Synthesis and characterisation of folic acid based lanthanide ion probes

Article abstract of DOI:10.1016/j.ica.2013.10.011

As a first step in the process of developing folate based visual probes and contrast agents, we have designed and synthesised a series of first generation lanthanide(III) molecular probes. The molecular probe structure included a lanthanide(III) (Eu(III), Tb(III), Gd(III)) chelate which was linked (2 or 3) to either a folic acid or pteroic acid targeting motif. We have defined the emission properties of the molecular probes at different pHs, the emission lifetimes, and the number of metal bound water molecules. The cellular uptake of the molecular probes was investigated in HeLa cells and the amount of Eu(III) internalisation quantified by inductively coupled plasma mass spectrometry. Our results highlighted several key features of probe design: a shorter linker was more optimal for both Eu(III) ion emission intensity and cellular uptake; the folic acid targeting motif exhibited higher cellular uptake when compared to pteroic acid; the emission intensity of the folic acid based probes was pH insensitive, whereas the pteroic acid based probes were pH sensitive. These first generation folate molecular probes displayed promising chemical and physical properties, suggesting that optical and MRI probes can potentially be developed, to enable the imaging of folate receptors in cancer cells and tissues.

Full text of DOI:10.1016/j.ica.2013.10.011

Accepted Manuscript
Synthesis and characterisation of folic acid based lanthanide ion probes
Zhangli Du, Glenn N. Borlace, Robert D. Brooks, Ross N. Butler, Doug A.
Brooks, Sally E. Plush
PII:
S0020-1693(13)00575-6
http://dx.doi.org/10.1016/j.ica.2013.10.011
ICA 15693
DOI:
Reference:
To appear in:
Inorganica Chimica Acta
Received Date:
Revised Date:
Accepted Date:
23 April 2013
9 October 2013
11 October 2013
Please cite this article as: Z. Du, G.N. Borlace, R.D. Brooks, R.N. Butler, D.A. Brooks, S.E. Plush, Synthesis and
characterisation of folic acid based lanthanide ion probes, Inorganica Chimica Acta (2013), doi: http://dx.doi.org/
1
0.1016/j.ica.2013.10.011
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Synthesis and characterisation of folic acid based lanthanide ion probes
a
Zhangli Du , Glenn N Borlace , Robert D Brooks , Ross N Butler , Doug A Brooks and Sally
a
a
a
a
a,
E Plush *
a
Mechanisms in Cell Biology and Disease Research Group, School of Pharmacy and
Medical Sciences, Sansom Institute for Health Research, University of South Australia, 5001,
Australia
*
Corresponding author: P5-28, Playford building, Frome Rd, Adelaide, SA, 5001, Australia
Tel: +61 8 830 22586. Fax: +61 8 830 22389. Email: Sally.Plush@unisa.edu.au
Abstract:
As a first step in the process of developing folate based visual probes and contrast agents, we
have designed and synthesised a series of first generation lanthanide(III) molecular probes.
The molecular probe structure included a lanthanide(III) (Eu(III), Tb(III), Gd(III)) chelate
which was linked (2 or 3) to either a folic acid or pteroic acid targeting motif. We have
defined the emission properties of the molecular probes at different pHs, the emission
lifetimes, and the number of metal bound water molecules. The cellular uptake of the
molecular probes was investigated in HeLa cells and the amount of Eu(III) internalisation
quantified by inductively coupled plasma mass spectrometry. Our results highlighted several
key features of probe design: a shorter linker was more optimal for both Eu(III) ion emission
intensity and cellular uptake; the folic acid targeting motif exhibited higher cellular uptake
when compared to pteroic acid; the emission intensity of the folic acid based probes was pH
insensitive, whereas the pteroic acid based probes were pH sensitive. These first generation
folate molecular probes displayed promising chemical and physical properties, suggesting
that optical and MRI probes can potentially be developed, to enable the imaging of folate
receptors in cancer cells and tissues.
Keywords:
Lanthanide(III), molecular probe, folic acid, pteroic acid, folate receptor
1

1
. Introduction
Molecular probes based on organic fluorophores [1-3], recombinant proteins [4],
semiconductor nanoparticles (quantum dots) [5-7] and metal ion complexes [8, 9] have been
vital to our understanding of the cellular and molecular mechanisms that occur in different
diseases [10-12]. These probes have been used in combination with imaging technologies
such as MRI, PET/SPECT, CT and confocal/super resolution microscopy to report on
structure and function at the level of individual cells through to whole organisms. There is
now a demand for new types of targeted molecular imaging probes that can discriminate
between different tissue and cell types. For example, probes that can distinguish between
tumour lesions and normal tissue/cells would be of substantial benefit in the detection and
treatment of cancer. This kind of specificity can be achieved by using labelled antibodies [13,
1
4], however there are drawbacks to this approach. For example, the relatively large size of
the antibody-conjugate complex compared to the target may affect the behaviour of the target
during receptor internalisation/endocytosis. A better approach may be to incorporate targeting
motifs directly into the probe structure [15-19]. While there are obvious advantages to this
approach with regard to probe specificity and size, the development of molecular imaging
probes coupled to specific targeting motifs presents key challenges in design and synthesis.
Firstly, the design of the probe should take into account that fluorescence emission can be
modulated by pH and or structural changes, and that the signal from the probe can be
contaminated by autofluorescence, particularly for live cell imaging applications. Secondly,
the synthetic protocols for the probe should ideally be adaptable so that different reporters
can be easily incorporated to enable different methods of detection e.g. magnetic resonance
vs confocal imaging.
Lanthanide ion complexes have a number of attractive properties that make them suitable
candidates for use in both MRI and optical imaging [20-22]. There are a range of
commercially available Gd(III) mono-aqua complexes that are routinely used as contrast
agents for MRI [23-26]. Other lanthanides (Ln(III) = Sm(III), Eu(III), Tb(III), Dy(III),
Yb(III)) are being investigated for use in optical imaging technologies [27-29]. These
optically active lanthanides a) possess large Stoke’s shifts, which facilitate the easy
2

