J=8.5 Hz, 2H; PhO), 6.60 (s,
(brs, 1H; CbzNH), 5.16 (d, Jgem =12.2 Hz, 1H; PhCH2O), 5.00 (d, Jgem
=
12.2 Hz, 1H; PhCH2O), 4.50 (brd, 1H; CH2), 4.30–4.00 (m, 33H;
OCH2Fc+Fc), 3.96–3.76 (m, 7H; OCH3 +CH+CH2CO+CH2O), 3.70
(s, 6H; C(CH2)3O), 3.50–3.00 (m, 7H; CH2N+ CH2Ph+CH2NHCbz+
R
CH2NHCONH), 2.60 (brs, 1H; CH2N), 1.84 ppm (s, 3H, CH3); 13C NMR
(300 MHz, CDCl3): d=170.7, 167, 164.1, 157.7–156.4, 151.7, 141, 136.6,
130.2, 129.4, 128.4, 128.3, 128.2, 114.9, 110.3, 83.6, 69.6, 69.3–68.2, 67.5,
66.9, 63.5, 58.8, 52.6, 48.9, 48.2, 39.6, 39.0, 33.1, 12.2 ppm; IR (CH2Cl2):
n˜ =1688, 1716 cmÀ1 (NHCO, COOMe); MS (ESI): m/z: 1345 [M+Na]+;
elemental analysis calcd (%) for C67H74Fe3N6O12: C 60.83, H, 5.64, N
6.35; found: C 61.02, H 5.65, N 6.37.
Tris-Fc tyrosine acidmonomer 21 : At room temperature Ba(OH)2·8H2O
(0.090 mg, 0.28 mmol) was added to a solution of 20 (0.15 g, 0.11 mmol)
in CH2Cl2 (1 mL), MeOH (0.5 mL) and H2O(0.10 mL). The mixture was
vigorously stirred for 2 h at the same temperature, and then CH2Cl2
(20 mL) was added and the organic phase was washed with KHSO4
(0.3m, 2”15 mL) (pH 2). The organic phase was then washed again with
H2O, dried over Na2SO4 and evaporated. The yellow residue produced
was dissolved in pentane (5 mL), cooled and filtered (96%). M.p. 225–
2308C; [a]2D0 =À32.26 (c=0.062 in CHCl3); 1H NMR (300 MHz, CDCl3):
d=10.2 (s, 1H; NH), 7.40–7.20 (m, 5H; Ph), 7.20–6.95 (m, 2H; PhOH),
6.90–6.70 (m, 3H; CH=+PhOH), 6.10 (brs, 1H; NH), 5.60 (brs, 1H;
NH), 5.10–4.90 (m, 3H; PhCH2O+NH), 4.50–3.76 (m, 38H; CH+
1H; CH=), 5.50 (brs, 1H; NHCbz), 5.16 (d,
Jgem =12.2 Hz, 1H;
PhCH2O), 5.00 (d, Jgem =12.2 Hz, 1H; PhCH2O), 4.50 (d, Jgem =16.2 Hz,
1H; CH2CO), 3.96 (t, J=5.18 Hz, 2H; CH2O), 3.90–3.76 (m, 5H;
OCH3 +CH+CH2CO), 3.50–3.00 (m, 7H; CH2N
+
CH2PhO
+
CH2NHCbz + CH2NHBoc), 2.60 (brd, Jgem =15.5 Hz, 1H; CH2N), 1.84
(s, 3H; CH3), 1.44 ppm (s, 9H; tBu); 13C NMR (300 MHz, CDCl3): d=
170.7, 167, 164.5, 157.4, 156.4, 155.7, 151.6, 141, 136.6, 130.2, 129.5, 128.4–
128.2, 114.7, 110.3, 79.4, 66.9, 66.7, 63.5, 52.6, 48.7, 48.2, 39.9, 39.0, 33.1,
28.3, 12.2 ppm; IR (Nujol): n˜ =1664–1719 cmÀ1 (NHCO); MS (ESI): m/z:
704.2 [M+Na]+; elemental analysis calcd (%) for C34H43N5O10: C 59.90,
H 6.36, N 10.27; found: C 61.02, H 6.35, N 10.25.
