π-Expanded Thioxanthones – Engineering the Triplet Level of Thioxanthone Sensitizers for Lanthanide-Based Luminescent Probes with Visible Excitation

Article abstract of DOI:10.1002/cplu.201900309

Bright lanthanide based probes for optical bioimaging must rely on the antenna principle, where the lanthanide-centred excited state is formed by a complex sensitization process. Efficient sensitization of lanthanide-centred emission occurs via triplet states centred on the sensitizing chromophore. Here, the triplet state of thioxanthone chromophores is modulated by extending the π-system. Three thioxanthone chromophores-thioxanthone, benzo[c]thioxanthone, and naphtho[2,3-c]thioxanthone were synthesised and characterised. The triplet state energies and lifetimes is found to change as expected, and two dyes are found to be suitable sensitizers for europium(iii) luminescence. Reactive derivatives of thioxanthone and benzo[c]thioxanthone were prepared and coupled to a 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A) lanthanide binding pocket. The photophysics and the performance in optical bioimaging of the resulting europium(iii) complexes were investigated. It is concluded that while the energetics favour efficient sensitization, the solution structure does not. While it was found that the complexes are too lipophilic to be efficient luminescent probes for optical bioimaging, we successfully demonstrated bioimaging using europium(iii) luminescence following 405 nm excitation.

Full text of DOI:10.1002/cplu.201900309

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Accepted Article
Title: π-Expanded Thioxanthones – Engineering the Triplet Level of
Thioxanthone Sensitizers for Lanthanide-Based Luminescent
Probes with Visible Excitation
Authors: Charlotte Nybro Dansholm, Anne Kathrine R Junker, Lea
Gundorff Nielsen, Nicolaj Kofod, ROBERT PAL, and Thomas
Just Sørensen
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π-Expanded Thioxanthones – Engineering the Triplet Level of
Thioxanthone Sensitizers for Lanthanide-Based Luminescent
Probes with Visible Excitation
Charlotte Nybro Dansholm[a], Anne Kathrine R. Junker[a], Lea G. Nielsen[a], Nicolaj Kofod[a], Robert
Pal*[b] and Thomas Just Sørensen*[a]
Abstract: Bright lanthanide based probes for optical bioimaging must
rely on the antenna principle, where the lanthanide centred excited
state is formed via a complex sensitization process. It is established
that efficient sensitization of lanthanide centred emission occur via
triplet states centred on the sensitizing chromophore. Here, the triplet
state of thioxanthone chromophores is modulated by extending the π-
engineering.[3] The goal is to make bright luminescent probes that
can be excited with the blue (405 nm) laser, thus using the highest
energy line in commercial microscopes, rather than the
specialised costly UV laser that currently is needed to do
bioimaging using lanthanide centred luminescence. This requires
a narrow singlet-triplet energy splitting, and a redshift of the
absorption of commonly used sensitizers above 400 nm.[2, 3]
Sensitized lanthanide luminescence can result in bright
lanthanide luminescent probes. This is also known as the antenna
principle, where the sensitizer is considered a light harvesting
antenna chromophore. Direct excitation of the Ln3+ ions yields a
low luminescence intensity due to their low molar absorption
coefficients (ε ≈ 0.1 - 1 M-1cm-1).[2a, 2i] While it is possible to do
optical microscopy following direct excitation,[4] bright lanthanide
based probes must rely on sensitization via an antenna
chromophore.[2, 5]
system.
Three
thioxanthone
chromophores—thioxanthone,
naphtho[2,3-c]thioxanthone are
benzo[c]thioxanthone,
and
synthesised and characterised. The general photophysics, triplet
state energies, and lifetimes is found to change as expected, and two
dyes are found to be suitable sensitizers for europium(III)
luminescence. Reactive derivatives of thioxanthone and
benzo[c]thioxanthone are prepared and coupled to a 1,4,7,10-
tetraazacyclododecane-1,4,7-triacetic acid (DO3A) lanthanide
binding pocket. The photophysics and the performance in optical
bioimaging of the resulting europium(III) complexes are investigated.
