Sequential insertion of three different organometallics into a versatile building block containing a PNA backbone

Article abstract of DOI:10.1039/c003598j

In the view of developing a synthetic route for the controlled insertion of distinct organometallic moieties into peptide nucleic acid (PNA) oligomers, a proof-of-principle study of the chemoselective insertion of three different organometallics into a building block containing both a PNA backbone and an alkyne side-chain is presented in this study.

Full text of DOI:10.1039/c003598j

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Sequential insertion of three different organometallics into a versatile building
block containing a PNA backbone†
Malay Patra,a Gilles Gasser,*‡a Dmytro Bobukhov,b Klaus Merz,a Alexander V. Shtemenkob and Nils
Metzler-Nolte*a
Received 24th February 2010, Accepted 6th May 2010
First published as an Advance Article on the web 19th May 2010
DOI: 10.1039/c003598j
In the view of developing a synthetic route for the controlled
insertion of distinct organometallic moieties into peptide nu-
cleic acid (PNA) oligomers, a proof-of-principle study of the
chemoselective insertion of three different organometallics
into a building block containing both a PNA backbone and
an alkyne side-chain is presented in this study.
example, application of the [M(CO)3]+ core has become a popular
technique in radiolabelling, with M = 99mTc for radio imaging
or M = 186/188Re for therapeutic applications.5 Also, the non-
radioactive Re is often used as a surrogate for 99mTc.
As
a
first step towards multi-modal imaging with
organometallics, the combination of several of these proper-
ties into the same biomolecule would be of high interest and
consequently novel synthetic routes for the incorporation of
different organometallic entities into the same biomolecule are
highly demanded. The preparation, in solution, of compounds
containing up to six different metal complexes was recently
reported by Packheiser and Lang.6 Heinze and co-workers have
described the multistep synthesis of transition metal oligomers by
solid phase synthesis.7 However, reports of such compounds for
biological purposes are rare. Beck et al. have synthesised hetero-
biometallic amino acid derivatives for peptidomimetic purposes.8
Our group recently reported the preparation of mono and bis-
ferrocene containing peptide nucleic acid (PNA) oligomers9 as
well as the study of their thermal stability upon hybridisation
with DNA sequences.10 Also, we reported the preparation and
characterisation of hetero-bimetallic organometallic phenylala-
nine and PNA monomer derivatives, in which the N-terminus
and the modified C-terminus of these biomolecules were la-
beled with ferrocene and a bis(triphenylphosphine)-platinum(0)
complex, respectively.11 With the strive of developing a reaction
sequence for the controlled and sequential insertion of distinct
organometallics into a PNA oligomer, we have chosen, as a model
compound, a building block 49 (Scheme 1) containing both a PNA
backbone and an alkyne side-chain and investigated the feasibility
of attaching three different organometallics to the modified
alkyne side chain, N-terminus and modified C-terminus of 4,
respectively.
Multi-modal imaging is becoming an important addition to
the chemical biology toolbox for the study of complex biological
systems. As a typical example, an (organic) fluorescent dye could
be combined with a lanthanide magnetic resonance imaging
(MRI) agent. In this toolbox, organometallic compounds are not
well represented yet. This is certainly an unfounded omission, since
many organometallic compounds are stable under physiological
conditions.1 Moreover, organometallic complexes may impart
specific properties thereby enhancing already existing possibilities
for detection. Examples include the favourable electrochemical
properties of ferrocene, which make it ideal for electrochemical
detection.2 The characteristic CO stretching bands of metal
carbonyl complexes have been exploited by Jaouen, Salmain
and co-workers in the so-called carbonyl metallo immuno assay
(CMIA)3 to detect and quantify different analytes in biological
samples. Moreover, we have recently introduced metal carbonyl
complexes as novel entities for RAMAN microscopy.4 As a third
aLehrstuhl fu¨r Anorganische Chemie I, Fakulta¨t fu¨r Chemie und Biochemie,
Ruhr-Universita¨t Bochum, Geba¨ude NC 3 Nord, Universita¨tsstr. 150, 44801,
Bochum, Germany. E-mail: nils.metzler-nolte@rub.de; Fax: 0234 32-14378
bDepartment of Inorganic Chemistry, Ukrainian State Chemical Technolo-
gical University, Gagarin Avenue 8, Dnipropetrovs’k, 49005, Ukraine
† Electronic supplementary information (ESI) available: Full experimental
details, 1H and 13C NMR spectra of all the compounds, IR spectrum and
cyclic voltammogram of 1 and X-ray single crystal structure of 6c. CCDC
reference numbers 755697 and 755698. For ESI and crystallographic data
in CIF or other electronic format see DOI: 10.1039/c003598j
‡ Please note the new address: Institute of Inorganic Chemistry,
University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich,
Switzerland. Tel.: +41 (0)44 635 46 11. Fax: +41 (0)44 635 68 02; E-mail:
gilles.gasser@aci.uzh.ch.
With this in mind, we report herein the synthesis of the first
hetero-triorganometallic derivative (1, Fig. 1) of 4.9 To insert
three different organometallics into 4, we used click chemistry,
amide bond formation, and Sonogashira coupling as chemically
orthogonal conjunction methods.
