Fluorescent rhenium-naphthalimide conjugates as cellular imaging agents

Article abstract of DOI:10.1021/ic500142z

A range of biologically compatible, fluorescent rhenium-naphthalimide conjugates, based upon the rhenium fac-tricarbonyl core, has been synthesized. The fluorescent ligands are based upon a N-functionalized, 4-amino-derived 1,8-naphthalimide core and incorporate a dipicolyl amine binding unit to chelate Re(I); the structural variations accord to the nature of the alkylated imide with ethyl ester glycine (L¹), 3-propanol (L²), diethylene glycol (L³), and benzyl alcohol (L⁴) variants. The species are fluorescent in the visible region between 505 and 537 nm through a naphthalimide-localized intramolecular charge transfer, with corresponding fluorescent lifetimes of up to 9.8 ns. The ligands and complexes were investigated for their potential as imaging agents for human osteoarthritic cells and protistan fish parasite Spironucleus vortens using confocal fluorescence microscopy. The results show that the specific nature of the naphthalimide structure serves to control the uptake and intracellular localization of these imaging agents. Significant differences were noted between the free ligands and complexes, with the Re(I) complex of L² showing hydrogenosomal localization in S. vortens.

Full text of DOI:10.1021/ic500142z

Article
pubs.acs.org/IC
Fluorescent Rhenium-Naphthalimide Conjugates as Cellular Imaging
Agents
Emily E. Langdon-Jones,† Nadine O. Symonds,† Sara E. Yates,† Anthony J. Hayes,‡ David Lloyd,‡
Rebecca Williams,‡ Simon J. Coles,§ Peter N. Horton,§ and Simon J.A. Pope*,†
†School of Chemistry, Main Building, Cardiff University, Cardiff CF10 3AT, Cymru/Wales, U.K.
‡School of Biosciences, Main Building, Cardiff University, Cardiff CF10 3AT, Cymru/Wales, U.K.
§National Crystallographic Service, Chemistry, Faculty of Natural and Environmental Sciences, University of Southampton, Highfield,
Southampton, SO17 1BJ, England, U.K.
S
* Supporting Information
ABSTRACT: A range of biologically compatible, fluorescent
rhenium-naphthalimide conjugates, based upon the rhenium fac-
tricarbonyl core, has been synthesized. The fluorescent ligands
are based upon a N-functionalized, 4-amino-derived 1,8-
naphthalimide core and incorporate a dipicolyl amine binding
unit to chelate Re(I); the structural variations accord to the
nature of the alkylated imide with ethyl ester glycine (L1), 3-
propanol (L2), diethylene glycol (L3), and benzyl alcohol (L4)
variants. The species are fluorescent in the visible region between
505 and 537 nm through a naphthalimide-localized intra-
molecular charge transfer, with corresponding fluorescent
lifetimes of up to 9.8 ns. The ligands and complexes were
investigated for their potential as imaging agents for human
osteoarthritic cells and protistan fish parasite Spironucleus vortens using confocal fluorescence microscopy. The results show that
the specific nature of the naphthalimide structure serves to control the uptake and intracellular localization of these imaging
agents. Significant differences were noted between the free ligands and complexes, with the Re(I) complex of L2 showing
hydrogenosomal localization in S. vortens.
Luminescent organometallic coordination complexes are
now well established as viable options for bioimaging
applications using confocal fluorescence microscopy.4 Re(I),5
Ir(III),6 and Pt(II)7 complexes in particular have shown
remarkable capability in cellular imaging, with intracellular
localization patterns that can be rationalized, and organelle
targeting. The added value of a single molecule agent that can
simultaneously image and deliver therapeutic activity is
significant as such an approach can provide an insight into
uptake, distribution, and therapeutic action.8 For example,
coupling anthraquinone fluorophores to Pt(II) complexes has
provided models for the investigation of compound distribution
in tumors: the anthraquinone fluorophore allows fluorescence
microscopy to track the distribution of the complex in cell
culture spheroids.9 Cytotoxic Au(I) complexes have also been
functionalized with alkynyl-adorned anthraquinone units and
are shown to be very effective cellular imaging agents with
modulated antiproliferative activity against a range of cancer
cell lines.10
INTRODUCTION
■
The use of metallo-radionuclides in positron emission
tomography (PET) and single photon emission computer
tomography (SPECT) is well established, with the metastable
isotope 99mTc (t1/2 = 6.01 h, γ = 142.7 keV) a particularly
popular choice for the latter. Importantly rhenium also
possesses radionuclides 186/188Re (186Re t1/2 = 3.68 d, β =
1.07 MeV, γ = 137 keV; 188Re t1/2 = 16.98 h, β = 2.12 MeV, γ =
155 keV), which have promise in radiopharmaceutical
therapies.1 For the application of these radionuclides in
synthesis, fac-[M(CO)3(H2O)3]+ (M = Tc, Re) is a very
convenient precursor for a range of M(I) coordination
chemistry and agent production.2 It is well-known that rhenium
is the closest chemical analogue for 99mTc, and the use of
nonradioactive fluorescent Re(I) analogues can obviously help
to provide valuable insight with respect to in vivo and in vitro
localization characteristics of 99mTc(I) agents, which are
particularly important at the cellular level.3 The challenge of
such an approach is the appropriate design of a biocompatible
and kinetically inert complex functionalized with a suitable
fluorophore that can be exploited in confocal fluorescence
microscopy.
Received: January 21, 2014
Published: March 13, 2014
© 2014 American Chemical Society
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Article
using a trypsin−ethylenediaminetetraacetic acid (EDTA) solution and
suspended in an excess volume of growth medium. The homogeneous
cell suspension was then distributed into 1 mL aliquots, with each
aliquot being subject to incubation with a different imaging probe.
These luminescent probes were initially dissolved in dimethyl sulfoxide
(DMSO) (5 mg/mL) before being added to the cell suspensions, with
a final concentration of 100 μg ml−1 (corresponding to about 110−190
μM) before incubation at 20 °C for 30 min. Cells were finally washed
in phosphate buffer saline (PBS, pH 7.2), removing agent from the
medium, then harvested by centrifugation (5 min, 800 g) and mounted
on a slide for imaging. Preparations were viewed using a Leica TCS
SP2 AOBS confocal laser microscope using ×63 or ×100 objective,
with excitation at 405, 488, or 543 nm and detection at 515−600 nm.
General. 1H and 13C{1H} NMR spectra were recorded on an
NMR-Fourier transform (FT) Bruker 400 and 250 MHz or Joel
Eclipse 300 MHz spectrometer and recorded in CDCl3, CD3CN,
Here we report the use of functionalized naphthalimide-
rhenium metal complex fluorophores as excellent agents for cell
imaging. The ease with which the substituted 1,8-naphthalimide
unit can be functionalized in a stepwise manner should allow
exquisite control over the physical properties of the
chromophore, including the luminescence characteristics.11
Critically, the optical properties can be tuned to the visible
part of the spectrum by increasing the degree of charge transfer
(CT) character in the excited state. Thus, amino-substituted
1,8-naphthalimide fluorophores absorb and emit in the visible
region due to N-to-imide CT.12 The CT character also
increases the Stokes shift of the fluorophore, which is
advantageous in removing autofluorescence from endogenous
fluorophores. The examples of the application of such species
to confocal fluorescence microscopy and cellular imaging are
rapidly expanding as the broad utility of these fluorophores is
realized.13 A recent comprehensive review by Gunnlaugsson
and Kelly has highlighted the breadth of applicability of 1,8-
naphthalimide species, including as anticancer agents, DNA
binders, and cellular imaging agents.14 Recent reports have
shown that the utility in a biological context is expanding with a
number of groups pursuing sensing platforms15 based upon the
1,8-naphthalimide motif: Zn(II)16 and Cu(I)17 imaging agents
have been reported using this fluorophore core, albeit relying
upon fluorescence intensity change as the prime indicator of
sensing.
