Luminescent conjugates between dinuclear rhenium(i) complexes and peptide nucleic acids (PNA) for cell imaging and DNA targeting

Article abstract of DOI:10.1039/c0cc00450b

New luminescent dinuclear rhenium(i) tricarbonyl complex-PNA conjugates have been synthesized through a reliable solid-phase synthetic methodology. Their photophysical properties have been measured. The most luminescent Re-PNA conjugate 7 showed interesting two-photon absorption (TPA) properties, that were exploited for imaging experiments, to demonstrate its easy uptake into living cells.

Full text of DOI:10.1039/c0cc00450b

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Luminescent conjugates between dinuclear rhenium(I) complexes and
peptide nucleic acids (PNA) for cell imaging and DNA targetingw
Elena Ferri,a Daniela Donghi,b Monica Panigati,b Giuseppe Prencipe,a Laura D’Alfonso,c
Ivan Zanoni,d Clara Baldoli,e Stefano Maiorana,a Giuseppe D’Alfonso*b and
Emanuela Licandro*a
Received 17th March 2010, Accepted 30th June 2010
DOI: 10.1039/c0cc00450b
New luminescent dinuclear rhenium(I) tricarbonyl complex–
PNA conjugates have been synthesized through a reliable
solid-phase synthetic methodology. Their photophysical properties
have been measured. The most luminescent Re–PNA conjugate 7
showed interesting two-photon absorption (TPA) properties,
that were exploited for imaging experiments, to demonstrate
its easy uptake into living cells.
2000 cmÀ1, that allows the use of organometalcarbonyl
compounds as bioprobes.11
A number of metal complexes have been conjugated to PNA
oligomers with the aim of imparting to them new biochemical
and spectroscopic properties.3,4,12,13 Some examples of mono-
nuclear rhenium complex–PNA conjugates have been reported
in the literature14–16 but, as far as we know, no luminescent
and no dinuclear rhenium complexes have been used in PNA
labelling.
Peptide nucleic acid (PNA) is a DNA mimic,1 in which
N-(2-aminoethyl)glycine units form a pseudo-peptide chain
bearing the four nucleobases. Because of its neutral chain,
PNA exhibits stronger and more selective binding affinity for
complementary nucleic acid (DNA and RNA) strands than
natural nucleic acids. PNA also shows higher mismatch
selectivity and noticeable chemical and enzymatic stability
compared to natural oligonucleotides.2
In the present work we focused our attention on two
dinuclear rhenium(I) complexes, namely 1 and 2 of Fig. 1,
containing a carboxyl group on the diazine ligand, for the
conjugation to PNA oligomers through an amide bond with
the terminus NH2 group. A reliable solid-phase synthetic
methodology has been established, which provided
a
luminescent rhenium–PNA decamer conjugate, whose easy
uptake into living cells has been demonstrated by imaging
through two-photon excitation.
The above advantages have addressed many research efforts
to the use of PNA for DNA detection, by providing PNA of
appropriate analytical probes (electrochemical,3,4 fluorescent,5
radioactive,6 etc.). Recently some of us have used magnetic
PNA as biosensor for the rapid and reliable assessment of
DNA hybridization by T2 relaxation measurements.7 In any
case luminescence detection is one of the most widely used
techniques to identify the DNA/PNA hybridization event.5
Some of us are currently developing a novel family of
dinuclear tricarbonyl rhenium(I) derivatives, of general
formula [Re2(m-X)(m-Y)(m-1,2-diazine)(CO)6], which exhibit
intense emission in the range 550–620 nm, originating from
triplet metal-to-ligand-charge transfer (3MLCT) excited
states.8–10 Thanks to their photophysical properties, this new
family of luminescent complexes appears a promising tool for
PNA labelling, in view of biomedical applications. In addition,
these complexes show the characteristic intense IR absorption
bands due to the stretching of the CO ligands, about
The new complexes 1 and 2, which are air stable orange and
yellow solids, were prepared in 60% and 90% yields,
respectively, by refluxing [Re(CO)5Cl] with 0.5 equivalents of
the corresponding diazine for 2 h in toluene solution.9,10 The
pyridazine-4-carboxylic acid ligand is commercially available,
while the 4-(pyridazin-4-yl)butanoic acid ligand was prepared
by a [4+2] Diels–Alder cycloaddition reaction between
1,2,4,5-tetrazine and 1-hexynoic acid.17 In order to find
the best conditions for binding these complexes to a PNA
oligomer, the model reactions between the thymine monomer
3 and complex 1 or the free pyridazine-4-carboxylic acid ligand
were studied (see ESI for details), showing that the best route
to obtain the conjugate 4 consists in binding the free diazine
ligand to the PNA monomer 3 to give the modified monomer
5, which was then reacted with [Re(CO)5Cl] (Scheme 1).
