Two-photon absorption and photoluminescence of europium based emissive probes for bioactive systems

Article abstract of DOI:10.1039/b710717j

Observation of two-photon excitation (760 nm) and emission of two responsive water soluble europium complexes is reported with cross-sections of up to 2 GM. Two-photon excitation spectra have also been measured, acquisition being achieved by the use of a cavity-dumped mode locked Ti-sapphire laser. Time-gated detection is used to differentiate the ligand fluorescence and metal centred emission in these europium complexes. This journal is The Royal Society of Chemistry.

Full text of DOI:10.1039/b710717j

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PAPER
www.rsc.org/dalton | Dalton Transactions
Two-photon absorption and photoluminescence of europium based emissive
probes for bioactive systems
Lars-Olof Pa˚lsson,* Robert Pal, Benjamin S. Murray, David Parker and Andrew Beeby
Received 12th July 2007, Accepted 9th October 2007
First published as an Advance Article on the web 16th October 2007
DOI: 10.1039/b710717j
Observation of two-photon excitation (760 nm) and emission of two responsive water soluble europium
complexes is reported with cross-sections of up to 2 GM. Two-photon excitation spectra have also been
measured, acquisition being achieved by the use of a cavity-dumped mode locked Ti-sapphire laser.
Time-gated detection is used to differentiate the ligand fluorescence and metal centred emission in these
europium complexes.
excitation of lanthanide ions9 and of various complexes following
Introduction
multi-photon absorption.10–12
Lanthanide (LnIII) ions are rapidly gaining ground as emissve
The high intensities required for two-photon absorption necessi-
probes or as components of bio-assays due to their unique
tates tight focusing of the excitation light, giving the method a high
spectral signatures and inherent sensitivity to enviromental fac-
spatial resolution. Here, we show that two-photon excited (TPE)
tors such as pH, pM and pX.1,2 Recent work has shown that
efficient sensitisation of the LnIII ion and subsequent intense
photoluminescence (PL) may be obtained by incorporating LnIII
ions into functionalised complexes bearing suitable sensitising
chromophores.3 These complexes permeate a variety of living
cells and exhibit distinctive compartmentalisation profiles within
cells.4 Emissive LnIII ions can be excellent probes of the local
physiological conditions. In particular, for the Eu(III) ion, changes
in the coordination environment can have a dramatic impact on
the PL properties, including the form of the emission spectrum
and the PL quantum yield (PLQY). In the latter context variation
in the nature and number of proximate X–H oscillators5e.g. N–H
and O–H groups and changes in the facility of electron transfer
processes are often responsible for modulation of the PLQY.6
The scope and utility of functionalised LnIII complexes have re-
cently been demonstrated in the development of ratiometric probes
for pH, bicarbonate, urate and citrate, through reversible binding
of the analyte to the ligand or metal centre.7 Measurements in
living systems using these probes are currently being pursued
using conventional microscopy techniques such as confocal lumi-
nescence microscopy. These rely upon a single photon excitation
process after absorption of light in the range 320–400 nm by the
chromophore (S0 → S1), followed by intersystem crossing (S1 →
T1) and intramolecular electronic energy transfer to the LnIII ion.
For some applications, this approach can be problematic due to
the non-selective nature of the excitation (UV-vis), leading to auto-
fluorescence and photo damage. It is desirable to avoid the UV-
vis range, and target the far visible/near-IR range (700–820 nm)
which penetrates biological media most effectively. Multiphoton
excitation spectroscopy enables the excitation of chromophores
with near-IR optical radiation to generate states that are normally
accessed by the absorption of a single UV-quantum.8 To date there
have been only limited reports of the observation of the direct
PL can be observed from the two functionalised LnIII complexes
shown in Scheme 1 in aqueous solution following excitation from
the output of a cavity dumped mode-locked Ti-sapphire laser.
We obtain strong and well resolved fluorescence from both the
7
ligand and long lived emission from the Eu 5D0 → FJ transitions
upon excitation in the near IR range. We therefore propose that
such functionalised LnIII complexes may be used in the future as
emissive probes for two-photon microscopy of biological systems,
and that time-gated imaging may be used to effectively eliminate
auto-fluorescence.
Scheme 1 Changes in metal ion ligation upon binding HCO−
.
3
Results and discussion
Fig. 1 shows TPE-PL from [Eu·L1(H2O)]3+ in D2O after excitation
at 758 nm. The broad feature centred at 440 nm is fluorescence
emission from the ligand and the sharp bands at lower energy are
5
7
the D0 → F0–3 emission bands of Eu(III). We note that due to
the presence of a dichroic mirror in the optical path of the two-
photon excitation spectrometer the 7F4 band cannot be detected.
A plot of the log(integrated emission intensity) plotted against
Department of Chemistry, Durham University, South Road, Durham, UK
DH1 3LE. E-mail: lars-olof.palsson@dur.ac.uk; Tel: +44-(0)-191-3342135
5726 | Dalton Trans., 2007, 5726–5734
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the Eu emission is much stronger relative to the chromophore
fluorescence when HCO− is added.
3
Fig. 3 shows the integrated emission intensity as a function of
excitation energy on a logarithmic scale, confirming the emission
arises from a two-photon process. Gradients were calculated using
a composite emission intensities of the ligand fluorescence and the
metal emission and are reported in Table 1. As the LnIII emission
is dependent upon the efficiency of the intra-molecular energy
transfer step, non-linear effects may occur that result in small
deviations from the ideal TPE gradient. A summary of the photo-
physical parameters measured for compounds [Eu·L1(H2O)]3+ and
[Eu·L2(H2O)]3+ is shown in Table 1.
Fig. 1 Emission spectrum of 60 lM [Eu·L1(H2O)]3+ in D2O excited under
two-photon absorption conditions at 758 nm, pD = 7.8.
log (excitation power) shows a gradient of 2.1 0.1, confirming
that the emission is due to a two-photon absorption process.
Importantly the spectrum is identical to that obtained by UV
excitation (data not shown), in contrast to what has been reported
previously.9
The complex [Eu·L2(H2O)]3+ exhibits a dramatic change in
its emission spectrum upon reversible binding to bicarbonate
13
(HCO−
,
Scheme 1. Displacement of the coordinated water
3
Fig. 3 Excitation power dependency on the integrated luminescence
intensity. (A) is [Eu·L1(H2O)]3+ which has a slope of 2.1 0.1(R = 0.99),
(B) is [Eu·L2(H2O)]3+ which has a slope of 1.9 0.1 (R = 0.98).
enhances the intensity of the Eu(III) emission and increases
the (DJ = 2)/(DJ = 1) ratio by more than 100%, Fig. 2.
The concentration and optical density of the two samples with
and without the HCO− are nearly equal in these experiments;
3
The near IR excitation spectra of the two complexes and
their corresponding one-photon absorption spectra are shown in
Fig. 4 and 5. The TPE profiles follow the red edge of the one-
photon absorption spectra rather well. For [Eu·L2(H2O)]3+ the
TPE maximum is located around 760 nm while [Eu·L1(H2O)]3+
has a TPE maximum at 770 nm. These observations are consistent
with the absorption spectra which show a slight blue shift for
[Eu·L2(H2O)]3+ compared to [Eu·L1(H2O)]3+. The observations
are also in line with Fu et al’s observations for another Eu(III)
complex.16 On the blue side of the absorption spectra, in each
case, the TPE does not follow the absorption so well, with a rapid
decrease in TPE intensity for shorter excitation wavelengths.