separation of excitation from emission wavelengths, b) avoid concentration-dependant self-
absorption, which means that the strength of the signal will be proportional to probe
concentration, and c) have long emission lifetimes which enables the removal of background
autofluorescence in biological samples by using time gated measurements. The lanthanides
are also considered to be iso-structural [27], meaning that any single ligand complex
combined with any lanthanide ion will retain the same stability and coordination geometry
[
27, 30]. These qualities provide the capability of generating iso-structural probe sets with a
single ligand and a mixed population of lanthanide ions. Thus, administering a “cocktail” of
complexes, comprised of a mixture of Gd(III) and Eu(III) ions incorporated with a single
ligand, could enable dual detection by MRI and optical imaging. Furthermore, the coupling of
a site specific targeting moiety to a lanthanide chelate would enable selective delivery to any
cell or tissue with an appropriate receptor. The development of adaptable lanthanide ion
based molecular probes with specific targeting motifs may therefore provide a new
generation of imaging reagents to visualise disease processes at the molecular level.
However, due to the emerging nature of this field, the first priority is to systematically study
the effect of structural changes on probe uptake and emission intensity, thereby identifying
critical parameters in probe design.
The folate receptor provides an effective model system to evaluate site specific targeting of
molecular probes and to investigate the potential of lanthanide molecular probes in biology.
This receptor is a cell surface glycosyl-phosphatidylinositol-linked membrane glycoprotein,
which has increased expression in several cancers; including colorectal, ovarian, breast, lung,
cervical, renal and nasopharyngeal cancers [31-33]. A wide variety of folate receptor targeted
systems have already shown promise in cancer biology [18, 34-36]; for example as selective
delivery systems for cyctotoxic proteins and as real time interoperative fluorescence imaging
agents. However, no single system has proven effective both in vitro and in vivo without
concerns over specificity, targeting, side effects or quality of image generated. There are also
significant questions regarding the ideal structure of these delivery systems that need to be
addressed in terms of; what is the essential binding component of folate and does the
glutamic acid residue have a role in folate receptor binding and what is the ideal distance
between the folate receptor targeting motif and the imaging agent for example.
3

Herein, we describe the synthesis and characterisation of a series of first generation folic acid
based lanthanide molecular probes [Ln(III) = Eu(III), Tb(III) or Gd(III)]. The basic probe
design consisted of a lanthanide chelate coupled to a folate receptor targeting moiety via a
linker. A series of probes were synthesised with different linker lengths and different
targeting moieties to examine the effect on probe emission and cellular uptake: first, the
length of the linker between the folate receptor targeting moiety and the lanthanide chelate
was varied, to gain information on the optimal distance for receptor mediated uptake; second,
pteroic acid (PTE) was substituted for folic acid as the folate receptor targeting moiety. PTE
is a sub-component of folic acid (Figure 1), which has been shown to target tumour cells
with varying efficiency [37, 38]. While synthetic preparations of folic acid are known to
produce two regio-isomers, due to the presence of the two carboxylic acid groups in the
glutamic acid residue (Figure 1a), PTE based probes are synthetically simpler to prepare and
still enabled us to examine the efficiency of cellular uptake in response to changes in probe
design.
2
2
1
. Experimental
.1. General materials and methods
,4,7,10-tetraazacyclododecane, Cyclen, was obtained commercially from Strem USA. All
other solvents and chemicals used were obtained from Sigma-Aldrich or Merck, Australia.
Dowex 1-X8 (CI) and Sephadex G10 resins were purchased from Sigma-Aldrich, Australia.
DO3AtBu (1) [39], N-(tert-butoxycarbonyl)-1,2-diaminoethane [40], N-(tert-
butoxycarbonyl)-1,6-diaminohexane [40, 41], N-(2-((2-chloroacetyl)amino)ethyl)-,1,1-
dimethylethyl ester (2) [30] , N-(6-((2-chloroacetyl)aminohexyl)-,1,1-dimethylethyl ester (3)
were prepared as previously described [30]. Thin layer chromatography (TLC) was
performed on silica gel 60 F254 plates obtained from Merck, Australia. The infrared
spectroscopy (IR) was recorded on a Shimadzu FTIR-8400S. Electrospray ionization mass
spectrometry (ESI-MS) was recorded using a Perkin-Elmer Scoex API 3000. Inductively
1
coupled plasma mass spectrometry (ICP-MS) was conducted on an Agilent 7500 CS. H
13
NMR and C NMR were recorded on a Bruker 500 MHz NMR spectrometer. All chemical
4

shifts are given in ppm with coupling constants in Hz. All pH measurements were conducted
on an Orion Ross pH meter. Optical spectroscopy experiments were recorded in 100% water
at constant ionic strength (I = 0.01 (NaCl)) using a Varian CARY 50 UV-Vis
spectrophotometer or a Varian Cary Eclipse spectrophotometer at room temperature. High
pressure liquid chromatography (HPLC) was performed on a Shimadzu LC-20AD with
manual injection fitted with a Nova-Pak phenyl analytical column (3.9 x 150 mm) with a
flow rate of 0.8ml/min and was analysed using a Shimadzu SPD-20A detector at 280 nm.
Isocratic elution was used with the mobile phases 70% A (H
CH OH+0.1% TFA). The ICP-MS was performed at the University of Adelaide; CNH
analysis was conducted in Campbell Microanalytical Laboratory, University of Otago.
2
O+0.1% TFA) and 30% B
(
3
2
.2. Synthesis
2
.2.1. 1,4,7-Tris(tert-butoxycarbonylmethyl)-10-(N-(2-tert-butoxycarbonylaminoethyl)
acetamide)-1,4,7,10-tetraazacyclododecane (4)
2 3
A solution of 1 (294 mg, 0.57 mmol), 2 (139 mg, 0.59 mmol) and Cs CO (326 mg, 1.2
°
CN (20 ml) at 65 C for 24 h. The reaction mixture was filtered
mmol) were stirred in CH
3
through celite and the solvent was removed under reduced pressure. DCM (30 ml) was added
to the residue and the organic layers washed with water (3 x 20 ml), dried over Na SO and
2
4
the solvent removed under reduced pressure to yield the product as a yellow oil. Yield: 380
mg (93%). IR (KBr) v = 3341 (br), 2978, 2936, 2870, 2828, 1728, 1670, 1524, 1454, 1431,
-
1 1
1
370, 1250, 1227, 1161, 1103, 1007 cm . H NMR (CDCl
supplementary materials): δ 3.36 to 3.25 (br m, approx. 16H (not resolved), CH
.50 (br m, approx. 12H (not resolved), CH ), 1.45 (t, approx. 36H (not resolved), CH
NMR (CDCl , 125.8 MHz,): δ 172.2 (C=O), 170.3 (C=O), 156.6 (C=O), 156.1 (C=O), 82.0
C), 81.4 (C), 79.0 (C), 56.4 (CH ), 56.0 (CH ), 55.7 (CH ), 52.5 (CH ), 40.9 (CH ), 40.1
CH ), 39.8 (CH ), 30.9 (CH ), 28.5 (CH ), 28.2 (br, CH ). ESI-MS : m/z = 715.7 (M+1) ,
99); 659.4 (100); 603.6 (99).
3
, 500 MHz) (Fig. S1 in the
2
), 2.89 to
1
3
2
2
3
); C
3
(
(
(
2
2
2
2
2
+
+
2
2
2
2
3
2
.2.2. 1,4,7-Tris(tert-butoxycarbonylmethyl)-10-(N-(2-tert-butoxycarbonylaminohexyl)
acetamide)-1,4,7,10-tetraazacyclododecane (5)
5