It is concluded that while the energetics favour efficient sensitization,
the solution structure does not. While it was found that the complexes
are too lipophilic to be efficient luminescent probes for optical
bioimaging, we successfully demonstrate bioimaging using
europium(III) luminescence following 405 nm excitation.
Introduction
To improve lanthanide-based molecular probes for bioimaging the
sensitization pathway must be optimized.[1] As the most efficient
sensitization pathway goes through the triplet state of a strongly
absorbing chromophore,[2] the next natural step in optimizing
lanthanide luminescent probes for bioimaging is triplet
Chart 1. Model compounds thioxanthone 1, and 3-methyl-thioxanthone 8; π-
extended thioxanthones benzo[c]thioxanthone 2, and naphtho[2,3-
c]thioxanthone 3, and 10-methyl benzo[c]thioxanthone 9; and 3-methyl-
azathioxanthone 8a, donor substituted 7-methoxy-3-methyl-1-azathioxanthone
8b and 8-methoxy-3-methyl-1-azathioxanthone 8c.
[a]
C. N. Dansholm, Dr A. K. R. Junker, L. G. Nielsen, N. Kofod, Dr T. J.
Sørensen
Nano-Science Center & Department of Chemistry
University of Copenhagen
Triplet engineering has been done for triplet sensitizers used in
photo- and polymer chemistry,[6] and in the more esoteric pursuit
of excited state aromaticity.[7] One of the most explored
chromophores is thioxanthone (1, Chart 1),[6d, 6g-j, 8] a chromophore
that has also been shown to be one of the best as a sensitizer for
lanthanide centred emission, in particular the coordinating
azathioxanthones.[8d, 9] Methyl substitution and addition of aza-
bridges does not change the photophysics of the parent
thioxanthone chromophore.[10] We have previously explored the
effect on the triplet state of adding electron donating groups to the
Universitetsparken 5, 2100 København Ø, Denmark
E-mail: tjs@chem.ku.dk
Web: sites.google.com/view/tjsgroup/home
[b]
Dr R. Pal
Department of Chemistry
Durham Univeristy
Lower Mountjoy, Stockton Road, Durham DH1 3LE, UK
E-mail: robert.pal@durham.ac.uk
Web: www.dur.ac.uk/research/directory/staff/?mode=staff&id=6011
Supporting information for this article is given via a link at the end of
the document.
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azathioxanthone scaffold, see Chart 1 compounds 8a-c.[10] The
results confirmed that the absorption and fluorescent properties
of the thioxanthone chromophore (S1) behave as predicted by
extended Hückel theory (see the supporting information for
details). Unfortunately, the properties of the phosphorescent T1
state are less easily predicted, and the addition of an electron
donating group in 8b results in the desired redshift, but also
unfavorable strong fluorescence.[10] Here, we chose to modulate
the photophysical properties of thioxanthone by expanding the
conjugated system of thioxanthone as illustrated in Chart 1
compounds 2, 3 and 9. The target is absorption in the blue, a small
singlet-triplet splitting, and efficient population of first the triplet
state and then the lanthanide(III) centre when the chromophore
is incorporated in a kinetically inert lanthanide(III) complex.[2i, 11]
To explore the photophysics of thioxanthones with an extended
aromatic system we first synthesized the model compounds 1, 2,
and 3 shown in Chart 1. After a full characterization of the
photophysical properties of 1-3, we concluded that only 1 and 2
would give rise to luminescent lanthanide complexes as the
energetics of 3 were a poor match with the excited lanthanide
states. Thus we synthesised DO3A ligands L1 and L2 appended
Scheme 1. Synthesis of thioxanthone 1, benzo[c]thioxanthone 2, naphtho[2,3-
c]thioxanthone 3, 3-methylthioxanthone 8, and 10-methyl-benzo[c]-
thioxanthone 9.