The origin of the differences between the linear absorption and
the TPE on the blue side of the spectra is not totally clear. However,
the linear absorption spectrum is a composite band of many
different origins (e.g. multiple excited states, vibronic components
and charge transfer bands) and due to different selection rules
there could clearly be relative differences in oscillator strength in
this part of the spectrum.
Fig. 2 Emission spectrum of 80 lM [Eu·L2(H2O)]3+ in D2O excited under
two-photon absorption conditions at 758 nm, without HCO− ( · · · ) and
3
with 20 mM HCO− added (
), pD = 7.8.
3
Table 1 Spectroscopic data for complexes
a
b
b
Complex
Uf
UEu (H2O)
UEu (D2O)
Gradient
r2/GMc
[Eu·L1]3+
[Eu·L2]3+
0.23
0.11
0.13
0.04
0.008
0.024
0.08
0.016
0.050
2.1 0.1
1.9 0.1
1.9 0.1
1.7
0.4
0.4
[Eu·L2]3+ + 20 mM HCO−
3
a Ligand centred fluorescence quantum yield, measured according to ref. 14. b Quantum yield of europium based emission. The gradient value is the
excitation power dependence on the PL intensity on logarithmic scales. c 1 GM = 10−50 (cm4 s photon−1). Samples were recorded at pH = 7.4 and pD =
7.8 respectively.
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The results of this study have several important implications.
First, it is noteworthy that these water soluble LnIII complexes
have sufficiently high TPE cross-sections and PL for strong and
well-defined signals to be observed. We stress that these complexes
were not designed to have high TPE cross-sections, and inclusion
of this design criterion in future complex synthesis should result
in materials with values of r2 improved by orders of magnitude.
There are reports in the literature of Eu(III) complexes with higher
TPE cross sections, but importantly these are either not water
soluble, dissociate in dilute solution or are not designed to show a
responsive behaviour, making them inappropriate for bioimaging
applications.16 We also point out that while cross-sections of
the compounds and emission efficiencies reported in the present
study are relatively low, time-gating of the PL can be applied as
demonstrated in Fig. 6. This technique allows for enhancement of
the metal centred emission relative to auto-fluorescence.
Fig. 4 Two-photon excitation spectrum (ꢀ and bottom abscissa) and
one-photon absorption (
and upper abscissa) of [Eu·L1(H2O)]3+
.
Secondly, as expected, identical spectra are obtained for one-
and two-photon excitation, contrary to earlier work.8 This means
that the well characterised response of these complexes is retained
and that these can be used as sensors, and illustrates that in these
materials we are observing two-photon excitation of the antenna
chromophore, which undergoes subsequent intersystem crossing
and energy transfer to the metal ion. This allows us to build
upon the established principles of design of responsive molecular
probes based upon this class of complex. The cellular uptake and
compartmentalisation of related LnIII complexes within different
cell organelles has already been demonstrated by us elsewhere.4,7
Finally, the choice of excitation source is particularly important
for this measurement. Using a cavity dumped mode-locked Ti-
sapphire laser operating at 4 MHz repetition rate (variable) allows
a sufficiently long time-gate to be used to efficiently discriminate
the short lived auto-fluorescence or other fluorescent probes that
are being used, s ∼ 10 ns, and the long lived Ln emission,
s ∼ ms, providing a high duty cycle. A cavity dumped Ti-sapphire
laser provides higher pulse energies than a comparable mode-
locked laser; for example the laser system used in this work
provides ca. 10 nJ per pulse when operated in a conventional
mode-locked configuration, and up to 60 nJ per pulse when cavity
dumped. This output energy is much lower than that provided
by amplified laser systems as used elsewhere, where much higher
pulse energies of > lJ per pulse are used, with the potential for
sample damage.10 Also, the repetition rate of the cavity dumped
system remains sufficiently high to permit rapid scanning of the
sample for microscopic imaging.
Fig. 5 Two-photon excitation spectrum (ꢀ and bottom abscissa) and
one-photon absorption (
line and upper abscissa) of [Eu·L2(H2O)]3+
.
Fig. 6 shows the time-gated and total PL of [Eu·L1(H2O)]3+. As
can be seen from the figure, the broad emission around 450 nm
originating from the ligand chromophore is significantly reduced
in intensity relative to the Eu(III) emission. The time-gated PL does
show a small amount of chromophore PL and we attribute this
to the leakage of mode-locked optical pulses which are not fully
suppressed by the cavity dumper of the mode-locked laser system.
Taken together, these different aspects make functionalised
LnIII complexes potential candidates for future TPE microscopy
studies. The next generation of functionalised LnIII complexes will
permit the in cellulo study of analytes of biological importance
by two-photon luminescence microscopy. Multi-photon excitation
microscopy has established itself as a powerful tool in the life
sciences; the enhanced spatial resolution and reduced sample
damage are particularly attractive attributes. Future studies will
set out to explore this possibility in more detail.
Experimental
Fig.
6
Time-gated emission
(
)
and total emission ( · · · ) of
Two new complexes, Eu·L1(H2O)]3+ and [Eu·L2(H2O)]3+, designed
[Eu·L1(H2O)]3. The time-gate had a duration of 480 ns and was delayed
75 ns from the laser pulse.
to probe local changes in bicarbonate concentration (Scheme 1)
5728 | Dalton Trans., 2007, 5726–5734
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have been prepared. Schemes 2 and 3 below summarise the
synthetic pathway to [Eu·L1(H2O)]3+ and [Eu·L2(H2O)]3+.
(10 ml) then left to cool to room temperature. The desired product
precipitated as a crystalline yellow solid that was collected by
filtration, washed with cold ethyl acetate (3 ml), then dried◦under
reduced pressure (0.136 g, 0.316 mmol, 92%); mp 246–247 C; 1H
NMR (CDCl3, 500 MHz) d 9.19 (d, 1H, J = 2.0 Hz, HO), 8.79
(d, 1H, J = 8.0 Hz, HK), 8.26 (dd, 1H, J = 8.5, 2.0 Hz, HD), 7.93
(dd, 2H, J = 5.5, 3.0 Hz, HR), 7.79 (dd, 2H, J = 5.5, 3.0 Hz, HS),
7.65 (d, 1H, J = 8.5 Hz, HE), 7.40 (d, 1H, J = 8.0 Hz, HJ), 5.13 (s,
2H, HI), 3.98 (s, 3H, HA); 13C NMR (CDCl3, 125 MHz) d 180.1
(1C, CM), 168.3 (2C, CP), 166.2 (1C, CB), 160.5 (1C, CH), 158.3 (1C,
CG), 142.7 (1C, CF), 139.1 (1C, CK), 134.7 (2C, CS), 133.2 (1C, CD),
132.4 (2C, CQ), 131.8 (1C, CO), 129.1 (2C, CC,N), 127.1 (1C, CE),
125.7 (1C, CL), 124.1 (2C, CR), 120.1 (1C, CJ), 52.9 (1C, CA), 43.1
(1C, CI); MS (MALDI, pencil matrix, FTMS+) m/z 430.9 [M +
H]+, 452.9 [M + Na]+; HRMS (MALDI, pencil matrix, FTMS+)
m/z found 431.0700 [M + H]+ C23H15O5N232S requires 431.0696;
C23H14N2O5S·1/6 C4H8O2 (%): calcd C 63.86 H 3.47 N 6.29; found
C 63.70 H 3.26 N 6.25; the constitution was confirmed by a single
crystal X-ray diffraction study.