Compound 5 was prepared as described for 4 except that 3 (259 mg, 0.88 mol) was reacted
with 1 (463 mg, 0.90 mol) and Cs CO (69 mg, 0.21 mmol) instead of 2. The product was
obtained as a yellow oil. Yield: 560 mg (98%). IR (KBr) v = 3383 (br), 2978, 2932, 2855,
2
3
-
1 1
2
5
2
824, 1728, 1663, 1524, 1458, 1370, 1308, 1250, 1227, 1161, 1107 cm . H NMR (CDCl
00 MHz) (Fig. S2 in the supplementary materials): δ 3.12 (m, 8H, CH ), 3.06 (br, 4H, CH
.79 (m, 16H, ) 1.41-1.29 (br m, approx. 44H (not resolved), CH and CH
, 125.8 MHz): δ 172.3 (C=O), 171.5 (C=O), 170.6 (C=O), 156.0 (C=O), 81.8
), 81.1 (C(CH ), 78.9 (C(CH ), 56.6 (br, CH ), 56.2 (CH ), 52.6 (CH ), 42.7 (br,
), 40.5 (CH ), 40.0 (CH ), 39.2 (CH ), 30.0 (br, CH ), 28.4 (CH ), 28.2 (CH ), 26.7 (br,
). ESI-MS : m/z = 771.8 (M+1) , (40); 715.8 (100); 659.8 (15); 615.8 (70).
3
,
2
2
),
13
2
3
); C NMR
(
(
CDCl
3
C(CH
3
)
3
3
)
3
3
)
3
2
2
2
CH
2
2
2
2
2
3
3
+
+
CH
2
2
.2.3. 1,4,7-Tris(carbonylmethyl)-10-(aminoethyl-N’-acetyl)-1,4,7,10-tetraazacyclododecane
(
6)
Compound 4 (360 mg, 0.50 mmol) was dissolved in a DCM/TFA (1/1) solution and stirred at
room temperature overnight. The solvent was then removed under reduced pressure to yield a
brown residue. The residue was then re-dissolved in water and the pH of the solution adjusted
to pH 7 using 1 M NaOH. The solvent was removed under reduced pressure to yield the
product as a hygroscopic solid in quantitative yield. This was used in the next step without
-1 1
further purification. IR (KBr) v = 3449 (br), 1686, 1640, 1435, 1404, 1207, 1134 cm . H
NMR (D O, 500 MHz) (Fig. S3 in the supplementary materials): δ 3.84 (m, 4H, CH ), 3.64
br, 2H, CH ), 3.54 – 3.42 (m, 10H, CH ), 3.35 (s, 2H, CH ), 3.21 (m, 2H, CH ), 3.13 (br,
H, CH ), 2.95 (br, 2H, CONHCH ), 2.88 (br, 2H, NH CH , 125.8 MHz)
Fig. S4 in the supplementary materials): δ 178.9 (C=O), 173.2 (C=O), 170.1 (C=O), 57.3
CH ), 56.3 (CH ), 56.2 (CH ), 52.1 (CH ), 50.4 (CH ), 48.8 (CH ), 48.4 (CH ), 39.1 (CH
6.8 (CH ). ESI-MS : m/z = 447.5 (M+1) .
2
2
(
2
2
2
2
1
3
4
2
2
2
2 3
); C NMR (CDCl
(
(
2
2
2
2
2
2
2
2
),
+
+
3
2
2
.2.4. 1,4,7-Tris(carbonylmethyl)-10-(aminohexyl-N’-acetyl)-1,4,7,10-tetraazacyclododecane
(
7)
Compound 7 was prepared using the same methodology as described for 6 except that 5 (580
mg, 0.75 mmol) was used instead of 4. The product was obtained as a hygroscopic solid
6

quantitatively and used for next step without further purification. IR (KBr) v = 3449 (br),
-
1 1
3
094 (br), 2947, 2870, 1732, 1674, 1462, 1427, 1389, 1354, 1316, 1200, 1022 (br) cm . H
NMR (D O, 500 MHz) (Fig. S5 in the supplementary materials): δ 3.73 (m, 8H, CH ), 3.40
m, 10H, CH ), 3.02 (m, 10H, CH ), 1.59 (m, 2H, CH CH CH ), 1.46 (m, 2H, CH CH
); C NMR (CDCl , 125.8 MHz) (Fig. S6 in the supplementary
), 56.2 (CH ), 55.9 (CH
), 28.0 (CH ), 26.5 (d, CH
2
2
(
2
2
2
2
2
2
2
CH
2
),
1
3
1
.30 (m, 4H, CH
2
CH
2
CH
2
3
materials): δ 178.2 (C=O), 171.8 (C=O), 170.0 (C=O), 56.6 (CH
1.8 (CH ), 50.9 (CH ), 48.3 (CH ), 39.4 (CH ), 39.1 (CH
CH ), 25.0 (CH ). ESI-MS : m/z = 503.7 (M+1) .
2
2
2
),
5
2
2
2
2
2
2
2
), 25.4
+
+
(
2
2
2
.2.5. 1,4,7-Tris(carbonylmethyl)-10-(aminoethyl-N’-acetyl)-1,4,7,10-
tetraazacyclododecane.Eu (6.Eu, 8)
Compound 6 (484 mg, 1.1 mmol) and Eu(CF
3
SO
3
)
3
(649 mg, 1.1 mmol) were dissolved in
. The solvent was then removed under
and any excess salt from the previous step
were removed by a Sephadex G10 column in CH OH to yield the product as a yellow powder.
Yield: 300 mg (47%). IR (KBr) v = 3426 (br), 2994, 2924, 2878, 1586, 1443, 1408, 1273,
°
CH
3
OH (8 ml) and stirred at 65 C for 24 h under N
2
3 3 3
reduced pressure. The unreacted Eu(CF SO )
3
-
1
3+
3-
1
3
6 7 3 3 2
254, 1173, 1084, 1030 cm . Calculated for C18H34EuN O .3CF SO .2H O: C, 23.32; H,
.54; N, 7.77. Found: C, 23.20; H, 3.76; N, 7.57. For elemental analysis complex 8 was
1
O) and freeze dried. H NMR (D
dissolved in water (H
2
2
O, 500 MHz) (Fig. S7 in the
supplementary materials): δ 33.68, 31.61, 30.48, 12.15, 11.03, 10.42, 7.07, 5.95, 4.91 to 1.40
(
m), 0.48, -0.47, -2.22, -2.45, -3.32, -4.97, -5.49, -7.40, -7.73, -8.03, -10.99, -11.39, 12.55, -
+
3.66, -14.00, -14.86, -15.64, -16.74. ESI-MS : m/z = 597.4 (M+1) . HPLC: method A, t =
+
1
2
.2 min (Fig. S15 in the supplementary materials).
2
.2.6. 1,4,7-Tris(carbonylmethyl)-10-(aminoethyl-N’-acetyl)-1,4,7,10-
tetraazacyclododecane.Tb (6.Tb, 9)
The lanthanide complex, 9 was prepared in a similar manner as described for complex 8; 6
(
135 mg, 0.30 mmol) was reacted with Tb(CF
3 3 3 3
SO ) (185 mg, 0.31 mmol) in CH OH.
Complex 9 was obtained as a yellow powder. Yield: 76 mg (41%). IR (KBr) v = 3487 (br),
-
1 1
1
2
609, 1261, 1234, 1173, 1038 cm . H NMR (D O, 500 MHz) (Fig. S8a – S8b in the
7

