Synthesis and characterisation of highly emissive and kinetically stable lanthanide complexes suitable for usage 'in cellulo'

Article abstract of DOI:10.1039/b418964g

The synthesis and photophysical characterisation are reported of a series of cationic, neutral and anionic europium and terbium complexes based on structurally related, nonadentate ligands based on the cyclen macrocycle. Each complex incorporates a tetraazatriphenylene moiety and overall absolute emission quantum yields are in the range 15-40% in aerated aqueous media. Dynamic quenching of the lanthanide excited state occurs with electron-rich donors, e.g. iodide, ascorbate and urate, and a mechanistic interpretation is put forward involving an electron transfer process. The cationic lanthanide complexes are taken up by N1H/3T3 cells and tend to localise inside the cell nucleus. The Royal Society of Chemistry 2005.

Full text of DOI:10.1039/b418964g

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A R T I C L E
Synthesis and characterisation of highly emissive and kinetically
stable lanthanide complexes suitable for usage ‘in cellulo’
Robert A. Poole,a Gabriella Bobba,a Martin J. Cann,b Juan-Carlos Frias,a David Parker*a and
Robert D. Peacockc
a Department of Chemistry, University of Durham, South Road, Durham, UK DH1 3LE
b Department of Biological Sciences, University of Durham, South Road, Durham,
UK DH1 3LE
c Department of Chemistry, University of Glasgow, Glasgow, UK G12 8QQ
Received 20th December 2004, Accepted 19th January 2005
First published as an Advance Article on the web 2nd March 2005
The synthesis and photophysical characterisation are reported of a series of cationic, neutral and anionic europium
and terbium complexes based on structurally related, nonadentate ligands based on the cyclen macrocycle. Each
complex incorporates a tetraazatriphenylene moiety and overall absolute emission quantum yields are in the range
15–40% in aerated aqueous media. Dynamic quenching of the lanthanide excited state occurs with electron-rich
donors, e.g. iodide, ascorbate and urate, and a mechanistic interpretation is put forward involving an electron transfer
process. The cationic lanthanide complexes are taken up by NlH/3T3 cells and tend to localise inside the cell nucleus.
Introduction
Our understanding of the photophysical principles involved in
devising emissive lanthanide probe complexes has developed to
such an extent that we are now able to relate complex structure to
spectral property with sufficient confidence that the development
of practicable probes can be undertaken more assertively.1–3
This is evident in the increasing usage of luminescent europium
cryptate complexes as donors in a variety of time-resolved
homogeneous assays. These are of interest, for example, to bio-
scientists using FRET techniques to study G-protein-coupled
receptors and to chemists more interested in their application
as luminescent tags in high-throughput technology.4 It has also
driven the development of ‘responsive’ lanthanide complexes in
which changes in the emission profile have been used to signal
variations in the local environment, e.g. pH, pO2, pX and pM,
allowing the definition of luminescent sensors.5,6 Most work
has concentrated on the more emissive Eu and Tb complexes
emitting in the red and green.
Scheme 1 Photophysical processes following light absorption.
the quenching processes (i.e. via electron or energy transfer)
that may limit the overall emission quantum yield.5 Examples
of such processes include singlet excited state quenching by
halide anions or by Eu3+ ion (minimised by systems with a fast
rate of inter-system crossing and a high oxidation potential);
triplet quenching by oxygen (averted in systems with fast energy
transfer from the triplet sensitiser to the lanthanide ion) and
electron/charge transfer quenching of the long-lived lanthanide
excited state by electron-rich species. The latter case is more
likely to be prevalent in cationic complexes or with systems in
which the ligand has a low-lying LUMO.
The tetraazatriphenylene chromophore is an established sen-
sitiser for lanthanide luminescence.9 It possesses a fast rate of
inter-system crossing, so that the triplet excited state may be
populated efficiently in polar media. It has a triplet energy of the
order of 24 000 cm−1 (singlet energy ca. 29 000 cm−1) and, being
electron-poor, it is difficult to oxidise. It may serve as a bidentate
ligand and several examples of ruthenium (and related d-block)
complexes incorporating this ligand have been examined as
luminescent probes for nucleic acids.10,11 Indeed, examples of
enantiopure cationic complexes have been postulated to bind
with some selectivity to DNA, with contributions from electro-
static attraction, groove and intercalative binding. Analogous
complexes of the ‘f’-block ions are virtually unexplored. The
first examples were based on phenanthridinium conjugates12 but
were of limited utility as the sensitiser singlet excited state was
quenched by electron transfer, associated with intercalation with
GC-rich DNA.
An essential step in continuing to extend their utility is to
devise probes that may be used for live cell imaging and related
‘in vivo’ applications. The key issues to address are: complex
stability/toxicity; excitation wavelength and the product of
overall emission quantum yield and the extinction coefficient
at that wavelength; absence of competitive quenching processes
from endogenous ions/biomolecules. Thus, the lanthanide com-
plex must possess sufficient kinetic stability to resist premature
decomplexation or chemical degradation. Fortunately, there are
obvious parallels to be drawn in the chemistry of gadolinium
contrast agents in MRI and in radiolabelled lanthanide conju-
gates being used in clinical imaging and therapy.7 As lanthanide
f–f transitions are parity-forbidden and exceedingly weak, an
appropriate sensitiser needs to be engineered into the complex
capable of absorbing light efficiently (high e) and transferring
its excited state energy to the lanthanide ion8 (Scheme 1). The
excitation wavelength should preferably be in the range 340–
420 nm. The lower limit marks the wavelength below which
quartz optics are required and, also, includes the tail absorption
wavelength for several common biomolecules. The upper limit is
set by the limited number of chromophores with a small singlet–
triplet energy gap that possess a triplet energy of more than
22 000 cm−1 (cf. the lowest energy 5D4 Tb ‘acceptor’ excited state
at 20 400 cm−1). Finally, each of the excited states along the
photophysical reaction pathway (singlet and triplet states of the
sensitiser and the lanthanide excited state itself) should resist
Here, we report the synthesis, and aspects of the charac-
terisation, of an emerging family of enantiopure lanthanide
complexes. A series of nine-coordinate ligands has been prepared
based on the ‘cyclen’ ring skeleton. Each ligand includes a
T h i s j o u r n a l i s
T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 0 1 3 – 1 0 2 4
1 0 1 3
©

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2-substituted tetraazatriphenylene moiety. Some parts of this
work have appeared in preliminary communications.13,14
were not very successful, and were compromised by competitive
perhalogenation, as had been noted in the attempted functional-
isation of 2,9-dimethyl-1,10-phenanthroline.17 Accordingly, an
alternative route was used, involving stepwise oxidation with
SeO2 16 (13, 14), reduction with sodium borohydride17 in ethanol
(to give 15 and 16) and chlorination with PCl3 to yield 8
and 9.
Ligand and complex synthesis
The target series of complexes, [Ln.1]3+ to [Ln.7]3−, is based
on structurally related nonadentate ligands, designed to form
coordinatively saturated lanthanide complexes. The variation of
overall charge on the complex was considered to be an important
issue as this affords (inter alia) a simple means of modulating
the quenching effect of anionic or electron-rich species. The
commonality of the 2-tetraazatriphenylene structural motif led
to the initial requirement of an effective synthesis of the 2-
halomethyl compounds, 8 and 9.
Reaction of 1,10-phenanthroline with one equivalent of
methyl lithium, followed by oxidation with MnO2 in the presence
of MgSO4 afforded the 2-methyl derivative in 51% yield.
Oxidation of the 9,10-double bond with molecular bromine
under strongly acidic conditions15 gave the dione, 10, in 77%
yield. Condensation of 10 with 1,2-diaminoethane or 1,2-
diaminocyclohexylamine was more effective in ethanol16 than
THF9 and yielded 11 (2-Me dpq) and 12 (2-Me dpqC) in good
yield, each of which was readily recrystallised from hot ethanol.
Attempts to introduce a bromine or chlorine atom selectively
at the a-Me site, by radical halogenation with NBS or NCS,
18
Selective mono-substitution of the cyclen ring was achieved
via alkylation of tri-BOC-cyclen, 17,19 with 8 or 9 in ace-
tonitrile to yield the carbonates 18 and 19. Deprotection of
the BOC protecting groups (CF3CO2H/CH2Cl2) yielded the
tetramines 20 and 21. Alkylation with (S)-N-2-chloroethanoyl-
2-phenylethylamine (MeCN, K2CO3, cat. KI) afforded the
triamides 1 and 2 which were purified by chromatography
on neutral alumina. Similar procedures for 21 using the N-2-
chloroethanoyl derivatives of ethyl alaninate and diethyl gluta-
mate, yielded the related ligands 4 and 5. For the synthesis of
the tris(carboxymethyl) ligand, 3, an alternative procedure was
used. Direct alkylation of 1,4,7-tris(t-butoxycarbonylmethyl)-
1,4,7,10-tetraazacylododecane, 22, with 9 under SN2 conditions
afforded the tri-ester 23. Deprotection with TFA gave the ligand
3, and complexation with the LnCl3 salt at pH 5.5 gave the
neutral species, [Ln.3], which was amenable to purification by
column chromatography. For the cationic triamide complexes,
reaction of the ligand with one equivalent of Ln(CF3SO3)3
1 0 1 4
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the cationic amide complexes [Ln.4]3+ and [Ln.5]3+ afforded the
zwitterionic and anionic complexes, [Ln.6] and [Ln.7]3−, which
were purified by ion exchange chromatography.
Complex characterisation
The absorption spectra of (RRR)- and (SSS)-[Ln.1]3+ in water
were identical, with major bands at 262 (e = 19 000 dm3 mol−1
cm−1), 305 (e 10 900 M−1 cm−1) and 340 nm (e 3500 M−1 cm−1).
For the lanthanide complexes of 2, and for each complex bearing
a coordinated ‘dpqC’ chromophore, the absorption spectra
were slightly red-shifted, with the longest wavelength band at
348 nm (e 8300 M−1 cm−1). Mirror image circularly polarised
in dry MeCN gave the desired triflate salt. This could be
converted to the more water soluble chloride salt by anion
exchange chromatography. Finally, controlled base hydrolysis of
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Table 2 Hydration states, q,24 and rate constants (kobs/ms−1
; 10%) for
depopulation of the lanthanide excited state for [Ln.1]3+ (kexc = 340 nm)
and [Ln.2]3+ (kexc = 350 nm) (pH 7.4, 100 mM HEPES, 10 mM NaCl)
Complexa
kH
kD
q
2O
2O
[Eu.1]3+
[Eu.2]3+
[Tb.1]3+
[Tb.2]3+
0.95
0.96
0.54
0.64
0.61
0.63
0.42
0.58
0
0
0.3
0
a Similar sets of values were obtained for [Ln.3], [Ln.4]3+, [Ln.5]3+, [Ln.6]
and [Ln.7]3− and were unchanged in deoxygenated samples.
coordination environment.2,3 Indeed, a sign reversal was also
observed when changing from a hard donor (e.g. OH2) to a more
polarisable P–O bond (e.g. HMPA) with [Eu.23]3+. In [Ln.1]3+,
it is very likely that the axial site in the putative nine-coordinate
complex is occupied by a pyridyl N atom. Support for this
premise comes from the 1H NMR assignment of [Eu.1]3+ in D2O
or CD3OD. The proton a to this coordinated nitrogen (H-10)
was assigned (COSY) at +31 ppm (243 K, CD3OD), consistent
with its location within the large paramagnetic shielding effect
associated with the ‘McConnell cone’. This is usually defined
in relation to a principal (2) axis, passing through the apical
substituent. A similarly large coordination shift for an analogous
proton has been reported by Botta, Quici and co-workers,
in a related 2-substituted phenanthridinyl complex23 based on
cyclen.