A small number of naphthalimide-metal complex species
have also been reported, often focused upon the development
of cancer chemotherapeutic agents. Examples with Pt(II)18 and
Au(I)19 are known, as well as a number of luminescent Ru(II)
species that target DNA binding.20 Some Ru(II) arene
complexes appended with a 1,8-naphthalimide fluorophore
also possess very good anticancer activity in drug-resistant
cancer cells.21
In this Paper we discuss the development of some new
dipicolyl amine ligands, which are appended with a N-
substituted 1,8-naphthalimide fluorophore, together with their
corresponding organometallic Re(I) complexes. These four
new complexes are highly fluorescent, kinetically inert, and
biologically compatible and have been successfully applied for
cellular imaging using confocal fluorescence microscopy.
1
CD3OD, or (CD3)2CO. H and 13C{1H} NMR chemical shifts (δ)
were determined relative to residual solvent peaks with digital locking
and are given in ppm. Low-resolution mass spectra were obtained by
the staff at Cardiff University. High-resolution mass spectra were
carried out at the EPSRC National Mass Spectrometry Service at
Swansea University. UV−visible (UV−vis) studies were performed on
a Jasco V-570 spectrophotometer as MeCN and MeOH solutions (2.5
or 5 × 10−5 M). Photophysical data were obtained on a JobinYvon−
Horiba Fluorolog spectrometer fitted with a JY TBX picosecond
photodetection module as MeCN solutions. Emission spectra were
uncorrected, and excitation spectra were instrument-corrected. The
pulsed source was a Nano-LED configured for 295 nm output
operating at 1 MHz. Luminescence lifetime profiles were obtained
using the JobinYvon−Horiba FluoroHub single photon counting
module, and the data fits yielded the lifetime values using the provided
DAS6 deconvolution software. Quantum yield measurements were
obtained on aerated MeCN solutions of the complexes using
[Ru(bpy)3](PF6)2 in aerated MeCN as a standard (Φ = 0.016).25
All reactions were performed with the use of vacuum line and
Schlenk techniques. Reagents were commercial grade and used
26
without further purification. fac-[Re(CO)3(MeCN)3]BF4 and N-
tert-Boc-ethylenediamine27 were prepared according to the literature.
Synthesis of Compound Cl1.28 Triethylamine (1 mL, 7.16 mmol)
and 4-chloro-1,8-naphthalic anhydride (1.110 g, 4.77 mmol) were
added to a solution of glycine ethyl ester hydrochloride (0.999 g, 7.16
mmol) in ethanol (50 mL). The orange solution was heated at reflux
under a dinitrogen atmosphere for 12 h, resulting in the formation of a
precipitate. The reaction solution was then cooled, and the solvent was
removed in vacuo. The solids were then extracted into dichloro-
methane and washed with 0.1 M HCl (3 × 20 mL) and water (3 × 20
mL). The organic phase was then dried over MgSO4, filtered, and
reduced to a minimal volume. Precipitation with diethyl ether and
subsequent filtration and drying afforded Cl1 as an orange solid. Yield:
0.931 g, 2.93 mmol, 61%. 1H NMR (400 MHz, CDCl3): δH = 8.43 (d,
1H, 3JHH = 7.0 Hz, nap), 8.33 (d, 1H, 3JHH = 8.5 Hz, nap), 8.28 (d, 1H,
3JHH = 8.0 Hz, nap), 7.64 (dd, 2H, JHH = 8.0 Hz, 7.5 Hz, nap), 4.81 (s,
2H, NCH2), 4.17 (q, 2H, 3JHH = 7.0 Hz, OCH2CH3), 1.22 (t, 3H, 3JHH
= 7.0 Hz, OCH2CH3) ppm.
EXPERIMENTAL SECTION
■
Diffraction Data Collection and Processing. Diffraction data
for L1 were collected using a standard method22 on a Rigaku AFC12
goniometer equipped with an enhanced sensitivity (HG) Saturn724+
detector mounted at the window of an FR-E+ SuperBright
molybdenum rotating anode generator with HF Varimax optics (100
μm focus) at 100 K. Data collection, data reduction, cell refinement,
and absorption correction using CrystalClear-SM Expert 3.1 b26
(Rigaku, 2013). CCDC reference number 981267 contains the
supplementary crystallographic data (see Supporting Information)
for this Paper.
Synthesis of Compound N1.29 Compound Cl1 (0.630 g, 1.98
mmol) and N-tert-Boc-ethylenediamine (0.953 g, 5.95 mmol) were
heated in dimethylsulfoxide (3 mL) at 70 °C under a nitrogen
atmosphere for 12 h. The solution was then allowed to cool and was
neutralized with 0.1 M HCl, which induced precipitation of a yellow
solid. The crude product was extracted into dichloromethane, washed
with water, dried over MgSO4, and filtered. The solvent was reduced
to a minimal volume, and precipitation was induced with petroleum
ether, allowing subsequent filtration and drying to afford the
intermediate product as an orange solid. Yield: 0.521 g, 1.18 mmol,
Structure Analysis and Refinement. The structure was solved
by direct methods using SHELXS-9723 and was completed by iterative
cycles of ΔF syntheses and full-matrix least-squares refinement in
SHELKX-2013.23 All non-H atoms were refined anisotropically, and
difference Fourier syntheses were employed in positioning idealized
hydrogen atoms, which were allowed to ride on their parent C-atoms.
The figures was created using the ORTEP3 for Windows.24
Human Cell Incubation and Confocal Microscopy. An
osteoporetic cell line was maintained in N-(2-hydroxyethyl)-
piperazine-N′-ethanesulfonic acid (Hepes)-modified minimum essen-
tial medium (HMEM) supplemented with 10% fetal bovine serum,
penicillin, and streptomycin. Cells were detached from the plastic flask
1
3
60%. H NMR (400 MHz, CDCl3): δH = 8.38 (d, 1H, JHH = 8.0 Hz,
3
3
nap), 8.29 (d, 1H, JHH = 8.0 Hz, nap), 8.17 (d, 1H, JHH = 8.5 Hz,
nap), 7.42 (t, 1H, 3JHH = 8.0 Hz, NH), 6.95 (s, 1H, nap), 6.44 (d, 1H,
3JHH = 8.0 Hz, nap), 5.30 (t, 1H, 3JHH = 7.5 Hz, CONH), 4.87 (s, 2H,
3
NCH2), 4.20 (q, 2H, JHH = 7.0 Hz, COCH2CH3), 3.53 (broad app.
quin, 2H, NHCH2), 3.36 (broad t, 2H, CH2CH2), 1.42 (s, 9H, Bu),
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Inorganic Chemistry
Article
3
1.25 (t, 3H, JHH = 7.0 Hz, OCH2CH3) ppm. 13C{1H} NMR (75
Hz, NCH2), 4.20−4.10 (broad m, 2H, NCH2), 4.07 (q, 2H, 3JHH = 8.5
Hz, OCH2CH3), 1.13 (t, 3H, 3JHH = 8.5 Hz, OCH2CH3) ppm. LRMS
(ES+) found m/z 794.2 for [C33H29N5O7Re]+. HRMS (ES+) found m/
z 792.1587; calculated 792.1591 for [C33H29N5O7Re]+. UV−vis
(MeCN): λmax (ε/M−1 cm−1) 421 (12 800), 338 (2300), 321
(4100), 277 (26 300), 255 (23 700), 228 (24 600) nm. UV−vis
(MeOH): λmax (ε/M−1 cm−1) 427 (13 100), 339 (2500), 322 (4400),
277 (25 900), 255 (24 200), 228 (24 100), 203 (55 900) nm. IR
(solid) ν(CO) = 2031, 1930, 1903, 1676, 1639, 1587 cm−1.