supplementary materials): δ 252.38, 242.72, 204.38, 196.23, 120.76, 111.40, 73.06, 50 to -50
(
br), -62.59, -75.11, -82.23, -96.22, -109.23, -118.56, -134.03, -364.03, -376.05, -378.51, -
+
06.98. ESI-MS : m/z = 603.2 (M+1) .
+
4
2
.2.7. 1,4,7-Tris(carbonylmethyl)-10-(aminoethyl-N’-acetyl)-1,4,7,10-
tetraazacyclododecane.Gd (6.Gd,10)
The lanthanide complex, 10 was prepared in a similar manner as described for complex 8; 6
(
43 mg, 0.097 mmol) was reacted with Gd(CF
3 3 3
SO ) (62 mg, 0.10 mmol) to obtain 10 as a
yellow powder. Yield: 45 mg (77 %). IR (KBr) v = 3433 (br), 1686, 1609, 1451, 1412, 1261,
-
1
+
196, 1173, 1084, 1038 cm . ESI-MS : m/z 602.2 (M+1) .
+
1
2
.2.8. 1,4,7-Tris(carbonylmethyl)-10-(aminohexyl-N’-acetyl)-1,4,7,10-
tetraazacyclododecane.Eu (7.Eu, 11)
The lanthanide complex, 11 was prepared in a similar manner as described for complex 8,
except that 7 (368 mg, 0.73 mmol) was reacted with Eu(CF
CH OH instead of 6. Complex 11 was obtained as a yellow powder. Yield: 246 mg (52%). IR
KBr) v = 3426 (br), 3109, 2940, 2870, 1620, 1439, 1400, 1323, 1277, 1250, 1204, 1169,
3 3 3
SO ) (461 mg, 0.77 mmol) in
3
(
-
1
2+
2-
1
3
1
5
1
6
6 7 3 3 3 3 2
084, 1030, 1003 cm . Calculated for C22H41EuN O .2CF SO .2CH OH.COHN(CH ) : C,
1.99; H, 5.18; N, 9.00. Found: C, 32.13; H, 4.96; N, 8.99. For elemental analysis complex
1
) and freeze dried. H NMR (D
1 was dissolved in dimethylformamide (COHN(CH
3
)
2
2
O,
00 MHz) (Fig. S9 in the supplementary materials): δ 33.16, 32.80, 31.90, 31.66, 12.88 (d),
2.18 (d), 4.09 to 1.12 (m), 0.70, 0.39, -0.09, -0.30, -0.50, -1.69, -2.61, -4.25, -5.24, -6.00, -
+
.54, -7.20, -7.79, -9.87, -10.79, -12.66, -12.91, -15.10, -15.30, -16.03. ESMS : m/z 653.3
+
M+1) . HPLC: t = 2.7 min (Fig. S16 in the supplementary materials).
(
2
.2.9. 1,4,7-Tris(carbonylmethyl)-10-(aminohexyl-N’-acetyl)-1,4,7,10-
tetraazacyclododecane.Gd (7.Gd, 12)
The lanthanide complex, 12 was prepared in a similar manner as described for complex 8,
3 3 3
except that 7 (48 mg, 0.095mmol) was reacted with Gd(CF SO ) (61 mg, 0.10 mmol) in
8

3
CH OH instead of 6. 12 was obtained as a yellow powder. Yield: 51 mg (82%). IR (KBr) v =
-
422 (br), 2943, 2859, 1678, 1636, 1605, 1454, 1397, 1254, 1204, 1173, 1084, 1038 cm .
1
3
+
ESI-MS : m/z 657.8 (M+1) .
+
2
.2.10. [6.Eu]-pteroic acid (13)
Pteroic acid (50 mg, 0.16 mmol) was dissolved in DMSO (2ml) by the assistance of
sonication, after which N,N-Diisopropylethylamine (DIPEA) (42 mg, 0.32 mmol) was added.
The reaction mixture was stirred for 10 min, then 1-Hydroxybenzotriazole (HOBt) (23 mg,
0
.17 mmol) and (Benzotriazol-1-yloxy) tris (dimethylamino) phosphonium
hexafluorophosphate (BOP) (74 mg, 0.17 mmol) were added and the reaction mixture was
stirred for a further 2 hours. Following this, a solution of 8 (96 mg, 0.16 mmol) and DIPEA
(
21 mg, 0.16 mmol) in DMSO (1 ml) was added. The reaction mixture was left to stir at room
temperature, protected from the light, overnight. The crude product was precipitated out by
adding 10 ml of diethyl ether/acetone (7/3) and collected by centrifugation.
The crude product was dissolved in a minimal amount of NaOH (pH 10), and carefully
loaded onto a Dowex 1-X8 column (CI). Water was used to elute the product and fractions
were collected and analysed by MS and TLC. Fractions containing the product were
combined and the pH adjusted to pH 7. The solvent was then removed under reduced
pressure to yield a yellow residue. In order to extract the product from excess salt, DMSO (1
ml) was added to the residue, the solution was then centrifuged and the supernatant collected.
The product was then isolated from the DMSO solution through precipitation following the
3
addition of diethyl ether/acetone (7/3). The product was dried and washed by CH OH. The
solvent residue was removed under reduced pressure and the product was further dried by
vacuum to yield 13 as a yellow powder. Yield: 38 mg (21%). TLC: silica
isopropanol/ammonia (5 M) (7/3), Rf 0.2. IR (KBr) v = 3433 (br), 2920, 2866, 1609, 1512,
-
1 1
1
2
400, 1323, 1242, 1180, 1130, 1084 cm . H NMR (D O, 500 MHz) (Fig. S10 in the
supplementary materials): δ 34.00, 32.81, 31.35 (br), 30.66, 13.14, 12.10, 11.53, 10.91, 9.30
to 1.61 (m), 0.62, 0.38, -0.73 (br), -1.73 (br), -2.54 (br), -3.19, -3.77, -5.46, -5.88, -7.11 (d), -
7
.53, -7.95 (br), -8.68, -9.84, -10.99 (br), -11.64 (br), -12.02, -12.60, -14.56, -14.79, -15.37, -
9

