Simple polyphenyl zirconium and hafnium metallocene room-temperature lumophores for cell imaging

Article abstract of DOI:10.1021/om400212y

Zirconium and hafnium metallocene dihalides based on simple polyphenyl cyclopentadienes are stable room-temperature lumophores with large Stokes shifts, emitting from states with substantial LMCT character. Their synthesis is described, including by a rapid solvent-free approach compatible with the lifetime of PET-active isotopes (⁸⁹Zr). Preliminary experiments indicate the viability of these species as lumophores for fluorescence cell-imaging microscopy.

Full text of DOI:10.1021/om400212y

Note
pubs.acs.org/Organometallics
Simple Polyphenyl Zirconium and Hafnium Metallocene Room-
Temperature Lumophores for Cell Imaging
Victoria E. Pritchard,† Flora L. Thorp-Greenwood,† Rebeca G. Balasingham,† Catrin F. Williams,‡
Benson M. Kariuki,† James A. Platts,*,† Andrew J. Hallett,† and Michael P. Coogan*,§
†School of Chemistry, Cardiff University, Cardiff CF10 3AT, U.K.
‡School of Bioscience, Cardiff University, Cardiff CF10 3AX, U.K.
§Department of Chemistry, University of Lancaster, Lancashire LA1 4YB, U.K.
S
* Supporting Information
ABSTRACT: Zirconium and hafnium metallocene dihalides
based on simple polyphenyl cyclopentadienes are stable room-
temperature lumophores with large Stokes shifts, emitting from
states with substantial LMCT character. Their synthesis is
described, including by a rapid solvent-free approach compatible
with the lifetime of PET-active isotopes (89Zr). Preliminary
experiments indicate the viability of these species as lumophores
for fluorescence cell-imaging microscopy.
dichlorides,23−28 and there is a long-lived positron emitting
ate transition metals coupled with ligands based around
Laromatic heterocycles and other conjugated species form isotope of zirconium, 89Zr (t1/2 = 78 h),29−31 suggesting
an important class of photoactive complexes,1 which have
potential for a bimodal PET/fluorescence imaging agent with
integrated therapeutic ability.32
found a vast range of applications, including photocatalysis,2
light-emitting devices,3 photovoltaics,4 and sensing and
imaging.5 Energy losses through recombination of charge-
separated states can be reduced via triplet electronic
configurations, which are prevented from relaxing to the
ground (singlet) state by spin selection rules6 and can
themselves be accessed by spin−orbit coupling with heavy
second- and third-row transition metals (particularly Ru, Ir, Re,
and Pt).7−10 Almost all applications reported to date involve
these late transition metals in low oxidation states (0 → 3+)
with ligands which can easily be reduced, often in MLCT (or
related) processes.11,12 In contrast, early transition metals in
high oxidation states often absorb light through LMCT
processes, but these are rarely emissive.13−17 However, certain
metallocene derivatives of the heavier group IV metals emit
The synthesis of the ansa-metallocene 122 involves post-
coordination modification of ligands, undesirable with radio-
istopes. Noting that highly substituted cyclopentadienes may
also retard vibrational quenching and thus allow room-
temperature emission, following this initial seminal report, we
have now explored a series of penta- and tetraphenylcyclo-
pentadienyl Zr and Hf complexes; both are readily available and
easily substituted, and their metallocene halides are water-
stable, although their syntheses often require long reaction
times.33,34
Reaction of lithiated tetraphenylcyclopentadiene with
MCl4(THF)2 (M = Zr/Hf) gave the bis(cyclopentadienyl)
metallocene dichlorides (Scheme 1), and samples appropriate
for X-ray crystallography35 were grown of both (Ph4Cp)2ZrCl2
(2) and (Ph4Cp)2HfCl2 (3) (Figure 1; see the Supporting
Information for more details).36
18−21
3
from LMCT states at low temperatures,
and while these
species are underexplored, recently room-temperature emission
was reported from a rigid Zr−ansa-metallocene dichloride (1;
Scheme 1).22 This ground-breaking report from Loukova et al.