MHz, CDCl3): δC = 169.4 (CO), 164.3 (CO), 163.7 (CO), 157.9,
150.8, 135.0, 131.3, 127.6, 124.6, 122.1, 120.3, 108.7, 103.5, 99.6, 80.4,
61.7, 46.0, 41.3, 39.6, 31.0, 14.3 ppm. Low-resolution mass
spectrometry (LRMS) electrospray ionization (ES−) found: m/z =
440.2 for [M-H]− and 476.2 for [M+Cl]−.
Trifluoroacetic acid (TFA) (2 mL) was then added dropwise to a
solution of the Boc-protected intermediate (0.495 g, 1.12 mmol) in
dichloromethane (5 mL) under a nitrogen atmosphere and stirred for
24 h. The solvents were removed under vacuum, with further drying
over 30 min. The residue was dissolved in methanol (20 mL) and then
dried in vacuo, a process that was repeated in triplicate, to finally afford
N1 as a hygroscopic yellow solid. 1H NMR (400 MHz, CD3OD): δH =
Synthesis of Compound Cl2. Prepared as for compound Cl1 but
using 3-aminopropanol (0.48 mL, 6.13 mmol) and 4-chloro-1,8-
naphthalic anhydride (0.713 g, 3.06 mmol) to give Cl2 as an orange
1
solid. Yield: 0.662 g, 2.29 mmol, 75%. H NMR (250 MHz, CDCl3):
3
3
3
3
δH = 8.61 (d, 1H, JHH = 7.5 Hz, nap), 8.56 (d, 1H, JHH = 8.5 Hz,
8.40 (d, 1H, JHH = 8.0 Hz, nap), 8.36 (d, 1H, JHH = 7.0 Hz, nap),
nap), 8.45 (d, 1H, 3JHH = 8.0 Hz, nap), 7.83−7.76 (m, 2H, nap), 4.27
(t, 2H, JHH = 6.0 Hz, CH2OH), 3.53 (broad t, 2H, NCH2CH2), 3.04
(broad s, 1H, OH), 1.92 (app. quin, 2H, JHH = 6.0 Hz, NCH2CH2)
8.23 (d, 1H, 3JHH = 8.0 Hz, nap), 7.57 (app. t, 1H, 3JHH = 7.5 Hz, nap),
3
3
6.76 (d, 1H, JHH = 8.0 Hz, nap), 4.83 (s, 2H, NCH2), 4.26 (q, 2H,
3
3JHH = 7.0 Hz, OCH2CH3), 3.76 (t, 2H, JHH = 5.5 Hz, NHCH2),
3
3
ppm.
3.30−3.37 (m, 4H, NHCH2CH2), 1.32 (t, 3H, JHH = 7.0 Hz,
Synthesis of Compound N2. Prepared as for compound N1 but
using Cl2 (0.510 g, 1.76 mmol) and N-tert-Boc-ethylenediamine
(0.841 g, 5.25 mmol), giving the intermediate as an orange oil. Yield:
0.649 g, 1.57 mmol, 89%. 1H NMR (400 MHz, CDCl3): δH = 8.35 (d,
1H, 3JHH = 7.0 Hz, nap), 8.18 (d, 1H, 3JHH = 8.5 Hz, nap), 8.14 (d, 1H,
OCH2CH3) ppm. 13C{1H} NMR (75 MHz, CD3OD): δC = 169.3
(CO), 164.0 (CO), 163.5 (CO) 150.3, 134.2, 130.9, 129.2, 128.0,
124.4, 121.1, 120.3, 108.6, 103.8, 61.4, 40.8, 40.1, 38.2, 13.1 ppm.
LRMS (ES+) found m/z 342.2 for [M+H]+ and 383.2 for [M
+MeCN]+.
3
3JHH = 8.5 Hz), 7.43 (app. t, 1H, JHH = 7.5 Hz, nap), 7.15 (broad s,
Synthesis of Compound L1. Compound N1 (0.176 g, 0.51 mmol)
was added to a solution of 2-pyridinecarboxaldehyde (0.1 mL, 1.05
mmol) in 1,2-dichloroethane (15 mL) and stirred for 2 h at room
temperature under a nitrogen atmosphere. Sodium triacetoxyborohy-
dride (0.327 g, 1.54 mmol) was then added, and the mixture was
stirred for a further 16 h. The solution was then neutralized with
saturated aqueous (aq) NaHCO3, and the product was extracted using
CHCl3. The organic layer was washed with water (3 × 25 mL) and
brine (2 × 25 mL) and then dried over MgSO4. Following filtration,
the solvent was removed in vacuo to afford L1 as a yellow solid. Yield:
0.133 g, 2.55 mmol, 50%. 1H NMR (400 MHz, CDCl3): δH = 8.79 (d,
1H, 3JHH = 8.5 Hz, nap), 8.56 (d, 1H, 3JHH = 7.0 Hz, nap), 8.51 (d, 2H,
3
1H, NH), 6.38 (d, 1H, JHH = 8.5 Hz, nap), 5.97 (broad s, 1H,
3
NHCO), 4.21 (t, 2H, JHH = 6.0 Hz, CH2OH), 3.93 (broad s, 1H,
OH), 3.58−3.52 (broad m, 2H, naph-NHCH2), 3.52 (t, 2H, 3JHH = 5.5
Hz, NCH2), 3.37 (app. broad s, 2H, CH2NHCO), 1.91 (app. quin, 2H,
3JHH = 6.0 Hz, CH2CH2OH), 1.42 (s, 9H, Bu) ppm.
Deprotection of the intermediate with TFA in dichloromethane
yielded N2 as a hygroscopic yellow solid. 1H NMR (400 MHz,
CD3OD): δH = 8.52 (app. t, 2H, 3JHH = 8.0 Hz, nap), 8.38 (d, 1H, 3JHH
= 8.5 Hz, nap), 7.69 (app t, 1H, 3JHH = 7.5 Hz, nap), 6.89 (d, 1H, 3JHH
= 8.5 Hz, nap), 4.48 (t, 2H, 3JHH = 6.0 Hz, NHCH2), 4.28 (t, 2H, 3JHH
3
= 7.0 Hz, CH2OH), 3.81 (t, 2H, JHH = 6.5 Hz, NCH2), 3.36 (t, 2H,
3JHH = 4.5 Hz, py), 8.35 (d, 1H, JHH = 8.5 Hz, nap), 7.93 (br t, 1H,
3
3
3JHH = 6.5 Hz, CH2NH2), 2.20 (app. quin, 2H, JHH = 6.5 Hz,
NH), 7.63 (app. t, 1H, 3JHH = 8.0 Hz), 7.50 (app. t, 2H, 3JHH = 8.0 Hz,
CH2CH2OH) ppm.
3
3
Synthesis of Compound L2. Prepared as for compound L1 but
using 2-pyridinecarboxaldehyde (0.39 mL, 4.05 mmol), compound N2
(0.605 g, 1.94 mmol), and sodium triacetoxyborohydride (1.23 g, 5.82
mmol), yielding a crude product. The product was extracted into 0.1
M HCl, washed with dichloromethane (20 mL), neutralized, and then
re-extracted into dichloromethane, which was removed under vacuum
to give L2 as an orange oil. Yield: 366 mg, 0.74 mmol, 38%. 1H NMR
(400 MHz, CDCl3): δH = 8.73 (d, 1H, 3JHH = 8.5 Hz), 8.48−8.44 (m,
3H, nap, py), 8.23 (d, 1H, 3JHH = 8.5 Hz, nap), 7.92 (s, 1H, NH), 7.57
(app. t, 1H, 3JHH = 7.5 Hz, nap), 7.45 (app. t, 2H, 3JHH = 7.5 Hz, py),
py), 7.31 (d, 2H, JHH = 8.0 Hz, py), 7.09 (app. t, 2H, JHH = 5.5 Hz,
py), 6.47 (d, 1H, JHH = 8.5 Hz, nap), 4.87 (s, 2H, NCH2), 4.17 (q,
3
3
2H, JHH = 7.0 Hz, OCH2CH3), 3.95 (s, 4H, NCH2), 3.38−3.30
3
(broad m, 2H, HNCH2), 3.00 (t, 2H, JHH = 5.0 Hz, HNCH2CH2),
3
1.22 (t, 3H, JHH = 7.0 Hz, OCH2CH3) ppm. 13C{1H} NMR (75
MHz, CDCl3): δC = 199.3 (CO), 164.7 (CO), 163.8 (CO), 150.9,
149.3, 139.5, 136.8, 135.4, 131.5, 130.5, 128.2, 124.4, 123.5, 122.5,
117.3, 108.0, 104.1, 63.9, 61.4, 60.1, 59.8, 41.3, 41.0, 14.3 ppm. LRMS
(ES+) found m/z 524.5 for [M+H]+, 546.5 for [M+Na]+ and 587.6 for
[M+MeCN+Na]+. (High-resolution mass spectroscopy (HRMS)
(ES+) found m/z 524.2285; calculated 524.2292 for [C30H30O4N5]+.