Scheme 2 Synthetic pathway used for the preparation of [Eu·L1(H2O)]3+
.
2-Aminomethyl-7-methoxycarbonyl-1-azathioxanthone
A
solution of 2-phthalimidomethyl-7-methoxycarbonyl-1-
azathioxanthone (0.055 g, 0.128 mmol) and NH2NH2·H2O
(0.5 ml, 10.3 mmol) in DCM–MeOH (3 : 7, 10 ml) was heated at
50 ◦C for 2 h, at which point TLC analysis showed all starting
2-phthalimidomethyl-7-methoxycarbonyl-1-azathioxanthone had
been consumed. The clear yellow solution was allowed to cool
followed by the addition of conc. HCl(aq) until a pH of 2 was
reached. The resultant solution containing a white precipitate was
heated at 60 ◦C for 2 h then allowed to cool to room temperature,
The solution was filtered, followed by the drying of the yellow
filtrate under reduced pressure to yield a yellow powder. The
yellow solid was dissolved in H2O containing 1 eq. NaOMe then
extracted with DCM (5 ml), the organic phase was dried under
reduced pressure to yield a white powder (0.037 g, 0.123 mmol,
1
96%); H NMR (CDCl3, 500 MHz) d 9.21 (d, 1H, J = 2.0 Hz,
HO), 8.78 (d, 1H, J = 8.0 Hz, HK), 8.27 (dd, 1H, J = 8.0, 1.5 Hz,
HD), 7.71 (d, 1H, J = 8.0 Hz, HE), 7.47 (d, 1H, J = 8.0 Hz, HJ),
4.12 (s, 2H, HI), 3.99 (s, 3H, HA), 1.78 (s br, 2H, NH2); 13C NMR
(CDCl3, 125 MHz) d 180.3 (1C, CM), 167.4 (1C, CH), 166.3 (1C,
CB), 158.1 (1C, CG), 142.7 (1C, CF), 138.7 (1C, CK), 133.1 (1C,
CD), 131.8 (1C, CO), 129.2 (1C, CN), 129.0 (1C, CC), 127.1 (1C,
CE), 125.3 (1C, CL), 120.4 (1C, CJ), 52.8 (1C, CA), 48.0 (1C, CI);
MS (ES+) m/z 301.0 [M + H]+ 100%; HRMS (ES+) m/z found
301.0642 [M + H]+ C15H13O3N232S requires 301.0641.
Scheme 3 Synthetic pathway used for the preparation of [Eu·L2(H2O)]3+
.
2-Bromomethyl-7-methoxycarbonyl-1-azathioxanthone and
precursors were synthesised as described in ref. 17. 1,4,7,10-
Tetraaza-cyclododecane-1,7-dicarboxylic acid dibenzyl ester was
prepared as reported in ref. 18.
2-Phthalimidomethyl-7-methoxycarbonyl-1-azathioxanthone
4,10-Bis-[((S)-1-phenyl-ethylcarbamoyl)-methyl]-1,4,7,10-
tetraaza-cyclododecane-1,7-dicarboxylic acid dibenzyl ester
2-Bromomethyl-7-methoxycarbonyl-1-azathioxanthone (0.125 g,
0.343 mmol) and potassium phthalimide (0.191 g, 1.03 mmol)
in DMF (10 ml) were stirred, under argon, for 12 h at room
temperature. The resulting brown product mixture was poured
onto iced water (200 ml) forming a brown solution containing a
yellow oily precipitate that was extracted with dichloromethane
(5 × 50 ml). The crude organic extracts were combined and dried
under reduced pressure yielding the desired product as a crude
yellow-brown solid. The crude solid was dissolved in boiling ethyl
acetate (50 ml) then filtered hot, the solution volume was reduced
A
stirred mixture of 1,4,7,10-tetraaza-cyclododecane-1,7-
dicarboxylic acid dibenzyl ester (1.25 g, 2.84 mmol), 2-chloro-
N-[(S)-methylbenzyl]ethanamide (1.23 g, 6.22 mmol), Cs2CO3
(1.84 g, 5.65 mmol) and KI (10 mg) was boiled under refux in dry
MeCN (25 ml) for 24 h. The resultant orange tinged solution was
allowed to cool to room temperature then dried under reduced
pressure. The orange residue was taken up in dichloromethane
(15 ml), washed with sodium thiosulfate solution (10 ml, 0.01
M) then H2O (2 × 15 ml). The orange tinged organic layer was
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dried under reduced pressure to leave a viscous oil. The oil was
sonicated in diethyl ether (2 × 20 ml) and the insoluble material
was collected by centrifugation, dissolved in DCM (10 ml) then
dried under vacuum to give the desired product as an off-white
glassy solid (1.70 g, 2.23 mmol, 78%); mp 64–66 C; H NMR
(CDCl3, 500 MHz) d 7.59 (s br, 2H, NH), 7.20–7.35 (m, 20H,
amide arm and Cbz Ar), 5.11 (m, 2H, CH), 4.97 (s br, 4H, Cbz
CH2), 3.43 (m br, 8H, cyclen CH2), 3.15 (s br, 4H, CH2CO), 2.75
(m br, 8H, cyclen CH2), 1.44 (s br, 6H, CH3); 13C NMR (CDCl3,
CH), 28.8 (3C, tBOC CH3), 22.0 (2C, amide arm CH3); MS (ES+)
m/z 595.4 [M + H]+ 100%; HRMS (ES+) m/z found 595.3972
[M + H]+ C33H51O4N6 requires 595.3966.
◦
1
7-Ethoxycarbonylmethyl-4,10-bis-[((S)-1-phenyl-ethylcarbamoyl)-
methyl]-1,4,7,10-tetraaza-cyclododecane-1-carboxylic acid
tert-butyl ester
A boiling mixture of 4,10-bis-[((S)-1-phenyl-ethylcarbamoyl)-
methyl]-1,4,7,10-tetraaza-cyclododecane-1-carboxylic acid tert-
butyl ester (0.224 g, 0.377 mmol), ethyl bromoacetate (0.035 ml,
0.316 mmol) and Cs2CO3 (0.123 g, 0.378 mmol) in MeCN (10 ml)
was stirred vigorously for 12 h then allowed to cool to room
temperature. The mixture was dried under reduced pressure,
dissolved in DCM then extracted with saturated NaHCO3 (aq)
solution (10 ml) then H2O (2 × 10 ml). The yellow tinged organic
layer was dried under reduced pressure to leave a yellow tinged
oil. The desired product was isolated from the oil by column
chromatography on alumina (using DCM–MeOH starting from
100% DCM with 0.1% MeOH increments) as a clear glassy solid
(0.151 g, 0.222 mmol, 70%); RF (Alumina, DCM–MeOH, 95 : 5
v/v) : 0.61; mp 39–41 ◦C; 1H NMR (CDCl3, 500 MHz) d 7.87 (s
br, 1H, NH), 7.59 (s br, 1H, NH), 7.21–7.33 (m, 10H, Ar), 5.16 (m,
2H, CH), 4.07 (m, 2H, ethyl CH2), 2.18–3.36 (m, 22H, cyclen CH2,
amide arms CH2 and ester arm CH2), 1.51 (d br, 6H, J = 13.0 Hz,
amide arm CH3), 1.42 (m br, 9H, tBu CH3), 1.24 (t, 3H, J = 7 Hz,
=
=
125 MHz) d 170.2 (2C, amide arm C O), 157.0 (2C, Cbz C O),
143.8 (2C, amide arm Ar(q)), 136.5 (2C, Cbz Ar(q)), 128.8, 128.5,
128.4, 2 × 127.4, 126.5 (20C, amide arm and Cbz Ar), 67.5 (2C,
Cbz CH2), 59.2 (2C, CH2CO), 54.9–56.3 (br, 4C, cyclen CH2),
48.7 (2C, CH), 47.8–49.0 (br, 4C, cyclen CH2), 22.0 (2C, CH3);
MS (ES+) m/z 763.5 [M + H]+ 100%; HRMS (ES+) m/z found
763.4187 [M + H]+ C44H55O6N6 requires 763.4178.