demonstrated the potential for early-transition-metal lumo-
phores to compete with the better known late-TM species due
to its remarkable photophysical properties, with room-temper-
ature quantum yields up to 0.41 and room-temperature
lifetimes of up to 17 μs. In addition to their potential for
photophysical applications such as luminescent imaging, there
are reports of cytotoxicity in group IV metallocene
The UV−vis spectra of 2 and 3 showed ligand-based (π−π*)
transitions between 250 and 350 nm, shifted in comparison to
those of the free ligand, and a broad lower-energy band
assigned19,22 as LMCT (2, 404 nm; 3, 364 nm; see the
1
Supporting Information) extending into the visible region in
each case. Steady-state excitation and emission spectra
Received: March 14, 2013
© XXXX American Chemical Society
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Organometallics
Note
Scheme 1. Structure of 1 and Synthesis of 2 and 3
Figure 1. Crystal structures of 2 and 3.
confirmed the desired room-temperature luminescence, with
excitation spectra matching the UV−vis spectra, demonstrating
that the Zr species 2 is better excited at long wavelengths than
the Hf analogue 3 (λmax(excitation) 334 and 269 nm,
respectively) (Figure 2). The emission spectra were very
similar, with a single broad peak centered at 462 and 445 nm (a
Stokes shift of over 100 nm). Time-resolved studies showed
that both 2 and 3 had lifetimes too low to be accurately
measured, although the lifetimes extended to 5−10 ns in
degassed solutions, the extension of lifetime being typical of
triplet lumophores due to their sensitivity to quenching by 3O2.
3O2 is the archetypal quencher for triplet charge transfer states
3
of heavy-metal complexes which have been widely used as O2
sensors as a result of this behavior.37 A full description of the
procedures for the time-resolved spectroscopy is given in the
Supporting Information. Given that the explanation for the
long luminescence lifetime of 1 is related to its rigidity, while
less rigid simple species such as Cp2ZrCl2 are nonemissive
unless cooled to reduce vibration, it seems likely that complexes
2 and 3 have long intrinsic lifetimes but suffer from vibrational
quenching, which suggests that ligand substitution may offer
other simple species with long lifetimes. The quantum yields of
these complexes, while low in comparison to organic
fluorophores, are moderate to good by comparison with
many metallo or organometallic lumophores. The zirconium
species 2 has a quantum yield (room-temperature aerated
solution) of 0.5% and the hafnium analogue 3 of 1.2%, even
higher than for the archetypal [Ru(bpy)3] dication.
Tuning photophysics is vital to many applications, and so
mixed-ligand systems were examined to see whether only bis-
polyphenyl species were emissive and, if not, what the effect of
substituting for a simpler ligand would be. Reaction of CpZrCl3
with 1 equiv of lithiated tetraphenylcyclopentadiene gave 4
(Figure 3) in good yield, although this species shows ligand
Figure 2. Photophysical behavior of 2 and 3 (room temperature,
MeCN).
B
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Note
Figure 3. Structure of 4 and UV−vis spectrum.
Figure 4. Spin density in the triplet state (left) and difference in
density between singlet and triplet states (red denotes more density in
the triplet state).
redistribution at high temperatures to give 2 and Cp2ZrCl2 and
must be handled with care (Scheme S1, Supporting
Information). Interestingly, not only did 4 retain useful
luminescence (Figure S2, Supporting Information) but also it
showed the most intense absorption at low energy, with the
LMCT shoulder extending well into the visible region with
reasonable absorption up to 450 nm (Figure 3). Initially it was
assumed that the emission would emanate from the substituted
Cp ring, so that the effect of the other ligands would be low,
and therefore in order to understand why substitution of one of
the tetraphenyl-Cp rings with an unsubstituted Cp should have
such a marked effect on the photophysics, computational
studies were undertaken. As charge transfer species present
problems for some DFT methods and Zr and Hf are not
represented in standard basis sets, the computational method-
ology was chosen to reflect these systems (Hybrid DFT with
effective core potential (ECP)/basis set) and full details are
included in the Supporting Information.
For the simplest model complex Cp2ZrCl2, the lowest excited
triplet state in simulated CH3CN was calculated by TD-DFT to
be above the ground singlet state at the triplet geometry by 451
nm, which is in excellent agreement with the low-temperature
emission reported in the solid state (22120 cm−1, 452 nm)19
and frozen toluene solution (21800 cm−1, 459 nm), a pure
HOMO−LUMO transition.19 Interestingly, however, the
nature of the ground state was less clear than formal oxidation
states may suggest, with the HOMO mainly located on Cp π
orbitals, but with significant contribution from chlorides and
metal d orbitals (Figure S3, Supporting Information).