UV−vis (MeCN): λmax (ε/M−1 cm−1) = 439 (13 500), 340 (1100),
324 (1600), 282 (16 000), 269 (15 100), 256 (18 000), 228 (21 500)
nm; UV−vis (MeOH): λmax (ε/M−1cm−1) = 445 (13 800), 338 (700),
324 (1600), 282 (18 200), 268 (18 600), 257 (20 700), 228 (16 000),
205 (40 000) nm. IR (solid) νmax = 3302, 2987, 2899, 2848, 1737,
1685, 1641, 1586, 1568, 1549, 1471, 1427, 1400, 1371, 1321, 1302,
1236, 1209, 1136, 1112, 1010, 995, 952, 772, 752 cm−1.
3
3
7.26 (d, 2H, JHH = 8.0 Hz, py), 7.03 (app. t, 2H, JHH = 5.5 Hz, py),
6.37 (d, 1H, 3JHH = 8.5 Hz, nap), 4.20 (t, 2H, 3JHH = 5.5 Hz, CH2OH),
3.88 (s, 4H, CH2), 3.47 (t, 2H, JHH = 5.5 Hz), 3.30−3.25 (broad m,
3
2H, CH2), 2.92 (t, 2H, 3JHH = 5.0 Hz, CH2), 1.86 (app. quin, 2H, 3JHH
= 5.5 Hz, CH2CH2OH) ppm. 13C{1H} NMR (75 MHz, CDCl3): δC =
165.3 (CO), 164.7 (CO), 159.5, 158.7, 150.8, 149.1, 149.0, 136.7,
135.1, 131.3, 128.1, 124.3, 123.4, 122.4, 122.3, 120.7, 108.3, 104.0,
59.6, 58.9, 51.0, 41.0, 36.5, 31.1 ppm. LRMS (ES+) found m/z 495.2
for [M]+. HRMS (ES+) found m/z = 496.2335 for [C29H29N5O3 +
H]+, calculated 496.2343 for [C29H29N5O3 + H]+. UV−vis (MeCN):
λmax (ε/M−1 cm−1) 437 (13 100), 339 (700), 324 (1300), 281 (17
200), 260 (22 500), 228 (18 200) nm. UV−vis (MeOH): λmax (ε/M−1
cm−1) = 431 (12 900), 339 (1000), 323 (1400), 277 (13 100), 257
(21 600), 226 (16 000), 203 (46 800) nm. IR (solid) νmax = 3267 (br),
2852, 1680, 1641, 1584, 1549, 1476, 1429, 1395, 1331, 1259, 1242,
1140, 1115, 1049, 995, 922, 772, 750 cm−1.
Synthesis of fac-[Re(CO)3(L1)]BF4 (Re-L1). Compound L1 (57 mg,
0.11 mmol) and fac-[Re(CO)3(MeCN)3]BF4 (50 mg, 0.10 mmol)
were dissolved in CHCl3 (10 mL) and heated at reflux under a
nitrogen atmosphere for 18 h. The reaction mixture was allowed to
cool, the solvent was reduced to 1−2 mL, and precipitation was
induced with the addition of diethyl ether. The product was collected
by filtration and washed with diethyl ether, giving the complex as a
1
yellow solid. Yield: 16.7 mg, 19.0 μmol, 18%. H NMR (400 MHz,
(CD3)2CO): δH = 8.86 (d, 2H, 3JHH = 8.0 Hz, py), 8.52 (d, 1H, 3JHH
=
Synthesis of fac-[Re(CO)3(L2)]BF4 (Re-L2). Prepared as for fac-
[Re(CO)3(L1)]BF4 but using compound L2 (53.6 mg, 0.11 mmol)
and fac-[Re(CO)3(MeCN)3]BF4 (48.0 mg, 0.10 mmol) to give Re-L2
as a yellow solid. Yield: 61.9 mg, 72.6 μmol, 73%. 1H NMR (400 MHz,
CD3CN): δH = 8.82 (d, 2H, 3JHH = 5.5 Hz, py), 8.53 (d, 1H, 3JHH = 7.5
Hz, nap), 8.42 (app. d, 2H, 3JHH = 8.5 Hz, nap), 7.92 (app. t, 2H, 3JHH
= 8.0 Hz, py), 7.73 (app. t, 1H, 3JHH = 7.5 Hz, nap), 7.49 (d, 2H, 3JHH
8.0 Hz, nap), 8.40 (d, 1H, 3JHH = 8.0 Hz, nap), 8.28 (d, 1H, 3JHH = 7.5
Hz, nap), 7.95−7.85 (m, 2H, nap, py), 7.60 (app. t, 1H, 3JHH = 8.5 Hz,
nap), 7.56 (d, 2H, 3JHH = 8.0 Hz, py), 7.36 (app. t, 2H, 3JHH = 8.0 Hz,
3
3
py), 7.19 (br t, 1H, JHH = 4.5 Hz, NH), 7.02 (d, 1H, JHH = 8.0 Hz,
nap), 5.36 (d, 2H, JHH = 16.5 Hz, 1/2 × 2CH2), 5.18 (d, 2H, 2JHH
16.5 Hz, 1/2 × 2CH2), 4.71 (s, 2H, NCH2CO), 4.40 (t, 2H, 3JHH = 8.0
=
2
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= 8.0 Hz, py), 7.34 (app. t, 2H, 3JHH = 6.5 Hz, py), 6.95 (d, 1H, 3JHH
=
1H, 3JHH = 8.0 Hz, nap), 7.14 (app. t, 2H, 3JHH = 6.0 Hz, py), 6.78 (br
3
8.5 Hz, nap), 6.37 (br t, 1H, NH), 4.99 (d, 2H, 2JHH = 16.5 Hz, 1/2 ×
t, 1H, JHH = 8.5 Hz, NH), 6.67 (d, 1H, 3JHH = 8.0 Hz, nap), 5.53 (d,
2CH2), 4.90 (d, 2H, 2JHH = 16.5 Hz, 1/2 × 2CH2), 4.25 (t, 2H, 3JHH
=
2H, 2JHH = 16.5 Hz, 1/2 × 2CH2), 4.58 (d, 2H, 2JHH = 16.5 Hz, 1/2 ×
3
7.5 Hz, CH2), 4.19 (t, 2H, JHH = 5.5 Hz, CH2), 4.01 (app. q, 2H,
CH2), 3.55 (t, 2H, 3JHH = 6.0 Hz), 1.87 (app. quin, 2H, 3JHH = 6.0 Hz,
CH2CH2OH) ppm. LRMS (ES+) found m/z 766.1 for [M]+. HRMS
(ES+) found m/z 764.1639; calculated 764.1642 for
[C32H29N5O6Re]+. UV−vis (MeCN): λmax (ε/M−1 cm−1) = 421 (13
800), 338 (4400), 323 (6100), 272 (35 600), 258 (35 600); UV−vis
(MeOH): λmax (ε/M−1 cm−1) 427 (13 000), 337 (3300), 324 (4800),
272 (29 700), 258 (30 900), 227 (26 200), 203 (59 200) nm. IR
(solid) ν(CO) = 2025, 1921, 1898, 1690, 1629 cm−1.