Therefore this procedure was used for preparing the
rhenium PNA decamer conjugates 6 and 7 (Scheme 2). The
PNA homo-thymine decamer was prepared by an automated
solid phase synthesis,2 using standard Boc-procedure
conditions (see ESI). Then the corresponding free diazine
ligands were manually coupled to the terminal NH2 group of
a
`
Dipartimento di Chimica Organica e Industriale, Universita degli
Studi di Milano, via Venezian 21, I-20133 Milano, Italy.
E-mail: emanuela.licandro@unimi.it
b Dipartimento di Chimica Inorganica, Metallorganica e Analitica
`
‘‘L. Malatesta’’, Universita degli Studi di Milano, via Venezian 21,
I-20133 Milano, Italy. E-mail: giuseppe.dalfonso@unimi.it
c
`
Dipartimento di Fisica, Universita di Milano-Bicocca,
piazza della Scienza 3, I-20126 Milano, Italy
d Dipartimento di Biotecnologie e Bioscienze, Universita` di
Milano-Bicocca, piazza della Scienza 2, I-20126 Milano, Italy
e Istituto di Scienze e Tecnologie Molecolari, C.N.R., Via C. Golgi 19,
I-20133 Milano, Italy
w Electronic supplementary information (ESI) available: Acronyms of
reagents and solvents, details on the synthesis and characterization of
the compounds here described. See DOI: 10.1039/c0cc00450b
Fig. 1 Structure of complexes 1 and 2.
c
This journal is The Royal Society of Chemistry 2010
Chem. Commun., 2010, 46, 6255–6257 6255

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Table 1 Photophysical properties of complexes 1, 2, 4, 6 and 7 at
295 K
l
abs/nm (e/103)
l
em/nm
Fem  100
—
—
12(1)
1.7(1)
1.0(1)
0.4(1)
—
t/ns
a
b
a
b
c
1
2
409 (6.5)
355 (7.1)
370 (8.6)
335 (7.5)
335 (7.5)
390 (6.9)
364 (8.1)
360 (n.d.)
333 (7.9)
333 (7.9)
—
—
—
—
1600
535
320
20
—
—
546
316
586
610
610
660
—
—
610
610
a
b
b
b
c
4
6
7
—
1.7(2)
1.1(2)
Scheme 1 Synthesis of the Re–PNA monomer conjugate 4.
a
b
Deaerated toluene solution. Deaerated and aerated CH3CN–H2O
c
the resin-supported PNA 8, by forming the amide bond with
HATU as the condensing agent and DIPEA, in NMP as the
solvent. The resin supported PNA decamers 9 and 10, bearing
terminal diazine ligands, were then reacted with an excess of
[Re(CO)5Cl] in refluxing toluene. The results of this solid
phase complexation were excellent and the supported
Re–PNA decamer conjugates 11 and 12 could be easily
purified by washing the resin with toluene.
1 : 1 solution. In parentheses the uncertainty on the last digit of the
photoluminescence quantum yields (Fem). For the lifetimes uncertain-
ties see ESI.
Interestingly, complex 1 becomes weakly emitting when
bound to PNA monomer (conjugate 4, emission: lmax 660 nm,
Fem 0.4% in deaerated toluene), probably because, owing to
the presence of the amide group, in this complex the diazine is
less electron-poor than in 1. Such poor luminescence is
completely lost in aerated CH3CN–H2O solution, both for 4
and for 6 (which is insoluble in toluene).
Thanks to the stability of these Re complexes (which was
checked by treating 1 with a TFA/TFMSA solution from
which it was recovered unchanged), the final cleavage from the
resin was carried out in the standard acidic conditions.2
Therefore, 11 and 12 were treated with TFA/TMSA/
m-cresol/thioanisole 6 : 2 : 1 : 1 for 1 h and the corresponding
rhenium–PNA conjugates 6 and 7 were recovered, as orange
and pale yellow solids respectively, by precipitation with
diethyl ether. Both compounds were purified by means of
semipreparative RP-HPLC and their structures confirmed
with MALDI, LC-MS, and UV-vis spectroscopy (see ESI).
The photophysical properties of complexes 1 and 2 and of
their PNA conjugates 4, 6 and 7 are reported in Table 1.
