Fluorescent functionalised naphthalimides and their Au(i)-NHC complexes for potential use in cellular bioimaging

Article abstract of DOI:10.1039/c8dt04069a

A series of cationic, dihydroimidazolinium-functionalized 1,8-naphthalimide fluorophores have been isolated as their hexafluorophosphate salts, [HL]PF₆. These pro-ligands react with [AuCl(tht)] in the presence of base to form N-heterocyclic car

Full text of DOI:10.1039/c8dt04069a

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Fluorescent functionalised naphthalimides and
their Au(I)–NHC complexes for potential use in
cellular bioimaging†‡
Cite this: DOI: 10.1039/c8dt04069a
Lara M. Groves,a Catrin F. Williams,b Anthony J. Hayes,c Benjamin D. Ward,a
Marc D. Isaacs,c Nadine O. Symonds,a David Lloyd,c Peter N. Horton,d
a
Simon J. Coles d and Simon J. A. Pope
*
A series of cationic, dihydroimidazolinium-functionalized 1,8-naphthalimide fluorophores have been iso-
lated as their hexafluorophosphate salts, [HL]PF6. These pro-ligands react with [AuCl(tht)] in the presence
of base to form N-heterocyclic carbene (NHC) complexes, [AuCl(L)]. Two X-ray structures represent a
pro-ligand and complex pairing: the latter reveals the two-coordinate linear geometry of the NHC–Au(I)
species, as well as intermolecular interactions supported by both ligand π–π stacking and a weak auro-
philic interaction of 3.3205(6) Å. The luminescence properties of the pro-ligands and complexes are
dominated by the ICT character of the substituted fluorophore at ca. 500 nm, which is further modulated
via functionalization at the 4-position of the naphthalimide. Cytotoxicity assessments were performed for
all [HL]PF6 and [AuCl(L)] species against LOVO, MCF-7, A549 and PC3 cell lines; added lipophilicity seems
to correlate with increased cytotoxicity. Confocal fluorescence microscopy was undertaken on a selected
[HL]PF6 and [AuCl(L)] species and showed that the intracellular distribution is dependent upon the
specific ligand structure. More detailed co-localisation studies show that selected examples present a
predominant lysosomal staining pattern. FLIM studies exemplified the applicability of these probes, and
secondly suggest that fluorescence lifetime could be used to provide information on the integrity of the
complex and thus liberation of gold in a biological environment.
Received 10th October 2018,
Accepted 21st November 2018
DOI: 10.1039/c8dt04069a
rsc.li/dalton
and sodium aurothioglucose, all of which have been applied to
the treatment of inflammatory autoimmune conditions such
Introduction
Although the use of gold in medicinal chemistry (known as as rheumatoid arthritis.3 Auranofin is also under investigation
chrysotherapy)1 has been recognised for millennia, recent as a treatment to reduce the viral reservoir of HIV that lies
developments have renewed interest in gold’s use in metallo- latent in the body’s T-cells.4 The mechanism of biological
drugs. The variety of Au(I) complexes used in a clinical setting action of such clinical treatments remains extremely difficult
are those incorporating thiolate or phosphine ligands.2 The to unravel.5 Over the last two decades, there have been several
most widely utilised are auranofin, sodium aurothiomalate reports suggesting that a variety of Au(I) complexes possess
antiproliferative properties in vitro against selected human
tumour cell lines.6
aSchool of Chemistry, Main Building, Cardiff University, Cardiff CF10 3AT, UK.
One must consider that the gold agents used clinically are
E-mail: popesj@cardiff.ac.uk; Fax: +44 (0)29-20874030; Tel: +44 (0)29-20879316
bSchool of Engineering, Cardiff University, Cardiff, UK CF24 3AA
cSchool of Biosciences (and Bio-imaging Research Hub), Sir Martin Evans Building,
Cardiff University, Cardiff, UK CF19 3AX
dUK National Crystallographic Service, Chemistry, Faculty of Natural and
Environmental Sciences, University of Southampton, Highfield, Southampton,
SO17 1BJ England, UK
not luminescent compounds and thus other, invasive and
analytical techniques are required to understand the intra-
cellular distribution of the gold.7 To support such studies, it is
helpful therefore if the gold species in question is inherently
luminescent, thus enabling non-invasive visualisation of live
cells through fluorescence microscopy techniques.8
Whilst fluorescence wavelength and Stokes’ shift are impor-
tant parameters for the application of probes to fluorescence
microscopy, the use of time-resolved analyses via fluorescence
lifetime imaging microscopy (FLIM) can provide even more
detailed information. This current work describes the synthesis
and characterisation of a series of dihydroimidazolinium func-
†Information on the data underpinning the results presented here, including
how to access them, can be found in the Cardiff University data catalogue at
http://doi.org/10.17035/d.2018.0064817469.
‡Electronic supplementary information (ESI) available: DFT Cartesian coordi-
nates of all calculated structures in a format for convenient visualization; details
of all excited states and examples of NMR spectra. CCDC 1831224 and 1831225
for [HL]PF6 and [AuCl(L)]. For ESI and crystallographic data in CIF or other elec-
tronic format see DOI: 10.1039/c8dt04069a
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tionalised 1,8-naphthalimide fluorophores which can be depro-
tonated to act as N-heterocyclic carbene (NHC) donors for
coordination to Au(I). These new species are shown to be fluo-
rescent in the visible region and show excellent utility as cell
imaging agents including application to FLIM studies of MCF-7
cells. Importantly, the results suggest that fluorescence lifetime
is significantly modulated by the presence of the coordinated
Au(I), and thus provides further information on the integrity of
intracellular metal–ligand complexes.
Results and discussion
Synthesis
The intermediates Cl-Nap and N-Nap, pro-ligands [HL]PF6 and
complexes [AuCl(L)] are shown in Scheme 1. The di-substituted
dihydroimidazolinium pro-ligand salts, [HL]PF6, were syn-
thesized in three steps from commercially available 4-chloro-
1,8-naphthalic anhydride. Reaction at the naphthalimide
ring of Cl-Nap using either N-ethylethylenediamine or
N-phenylethylenediamine (thus R2 = Et or Ph) gave the inter-
mediate species, N-Nap. Subsequent ring closure of the N-Nap
species using triethylorthoformate (both as reagent and
solvent) in the presence of ammonium hexafluorophosphate
yielded the cationic dihydroimidazolinium pro-ligands as their
hexafluorophosphate salt, [HL]PF6. The corresponding gold(I)
complexes, [AuCl(L)], were synthesised from [AuCl(tht)] (tht =
tetrahydrothiophene) via deprotonation of the pro-ligand to
yield the NHC Au(I) complex. Despite repeated attempts we
were unable to isolate [AuCl(L5)] in sufficient quantities to
allow unequivocal characterisation and further study. The
yielded complexes (Scheme 2) are thus based upon an unsym-
metrical substitution of a NHC donor at Au(I) that incorporates
a conjugated 1,8-naphthalimide fluorophore.
Scheme 2 Structures of the fluorescent Au(I) complexes targeted in
this work.
istic NH resonance was noted ca. 6.0–6.5 ppm, which was
assigned to the naphthalimide secondary amine substituent.
Upon cyclisation to form the pro-ligands [HL1–8]PF6, the
1
corresponding H NMR spectra showed subtle changes in the
chemical shifts of the dihydroimidazolium ethyl backbone; in
most cases these were observed as multiplets ca. 4.0–4.5 ppm.
The NH resonance at 6.0–6.5 ppm also disappeared, and a new
downfield peak at 9.0–10.0 ppm was assigned to the
deshielded H2 position of the imidazolinium moiety: both
observations were consistent with cyclisation. IR spectra of
[HL1–8]PF6 showed ν(CvO) ca. 1650 cm−1 and ν(PF6) ca.
830 cm−1 consistent with the formation of the salts. HRMS
were obtained for all intermediates and pro-ligands.
Spectroscopic characterisation of ligands and complexes
1H NMR spectra for N-Nap1–8 provided confirmation of substi-
tution at the naphthalimide ring: in most cases, a character-
The Au(I) complexes were again characterised by a range of
1
spectroscopic and analytical techniques. H NMR spectra pro-
vided clear evidence of coordination to Au(I) through the
absence of the downfield H2 imidazolinium resonance, and
changes in the chemical shifts associated with the ethyl
protons in the backbone of the NHC unit. 13C{1H} NMR
spectra revealed resonances that correlated with the presence
of the different R1 and R2 groups. In addition, two unique car-
bonyl resonances around 162–165 ppm were observed, consist-
ent with the unsymmetrical nature of the 1,8-naphthalimide
unit. A downfield signal ca. 192–195 ppm (Fig. S1, ESI‡) was
assigned to the Au(I)-coordinated NHC carbon and is compar-
able with previous studies on Au(I)–NHC complexes which
incorporate a saturated NHC backbone.9
DFT calculations were employed to further support this
13C{1H} NMR assignment, in which the NMR shielding
constants were computed using the gauge including atomic
orbital (GIAO) method.10 Sarotti and Pellegrinet11 have
Scheme 1 Synthetic route: (i) EtNH(CH2)2NH2, DMSO, heat; (ii) CH
(OEt)3, NH4PF6, heat; (iii) [AuCl(tht)], KOtBu, MeOH.
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suggested that a judicious choice of reference compound gives
superior chemical shift predictions, and to this end, since the
carbene carbon can be considered sp2-hybridized, benzene
was used as the reference standard and its shielding constants
computed at the same level of theory as the carbene com-
plexes. Using these calculations, the δC values of the co-
ordinated carbene are predicted to be 192.1 ppm and
192.7 ppm for [AuCl(L3)] and [AuCl(L4)] respectively, both of
which compare very favorably to the experimental data thus
providing further evidence that the assignments are reliable.
HRMS data were also obtained for the complexes, often cor-
relating with the formation of a cationic complex fragment
(through loss of a chloride ligand, or addition of a cation such
as Na+ or NH4+). Each spectrum was consistent with retention
of the Au–NHC unit. IR spectra confirmed the presence of the
ligand, primarily via the naphthalimide ν(CvO) bands ca.
1600–1650 cm−1
.