2CH2), 4.34 (t, 2H, 3JHH = 5.0 Hz, NCH2), 4.18 (t, 2H, 3JHH = 5.0 Hz,
3
CH2), 4.10−4.02 (m, 2H, CH2), 3.83 (t, 2H, JHH = 5.5 Hz, NCH2),
3.70−3.48 (m, 4H, OCH2). LRMS (ES+) found m/z 796.4 for [M]+.
HRMS (ES+) found m/z 794.1741; calculated 794.1748 for
[C33H31N5O7Re]+. UV−vis (MeCN): λmax (ε/M−1 cm−1) = 421 (12
800), 338 (2300), 321 (4100), 277 (26 300), 255 (23 700), 228 (24
600) nm. UV−vis (MeOH): λmax (ε/M−1 cm−1) = 427 (15 200), 338
(5000), 323 (6600), 277 (28 000), 258 (29 600), 227 (28 000), 203
(62 300) nm. IR (solid) ν(CO) = 2031, 1910 (br), 1690, 1643 cm−1.
Synthesis of Compound Cl4. Prepared as for compound Cl1, but
using 4-chloro-1,8-naphthalic anhydride (0.144 g, 0.62 mmol), 3-
aminobenzyl alcohol (0.304 g, 2.47 mmol), and triethylamine (0.14
mL, 0.926 mmol) to afford Cl4 as a yellow solid. Yield: 132 mg, 0.39
Synthesis of Compound Cl3. Prepared as for compound Cl1, using
2-(2-aminoethyl)ethanol (0.80 mL, 8.04 mmol) and 4-chloro-1,8-
naphthalic anhydride (0.935 g, 4.02 mmol) to give Cl3 as an orange
1
solid. Yield: 1.065 g, 3.33 mmol, 83%. H NMR (250 MHz, CDCl3):
3
3
1
3
δH = 8.53 (d, 1H, JHH = 8.5 Hz, nap), 8.45 (d, 1H, JHH = 9.5 Hz,
mmol, 63%. H NMR (250 MHz, CDCl3): δH = 8.69 (d, 1H, JHH
=
nap), 8.37 (d, 1H, 3JHH = 8.0 Hz, nap), 7.72 (app. t, 1H, 3JHH = 7.5 Hz,
7.5 Hz, nap), 8.60 (d, 1H, 3JHH = 7.5 Hz, nap), 8.44 (d, 1H, 3JHH = 8.0
Hz, nap), 7.90−7.76 (m, 2H, Ph), 7.51−7.35 (m, 2H, nap, Ph), 7.26
(s, 1H, Ph), 7.20−7.08 (d, 1H, nap), 4.68 (s, 2H, CH2OH) ppm.
Synthesis of Compound N4. Prepared as for compound N1, but
using Cl4 (0.619 g, 1.83 mmol) and N-tert-Boc-ethylenediamine
(0.881 g, 5.50 mmol). The intermediate was isolated as a sticky orange
3
3
nap), 7.69 (d, 1H, JH3H = 8.0 Hz, nap), 4.34 (t, 2H, JHH = 5.5 Hz,
NCH2), 3.77 (t, 2H, JHH = 5.5 Hz, CH2OH), 3.62−3.55 (m, 4H,
CH2OCH2), 2.56 (s, 1H, OH) ppm.
Synthesis of Compound N3. Prepared as for compound N1, but
using Cl3 (0.401 g, 1.25 mmol) and N-tert-Boc-ethylenediamine
(0.601 g, 3.75 mmol), affording the intermediate as a solid. Yield:
0.492 g, 1.11 mmol, 89%. 1H NMR (400 MHz, CDCl3): δH = 8.00 (d,
1H, 3JHH = 8.0 Hz, nap), 7.90 (d, 1H, 3JHH = 7.0 Hz, nap), 7.84 (d, 1H,
1
solid. Yield: 0.810 g, 1.76 mmol, 96%. H NMR (250 MHz, CDCl3):
3
3
δH = 8.59 (d, 1H, JHH = 7.0 Hz, nap), 8.46 (d, 1H, JHH = 8.5 Hz,
nap), 8.33 (d, 1H, 3JHH = 8.5 Hz, nap), 7.61 (app. t, 1H, 3JHH = 8.0 Hz,
nap), 7.52−7.40 (m, 2H, Ph), 7.35 (s, 1H, Ph), 7.28 (d, 1H, 3JHH = 8.0
Hz, Ph), 7.22 (br t, 1H, NH), 6.59 (d, 1H, 3JHH = 8.5 Hz, nap), 5.23 (t,
1H, 3JHH = 6.5 Hz, NHCO), 4.77 (s, 2H, CH2OH), 3.73−3.63 (br m,
2H, NHCH2), 3.50−3.46 (br m, 2H, CH2NHCO), 2.27 (br s, 1H,
OH), 1.51 (s, 9H, Bu) ppm.
3JHH = 8.0 Hz), 7.12 (app. t, 1H, JHH = 7.0 Hz, NH), 6.79 (s, 1H,
3
nap), 6.24 (app. s, 1H, NHCO), 6.11 (d, 1H, 3JHH = 8.0 Hz, nap), 4.20
(app. br s, 2H, CH2), 3.73 (br t, 2H, CH2), 3.66 (app. br s, 2H, CH2),
3.59 (app. br s, 2H, CH2), 3.42 (app. br s, 2H, CH2), 3.19 (app. br s,
2H, CH2), 1.36 (s, 9H, Bu) ppm.
Deprotection of the intermediate with TFA, followed by work up
Deprotection of the intermediate with TFA, followed by work up
yielded N2 as a hygroscopic yellow oil. 1H NMR (400 MHz, CD3OD):
yielded N3 as a hygroscopic orange solid. 1H NMR (400 MHz,
3
3
3
CD3OD): δH = 8.51 (d, 1H, 3JHH = 8.0 Hz, nap), 8.45 (d, 1H, JHH
8.0 Hz, nap), 8.26 (d, 1H, 3JHH = 8.5 Hz, nap), 7.59 (app. t, 1H, 3JHH
8.0 Hz, nap), 6.80 (d, 1H, 3JHH = 8.5 Hz, nap), 4.47 (app. t, 2H, 3JHH
=
=
=
δH = 8.27 (d, 1H, JHH = 8.5 Hz, nap), 8.18 (d, 1H, JHH = 8.0 Hz,
3
nap), 8.09 (d, 1H, JHH = 8.5 Hz, nap), 7.48−7.23 (m, 3H, nap, Ph),
7.17 (s, 1H, Ph), 7.05 (d, 1H, 3JHH = 7.0 Hz, Ph), 6.60 (d, 1H, 3JHH
=
3
9.0 Hz, nap), 4.55 (s, 2H, CH2OH), 3.62 (t, 2H, JHH = 6.0 Hz,
4.5 Hz, CH2), 4.32−4.28 (m, 4H, CH2), 3.84−3.77 (m, 4H, CH2),
3
3.36 (t, 2H, JHH = 7.5 Hz, CH2) ppm. 13C{1H} NMR (75 MHz,
CH2CH2), 3.19 (br s, 2H) ppm.