Complexes 1 and 2 show properties typical of excited states
arising from dp(Re)–p*(diazine) charge transfer:9,10 (i) blue
shift of the absorption and red shift of the emission by
increasing solvent polarity, (ii) modulation of the emission
on varying the diazine substituents (i.e. bathochromic shift
and quenching of the emission by introducing the electron-
withdrawing carboxyl group). Then complex 1 is completely
non-emitting, whilst 2, owing to the presence of the alkyl
spacer between pyridazine and the carboxyl group, gives
intense broad emission (quantum yields 12%), centred at
586 nm in toluene solution, with the long lifetime typical of
triplet emission (1600 ns in deareated solution).
In contrast, the binding with PNA does not change the
emission properties of complex 2, since the carboxyl group is
well spaced from the diazine ring. So, both 2 and its conjugate
7 show almost identical absorption and emission features in
aerated CH3CN–H2O solution, as well as identical PLQY and
similar lifetimes (lmax absorption 333–335 nm, lmax emission
610 nm, Fem 1.0–1.1%, t 316–320 ns, Table 1). Both lifetimes
and quantum yields increase in deaerated solution, in agreement
with a substantial triplet character of the excited state.
In addition, the ability of the new Re–PNA conjugate 7 to
recognize and bind the complementary DNA strand was
evaluated by measuring the Tm (see ESI).
Compound 7 possesses therefore a series of very favourable
properties for biological applications. It not only maintains a
good emission in the presence of oxygen and water, but also
exhibits a large Stokes shift (277 nm) and long lifetime of the
excited state, that eliminate self-quenching problems and allow
easy distinction from the cell background auto-fluorescence,
which is normally very short lived (less than 10 ns).18
Moreover, both complexes 2 and 7 can be excited by two
photon absorption using near IR laser light, with a maximum
effect around 750 nm (see ESI). This red shifted excitation
profile (already established for mononuclear Re complexes,
but rarely exploited)19 is advantageous over one-photon
absorption, since it allows the achievement of deeper tissue
penetration with less damage to the cell tissues.20
Recently, many papers have appeared dealing with the use
of phosphorescent metal complexes as targeting molecules in
cell imaging.18 Moreover, a few studies reported enhanced
cellular uptake of PNA oligomers containing N-terminally
attached Zn2+ or Co2+ complexes.21,22 We have therefore
investigated the ability of the conjugate 7 to permeate cell
membranes, by measuring the cellular uptake from HEK-293
cells in tissue plated cells. A small aliquot (50 mL) of a
Scheme 2 Solid phase synthesis of the Re–PNA conjugates 6, 7.
6256 Chem. Commun., 2010, 46, 6255–6257
c
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provides a new bioorganometallic system with many favour-
able properties: it is photostable and non cytotoxic, it very
quickly permeates living cells, in the presence of small amounts
of DMSO and it stains with different colours nucleus and
cytoplasm (suggesting a possible use for highlighting environ-
ments with different lipophilicity or rigidity). So far, no PNA
with such photophysical property has been reported and we
think that this study can open new perspectives for biological
PNA applications.
Fig. 2 Images of HEK-293 cells stained with the Re–PNA conjugate 7,
recorded about 10 min after the addition of the complex, through a
485/30 (left) and a 600/40 (center) band pass filter and their super-
position (final 7 concentration 3 mM; field of view: 78 Â 78 mm2).
Analogous images through the 485 and 535 filters are shown in Fig. S7
of ESI.
This work was supported by MIUR and University of
Milan, PRIN 2007 (2007F9TWKE_002), PUR 2008,
Fondazione ‘‘Romeo ed Enrica Invernizzi’’, and CNR- Regione
Lombardia ‘‘Mind in Italy’’ project.
0.12 mM DMSO solution of 7 was added to 2 mL of
phosphate buffer solution (PBS) in different wells of the plate,
giving a 3 mM concentration of 7 in the wells. The samples
were imaged using two photon excitation at 770 nm and the
luminescence of the complex was detected in a wide spectral
range, through 485/30, 535/50 and 600/40 band pass filters.