The two-step, one-pot synthesis of thioxanthones 1-3 from 2-
thiobenzoic acid is shown in Scheme 1.[13] The first step is an
electrophilic aromatic substitution reaction yielding 2-carboxy-3’-
methyldiaryl sulfide, followed by a cyclisation. The conditions
shown in Scheme 1 are generally applicable, yet several other
combinations of starting materials and reagents have been
reported.[13] The reported method of purification is recrystallization
from dioxane:water,[12a-d] but to obtain analytically pure samples
column chromatography (toluene:DCM or DCM, see the
supporting information) was required, and the product were
isolated as pale yellow to yellow powders in yields from 8 to
32%.[12b, 14]
with chromophore
1
and
2
respectively and prepared
europium(III), terbium(III), yttrium(III) complexes of these, see
Chart 2. We found that the complexes have an ill-defined solution
structure that resulted in inefficient sensitization of the
lanthanide(III) centre. Further, the low solubility of the ligands and
complexes in aqueous media resulted in less than ideal behavior
in biological systems. Despite these complications, we succeeded
in demonstrating that the triplet level of the thioxanthone
chromophore could be engineered exclusively, and that the
chromophore can sensitize europium(III) luminescence so that
molecular probes based on lanthanide luminescence are possible
for bioimaging following excitation at 405 nm.
While the mechanism for the formation of the thioxanthones
13c, 15]
from thiosalicylic acid has been investigated,[12d,
the
constitution of the products are inconsistently reported. By
extensive NMR analysis (see the supporting information) it was
verified that compounds 2, 3, and 9 are chevron-shaped as
indicated in Scheme 1.
To make thioxanthones that can be linked to the 1,4,7,10-
tetraazadodecane-1,4,7-triacetic acid (DO3A) lanthanide binding
pocket, a methyl group is introduced in the scaffold by starting
from 4-methylthiosalicylic acid 7, see Scheme 2. To make the 4-
methylthiosalicylic acid 7 a four-step synthesis starting from 4-
methylsalicylic acid was used, see Scheme 2.[16] First step is
protecting the carboxylic acid as the methyl ester 4. Unreacted 4-
methylsalicylic acid was removed by extraction with alkaline water
and 4 was isolated as an oil in a yield of 79 %. The phenol was
reacted with N,N-dimethylthiocarbamoyl chloride in the presence
of DABCO that upon recrystallization gave the pure N,N-
dimethylthiocarbamate ester 5 as a white powder in a 83 % yield.
Following a Newman-Kwart rearrangement under an inert
atmosphere of nitrogen at 190˚C 5 was converted to methyl 2-
((dimethylcarbamoyl)thio)-4-methylbenzoate 6 that was isolated
as an off-white product in a yield of 75 %. The Newman-Kwart
rearrangement is known to give pure products.[16b] Here, the
product took on a brown colour, in part due to the 20 hours
required for full conversion thus, necessitating recrystallization.
The final step is a double hydrolysis of 6 in alkaline water under
reflux. Upon hydrolysis 7 was isolated as a white powder in 65 %
Chart 2. Lanthanide(III) complexes of thioxanthone and benzo[c]thioxanthone
appended 1,4,7,10-tetraazadodecane-1,4,7-triacetic acid (DO3A) ligands L1
and L2 drawn in a form with q = 2.[1]
Results and Discussion
Synthesis
Thioxanthone 1, benzo[c]thioxanthone 2 and naphtho[2,3-
c]thioxanthone 3 were synthesised from the commercially
available thiosalicylic acid and benzene, naphthalene or
anthracene as outlined in scheme 1. The chosen synthetic
approach was selected as it has been shown to be efficient for a
variety of aromatic hydrocarbons.[12]
yield.
3-methyl-thioxanthone
8
and
the
10-methyl-
benzo[c]thioxanthone 9 were synthesised from 7 using the same
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procedures as described for the synthesis of 1 and 2 and with
similar appearance and yields, see Scheme 1.
described,[1b] following the procedure developed by Sammes and
Faulkner.[19] Bromomethyl thioxanthone derivatives 10 or 11 was
used to alkylate the secondary amine of the tert-butyl triester of
DO3A 12 in MeCN at 60˚C with K2CO3. The preligands 13 and 14
were isolated as off-white and pale yellow powders in moderate
yields of 60
% and 55 % after purification by column
chromatography (2-5 % MeOH:DCM). Compounds 10 and 11
were used in excess (1.1 eq.), as they are easier to seperate from
the products compared to compound 12.