+
5.60, -16.21, -17.02, -17.48. ESI-MS : m/z = 891.4 (M+1) . HPLC: method A, t = 4.9 min
+
1
(
Fig. S17 in the supplementary materials).
2
.2.11. [7.Eu]-pteroic acid (14)
The Eu(III) complex, 14, was prepared and purified in a similar manner as described for
Eu(III) complex 13; Pteroic acid (75 mg, 0.24 mmol), BOP (119 mg, 0.27 mmol), HOBt (33
mg, 0.25 mmol), DIPEA (93 mg, 0.72 mmol) and 11 (157 mg, 0.24 mmol) in DMSO (3 ml)
and purified by Dowex 1-X8 (CI) column to yield a yellow powder. Yield: 42 mg (22%).
TLC: silica isopropanol/ammonia (5 M) (7/3), R
613, 1512, 1389, 1261, 1173, 1134, 1084, 1034 cm . H NMR (D
in the supplementary materials): δ 32.21, 32.69, 31.78, 12.50 (br), 8.47 to 1.07 (m), -0.09, -
.39, -1.73, -2.74, -4.13, -5.18, -6.03, -6.67, -7.28, -7.82 (br), -10.09 (br), -10.77 (br), -12.56,
f
0.3. IR (KBr) v = 3437 (br), 2920, 2866,
-
1 1
1
2
O, 500 MHz) (Fig. S11
0
+
12.87, -15.27, -16.15. ESI-MS : m/z = 947.5 (M+1) . HPLC: method A, t = 13.7 min (Fig.
+
-
S18 in the supplementary materials).
2
.2.12. [6.Eu]-folic acid (15)
The Eu(III) complex, 15, was prepared and purified in a similar manner as described for
Eu(III) complex 13; by using folic acid (74 mg, 0.17 mmol), BOP (76 mg, 0.17 mmol), HOBt
(
23 mg, 0.17 mmol), DIPEA (65 mg, 0.50 mmol) and 8 (101 mg, 0.17 mmol) in DMSO (3 ml)
to yield the crude product. However, the following purification was used instead: The crude
product was dissolved in a minimal amount of 20 mM sodium phosphate buffer (pH 10), and
carefully loaded onto a Sephadex G10 column that had been pre-equilibrated by the same
buffer system overnight. The fractions were collected, checked by TLC and altered to pH 7,
then dried under reduced pressure. DMSO was added to extract the product from salt, the
solution centrifuged, the supernatant collected and the product precipitated out by the
3
addition of diethyl ether /acetone (7/3). The product was dried and washed by CH OH. The
solvent was removed under reduced pressure and the product further dried by vacuum line to
yield a yellow powder. Yield: 73 mg (43%). TLC: silica isopropanol/ammonia (5 M) (7/3), R
f
0
1
.2. IR (KBr) v = 3383 (br), 2920, 2866, 2805, 1728, 1690, 1605, 1512, 1409, 1319, 1296,
-1 1
2
177, 1130, 1084, 1018 cm . H NMR (D O, 300 MHz) (Fig. S12 in the supplementary
materials): δ 33.55, 33.10, 31.48, 30.64, 12.05 (br), 11.41 (br), 8.66 to 1.26
1
0

(
m), -0.04, -0.56, -2.31, -3.27, -5.00, -5.64, -7.27, -7.54, -10.76, -11.29, -11.67, -11.95, -
+
2.63, -13.97, -14.26, -14.95, -15.49, -16.58. ESI-MS : m/z = 1020.7 (M+1) . HPLC: method
+
1
A, t = 4.8 min, 5.6 min (Fig. S19 in the supplementary materials).
2
.2.13. [7.Eu]-folic acid (16)
The Eu(III) complex, 16 was synthesised and purified as describe for complex 15 except that
Eu(III) complex 11 (101 mg, 0.16 mmol) was used instead of the Eu(III) complex 8 with folic
acid (70 mg, 0.15 mmol), BOP (68 mg, 0.15 mmol), HOBt (21 mg, 0.15 mmol), DIPEA (59
mg, 0.46 mmol) in DMSO (3 ml) and purified with a Sephadex G10 column to yield the
f
product. Yield: 66 mg (40%). TLC: silica isopropanol/ammonia (5 M) (7/3), R 0.3. IR (KBr)
-1
v = 3433 (br), 2932, 2862, 1616, 1508, 1400, 1323, 1242, 1204, 1180, 1130, 1084, 1018 cm .
1
H NMR (D O, 500 MHz) (Fig. S13 in the supplementary materials): δ 33.00 (d), 31.81 (d),
2
1
5
1
2.91 (br), 12.11 (br), 9.06 to 0.89 (m), 0.44, -0.07, -0.29, -0.48, -1.72, -2.64, -4.26 (br), -
.17, -5.98, -6.49 (br), -7.19, -7.78 (br), -9.80 (br), -10.75 (br), -12.62 (br), -12.84, -15.15, -
+
6.07. ESI-MS : m/z = 1076.7 (M+1) . HPLC: method A, t = 14.3 min, 19.6 min (Fig. S20 in
+
the supplementary materials).
2
.2.14. [6.Tb]-pteroic acid (17)
The Tb(III) complex, 17, was prepared and purified in a similar manner as described for
Eu(III) complex 13; Pteroic acid (13 mg, 0.042 mmol), BOP (20 mg, 0.045 mmol), HOBt (6
mg, 0.047 mmol), DIPEA (16 mg, 0.13 mmol) and 9 (25 mg, 0.042 mmol) in DMSO (3 ml)
and purified by Dowex 1-X8 (CI) column to yield a yellow powder. Yield: 12 mg (32%).
f
TLC: silica isopropanol/ammonia (5 M) (7/3), R 0.4. IR (KBr) v = 3426 (br), 2924, 2874,
-
1 1
1
5
1
1
605, 1512, 1400, 1319, 1285, 1242, 1188, 1161, 1119, 1084, 1003 cm . H NMR (D
00MH) (Fig. S14a – S14b in the supplementary materials): δ 257.43, 242.17, 202.83,
93.15, 111.25, 104.79, 50 to -50 (br), -68.41, -76.42, -79.51, -101.39, -110.32, -116.18, -
2
O,
+
25.11, -369.18, -378.42, -381.81, -398.15. ESI-MS : m/z = 897.8 (M+1) . HPLC: method
+
A, t = 5.1 min (Fig. S21 in the supplementary materials).
1
1