N-((S)-1-Phenyl-ethyl)-2-(7-[((S)-1-phenyl-ethylcarbamoyl)-
methyl]-1,4,7,10-tetraaza-cyclododec-1-yl)-acetamide
4,10-Bis-[((S)-1-phenyl-ethylcarbamoyl)-methyl]-1,4,7,10-tetraaza-
cyclododecane-1,7-dicarboxylic acid dibenzyl ester (1.69 g,
2.22 mmol) in MeOH (30 ml) was shaken in a Parr hydrogenation
flask at 40 psi H2 over Pd(OH)2/C (0.15 g) for 24 h. The resulting
mixture was filtered through Celite to leave a clear solution which
was dried under reduced pressure to give the desired product as a
◦
1
glassy white solid (1.05 g, 2.12 mmol, 96%); mp 140–143 C; H
NMR (CD3OD, 500 MHz) d 7.28–7.32 and 7.21–7.23 (m, 10H,
Ar), 5.04 (m, 2H, CH), 3.54 (d, 2H, J = 17.0 Hz, CHCO), 3.41
(d, 2H, J = 16.5 Hz, CHCO), 2.88–3.18 (m br, 16H, cyclen CH2),
1.46 (d, 6H, J = 7.0 Hz, CH3); 13C NMR (CD3OD, 125 MHz)
ethyl CH3); 13C NMR (CDCl3, 125 MHz) d 171.0 (1C, ethyl ester
t
=
=
=
C O), 170.9, 170.3 (2C, amide C O), 156.2 (1C, BOC C O),
144.2, 143.1 (2C, Ar(q)), 129.0, 128.8, 127.9, 127.3, 127.0, 126.6
(10C, Ar), 80.1 (1C, tBOC(q)), 60.7 (1C, ethyl CH2), 59.5 (1C, ethyl
ester CH2CO), 53.3–55.0 (m br, 8C, cyclen CH2), 54.3 (2C, amide
t
=
d 172.7 (2C, C O), 145.8 (2C, Ar(q)), 130.5, 129.0, 128.0 (10C,
arm CH2CO), 48.5, 48.3 (2C, CH), 28.8 (3C, BOC CH3), 22.3,
Ar), 57.2 (2C, CH2CO), 52.4 (2C, CH), 51.1, 50.7, 45.2 (br, 8C,
cyclen CH2), 23.5 (2C, CH3); MS (ES+) m/z 495.3 [M + H]+
100%; HRMS (ES+) m/z 495.3447 [M + H]+ C28H43O2N6 requires
495.3442.
21.2 (2C, amide arm CH3), 14.6 (1C, ethyl CH3); MS (ES+) m/z
344.1 [M + H + Li]2+ 100%, 681.4 [M + H]+ 10%; HRMS (ES+)
m/z found 681.4341 [M + H]+ C37H57O6N6 requires 681.4334.
7-Carboxymethyl-4,10-bis-[((S)-1-phenyl-ethylcarbamoyl)-
methyl]-1,4,7,10-tetraaza-cyclododecane-1-carboxylic acid
tert-butyl ester
4,10-Bis-[((S)-1-phenyl-ethylcarbamoyl)-methyl]-1,4,7,10-
tetraaza-cyclododecane-1-carboxylic acid tert-butyl ester
A solution of di-tert-butyl dicarbonate (0.353 g, 1.61 mmol)
in dry MeOH (6 ml) was added dropwise over 1 h, at room
temperature, to a stirring solution of N-((S)-1-phenyl-ethyl)-2-(7-
[((S)-1-phenyl-ethylcarbamoyl)-methyl]-1,4,7,10-tetraaza-cyclo-
dodec-1-yl)-acetamide (0.998 g, 2.02 mmol) and NEt3 (0.560 ml,
4.04 mmol) in dry MeOH (30 ml). The reaction mixture was left to
stir overnight then dried under reduced pressure. The crude yellow
tinged viscous oil was purified by column chromatography on
alumina (utilising an incremental solvent system of DCM–MeOH
starting from 100% DCM with 0.1% MeOH increments) to yield
the desired product as a glassy white solid (0.573 g, 0.963 mmol,
60%); Rf (Alumina, DCM–MeOH, 49 : 1 v/v): 0.46; mp 98–101 ◦C;
1H NMR (CDCl3, 500 MHz) d 7.86 (s br, 1H, cyclen NH), 7.20–
7.33 (m, 10H, Ar), 5.13 (m br, 2H, CH), 3.09 (m br, 4H, CH2CO),
2.53–3.37 (m br, 16H, cyclen CH2), 1.50 (d, 6H, J = 7.0 Hz, amide
7-Ethoxycarbonylmethyl-4,10-bis-[((S)-1-phenyl-ethylcarbamoyl)-
methyl]-1,4,7,10-tetraaza-cyclododecane-1-carboxylic acid tert-
butyl ester (0.135 g, 0.198 mmol) and LiOH·H2O (0.025 g,
0.596 mmol) were boiled under reflux in H2O–MeOH (100 ml,
50 : 50 v/v) at 110 ◦C for 12 h. The solution was allowed to cool
then MeOH was removed under reduced pressure. The aqueous
solution was neutralised with dilute HCl(aq) then extracted with
DCM (3 × 20 ml) after the addition of brine (20 ml). The
combined organic washings were dried under reduced pressure
to leave the desired product as a glassy white solid (0.127 g,
0.195 mmol, 98%). 1H NMR (CDCl3, 400 MHz) d 7.18–7.32 (m,
10H, Ar), 5.10 (m br, 2H, CH), 2.31–3.22 (m br, 22H, cyclen CH2,
amide arms CH2CO and acid arm CH2CO), 1.46 (d, 6H, J =
t
4.8 Hz, amide arm CH3), 1.35 (m br, 9H, Bu CH3); 13C NMR
=
(CDCl3, 100 MHz) d 170.7 (br, 2C, amide C O), 159.9 (br, 1C,
t
t
arm CH3), 1.39–1.41 (m br, 9H, Bu CH3); 13C NMR (CDCl3,
BOC C O), 143.9 (br, 2C, Ar(q)), 128.9, 127.4, 126.8 (10C, Ar),
=
t
125 MHz) d 170.5 (2C, amide C O), 156.7 (1C, BOC C O),
143.8 (2C, Ar(q)), 128.8, 127.5, 126.8 (10C, Ar), 80.3 (1C, tBOC(q)),
59.6 (2C, CH2CO), 49.0–53.8 (m br, 8C, cyclen CH2), 48.7 (2C,
80.4 (1C, tBOC(q)), 58.4 (1C, acid arm CH2CO), 53.7 (m br, 10C,
cyclen CH2 and amide arm CH2CO), 49.1 (2C, CH), 28.7 (3C,
tBOC CH3), 22.2 (2C, amide arm CH3); MS (ES+) m/z 653.4
=
=
5730 | Dalton Trans., 2007, 5726–5734
This journal is
The Royal Society of Chemistry 2007
©

View Article Online
[M + H]+ 100%; HRMS (ES+) m/z found 653.4029 [M + H]+
C35H53O6N6 requires 653.4021.