X-ray crystallography
During the synthesis of the ligands and complexes, diffraction
quality crystals of [HL3]PF6 and [AuCl(L3)] were isolated. The
crystals were obtained by the vapour diffusion of diethyl ether
into either a concentrated solution of [HL3]PF6 (acetone) or
[AuCl(L3)] (acetonitrile). Data collection parameters are shown
in Table S1 (ESI‡), together with supporting bond length and
bond angle data (Table 1). The resultant structures of [HL3]PF6
and [AuCl(L3)] are shown in Fig. 1 and 2. The structure of the
complex (Fig. 1) shows an approximately linear (the C1–Au1–
Cl1 angle is 173.76(6)°) two-coordinate geometry at Au(I). As
expected, the C–N bond lengths within the dihydroimidazoli-
nium moiety are extended upon coordination to Au(I). The Au–
C bond length is 1.984(2) Å and thus comparable with structu-
rally related NHC complexes of Au(I).9 The structure of [AuCl
(L)] also reveals intermolecular π–π stacking interactions
between neighbouring naphthalimide units (Fig. 2).
Additional intermolecular interactions are also evident
through an Au(I)⋯Au(I) distance of 3.31459(15) Å, which is at
the weaker limit of an aurophilic interaction.12
Fig. 1 Crystal structures of [HL3]PF6 (top) and [AuCl(L3)] (bottom).
Ellipsoids drawn at 50% probability and counterion omitted for [HL3]PF6.
Electronic properties and DFT calculations
Fig. 2 Representations of the crystal structure of [AuCl(L3)]. The π–π
packing interactions where the angle between these two planes is 0.00
(19)°, the centroid–centroid distance is 3.8001(17) Å and the shift dis-
tance is 1.644(3) Å.
Solution state UV-vis. absorption and luminescence spectra
were obtained for all pro-ligands and complexes (Table 2). The
absorption spectra (MeCN) of the pro-ligands were character-
ised by shorter wavelength peaks (<300 nm) which are
assigned to π → π* transitions associated with the naphthali-
mide chromophore and phenyl substituents. Between
300–450 nm the spectra also revealed weaker features due to
different n → π* transitions including an intramolecular
charge transfer (ICT) localised on the naphthalimide unit. The
wavelength of the ICT band was relatively insensitive, firstly, to
the nature of the imide (R1) substituent, and secondly, the R2
group on the dihydroimidazolinium group (Fig. 3). However,
the ethyl variants ([HL1]PF6, [HL3]PF6, [HL5]PF6, [HL7]PF6) all
showed substantial hyperchromic shifts in the ICT band rela-
tive to their phenyl ([HL2]PF6, [HL4]PF6, [HL6]PF6, [HL8]PF6)
Table 1 Selected bond lengths for [HL3]PF6 and [AuCl(L3)]
[HL3]PF6
Bond
[AuCl(L3)]
Bond
N(2)–C(16)
N(3)–C(16)
1.318(7)
1.299(7)
N(1)–C(1)
N(2)–C(1)
Au(1)–C(1)
Au(1)–Cl(1)
Au(1)–Au(1′)
1.359(2)
1.318(3)
1.984(2)
2.2933 (5)
3.31459 (15)
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Table 2 Absorption and emission data for the proligands and
electronic basis for the electronic absorption spectra. The
structure of the ethyl congener [HL3]+ was compared to that
obtained using X-ray data, and was found to provide good
agreement between experiment and theory.
complexesa
λabs a /nm
λemb/nm τobsc /ns
ϕd/%
[HL1]PF6
[HL2]PF6
[HL3]PF6
[HL4]PF6
[HL5]PF6
[HL6]PF6
[HL7]PF6
[HL8]PF6
344, 426
342, 429
344, 426
504
507
502
7.3
8.7
9.1
7.2
8.4
8.8
4.7
9.0
4
28
4
Using these optimized structures for TD-DFT calculations
gave a particularly noteworthy result: the low energy ICT band,
experimentally observed at 425–430 nm, was not obtained in
the calculations. We initially rationalized that the band ca.
350 nm might correspond to this transition, but with a large
computational-experimental discrepancy. TD-DFT often under-
344, 351, 432 503
4
344, 428
343, 435
344, 427
345, 433
502
504
501
503
31
17
5
12
2
9
1
1
2
1
8
[AuCl(L1)] 344, 351, 436 507
7.8 (45%), <1.0 (55%)
7.2 (78%), 2.8 (22%)
1.1 (76%), <1.0 (24%)
6.9 (33%), <1.0 (67%)
8.3 (82%), 2.6 (18%)
4.3 (9%), 1.0 (91%)
<1.0
estimates transitions with
a
significant CT character,14
[AuCl(L2)] 349, 433
487
however, this hypothesis would involve an overestimation of
the excited state energy, which is apparently contrary to known
limitations in this area. Moreover, whilst the phenyl derivative
[HL4]+ contains some CT character (vide infra), the ethyl deriva-
tive does not. In our earlier work, we have noticed that the
accuracy of excited state calculations is only slightly dependent
on basis set, but varies considerably with functional.
Therefore, the calculations were performed using a number of
functionals, namely CAM-B3LYP, B3LYP, PBE0, LC-wPBE,
B3PW91, M06, and M06-2X. The lowest energy excited state
varied between 291 nm (LC-wPBE) and 353 (B3LYP) for the
ethyl derivative [HL3]+.
[AuCl(L3)] 345, 351, 439 500
[AuCl(L4)] 354, 352
[AuCl(L6)] 343, 436
504
503
[AuCl(L7)] 345, 351, 439 499
[AuCl(L8)] 344, 351, 441 504
a Measurements obtained in aerated 10−5 M acetone solutions. λex
=
b
405 nm. λex = 372 nm. d Using aerated MeCN solution of [Ru(bipy)3]
c
(PF6)2 as a reference.
Upon comparison with the experimental spectra, it became
apparent that the lowest energy transition corresponded to the
experimental band at ca. 340–350 nm, and the low-energy
band at 425 nm is not replicated in the TD-DFT calculations.
One of the significant limitations of DFT calculations can be
the lack of counterion or discrete solvent interactions with the
substrate; attempts were made to model these, by introducing
the anion, and solvent molecules (water and acetone) hydrogen
bonding to the appropriate polar groups in the naphthalimide
cation, but in every case only a slight (up to 10 nm) variation
in the low energy excitation energy was observed.
Fig. 3 A comparison of the UV-vis. absorption spectra for selected
pro-ligands (recorded in acetone).
TD-DFT calculations were performed using the PBE0 hybrid
functional, since this allows a closer comparison to related
theoretical studies on 1,8-naphthalimides.15 In this regard, the
calculations were in good agreement with the experimental
data, since the computed energies were 342 nm ([HL3]+) and
348 nm ([HL4]+) versus 344 nm (for [HL3]PF6) and 351 nm (for
[HL4]PF6). These maxima are in excellent agreement with the
experimental data, and moreover the slight red-shift, experi-
mentally observed for [HL4]+, is replicated (simulated spectra
are provided in Fig. 4). The molar absorption coefficients have
been estimated from the oscillator strengths using established
methods,16 and their magnitudes agree with the experi-
mentally observed values and are consistent with the expected
π–π* transitions. Experimentally, the phenyl derivative has a
higher absorptivity coefficient than the ethyl, which is repli-
cated in the calculations. A further analysis of the excited
states indicates that they correspond to π–π* transitions
between the HOMO and LUMO; the HOMO and LUMO of
analogues. This may be due to the relative electron donating
ability of the R2 substituent.
Overall, when compared to other 4-amino-substituted 1,8-
naphthalimides,13 the absorptivity of the ICT bands in these
pro-ligands is relatively diminished and hypsochromically
shifted. This is attributed to the cationic nature of the dihy-
droimidazolium moiety, which may well modulate the overall
donor ability of the nitrogen atom at the 4-position of the
naphthalimide chromophore. The absorption spectra of the
related Au(I) complexes were thus dominated by the ligand-
centred transitions. In general, a hypochromic shift of the ICT
band relative to the pro-ligand was noted, again probably relat-
ing to the net donating ability of the ligated NHC substituent.
DFT calculations
To further probe the electronic properties of the substituted [HL4]+ are displayed in Fig. 5. The principal difference between
naphthalimide species, the structures of two pro-ligands the HOMO of [HL3]+ and [HL4]+ is that there is a significant
[HL3]+ and [HL4]+ were calculated using density functional orbital coefficient corresponding to the phenyl group in
methods, and were subsequently used to probe the underlying [HL4]+; the LUMOs of [HL3]+ and [HL4]+ are essentially identi-
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LUMO energy difference decreases from 4.22 eV to 4.13 eV in
[AuCl(L4)].
Fluorescence properties
The fluorescence properties of the ligands and complexes were
assessed at room temperature in aerated acetone solution
(Table 1). Irradiation with 405 nm (corresponding to the exci-
tation line for confocal fluorescence microscopy) relates to
excitation of the ICT absorption band. For the pro-ligands this
yielded a broad structureless emission peak (Fig. 6) at around
490–510 nm with a corresponding lifetimes in the range 4–10
ns, consistent with previous reports on amino-functionalized
1,8-naphthalimides of this type.13 The variation in imide sub-
stituent had minimal influence upon emission wavelength
(Fig. 6). Quantum yields were 4–31% implying some relatively
bright fluorophores within the series. It was also noted that
these species showed excitation wavelength dependent emis-
sion wavelengths: excitation at 345 nm, which corresponds to
one of the π–π* absorptions gave a higher energy emission at
ca. 440 nm, which is assumed to correlate with emission from
a π–π* excited state.
Fig. 4 Simulated UV-vis. absorption spectra of [HL3]+ (black), [HL4]+
(red), [AuCl(L3)] (green), and [AuCl(L4)] (blue) using TD-DFT data [PBE0:
6-311+G(d,p)/SDD].
For the complexes, irradiation at 405 nm again generally
revealed a ligand-based ICT peak ca. 500 nm, but in some
cases an additional peak or shoulder at higher energies
(440–450 nm) was also noted (Fig. 7). Again, this shorter wave-
length peak was enhanced by using excitation at 345 nm.
Comparison of the fluorescent lifetimes and quantum yield
values for the complexes with the corresponding free ligands
generally indicated a partial quenching of the ICT emission in
the presence of a coordinated, heavy atom. For example, in the
case of [AuCl(L3)] the fluorescence lifetime was dominated by a
very short lifetime component ca. 1 ns, contrasting with, and
differentiated from, the value obtained for [HL3]PF6 (Fig. 7).
Fig. 5 Calculated HOMO and LUMO of [HL4]+ [PBE0/6-311+G(d,p)].