Fig. 1 Mirror image CPL spectra for (SSS)-D (green) and (RRR)-K
(purple) europium and terbium complexes of 1. The blue trace represents
the total emission spectrum (kexc = 340 nm, pH 7.4, 100 mM HEPES,
10 mM NaCl).
The number of coordinated water molecules in [Ln.1]3+ and
[Ln.2]3+ was assessed by measuring the radiative rate constants
associated with depopulation of the Eu 5D0 and Tb 5D4 excited
states in H2O and D2O and applying an established analysis
that allows for the quenching effect of second-sphere waters and
amide N–H oscillators.24 Values obtained (Table 2) are consistent
with the absence of any bound water molecule, supporting the
hypothesis of a nine-coordinate structure.
The stability of the complex with respect to acid-catalysed
dissociation or degradation was assessed in two series of exper-
iments. First, TFA was added to aqueous solutions of [Eu.1]3+
and [Eu.3], and the pH varied between 6 and 2 (298 K, 10 mM
NaCl). No observable change was found in the absorption and
emission spectra of each complex over a two week period,
consistent with the absence of protonation on the heterocyclic
chromophore and the resistance to decomplexation. Similar
stability studies were undertaken using [Tb.2]3+ over a 48 h
period at pH 4 (0.2 M acetate buffer) and in reconstituted human
serum (pH 7.2). In each case less than a 5% time-dependent
change was observed, consistent with high complex stability.
luminescence (CPL) emission spectra were obtained for the
enantiomeric Eu and Tb complexes of the ‘dpq’ ligand, 1 (Fig. 1).
A comparison of the sign and magnitude of the observed CPL
provides both information on the helicity about the metal centre
and the structure of the complex.20,21 The sign and sequence of
the transitions observed were the same as for the C-4 symmetric
(S)-D and (R)-K Tb- and Eu-tetramide analogues [Ln.24]3+,
Emission quantum yields
Overall emission quantum yields, φem (eqn (1), where φT is
the quantum yield for formation of the triplet state, gET is the
efficiency of the energy transfer process, k0 is the natural radiative
constant of the complex and sobs is the observed luminescence
lifetime of the lanthanide ion, i.e. the inverse of the radiative
decay constant), were measured in water,25 using an excitation
wavelength corresponding to the lowest energy maximum in the
absorption/excitation spectrum.
whose absolute configuration has been verified by X-ray
crystallography22 (Table 1). The exception to this correlation was
in the sign and form of the DJ = 2 manifold for the europium
complex. In this case, a sign reversal was noted. However,
this electric-dipole-allowed transition (DJ = 2) is particularly
sensitive to the nature and polarisability of the donor atom that
adopts an axial site in the mono-capped square anti-prismatic
φem = φTgETk0sobs
(1)
Table 1 Emission dissymmetry factors ( 10%) for [Eu.1]3+, [Tb.1]3+
The values found (Table 3) are amongst the highest observed
in aqueous media,26,27 consistent with the high triplet quantum
yield of the ‘dpq’ and ‘dpqC’ chromophore9 (no measurable
fluorescence) and the high efficiency of the energy transfer step.
This latter aspect is undoubtedly related to the minimisation
of the lanthanide/chromophore distance and the observed
insensitivity of the emission quantum yield and the rate of
decay of the Ln excited state to dissolved oxygen. The triplet
energy of the ‘dpq’ chromophore was measured for [Gd.1]3+
and related tetra-amide complexes, [Ln.23]3+ (H2O, 295 K, kexc
340 nm)
=
588
em
595
em
614
em
545
em
Complex
g
g
g
g
(SSS)-[Eu.1]3+
(S)-D-[Eu.23]3+
(SSS)-[Tb.1]3+
(S)-D-[Tb.23]3+
+0.18
+0.09
−016
−0.04
+0.08
−0.09
+0.12
+0.27
1 0 1 6
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 0 1 3 – 1 0 2 4

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Table 3 Absolute emission quantum yieldsa for aqueous solutions of
Eu and Tb complexes (kexc = 340 for [Eu.1]3+ and 348 nm for others)
(in our preliminary work13) to reduce the emission intensity and
the lifetime, with a Stern–Volmer quenching constant [eqn. (3)],
KSV−1, of 9 mM.
Complex
uH
uD
2O
2O
[Eu.1]3+
[Tb.1]3+
[Eu.2]3+
[Tb.2]3+
[Eu.3]
0.21
0.36
0.16
0.40
0.18
0.33
0.18
0.36
0.15
0.34
0.27
0.46
0.20
0.48
(3)
Given that the iodide oxidation potential is +0.54 V and
that both the reduction of related cationic Eu(III) tetra-
amide complexes5,25 (metal-centred at −1.1 V) and of related
[Rudpq(dpqC)2]2+ complexes (ligand-centred at −1.07 V)10 occur
at a similar potential, then [eqn. (4)] applies, giving a favourable
free energy change of −65 kJ mol−1.
[Tb.3]
[Eu.6]
[Tb.6]
[Eu.7]3−
[Tb.7]3−
DGET = 96.5[(0.54 + 1.07) − 2.13 − 0.15] = −65 kJ mol−1 (4)
a Determined as described earlier25 with reference to p-cresyl violet in
MeOH (φ = 0.54) and rhodamine-101 (φ = 1.0) in ethanol.
This analysis is in line with the insensitivity of the europium
emission intensity/lifetime ( 10%) to added chloride ions (up to
100 mM). The chloride oxidation potential is +1.36 V, rendering
the overall free energy change positive and unfavourable. The
magnitude of the second-order rate constant that defines the
iodide quenching process is of the order of 105 M−1 s−1. This
is a rather low value compared to the values of >109 M−1 s−1
that characterise dynamic quenching of aryl triplet states by O2.
This probably reflects the inhibition to collisional encounter on
diffusion (i.e. the longer range nature of the process) associated
with the relative inaccessibility of the encapsulated lanthanide
to the quenching species.
at 77 K in a frozen glass (MeOH–EtOH 4 : 1). The shortest
wavelength transition (0–0 band) (Fig. 2) was observed at 420 nm
(23 800 cm−1), sufficiently higher than the Eu (17 200 cm−1) and
Tb (20 400 cm−1) emissive excited state levels to preclude any
back energy transfer process at ambient temperature.
Stern–Volmer plots (Fig. 3) were obtained for a range
of complexes (Table 4). Quenchers included selected halide
anions, urate [serum concentration is typically 0.3 mM, with
an oxidation potential (in serum) of +0.42 V],29 ascorbate
and glutathione. The latter three species are well-established
low molecular-weight anti-oxidants.29 The concentration of
glutathione in serum or in selected cells is typically much lower
than that of ascorbate, which averages 60 lM in human serum,30
but is found at much higher concentrations (0.8 to 2 mM
typically) in oxygen-sensitive tissues such as the eye, adrenal
glands, and in the heart, liver and brain.31
Fig. 2 Phosphorescence spectrum of [Gd.1]3+ (CF3SO3)3 in a frozen
glass (77 K, MeOH–EtOH 4 : 1, kexc = 340 nm), compared to that of
7-methyl-tetraazatriphenylene, 11 (dashed line).
Dynamic quenching of the lanthanide excited state
In the photophysical pathway that leads to lanthanide emission,
there are three salient excited states to consider: the singlet and
triplet states of the sensitising chromophore and the lanthanide
excited state itself. The facility of the S1 to T1 transition with
the dpq and dpqC chromophores, coupled with the absence of
any overt triplet quenching process, allows – for the first time –
an assessment of the quenching of the lanthanide excited state
by a charge transfer mechanism. Prior studies have focused on
the quenching by X–H energy-matched oscillators (X = O, N,
C).6,9,24 Here, the absence of a bound water molecule and the
subsequent minimisation of quenching by vibrational energy
transfer to OH oscillators makes these complexes well suited for
this analysis.
Fig. 3 Stern–Volmer plots showing the differing sensitivity of com-
plexes to quenching by iodide (pH 7.4, 100 mM HEPES). Data for
[Tb.2]3+ (ꢀ), [Tb.3] (ꢁ), [Tb.6]3− (×) and [Eu.2]3+ (᭜) are illustrated with
−1
The Tb and Eu excited states lie at 2.52 and 2.13 eV
respectively and this free energy can be used to ‘drive’ the
charge transfer process in quenching by electron-rich species.
The Weller equation [eqn. (2)]28 can be used to assess the facility
of this process,
KSV values of 0.9, 2.1, 2.5 and 27 mM, respectively.
Analysis of the information in Table 4 reveals some common
trends. First, quenching of the Tb emission is greater than for the
Eu analogue, in line with the higher excited state energy of the
5D4 Tb state. Second, the sequence of sensitivity to quenching
generally follows the ease of oxidation of the quenching anion,
i.e. ascorbate ≈ urate >> I− >>> Br−/Cl−. Glutathione does
not fall readily into this sequence but is only partially ionised
under the pH conditions used (thiol pKa is 8.66). Third, the
facility of quenching decreases as the overall complex charge
varies from positive to neutral and anionic, consistent with the
sequence of enhanced Coulombic repulsion in formation of the
encounter complex. Given that the overall emission intensity
with [Eu.2]3+ and [Tb.2]3+ in 50% human serum decreased by
DGET = nF[(Eox − Ered) − ELn∗ − e2/er] J mol−1
(2)
where Eox is the oxidation potential (versus NHE) of the electron
donor (quencher, Q), Ered is the reduction potential of the
acceptor (ligand or metal based) in the complex, ELn∗ is the
energy of the lanthanide excited state, and e2/er is a Coulombic
attraction correction term associated with the formation of
the transient ion pair (or radical/ion). It is usually a small
term (<0.2 eV). As an example of this approach, consider the
quenching of [Eu.1]3+ by iodide anions, which has been reported
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Table 4 Stern–Volmer quenching constants (KSV−1/mM, 100%, pH 7.4, 100 mM HEPES) defining the deactivation of the lanthanide excited
state,a,b and relevant oxidation potential for quenching species (Eox vs. NHE)
Oxidation potential/V
Quenching species
+1.36
+1.07
+0.54
+0.42
+0.48
+0.19
+0.36
Cl−
Br−
I−
Urate
Ascorbate
Glutathione
Fe(CN)6
4−
−1
−1
−1
Complex
KSV (I−)/mM
KSV (urate)/mM
KSV (ascorbate)/mM
D-[Eu.1]3
D-[Eu.2]3+
D-[Tb.2]3+
[Tb.3]
9.0c
27.0d
0.9
0.93
0.25
0.006
0.012
0.39
0.18
0.30
0.38
2.1d
2.5
[Tb.6]3−
a KSV−1 values for glutathione were: [Eu.2]3+, 24 mM; [Tb.2]3+, 20 mM. b Data were analysed by examining the change in emission lifetime; analysis of
emission intensity variation gave values within 15% of those stated above. c Value was 10 mM for the K-complex; in the presence of poly(dGdC) (4
base pairs per complex), KSV−1 for the D-complex was 50 mM but was 9 mM for the K-complex, consistent with preferential inhibition to I− encounter
in the diastereoisomeric complex with the D-isomer. d Exhibited slight curvature in the Stern–Volmer plot at [I−] > 10 mM.