2
.2.15. [6.Gd]-pteroic acid (18)
The Gd(III) complex, 18, was prepared and purified in a similar manner as described for
Eu(III) complex 13; Pteroic acid (24 mg, 0.077 mmol), BOP (35 mg, 0.079 mmol), HOBt (10
mg, 0.077 mmol), DIPEA (29.1 mg, 0.23 mmol) and 10 (45 mg, 0.075 mmol) in DMSO (3
ml) and purified by Dowex 1-X8 (CI) column to yield a yellow powder. Yield: 30.5 mg
(
44%). TLC: silica isopropanol/ammonia (5M) (7/3), Rf 0.3. IR (KBr) v = 3418 (br), 2924,
-
1
+
870, 1605, 1508, 1439, 1404, 1319, 1242, 1192, 1161, 1123, 1084, 1003 cm . ESI-MS : m/z
2
8
+
96.3 (M+1) . HPLC: t = 5.0 min (Fig. S22 in the supplementary materials).
2
.2.16. [7.Gd]-pteroic acid (19)
The Gd(III) complex, 19, was prepared and purified in a similar manner as described for
Eu(III) complex 13; Pteroic acid (17.4 mg, 0.056 mmol), BOP (26.4 mg, 0.060 mmol), HOBt
(
9.2 mg, 0.068 mmol), DIPEA (22 mg, 0.17 mmol) and 12 (36.9 mg, 0.056 mmol) in DMSO
3 ml) and purified by Dowex 1-X8 (CI) column to yield a yellow powder. Yield: 19 mg
36%). TLC: silica isopropanol/ammonia (5M) (7/3), Rf 0.3. IR (KBr) v = 3426 (br), 2924,
(
(
-
1
+
+
862, 1609, 1404, 1389, 1319, 1246, 1180, 1084, 1018 cm . ESI-MS : m/z 952.6 (M+1) .
2
HPLC: t = 12.3 min (Fig. S23 in the supplementary materials).
2
.2.17. [6.Gd]-folic acid (20)
The Gd(III) complex, 20 was synthesised and purified as describe for the Eu(III) complex, 15
except that the Gd(III) complex 10 (40 mg, 0.067 mmol) was used instead of the Eu(III)
complex, 8 with folic acid (29 mg, 0.067 mmol), BOP (30 mg, 0.068 mmol), HOBt (11 mg,
0
.081 mmol), DIPEA (27 mg, 0.21 mmol) in DMSO (3 ml) and purified with a Sephadex
G10 column to yield the product. Yield: 24 mg (35%). TLC: silica isopropanol/ammonia
(
5M) (7/3), Rf 0.3. IR (KBr) v = 3403 (br), 3106 (br), 2893 (br), 1597, 1501, 1400, 1335,
+
126, 1042. ESI-MS : m/z 1025.7 (M+1) . HPLC: t = 5.0 min, 5.7 min (Fig. S24 in the
+
1
supplementary materials).
1
2

2
.3. Cellular uptake
HeLa cells (human endothelial carcinoma cell line, ATCC CCL-2), were cultured in
DMEM/F-12 (Dulbecco’s Modified Eagle Medium/F-12, Gibco, Life Technologies, USA)
supplemented with 10% Foetal Bovine Serum (In Vitro Technologies, Vic, Australia), in a
o
atmosphere at 37 C. Cells were cultured to ~80% confluence in T-75 flasks (In
5
% CO
2
Vitro Technologies) before overnight (16 hours) incubation with Eu(III) probes (13, 14, 15
and 16) dissolved in fresh cell culture medium. Cells were washed 3 times in Dulbecco’s
phosphate buffered saline (DPBS, SAFC Biosciences, USA) and detached from the flask by
o
treatment with TrypLE Express (Gibco, Life Technologies, USA) for 10 min at 37 C before
being pelleted (400xg, 5 min) and resuspended in 5 ml DPBS. A 0.5 ml aliquot was taken for
trypan blue exclusion cell counts and protein determination by the bicinchoninic acid (BCA)
protein assay. The protein assay aliquots were centrifuged (16,000xg, 3 min) and the pellets
resuspended in 35 µl lysis buffer (10 mM Tris pH 7.5, 100 mM NaCl, 1mM EDTA, 1% (v/v)
TritonX-100) containing protease inhibitors (Complete Mini EDTA-free Protease Inhibitor
o
Cocktail Tablets, Roche, USA) and incubated overnight (16 hours) at 4 C. Total protein
content was determined using the Micro BCA protein Assay kit according to the
manufacturer’s instructions (Thermo Scientific, USA). The remaining cells (4.5 ml) were
3
centrifuged (400xg, 5 min) and the pellet was resuspended in 5 ml 5% HNO . After 3 days
the acid treated cells were filter sterilised through a 0.22 µM syringe filter (Millipore, Ireland)
and the supernatant analysed by inductively coupled plasma mass spectrometry (ICP-MS) to
determine the Eu(III) concentration in parts per billion; using an Agilent 7500CS (Adelaide
Microscopy, University of Adelaide). The Eu(III) counts were normalised to the amount of
cellular protein (nmol Eu/mg of protein). Haemocytometer cell counts determined that 1 mg
6
of protein corresponded to 2.5 x 10 cells; and the final intracellular concentration of Eu(III)
3
was calculated using an assumed individual cell volume of 5000 µm [42].
3
3
. Results and Discussion
.1. Synthesis of the complexes
The synthetic pathway outlined in Scheme 1 describes the synthesis of the folate receptor
targeted lanthanide complexes 13-20 from the tri-Boc protected cyclen precursor (DO3AtBu)
1
3

(
1). This pathway was designed to be easily adapted to different targeting moieties, in this
work the two folate receptor targeting moieites, PTE and folic acid, were used. However, it is
anticipated that this pathway could be further adapted to any targeting moiety with a reactive
carboxylate functionality. Furthermore, it was designed to allow the facile introduction of
different Ln(III) ions to generate a series of isostructural Ln(III) ion complexes.
The Boc protected chloroacetamide linker derivatives 2 and 3 were prepared as described by
Fanning and co-workers [30]. Mono protection of both ethylene and hexane diamine was
°
achieved by the addition of di-tert-butyl dicarbonate at 0 C; which was then reacted with
°
chloroacetyl chloride in the presence of triethylamine in DCM at 0 C to yield the α-
chloroacetamide derivatives 2 and 3 in 62% and 54% yield, respectively. The tetra substituted
ligands 4 and 5 were formed by the reaction of (DO3AtBu) with 2 and 3, in dry CH
3
CN in
°
the presence of Cs CO at 65 C for 24 hours. This generated 4 and 5, respectively, in high
2 3
yield. The deprotection of both the t-butyl and carbamate esters was achieved using a 1/1
ratio of trifluoroacetic acid in DCM, generating the ligands 6 and 7 as hygroscopic solids.
The Ln(III) complexes (8-12), were prepared by refluxing each of the ligands with 1 to1.1
3
molar equivalents of the relevant lanthanide triflate in dry CH OH under nitrogen for 24
hours. The lanthanide complexes were then purified using a sephadex G10 column to ensure
complete removal of excess salt and lanthanide ions, isolating the lanthanide complexes 8-12
as yellow powders.
1
The H NMR spectra of the Eu(III) complexes (8 and 11) in D
2
O showed resonances between
+
34 ppm and -17 ppm, with peaks around +34 to +30 ppm and +14 to +10 ppm, which were
characteristic of square antiprismatic geometry and twisted square antiprismatic geometry
Fig. S7 and S9 in the supplementary materials). This was consistent with other cyclen
(
complexes in solution [43, 44]. The Eu(III) complexes (8 and 11), were fully characterised by
elemental analysis, HPLC, IR, ESI-MS and NMR spectroscopy. These Eu(III) complexes
were then reacted with either folic acid or PTE using the amide coupling reagents, 1-
Hydroxybenzotriazole (HOBt) and (Benzotriazol-1-yloxy) tris (dimethylamino) phosphonium
hexafluorophosphate (BOP) in the presence of N,N-diisopropylethylamine (DIPEA) in
DMSO. The PTE based Eu(III) probes, 13 and 14, were purified using an ion exchange
1
4