Scheme 1 Synthesis of hetero-triorganometallic PNA derivative 1. Abbreviations: Fc = Cp2Fe, Cy = CpMn(CO)3 (Cp = cyclopentadienyl). Reagents
and conditions: (a) Fc–CH2–N3 (3), CuSO4·5H2O, sodium ascorbate, acetone/water; 63%; (b) HATU, DIPEA, p-iodo-aniline, DMF; 76%; (c) (i) 20%
piperidine in DMF, (ii) HATU, DIPEA, 2, DMF; 61%; (d) 6b, CuBr, (PPh3)2PdCl2, DMF/NEt3 60%.
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Fig. 1 Structures of the hetero-triorganometallic PNA monomer derivative 1 and Re-tricarbonyl complexes 6a, 6b and 6c.
Three different organometallics were chosen in this study to
exemplify the range of physico–chemical properties that can
be added to the building block 4. First, a ferrocene deriva-
tive (azidomethyl ferrocene, Fc–CH2–N3, 3) was selected due
to the chemical stability and well-established redox activity of
ferrocene.12 Ferrocene was also the first organometallic moiety
used for the labeling of PNA monomers and oligomers,13 and has
been used for electrochemical PNA·DNA biosensors, including a
“four potential”detection mode.14 As a metalcarbonyl, weselected
a tricarbonyl manganese derivative (b-cymantrenoyl-propionic
acid, 2). Apart from the CMIA mentioned above, Schatzschneider
et al. have recently shown that the conjugation of cymantrene
to the cell-penetrating peptide (CPP) hCT(18–32)-k7 modulates
the intracellular distribution of the CPP and induces cytotoxicity
against MCF-7 cells.15 Finally, a rhenium tricarbonyl complex
(6b, Fig. 1) was chosen for its relation to radiolabelling studies.16
Furthermore, Alberto and co-workers have recently reported the
labelling of a PNA monomer and oligomer with Re and 99mTc
tricarbonyl complexes.17
Fig. 2 ORTEP plot (50% probability ◦level) of the [Re(CO)3(5)]+ of 6b.
˚
Selected bond distances (A) and angles ( ): Re–C (average) 1.91 (9), Re–N1
2.18 (6), Re–N2 2.23 (8), N–Re–N (average) 78.0 (2), C–Re–C (average)
87.3 (3).
As the Sonogashira building block, the rhenium-tricarbonyl
complex [Re(CO)3(5)]+Br- (6a) was first prepared by refluxing
a mixture of N,N-bis(pyridine-2-ylmethyl)prop-2-yn-1-amine (5)
and [NEt4]2[ReBr3(CO)3] in MeOH.18 In order to improve the
solubility of 6a in organic solvents, its bromide counter-anion
was exchanged with PF6 (6b) and BPh4 (6c) using KPF6 and
NaBPh4 respectively. Single crystals of 6b and 6c§ suitable for X-
ray diffraction were grown in CH2Cl2 at room temperature and the
ORTEP plot of the [Re(CO)3(5)]+ cation of 6b is shown in Fig. 2
(see ESI for ORTEP plot of 6c†). Both 6b and 6c consist of the
[Re(CO)3(5)]+ cation and of a PF6- or B(C6H5)4- anion respectively,
which are located in channels of a three-dimensional network.
The coordination number around each Re ion is six in a distorted
octahedral coordination environment. The bond distances of the
Fmoc-deprotection of the amino group of 8, followed by HATU-
mediated peptide coupling with b-cymantrenoyl-propionic acid
1
(2)20 yielded 9. H NMR spectroscopy confirmed the formation
of 9 with the presence of two apparent triplets at 4.89 and
5.48 ppm for the cyclopentadienyl protons of cymantrene. The last
synthesis step is incorporation of the third organometallic moiety
6b into the modified C-terminus of 9 by Pd-catalyzed Sonogashira
coupling. The hetero-triorganometallic building block with a PNA
backbone 1 was obtained in a good yield (60%). The presence
of 1 was confirmed by ESI(+)-MS with a signal at 1305.92
corresponding to [M - PF6]+ (Fig. 3). The isotope pattern is very
characteristic due to the presence of three different metal atoms
and matches well with the calculated pattern (Fig. 3).
-
-
1
A complete assignment of all H and 13C NMR was achieved
˚
≡
alkyne unit in 6b (C C 1.18; C–C 1.462 and C–N 1.48 A) and 6c
˚
≡
using 2D NMR techniques. As expected, strong IR bands were
observed at 2025 and 1908 cm-1 corresponding to the M–CO
stretching vibrations (see ESI†). Due to spectral overlap, the
individual bands from the two different moieties could not be
resolved. As 1 contains a ferrocene derivative, voltammetric
investigations of 1 were carried out. Cyclic voltammetry was
performed at a stationary glassy carbon (GC) electrode at a scan
rate of 50 mV s-1 in acetonitrile with tetrabutylammonium hex-
afluorophosphate (Bu4NPF6, 0.1 M) as the supporting electrolyte.