Cytotoxicity and bioimaging studies
Au(I) NHC complexes have been the focus of several studies
examining biological applications, including mitochondria-tar-
geted, anti-tumour,17 and antimicrobial18 examples. Fine-
tuning of the ligand architecture has allowed the development
cal, with no significant orbital coefficient over the phenyl ring
in [HL4]+. This difference in the HOMOs explains the subtle
difference in intensity and excitation energy, with the longer
excitation wavelength in [HL4]+ corresponding to the greater
HOMO delocalization in [HL4]+, compared to [HL3]+.
The nature of the low energy bands do not change signifi-
cantly upon coordination of the pro-ligands to Au(I), in that
they are still dominated by HOMO–LUMO π–π* transitions.
However, the maxima are predicted to undergo a small red-
shift, which replicates the experimentally observed spectra. A
comparison of the HOMO and LUMO orbital energies in the
pro-ligands and complexes reveals that both HOMO and
LUMO increase in energy as a result of coordination to the Au
centre, but that the increase in the HOMO energies is margin-
ally more than that found for the LUMO; for example in [HL3]+
the HOMO and LUMO lie at −7.32 eV and −3.06 eV respect-
ively, and at −6.91 eV and −2.73 eV in [AuCl(L3)]. This has the
effect of reducing the HOMO–LUMO difference in the complex
to 4.18 eV from 4.26 eV in the imidazolium precursor. A
similar observation is found for [HL4]+, for which the HOMO–
Fig. 6 Normalised steady state emission spectra of selected ligands
excited at 405 nm (recorded in aerated acetone).
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complexes were less toxic to LOVO, A549 and PC3 cells than
the corresponding cationic pro-ligands. This is in contrast to
our previous findings which have shown that gold complexes
of the type [Au(PPh3)(L)] can often have enhanced cytotoxicity
versus the free ligand, attributed to the presence of lipophili-
city enhancing co-ligands (e.g. PPh3) at Au(I).23 The importance
of charge has also been noted in cytotoxic iridium(III) com-
plexes, with cationic species proving more toxic to cell lines24
and Gram positive bacteria25 than neutral analogues. For the
pro-ligands, [HL6]PF6 was the least toxic with IC50 values
>100 μM for each cell type. In contrast, closely related [HL5]PF6
was the most toxic, with strong activity against LOVO, PC3 and
MCF-7s. Highly lipophilic [HL7]PF6 also showed strong activity
against LOVO and MCF-7. Overall the results showed that rela-
tively minor changes in the molecular structures had a pro-
found influence upon the cytotoxicity.
A limited number of Au(I) complexes incorporating fluo-
rescent ligands have been reported26 for cellular imaging,
although the fluorescent properties of such species are not
always optimal for microscopy. Therefore, cellular microscopy
studies including FLIM were undertaken with one of the pro-
ligand/complex pairings, [HL3]PF6 and [AuCl(L3)], using
MCF-7 cells. Fluorophore concentrations of 100 μg ml−1 (i.e.
comparable to the IC50 values) and more dilute 10 ng ml−1
(well below the obtained IC50 values) in DMSO were used for
[HL3]PF6 and [AuCl(L3)]. In all cases the cells were irradiated
with 405 nm excitation which matches well with the ICT
absorption band of these naphthalimide-based fluorophores;
detection wavelengths were 500–550 nm.
Fig. 7 Top: Normalised emission spectra of selected complexes excited
at 405 nm (recorded in aerated acetone). Bottom: Time-resolved
measurements showing the comparative decay profiles for ligand [HL3]
PF6 (red) and complex [AuCl(L3)] (blue) against the instrument response
(green).
of lipophilic, cationic Au(I)–NHC complexes that selectively
induce apoptosis in cancer cells and show selective targeting
of mitochondrial selenoproteins, such as TrxR.19 Fluorophore-
tethered Au(I)–NHC compounds have demonstrated apoptosis
in cancer cells,20 as well as DNA binding capability.21 Au(I)
NHC frameworks provide an excellent basis for the develop-
ment of anticancer agents.22
Prior to any imaging work, cytotoxicity assessments were
obtained for complexes and pro-ligands using the MTT assay
(Table 3). Data were recorded for four cancer cell lines: LOVO,
A549, PC3 and MCF-7, the latter for correlation with the
imaging studies. The data clearly revealed that the uncharged
Firstly, [HL3]PF6 demonstrated rapid uptake (<30 min) and
internalisation by the MCF-7 cells (Fig. 8). Cytoplasmic fluo-
rescence was concentrated in well-defined spheroidal volumes
consistent with organelle localisation. There was no evidence
of nuclear sequestration. The corresponding complex
[AuCl(L3)] also showed reasonable uptake internalisation with
bright foci of fluorescence observable within the cytoplasm,
but not the nucleus (Fig. S2, ESI‡).
More distinct localisation was observed for both [HL3]PF6
and [AuCl(L3)] at a diluted probe concentration of 10 ng ml−1
Table 3 IC50 (μM) values (standard deviation in parentheses) for the
pro-ligands and complexes against four cancer cell lines
Compound LOVO
A549
PC3
MCF-7
[HL1]PF6
[HL2]PF6
[HL3]PF6
[HL4]PF6
[HL5]PF6
[HL6]PF6
[HL7]PF6
[HL8]PF6
[AuCl(L1)]
[AuCl(L2)]
[AuCl(L3)]
[AuCl(L4)]
[AuCl(L6)]
[AuCl(L7)]
[AuCl(L8)]
30.07(3.19)
33.62(5.09)
>100
56.83(4.06)
75.68(1.10)
47.10(0.55)
>100
53.95(1.41)
>100
>100
>100
>100
>100
>100
56.40(3.08)
98.1(3.91)
71.27(2.89)
72.82(3.03)
8.58(0.03)
>100
42.00(5.69)
>100
>100
>100
>100
>100
>100
68.35(3.95)
66.83(2.85)
61.21(3.02)
56.38(2.84)
7.12(0.36)
>100
9.98(4.77)
33.23(5.84)
84.21(11.38)
62.88(5.58)
48.58(10.58)
27.79(11.89)
46.21(13.01)
41.75(9.34)
4.82(0.77)
73.36(2.36)
42.75(8.66)
51.81(5.34)
7.27(1.19)
>100
5.22(2.49)
>100
68.94(2.29)
91.64(3.20)
>100
41.54(2.88)
49.10(7.97)
50.92(6.20)
40.99(11.08)
Fig. 8 Confocal fluorescence microscopy images of MCF-7 cells incu-
bated with [HL3]PF6 showing localized signals (λex = 405 nm and λem
525 nm). Cells incubated with 100 μg ml−1 of the fluorophore.
=
>100
>100
46.63(0.71)
>100
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(1 : 10 000 dilution when compared to the preliminary
microscopy studies). Under these conditions, co-localisation
studies were investigated using commercial fluorophores for
staining of mitochondrial, endoplasmic reticulum (ER), Golgi
and lysosomal compartments (Fig. 9 and Fig. S3 and S4, ESI‡).
Fig. 9 shows the obtained superimposed images with [HL3]PF6
and [AuCl(L3)] respectively, and colocalisation is clearly
evident (as indicated by the yellow-orange colouration) only
with the lysosomal stain, LysoTracker Deep Red. A recently
reported acridine-functionalized Au(I) compound has also
shown lysosomal localisation.20
Fig. 10 FLIM images of MCF-7 cells incubated with [HL3]PF6 (left) and
[AuCl(L3)] (right) using pulsed excitation at 440 nm. Fitted parameters
for left image: τ1 = 9.2 ns (77%), τ2 = 3.4 ns (20%), τ1 = 0.4 ns (3%); for
right image, τ1 = 11.0 ns (68%), τ2 = 3.9 ns (25%), τ1 = 0.5 ns (7%).
Having fully elucidated the intracellular localisation of both
probes, FLIM analyses was applied to MCF-7 cells which had
been incubated with [HL3]PF6 and [AuCl(L3)]. For FLIM, the
cells were imaged via a pulsed 440 nm diode laser excitation
source, which is again well matched to the ICT absorption
band of the naphthalimide fluorophores, and data collection
was facilitated through time correlated single photon count-
ing. Decay curves were fitted using an n-exponential tail fit,
while applying an estimated instrument response function. In
all cases the lowest χ2 square value was obtained by modelling
three components to the exponential decay.
(Fig. 10). This value is significantly longer than that spectro-
scopically obtained (1.1 ns), but more comparable with the
fluorescence lifetime for [HL3]PF6. Therefore, the FLIM data
suggests that the gold complex may dissociate under biological
conditions to regenerate the dihydroimidazolinium-like fluoro-
phore. In these cases, the fluorescence lifetime obtained from
FLIM might be indicative of such an intracellular process.
The data from these microscopy measurements suggests
that further work is required to understand the speciation of
these Au(I) carbene complexes within a biological environ-
ment. In particular, it is important to consider the acidic
nature of lysosomal compartments and the effect that this may
have on such complexes. Since the solubility of [AuCl(L3)] is
limited to organic solvents it has not been possible to eluci-
date the pH dependent stability of this species, but previous
studies have noted how the gold–carbene bond can tolerate
acidic conditions at elevated temperatures.27 Further, our
studies have shown that such complexes are stable >6 h in
DMSO (incubated at 37 celcius) solutions in the absence of
light, as indicated by 13C NMR data (retention of coordinated
carbene). However, it is well known that Au(I) carbene com-
plexes can display photoinduced decomposition.28 Therefore
one must exercise caution when seeking to correlate cyto-
toxicity data with confocal fluorescence microscopy imaging.
Further work is required to unravel the influence that light
irradiation may have upon the speciation and toxicity of gold(I)
complexes within a cell.
For [HL3]PF6, the FLIM images (Fig. 10) show the localised
hot spots of fluorescence which were earlier attributed to
lysosomal localisation. From the data analyses of the image,
the dominant lifetime component was obtained as 9.2 ns. This
value compares favourably (cf. 9.1 ns) with the solution state
spectroscopic measurements (Table 1), validating the data
fitting protocol. This value also implies that the fluorescence
labelling of the lysosomal compartments of the cell can be
clearly attributed to [HL3]PF6 and that the intrinsic structure
of the fluorophore is intact following incubation with the cells.