Synthesis of Compound L4. Prepared as for compound L1 but
using 2-pyridinecarboxaldehyde (0.32 mL, 3.35 mmol), compound N4
(0.590 g, 1.63 mmol), and sodium triacetoxyborohydride (1.13 g, 4.85
mmol) to give crude L4 as an orange oil. The product was purified via
column chromatography, first eluting with dichloromethane and then
CD3OD): δC = 164.7 (CO), 164.2 (CO), 150.2 (CO), 134.2, 130.9,
127.9, 124.5, 122.0, 120.6, 109.4, 108.0, 104.1, 103.9, 84.1, 72.1, 67.9,
60.9, 39.8, 38.2, 37.6 ppm. LRMS (ES+) found m/z 344.3 for [M+H]+,
366.3 for [M+Na]+ and 407.3 for [M+MeCN+Na]+.
Synthesis of Compound L3. Prepared as for compound L1, but
using 2-pyridinecarboxaldehyde (70 μL, 0.76 mmol), compound N3
(0.128 g, 0.37 mmol), and sodium triacetoxyborohydride (0.237 g,
1.12 mmol) to give L3 as an orange solid. Yield: 35.6 mg, 67.5 μmol,
collecting the product by eluting with 3% MeOH in dichloromethane.
1
Yield: 149 mg, 0.74 mmol, 17%. H NMR (400 MHz, CDCl3): δH
=
3
3
8.77 (d, 1H, JHH = 8.5 Hz, nap), 8.53 (d, 1H, JHH = 7.0 Hz, nap),
8.46 (d, 2H, 3JHH = 4.5 Hz, py), 8.30 (d, 1H, 3JHH = 8.5 Hz, nap), 7.89
3
18%. 1H NMR (400 MHz, CDCl3): δH = 8.77 (d, 1H, JHH = 8.5 Hz,
3
3
3
(br s, 1H, NH), 7.61 (app. t, 1H, JHH = 8.0 Hz, nap), 7.50−7.31 (m,
nap), 8.56 (d, 1H, JHH = 6.5 Hz, nap), 8.51 (d, 2H, JHH = 4.0 Hz,
py), 8.34 (d, 1H, 3JHH = 8.5 Hz, nap), 7.88 (s, 1H, NH), 7.62 (app. t,
1H, 3JHH = 8.0 Hz, nap), 7.50 (app. t, 2H, 3JHH = 8.5 Hz, py), 7.31 (d,
3
4H, nap, Ph, py), 7.29 (d, 2H, JHH = 8.0 Hz, py), 7.25 (s, 1H, Ph),
7.15 (d, 1H, 3JHH = 7.5 Hz, nap), 7.05 (app. t, 2H, 3JHH = 6.5 Hz, py),
3
3
3
6.44 (d, 1H, JHH = 8.5 Hz, nap), 4.66 (s, 2H, HOCH2), 3.89 (s, 4H,
NCH2), 3.40−3.25 (broad m, 2H, CH2), 2.93 (t, 2H, JHH = 5.0 Hz,
2H, JHH = 8.0 Hz, py), 7.09 (app. t, 2H, JHH = 6.0 Hz, py), 6.47 (d,
1H, JHH = 8.5 Hz, nap), 4.37 (t, 2H, JHH = 5.5 Hz, NCH2), 3.95 (s,
3
3
3
CH2), 2.61 (broad s, 1H, OH) ppm. 13C{1H} NMR (75 MHz,
CDCl3): δC = 165.2, 164.4, 158.8, 150.9, 149.2, 142.8, 136.8, 135.3,
131.5, 129.4, 128.3, 127.9, 127.4, 126.9, 124.4, 123.5, 122.9, 122.5,
121.0, 108.7, 104.1, 64.7, 59.8, 51.2, 41.0 ppm. LRMS (ES+) found m/
z 526.2 for [M+H]+ and 548.2 for [M+Na]+. HRMS (ES+) found m/z
= 544.2338; calculated 544.2343 for [C33H29N5O3 + H]+. UV−vis
(MeCN): λmax (ε/M−1 cm−1) 436 (14 900), 341 (1000), 325 (1700),
282 (18 100), 265 (19 500), 226 (22 200) nm. UV−vis (MeOH): λmax
(ε/M−1 cm−1) 445 (14 200), 339 (900), 323 (2000), 282 (16 200),
268 (17 800), 261 (18 000), 227 (20 300), 203 (61 500) nm. IR
(solid) νmax = 3260 (br), 2830, 1688, 1641, 1576, 1539, 1433, 1358,
1260, 1240, 1146, 1003, 766, 754 cm−1.
3
4H, NCH2), 3.79 (t, 2H, JHH = 5.5 Hz), 3.65−3.61 (m, 4H, CH2),
3
3.40−3.30 (broad m, 2H, CH2), 2.99 (t, 2H, JHH = 5.5 Hz, CH2)
ppm. 13C{1H} NMR (75 MHz, CDCl3): δC = 187.6, 167.6 (CO),
164.2 (CO), 158.8, 151.5, 149.3, 143.9, 139.8, 137.3, 136.8, 135.7,
128.1, 123.4, 122.5, 107.5, 104.1, 81.3, 59.8, 23.0, 21.9, 20.5, 17.4, 13.8,
5.9 ppm. LRMS (ES+) found m/z 526.2 for [M+H]+ and 548.2 for [M
+Na]+. HRMS (ES+) found m/z 560.2053; calculated 560.2047 for
[C30H31O4N5 + Cl]. UV−vis (MeCN): λmax (ε/M−1 cm−1) 437 (14
400), 340 (1000), 324 (1500), 280 (20 800), 260 (24 400), 228 (18
700) nm. IR (solid) νmax = 3208 (br), 2893, 2864, 1684, 1641, 1589,
1556, 1476, 1429, 1368, 1327, 1304, 1234, 1117, 1051, 1003, 885, 772,
756 cm−1.
Synthesis of fac-[Re(CO)3(L3)]BF4 (Re-L3). Prepared as for fac-
[Re(CO)3(L1)]BF4, but using compound L3 (16.8 mg, 32.0 μmol) and
fac-[Re(CO)3(MeCN)3]BF4 (14.6 mg, 30.4 μmol) to give a yellow
Synthesis of fac-[Re(CO)3(L4)]BF4 (Re-L4). Prepared as for [Re-
(CO)3(L1)]BF4, but using compound L4 (30.2 mg, 55.6 μmol) and
[Re(CO)3(MeCN)3]BF4 (25.9 mg, 54.0 μmol) to give a yellow solid.
Yield: 33.4 mg, 37.1 μmol, 69%. 1H NMR (400 MHz, CD3CN): δH =
8.82 (d, 2H, 3JHH = 5.5 Hz, py), 8.56 (d, 1H, 3JHH = 8.0 Hz, nap), 8.49
1
solid. Yield: 15.5 mg, 17.6 μmol, 58%. H NMR (400 MHz, CDCl3):
3
δH = 8.59 (d, 2H, JHH = 5.5 Hz, py), 8.36−8.30 (m, 3H, nap, py),
3
3
7.79−7.69 (m, 2H, nap), 7.66 (d, 2H, 3JHH = 8.0 Hz, py), 7.49 (app. t,
(d, 1H, JHH = 8.5 Hz, nap), 8.45 (d, 1H, JHH = 8.0 Hz, nap), 7.92
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(app. t, 2H, 3JHH = 8.0 Hz, py), 7.78 (app. t, 1H, 3JHH = 7.5 Hz, nap),
7.55−7.40 (m, 4H, nap, Ph), 7.38−7.32 (m, 3H, py, Ph), 7.23 (d, 1H,
3JHH = 8.0 Hz, Ph), 7.01 (d, 1H, 3JHH = 8.5 Hz, nap), 6.42 (t, 1H, 3JHH
2
= 8.0 Hz, NH), 4.98 (d, 2H, JHH = 17.0 Hz, 1/2 × 2CH2), 4.90 (d,
2H, 2JHH = 17.0 Hz, 1/2 × 2CH2), 4.68 (s, 2H), 4.26 (t, 2H, 3JHH = 6.0
Hz, CH2), 4.10−4.02 (m, 2H, CH2) ppm. LRMS (ES+) found: m/z
766.1 for [M]+. HRMS (ES+) found m/z 812.1642; calculated
812.1642 for [C36H29O6N5Re]+. UV−vis (MeCN): λmax (ε/M−1 cm−1)
420 (15 200), 338 (3200), 321 (4800), 312 (4600), 276 (11 100), 256
(13 400), 226 (33 500) nm. UV−vis (MeOH): λmax (ε/M−1 cm−1)
427 (17 900), 336 (4100), 322 (6100), 270 (32 600), 205 (81 400)
nm. IR (solid) ν(CO) = 2029, 1906, 1682, 1640 cm−1.