A cyclic voltammogram of 1 and Cp*2Fe as secondary standard
is presented in the ESI.† Compound 1 exhibits a single reversible
(C C 1.16; C–C 1.45 and C–N 1.496 A) are similar and match
the expectation.
The synthesis of 1 (Scheme 1) starts with the CuI-catalysed
cycloaddition reaction of azidomethyl ferrocene (3)19 to the side
chain of the alkyne-containing building block 49 (Cu–AAC, often
considered the prototypical “click reaction”). Formation of the
ferrocene-labeled building block 7 was confirmed by ESI(-)-MS
with a peak at m/z = 660.1 corresponding to [M - H]-. The
carboxylic acid group of 7 was then coupled to p-iodo-aniline to
give 8 (Scheme 1). The 1H NMR spectrum of 8 shows two expected
multiplets at 7.25 and 7.82 ppm, respectively, corresponding to the
AA¢BB¢ spin system of the para-substituted aromatic ring. The
one-electron wave with a potential of E1/2 = +97 mV vs. FcH0/+
.
5618 | Dalton Trans., 2010, 39, 5617–5619
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◦
3
˚
a = 108.477(9), b = 91.25(1), g = 101.914(9) , V = 2075(1) A , Z = 2,
2qmax = 49.96◦, 11212 measured reflections, 6947 independent reflections,
452 parameters, m = 2.965 mm -1, R1 = 0.0476 for 6075 observed reflections
(I > 2s(I)), wR2 = 0.1355 for all reflections, CCDC 755698.
Bruker-axs-SMART 1000 CCD. Structure solution with direct methods
(SHELXS97), and refined against F2 with all measured reflections
(SHELXL97,21 the disorderd CH2Cl2 and water molecules in 6c were
handled using Platon/Squeeze22).
1 G. Jaouen, Bioorganometallics: Biomolecules, Labelling, Medicine,
Wiley-VCH, Weinheim, 2006; M. Metzler-Nolte and K. Severin, in
Concepts and Models in Bioinorganic Chemistry, ed. H.-B. Kraatz and
N. Metzler-Nolte, Wiley-VCH, Weinheim, 2006, pp. 113–136.
2 N. Metzler-Nolte and M. Salmain, in Ferrocenes: Ligands, Materials
and Biomolecules, ed. P. Stepnicka, John Wiley & Sons Ltd., Chichester,
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Fig. 3 ESI(+) mass spectrum of 1. Inserts: (a) experimental isotopic
pattern of the [M - PF6]+ peak of 1; (b) simulated isotopic pattern of
the [M - PF6]+ peak of 1.
The 10/+ process is assigned to the reversible oxidation/reduction
of the ferrocene redox couple of 1.
The sequential insertion of three different organometallics into
the building block 4 containing a PNA backbone has been
demonstrated in this study using three distinct types of orthogonal
chemical reactions. The preparation of 1 is an excellent proof
of the feasibility of the synthesis of hetero-triorganometallic
PNA oligomers and other suitable biomolecules. Indeed, similar
chemistries as those applied in this study can be undertaken on the
solid-phase, as required during the preparation of PNA oligomers.
The preparation of such PNA oligomers is currently under way in
our laboratories and will be published in due course.
Financial support from the International Max Planck Research
School for Chemical Biology (fellowship to M.P.), the Alexander
von Humboldt Foundation (fellowship to G.G.), the Research
Department Interfacial Systems Chemistry at Ruhr-University
Bochum and the DFG (Research Unit “Biological Function of
Organometallic Compounds”, FOR 630, www.rub.de/for630) are
all gratefully acknowledged. G.G. thanks Prof. Roger Alberto
for generous access to all facilities at the Institute of Inorganic
Chemistry of the University of Zurich.
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Notes and references
19 J. M. Casas-Solvas, E. Ortiz-Salmero´n, J. J. Gime´nez-Mart´ınez, L.
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Vargas-Berenguel, Chem.–Eur. J., 2009, 15, 710–725.
20 H. W. P. N’Dongo, I. Neundorf, K. Merz and U. Schatzschneider,
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§ 6b: C18H15F6N3O3PRe, M = 652.51, light brown prisms, orthorhombic,
space group Pnma, T = 223 K, a = 7.852(4), b = 10.081(5), c =
26.274(13) A, V = 2079(2) A , Z = 4, 2qmax = 50.00◦, 10532 measured
reflections, 1922 independent reflections, 160 parameters, m = 5.999 mm-1,
R1 = 0.0477 for 1693 observed reflections (I > 2s(I)), wR2 = 0.1275 for
all reflections, CCDC 755697.
3
˚
˚
21 G. M. Sheldrick, in SHELXL-97, University of Go¨ttingen, Germany,
1997.
6c: C42H35BN3O3Re, M = 826.75, light brown needles, triclinic, space
22 P. Van der Sluis and A. L. Spek, Acta Crystallogr., Sect. A: Found.
¯
˚
group P1, T = 223 K, a = 9.135(4), b = 12.789(6), c = 19.230(9) A,
Crystallogr., 1990, 46, 194.
This journal is
The Royal Society of Chemistry 2010
Dalton Trans., 2010, 39, 5617–5619 | 5619
©