Assignment of the transition as having predominantly LMCT
nature is supported by calculated natural bond orbital (NBO)
charges: on Zr these are +0.81 and +0.50 e in the singlet and
triplet states, respectively (both at triplet optimum geometry);
each Cp has a charge of −0.11 (singlet) and +0.13 (triplet), and
chloride charges are −0.30 (singlet) and −0.38 (triplet). Thus,
Zr accepts electron density when the complex is excited to the
emissive state, meaning that emission involves transfer of
density back from the metal to the ligand, which as an emissive
process is the direction of charge transfer associated with
LMCT (Figure 4). However, as suggested from orbital plots,
chloride is also involved in this transition, increasing its negative
charge in the triplet state over the singlet state: i.e., charge
transfer to the ligand. The NBO electron configuration suggests
a more complex picture: the description of Zr in the singlet
ground state [core] 4d2.265p0.596s0.25 does not indicate formal
zero d orbital population in the ground state. In the triplet state,
the NBO configuration of [core] 4d2.625p0.546s0.12 indicates not
only charge transfer from Cp into Zr d orbitals but also reduced
population of valence s and p orbitals. (Analogous data for
Cp2HfCl2 (3) are given in Table S2 (Supporting Information)).
Similar calculations on 2 and 4 were then performed using
the same methodology (summarized in Table S1, Supporting
Information) and indicate that the HOMO (CpPh4 based) is
noticeably higher in energy than in Cp2ZrCl2, whereas LUMO
(metal-based) energies are barely affected. In free [CpPh4]−,
conjugation lowers the HOMO, whereas in the metal
complexes steric crowding apparently prevents efficient overlap
between Cp π and metal d orbitals, increasing the HOMO
energy (Figure S4, Supporting Information). Calculations on 4
indicate that emission maxima with λ 430−460 nm are pure
HOMO−LUMO transitions, and excitation singlet states were
found at 359 and 339 nm but not the low-energy shoulder.
However, an excited triplet state was calculated to lie at 435 nm
3
relative to the ground state, representing a rare direct LMCT
absorption, which is formally forbidden but occasionally
observed in heavy-metal systems which show strong spin−
orbit coupling.38−41 A spin−orbit coupled TD-DFT calculation
using the zeroth-order regular approximation (ZORA)
approach found a triplet absorption with low but nonzero
intensity (f = 1.84 × 10−5 au) at 439 nm, as well as a more
intense singlet band (0.120 au) at 386 nm, both purely
HOMO−LUMO transitions, that match the experimental
spectra well. Triplet excitations are not observed or calculated
for 2 or 3, demonstrating a remarkable richness in the
electronic transitions available in these complexes.
As a very preliminary example of applications of these
lumophores, cell imaging studies in MCF-7 human adenocarci-
noma cells were undertaken with 4, as this complex has the best
3
absorption at 405 nm, exploiting the LMCT excitation, and
imaging studies with metal complexes are now gaining in
importance.42,43 The cell sample showed good uptake (Figure
5A), even though 4 is neutral (many metal complexes show
good uptake only as cations),11 across most of the sample
(Figure 5B) and a healthy morphology, indicating low toxicity.
4 shows localization in a range of perinuclear organelles (Figure
5C) (but has not been shown to be specific by colocalization),
with little in the nucleus or plasma membranes.
In summary, Zr and Hf complexes of the general formula
Cp2MCl2, in which at least one of the Cp rings bears aromatic
substituents, offer a family of easily accessible room-temper-
ature lumophores which emit from triplet excited states, are air-
and water-stable, and offer a new concept in metal-based cell
C
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ASSOCIATED CONTENT
* Supporting Information
Figures, tables, text, and CIF files giving structural parameters
relating to the crystallography, additional photophysical data,
and computational details. This material is available free of
charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*J.A.P.: tel, +44 (0)2920874950; e-mail, platts@cardiff.ac.uk.
M.P.C.: tel, +44 (0)1524 592342; e-mail, m.coogan@lancs.ac.
uk.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the EPSRC National Mass Spectrometry Service,
Swansea, U.K., and Mr. R. Higgs (Cardiff University) for
invaluable assistance in obtaining mass ions on 2−4 and Drs.
Simon Coles and Claire Wilson of the EPSRC National
Crystallography Service, Southampton, U.K., for the data
collection of 2.
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