The tert-butyl-ester groups of preligands 13 and 14 were
removed in TFA and DCM in a 1:1 mixture at r.t.[20] The
thioxanthone
appended
DO3A
ligand
H3L1
and
benzo[c]thioxanthone appended DO3A ligand H3L2 were purified
by trituration with diethyl ether from MeOH and isolated in yields
of 56 % and 71 % as pale-yellow powders.
Scheme 2. Synthesis of 4-methylthiosalicylic acid 7.
The ligands H3L1 and H3L2 were used to form complexes with
europium(III), terbium(III), and yttrium(III). Ln.L1 (Ln = Y3+, Eu3+
and Tb3+). The complexes proved difficult to isolate and purify,
presumably due to the low solubility. Thus, the complexes Ln.L1
and Ln.L2 (Ln = Y3+, Eu3+ and Tb3+) were prepared in situ from
stock solutions by mixing 1:1 equivalent of H3L1 or H3L2 and the
corresponding lanthanide(III) triflate. Ln.L1 and Ln.L2 (See Chart
In order to link thionxanthones 8 and 9 to a DO3A binding
pocket a leaving group must be introduced on the α-methyl group.
Wohl-Ziegler bromination of several methyl thioxanthone
17]-[18]
derivatives have previosly been reported.[13b,
Radical
bromination was first tested in carbon tetrachloride, however, the
more accessible and less toxic cyclohexane was used for the
synthesis of 3-bromomethyl-thioxanthone 10 and the 10-
bromomethyl-benzo[c]thioxanthone 11, see Scheme 3. The
reactant was suspended in cyclohexane with NBS. Benzoyl
peroxide was used as initiator. Successive additions of NBS
during the reaction proved to give the highest yield of the final
product. For 10, the product was purified by column
chromatography (DCM) and isolated as a pale yellow powder in a
yield of 20 %. For 11, column chromatography (DCM) resulted in
a mixture of 11 and 9. As only 11 will react in the next synthesis
step, the crude product was taken forward. A yield of 8 % for
compound 11 was calculated based in the integrals in the 1H-
NMR spectrum.
1
2) were characterised using HRMS, H NMR and luminescence,
see the supporting information for details.
Table 1. Photophysical properties in the form of absorption maximum λmax
absorption cross section ε, fluorescence maximum λfl, fluorescence quantum
yield φfl, fluorescence lifetime τfl, phosphorescence maximum λPhos, and
phosphorescence lifetime τPhos of thioxanthone 1, benzo[c]thioxanthone 2,
naphtho[2,3-c]thioxanthone 3, 3-methyl-thioxanthone 8, and 10-methyl
benzo[c]thioxanthone 9 in methanol.
,
[a]
Compound
λmax
nm
log(ε)
λfl
nm
φfl
%
τfl
ns
λPhos
nm
τPhos
ms
ΔES-T
cm-1
1
8
2
9
3
378
256
3.99
4.85
434
431
440
434
507
11
2.8
454
450
512
507
672
161±1
146±6
269±5
268±1
24±4
1890
1860
4100
4160
6545
376
258
3.84
4.77
12
3.07
0.80
0.64
0.71
387
287
3.88
4.56
3.2
3.0
1.7
385
288
3.91
4.72
426
326
3.80
4.57
[a] Estimated error relative 10%.
Scheme 3. Synthesis of thioxanthone appendend DO3A ligands H3L1 and H3L2.
Chromophore photophysics
Even though the thioxanthone chromophore has been explored
as a sensitizer for lanthanide(III) centre emission,[4c, 10, 21] and
though thioxanthones with various substitution patterns have
The synthesis of ligands H3L1 and H3L2 were performed
following a two-step synthetic route shown in Scheme 3. The tert-
butyl triester of DO3A 12 was prepared as previously
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