40 and 70% respectively, compared to a 10 mM HEPES buffer
standard solution, it seems very likely that ascorbate/urate are
the major quenching species.
Earlier work had revealed that both [Tb.1]3+ and [Eu.1]3+ bind
to poly(dGdC) and to calf-thymus DNA. Similar behaviour was
noted with [Ln.3]3+, and DNA-binding was characterised by
a decrease in the lifetime of the lanthanide emission, with the
effect being bigger for Tb compared to Eu, and for poly(dGdC)
with respect to poly(dAdT).13 Such behaviour accords with the
charge transfer mechanism postulated above, with the electron-
rich guanines the likely reductant. Competitive quenching
experiments with I− were performed with D- and K-[Eu.1]3+ in
the presence of polynucleotides, in order to deduce information
concerning their relative sensitivity to dynamic quenching.
Addition of iodide to the poly(dAdT)-bound complex reduced
the apparent KSV values by about 50% for both the D- and the
K-complex. In the presence of poly(dGdC), the Stern–Volmer
constant was unchanged for K-[Eu.1]3+, but KSV−1 changed from
Fig. 4 Fluorescence microscope image showing a live NIH/3T3 cell
stained with [Tb.2]3+ (0.3 mM in growth medium), 4 h post-incubation.
9 to 50 mM for the D-isomer. It appears that quenching by iodide
is suppressed by the DNA-binding interaction and that the K-
isomer must be equally exposed to I− quenching in the free and
DNA-bound states. This is likely to occur only if it is neither
intercalated nor ‘groove’-bound. In the diastereoisomeric com-
plex with the D-isomer, the strong inhibition of I− quenching
suggests a binding interaction in which the dpq chromophore is
shielded much more effectively from iodide encounter.
Preliminary microscopy experiments
Some preliminary experiments have been carried out monitoring
the spatio-temporal localisation of [Tb.2]3+ in mouse skin
fibroblasts (NlH/3T3 cells). Cultured cells were exposed to a
growth medium containing between a 50 lM and 1 mM solution
of the terbium complex. The living cells were then examined
by fluorescence microscopy at time intervals in the range of
30 min to 48 h, monitoring the green terbium luminescence
selectively, with the aid of appropriate excitation and emission
cut-off filters. Selected cell populations were exposed to the
complex for periods of up to 24 h, then the growth medium
was replaced (after washing with phosphate buffered saline) to
allow the examination of the localised complex in the absence
of a concentration gradient.
Representative images are shown in Figs. 4, 5 and 6. The
first image, taken after 4 h, for a cell exposed to a 0.3 mM
solution complex, highlights the distribution of the complex
through the cytoplasm, with brighter regions near the centre
of the cell. The cell nucleus remains dark. As the cell remains
exposed to the complex, most of the luminescence is observed in
the cell nucleus and around the cell membrane. Similar results
were obtained for the analogous Eu complex. For example, in
Fig. 5 Fluorescence microscope image revealing the localisation of
[Tb.2]3+ (1 mM in medium, 24 h post-incubation) in the cell nucleus and
defining the nuclear cell membrane.
Fig. 5, an image is shown (1 mM complex in medium, 24 h)
clearly highlighting the localisation of the complex inside the
nucleus of the cell and in certain compartments within the
1 0 1 8
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Platform II (Fisons instrument), operating in positive or negative
ion mode as stated, with methanol as the carrier solvent.
Accurate mass spectra were recorded at the EPSRC Mass
Spectrometry Service at the University of Wales, Swansea. UV-
Visible absorbance spectra were recorded on a Perkin Elmer
Lambda 900 UV/Vis/NIR spectrometer and emission spectra
and lifetimes were measured on a Perkin Elmer LS55 lumines-
cence spectrometer. Lifetimes were measured by excitation of the
sample by a short pulse of light (348 nm) followed by monitoring
the integrated intensity of light (545 nm for terbium, 620 nm for
europium) emitted during a fixed gate time, tg, and a delay time,
td, later. At least 20 delay times were used covering three or more
lifetimes. A gate time of 0.1 ms was used, and the excitation and
emission slits were set to 10 nm band-pass. The obtained decay
curves were fitted to the equation below using Microcal Origin
or Microsoft Excel,
Fig. 6 Fluorescence microscope image highlighting the egress of the
[Tb.2]3+ complex out of the cell nucleus and back into the extra-nuclear
compartments, 24 h after removal of the external concentration gradient.
I = A0 + A1 exp(−kt)
where I = intensity at time t after the flash, A0 = intensity after
the decay has finished, A1 = pre-exponential factor, k = rate
constant for decay of the excited state.
nucleus. The nuclear membrane is also delineated, with a residual
diffuse luminescence in the cytosol. In the absence of complex
in the external medium, the luminescence in the cell nucleus
becomes progressively less intense and the complex was again to
be found distributed mostly outside the cell nucleus. Thus, 24 h
after sequential incubation with complex followed by removal
of the medium containing the complex, the images obtained
(Fig. 6) revealed the complex in the cytosol once more. The
slightly ‘spotty’ nature of the image suggested some preferential
localisation in a particular cell organelle. This might be the
cellular mitochondria, which possess a negative surface potential
and contain DNA. Co-localisation studies are underway to
verify this hypothesis, using a known mitochondrial stain, such
as ‘Mitotracker Red’.
These experiments confirm that the complex is able to pen-
etrate first the cell membrane and then the nuclear membrane,
and can localise inside the cell nucleus, in the presence of a
sufficient external concentration gradient. Transport in and out
of the cell nucleus appears to be reversible. The neutral [Tb.3] and
anionic [Tb.6]3− complexes were examined in a parallel manner,
but were very poorly taken up by the cells, even at the highest
concentration of added complex. The complex localises inside
the cell rapidly (within 15–30 min), and this rate of ingress
appeared to be similar, in an incubation carried out at 4 ◦C,
monitored after 30 mins, 1 h and 3 h. Such behaviour is typical
of an uptake mechanism involving a channel or pore in the
cell membrane rather than by an activated process. Certainly,
these preliminary findings indicate that these emissive lanthanide
complexes offer much scope as luminescent probes in biological
systems, and are suitable for further study ‘in cellulo’.
Quenching constants were determined by measuring the
emissive lifetime, s, of each of the complexes with varying
concentrations of the quenching species (Q). A plot of s0/s
versus [Q] (as seen previously in Fig. 3) was used to determine
the Stern–Volmer quenching constant, KSV, as the slope of this
plot. In each case, a 10 lM solution of the complex buffered to
pH 7.4 with HEPES (100 mM) in the presence of NaCl (10 mM)
was used.
Quantum yield measurements were made relative to two stan-
dards as reported earlier.25 Five solutions with an absorbance of
between 0.02 and 0.1 were used. For each of these solutions
the absorbance at the excitation wavelength and the total
integrated emission was determined. A plot of total integrated
emission against absorbance gives a straight line with slope E/A.
The unknown quantum yield can thus be calculated from the
following equation.
Since values for both the sample (x) and reference (r) compounds
were recorded in water the refractive index term (n) cancels out.
Errors in quantum yield determinations can arise due to the
inner filter effect or errors in the amount of absorbed light. This
effect was minimised by only using samples with an absorbance
below 0.1. Errors in the amount of light absorbed by each
sample were minimised by choosing an excitation wavelength
on a relatively flat area of the absorption curve and by using a
small band-pass for excitation.
Experimental
Cell culture and microscopy
Reactions requiring anhydrous conditions were carried out using
Schlenk-line techniques under an atmosphere of dry argon.
Solvents were dried from an appropriate drying agent when
required and water was purified by the ‘PuriteSTILL plus’ system.
Thin-layer chromatography was carried out on neutral alumina
plates (Merck Art 5550) or silica plates (Merck 5554) and
visualised under UV (254 nm) or by staining with iodine.
Preparative column chromatography was carried out using
neutral alumina (Merck Aluminium Oxide 90, activity II–III,
70–230 mesh), pre-soaked in ethyl acetate, or silica (Merck Silica
Gel 60, 230 400 mesh).
Mouse skin fibroblasts (NIH-3T3 cells) were cultured to 50%
confluence on a cover slip in a DMEM (Dulbeccos Modified
Eagles Medium) solution supplemented with 10% foetal bovine
serum and a 1% penicillin/streptomycin mixture. The cells
were washed with phosphate buffered saline and the lanthanide
complexes added in a clean medium. The incubation time
was varied from 30 min to 48 h, and the concentration of
added complex was examined in the range 0.05 to 1 mM, For
examination by fluorescence microscopy, the cells were washed
with phosphate buffered saline and mounted onto slides (Zeiss
Axiovert 200 M). For the imaging of the terbium complexes, the
excitation wavelength was 348 nm.
1H and 13C NMR spectra were recorded on a Varian Mercury
200 (1H at 199.975 MHz, 13C at 50.289 MHz), a Varian Unity
300 (1H at 299.908 MHz, 13C at 75.412 MHz), a Varian VXR
400 (1H at 399.968 MHz, 13C at 100.572 MHz), or a Bruker
AMX 500 spectrometer. Spectra were referenced internally to
the residual protio-solvent resonances and are reported relative
to TMS. Electrospray mass spectra were recorded on a VG
Ligand and complex synthesis
2-Methyl-1,10-phenanthroline. A solution of 1,10-phenan-
throline (10 g, 55 mmol) in dry THF (100 ml) was added
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 0 1 3 – 1 0 2 4
1 0 1 9

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dropwise to a stirred solution of methyl lithium (1.6 mol dm−3
in Et2O, 34.5 ml, 55 mmol) in dry THF (150 ml) at 0 ◦C under
argon. The mixture was stirred overnight at room temperature
under argon to give a dark green solution. The reaction was
247 (30%, MH+), 269 (33%, MNa+), 515 (100%, M2Na+); mp
192–194 ◦C. Elemental analysis: found C 71.8, H 3.99, N 22.7;
C15H10N4·0.25H2O requires C 71.9, H 4.19, N 22.4%.
3-Methyl-10,11,12,13-tetrahydrodipyrido-[3,2a:2ꢀ,3ꢀ-c]phena-
◦
quenched by careful addition of water (100 ml) at 0 C. THF
zine, 12. To
a
solution of 2-methyl-1,10-phenanthro-
was removed under reduced pressure using a rotary evaporator
and the product extracted into diethyl ether (3 × 150 ml).