Measurements performed under the same laser power in the
absence of 7 showed that cells autofluorescence was negligible
(see ESI). After incubation for 10 min at 37 1C, intense
luminescence was observed, indicating that 7 penetrates the
cell membrane, staining both the cytoplasm and the nucleus
(see Fig. 2, whose image plane was chosen through a z-scan of
the cells in a 40 mm range). The emission of 7 inside the nucleus
appears to be blue shifted with respect to that in the cytoplasm
(enhanced visibility of the nucleus in the blue filtered image of
Fig. 2). Both the reduced mobility and the more hydrophobic
character of the nuclear environment, with respect to the
cytoplasm, could be responsible for this hypsochromic shift
Notes and references
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2 Peptide Nucleic Acids: Protocols and Applications, ed. P. E. Nielsen,
Horizon Bioscience, Wymondham, UK, 2nd edn, 2004.
3 C. Baldoli, C. Rigamonti, S. Maiorana, E. Licandro, L. Falciola
and P. Mussini, Chem.–Eur. J., 2006, 12, 4091.
4 N. Metzler-Nolte, in Bioorganometallics, ed. G. Jaouen, Wiley-VCH,
Weinheim, 2006, ch. 5, p. 125.
5 B. Y. Oquare and J.-S. Taylor, Bioconjugate Chem., 2008, 19, 2196
and references therein.
6 E. Wickstrom, X. Tian, N. V. Amirkhanov, A. Chakrabarti,
M. R. Aruva, P. S. Rao, W. Qin, W. Zhu, E. R. Sauter and
M. L. Thakur, Methods Mol. Med., 2005, 106, 135–191 and ref.
cited therein; F. Gallazzi, Y. Wang, F. Jia, N. Shenoy,
L. A. Landon, M. Hannink, S. Z. Lever and M. R. Lewis,
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7 G. Prencipe, S. Maiorana, P. Verderio, M. Colombo, P. Fermo,
E. Caneva, D. Prosperi and E. Licandro, Chem. Commun., 2009,
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8 M. Panigati, D. Donghi, G. D’Alfonso, P. Mercandelli, A. Sironi,
P. Mussini and L. D’Alfonso, Inorg. Chem., 2006, 45, 10909.
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D. Donghi, M. Panigati, P. Mercandelli, P. Mussini, G. D’Alfonso
and L. De Cola, Adv. Funct. Mater., 2009, 19, 26074.
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12 For exhaustive references see: A. M. Sosniak, G. Gasser and
N. Metzler-Nolte, Org. Biomol. Chem., 2009, 7, 4992.
13 C. Baldoli, P. Cerea, C. Giannini, E. Licandro, C. Rigamonti and
S. Maiorana, Synlett, 2005, 1984.
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18 V. Fernandez-Moreira, F. L. Thorp-Greenwood and M. P. Coogan,
Chem. Commun., 2010, 46, 186 and references therein.
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3
of the MLCT emission. Interestingly, in the same conditions
the homothymine PNA decamer labeled with fluorescein
appears to not permeate small vesicles inside the cytoplasm
region of cells (see ESI), whilst the neutral complex 2 alone
shows roughly the same behaviour as 7 (see ESI).
Wide field transmitted light images confirmed that the cells
were alive during all the imaging experiments, ruling out the
hypothesis that cellular uptake was promoted by the onset of
cell apoptosis, due to singlet oxygen formation (triplet emitters
being potential sensitizers of singlet oxygen).
These preliminary results indicate that 7 is viable as a
fluorophore for cell imaging, although more detailed experi-
ments are needed in order to establish the kinetics and
mechanism of the process, the influence of the kind of cells
used and the lowest limits of complex and DMSO concentration.
In fact, a role of DMSO in enhancing membrane permeability
can be anticipated (in line with recent studies on cellular
delivery of proteins, drugs and organometallic complexes),23
since no staining of the cells was detected using an aqueous
solution of 7. At the very low concentrations used in
the present study (2.5% v/v), DMSO does not appear to
significantly affect cells morphology and viability (see ESI).
Moreover, viability studies have indicated that neither 7 nor 2
shows any specific toxicity (see ESI).
21 A. Fussl, A. Schleifenbaum, M. Goritz, A. Riddell, C. Schultz and
¨
¨
R. Kra
¨
mer, J. Am. Chem. Soc., 2006, 128, 5986.
22 S. I. Kirin, I. Ott, R. Gust, W. Mier, T. Weyhermuller and
N. Metzler-Nolte, Angew. Chem., Int. Ed., 2008, 47, 955.
23 H. Wang, C.-Y. Zhong, J.-F. Wu, Y.-B. Huang and C.-B. Liu,
J. Controlled Release, 2010, 143, 64.
In conclusion, in this paper we have shown that the
conjugation of PNA with dinuclear rhenium(I) complexes
c
This journal is The Royal Society of Chemistry 2010
Chem. Commun., 2010, 46, 6255–6257 6257