In the case of MCF-7 cells incubated with [AuCl(L3)], good
quality FLIM images were again obtained. In this case, the
fitted parameters suggest a dominant lifetime of ca. 11 ns
Conclusions
The stepwise synthesis of a range of 4-substituted 1,8-naphtha-
limide derivatives that incorporate a dihydroimidazolium cat-
ionic unit give fluorescent species with emission dominated by
an ICT emission band around 500 nm. Once deprotonated,
these dihydroimidazolium salts can be utilised as NHC
ligands for Au(I) to form complexes. In all cases, the fluo-
rescence of these complexes is again dominated by the ligand,
but often partially quenched by the presence of Au(I) leading
to the possibility of discriminating ligand/complex pairings on
the basis of fluorescence lifetime. The varying functionality of
the ligands led to different cytotoxicity profiles across cancer
Fig. 9 Confocal fluorescence microscopy images of MCF-7 cells incu-
bated with [HL3]PF6 (top) and [AuCl(L3)] (bottom). Representative super-
imposed images where yellow-orange signifies co-localisation (from left
to right) with an ER stain, Golgi stain and lysosomal stain. Note strong
lysosomal fluorescence co-localisation.
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cell lines, with the cationic pro-ligands proving generally more kV, 55.0 mA) equipped with HF Varimax confocal mirrors
toxic than the corresponding complexes. Selected ligands and (70 µm focus) and an AFC12 goniometer and HG Saturn 724+
complexes were incubated with MCF-7 cells and fluorescence detector ([HL3]PF6) or a HyPix 6000 detector ([AuCl(L3)]). The
microscopy was undertaken, showing that these fluorophores crystals were kept at T = 100(2) K during data collection. Data
are biocompatible. More detailed colocalisation studies were were measured using profile data from ω-scans using MoKα
conducted on a ligand/complex pairing and identified that the radiation. Cell determination and data collection were carried
species show definitive localisation in lysosomes. Finally, we out using CrystalClear31 ([HL3]PF6) or CrystalisPro32 ([AuCl
have shown that the ICT fluorescence lifetime of these species (L3)]). With the data reduction, cell refinement and absorption
can be utilised in FLIM. Lifetime imaging studies on correction using CrystalisPro. Using Olex2,33 the structures
[AuCl(L3)] indicate that this species probably dissociates from were solved with the ShelXT34 structure solution program and
gold within the cell. Further studies are now required to fully the models were refined with version 2014/7 of ShelXL35 using
elucidate the possible mechanism for this process, but FLIM Least Squares minimisation. All non-hydrogen atoms were
appears to be a potential imaging tool for supporting such refined anisotropically. Hydrogen atom positions were calcu-
investigations.
lated geometrically and refined using the riding model. For
sample [HL3]PF6, the PF6 anion was disordered over two posi-
tions and for [AuCl(L3)] the propyl chain was disordered over
two positions. As such various geometrical (SADI) and displa-
cement (SIMU, RIGU) restraints were applied to the disordered
atoms. Diffractometer: 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 VHF Varimax optics (70 µm
focus). Cell determination and data collection: CrystalClear-
SM Expert 3.1 b27 (Rigaku, 2013). Data reduction, cell refine-
ment and absorption correction: CrystAlisPro. Structure solu-
tion: SUPERFLIP.36 For sample L3, the PF6 anion was dis-
ordered over two positions. As such various geometrical
(SAME) and displacement (SIMU) restraints were applied.
Experimental
General considerations
All reagents and solvents were commercially available and were
used without further purification if not stated otherwise. For
the measurement of 1H, 31P, and 13C NMR spectra a Bruker
Fourier300 (300 MHz), Bruker AVANCE HD III equipped with a
BFFO SmartProbe™ (400 MHz) or Bruker AVANCE III HD with
BBO Prodigy CryoProbe (500 MHz) was used. The obtained
chemical shifts δ are reported in ppm and are referenced to
the residual solvent signal. Spin–spin coupling constants J are
given in Hz.
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 Facility at
Swansea University. High resolution mass spectral (HRMS)
data were obtained on a Waters MALDI-TOF mx at Cardiff
University or on a Thermo Scientific LTQ Orbitrap XL by the
EPSRC UK National Mass Spectrometry Facility at Swansea
Cytotoxicity assessment via MTT assay
The cytotoxicity of the complexes was assessed using the col-
ourimetric and quantitative MTT [3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide] assay, first reported by
Mosmann.37 Quantification was achieved using a multi-well
scanning spectrophotometer and reported as an IC50 value.
University. IR spectra were obtained from
a Shimadzu
Method for cytotoxicity analysis
IR-Affinity-1S FTIR. Reference to spectroscopic data are given
for known compounds. UV-Vis studies were performed on a
Shimadzu UV-1800 spectrophotometer as MeCN 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.
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).29 Emission spectra were
uncorrected and excitation spectra were instrument corrected.
The pulsed source was a Nano-LED configured for 295 nm or
372 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.
Anti-tumor evaluation in MCF7, LOVO, A549 and PC3 cell lines
was performed by MTT assay. Compounds were prepared as
0.1–100 mM stock solutions dissolved in DMSO and stored at
−20 °C. Cells were seeded into 96-well microtitre plates at a
density of 5 × 103 cells per well and allowed 24 h to adhere.
Decimal compound dilutions were prepared in medium
immediately prior to each assay (final concentration
0.1–100 µM). Experimental medium was DMEM +10% FCS
(PC3 and Lovo) or RPMI +10% heat inactivated FCS (A549 and
MCF7). Following 96 h compound exposure at 37 °C, 5% CO2,
MTT reagent (Sigma Aldrich) was added to each well (final
concentration 0.5 mg ml−1). Incubation at 37 °C for 4 h
allowed reduction of MTT by viable cells to an insoluble forma-
zan product. MTT was removed and formazan solubilized by
addition of 10% Triton X-100 in PBS. Absorbance was read on
a Tecan Sunrise spectrophotometer at 540 nm as a measure of
cell viability; thus inhibition relative to control was determined
X-ray diffraction
For both samples, a suitable crystal30 was selected and (IC50) from four independent sets of data and the standard
mounted on a MITIGEN holder in oil on a Rigaku FRE+ (45.0 deviations calculated from these data sets.
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Confocal imaging of MCF-7 cells
included in all calculations as the polarized continuum model,
with the molecular cavity defined by a united atom model that
incorporates hydrogen into the parent heavy atom.42 TD-DFT
calculations were carried out with at the same level of theory
as the geometry optimizations; the first 24 excited states were
calculated. NMR shielding constants were computed using the
gauge including atomic orbital method incorporated into
Gaussian 09;10 13C chemical shifts were computed relative to
benzene (δC = 129.2 ppm).
Cell culture. All cell culture reagents were purchased
through Sigma Aldrich. MCF-7 cells were grown on circular
glass coverslips for 5–7 days in RPMI-1640 medium, sup-
plemented with heat inactivated 5% foetal bovine serum and
100 U ml−1 penicillin/streptomycin solution, until around
70–90% confluent. For experiments, culture medium was
replaced with 1 ml fresh RPMI-1640 and the cells then incu-
bated with either 100 µg ml−1 or 10 ng ml−1 of probe in
DMSO. A range of commercially available organelle probes
(ThermoFisher) were used for co-localisation studies with the
above compounds in live cells: mitochondria were labelled
Reagents and precursors
Cl-Nap derivatives43 and [AuCl(tht)]44 were prepared according
to the literature. All other reagents were used as received and
general and standard precautions were taken during their
handling and manipulation.
with TMRE (tetramethylrhodamine ethyl ester) 200 nM; λex
=
549 nm: λem = 575 nm; cell membranes and lipid droplets with
HCS LipidTOX Red Neutral Lipid Stain (1 : 1000 dilution from
the commercial kit; λex = 577 nm; λem = 609 nm); the endoplas-
Ligands and precursors
mic reticulum with ER Tracker red (1 µM; λex = 587 nm; λem
=
Synthesis of N-Nap1. Cl-Nap1 (502 mg, 1.6 mmol) and
N-ethylethylenediamine (0.64 ml, 6.3 mmol) were heated for
48 hours at 100 °C in DMSO under a nitrogen atmosphere.
The solution was cooled and water (15 ml) added. The solution
was neutralised using 1 M HCl and then extracted into DCM.
The organic phase was washed with water, dried over MgSO4,
and the solvent removed to yield the product as an orange
solid (yield: 125 mg, 22%). 1H NMR (400 MHz, CDCl3): δH 8.51
615 nm); the Golgi apparatus with BODIPY TR ceramide
(5 µM; λex = 590 nm; λem = 620 nm) and lysosomes with
LysoTracker Red (1 µM; λex = 577 nm; λem = 590 nm). All fluo-
rescent compounds were incubated for 30 minutes at 37 °C
then washed in warmed PBS before imaging.
Confocal microscopy. MCF-7 cells were imaged using an
LSM880 Airyscan confocal microscope system (Zeiss, Germany)
equipped with time correlated single photon counting (TCSPC)
for fluorescence lifetime imaging (FLIM; PicoQuant GmbH,
Germany). For fluorescence localisation studies, cells were
imaged using appropriate excitation and emission settings for
detection of either the naphthalimide-based probes alone, or
either probe together with a commercial co-localisation agent
(see above) using Zen software (Zeiss, Germany). Differential
interference contrast (DIC) optics were used to simultaneously
record cell morphology alongside the fluorescence signal. For
co-localisation studies, sequential fluorescence image acqui-
sition was used to avoid spectral crosstalk between the dyes.
For FLIM, the probes were imaged via a pulsed 440 nm diode
laser using SymphoTime software (PicoQuant GmbH,
Germany) for TCSPC. Decay curves were fitted using an n-expo-
nential tail fit, applying an estimated instrument response
function (IRF). A decent fit was accepted provided the fitted
curve overlaid well with the decay curve; the χ2 square value
was ∼1 and the residual values spread randomly around 0;
selecting the least number of model parameters (n) in each
case. Fluorescence lifetime measurements of the probes were
displayed as fitted lifetime image maps.
3
3
(d, 1H, JHH = 7.2 Hz), 8.30 (d, 1H, JHH = 8.4 Hz), 8.09 (d, 1H,
3JHH = 8.5 Hz), 7.48 (app. t, 1H, JHH = 7.7 Hz), 6.51 (d, 1H,
3
3JHH = 8.4 Hz), 6.35 (br. t, 1H), 4.36 (t, 2H, JHH = 5.2 Hz), 3.81
3
3
3
(t, 2H, JHH = 8.1 Hz), 3.70–3.60 (m, 4H), 3.35 (t, 2H, JHH = 5.8
Hz), 3.02 (t, 2H, 3JHH = 5.8 Hz), 2.69 (q, 2H, 3JHH = 7.1 Hz), 1.13
3
(t, 3H, JHH = 7.1 Hz) ppm. HRMS found m/z 372.1919, calcd
m/z 372.1918 for [C20H26N3O4]+.