CH2CO+CH2O , CHFc+Fc), 3.70–2.30 (m, 14H; CH2N + CH2PhO +
2
CH2NH-Cbz
+
CH2NHCONH; C(CH2)3O), 1.84 ppm (s, 3H; CH3);
G
PNA monomer 18: At room temperature
a solution of TFA (1m,
13C NMR (300 MHz, CDCl3); d=170.7, 167.0, 164.1, 158.2, 157.5, 156.6,
151.9, 141, 136.4, 130.2, 129.4, 128.4–128.1, 114.7, 110.3, 83.6, 69.5, 69.2–
68.4, 67.3, 66.8, 63.3, 58.8, 48.7, 39.6, 38.6, 33.1, 12.2 ppm; IR (CH2Cl2)
n˜ =3050 cmÀ1 (COOH), 1688, 1716 cmÀ1 (NHCO); MS (ESI): m/z: 1308
0.35 mL, 4.4 mmol) in CH2Cl2 was added to a solution of 17 (0.5 g,
0.73 mmol) in CH2Cl2 (3 mL), and the mixture was stirred at room tem-
perature for 48 h. After this time, the solvent was evaporated and the res-
idue was dissolved in AcOEt (30 mL) and evaporated. The crude product
was precipitated from pentane (5 mL) and filtered to give 18 (97%).
1H NMR (300 MHz, CD3OD): d=7.4–7.2 (m, 6H; Ph + CH=), 7.17 (d,
[M]+, 1331 [M+Na]+; elemental analysis calcd (%) for C66H72Fe3N6O12
C 60.57, H 5.54, N 6.42; found: C 60.53, H 5.55, N 6.44.
:
Chem. Eur. J. 2006, 12, 4091 – 4100
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
4099

S. Maiorana, L. Falciola et al.
Tris-Fc aminoethylglycine monomer 23:
A
solution of
2
(0.21 g,
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0.30 mmol) and DIPEA (0.13 mL, 0.74 mmol) in DMF (0.5 mL) was
added at 08C to a solution of pentafluorophenyl carbonate (0.115 g,
0.30 mmol) in N-methyl pyrrolidone (1.5 mL) and the mixture was stirred
for 1 h at the same temperature. This solution was then slowly added to a
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was purified by column chromatography on silica gel (AcOEt/light petro-
leum 4:6, increasing progressively the polarity of the solvent) to afford 23
1
(75%) as a yellow solid. M.p. 176–1788C (pentane); H NMR (300 MHz,
CDCl3): d=8.11 (brs, 1H; NH), 6.76 (s, 1H; CH=), 5.70 (brs, 2H; NH),
4.28 (s, 2H; CH2CO), 4.20 (s, 6H; CH2Fc), 4.17 (t, 6H; Fc), 4.12 (t, 6H;
Fc), 4.10 (s, 15H; Fc), 3.96 (s, 2H; CH2COO), 3.72 (s, 3H; OCH3), 3.62
(s, 6H;
C
A
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CH3CH=); 13C NMR (300 MHz, CDCl3); d=170.1, 167.6, 163.9, 157.2,
150.9, 141.9, 110.0, 83.5, 69.7, 69.4, 69.3, 68.5–68.4, 58.9, 52.5, 49.5, 49.0,
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(Nujol): n˜ =1651 (CO), 1455 cmÀ1 (NHCONH); elemental analysis calcd
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Acknowledgements
This study was financially supported by the Italian Ministero dell’Istru-
zione e della Ricerca (MIUR) and National Research Council (CNR).
We would like to thank Drs. Clelia Giannini and Dario Resemini for
NMR and experimental support.
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Received: November 25, 2005
Published online: March 17, 2006
4100
www.chemeurj.org
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2006, 12, 4091 – 4100