column loaded with Dowex 1-X8 (Cl); probes were obtained with a 20-40% yield. As folic
acid has two carboxylic acid groups at the α and γ positions, the coupling of the Eu(III)
complexes 8 and 11 with folic acid can produce both the expected regio-isomers and a bis-
coupled product. There was, however, no evidence for the formation of the bis-coupled
product in the mass spectra of either the reaction mixtures or the purified products, which was
attributed to steric hindrance. Purification of the folic acid based Eu(III) based probes (15 and
1
6) was achieved using a sephadex G10 column with 20 mM phosphate buffer at pH 10. The
1
2
H NMR spectra of the folic acid and PTE based Eu(III) probes (13-16) in D O were
consistent with the molecular probes adopting square antiprismatic geometry and twisted
square antiprismatic geometry (Fig. S10 - S13 in the supplementary materials). The Tb(III)
and Gd(III) probes were prepared and purified using the same methodologies with the
appropriate lanthanide triflate salt (see Fig. S8 and S14 in the supplementary materials for the
1
H NMR spectra of the Tb(III) ion complexes). All complexes were analysed for purity using
analytical HPLC, conducted on a phenyl analytical column with isocratic elution. For the
PTE based lanthanide probes, 13, 14, 17, 18 and 19 a single peak was observed (Fig. S17,
S18 and S21 – S23 in the supplementary materials). The retention times of these peaks were
significantly different to that of the starting materials (Fig. S15 – S16 in the supplementary
materials), confirming the formation and isolation of a single pure compound in each case.
Two peaks with similar retention times were observed in all the HPLC traces of the folic acid
based lanthanide probes, 15, 16 and 20 (Fig. S19, S20 and S24 in the supplementary
materials) which were attributed to the formation of the two regio-isomers (α and γ). The area
of the first peak was approximately twice the area of the second peak in all of the HPLC
traces recorded, which is consistent with literature reports for the formation of the regio-
isomers [45]; the α-isomer is known to form at approximately half the rate of the γ-isomer
due to the increased steric hindrance present at this carboxylic acid moiety. In line with
common practice, the regio-isomers of the folic acid based probes were not separated into
individual components [45-48]. The effect that the different isomers have on cell uptake in
relation to probe structure is currently being investigated.
1
5

3
.2.
Lifetimes and q values
The emission lifetimes of complexes 13-17 were determined to provide information for future
time-gated measurements and cell imaging. The lifetimes also allow the q value (the number
of metal bound waters) to be quantified for each probe, to confirm the coordination
environment around the lanthanide ion. The inner sphere hydration state (q) (i.e. the number
of metal bound water molecules) was defined by measuring the excited state lifetimes
(
2 2
measured as taui (τ) values) of the Eu(III) and Tb(III) probes, 13-17, in H O and D O,
respectively. The q values were calculated using q = 1.11 (kH2O-kD2O-0.31)[49] for Eu(III)
probes and q = 4.2 (kH2O-kD2O)[50] for Tb(III) probes (Table 1). The taui (τ) values found in
Table 1 are the reciprocal of k used in the two equations and were determined using the Cary
Eclipse Win FLR lifetime program. A q ~ 1 was found for all probes, indicating that each of
the Eu(III) and Tb(III) probes possessed one metal bound water molecule, supporting the
formation of an 8 coordination complex with the ligand. Due to the similar solution chemistry
exhibited by the lanthanides it was reasonable to assume that the Gd(III) probes, 18-20, also
possessed one metal bound water. The presence of the metal bound water is important for
Gd(III) complexes to be used as contrast agents for MRI applications. The relaxivity of these
complexes could not be accurately determined due to issues associated with the long term
stability of these probes in their solid state during transport; long term stability is associated
with the coupling of folate/PTE to the linker and not the chelate. Elemental analysis results of
the degraded products following transport supported this; results could only be fitted to Ln(III)
chelate and not to Ln(III) chelate-folate/PTE conjugates. Degradation of the probes was also
easily identifiable due to a change in colour of the solid. However it is important to note that
all complexes retained sufficient aqueous stability for use in the analysis of photophysical
properties and cell uptake studies. These studies were performed immediately following
1
synthesis to minimise the potential for degradation. H NMR and HPLC analysis of the probe
solution following photophysical analysis confirmed that the probe structure was only
affected during transport.
3
.3 UV-Visible, fluorescence and lanthanide ion emission spectroscopy
After the synthesis, the probes photophysical properties were evaluated, including the UV-
Visible, fluorescence and lanthanide ion emission responses of the probes over physiological
1
6

pH ranges. The photophysical studies were designed to provide information on the optimal
excitation wavelength, the effect of pH on the probes and how the structure of the probes
affects the photophysical properties. The UV-Visible absorption, fluorescence and lanthanide
luminescence emission of the probes was measured at 298 K as a function of pH, in 100%
aqueous solution [I = 0.01 M (NaCl)] (see Figure 2 for the UV-Visible absorption,
fluorescence and Eu(III) emission spectra and a plot of the changes vs pH of 16 as an
example (see supplementary materials Fig. S25-S28 for corresponding data for 13, 14, 15 and
1
7)).
All of the folic acid and PTE based probes at pH 3 showed both an intense absorption band
centred around 280 nm, which upon titration with base underwent a bathochromic shift to
approximately 260 nm or less, and a small absorption band centred around 360 nm, which
increased in molar absorptivity and underwent a hypsochromic shift upon titration with base
[
51]. A plot of the molar absorbance changes vs pH for 13, 14, 15, 16 and 17 showed that the
molar absorptivity was significantly altered between pH 6 – 8 and only slightly altered below
pH 4. The molar absorptivity changes between pH 6 – 8 can be attributed to the protonation
3
3
a
of N H/CO of the pteridine ring in both targeting moieties; pK 8.0 (N H/CO) for both folic
acid and pteroic acid. The shift of the pKa to a lower value was assigned to the electron
withdrawing effect of the Eu(III) complex [52]. The influence on molar absorptivity below
pH 4 is unlikely to be replicated in a cellular environment and hence will not be discussed in
detail here.
The molar absorbance changes as a function of pH were measured to inform the choice of
appropriate excitation wavelength(s) for the florescence and Ln(III) emission spectroscopy.
Excitation wavelengths are commonly chosen from absorption maxima(s). Furthermore,
excitation wavelengths chosen for biological applications will ideally not cause cell damage
(
wavelengths below 280 nm are known to cause cell damage [53, 54]) and will match
available confocal microscopy lasers (360 nm and 720 nm using two photon). However, the
low molar absorbance of the probes at 360 nm may only result in low energy emissions and
this might not be ideal for cellular imaging (low energy emissions are difficult to observe at
concentrations required for cell studies) or to investigate any trends between structure and
1
7