Synthesis of [HL1]PF6. N-Nap1 (100 mg, 0.27 mmol) and
NH4PF6 (49 mg, 0.3 mmol) were heated at 100 °C in HC(OEt)3
for 3 h under a nitrogen atmosphere. The solution was cooled
and the solvent decanted to leave an orange colored oil. Et2O
(2 × 10 ml) was then added and slowly decanted and the
remaining oil dried in vacuo to yield [HL1]PF6 as an orange oil
1
(77 mg, 54%). H NMR (300 MHz, (CD3)2CO): δH 8.97 (s, 1H),
8.59 (dd, 1H, JHH = 8.5, 3.4 Hz), 8.55–8.46 (m, 2H), 8.00 (dd,
3
1H, JHH = 7.5, 2.3 Hz), 7.95–7.86 (m, 1H), 4.80 (t, 2H, JHH
=
9.9 Hz), 4.50 (t, 2H, 3JHH = 10.5 Hz), 4.24 (t, 2H, 3JHH = 6.2 Hz),
3.90 (q, 2H, JHH = 7.3 Hz), 3.73–3.66 (m, 2H), 3.58–3.52 (m,
3
3
2H), 2.03–1.99 (m, 2H), 1.47 (t, 3H, JHH = 7.3 Hz) ppm.
13C{1H} NMR (101 MHz, (CD3)2CO): δC 158.2, 138.6, 131.7,
130.8, 128.7, 128.4, 127.1, 124.1, 123.4, 72.4, 70.2, 69.6, 67.3,
61.1, 53.0, 49.3, 44.1, 39.2, 39.1, 14.6, 12.0 ppm. UV/Vis
((CH3)2CO): λmax/nm (ε/M−1 cm−1) = 426 (4960), 344 (10 920).
Density functional calculations
Calculations were performed using the Gaussian 09 HRMS found m/z 382.1752, calcd m/z 382.1761 for
program.38 Calculated structures were optimized without sym- [C21H24N3O4]+. IR (solid) ν/cm−1: 2978, 2947, 2875, 1697, 1651,
metry constraints and the nature of the stationary point veri- 1581, 1503, 1462, 1450, 1346, 1157, 1123, 1090, 1057, 825, 783,
fied using a frequency calculation; in all cases no imaginary 754, 739, 556, 422, 405, 392.
frequencies were observed. The PBE039 functional was used
Synthesis of N-Nap2. As for N-Nap1, but using Cl-Nap1
along with the 6-311+G(d,p) triple-ζ basis set40 on all centres (502 mg, 1.6 mmol) and N-phenylethylenediamine (0.82 ml,
except for Au, for which the Stuttgart-Dresden basis set was 6.2 mmol). The product was isolated as a yellow solid (yield:
used with a relativistic effective core potential.41 Solvent was 260 mg, 66%). 1H NMR (400 MHz, CDCl3): δH 8.37 (d, 1H,
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3
3
3JHH = 7.3 Hz), 8.28 (d, 1H, JHH = 8.7 Hz), 8.09 (d, 1H, JHH
=
2968, 1697, 1651, 1591, 1514, 1443, 1429, 1408, 1389, 1350,
3
8.3 Hz), 7.44 (app. t, 1H, JHH = 7.4 Hz), 7.30 (m, 2H), 1269, 1234, 1201, 1155, 1086, 1069, 962, 891, 876, 825, 789,
7.01–6.93 (m, 2H), 6.54 (d, 1H, 3JHH = 8.3 Hz), 6.11 (s, 1H), 4.38 760, 738, 556, 463, 434, 409.
(t, 2H, JHH = 4.9 Hz), 3.87 (t, 2H, JHH = 4.7 Hz), 3.77–3.62
3
3
Synthesis of N-Nap4. As for N-Nap1, but using Cl-Nap2
(767 mg, 2.8 mmol) and N-phenylethylenediamine (1.5 ml,
(overlapping m, 8H) ppm.
Synthesis of [HL2]PF6. As for [HL1]PF6, but using N-Nap2 11.2 mmol). The product was yielded as an orange solid (yield:
(198 mg, 0.47 mmol) and NH4PF6 (82 mg, 0.52 mmol). The 1.00 g, 97%). 1H NMR (400 MHz, (CD3)2CO): δH 8.42 (d, 1H,
product was precipitated from CHCl3 and Et2O giving a yellow 3JHH = 8.5 Hz), 8.35 (d, 1H, JHH = 7.2 Hz), 8.21 (d, 1H, JHH
=
=
3
3
powder (yield: 129 mg, 64%). H NMR (400 MHz, (CD3)2CO): 8.5 Hz), 7.52 (app. t, 1H, 3JHH = 8.2 Hz), 6.98 (app. t, 2H, 3JHH
1
3
δH 9.85 (s, 1H), 8.85 (d, 1H, JHH = 8.5 Hz), 8.77–8.62 (m, 2H), 8.2 Hz), 6.76 (d, 1H, 3JHH = 8.5 Hz), 6.58 (d, 2H, 3JHH = 8.0 Hz),
8.30 (d, 1H, JHH = 7.8 Hz), 8.06 (dd, 1H, JHH = 15.5, 7.9 Hz), 6.48 (br. t, 1H), 5.15 (br. t, 1H, NH), 3.93 (t, 2H, 3JHH = 7.5 Hz),
3
3
3
3
3
7.72 (d, 2H, JHH = 7.7 Hz), 7.63 (d, 2H, JHH = 7.5 Hz), 3.62 (t, 2H, JHH = 5.8 Hz), 3.47 (t, 2H, JHH = 6.0 Hz), 1.58 (q,
3
7.56–7.43 (m, 1H), 5.23–4.98 (m, 4H), 4.37 (t, 2H, JHH
=
2H, 3JHH = 7.5 Hz), 0.81 (t, 3H, 3JHH = 7.5 Hz) ppm.
6.2 Hz), 3.89–3.71 (overlapping m, 8H) ppm. 13C{1H} NMR
Synthesis of [HL4]PF6. As for [HL1]PF6, but using N-Nap4
(101 MHz, (CD3)2CO): δC 164.2, 163.7, 157.2, 138.8, 137.0, (750 mg, 2.0 mmol) and NH4PF6 (360 mg, 2.2 mmol). Product
1
132.7, 131.6, 130.9, 130.5, 129.9, 129.7, 129.6, 128.9, 128.0, was obtained as a yellow solid (yield: 860 mg, 81%). H NMR
3
125.9, 124.9, 124.3, 119.8, 73.3, 71.0, 68.20, 61.9, 54.6, 50.8, (400 MHz, (CD3)2CO): δH 9.87 (s, 1H), 8.86 (d, 1H, JHH = 8.5
3
40.2 ppm. UV/Vis ((CH3)2CO): λmax/nm (ε/M−1 cm−1) 429 Hz), 8.71 (overlapping d, 2H), 8.31 (d, 1H, JHH = 7.8 Hz), 8.06
3
3
(1000), 342 (9700). HRMS ES− found m/z 430.1756, calcd m/z (app. t, 1H, JHH = 7.9 Hz), 7.73 (d, 2H, JHH = 7.6 Hz), 7.62
3
3
430.1761 for [C25H24N3O4]+. IR (solid) ν/cm−1: 3010, 2932, (app. t, 2H, JHH = 7.5 Hz), 7.50 (app. t, 1H, JHH = 7.4 Hz),
3
3
2810, 1699, 1654, 1626, 1581, 1492, 1379, 1348, 1269, 1234, 5.17–5.08 (m, 4H), 4.12 (t, 2H, JHH = 7.5 Hz), 1.78 (q, 2H, JHH
3
1142, 1128, 1097, 1057, 829, 785, 754, 736, 689, 556, 410.
= 7.5 Hz), 1.00 (t, 3H, JHH = 7.4 Hz) ppm. 13C{1H} NMR
Synthesis of Cl-Nap2. 4-Chloro-1,8-naphthalic anhydride (101 MHz, (CD3)2CO): δC 164.1, 163.6, 157.2, 138.7, 137.0,
(3 g, 12.9 mmol) and propylamine (1.9 ml, 25.8 mmol) were 132.6, 131.5, 130.9, 130.8, 129.9, 129.6, 128.9, 128.0, 125.9,
heated at reflux in EtOH for 24 hours under a nitrogen atmo- 125.1, 124.4, 119.8, 119.6, 119.3, 54.6, 50.9, 42.5, 22.0,
sphere. The solution was cooled to room temperature and 11.7 ppm. UV/Vis ((CH3)2CO): λmax/nm (ε/M−1 cm−1) = 351
stored at −20 °C for 24 hours. The resulting precipitate was (14 320), 344 (0.599). HRMS found m/z 384.1700, calcd m/z
filtered to yield Cl-Nap2 as a bright yellow solid (yield: 2.74 g, 384.1707 for [C24H22N3O2]+. IR (solid) ν/cm−1: 2974, 1699,
3
78%). 1H NMR (400 MHz, CDCl3): δH 8.59 (d, 1H, JHH = 7.3 1651, 1626, 1585, 1494, 1391, 1364, 1350, 1271, 1236, 1072,
3
3
Hz), 8.53 (d, 1H, JHH = 8.5 Hz), 8.42 (d, 1H, JHH = 7.9 Hz), 8423, 829, 787, 758, 689, 557, 469, 434.
3
7.82–7.73 (m, 2H), 4.08 (t, 2H, JHH = 7.6 Hz), 1.70 (app. sext.,
2H, 3JHH = 7.5 Hz), 0.95 (t, 3H, 3JHH = 7.4 Hz) ppm.