RESULTS AND DISCUSSION
■
Synthesis and Characterization. The ligands (L1−4) were
isolated in four steps (Scheme 1) from commercially available
a
Scheme 1. Synthetic Route to the Ligands and Complexes
Figure 1. X-ray crystal structure of L1 with ellipsoids at 50%
occupancy.
desired rhenium complexes in the form of fac-[Re(CO)3(Ln)]-
(BF4) as yellow air-stable powders. Again the complexes were
characterized using a range of appropriate techniques. IR
spectroscopy confirmed the facial geometry (pseudo Cs) of the
complexes via three CO stretching frequencies at 2030−1890
cm−1, as well as the carbonyls of the coordinated ligand. HRMS
produced the correct isotopic pattern expected for 185/187Re. In
1
the H NMR spectra both the methylene and ethylene linkers
of the dipicolyl amine receptor unit shift from the
corresponding free ligand. Among the aliphatic proton signals,
two sets of characteristic resonances were assigned to the
diastereotopic protons of the dipicolyl amine methylene linkers,
possessing a coupling constant of ca. 16−18 Hz consistent with
a 2JHH geminal coupling.31 Remarkably, the separation between
the two diastereotopic resonances was strongly dependent
upon the naphthalimide structure, with L3 inducing the largest
separation. The observation of diastereotopic protons provides
categorical evidence for the proposed binding mode, rendering
coordinative participation of the naphthalimide nitrogen donor
unlikely.
The solution absorption spectra of L1−4 (data recorded in
Table 1) were composed of at least two dominant absorptions:
the pyridyl units absorb at <290 nm, while the naphthalimide
chromophore absorbs at 330−450 nm. The lowest energy
absorption appeared as a broad, unstructured band at ca. 437
nm (ε ≈ 104 M−1 cm−1). Comparison with the unsubstituted
precursors (Cl1−4) indicated that the addition of the amine
substituent clearly induced a significant bathochromic shift of
the lowest energy absorption band, which was also found to be
sensitive to solvent polarity. The solubility of L3 enabled
additional measurements to be conducted on aqueous
solutions, with the visible band further shifted to 449 nm.
These observations are consistent with a significant intraligand
charge transfer (ICT) (N-to-π*) contribution to the transition
arising from the donating amino substituent and the accepting
naphthalimide unit.
a
(i) RNH2, EtOH; (ii) Boc-NH(CH2)2NH2, DMSO; (iii) TFA,
dichloromethane; (iv) 2-pyridinecarboxaldehyde, NaBH(OAc)3, 1,2-
dichloroethane; (v) fac-[Re(CO)3(MeCN)3](BF4), CHCl3.
4-chloro-1,8-naphthalic anhydride. First, conversion to the
chloro-substituted naphthalimide species (Cl1−4) was easily
achieved by heating the appropriate amine in ethanol.
Subsequent reaction of this intermediate with N-tert-Boc-
ethylenediamine in DMSO, followed by TFA-mediated
deprotection, yielded the amino-derived pro-ligand, N1−4. A
one-pot reductive amination procedure using 2-pyridinecarbox-
aldehyde gave the desired dipicolylamine binding site.30 The
four ligands, incorporating glycine ethyl ester (L1), propyl
alcohol (L2), ethylene glycol (L3), and benzyl alcohol (L4),
were fully characterized via NMR spectroscopy and MS studies.
In addition to the solution-state characterization, a single-
crystal structural determination (Figure 1) was obtained for L1.
The data collection parameters (Table S1) are presented in the
Supporting Information. The data shows the anticipated
structure.
Solutions of L1−4 were also found to be fluorescent (Table
1). Using an excitation wavelength of 345−425 nm on aerated
MeCN and MeOH solutions of the ligands revealed a visible
emission band at ca. 520 nm, which was largely insensitive to
the nature of imide functionalization (Scheme 1). Again, the
emission of L3 was also obtained in water, fluorescing at 537
nm. The moderate Stokes shift and sensitivity of λem to solvent
The four ligands were each reacted with fac-[Re-
(CO)3(MeCN)3](BF4) in refluxing chloroform to yield the
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Table 1. Absorption and Fluorescence Properties of the Ligands and Complexes
a
b
c
d
e
f
g
structure
λmax/nm (ε/M−1cm−1
)
λmax/nm (ε/M−1cm−1
)
λem/nm
λem/nm
τ
τ
Φ
L1
439 (13 500)
437 (13 100)
437 (14 400)
436 (14 900)
421 (12 800)
421 (13 800)
421 (12 800)
420 (15 200)
445 (13 800)
431 (12 900)
442 (13 900)
445 (14 200)
427 (13 100)
427 (13 000)
427 (15 200)
523
529
523
524
505
510
505
506
534
537
530
533
518
514
516
520
2.6, 7.6 (97%)
2.6, 6.4 (89%)
3.7, 8.2 (90%)
4.5, 8.5 (93%)
3.1, 9.8 (98%)
3.2, 9.7 (96%)
4.1, 9.8 (95%)
2.8, 8.7 (83%)
1.7, 5.4 (92%)
2.9, 7.1 (80%)
1.8, 5.1 (90%)
3.8, 8.8 (98%)
3.3, 8.8 (95%)
2.2, 8.4 (97%)
L2
L3
L4
Re-L1
Re-L2
Re-L3
Re-L4
0.83
0.57
0.58
0.89
427 (17 900)
9.1
8.1
a
b
c
d
e
f
g
10−5 M MeCN. 10−5 M MeOH. MeCN, λex = 425 nm. MeOH, λex = 425 nm. MeCN, λex = 372 nm. MeOH, λex = 372 nm. Aerated MeCN.
Figure 2. (left) UV−vis absorption for L2 (black) and Re-L2 (gray) (recorded in MeCN). (right) Normalized excitation and emission spectra for L4
and Re-L4 (recorded in MeCN).
Figure 3. Confocal fluorescence microscopy of osteoarthritic cells imaged using (left to right) L1 (poor uptake, indicative of limiting permeability),
L2 (excellent uptake, with mitochondrial localization), and L3 (good uptake, diffusely distributed with cytoplasmic binding). All images obtained
using λex = 405 nm and λem = 535 nm.
polarity again suggests an excited state of ICT character. Time-
resolved measurements showed that in the majority of cases the
decay profiles fitted best to a biexponential yielding two lifetime
components all of which were indicative of fluorescence (<10
ns). The dual-component decay suggests the presence of a
minor, quenched species, since in all cases the longer
component dominates (>80%).
Crucially, upon coordination to Re(I) each of the ligands
retained these characteristic absorption and emission properties
(see example spectra in Figure 2). For the complexes the
absorption maximum was slightly blue-shifted to around 427
nm and thus was highly compatible with confocal fluorescence
microscopy. Relative to the free ligands, hypsochromic shifts
were noted for the corresponding emission wavelengths, and
fluorescence lifetimes were generally extended for the
complexes (Table 1). This shift may be a consequence of the
overall cationic charge of the complex modulating the stability
of the emitting ICT excited state.32,33 The quantum yields for
the complexes were uniformly excellent (>50%) in aerated
solution, suggesting that potential quenching pathways are
suppressed. The occupancy of the dipicolylamine receptor
probably inhibits photoinduced electron transfer, which can
account for significant quenching in amino-substituted
naphthalimide chromophores, as well as enhancing the rigidity
of the complex and limiting vibrational deactivating pathways.