CH), 4.57 (m br, 2H, HI), 3.95 (s, 3H, HA), 2.91–3.57 (m br, 22H,
cyclen, arm and linker CH2), 1.40 (d, 6H, J = 7.0 Hz, amide arms
CH3); 13C NMR (CD3CN, 125 MHz) d 180.5 (1C, CM), 171.3 (1C,
=
=
linker arm C O), 170.9, 170.4 (2C, amide arm C O), 166.6 (1C,
2-[(2-(7-tert-Butoxycarbonyl-4,10-bis-[((S)-1-phenyl-
ethylcarbamoyl)-methyl]-1-yl)-acetylamino)-methyl]-5-oxo-5H-
[1]benzothiopyrano[2,3-b]pyridine-7-carboxylic acid methyl ester
=
CB), 162.9 (1C, CH), 160.3 (q, 1C, TFA C O), 158.3 (1C, CG),
144.8, 144.5 (2C, arm Ar(q)), 142.9 (1C, CF) 139.1 (1C, CK), 133.6
(1C, CD), 131.3 (1C, CO), 129.8, 129.7 (2C, CC and CN) 129.4 (2C,
arm Ar), 128.2 (1C, CE), 127.9, 126.9 (8C, arm Ar), 126.1 (1C,
CL), 121.3 (1C, CJ), 116.9 (q, 1C, TFA CF3), 55.5, 51.0, 50.0 (br,
8C, cyclen CH2), 53.2 (1C, CA), 49.8 (br, 2C, arm CH), 45.6 (1C,
CI), 43.7 (br, 3C, arm and linker CH2), 22.7, 22.6 (br, 2C, amide
arm CH3); MS (ES+) m/z 835.4 [M + H]+ 25%; HRMS (ES+) m/z
found 835.3959 [M + H]+ C45H55O6N832S requires 835.3960.
7-Carboxymethyl-4,10-bis-[((S)-1-phenyl-ethylcarbamoyl)-methyl]-
1,4,7,10-tetraaza-cyclododecane-1-carboxylic acid tert-butyl
ester (0.109 g, 0.167 mmol), EDC·HCl (EDC = N-ethyl-Nꢀ-(3-
dimethylaminopropyl)diimide; 0.031 g, 0.162 mmol), HOBt·H2O
(5 mg) and NEt3 (0.093 ml, 0.668 mmol) in CHCl3 (5 ml) were
stirred for 45 min at room temperature to form a yellow tinged
solution. 2-Aminomethyl-7-methoxycarbonyl-1-azathioxanthone
(0.033 g, 0.110 mmol) in CHCl3 (5 ml) was then added to form
a dark yellow solution that was left to stir for 12 h at room
temperature. The resulting yellow solution was extracted with
HCl(aq) (10 ml, 0.025 M) then H2O (5 ml), the combined aqueous
fractions were backwashed with CHCl3 (5 ml) then the combined
organic layers were dried under reduced pressure to leave the
crude product as a yellow-brown foam. The desired product was
isolated via alumina column chromatography (using a graduated
solvent system starting from 100% DCM with 0.1% MeOH
increments every 50 ml) as a pale yellow glassy solid (0.060 g,
0.064 mmol, 58%); Rf (alumina, DCM–MeOH, 98.5 : 1.5 v/v):
Eu complex of 2-[(2-(4,10-Bis-[((S)-1-phenyl-
ethylcarbamoyl)methyl]-1-yl)acetylamino)-methyl]-5-oxo-5H-
[1]benzothiopyrano[2,3-b]pyridine-7-carboxylic acid methyl ester:
[Eu·L2(H2O)]3+
A
solution of 2-[(2-(4,10-bis-[((S)-1-phenyl-ethylcarbamoyl)-
methyl]-1-yl)acetylamino)-methyl]-5-oxo-5H-[1]benzothiopyrano-
[2,3-b]pyridine-7-carboxylic acid methyl ester (43 mg, 0.051 mmol)
and Eu(OTf)3·6H2O (21 mg, 0.030 mmol) in MeCN (1.5 cm3)
was refluxed under argon, in a Schlenk tube, for 48 h. Solvent
was removed under reduced pressure to leave a yellow glassy
solid. Dichloromethane (5 cm3) was added to the solid which
was sonicated for 5 min, solvent was then decanted leaving a
orange residue. The sonication process was repeated followed by
drying of the remaining residue, under reduced pressure, to yield
the triflate salt of the desired product as a fine yellow coloured
powder (36 mg, 0.025 mmol, 83%). The solid was made water
soluble by the exchange of triflate anions for chloride anions
using ‘DOWEX 1 × 8 200–400 mesh Cl’ ion exchange resin.
The resin was prepared by reflux in MeOH overnight followed
by washing with H2O (500 ml), 0.1 M HCl (200 ml) then H2O
(500 ml) at which point the washings were neutral pH. The
procedure involved the dissolving of the solid in MeOH (1 ml)
followed by the addition of H2O (1 ml), this complex solution
was added to a mixture of the resin (0.2 g) in H2O–MeOH (50 :
50 v/v, 5 ml) then stirred for 2 h. The resin was then removed by
filtration followed by the drying of the solution under reduced
1
0.25; H NMR (CDCl3, 500 MHz) d 9.14 (s, 1H, HO), 8.68 (d,
1H, J = 8.5 Hz, HK), 8.22 (dd, 1H, J = 8.5, 1.5 Hz, HD), 7.65
(d, 1H, J = 8.5 Hz, HE), 7.15–7.30 (m, 13H, amide arms Ar,
amide arms NH and HJ), 5.93 (s br, 1H, linker amide NH), 5.07
(m, 2H, arm CH), 4.49 (m, 2H, HI), 3.94 (s, 3H, HA), 2.40–3.30
(m br, 22H, cyclen CH2 and amide arms CH2CO), 1.43 (d, 6H,
t
J = 7.0 Hz, amide arms CH3), 1.37 (s, 9H, Bu CH3); 13C NMR
(CDCl3, 125 MHz) d 179.9 (1C, CM), 170.1 (3C, amide arms and
=
linker C O), 166.1 (1C, CB), 162.6 (1C, CH), 157.8 (1C, CG), 156.1
t
=
(1C, BOC C O), 143.5 (2C br, amide arms Ar(q)), 142.1 (1C,
CF), 138.8 (1C, CK), 133.1 (1C, CD), 131.7 (1C, CO), 129.2 (1C,
CN), 129.1 (1C, CC), 128.9, 127.6, 126.5 (10C, amide arms Ar),
127.1 (1C, CE), 125.4 (1C, CL), 120.9 (1C, CJ), 80.2 (1C, tBOC(q)),
60.0, 58.0, 54.7, 53.6 (8C, cyclen CH2), 52.8 (1C, CA), 49.0 (2C,
amide arms CH), 48.7, 48.2 (3C, amide arms and linker CH2CO),
44.6 (1C, CI), 28.8 (3C, tBOC CH3), 22.0 (2C, amide arms CH3);
MS (ES+) m/z 935.2 [M + H]+ 100%; HRMS (ES+) m/z found
935.4495 [M + H]+ C50H63O8N832S requires 935.4484.