Synthesis of Cl-Nap3. 4-Chloro-1,8-naphthalic anhydride
(1.00 g, 4.3 mmol) and benzylamine (1 ml, 9.2 mmol) were
Synthesis of N-Nap3. As for N-Nap1, but using Cl-Nap2 heated in ethanol (70 ml) at reflux under nitrogen for
(491 mg, 1.8 mmol) and N-ethylethylenediamine (0.76 ml, 12 hours. The solution was cooled to room temperature
7.2 mmol). The product was obtained as an orange solid forming a yellow precipitate immediately on freezing. This was
(yield: 0.573 g, 98%). H NMR (500 MHz, CDCl3): δH 8.51 (d, filtered and washed with Et2O (10 ml) to give Cl-Nap3 as a
1
1H, 3JHH = 8.2 Hz), 8.38 (d, 1H, 3JHH = 8.4 Hz), 8.14 (d, 1H, 3JHH yellow solid (yield: 1.2 g, 88%). H NMR (500 MHz, CDCl3): δH
1
3
3
3
3
= 8.4 Hz), 7.55 (app. t, 1H, JHH = 8.2 Hz), 6.61 (d, 1H, JHH
=
8.61 (d, 1H, JHH = 7.3 Hz), 8.53 (d, 1H, JHH = 8.5 Hz), 8.45 (d,
3
8.4 Hz), 6.29 (br. t, 1H), 4.08–4.03 (m, 2H), 3.41–3.36 (m, 2H), 1H, JHH = 7.8 Hz), 7.77 (dd, 2H, JHH = 17.4, 8.2 Hz), 7.49 (d,
3.06–3.01 (m, 2H), 2.69 (q, 2H, 3JHH = 7.1 Hz), 1.69 (q, 2H, 3JHH 2H, JHH = 7.6 Hz), 7.26 (app. t, 1H, JHH = 7.4 Hz), 7.23–7.16
3
3
= 7.5 Hz), 1.12 (t, 3H, 3JHH = 5.1 Hz), 0.94 (t, 3H, 3JHH = 7.4 Hz) (m, 2H), 5.32 (s, 2H) ppm.
ppm. HRMS found m/z 326.1866, calcd m/z 326.1863 for
Synthesis of N-Nap5. As for N-Nap1, but using Cl-Nap3
(500 mg, 1.6 mmol) and N-ethylethylenediamine (0.65 ml,
[C29H24N3O2]+.
Synthesis of [HL3]PF6. As for [HL1]PF6, but using N-Nap3 6.2 mmol). N-Nap5 was obtained as an orange solid (574 mg,
(346 mg, 1.1 mmol) and NH4PF6 (190 mg, 1.2 mmol). The 94%). H NMR (300 MHz, CDCl3): δH 8.41 (d, 1H, J = 7.3 Hz),
1
product was obtained as an orange coloured oil (yield: 0.254 g, 8.29 (d, 1H, J = 8.4 Hz), 7.97 (d, 1H, J = 8.3 Hz), 7.52–7.37 (m,
50%). 1H NMR (400 MHz, (CD3)2CO): δH 8.94 (s, 1H), 8.58–8.47 3H), 7.25–7.10 (m, 3H), 6.48 (d, 1H, J = 8.4 Hz), 6.25 (br. t, 1H),
3
3
(m, 3H), 7.97 (d, 1H, JHH = 7.8 Hz) 7.88 (app. t, 1H, JHH = 7.9 5.28 (s, 2H), 3.27 (dd, 2H, J = 10.3, 5.0 Hz), 2.97 (t, 2H, J = 5.7
3
3
Hz), 4.65 (t, 2H, JHH = 10.0 Hz), 4.45 (t, 2H, JHH = 11.1 Hz), Hz), 2.65 (q, 2H, J = 7.1 Hz), 1.09 (t, 3H, J = 7.1 Hz) ppm.
3
3.82 (q, 2H, JHH = 7.3 Hz), 1.66–1.52 (m, 2H), 1.07–0.96 (m,
Synthesis of [HL5]PF6. As for [HL1]PF6, but using N-Nap5
3H), 0.85 (t, 3H, JHH = 7.5 Hz) ppm. 13C{1H} NMR (101 MHz, (389 mg, 1.0 mmol) and ammonium hexafluorophosphate
(CD3)2CO): δC 164.0, 163.5, 156.0, 139.3, 132.4, 131.5, 129.8, (187 mg, 1.15 mmol). The product [HL5]PF6 was obtained as
129.6, 129.3, 127.9, 125.0, 124.2, 124.2, 53.9, 50.1, 45.0, 42.4, an orange oil (371 mg, 67%). 1H NMR (300 MHz, (CD3)2CO):
3
21.9, 12.9, 11.7 ppm. UV/Vis ((CH3)2CO): λmax/nm (ε/M−1 cm−1
)
δH 9.00 (s, 1H), 8.63 (d, 1H, J = 8.5 Hz), 8.54 (overlapping d,
= 426 (2520), 344 (11 980). HRMS found m/z 336.1709, calcd 2H), 8.01 (d, 1H, J = 7.8 Hz), 7.94 (app. t, 1H, J = 7.9 Hz), 7.44
m/z 336.1707 for [C20H22N3O2]+. IR (solid) ν/cm−1: 3381, 3089, (d, 2H, J = 7.3 Hz), 7.33–7.20 (m, 3H), 5.25 (s, 2H), 4.82 (t, 2H,
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J = 10.4 Hz), 4.53 (t, 2H, J = 10.5 Hz), 3.94 (q, 2H, J = 7.2 Hz),
Synthesis of N-Nap7. As for N-Nap1, but using Cl-Nap4
1.52 (t, 3H, J = 7.4 Hz) ppm. 13C{1H} NMR (126 MHz, (600 mg, 1.7 mmol) and N-ethylethylenediamine (0.71 ml,
(CD3)2CO): δC 163.3, 162.8, 158.0, 138.7, 137.5, 131.8, 131.0, 7.0 mmol). The product was obtained as an orange solid
1
129.0, 128.5, 128.3, 128.2, 127.2, 127.0, 124.1, 123.1, 123.1, (yield: 582 mg, 84%). H NMR (400 MHz, CDCl3): δH 8.60 (d,
3
3
53.0, 49.3, 44.1, 43.2, 35.2, 23.6, 20.6, 12.0 ppm. UV/Vis 1H, JHH = 6.5 Hz), 8.37 (d, 1H, JHH = 8.4 Hz), 8.15 (d, 1H,
((CH3)2CO): λmax/nm (ε/M−1 cm−1): 428 (3700), 344 (14 560). 3JHH = 8.2 Hz), 7.55 (app. t, 1H, JHH = 7.6 Hz), 6.59 (d, 1H,
3
HRMS found m/z 384.1718, calcd m/z 384.1712 for 3JHH = 8.4 Hz), 6.32 (br. t, 1H), 4.07 (t, 2H, JHH = 7.6 Hz), 3.40
3
[C24H22N3O2]+. IR (solid) ν/cm−1: 2372, 2341, 1699, 1651, 1582, (q, 2H, JHH = 4.9 Hz), 3.05 (t, 2H, JHH = 5.4 Hz), 2.71 (q, 2H,
3
3
1549, 1497, 1454, 1423, 1381, 1346, 1290, 1234, 1182, 1155, 3JHH = 7.1 Hz), 1.66 (m, 2H), 1.40–1.11 (overlapping m, 13H),
3
1072, 1028, 970, 876, 827, 783, 754, 734, 702, 665, 613, 583, 0.78 (t, 3H, JHH = 7.0 Hz) ppm. HRMS found m/z 396.2644,
556, 469, 407.
calcd m/z 396.2646 for [C24H34N3O2]+.
Synthesis of N-Nap6. Cl-Nap3 (513 mg, 1.65 mmol) and
Synthesis of [HL7]PF6. As for [HL1]PF6, but using Pro7
N-phenylethylenediamine (0.83 ml, 6.34 mmol) were heated in (476 mg, 1.2 mmol) and NH4PF6 (217 mg, 1.3 mmol). The
DMSO (6 ml) at reflux under nitrogen for 48 hours. The product was obtained as a yellow colored powder (496 mg,
product was cooled and water (6 ml) was added to form yellow 74%). 1H NMR (500 MHz, (CD3)2CO): δH 9.06 (s, 1H), 8.69–8.58
precipitate. The reaction solution was neutralised with hydro- (m, 3H), 8.08 (d, 1H, 3JHH = 7.8 Hz), 7.99 (dd, 1H, JHH = 8.5, 7.3
3
3
chloric acid (3 ml, 0.1 M) and filtered. Product was re-precipi- Hz), 4.86 (t, 2H, JHH = 10 Hz), 4.57 (t, 2H, JHH = 10 Hz),
tated using dichloromethane and Et2O and then filtered to 4.13–4.09 (m, 2H), 3.95 (q, 2H, 3JHH = 7.2 Hz), 2.87 (d, 3H, 3JHH
give a yellow solid (yield: 551 mg, 87%). 1H NMR (400 MHz, = 15.7 Hz), 1.70 (dt, 2H, 3JHH = 15.0, 7.5 Hz), 1.51 (t, 2H, 3JHH
=
C
3
3
CDCl3): δH 8.51 (d, 1H, JHH = 6.6 Hz), 8.41 (d, 1H, JHH
=
7.3 Hz), 1.33–1.24 (m, 6H), 0.87 (t, 3H, 3JHH = 6.9 Hz) ppm. 13
8.3 Hz), 7.88 (d, 1H, 3JHH = 8.3 Hz), 7.56 (d, 2H, 3JHH = 7.1 Hz), {1H} NMR (101 MHz, (CD3)2CO): δC 164.0, 163.5, 158.9, 139.3,
7.54–7.48 (m, 1H), 7.30 (app. t, 2H, 3JHH = 7.3 Hz), 7.23 (d, 3H, 132.4, 131.5, 129.7 (two overlapping environments), 129.5,
3
3JHH = 7.4 Hz), 6.81 (app. t, 1H, JHH = 7.4 Hz), 6.76 (d, 2H, 129.3, 127.9, 125.0, 124.1, 53.9, 50.1, 45.0, 40.9, 32.5, 30.0,
3JHH = 7.6 Hz), 6.66 (d, 1H, 3JHH = 8.4 Hz), 5.57 (br. t, 1H), 5.35 29.9, 28.6, 27.8, 23.3, 14.3, 12.8 ppm. UV/Vis ((CH3)2CO): λmax
/
(s, 2H), 3.61 (overlapping, 4H) ppm.
nm (ε/M−1 cm−1) = 427 (1820), 344 (13 620). HRMS found m/z
Synthesis of [HL6]PF6. As for [HL1]PF6, but using N-Nap6 406.2482, calcd m/z 406.2489 for [C25H32N3O2]+. IR (solid) ν/
(346 mg, 0.87 mmol) and NH4PF6 (156 mg, 0.97 mmol). The cm−1: 2959, 2924, 2855, 1703, 1651, 1589, 1508, 1391, 1354,
product was obtained as a yellow powder (yield: 322 mg, 85%). 1254, 1232, 1155, 1093, 827, 785, 754, 555, 416.