emission properties. Therefore, both 280 nm and 360 nm wavelengths were chosen to
monitor the fluorescence and Ln(III) emission from the probes as a function of pH. Both
excitation wavelengths resulted in a large fluorescence emission band centred at ~ 450 nm. A
plot of the fluorescence emission at 450 nm vs pH shows that the fluorescence is significantly
modulated above pH 6. This is similar to the molar absorptivity and hence the change in
3
fluorescence emission intensity was attributed to the protonation of N H/CO of the pteridine
ring in both targeting moieties. Below pH 4 the probes were shown to differ in the extent to
which fluorescence emission was sensitive to pH changes. However as it is unlikely that the
probes will encounter conditions as acidic as this in cells (cancer cells vary pH 5 – 8) [55]
fluorescence change below pH 4 will not be discussed herein.
The most important photophysical response to be investigated against changes in pH is the
Ln(III) emission response; it is expected that only the Ln(III) ion emission that will be
detected using confocal microscopy. Characteristic Ln(III) emission bands were observed
from the lanthanide ions when excited indirectly at either 280 nm or 360 nm for all the probes.
The Tb(III) probes exhibited higher emission intensities compared to the Eu(III) probes. This
is as expected as the excited states of Eu(III) are known to be more sensitive to quenching via
-
OH vibration deactivation pathways [56]. Therefore, Tb(III) ion probes may be more ideally
suited to investigating molecules of interest or targeting receptors which are present in low
concentration in cellular applications.
Interestingly, the type of targeting moiety was found to alter the Ln(III) emission response to
pH; the Ln(III) emission from the folic acid based probes, 15 and 16, was found to be
relatively pH insensitive, whereas for the PTE based probes, 13, 14 and 17, some Ln(III) ion
emission quenching under acidic conditions was observed. This is in contrast to both the
molar absorbance and fluorescence emission where no difference in pH response was
observed between targeting moiety.
The lack of pH sensitivity in the Eu(III) emission as a function of pH of the folic acid based
probes, 15 and 16 can be attributed to the extra glutamic acid residue in folic acid which acts
1
8

to increase the distance between the aromatic moiety and the Eu(III) ion. This was consistent
with the observation that the effective population of the Eu(III) ion via either the Dexter or
Förster energy transfer mechanisms reduces as distance increases. Therefore, the effect from
3
the protonation at the N H/CO of the pteridine ring of folic acid is to far removed from the
Eu(III) ion to affect the sensitisation process in the folic acid based probes. Therefore the
emission from folic acid based probes will directly reflect the local probe concentration
regardless of the pH variations that are known to occur within cancer tissues [55, 57].
A comparison of the PTE and folate based Eu(III) probes, 13, 14, 15 and 16 , showed that the
length of the linker influenced the spectral properties of the probes. As the distance between
the aromatic moiety and the Eu(III) ion increased, the emission from the Eu(III) decreased.
This is further evidence that the Dexter or Förster energy transfer mechanisms reduces as
distance increases.
3
.4. Cellular uptake
The cellular uptake of Eu(III) probes was investigated using the human endothelial carcinoma
cell line HeLa, which is known to express high amounts of the folate receptor [58]. Cells
were incubated with the probes overnight, washed and lysed by HNO
concentrations of the probes were determined by the measurement of Eu(III) ion by ICP-MS
Table 2). The viability of the cells (as assessed by trypan blue exclusion) was not affected
3
. The intracellular
(
by incubation with the probes at the tested concentration. This indicates that the probes were
not toxic to cells; an essential property for live cell imaging applications.
The folic acid based probes exhibited a higher intracellular concentration compared to the
PTE based probes containing the same linker group (15 > 13, 16 > 14). This indicates that the
affinity of the receptor for PTE is reduced when compared with folic acid, and therefore the
use of the synthetically simpler PTE is outweighed by the reduced uptake efficiency.
1
9

The ICP-MS cell uptake studies also showed that when a smaller distance between the
targeting motif and the lanthanide chelate was employed that the percentage of probe
internalised was increased. This is an interesting finding, when considered contextually in
relation to the folate receptor targeting literature. The most commonly used linker structure
for probe development includes long and structurally complicated linkers such as PEG,
peptides and carbohydrates. The rationale behind the use of such long linkers is that research
has shown that the size of the linker is associated with the flexibility of the probe; and
reduced cell binding has been traditionally attributed to linkers with poor flexibility [59].
Hence, these larger linkers have been employed even though they significantly increase the
synthetic difficulty of probe preparation and the overall molecular weight of a probe. These
findings suggest that there is no need to employ such large and complicated linker structures,
especially as increased molecular weight is often associated with macropinocytic uptake [60].
Therefore, the size of the linker group can be kept to a minimum for future applications.
4
. Conclusion
In conclusion, we have designed, synthesised and characterised a series of water-soluble
folate receptor targeted lanthanide probes, which demonstrated cellular uptake in a HeLa cell
line. All probes were found to have a q ~ 1, with eight binding sites for the core lanthanide
ion. In addition, the probes were found to not perturb cell viability even at high
concentrations. Compared to fluorescent probes targeted to the folate receptor, these new
probes have the advantage of being easily adapted to enable the detection through MRI and
time-gated luminescence methodologies for future in vivo applications.
We demonstrated that the targeting moiety was important for the efficacy of probe uptake:
with folic acid based probes exhibiting more cellular uptake than the corresponding PTE
based probes. Furthermore, the increased uptake was not associated with the increased
flexibility of a longer linker; and thus a shorter linker was optimal for both cellular uptake
and emission from the Eu(III) ion.
2
0

The folic acid based probes were pH insensitive and this makes them ideal for localisation in
cancers where the pH is known to vary. The information gained in this work is currently
being applied to the development of new lanthanide molecular probes with higher emission
quantum yields, which will support live cell imaging and have the potential to provide new
reagents for cancer detection and imaging.
Acknowledgements:
David Parker, University of Durham for advice on coupling marocycles to folic acid. A. Dean
Sherry, Advanced Medical Imaging Center, Southwestern Medical Center, University of
Texas at Dallas for relaxivity measurements. University of South Australia for funding this
research.
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4

Table 1 Luminescence lifetimes (τ) and inner sphere hydration numbers (q).
Table 2 The intracellular concentration of each Eu(III) probe.
Figure 1. The structure of a) folic acid (with α and γ carboxyls) and b) PTE.
-
5
Figure 2. The spectra of compound 16 (3.2 x 10 M), changes upon incremental addition of
base (NaOH) in the presence of 0.01 M NaCl a) The UV-Vis spectra. b) The Fluorescence
spectra (λex = 280 nm). c) The Eu(III) emission spectra (λex = 280 nm). d) The normalised
changes of 15 as a function of pH in the presence of 0.01 M NaCl. The UV-Vis absorbance
changes in the 260 nm transition (squares). The fluorescence emission intensity changes at
4
50 nm transition (λex = 280 nm, triangles). The Eu(III) emission intensity changes at 615 nm
(
λ
ex = 280 nm, circles).
Scheme 1. The synthesis of the probes.
2
5

Figure(1)

Figure(2)
a
b
c
d