1
pressure to yield the complex in quantitative yield; H NMR (as
tri-chloride salt, D2O, 500 MHz, partial data and assignment) d
24.7 (1H, NH), 16.6 (1H, Hax), 16.2 (1H, Hax), 13.7 (1H, Hax), 11.5
(1H, Hax); MS (ES+) m/z 515.3 [M + HCO2]2+ 50%; HRMS (ES+)
m/z found 1043.3146 [M + CH3CO2 − H]+ C47H56O8N8151Eu32S
requires 1043.3135; kabs(H2O): 372 nm; s (H2O): 0.33 ms, s (D2O):
0.65 ms.
2-[(2-(4,10-Bis-[((S)-1-phenyl-ethylcarbamoyl)methyl]-1-
yl)acetylamino)-methyl]-5-oxo-5H-[1]benzothiopyrano[2,3-
b]pyridine-7-carboxylic acid methyl ester
2-[(2-(7-tert-Butoxycarbonyl-4,10-bis-[((S)-1-phenyl-ethylcarba-
moyl)-methyl]-1-yl)-acetylamino)-methyl]-5-oxo-5H-[1]benzothio-
pyrano[2,3-b]pyridine-7-carboxylic acid methyl ester (36 mg,
0.038 mmol) in DCM–TFA (1 : 1 v/v, 2 ml) was stirred in a
sealed flask overnight at room temperature yielding a dark yellow
solution. Solvent was then removed under reduced pressure to
yield the desired product as its TFA salt as a glassy orange solid
in quantitative yield; 1H NMR (CD3CN, 500 MHz) d 8.99 (s, 1H,
HO), 8.67 (d, 1H, J = 8.0 Hz, HK), 8.24 (dd, 1H, J = 8.5 Hz
1.5 Hz, HD), 7.81 (d, 1H, J = 8.5 Hz, HE), 7.47 (d, 1H, J = 8.0 Hz,
HJ), 7.24–7.34 (m br, 10H, arm Ar), 4.88–4.96 (m br, 2H, arm
1,7-Bis(benzoyloxycarbonyl)-4,10-bis(N-acetyl-L-alanine ethyl
ester)-1,4,7,10-tetraazacyclododecane
1, 7 - Bis ( benzyloxycarbonyl ) - 1, 4, 7, 10 - tetraazacyclododecane
(2.40 g, 5.45 mmol) and N-bromoacetyl(L)alanine ethyl ester
(2.74 g, 11.5 mmol) were dissolved in dry MeCN (50 mL) followed
by the addition of K2CO3 (4.50 g, 32.61 mmol). The mixture was
stirred at 60 ◦C for 5 d under argon and the reaction was monitored
by ESMS and TLC (DCM 3% MeOH, alumina) to confirm that
all N-bromoacetyl-L-alanine ethyl ester starting material had been
This journal is
The Royal Society of Chemistry 2007
Dalton Trans., 2007, 5726–5734 | 5731
©

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consumed. The potassium salts were filtered off and MeCN was
removed by rotary evaporation to give a dark yellow oil as a crude
product, which was purified by column chromatography (alumina,
DCM–5% MeOH), to yield the title compound as a pale yellow
oil, (3.62 g, 88%). dH (CDCl3) 7.60 (H3, d, 2H, J 6.0 Hz), 7.33
(H13 br.s, 10 H), 5.12 (H12, s, 4H), 4.60 (H4, p, 2H, J = 7.2 Hz),
4.17 (H7, h, 4H, J = 7.2 Hz), 3.74 (H1, m, 4H), 2.89 (H9,10, br.m,
18H) 1.41 (H5, d, 6H, J = 7.2 Hz), 1.28 (H8, t, 6H, J = 7.1 Hz).
dc (CDCl3) 173.9 (C6), 170.4 (C2), 136.8; 128.4; 127.9; 127.8 (C13),
61.9 (C7), 61.5 (C1), 54.1, 48.2 (C9,10 + C4), 18.6 (C5), 14.4 (C8), m/z
(ESMS+) 755 (M + H), 777 (M + Na). Rf 0.62 (alumina, DCM–3%
MeOH).
7-Aminomethyl-1-azathioxanthone
A solution of 7-phthalimidomethyl-1-azathioxanthone (300 mg,
807 lmol) in DCM (20 ml) was mixed with NH2NH2·H2O (117 ll,
2.42 mmol) and MeOH (5 ml). The mixture was heated at 50 ◦C
followed by further additions of NH2NH2·H2O (80 ll, 1.6 mmol)
at 3 h intervals, until TLC analysis (silica, DCM–2.5% MeOH)
showed that all starting 7-phthalimidomethyl-1-azathioxanthone
had been consumed. The white/pale yellow cloudy mixture was
allowed to cool followed by the addition of 6 M HCl(aq.) until
the pH of 2 was reached. The resultant solution containing a
white precipitate was heated for 1 h then allowed to cool to room
temperature and further cooled in the fridge for 1 h. The solution
was filtered, followed by drying of the yellow filtrate under vacuum
to yield the title compound as the hydrochloride salt (confirmed
1,7-Bis(N-acetyl-L-alanine ethyl ester)-1,4,7,10-tetraaza-
cyclododecane
1
by H NMR). This was dissolved in H2O (25 ml containing 1.5
eq. NaOMe) then extracted with CHCl3 (3 × 50 ml). The organic
phase was dried under reduced pressure to leave the title product
as a bright yellow solid (58 mg, 240 lmol, 37%); mp 114–115 ◦C
(found C, 64.21; H, 4.60; N, 11.86; S, 13.10. C13H10N2OS requires
C, 64.46; H, 4.13; N, 11.57; S, 13.22%); dH (CDCl3) 8.83 (1H, dd, J
7.8 Hz, H4), 8.80 (1H, dd, J 7.8 Hz, H2), 8.51 (1H, s, H6), 7.68 (1H,
d, J 8.0 Hz H8), 7.62 (1H, d J 8.0 Hz, H9), 7.45 (1H, q, J 7.8 Hz, H3),
4.03 (2H, s, H10), 1.67 (2H, br.s, H11); dc (CDCl3) 46.2 (C10), 121.8
(C3), 126.6 (C1ꢀ ), 127.0 (C9), 128.0 (C6), 129.6 (C6ꢀ ), 132.6 (C8),
136.0 (C7), 138.1 (C2), 142.5 (C9ꢀ ), 153.5 (C4), 159.0 (C4ꢀ ), 180.9
(C5); m/z (ESMS+) 243 (M + H), 265 (M + Na); IR(CDCl3)[cm−1]:
1,7-Bis(benzoyloxycarbonyl)-4,10-bis(N-acetyl-L-alanine
ethyl
ester)-1,4,7,10-tetraaza-cyclododecane (3.60 g, 47.52 mmol)
was dissolved in absolute EtOH (100 mL) and Pd(OH)2/C (Pd
content 10%, 170 mg) was added. The mixture was hydrogenated
in a Parr hydrogenation apparatus (40 psi) for 5 d. The catalyst
was filtered off and EtOH was removed under reduced pressure.