Activated manganese dioxide (53 g, 0.61 mol) was added and
the solution stirred for 1.5 h; magnesium sulfate was added
and the solution stirred for a further 2 h. Filtration through
a Celite plug yielded a clear yellow solution. The solvent was
evaporated to dryness to yield the crude product, which was
purified by recrystallisation from ethyl acetate/hexane to give
line-5,6-dione (0.65 g, 2.90 mmol) in absolute ethanol (50 ml)
was added trans-1,2-diaminocyclohexane (0.33 g, 2.90 mmol)
and the resulting mixture boiled under reflux for 5 h. The
solution was allowed to cool and the solvent was removed
under reduced pressure. The product was re◦crystallised from
1
ethanol (0.81 g, 2.90 mmol, 93%), mp >220 C (decomp). H
NMR (300 MHz, CDCl3): d 2.09 (4H, m, H10, 13), 2.98 (3H, s,
H15), 3.24 (4H, m, H11, 12), 7.63 (1H, d, J = 8.3 Hz, H2), 7.73
(1H, dd, J = 8.2, 4.4, H7), 9.26 (1H, dd, J = 4.4, 1.8, H6),
9.35 (1H, d, J = 8.3, H1), 9.47 (1H, dd, J = 8.2, 1.8, H8). 13C
NMR (75 MHz, CDCl3): d 22.7 (C11), 22.8 (C12), 25.7 (CH3),
32.7 (C10), 32.8 (C13), 123.4 (C7), 124.2 (C2), 124.8 (q Ar),
151.5 (C6), 153.2 (q Ar), 153.6 (q Ar), 160.9 (q Ar). HRMS (+
m/z): [M + H]+ calculated for C19H16N4Na, 323.1273; found,
323.1269.
1
a white product (5.50 g, 28 mmol, 51%). H NMR (300 MHz,
CDCl3): d 2.97 (3H, s, CH3), 7.54 (1H, d, J = 8.1 Hz, H3),
7.63 (1H, dd, J = 8.1, 4.2, H8), 7.75 (1H, d, J = 8.7, H6), 7.79
(1H, d, J = 8.7, H5), 8.16 (1H, d, J = 8.1, H4), 8.26 (1H, dd,
J = 8.1, 1.5, H7), 9.22 (1H, dd, J = 4.2, 1.2, H9). 13C NMR
(75 MHz, CDCl3): d 25.9 (CH3), 122.8 (C8), 123.7 (C3), 125.5
(C6), 126.5 (C5), 126.7 (q Ar), 128.8 (q Ar), 136.1 (C7), 136.2
(C4), 145.7 (q Ar), 146.0 (q Ar), 150.3 (C9), 159.5 (C2). m/z
(ES+) 195 (MH+), 217 (MNa+), 411 (M2Na+); mp 75–76 ◦C
(lit.32 78 ◦C). Elemental analysis: found C 79.5, H 5.44, N 14.5;
C13H10N2·0.17H2O requires C 79.2, H 5.23, N 14.2%.
Dipyrido[3,2-f :2ꢀ,3ꢀ -h]quinoxaline-7-carboxyaldehyde, 13.
Selenium dioxide (0.77 g, 6.97 mmol) was added to a solution of
the 3-methyl phenazine (0.78 g, 3.17 mmol) in dioxane (100 ml).
The mixture was heated under reflux for 2 h, then filtered
through Celite. Evaporation of the solvent afforded a white
solid. Some more product was obtained by washing the Celite
2-Methyl-1,10-phenanthroline-5,6-dione, 10. A mixture of 2-
methyl-1,10-phenanthroline (2.00 g, 10.29 mmol) and potassium
bromide (12.25 g, 102.9 mmol) was placed in an ice bath. Cold
sulfuric acid (43 ml) was added carefully to the flask in a dropwise
fashion, which turned the solution to a dark reddish colour.
Concentrated nitric acid (21.5 ml) was then added in a similar
way and the solution was boiled under reflux overnight. The
mixture was cooled, poured into water (500 ml) and neutralised
by addition of sodium hydroxide pellets. The product was
extracted into dichloromethane (3 × 300 ml), which was dried
over K2CO3 and removed under reduced pressure to yield a
yellow solid. Purification was achieved by chromatography on
silica [1.79 g, 7.98 mmol, 77%; gradient elution: CH2Cl2 to 1%
1
with CH2Cl2 (0.77 g, 2.96 mmol, 93%). H NMR (300 MHz,
CDCl3): d 7.89 (1H, dd, J = 8.1, 4.5 Hz, H11), 8.46 (1H, d, J =
8.4, H5), 9.06 (1H, d, J = 1.8, H2), 9.08 (1H, d, J = 1.8, H3),
9.39 (1H, dd, J = 4.2, 1.8, H10), 9.57 (1H, dd, J = 8.1, 1.8,
H12), 9.73 (1H, dd, J = 8.4, 0.73, H6), 10.61 (1H, d, J = 0.73,
CHO). 13C NMR (75 MHz, CDCl3): d 121.0 (C5), 124.8 (C11),
127.8 (q Ar), 130.2 (q Ar), 133.7 (C12), 135.1 (C6), 140.0 (q Ar),
141.6 (q Ar), 145.2 (C2), 145.8 (C3), 147.1 (q Ar), 147.7 (q Ar),
152.9 (C10), 154.1 (C7), 194.0 (CHO). m/z (ES+) 283 (60%,
MNa+), 315 (100%, MNa+ + MeOH), 543 (60%, M2Na+), 575
(10%, M2Na+ + MeOH), 607 (40%, M2Na+ + 2MeOH). IR
1
CH3OH–CH2Cl2, Rf = 0.34 (5% MeOH–CH2Cl2)]. H NMR
=
(KBr): 3074, 3029 (C–H Ar), 2865 (C–H in CHO), 1703 (C O),
(300 MHz, CDCl3): d 2.86 (3H, s, CH3), 7.44 (1H, d, J = 7.8 Hz,
H3), 7.58 (1H, dd, J = 7.8, 4.8, H8), 8.40 (1H, d, J = 8.1, H4),
8.50 (1H, dd, J = 7.8, 2.1, H7), 9.14 (1H, dd, J = 4.8, 1.8,
H9). 13C NMR (75 MHz, CDCl3): d 26.2 (CH3), 125.6 (C8),
125.9 (C3), 126.2 (q Ar), 128.3 (q Ar), 137.5 (C7), 137.7 (C4),
152.7 (q Ar), 153.2 (q Ar), 156.6 (C9), 167.4 (C2), 178.7 (C5),
179.2 (C6). m/z (ES+) 246 (MNa+), 279 (MNa+ + MeOH), 471
(M2Na+), 503 (M2Na+ + MeOH), 535 (M2Na + 2MeOH). IR
=
1566, 1467 and 1449 (C C py), 1391 (C–H CHO), 1362, 1207,
1077, 767 and 748 cm−1; mp > 220 ◦C (decomp.). Elemental
analysis: found C 67.3, H 3.08, N 20.9; C15H8N4O·0.33H2O
requires C 67.7, H 3.27, N 21.0%.
3-Carboxaldehyde-10,11,12,13-tetrahydrodipyrido-[3,2a:2ꢀ,3ꢀ-
c]phenazine, 14. Selenium dioxide (0.96 g, 8.66 mmol) was
added to a solution of 3-methyl-10,11,12,13-tetrahydrodipyrido-
[3,2a:2ꢀ,3ꢀ-c]phenazine (1.30 g, 4.33 mmol) in dioxane (200 ml)
and the mixture boiled under reflux for 4 h. The solution was
cooled to room temperature and filtered through a Celite plug
to afford a crude product that was used directly in the next step.
1H NMR (300 MHz, CDCl3): d 2.11 (4H, m, H10, 13), 3.28
(4H, m, H11, 12), 7.83 (1H, dd, J = 8.2, 4.4 Hz, H7), 8.41 (1H,
d, J = 8.3, H1), 9.33 (1H, dd, J = 4.5, 1.8, H6), 9.53 (1H, dd,
J = 8.2, 1.7, H8), 9.68 (1H, dd, J = 8.3, 0.9, H2), 10.60 (1H, d,
J = 0.9, CHO). 13C NMR (75 MHz, CDCl3): d 22.5 (C11), 22.6
(C12), 32.7 (C10), 32.8 (C13), 120.2 (C7), 124.1 (c1), 127.5 (q
Ar), 130.0 (q Ar), 133.0 (c8), 134.1 (C2), 136.6 (q Ar), 138.3 (q
Ar), 146.2 (q Ar), 146.7 (q Ar), 151.7 (C6), 153.1 (q Ar), 154.4
(q Ar), 155.1 (q Ar), 193.9 (CHO). EI (+ m/z): [M+] 314.
=
(KBr): 3074 (C–H Ar), 1681 (C O), 1568 (py ring vibration),
−1
=
1436 and 1301 (C C py), 1216, 1123, 1013, 925, 829, 739 cm ;
mp 98–100 ◦C. Elemental analysis: found, C 66.3, H 3.46, N
12.2; C13H8N2O2·0.67H2O requires C 66.1, H 3.94, N 11.9%.
7-Methyldipyrido[3,2-f :2ꢀ,3ꢀ-h]quinoxaline, 11. A solution of
ethylenediamine (0.23 ml, 3.48 mmol) in THF (15 ml) was
added in a dropwise fashion to a solution of the dione (0.78 g,
3.48 mmol) in THF (400 ml) and the resulting mixture was boiled
under reflux overnight. The solvent was removed by evaporation
and the dark red solid taken into CH2Cl2 (100 ml) and washed
with H2O (3 × 100 ml). The organic layer was dried over
K2CO3. Removal of the solvent under reduced pressure afforded
a yellow solid (0.82 g, 0.33 mmol, 96%). Further purification was
achieved by chromatography on silica (gradient elution: CH2Cl2
to 1% CH3OH containing 0.5% NH3–CH2Cl2, Rf = 0.18, 10%
7-(Hydroxymethyl)dipyrido[3,2-f :2ꢀ,3ꢀ-h]quinoxaline, 15. So-
dium borohydride (54 mg, 1.43 mmol) was added to a solution
of the precursor aldehyde (300 mg, 1.15 mmol) in CH2Cl2–EtOH
7 : 1 (120 ml). The mixture was heated at reflux for 2 h.
The solvent was evaporated and the orange solid taken into a
saturated solution of Na2CO3 (50 ml) and extracted into CH2Cl2
(3 × 70 ml). The organic layer was dried over K2CO3. Removal
of the solvent under reduced pressure afforded a yellow solid
(207 mg, 0.79 mmol, 68%), which was used without further
purification. 1H NMR (300 MHz, CDCl3): d 5.20 (2H, s, CH2),
1
CH3OH containing 0.5% NH3–CH2Cl2). H NMR (300 MHz,
CDCl3): d 2.96 (3H, s, CH3), 7.64 (1H, d, J = 8.4 Hz, H6), 7.74
(1H, dd, J = 8.1, 4.5, H11), 8.91 (2H, m, H2, H3), 9.28 (1H,
dd, J = 4.5, 1.8, H10), 9.31 (1H, d, J = 8.4, H5), 9.44 (1H, dd,
J = 8.4, 1.8, H12). 13C NMR (75 MHz, CDCl3): d 26.1 (CH3),
123.9 (C11), 124.7 (C6), 124.9 (q Ar), 127.1 (q Ar), 133.3 (C12),
133.4 (C5), 140.2 (q Ar), 140.8 (q Ar), 144.2 (C2), 144.6 (C3),
147.1 (q Ar), 147.5 (q Ar), 152.5 (C10), 162.1 (C7). m/z (ES+)
1 0 2 0
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 0 1 3 – 1 0 2 4

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7.82 (1H, dd, J = 8.1, 4.5 Hz, H11), 7.87 (1H, d, J = 8.7, H6),
9.00 (2H, s, H2, H3), 9.27 (1H, dd, J = 4.5, 1.8, H10), 9.51 (1H,
d, J = 8.4, H5), 9.54 (1H, dd, J = 8.1, 1.8, H12). 13C NMR
(75 MHz, CDCl3): d 65.4 (CH2), 121.5 (C11), 124.0 (C6), 126.0
(q Ar), 127.3 (q Ar), 133.5 (C12), 133.9 (C5), 140.2 (q Ar), 140.5
(q Ar), 144.4 (C2), 144.7 (C3), 146.3 (q Ar), 147.0 (q Ar), 152.0
(C10), 163.0 (C7). m/z (ES+) 262 (100%, M+), 284 (30%, MNa+),
546 (20%, M2Na+).