Confocal Fluorescence Microscopy. Confocal fluores-
cence microscopy, used to study the fluorescence imaging
capability of the free ligands (L1−4) and corresponding
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Figure 4. Confocal fluorescence microscopy of osteoarthritic cells imaged using (left to right) Re-L1, Re-L2, and Re-L4 (λex = 405 nm; λem = 515
nm).
Figure 5. Microscopy images of human osteoarthritic cell undergoing apoptosis. (left) Imaged using fluorescence from Re-L2 (λex = 405 nm; λem
=
515 nm). (right) Imaged using trasmitted light.
complexes (Re-L1−4), was initially performed using human
osteoarthritic cells. In all cases an excitation wavelength of 405
or 488 nm was used together with a detection wavelength
between 515 and 535 nm (see Supporting Information, Figures
S2 and S3 for autofluorescence assessments).
L3 surprisingly revealed the poorest uptake into the cells and
was not an effective imaging fluorophore. However, the benzyl
alcohol-appended Re-L4 showed very good uptake into the cells
and bright emission, in stark contrast to L4, but the images
show that Re-L4 does not cross the nuclear membrane. Re-L1
also showed better uptake than the corresponding free ligand,
with bright emission and clear evidence of cytoplasmic
membrane staining together with the selective staining of
other organelles (possibly mitochondria and vacuoles). As with
L2, the propanol-derived complex Re-L2 showed the most
impressive uptake, yielding very bright, high-quality images.
Although the complex did not detectably cross the nuclear
membrane, it revealed labeling of smooth endoplasmic
membranes, including those of the Golgi apparatus.
Comparison of the free ligands showed some remarkable
differences in behavior (Figure 3). First, L4, which incorporates
the benzyl alcohol group, did not show any uptake into the
cells. L1 showed a reasonable uptake and a distinct granular
staining pattern, possibly indicative of mitochondrial local-
ization; no uptake in the nucleus was observed. The
functionalized ligands L2 and L3 both revealed good uptake;
however, no specific foci of fluorescence were noted for L3.
This behavior contrasts with cell imaging work conducted with
HepG2 (liver cancer) and HeLa cells,16b where L3 has been
shown to penetrate the nuclear envelope, which may be
characteristic of cells that are dying or dead.34 The present
studies clearly show that this behavior is not replicated with the
osteoarthritic cell line. In comparison, L2 showed very distinct
localization patterns with high-intensity fluorescence from
mitochondrial regions. It is noteworthy that recent studies on
Cu(I)-targeted detection using 4,5-disubstituted 1,8-naphthali-
mide probes demonstrated trans-Golgi and mitochondrial
localization.17
The successful identification and application of Re-L2 as a
promising cell imaging agent led to additional studies
investigating higher concentrations (4 times greater) of agent,
yielding remarkably detailed images of osteoarthritic cells
(Figure 5). The images provide clear evidence for rapid binding
to mitochondrial membranes and other ultrastructural elements
in cells undergoing apoptosis, as indicated by the extensive
plasma membrane blebbing (zeiosis), cytoplasmic vacuolation,
loss of cellular integrity, and, in some cases, nuclear
fragmentation.
Once incorporated into the architecture of the cationic
rhenium complex, the variation in structures again showed
vastly different imaging capability across the series (Figure 4).
Despite the imaging utility of the corresponding free ligand, Re-
Given the quality of its imaging capability, Re-L2 was also
selected for imaging the anaerobically grown aerotolerant
protistan fish parasite Spironucleus vortens, where it again
showed good uptake (Figure 6) and localization in the
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Figure 6. Microscope images of S. vortens incubated with Re-L2. (left) Normal transmission. (right) Confocal fluorescence (λex = 405 nm; λem = 515
nm).
hydrogenosomes (redox-active organelles equivalent to the
mitochondria of aerobic eukaryotic organisms). Re-L2 was not
evidently toxic to these organisms over the duration of the
entire experiment (4 h), as its rapid flagellar-driven motility and
forward propulsive swimming was maintained across the entire
population. Consequently, to obtain sharply focused images, it
was necessary to anaesthetize using 280 mM 2,2,2-trichloro-
ethane-1,1-diol (chloral hydrate) immediately before micro-
scopical examination. In addition, colocalization experiments
using Re-L2 and tetramethylrhodamine ethyl ester (TMRE)
clearly showed (Figure 7) that both agents colocalize in the
hydrogenosomes (secondarily derived mitochondria) of the
flagellated S. vortens.
the imide moiety can be used to tune the imaging capability of
the probes, from poor uptake (L4) to good uptake/poor
localization (L3), to excellent uptake and distinct granular hot
spots of fluorescence (L2). With the exception of Re-L3, the
cationic nature of the complexes appears to enhance cell uptake
in all cases. Taken together the imaging results clearly show
that simple tuning of the amphiphilic nature of the N-alkyl
imide group can fundamentally alter both the uptake and
localization patterns of the probes: the 3-propanol moiety
appears to offer the best balance, facilitating excellent imaging
capability, especially in the guise of the cationic complex Re-L2.
In conclusion, this Paper has described the multistep
synthetic development of the 1,8-naphthalimide fluorophore,
generating four ligands allowing the formation of biocompatible
cationic tricarbonyl rhenium complexes, where the metal ion is
added at the final step. The structures vary by the amphiphilic
nature of the N-alkylation of the imide compartment of the
ligand structure. The optical properties are dictated by ligand-
centered, intramolecular charge transfer dominated transitions
that facilitate visible absorption and efficient emission character-
istics, which are highly compatible with confocal fluorescence
microscopy. Cellular imaging studies were conducted on both
human osteoarthritic cells and S. vortens and showed the
excellent imaging capabilities of both the ligands and
complexes. Critically, the specific cellular uptake and local-
ization characteristics can be controlled by the structure of the
ligand. For the series of compounds studied here, the 3-
propanol moiety of L2 offers the most favorable attributes
regarding uptake and mitochondrial localization. Extensive
cellular imaging studies with emissive lanthanide complexes
have also shown that the nature of a fluorophore substituent
can influence their intracellular uptake, distributions, and
cytotoxicity, perhaps through varied affinities for proteins.35
Our future studies will investigate the biological impact of
further structural variations on the naphthalimide fluorophore.
To summarize, these naphthalimide-derived ligands provide a
chelating site that is ideally suited to radioimaging nuclides such
as 99mTc(I), while simultaneously offering the desirable physical
characteristics that are biocompatible with optical cellular
imaging techniques such as confocal fluorescence microscopy.
These imaging results present clear evidence of the
biocompatible utility of these amine-substituted naphthalimide
fluorophores. Importantly, it appears that the specific nature of
Figure 7. Microscope images of S. vortens incubated with Re-L2 and
TMRE showing (clockwise from top left): green fluorescence from Re-
ASSOCIATED CONTENT
■
S
L2 (λex = 488 nm; λem = 515 nm); red fluorescence from TMRE (λex
=
* Supporting Information
Parameters associated with the single-crystal diffraction data
collection, selected bond lengths and angles, and additional
543 nm; λem = 600 nm); overlaid images showing colocalization;
transmission image.
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confocal fluorescence microscopy imaging data. This material is
free of charge via the Internet at http://pubs.acs.org.
Supplementary crystallographic data are also available. These
data can be obtained free of charge from the Cambridge
Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_
request/cif.
AUTHOR INFORMATION
Corresponding Author
*E-mail: popesj@cardiff.ac.uk.
■
Funding
We thank Cardiff University for financial support and the staff
of the EPSRC Mass Spectrometry National Service (University
of Swansea) for providing MS data. We thank the EPSRC for
the use of the National Crystallographic Service at the
University of Southampton.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
■
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