The resulting oil was dissolved in DCM (50 mL) and the solution
was extracted with cold aq. NaOH solution (20% (w/v), 5 mL)
followed by extraction with H2O (3 × 30 ml). The organic phase
was dried and the solvent removed under vacuum to give the
title compound as a pale yellow oil (2.11 g, 91%). dH (CDCl3)
8.15 (H11, d, 2H, J = 6.0 Hz), 7.60 (H3, d, 2H, J 6.0 Hz), 4.61
(H4, p, 2H, J = 7.2 Hz), 4.17 (H7, h, 4H, J = 7.2 Hz), 3.75 (H1,
m, 4H), 2.91 (H9,10, m, 18H) 1.41 (H5, d, 6H, J = 7.2 Hz), 1.28
(H8, t, 6H, J = 7.1 Hz). dc (CDCl3) 173.9 (C6), 170.6 (C2), 61.9
(C7), 61.6 (C1), 53.0, 47.4 (C9,10), 48.1 (C4), 18.6 (C5), 14.4 (C8),
m/z (HRMS+) 487.3240 (C22H42O6N6 requires 487.3239), Rf 0.26
(alumina, DCM–7% MeOH).
=
808 (tri.sub.benzol), 826 (mNH2), 1076 (frame C O), 1159 (mC–N),
(dsAr–CH2), 1400 (mC–S), 1448 (dsCO–CH2), 1474 (dasAr–CH2),
=
1580 (mNH2), 1602 (frame Ar(C–C)), 1641 (mC O in conjugated
aromatic ring system), 2930 (aliphatic-CH2), 3048 (m aromatic-
CH2), 3384 (mNH2). Rf = 0.06 (silica, DCM–2.5% MeOH).
7-(N-Methyl-2-chloro-acetamide)-1-azathioxanthone
7-Aminomethyl-1-azathioxanthone (40 mg, 165 lmol) and 2,5-
dioxo-pyrrolidin-1-yl chloroacetate (60 mg, 248 lmol)) in DMF
(3 ml) were stirred, under argon, for 2 h at room temperature,
until TLC analysis (silica, DCM–3.5% MeOH) showed that all
starting 7-aminomethyl-1-azathioxanthone had been consumed.
The solvent was evaporated under reduced pressure and the
remaining bright yellow solid was sonicated in H2O (pH = 5,
trace amount of LiCl). The mixture was extracted with DCM (3 ×
100 ml) and the organic phase was dried and the solvent removed
under reduced pressure to give the product as a pale yellow powder
which was purified by column chromatography (silica, DCM–3%
MeOH) to yield the title compound as a pale yellow crystalline
solid (45 mg, 141 lmol, 85%); mp 188–190 ◦C (found C, 56.68; H,
3.51; N, 8.70; S, 10.01. C15H11N2O2SCl requires C, 56.52; H, 3.45;
N, 8.79; S, 10.04%); dH (CDCl3) 8.86 (1H, dd, J 8.0 Hz, H4), 8.83
(1H, dd, J 8.0 Hz, H2), 8.51 (1H, s, H6), 7.67 (2H, d, H8,9), 7.50 (1H,
q J 8.0 Hz, H3), 7.06 (1H, s, H11), 4.66 (2H, d, J 6.1 Hz H10), 4.17
(2H, s, H13); dc (CDCl3) 42.8 (C13), 43.5 (C10), 122.1 (C3), 126.6
(C1ꢀ ), 127.4 (C9), 128.8 (C6), 129.1 (C6ꢀ ), 133.0 (C8), 136.7 (C9ꢀ ),
137.2 (C7), 138.3 (C2), 153.7 (C4), 158.8 (C4ꢀ ), 166.6 (C12), 180.7
(C5); m/z (ESMS+) 341, 343 (1 : 3) (M + Na); IR(CDCl3)[cm−1] :
7-Phthalimidomethyl-1-azathioxanthone
7-Methyl-1-azathioxanthone (200 mg, 885 lmol) and potassium
phthalimide (655mg, 3.54 mmol) in DMF (20 ml) were stirred,
under argon, for 16 h. at room temperature. The solution was
poured into ice/water (300 ml) and a white/pale yellow precipitate
was formed, the mixture was cooled in the fridge for 2 h. The
mixture was extracted with DCM (3 × 300 ml) and the organic
phase was dried and the solvent removed under reduced pressure
to give a pale yellow powder which was dissolved in DCM
(100 ml) and washed with water (pH 10, 2 × 100 ml). The organic
phase evaporated and the remaining pale yellow powder was
recrystallised from DCM–EtOH to yield the title product as a
pale yellow solid (310 mg, 831 lmol, 94%); mp >240 ◦C (Found
C, 67.53; H, 3.29; N, 7.87; S, 8.71. C21H12N2O3S requires C, 67.74;
H, 3.23; N, 7.53; S, 8.60%); dH (CDCl3) 8.82 (1H, dd, J 7.9 Hz,
H4), 8.80 (1H, dd, J 7.9 Hz, H2), 8.61 (1H, s, H6), 7.88 (1H, m,
H13), 7.75 (2H, m, H8+14), 7.62 (1H, d J 8.0 Hz, H9), 7.45 (1H,
q, J 8.1 Hz, H3), 5.00 (2H, s, H10); dc (CDCl3) 41.3 (C10), 122.2
(C3), 123.8, 123.9 (C13), 126.6 (C1ꢀ ), 127.3 (C9), 129.2 (C6ꢀ ), 129.6
ꢀ
(C6ꢀ), 132.9 (C12 ), 133.3 (C8), 134.5, 134.6 (C14), 135.6 (C7), 137.2
(C9 ), 138.1 (C2), 153.6 (C4), 158.8 (C4ꢀ ), 168.1 (C11), 180.5 (C5);
m/z (ESMS+) 373 (M + H), 395 (M + Na), 411 (M + K), Rf = 0.8
(silica, DCM–2.5% MeOH).