32.2 (C10, 13), 47.0 (CH2), 122.3 (C7), 123.2 (C2), 125.7 (q Ar),
126.7 (q Ar), 128.9 (q Ar), 130.1 (q Ar), 132.2 (C8), 133.4 (C1),
145.4 (q Ar), 145.9 (q Ar), 151.1 (q Ar), 153.3 (C6), 157.9 (q
Ar). HRMS (+ m/z): [M + Na]+ calculated for C19H15N4NaCl,
357.0883; found, 357.0885.
1-(7ꢀ-Methyldipyrido[3,2-f :2ꢀ,3ꢀ-h]quinoxaline)-4,7,10-tris(tert-
butoxycarbonyl)-1,4,7,10-tetraazacyclododecane, 18. K2CO3
(218 mg, 1.58 mmol) and a catalytic amount of KI were
added to a solution of 1,4,7-tris(tert-butoxycarbonyl)-1,4,7,10-
tetraazacyclododecane, 17, (148 mg, 0.31 mmol) in CH3CN
(5 ml). The mixture was heated at 60 ◦C and a solution of
the3-chloromethylphenazine (88 mg, 0.31 mmol) in CH2Cl2
(5 ml) was added. The reaction mixture was boiled under
reflux under argon overnight. The solution was filtered and the
salts washed with CH2Cl2. Evaporation of the solvent afforded
a crude residue, which was purified by chromatography on
silica (gradient elution: CH2Cl2 to 3% CH3OH–CH2Cl2, Rf =
0.23, 10% CH3OH–CH2Cl2) to yield a colourless oil (160 mg,
7-(Chloromethyl)dipyrido[3,2-f :2ꢀ,3ꢀ-h]quinoxaline, 8. Phos-
phorus trichloride (0.82 ml, 9.36 mmol) in CHCl3 (5 ml)
was added in a dropwise fashion to a stirred solution of
the hydroxymethyl compound (206 mg, 0.78 mmol) in CHCl3
(100 ml). The mixture was heated at reflux for 4 h. The solution
was neutralised by addition of a saturated aqueous Na2CO3
solution (100 ml). The organic layer was separated and dried
over K2CO3. Removal of the solvent under reduced pressure
afforded a pink solid. Purification by chromatography on silica
gel (gradient elution: CH2Cl2 to 1% CH3OH–CH2Cl2, Rf =
0.47, 10% CH3OH–CH2Cl2) gave a colourless solid (95 mg,
1
0.22 mmol, 71%). H NMR (300 MHz, CDCl3): d 1.29 (27H,
1
0.34 mmol, 43%). H NMR (200 MHz, CDCl3): d 5.14 (2H, s,
m, CH3), 2.78 (4H, br, ring CH2), 3.50 (12H, m, ring CH2), 4.27
(2H, s, CH2), 7.75 (1H, dd, J = 8.1, 4.5 Hz, H11), 7.89 (1H, br,
H6), 8.94 (2H, m, H2, H3), 9.25 (1H, d, J = 3.0, H10), 9.39
(1H, d, J = 6.6, H5), 9.46 (1H, dd, J = 8.1, 1.5, H12). m/z
(ES+) 716 (3%, M+), 739 (100%, MNa+), 1455 (5%, M2Na+).
CH2), 7.81 (1H, dd, J = 8.2, 4.4 Hz, H11), 8.08 (1H, d, J = 8.4,
H6), 8.99 (2H, s, H2, H3), 9.32 (1H, dd, J = 4.4, 1.8, H10), 9.51
(1H, dd, J = 8.2, 1.8, H12), 9.54 (1H, d, J = 8.4, H5). 13C NMR
(75 MHz, CDCl3): d 47.1 (CH2), 123.1 (C11), 124.9 (C6), 126.1
(q Ar), 127.0 (q Ar), 133.0 (C12), 134.2 (C5), 139.8 (q Ar), 140.1
(q Ar), 144.4 (C2, C3), 146.2 (q Ar), 146.7 (q Ar), 152.2 (C10),
159.2 (C7). m/z (ES+) 268 (20%, MNa+ − Cl), 302 (95%, MNa+),
1-(7ꢀ-Methyldipyrido[3,2-f :2ꢀ,3ꢀ-h]quinoxaline)-1,4,7,10-tetra-
azacyclododecane, 20. Trifluoroacetic acid (10 ml) was added
to a solution of the tricarbamate (160 mg, 0.22 mmol) in CH2Cl2
(3 ml). The mixture was stirred for 2 h at room temperature.
The solvent was evaporated and the residue re-dissolved in
CH2Cl2 three times to facilitate elimination of excess acid and
tert-butyl alcohol. The resultant orange oil was taken into a
KOH solution (5 ml) and the product extracted into CH2Cl2
(3 × 5 ml). The organic layer was dried over K2CO3. Removal
of the solvent under reduced pressure afforded a very pale
◦
548 (50%, M2Na+ − Cl), 582 (100%, M2Na+); mp 194–196 C
(decomp.). Accurate mass: found MH+ 281.0593; C15H10ClN4
requires 281.0594.
3-Hydroxymethyl-10,11,12,13-tetrahydrodipyrido-[3,2a:2ꢀ,3ꢀ -
c]phenazine, 16. Sodium borohydride (164 mg, 4.33 mmol)
was added to a solution of 3-carboxaldehyde-10,11,12,13-
tetrahydrodipyrido-[3,2a:2ꢀ,3ꢀ-c]phenazine (1.36 g, 4.33 mmol)
in CHCl3–EtOH (200 ml). The mixture was heated under
reflux for 2 h. The solvent was evaporated and the orange
solution taken into a saturated solution of Na2CO3 (200 ml)
and extracted with CH2Cl2 (3 × 150 ml) then CHCl3 (1 ×
150 ml). The organic phase was dried over Na2SO4 filtered and
the solvent removed under reduced pre◦ssure to afford a yellow
solid (0.89 g, 2.81 mmol, 65%); mp 210 C (decomp.). 1H NMR
(300 MHz, CDCl3): d 2.10 (4H, m, H10, 13), 3.25 (4H, m, H11,
12), 5.18 (2H, s, CH2OH), 7.75 (1H, dd, J = 8.2, 4.4 Hz, H7),
7.80 (1H, d, J = 8.3, H2), 9.20 (1H, dd, J = 4.4, 1.7, H6), 9.47
(1H, d, J = 8.3, H1), 9.49 (1H, dd, J = 8.3, 1.7, H8). 13C NMR
(75 MHz, CDCl3): d 22.7 (C11, 12), 32.8 (C10, 13), 65.7 (CH2),
121.0 (C7), 123.6 (C2), 125.9 (q Ar), 127.2 (q Ar), 132.9 (C8),
133.4 (C1), 137.4 (q Ar), 151.1 (C6), 153.5 (q Ar), 153.8 (q
Ar), 162.3 (q Ar). HRMS (+ m/z): [M + Na]+ calculated for
C19H16N4ONa, 339.1222; found, 339.1219.
1
orange solid (65 mg, 0.16 mmol, 71%). H NMR (300 MHz,
CDCl3): d 2.57 (4H, m, ring CH2), 2.70 (8H, s, ring CH2), 2.80
(4H, m, ring CH2), 4.20 (2H, s, CH2), 7.71 (1H, dd, J = 8.1,
4.5 Hz, H11), 8.07 (1H, d, J = 8.4, H6), 8.87 (1H, d, J = 2.1,
H2), 8.88 (1H, d, J = 2.1, H3), 9.25 (1H, dd, J = 4.5, 1.8, H10),
9.41 (1H, dd, J = 8.4, 1.8, H12), 9.43 (1H, d, J = 8.4, H5). 13
C
NMR (75 MHz, CDCl3): d 45.1, 46.4, 47.1, 52.0 (ring CH2),
61.9 (CH2), 123.1 (C11), 123.5 (C6), 125.8 (q Ar), 126.9 (q Ar),
133.0 (C12), 133.8 (C5), 140.0 (q Ar), 140.5 (q Ar), 144.0 (C2),
144.4 (C3), 146.4 (q Ar), 147.2 (q Ar), 152.2 (C10), 163.5 (C7).
m/z (ES+) 228 (10%, MCa2+), 417 (100%, MH+), 439 (10%,
MNa+), 833 (3%, M2H+); mp 175–178 ◦C (decomp.). Accurate
mass: found MH+ 417.2515; C23H29N8 requires 417.2515.