1H NMR (400 MHz, (CD3)2CO): δH 9.87 (s, 1H), 8.86 (d, 1H,
Synthesis of N-Nap8. As for N-Nap1, but using Cl-Nap4
3JHH = 8.5 Hz), 8.74–8.68 (m, 2H), 8.30 (d, 1H, JHH = 7.6 Hz), (503 mg, 1.4 mmol) and N-phenylethylenediamine (0.76 ml,
3
3
3
8.06 (app. t, 1H, JHH = 7.7 Hz), 7.72 (d, 2H, JHH = 8.4 Hz), 5.8 mmol). The product was isolated as a yellow coloured solid
3
3
1
7.62 (app. t, 2H, JHH = 8.2 Hz), 7.48 (d, 3H, JHH = 6.7 Hz), (yield: 601 mg, 65%). H NMR (400 MHz, CDCl3): δH 8.55 (d,
7.36–7.24 (m, 3H), 5.35 (s, 2H), 5.15–3.90 (m, 6H) ppm. 1H, 3JHH = 6.6 Hz), 8.43 (d, 1H, 3JHH = 8.4 Hz), 8.03 (d, 1H, 3JHH
13C{1H} NMR (101 MHz, (CD3)2CO): δC 164.2, 163.7, 157.2, = 8.5 Hz), 7.61–7.55 (m, 1H), 7.25–7.19 (m, 2H), 6.83 (app. t,
138.9, 138.4, 137.0, 132.9, 131.8, 130.9, 130.2, 130.0, 129.8, 1H, 3JHH = 7.4 Hz), 6.78 (d, 2H, 3JHH = 8.5 Hz), 6.72 (d, 1H, 3JHH
129.6, 129.2, 129.1, 128.9, 128.2, 128.1, 125.9, 125.2, 125.0, = 8.4 Hz), 5.70 (br. s, 1H), 4.17–4.09 (m, 2H), 3.65 (t, 4H, 3JHH
=
124.3, 119.8, 54.6, 50.9, 44.2 ppm. UV/Vis ((CH3)2CO): λmax/nm 4.3 Hz), 1.72 (dd, 2H, JHH = 15.2, 7.3 Hz), 1.46–1.20 (m, 10H),
(ε/M−1 cm−1) 435 (400), 343 (5700). HRMS ES− found m/z 0.86 (t, 3H, 3JHH = 6.8 Hz) ppm.
432.1701, calcd m/z 432.1707 for [C28H22N3O2]+. IR (solid)
Synthesis of [HL8]PF6. As for [HL1]PF6, but using N-Nap8
ν/cm−1: 1699, 1655, 1637, 1589, 1495, 1452, 1387, 1350, 1337, (497 mg, 1.1 mmol) and NH4PF6 (200 mg, 1.2 mmol). The
1290, 1277, 1232, 1182, 959, 881, 839, 825, 791, 756, 748, 700, product was obtained as a yellow coloured solid (yield:
1
689, 557, 472.
274 mg, 54%). H NMR (400 MHz, (CD3)2CO): δH 9.85 (s, 1H),
Synthesis of Cl-Nap4. 4-Chloro-1,8-naphthalic anhydride 8.84 (d, 1H, JHH = 8.6 Hz), 8.70 (dd, 2H, JHH = 7.6, 2.6 Hz),
3
3
3
(3 g, 12.9 mmol) and octylamine (4.26 ml, 25.8 mmol) were 8.29 (d, 1H, JHH = 7.8 Hz), 8.08–8.03 (m, 1H), 7.72 (d, 2H,
heated at reflux in EtOH for 24 hours under a nitrogen atmo- 3JHH = 7.7 Hz), 7.62 (app. t, 2H, 3JHH = 8.0 Hz), 7.49 (app. t, 1H,
sphere. The solution was cooled to room temperature and 3JHH = 7.4 Hz), 5.18–4.70 (m, 6H), 4.14 (t, 2H, JHH = 7.2 Hz),
3
3
stored at −20 °C for 24 hours and the resulting precipitate fil- 1.77–1.66 (m, 2H), 1.45–1.24 (m, 10H), 0.87 (t, 3H, JHH
=
tered. The crude product was dissolved in dichloromethane 6.9 Hz) ppm. 13C{1H} NMR (101 MHz, (CD3)2CO): δC 164.0,
(30 ml) and washed with 0.1 M HCl (30 ml), followed by H2O 163.5, 157.1, 138.7, 137.0, 132.6, 131.5, 130.9, 129.8, 129.6,
(30 ml) and brine (30 ml). The collected organic phase was 128.9, 128.0, 125.9, 125.0, 124.4, 119.8, 54.6, 50.8, 41.0, 32.5,
dried over MgSO4, and the solvent removed to yield a yellow 30.5, 30.3, 30.1, 29.9, 28.7, 27.8, 23.3, 14.3 ppm. UV/Vis
solid (yield: 3.78 g, 85%). H NMR (400 MHz, CDCl3): δH 8.60 ((CH3)2CO): λmax/nm (ε/M−1 cm−1) 433 (200), 345 (7200). HRMS
1
(d, 1H, JHH = 7.3 Hz), 8.53 (d, 1H, JHH = 8.5 Hz), 8.45 (d, 1H, found m/z 454.2478, calcd m/z 454.2489 for [C29H32N3O2]+. IR
3
3
3JHH = 7.9 Hz), 7.82–7.75 (m, 2H), 4.11–4.05 (m, 2H), 1.69–1.50 (solid) ν/cm−1: 2922, 2850, 1708, 1697, 1653, 1536, 1589, 1492,
3
(m, 2H), 1.47–1.12 (overlapping m, 10H), 0.80 (t, 3H, JHH
6.7 Hz) ppm.
=
1390, 1352, 1267, 1232, 1095, 831, 785, 754, 734, 686, 557, 464,
405.
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Synthesis of [AuCl(L1)]. [HL1]PF6 (81 mg, 0.15 mmol), KOtBu
Synthesis of [AuCl(L4)]. As for [AuCl(L1)], but using [HL4]PF6
(17 mg, 0.15 mmol) and [AuCl(tht)] (49 mg, 0.15 mmol) were (98 mg, 0.17 mmol), KOtBu (19 mg, 0.17 mmol) and [AuCl(tht)]
dissolved in CH3OH and then heated to reflux, under nitrogen, (53 mg, 0.17 mmol). The product was obtained as a grey solid
overnight in the absence of light. The solution was cooled, and (yield: 86 mg, 85%). 1H NMR (500 MHz, CDCl3): δH 8.56 (d,
3
3
then concentrated in vacuo, and then Et2O added dropwise to 1H, JHH = 7.3 Hz), 8.50 (d, 1H, JHH = 7.7 Hz), 8.25 (d, 1H,
induce precipitation. [AuCl(L1)] was yielded as a grey colored 3JHH = 8.4 Hz), 7.83 (d, 1H, JHH = 7.7 Hz), 7.73–7.69 (m, 1H),
3
solid (yield: 76 mg, 81%). 1H NMR (400 MHz, CDCl3): δH 7.65 (d, 2H, JHH = 7.2 Hz), 7.31 (m, 3H), 4.40 (br. t, 2H),
3
8.68–8.59 (m, 2H), 8.25 (d, 1H, 3JHH = 8.7 Hz), 7.83 (app. d, 2H, 4.15–3.80 (m, 4H), 1.78–1.69 (overlapping m, 2H), 0.98 (t, 3H,
3
3JHH = 7.8 Hz), 4.46 (t, 2H, JHH = 4.6 Hz), 4.04–3.81 (m, 6H), 3JHH = 7.4 Hz) ppm. 13C NMR (126 MHz, CDCl3): δC 192.3,
3
3.73–3.63 (m, 2H), 3.56 (br s, 2H), 3.47 (q, 2H, JHH = 6.8 Hz), 163.8, 163.3, 142.6, 140.0, 131.8, 131.3, 129.6, 129.2, 128.8,
3
1.42 (t, 3H, JHH = 7.3 Hz), 1.25 (br s, 1H) ppm. 13C{1H} NMR 128.0, 127.9, 127.8, 126.4, 123.5, 123.4, 123.2, 53.8, 52.5, 42.3,
(126 MHz, CDCl3): δC 193.9, 163.9, 163.7, 132.3, 131.6, 129.1, 21.5, 11.7 ppm. UV/Vis ((CH3)2CO): λmax/nm (ε/M−1 cm−1) 352
128.5, 128.0, 126.2, 123.0, 72.4, 70.4, 70.0, 68.5, 66.8, 62.0, (11 760), 345 (11 860). HRMS found m/z 597.1551, calcd m/z
53.7, 48.7, 46.3, 39.9, 39.4, 15.3, 13.8 ppm. UV/Vis ((CH3)2CO): 597.1559 for [C24H21N3O2AuCl–HCl + NH4]+. IR (solid) ν/cm−1
:
λmax/nm (ε/M−1 cm−1) 436 (1420), 351 (4620), 344 (4660). 2967, 2955, 2873, 1703, 1655, 1622, 1591, 1506, 1475, 1433,
HRMS found m/z 636.0918, calcd m/z 636.0935 for 1389, 1354, 1340, 1286, 1276, 1238, 1068, 1018, 958, 908, 858,
[C21H23AuClN3O4 + Na]+. IR (solid) ν/cm−1: 2899, 2666, 2360, 785, 764, 754, 692, 673, 588, 546.
1695, 1653, 1582, 1537, 1510, 1477, 1427, 1379, 1348, 1341,
1265, 1230, 1105, 1043, 835, 785, 737, 669, 600, 581, 556, 455, (62 mg, 0.16 mmol), [AuCl(tht)] (57 mg, 0.16 mmol) and KOtBu
Synthesis of [AuCl(L6)]. As for [AuCl(L1)], but using [HL6]PF6
444, 436, 428, 420, 409.