762 (mC–Cl), 807 (tri.sub.benzol), 1077, 1160 (frame C O), 1136
=
=
(mC–N), 1200 (mC–C( O)), 1263, 1309 (mC–N), 1385 (dsAr–
CH2), 1400 (mC–S), 1448 (dsCO–CH2), 1474 (dasAr–CH2), 1581
5732 | Dalton Trans., 2007, 5726–5734
This journal is
The Royal Society of Chemistry 2007
©

View Article Online
=
(AMID-II), 1605 (frame Ar(C–C)), 1643 (mC O in conju-
Photoluminescence studies
=
gated aromatic ring system), 1676 (mC Oamid), 2889 (aliphatic-
The theoretical framework and experimental protocol for TPE
measurements has been outlined by Webb et al.15 In this approach
the ratio of the reference and sample TPE-PL is given by,
CH2), 3049 (mAr–CH2), 3134 (AMID-II), 3278 (mNHamid), 3423
(mNHSec.amid). Rf = 0.61 (silica, DCM–3% MeOH).
rS2 · φS
CR · nS · FS (k)
=
rR2 · φR
CS · nR · F (k)
R
4-[7-(N-Methyl-2-chloro-acetamide)-1-azathioxanthone]-1,7-
bis(N-acetyl-L-alanine ethyl ester)-1,4,7,10-tetraaza-
cyclododecane
where φ is the total emission quantum yield of the compound,
composite PL of both chromophore and Eu(III) ion for φS, C is
the concentration, n the refractive index and F(k) is the integrated
PL spectrum. In these experiments we have ensured that the
excitation flux and excitation wavelengths are the same for both
sample (S) and reference (R). The two-photon excitation source
consisted of a mode-locked cavity dumped (APE(tm) Pulse switch)
Ti:sapphire laser (MIRA(tm), Coherent) producing near infrared
optical pulses with a temporal width of ∼150 fs (FWHM) in the
wavelength range 740–810 nm, and a repetition rate of 4 MHz.
The excitation light was focused into the sample solution using
an Olympus LWD C A20 (20×) objective. The emission was
subsequently collected using a dichroic mirror (Semrock FF735)
and detected using a fiber coupled CCD spectrograph (Avantes
Avaspec 2048FT) with a 100 lm core fibre. The laser power was
monitored using a photodiode with a known response curve,
calibrated against a free standing power meter. The intensity
of the excitation light was varied using a variable ND filter
(Edmund Optics) mounted on a translation stage. Typical average
excitation power was 5–50 mW corresponding to pulse energies
of 1.25–12.5 nJ pulse−1. Spectra from the CCD spectrograph were
corrected for background and spectral response. Validation of the
spectrometer was performed using Rhodamine B in methanol and
Fluorescein in 0.1 M aqueous NaOH. Two photon absorption
cross sections of 60 lM Eu·L1(H2O)]3+ and 80 lM [Eu·L2(H2O)]3+
were calculated relative to 2 lM Fluorescein in 0.1 M NaOH, at
an excitation wavelength of 760 nm, where r2 = 36 GM and φf =
0.9.1
1,7-Bis(N-acetyl-L-alanine ethyl ester)-1,4,7,10-tetraaza-cyclo-
dodecane (200 mg, 411 lmol) was combined with 7-(N-methyl-
2-chloro-acetamide)-1-azathioxanthone (100 mg, 314 lmol) and
KHCO3 (1 eq., 32 mg) and the mixture stirred in dry MeCN
(10 mL), which was heated initially at 50 ◦C for 2 h followed
by 70 ◦C for 22 h under argon. The reaction was monitored
by TLC (DCM, 5% MeOH, alumina) and ESMS+ to confirm
that the chlorinated starting material had been consumed. The
solvent was removed under reduced pressure and the resulting
solid was dissolved in a small volume of DCM (5 mL) and
the potassium salts removed by filtration. The crude mixture
was purified by column chromatography (DCM → 3% MeOH,
alumina); fractions containing clean product were combined and
the solvents were removed under reduced pressure to yield the title
compound as a pale yellow solid (80 mg, 104 lmol, 33%); mp 138–
140 ◦C. dH (CDCl3) 8.76 (H2,4, m, 2H), 8.35 (H6, s, 1H), 8.11 (H16, d,
2H, J = 6.2 Hz), 7.72 (H8,24, m, 3H), 7.56 (H9, q, 1H, J = 8.1 Hz),
7.43 (H3, m, 1H), 7.35 (H11, d, 1H, J = 6.2 Hz), 4.48 (H17, 2H, m),
ꢀ
ꢀ
4.10 (H10,20, m, 6H), 3.63 (H14, m, 4H), 3.10 (H13,22,22 ,23,23 , m, 18H),
1.39 (H18, m, 6H), 1.24 (H21, t, 6H, J = 7.2 Hz). dc (CDCl3) 180.5
(C5), 173.0 (C19), 171.7 (C15), 171.1 (C12), 158.9 (C4ꢀ ), 153.6 (C4),
138.8 (C2), 138.0 (C7), 136.3 (C9ꢀ ), 133.0 (C8), 128.8 (C6ꢀ ), 128.2
(C6), 127.0 (C9ꢀ), 126.4 (C11), 121.9 (C3) 61.7 (C20), 55.9, 53.7, 53.1,
ꢀ
ꢀ
47.3 (C22,22 ,23 23 ), 46.1 (C13), 42.7 (C10), 17.6 (C18), 14.4 (C21). m/z
(HRMS+) 739.3690 (C37H52O8N8S requires 769.37016). Rf 0.15
(alumina, DCM–5% MeOH).
The time-gated PL spectra were aqcuired using a repetition
rate of 1.8 MHz with an energy of 20 nJ pulse−1. The lumine-
cence was detected using a single photon avalanche photodiode
(idQuantique id100–50) connected to a scanning monochromator
(IBH 5000 M). The TTL signal from the detector was gated using
an AND logic gate, the timing being achieved using a variable
duration TTL pulse synchronised with the laser cavity dumper
driver.
[Eu·L1(H2O)]3+
4-[7-(N-Methyl-2-chloro-acetamide)-1-azathioxanthone]-1,7-bis-
(N-acetyl-L-alanine ethyl ester)-1,4,7,10-tetraaza-cyclododecane
(25 mg, 55 lmol) was added to Eu(CF3SO3)3 (1 eq., 33 mg) and
the solids dissolved in dry MeCN (2 mL) and the reaction left
◦
to stir at 70 C for 72 h. After the reaction was cooled to room
Acknowledgements
temperature, the solvents were removed under reduced pressure
and the remaining residue was dissolved in 0.1 mL dry MeCN and
the mixture was dropped onto anhydrous Et2O which resulted in
precipitation of the title compound as a triflate salt. The precipitate
was separated by centrifugation and dissolved in 5 ml H2O. The
pH was then adjusted carefully to 10 by addition of conc. NaOH
solution (in order to remove the excess Eu as Eu(OH)3) resulting
in a white precipitate, removed via a fine syringe filter. The pH
was adjusted back to neutral and the mixture lyophilised to give
a bright yellow solid (60 mg, 49 lmol). m/z (HRMS−) 1221.2056
(C37H53O8N8S2Eu(CF3SO3)2 requires 1221.2033); kmax(H2O) 384
(4070 dm3 mol−1 cm−1); sEu(H2O, pH = 7.4): 440 ms; sEu(D2O, pD = 7.1): 899 ms;
We thank EPSRC, the Royal Society, Durham University and the
EC networks EMIL and DiMI for support.
Notes and references
1 D. Parker and R. Pal, Chem. Commun., 2007, 474; S. Pandya, J.
Yu and D. Parker, Dalton Trans., 2006, 2757; T. Gunnlaugsson and
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245.
φEu
= 5.1%.
(pH = 7.4)
This journal is
The Royal Society of Chemistry 2007
Dalton Trans., 2007, 5726–5734 | 5733
©

View Article Online
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5734 | Dalton Trans., 2007, 5726–5734
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The Royal Society of Chemistry 2007
©