(RRR)-1-(7ꢀ-Methyldipyrido[3,2-f :2ꢀ,3ꢀ-h]quinoxaline)-4,7,10-
tris[1-(1-phenyl)ethylcarbamoylmethyl]-1,4,7,10-tetraazacyclodo-
decane, 1. K2CO3 (83 mg, 0.6 mmol) and a catalytic amount
of KI were added to a solution of 1-(7ꢀ-methyldipyrido[3,2-
f :2ꢀ,3ꢀ-h]quinoxaline)-1,4,7,10-tetraazacyclododecane (50 mg,
0.12 mmol) in CH3CN (4 ml). The mixture was heated at 60 ◦C
and a solution of compound 7 (71 mg, 0.36 mmol) in CH2Cl2
(4 ml) was added. The reaction mixture was boiled under
reflux under argon overnight. The solution was filtered and the
salts washed with CH2Cl2. Evaporation of the solvent afforded
a crude residue, which was purified by chromatography on
alumina (gradient elution: CH2Cl2 to 2% CH3OH–CH2Cl2,
Rf = 0.31, 10% CH3OH–CH2Cl2) to yield a colourless solid
(50 mg, 0.06 mmol, 50%). 1H NMR (300 MHz, CDCl3): d 1.46
(H, d, J = 2.7 Hz, CH3), 1.48 (H, d, J = 2.7, CH3), 2.64 (16H,
m, ring CH2), 2.90 (2H, d, J = 6, CH2), 2.96 (4H, d, J = 6,
CH2), 4.08 (2H, s, CH2-dpq), 5.14 (3H, m, J = 7.5, CH), 6.88
(2H, br, NH), 7.05 (1H, br, NH), 7.16–7.37 (15H, m, Ph), 7.80
(1H, dd, J = 8.1, 4.2, H11), 7.91 (1H, d, J = 8.4, H6), 9.01 (2H,
m, H2, H3), 9.28 (1H, dd, J = 4.5, 1.8, H10), 9.41 (1H, d, J =
8.4, H5), 9.54 (1H, dd, J = 8.1, 1.8, H12). 13C NMR (75 MHz,
3-Chloromethyl-10,11,12,13-tetrahydrodipyrido-[3,2a:2ꢀ,3ꢀ-c]-
phenazine, 9. To a solution of 3-hydroxymethyl-10,11,12,13-
tetrahydrodipyrido-[3,2a:2ꢀ,3ꢀ-c]phenazine (0.84 g, 2.66 mmol)
in CHCl3 (450 ml) was added phosphorus trichloride (1.46 g,
10.6 mmol). The mixture was heated under reflux for 4 h. The
solution was neutralised by addition of a saturated aqueous
solution of Na2CO3 (500 ml). The organic layer was separated
and the aqueous layer extracted with CH2Cl2 (2 × 400 ml)
and CHCl3 (1 × 400 ml). The combined organic layers were
dried over K2CO3 and the solvent evaporated to afford a
yellow solid. Purification by chromatography on silica (gradient
elution: CH2Cl2 to 1% CH3OH–CH2Cl2) gave a pale yellow
solid (Rf 0.38, 5% MeOH–CH2Cl2; 0.75 g, 2.25 mmol, 84%),
◦
1
mp 207–208 C. H NMR (300 MHz, CDCl3): d 2.05 (4H, m,
H10, 13), 3.17 (4H, m, H11, 12), 5.10 (2H, s, CH2Cl), 7.70 (1H,
dd, J = 8.2, 4.4 Hz, H7), 7.97 (1H, d, J = 8.3, H2), 9.23 (1H,
dd, J = 4.5, 1.8, H6), 9.37 (1H, dd, J = 8.2, 1.8, H8), 9.40 (1H,
d, J = 8.3, H1). 13C NMR (75 MHz, CDCl3): d 22.7 (C11, 12),
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 0 1 3 – 1 0 2 4
1 0 2 1

View Article Online
CDCl3): d 21.4 (CH3), 21.9 (CH3), 48.2 (CH), 48.3 (CH), 52.8
(CH2N), 53.3 (CH2N), 53.6 (CH2N), 58.3 (NCH2CO), 58.9
(NCH2CO), 62.3 (CH2-dpq), 123.3 (C11), 123.9 (C6), 126.2
(o-Ar), 127.3 (p-Ar), 128.6 (m-Ar), 133.2 (C12), 133.6 (C5),
143.1 (q Ar), 144.6 (C2, C3), 140.4 (q-dpq), 152.3 (C10), 161.0
(C7), 169.5 (CO), 170.4 (CO). m/z (ES+) 469 (40%, MCa2+),
899 (5%, M+), 922 (100%, MNa+); mp 136–138 ◦C. Accurate
mass: found MH+ 900.5035; C53H62N11O3 requires 900.5037.
The (SSS) ligand was synthesised with the same procedure.
under argon. The solution was filtered and the salts washed
with CH2Cl2. The product was purified by chromatography
on neutral alumina (gradient elution: CH2Cl2 to 1% CH3OH–
CH2Cl2, Rf = 0.54, 7.5% CH3OH–CH2Cl2) to yield a pale yellow
solid (105 mg, 0.089 mmol, 48%), mp 82–84 ◦C. 1H NMR
(300 MHz, CDCl3): d 0.96–1.34 (18H, m, CH3), 1.74–2.56 (16H,
m, CH2CH3, H10, H13), 2.82–3.42 (20H, br m, CH2 ring, H11,
H12), 3.72–4.64 (21H, m, CH2CO, CH2-dpqC, CHCH2CH2,
CHCH2CH2, CHCH2CH2), 7.68 (1H, m, H2), 7.76 (1H, br
m, H7), 7.95 (1H, br m, H6), 9.35 (1H, m, H1), 9.45 (1H, m,
H8). 13C NMR (75 MHz, CDCl3): d 14.1 (CH3), 14.2 (CH3),
22.8 (C10, C13), 26.2 (CH2), 30.4 (CH2), 30.6 (CH2), 32.9
(C11, C12), 50.4–60.7 (CH2 ring, CH2CO, CH2CH3), 61.6
(CH2-dpqC), 124.2 (C2), 124.7 (C Ar), 126.7 (C Ar), 127.7
(C7), 133.2 (C8), 134.1 (C1), 137.4 (C Ar), 146.3 (C Ar), 146.9
(C Ar), 151.9 (C6), 154.2 (C Ar), 157.5 (C Ar), 160.9 (C Ar),
1-(3-Methyl-10,11,12,13-tetrahydrodipyrido[3,2-a:2ꢀ,3ꢀ-c]phen-
azine)-4,7,10-tris(tert-butoxycarbonyl)-1,4,7,10-tetraazacyclodo-
decane, 19. Potassium carbonate (124 mg, 0.716 mmol) and
a catalytic amount (2 mg) of KI were added to a solution of
1,4,7-tris-tert-butoxycarbonyl-1,4,7,10-tetraazacyclododecane,
17, (77 mg, 0.179 mmol) in CH3CN (3 ml). The mixture was
◦
heated at 60 C and a solution of 3-chloromethyl-10,11,12,13-
=
=
=
=
169.4 (C O), 171.3 (C O), 171.6 (C O), 172.4 (C O), 172.8
tetrahydrodipyrido-[3,2a:2ꢀ,3ꢀ-c]phenazine (60 mg, 0.179 mmol)
in CH2Cl2 (3 ml) was added. The reaction mixture was boiled
under reflux under argon overnight. The solution was filtered
and the salts washed with CH2Cl2. The residue was purified by
chromatography on silica gel (gradient elution: CH2Cl2 to 3%
CH3OH–CH2Cl2, Rf = 0.44, 10% CH3OH–CH2Cl2) to yield a
+
2+
2+
+
=
(C O). m/z (ES ): 620 (MCa ), 632 (MCu ), 1223 (MNa ).
1-(3-Methyl-10,11,12,13-tetrahydrodipyrido[3,2-a:2ꢀ,3ꢀ-c]phe-
nazine)-4,7,10-tris(N-acetyl-L-alanine diethyl ester)-1,4,7,10-
tetraazacyclododecane, 4. Potassium carbonate (116 mg,
0.840 mmol) and a catalytic amount of KI were added to a
solution of 1-(3-methyl-10,11,12,13-tetrahydrodipyrido[3,2-
a:2ꢀ,3ꢀ-c]phenazine)-1,4,7,10-tetraazacyclododecane (79.1 mg,
0.168 mmol) in CH3CN (6 ml). The reaction mixture was heated
at 60 ◦C and a solution of N-chloroacetyl-L-alanine ethyl ester
(97.6 mg, 0.504 mmol) in CH2Cl2 (6 ml) was added. The reaction
mixture was boiled under reflux overnight, under argon. The
solution was filtered and the salts washed with CH2Cl2. The
product was purified by chromatography on neutral alumina
(gradient elution: CH2Cl2 to 1% CH3OH–CH2Cl2, Rf = 0.55,
◦
pale yellow solid (105 mg, 0.136 mmol, 76%), mp 114–116 C.
1H NMR (300 MHz, CDCl3): d 0.92–0.71 (27H, br m, CH3),
2.04 (4H, m, H10, H13), 2.42–2.99 (4H, m, CH2 ring), 3.07–3.87
(16H, br m, CH2 ring + H11, H12), 4.24 (2H, s, CH2-dpqC),
7.67 (1H, dd, J = 8.2, 4.4 Hz, H7), 7.72–7.97 (1H, br s, H2),
9.71 (1H, br s, H6), 9.26–9.44 (2H, br m, H8, H1). 13C NMR
(75 MHz, CDCl3): d 22.7 (C10, C13), 28.3 (CH3), 28.5 (CH3),
32.7 (C11, C12), 46.2–51.3 (C ring), 60.0–61.2 (CH2-dpqC),
79.3 (CHN), 123.3 (C7), 123.8 (C Ar), 125.9 (C Ar), 127.0 (C2),
129.3 (C Ar), 130.4 (C Ar), 130.5 (C Ar), 137.6 (C Ar), 142.2
1
7.5% CH3OH–CH2Cl2) (94 mg, 0.10 mmol, 59%). H NMR
(400 MHz, CDCl3): d 0.90–1.58 (18H, m, CH2CH3, CHCH3),
2.06 (4H, m, H10, H13), 2.24–3.18 (16H, br m, CH2 ring), 3.21
(4H, m, H11, H12), 3.42–4.45 (17H, m, CH2CO, CH2-dpqC,
CH), 7.60 (1H, m, H2), 7.77 (1H, br m, H7), 7.89 (1H, br m,
H6), 9.24 (1H, m, H1), 9.43 (1H, m, H8). 13C NMR (75 MHz,
CDCl3): d 14.1 (CH3), 14.2 (CH3), 17.1 (CH3), 17.1 (CH3), 17.5
(CH3), 22.8 (C10, C13), 32.9 (C11, C12), 48.3–57.4 (CH2 ring,
CH2CO, CH2CH3), 61.3 (CH2-dpqC), 124.2 (C2), 124.9 (C Ar),
126.7 (C Ar), 127.6 (C7), 133.2 (C8), 134.1 (C1), 137.4 (C Ar),
146.4 (C Ar), 146.9 (C Ar), 151.6 (C6), 154.3 (C Ar), 157.5 (C
(C Ar), 146.4 (C Ar), 146.8 (C Ar), 151.3 (C Ar), 153.3 (C Ar),
+
=
154.7–157.0 (C O). HRMS (+ m/z): [M + Na] calculated for
C42H58N8O6Na, 793.4377; found, 793.4379.
1-(3-Methyl-10,11,12,13-tetrahydrodipyrido[3,2-a:2ꢀ,3ꢀ-c]phe-
nazine)-4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-
ane, 21. Trifluoroacetic acid (10 ml) was added to
a
solution of 1-(3-methyl-10,11,12,13-tetrahydrodipyrido[3,2-
a:2ꢀ,3ꢀ-c]phenazine)-4,7,10-tris(tert-butoxycarbonyl)-1,4,7,10-
tetraazacyclododecane (104.7 mg, 0.136 mmol) in CH2Cl2
(3 ml). The mixture was stirred for 2 h at room temperature. The
solvent was evaporated and the residue re-dissolved three times
to facilitate elimination of excess acid and tert-butyl alcohol.
The residue was taken into a 1 M KOH solution (5 ml) and the
product extracted into CH2Cl2 (3 × 5 ml). The organic layer
was dried over K2CO3. Removal of the solvent under reduced
pressure yielded an orange solid, mp > 240 ◦C (52.5 mg,
=
=
=
Ar), 160.9 (C Ar), 169.0 (C O), 172.5 (C O), 172.7 (C O).
m/z (ES+): 491 (MCa2+), 508 (MCu2+) 965 (MNa+).