(17 mg, 0.16 mmol). The product was obtained as a grey solid
Synthesis of [AuCl(L2)]. As for [AuCl(L1)], but using [HL2]PF6 (yield: 49 mg, 49%). 1H NMR (400 MHz, CDCl3): δH 8.67 (d,
(68 mg, 0.16 mmol), [AuCl(tht)] (51 mg, 0.16 mmol) and KOtBu 1H, 3JHH = 6.2 Hz), 8.62 (d, 1H, 3JHH = 7.7 Hz), 8.32 (d, 2H, 3JHH
3
(18 mg, 0.16 mmol). The product was obtained as a grey-yellow = 7.7 Hz), 7.91 (d, 1H, JHH = 7.8 Hz), 7.86–7.76 (m, 1H), 7.73
solid (yield: 88 mg, 84%). 1H NMR (500 MHz, CD3CN): δH (d, 2H, JHH = 8.0 Hz), 7.54 (d, 2H, JHH = 7.1 Hz), 7.47–7.30
3
3
8.66–8.59 (m, 2H), 8.01 (d, 1H, JHH = 7.9 Hz), 7.94 (app. t, 1H, (m, 5H), 5.42 (s, 2H), 4.48 (s, 2H), 4.10–3.65 (m, 4H) ppm. 13C
3
3JHH = 7.9 Hz), 7.82 (d, 2H, JHH = 7.9 Hz), 7.53 (d, 2H, JHH
=
{1H} NMR (126 MHz, CDCl3): δC 192.5, 164.8, 164.2, 163.8,
3
3
3
7.8 Hz), 7.43 (d, 2H, JHH = 7.6 Hz), 4.39–4.21 (m, 4H), 163.3, 149.4, 142.7, 139.9, 138.0, 137.2, 134.4, 132.2, 131.6,
3.81–3.19 (overlapping m, 10H) ppm. 13C{1H} NMR (126 MHz, 131.5, 130.3, 129.6, 129.0, 128.9, 128.6, 128.5, 128.1, 127.7,
CDCl3): δC 192.7, 163.8, 163.6, 132.2, 131.5, 131.4, 129.7, 129.0, 127.4, 126.4, 125.1, 125.0, 123.3, 122.7, 121.6, 120.9, 110.8,
128.2, 128.1, 126.5, 123.4, 123.3, 116.5, 113.9, 72.4, 70.4, 70.0, 104.0, 54.6, 54.5, 53.9, 53.8, 53.7, 52.3, 50.1, 45.4, 43.9, 43.5,
68.5, 66.8, 66.0, 62.0, 53.7, 52.3, 52.2, 39.9, 15.4, 15.3 ppm. UV/ 40.2, 25.7 ppm. UV/Vis ((CH3)2CO): λmax/nm (ε/M−1 cm−1) 436
Vis ((CH3)2CO): λmax/nm (ε/M−1 cm−1) 433 (900), 349 (8700). (700), 343 (1600). HRMS found m/z 645.1549, calcd m/z
HRMS found m/z 643.1608, calcd m/z 643.1614 for 645.1559 for [C28H20AuN3O2 + NH4]+. IR (solid) ν/cm−1: 2961,
[C25H23AuN3O4 + NH3]+. IR (solid) ν/cm−1: 2990, 2880, 1705, 2914, 2845, 1703, 1682, 1655, 1580, 1522, 1508, 1472, 1454,
1659, 1593, 1487, 1437, 1408, 1381, 1346, 1337, 1321, 1288, 1425, 1402, 1383, 1312, 1260, 1236, 1177, 1136, 1069, 1016,
1234, 1188, 1121, 1049, 887, 843, 789, 756, 692, 559, 482, 451, 964, 845, 799, 785, 737, 704, 692, 617, 594, 583, 557, 544, 500,
438, 426, 419, 405.
480, 459, 436, 428, 417.
Synthesis of [AuCl(L3)]. As for [AuCl(L1)], but using [HL3]PF6
Synthesis of [AuCl(L7)]. As for [AuCl(L1)], but using [HL7]PF6
(80 mg, 0.17 mmol), KOtBu (19 mg, 0.17 mmol) and [AuCl(tht)] (86 mg, 0.16 mmol), KOtBu (18 mg, 0.16 mmol) and [AuCl(tht)]
(53 mg, 0.17 mmol). The reaction mixture was heated over- (50 mg, 0.16 mmol). The product was obtained as a grey-yellow
night at reflux under a nitrogen atmosphere. The product was solid (yield: 68 mg, 68%). 1H NMR (400 MHz, (CD3)2CO): δH
obtained as an off-white solid (yield: 40 mg, 45%). 1H NMR 8.39 (app. d, 2H, JHH = 7.5 Hz), 8.33 (d, 1H, JHH = 7.6 Hz),
3
3
3
3
(500 MHz, CDCl3): δH 8.55 (dd, 1H, JHH = 7.3, 1.0 Hz), 8.52 (d, 7.80 (d, 1H, JHH = 8.7 Hz), 7.71 (app. t, 1H, JHH = 7.5 Hz),
1H, 3JHH = 7.7 Hz), 8.21 (d, 1H, 3JHH = 8.4 Hz), 7.76 (d, 1H, 3JHH 5.03–4.89 (m, 4H), 2.95–2.75 (m, 2H), 1.65–1.20 (m, 15H), 0.91
= 7.7 Hz), 7.73–7.68 (m, 1H), 4.20–4.05 (m, 4H), 3.93 (t, 2H, (t, 3H, 3JHH = 6.0 Hz) ppm. 13C{1H} NMR (126 MHz, (CD3)2CO):
3JHH = 9.3 Hz), 3.83 (br s, 2H), 1.76–1.76 (m, 2H), 1.29 (t, 3H, δC 193.8, 164.3, 163.8, 144.4, 132.1, 131.7, 130.4, 130.0, 129.7,
3JHH = 7.2 Hz), 0.96 (t, 3H, JHH = 7.4 Hz) ppm. 13C{1H} NMR 128.5, 127.1, 124.4, 123.7, 54.6, 49.5, 46.6, 40.9, 32.6, 30.2,
3
(126 MHz, CDCl3): δC 193.5, 163.9, 163.4, 142.9, 131.9, 131.3, 30.1, 28.7, 27.9, 23.3, 14.4, 13.9 ppm. UV/Vis ((CH3)2CO): λmax
/
129.4, 129.0, 128.4, 127.9, 126.3, 123.5, 123.1, 53.8, 48.7, 46.2, nm (ε/M−1 cm−1) 439 (340), 351 (4360), 345 (4360). HRMS
42.2, 21.5, 13.7, 11.7 ppm. UV/Vis ((CH3)2CO): λmax/nm (ε/M−1 found m/z 660.1678, calcd m/z 660.1663 for [C25H31AuClN3O2 +
cm−1) 439 (280), 351 (6900), 345 (6900). HRMS found m/z Na]+. IR (solid) ν/cm−1: 3341, 2920, 2854, 2110, 1703, 1657,
602.0682, calcd m/z 602.0682 for [C20H21N3O2AuCl + Cl]+. IR 1585, 1531, 1470, 1429, 1391, 1350, 1275, 1234, 1096, 856, 729,
(solid) ν/cm−1: 2959, 2882, 2357, 1691, 1651, 1585, 1520, 1470, 478.
1423, 1383, 1362, 1344, 1333, 1267, 1234, 1190, 1065, 907, 833,
789, 644, 557, 482, 405, 382, 359, 334, 324, 318, 312, 301, 293, (70 mg, 0.16 mmol), [AuCl(tht)] (50 mg, 0.16 mmol) and KOtBu
Synthesis of [AuCl(L8)]. As for [AuCl(L1)], but using [HL8]PF6
284, 276, 257, 231, 220, 214.
(18 mg, 0.16 mmol). The product was obtained as a grey
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coloured solid (yield: 31 mg, 79%). 1H NMR (500 MHz,
CD3CN): δH 8.63 (d, 2H, JHH = 7.4 Hz,), 8.57 (d, 1H, JHH = 8.6
7 (a) L. E. Wedlock and S. J. Berners-Price, Aust. J. Chem.,
2011, 64, 692; (b) L. E. Wedlock, M. R. Kilburn, J. B. Cliff,
L. Filgueira, M. Saunders and S. J. Berners-Price,
Metallomics, 2011, 3, 917; (c) O. Karaca, V. Scalcon,
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3
3
3
3
Hz,), 8.00 (d, 1H, JHH = 7.2 Hz), 7.93 (app. t, 1H, JHH = 7.8
3
3
Hz), 7.82 (d, 2H, JHH = 7.8 Hz), 7.52 (app. t, 2H, JHH = 7.4
3
Hz), 7.42 (app. t, 1H, JHH = 7.1 Hz), 5.18 (br s, 2H), 4.45 (br. t,
3
2H), 4.32 (br. t, 2H), 4.10 (t, 2H, JHH = 7.5 Hz), 2.85–2.75 (m,
2H), 1.44–1.22 (m, 10H), 0.88 (t, 3H, JHH = 6.0 Hz) ppm. 13C
3
{1H} NMR (126 MHz, CDCl3): δC 192.8, 163.8, 163.3, 142.4,
140.0, 132.0, 131.3, 129.7, 129.4, 128.6, 128.1, 128.1, 128.0,
126.4, 123.7, 123.5, 123.3, 66.0, 53.7, 52.3, 40.9, 32.0, 29.5,
29.4, 28.3, 27.3, 22.8, 15.4, 14.2 ppm. UV/Vis ((CH3)2CO): λmax
/
nm (ε/M−1 cm−1) 441 (3380), 351 (14 160), 344 (14 560). HRMS
found m/z 650.2071, calcd m/z 650.2076 for [C29H31AuN3O2–
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370.
Conflicts of interest
13 (a) S. Banerjee, E. B. Veale, C. M. Phelan, S. A. Murphy,
G. M. Tocci, L. J. Gillespie, D. O. Frimannsson, J. M. Kelly
and T. Gunnlaugsson, Chem. Soc. Rev., 2013, 42, 1601.
14 A. Vlček and S. Zalis, Coord. Chem. Rev., 2007, 251, 258.
15 D. Jacquemin, E. A. Perpète, G. Scalmani, I. Ciofini,
C. Peltier and C. Adamo, Chem. Phys., 2010, 372, 61.
16 P. J. Stephens and H. Nobuyuki, Chirality, 2010, 22, 229.
17 P. J. Barnard, M. V. Baker, B. W. Skelton, A. H. White and
S. J. Berners-Price, J. Inorg. Biochem., 2003, 96, 99;
P. J. Barnard, M. V. Baker, S. J. Berners-Price and D. A. Day,
J. Inorg. Biochem., 2004, 98, 1642.
There are no conflicts to declare.
Acknowledgements
We thank Cardiff University for an Endowment Studentship
and EPSRC for DTP funding (L. M. G). Dr C. Williams holds a
Sêr Cymru II Fellowship part funded by the European Regional
Development Fund through the Welsh Government. The
EPSRC Mass Spectrometry National Service (Swansea
University) and EPSRC National Crystallographic Service
(University of Southampton) are also thanked. Dr E. Lloyd-
Evans and Dr P. Watson (Cardiff University) are thanked for
commercial fluorophores and Mr H. Mottram for cytotoxicity
data.
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