1-(3-Methyl-10,11,12,13-tetrahydrodipyrido[3,2-a:2ꢀ,3ꢀ-c]phe-
nazine)-4,7,10-tris[(S)-1-(1-phenyl)ethylcarbamoylmethyl]-1,4,7,
10-tetraazacyclododecane, 2. Potassium carbonate (138 mg,
0.995 mmol) and a catalytic amount of KI were added to a
solution of 1-(3-methyl-10,11,12,13-tetrahydrodipyrido[3,2-
a:2ꢀ,3ꢀ-c]phenazine)-1,4,7,10-tetraazacyclododecane (94.1 mg,
0.199 mmol) in CH3CN (7 ml). The reaction mixture was
heated at 60 ◦C and a solution of (S)-N-(1-phenyleth-1-
yl)chloroacetamide (118 mg, 0.597 mmol) in CH2Cl2 (7 ml) was
added. The reaction mixture was boiled under reflux overnight,
under argon. The solution was filtered and the salts washed
with CH2Cl2. The product was purified by chromatography
on neutral alumina (gradient elution: CH2Cl2 to 1% CH3OH–
CH2Cl2, Rf = 0.55, 7.5% CH3OH–CH2Cl2) to yield a colourless
1
0.112 mmol, 82%). H NMR (300 MHz, CDCl3): d 2.09 (4H,
m, H10, 13), 2.60–3.00 (16H, m, CH2 ring), 3.24 (4H, m, H11,
H12), 4.16 (2H, s, CH2-dpqC), 7.69 (1H, d, J = 8.0 Hz, H2),
7.76 (1H, dd, J = 8.2, 4.4, H7), 9.26 (1H, dd, J = 4.4, 1.4, H6),
9.40 (1H, d, J = 8.0, H1), 9.46 (1H, dd, J = 8.2, 1.4, H8). 13C
NMR (75 MHz, CDCl3): d 22.6 (CH2), 32.6 (CH2), 45.2, 46.3,
47.1, 52.0 (CH2 ring), 61.7 (CH2), 122.8 (C7), 123.2 (C2), 125.9
(q Ar), 127.0 (q Ar), 132.7 (C8), 133.5 (C1), 137.1 (q Ar), 146.0
(q Ar), 146.8 (q Ar), 151.3 (C6), 153.3 (q Ar), 153.6 (q Ar),
162.3 (q Ar).
◦
1
solid (91 mg, 0.096 mmol, 48%), mp 148–150 C. H NMR
(300 MHz, CDCl3): d 1.35 (6H, d, J = 6.7 Hz, CH3), 1.56 (3H,
d, J = 6.7, CH3), 2.06 (4H, m, CH2), 2.16–3.12 (16H, br m,
CH2 ring), 3.21 (4H, d, J = 4.0, CH2), 3.42–4.45 (8H, br m,
CH2CO, CH2-dpqC), 4.57 (1H, br m, CH), 4.75 (2H, br m,
CH), 6.70–7.49 (18H, m, Ar, NH), 7.53 (1H, d, J = 8.3, H2),
7.96 (1H, br m, H7), 8.30 (1H, br m, H6), 9.37 (1H, d, J = 8.3,
H1), 9.48 (1H, d, J = 8.2, H8). 13C NMR (75 MHz, CDCl3): d
22.2 (CH3), 22.7 (C10, C13), 22.8 (CH3), 32.7 (C11, C12), 49.5
(CHN), 49.8–52.4 (CH2 ring + CH2CO), 60.4 (CH2-dpqC),
123.4 (C2), 125.1 (C7), 126.3 (C Ar), 126.4 (C Ar), 126.8 (C Ar),
1-(3-Methyl-10,11,12,13-tetrahydrodipyrido[3,2-a:2ꢀ,3ꢀ-c]phe-
nazine)-4,7,10-tris(N-acetyl-L-glutamic acid diethyl ester)-1,4,
7,10-tetraazacyclododecane, 5. Potassium carbonate (129 mg,
0.935 mmol) and a catalytic amount of KI were added to a
solution of 1-(3-methyl-10,11,12,13-tetrahydrodipyrido[3,2-
a:2ꢀ,3ꢀ-c]phenazine)-1,4,7,10-tetraazacyclododecane (87.8 mg,
0.187 mmol) in CH3CN (7 ml). The reaction mixture was
heated at 60 ◦C and a solution of N-chloroacetyl-L-glutamic
acid diethyl ester (157 mg, 0.561 mmol) in CH2Cl2 (7 ml) was
added. The reaction mixture was boiled under reflux overnight,
1 0 2 2
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 0 1 3 – 1 0 2 4

View Article Online
126.9 (C Ar), 127.4 (C Ar), 128.1 (C Ar), 128.2 (C Ar), 128.4 (C
Ar), 133.2 (C8), 134.1 (C1), 137.0 (C Ar), 137.5 (C Ar), 143.5
(C Ar), 144.2 (C Ar), 145.6 (C Ar), 146.2 (C Ar), 153.6 (C Ar),
127.7 (C Ar), 133.5 (C Ar), 133.9 (C Ar), 137.2 (C8), 137.5 (C1),
145.8 (C Ar), 146.4 (C Ar), 151.0 (C6), 154.2 (C Ar), 154.5 (C
+
=
=
Ar), 160.4 (C Ar), 172.5 (C O), 172.9 (C O). m/z (ES ): 836
153.9 (C Ar), 154.1 (C Ar), 159.9 (C Ar), 169.1 (C O), 170.7
(MNa+).
=
+
=
(C O). HRMS (+ m/z): [M + H] calculated for C57H68N11O3,
EuDO3AdpqC, [Eu.3]. Trifluoroacetic acid (4 ml) was added
to a solution of 1-(3-methyl-10,11,12,13-tetrahydrodipyrido[3,2-
a:2ꢀ,3ꢀ-c]phenazine)-4,7,10-tris-tert-butoxycarbonylmethyl-1,4,7,
10-tetraazacyclododecane (33.8 mg, 0.042 mmol) in CH2Cl2
(2 ml). The mixture was stirred overnight at room temperature.
The solvent was evaporated and the residue re-dissolved in
CH2Cl2 three times to facilitate elimination of excess acid and
tert-butyl alcohol. The product, 3, was checked by 1H NMR to
ensure complete ester hydrolysis, and was used for complexation
954.5506; found, 954.5497.
EuPh3dpq (CF3SO3)3, [Eu.1]. A solution of ligand 1 (28 mg,
0.03 mmol) and Eu(OTf)3 (19 mg, 0.03 mmol) in dry CH3CN
(2 ml) was boiled under reflux under argon overnight at
◦
80 C. The solution was then added dropwise to diethyl ether
(100 ml) with stirring, the precipitate centrifuged and the
solvent decanted. The solid was redissolved in CH3CN and
the process repeated. A colourless solid was obtained (35 mg,
0.02 mmol, 75%). 1H NMR (300 MHz, CD3OD, −10 ◦C)
partial assignment: d 37.9 (1H, s, Hax), 37.3 (1H, s, Hax),
31.9 (1H, s, Hax), 27.7 (1H, s, Hax), 26.9 (H10-dpq), 15.7
(dpq), 14.9 (dpq), 11–5 (dpq and Ph signals), −1.8 (6H, s,
CH3), −2.3 (3H, s, CH3), −3.5 (4H, m, Heq), −7.2 (2H, s,
Hax), −7.6 (2H, s, Hax), −9.7 (1H, s, Heq), −10.2 (1H, s,
Heq), −15.7 (1H, s, Heq), −15.8 (1H, s, Heq), −16.4 (1H, s,
NCH2CO), −17.0 (1H, s, NCH2CO), −17.6 (1H, s, NCH2CO),
−17.9 (1H, s, NCH2CO), −24.0 (2H, s, NCH2CO). m/z (ES+)
350 (60%, M3+), 525 (80%, M2+), 534 (50%, M2+ + H2O), 600
(30%, M◦3+ + CF3SO3−), 1348 (3%, M3+ + 2CF3SO3−); mp
176–179 C. Accurate mass spectrum: found (M + 2CF3SO3)+
1350.3208; C55H61N11O9EuS2F6 requires 1350.3202. kmax (H2O)
340 nm (e = 3500 M−1 cm−1); s(H2O) = 1.05 ms; s(D2O) =
1.64 ms, φ(H2O) = 21%, φ(D2O) = 27%.
1
immediately. H NMR (300 MHz, CD3OD): d 2.06 (4H, m,
H10, H13), 2.90–3.96 (28 H, br m, CH2 ring, CH2, CH2-dpqC,
H11, H12), 8.21 (2H, m, H7, H2), 9.19 (1H, m, H6), 9.38 (1H,
d, H1), 9.72 (1H, m, H8). The ligand was dissolved in a mixture
of methanol and water (1 : 1, 10 ml) and the pH raised to 5.5
by addition of a 1 M solution of KOH. EuCl3·6H2O (15.4 mg,
0.042 mmol) was added and the mixture heated at 90 ◦C
overnight. After being allowed to cool to room temperature
the pH of the mixture was raised to 10.0 using dilute KOH
solution and the mixture further stirred for 1 h. The solid
in the suspension was removed by syringe filtration and the
pH of the resulting clear solution again reduced to 5.5. The
solution was freeze dried to yield a white solid product that
was purified by chromatography on neutral alumina, eluting
with CH2Cl2–MeOH (70 : 30), to give a colourless complex.
kmax (H2O) 348 nm (e = 6500 M−1 cm−1); s(H2O) = 1.06 ms;
φ(H2O) = 18%.
TbPh3dpq (CF3SO3)3, [Tb.1]. The ligand
1 (15 mg,
0.02 mmol) and Tb(Otf)3 (10 mg, 0.02 mmol) were dissolved in
dry CH3CN (1 ml). The procedure followed was identical to that
described for the europium complex above A colourless solid was
obtained (20 mg, 0.01 mmol, 78%). m/z (ES+) 352 (100%, M3+),
603 (60%, M3+ + CF3SO3−), 1356 (5%, M3+ + 2CF3SO3−); mp
TbDO3AdpqC, [Tb.3]. This complex was prepared and
purified as for the Eu complex above. kmax (H2O) 348 nm (e =
8300 M−1 cm−1); s(H2O) = 1.46 ms; φ(H2O) = 33%.
◦
177–179 C. Accurate mass spectrum: found (M + CF3SO3)2+
Eu(Glu-Et)3dpqC (CF3SO3)3, [Eu.5]. A solution of 1-(3-
methyl-10,11,12,13-tetrahydrodipyrido[3,2-a:2ꢀ,3ꢀ-c]phenazine)-
4,7,10-tris(N-acetyl-L-glutamic acid diethyl ester)-1,4,7,10-
tetraazacyclododecane (25.4 mg, 0.021 mmol) and Eu(OTf)3
(12.5 mg, 0.021 mmol) in dry CH3CN (5 ml) was boiled under
reflux under argon overnight at 80 ◦C. The solution was then
added in a dropwise manner to diethyl ether (ca. 20 ml) with
stirring, the precipitate centrifuged and the solvent decanted.
The solid was re-dissolved in CH3CN and the process repeated
to yield an off-white solid product (36 mg, 0.02 mmol, 94%).
m/z (ES+): 451 (M3+), 751 (MOTf2+).
603.6862; C54H61N11O6TbSF3 requires 603.6866. kmax (H2O)
340 nm (e = 3500 M−1 cm−1); s(H2O) = 1.85 ms; s(D2O) =
2.38 ms, φ(H2O) = 36%, φ(D2O) = 46%.
GdPh3dpq (CF3SO3)3, [Gd.1]. Ligand 1 (10 mg, 0.01 mmol)
and Gd(OTf)3 (7 mg, 0.01 mmol) were dissolved in dry CH3CN
(1 ml). The procedure followed was identical to that described
above. A colourless solid was obtained (7 mg, 0.02 mmol, 42%).
m/z (ES+) 352 (100%, M3+), 603 (60%◦, M3+ + CF3SO3−), 1354
(5%, M3+ + 2CF3SO3−); mp 210–212 C. Accurate mass spec-
trum: found (M + 2CF3SO3)+ 1355.3240; C55H61N11O9GdS2F6
requires 1355.3251.
Eu(Ala-Et)3dpqC (CF3SO3)3, [Eu.4]. A solution of 1-(3-
methyl-10,11,12,13-tetrahydrodipyrido[3,2-a:2ꢀ,3ꢀ-c]phenazine)-
1-(3-Methyl-10,11,12,13-tetrahydrodipyrido[3,2-a:2ꢀ,3ꢀ-c]phe-
nazine)-4,7,10-tris-tert-butoxycarbonylmethyl-1,4,7,10-tetraaza-
4,7,10-tris(N-acetyl-L-alanine
diethyl
ester)-1,4,7,10-
tetraazacyclododecane (27.7 mg, 0.029 mmol) and Eu(OTf)3
(17.4 mg, 0.029 mmol) in dry CH3CN (5 ml) was boiled under
reflux under argon overnight at 80 ◦C. The solution was then
added in a dropwise manner to diethyl ether (ca. 20 ml) with
stirring, the precipitate centrifuged and the solvent decanted.
The solid was redissolved in CH3CN and the process repeated
to yield an off-white solid product (23 mg, 0.015 mmol, 51%).
m/z (ES+): 365 (M3+), 621 (MOTf2+).
cyclododecane,
2.90 mmol) and a catalytic amount of KI were added to
solution of 1,4,7-tris-tert-butoxycarbonylmethyl-1,4,7,10-
23. Potassium
carbonate
(401
mg,
a
tetraazacyclododecane, 22, (299 mg, 0.580 mmol) in CH3CN
(15 ml). The mixture was heated at 60 ◦C and a solution
of 3-chloromethyl-10,11,12,13-tetrahydrodipyrido-[3,2a:2ꢀ,3ꢀ-
c]phenazine (223 mg, 0.580 mmol) in CH2Cl2 (20 ml) was
added. The reaction mixture was boiled under reflux overnight,
under argon. The solution was filtered and the salts washed
with CH2Cl2. Evaporation of the solvent afforded the crude
residue. The product ester was purified by chromatography on
silica gel (gradient elution: CH2Cl2 to 3.3% CH3OH–CH2Cl2,
Rf = 0.47, 10% CH3OH–CH2Cl2) to yield a very pale yellow
EuPh3dpqC (CF3SO3)3, [Eu.2]. A solution of 1-(3-methyl-
10,11,12,13-tetrahydrodipyrido[3,2-a:2ꢀ,3ꢀ-c]phenazine)-4,7,10-
tris[(S)-1-(1-phenyl)ethylcarbamoylmethyl]-1,4,7,10-tetraazacy-
clododecane (31.8 mg, 0.033 mmol) and Eu(OTf)3 (20.0 mg,
0.033 mmol) in dry CH3CN (5 ml) was boiled under reflux
◦
◦
1
solid (68 mg, 0.084 mmol, 15%), mp 137–139 C. H NMR
(300 MHz, CDCl3): d 1.08 (18H, s, CH3), 1.41 (9H, s, CH3),
2.05 (4H, m, H10, H13), 2.25–3.58 (28H, br m, CH2 ring, H11,
H12), 7.68 (1H, m, H7), 7.75 (1H, m, H2), 8.76 (1H, dd, J =
4.2, 1.2 Hz, H6), 9.42 (1H, m, H1), 9.48 (1H, m, H8). 13C
NMR (100 MHz, CDCl3): d 27.9 (CH3), 50.9–60.4 (CH2 ring),
81.6–82.7 (CH2CO), 123.6 (C2), 123.9 (C Ar), 126.2 (C Ar),
under argon overnight at 80 C. The solution was then added
in a dropwise manner to diethyl ether (ca. 20 ml) with stirring,
the precipitate centrifuged and the solvent decanted. The solid
was re-dissolved in CH3CN and the process repeated to yield an
off-white solid product (39 mg, 0.025 mmol, 76%). m/z (ES+):
628 (MOTf2+), 1402 (MOTf2+). This complex was converted
to the more water soluble chloride salt by ion exchange
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 0 1 3 – 1 0 2 4
1 0 2 3

View Article Online
chromatography using Amberlite IRA resin. kmax (H2O) 348 nm
(e = 8300 M−1 cm−1); s(H2O) = 1.04 ms; s(D2O) = 1.59 ms,
C. E. Mathien, D. Parker and P. K. Senanayake, Inorg. Chem., 2001,
40, 5860.
6 R. S. Dickins, C. Crossland, D. Parker, H. Puschmann and J. A. K.
Howard, Chem. Rev., 2002, 102, 1977.
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A. N. Eberle and H. R. Maecke, Curr. Med. Chem., 2000, 7, 791; for
MRI, see:; P. Caravan, J. Ellison, T. J. McMurry and R. B. Lauffer,
Chem. Rev., 1999, 99, 2293.
1
␾(H2O) = 16%, ␾(D2O) = 20%. H NMR (CD3OD): d 37.51,
36.88, 31.43, 27.03, 26.45 (axial ring protons + ortho aromatic),
15.17, 14.39, 10.27, 9.56, −2.06, −2.62, −3.78, −4.14, −7.40,
−7.71, −8.08, −15.71, −16.24, −16.84, −17.13, −17.85,
−18.49, −24.32. (m/z, ESMS): [M + CF3SO3]2+ calculated for
C58H68N11O6F3SEu, 627.7081; found, 627.7080.
8 D. Parker and J. A. G. Williams, J. Chem. Soc., Dalton Trans., 1996,
3613.
Tb(Glu-Et)3dpqC (CF3SO3)3, [Tb.5] and Tb(Ala-Et)3dpqC
(CF3SO3)3, [Tb.4]. Each of these complexes was prepared by a
similar method.
9 B. H. Bakker, M. Goes, N. Hoebe, H. J. van Ramesdonk, J. W.
Verhoeven, M. H. V. Werts and J. W. Hofstraat, Coord. Chem. Rev.,
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Inorg. Chem., 2002, 41, 1966.
11 J. C. Collins, J. R. Aldrich-Wright, I. D. Greguric and P. A. Pellegrini,
Inorg. Chem., 1999, 38, 5502; C. G. Coates, J. Olofsson, M. Coletti, J. J.
McGarvey, B. Onfelt, P. Lincoln, B. Norden, E. Tuite, P. Matousek
and A. W. Parker, J. Phys. Chem. B, 2001, 105, 12 653.
12 G. Bobba, R. S. Dickins, S. D. Kean, C. E. Mathieu, D. Parker, R. D.
Peacock, G. Siligardi, M. J. Smith, J. A. G. Williams and C. F. G. C.
Geraldes, J. Chem. Soc., Perkin Trans. 2, 2001, 1729; G. Bobba, S. D.
Kean, D. Parker, A. Beeby and G. Baker, J. Chem. Soc., Perkin Trans.
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13 G. Bobba, J.-C. Frias and D. Parker, Chem. Commun., 2002, 890.
14 J.-C. Frias, G. Bobba, M. J. Cann, D. Parker and C. J. Hutchinson,
Org. Biomol. Chem., 2003, 1, 905.
15 M. Yamonda, Y. Tanaka, Y. Yoshimoto, S. Kuroda and I. Shimao,
Bull. Chem. Soc. Jpn., 1992, 65, 1006; care needs to be exercised
during basification to avoid an exotherm that may promote decar-
boxylation to give a substituted diazafluorenone:; G. A. Shabir and
N. J. Forrow, J. Pharm. Biomed. Anal., 2003, 33, 219.
16 A. M. S. Garas and R. S. Vagg, J. Heterocycl. Chem., 2000, 37,
151.
A solution of 1-(3-methyl-10,11,12,13-tetrahydrodipyrido-
[3,2-a:2ꢀ,3ꢀ-c]phenazinyl)-4,7,10-tris(N-acetyl-L-alanine diethyl
ester)-1,4,7,10-tetraazacyclododecane, 4, (25.4 mg, 0.027 mmol)
and Tb(OTf)3 (16.4 mg, 0.027 mmol) in dry CH3CN (5 ml)
was boiled under reflux under argon overnight at 80 ◦C. The
solution was then added in a dropwise manner to diethyl ether
(ca. 20 ml) with stirring, the precipitate centrifuged and the
solvent decanted. The solid was re-dissolved in CH3CN and the
process repeated to yield the product (14.8 mg, 0.0957 mmol,
35%). m/z (ES+): 367 (M3+), 625 (MOTf2+), 1399 (MOTf2+).
TbPh3dpqC (CF3SO3)3, [Tb.2]. A solution of 1-(3-methyl-
10,11,12,13-tetrahydrodipyrido[3,2-a:2ꢀ,3ꢀ-c]phenazinyl)-4,7,10-
tris[(S)-1-(1-phenyl)ethylcarbamoylmethyl]-1,4,7,10-tetraazacy-
clododecane (30.9 mg, 0.032 mmol) and Tb(OTf)3 (19.6 mg,
0.032 mmol) in dry CH3CN (5 ml) was boiled under reflux
◦
under argon overnight at 80 C. The solution was then added
in a dropwise manner to diethyl ether (ca. 20 ml) with stirring,
the precipitate centrifuged and the solvent decanted. The solid
was re-dissolved in CH3CN and the process repeated to yield
the product (39.5 mg, 0.0253 mmol, 79%). m/z (ES+): 371
(M3+), 631 (MOTf2+), 1411 (MOTf2+). kmax (H2O) 348 nm
(e = 8300 M−1 cm−1); s(H2O) = 1.56 ms; s(D2O) = 1.72 ms,
φ(H2O) = 40%, φ(D2O) = 48%. 1H NMR (CD3OD): d 118.57,
93.49, 73.02, 67.19, 64.91, 63.33, 54.31, 37.96, −2.26, −7.03,
−7.28, −8.11, −9.07, −9.35, −15.62, −17.19, −41.84, −73.53,
−79.19, −110.18. ESMS (m/z): [M + CF3SO3]2+ calculated for
C58H68N11O6F3STb, 630.7101; found, 630.7099.
17 C. J. Chandler, L. W. Deady and J. A. Reiss, J. Heterocycl. Chem.,
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Other complexes reported were prepared and purified by
analogous methods.
Acknowledgements
We thank EPSRC for studentship support (R. P.), the ONE-
NE Durham County Partnership (STBE), EMIL (EC network
of Excellence) and COST Action D-18 for partial support and
the EPSRC Mass Spectroscopy Service (Swansea) for assistance
with MS spectral measurements.
25 I. M. Clarkson, A. Beeby, J. I. Bruce, L. J. Govenlock, M. P. Lowe,
C. E. Mathieu, D. Parker and K. Senanayake, New J. Chem., 2000,
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O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 0 1 3 – 1 0 2 4