Synthesis of lanthanide(III) complexes appended with a diphenylphosphinamide and their interaction with human serum albumin

Article abstract of DOI:10.1007/s10847-011-0009-4

Two DOTA-based proligands bearing a pendant diphenylphosphinamide 4a and 4b were synthesised. Their Eu(III) complexes exhibit sensitised emission when excited at 270 nm via the diphenylphosphinamide chromophore. Hydration states of q = 1.5 were determined

Full text of DOI:10.1007/s10847-011-0009-4

J Incl Phenom Macrocycl Chem (2011) 71:435–444
DOI 10.1007/s10847-011-0009-4
ORIGINAL ARTICLE
Synthesis of lanthanide(III) complexes appended
with a diphenylphosphinamide and their interaction
with human serum albumin
•
Marco Giardiello Mauro Botta Mark P. Lowe
•
Received: 12 May 2011 / Accepted: 20 June 2011 / Published online: 12 July 2011
Ó Springer Science+Business Media B.V. 2011
Abstract Two DOTA-based proligands bearing a pen-
dant diphenylphosphinamide 4a and 4b were synthesised.
Their Eu(III) complexes exhibit sensitised emission when
excited at 270 nm via the diphenylphosphinamide chro-
mophore. Hydration states of q = 1.5 were determined
Keywords Contrast agents ꢁ Magnetic resonance
imaging ꢁ Gadolinium(III) ꢁ Europium(III) ꢁ
Luminescence ꢁ Diphenylphosphinamides
from excited state lifetime measurements (Eu.4a kH O
2:14 msꢀ1; kD O ¼ 0:64 msꢀ1; Eu.4b kH O ¼ 2:67 msꢀ1
¼
Introduction
2
;
2
2
kD O ¼ 1:18 msꢀ1). In the presence of human serum albu-
The use of gadolinium-containing contrast agents for
Magnetic Resonance Imaging (MRI) has increased dra-
matically in recent years [1–4]. The introduction of che-
lates containing the highly paramagnetic Gd3? ion
enhances image contrast by increasing the longitudinal
relaxation rate, R1 (1/T1) of surrounding water protons due
to its high effective magnetic moment and long electron
spin relaxation time. In order to minimize the inherent
toxicity of the ‘free’ Gd3? ion at the doses administered to
patients, first generation contrast agents were developed
which encase the Gd3? ion in multidentate chelating
ligands to render it safe for in vivo use. They are derived
from diethylenetriaminepentaacetic acid (DTPA) or
1,4,7,10-tetraazacyclododecane-N,N0,N00,N000-tetraacetic acid
(DOTA) (these are non-specific perfusion agents). Since
the production of the first generation contrast agents there
has been intense research into the design of probes
whereby the water proton longitudinal relaxation rates are
tailored to respond to changes in their biological environ-
ment, such as pH [5, 6], oxygen delivery/consumption [7],
the presence of a biological molecule [8–10] or metal ions
[11].
2
min (HSA) (0.1 mM Eu.4a/b, 0.67 mM HSA, pH 7.4)
q = 0.4 for Eu.4a (kH O ¼ 1:34 msꢀ1; kD O ¼ 0:75 msꢀ1
)
2
2
and q = 0.6 for Eu.4b (kH O ¼ 1:83 msꢀ1; kD O
¼
2
2
1:05 msꢀ1). Relaxivites (pH 7.4, 298 K, 20 MHz) of the
Gd(III) complexes in the absence and presence of HSA
(0.1 mM Gd.4a/b, 0.67 mM HSA) were: Gd.4a (r1 =
7.6 mM-1 -1
s
and r1 = 11.7 mM-1 -1
and r1 = 16.0 mM-1 -1
s
)
and Gd.4b.
). These rel-
(r1 = 7.3 mM-1 -1
s
s
atively modest increases in r1 are consistent with the
change in inner-sphere hydration on binding to HSA shown
by luminescence measurements on Eu.4a/b. Binding con-
stants for HSA determined by the quenching of lumines-
cence (Eu) and enhancement of relaxivity (Gd) were
Eu.4a (27,000 M-1 12%), Eu.4b (32,000 M-1 14%),
Gd.4a (21,000 M-1 15%) and Gd.4b (26,000 M-1
15%).
M. Giardiello ꢁ M. P. Lowe (&)
Department of Chemistry, University Road, University
of Leicester, Leicester LE1 7RH, UK
e-mail: mplowe@le.ac.uk
The efficacy of an MRI contrast agent is defined by its
relaxivity, r1p; the total paramagnetic relaxation rate
enhancement of the water protons per unit concentration of
the contrast agent (mM-1 -1
s ) [12]. The design of contrast
M. Botta
Dipartimento di Scienze dell’Ambiente e della Vita, Universita
`
agent ligands is such that one or two coordination sites of
the usually nine-coordinate Gd(III) ion are reserved for
del Piemonte Orientale ‘‘Amedeo Avogadro’’, Via Bellini 25/G,
15100 Alessandria, Italy
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J Incl Phenom Macrocycl Chem (2011) 71:435–444
water to bind and exchange with the bulk water. The most
significant andcontrollable relaxationcontributionwithregards
to contrast agent design comes from the inner-sphere. The
longitudinal inner-sphere relaxation rate is expressed by Eq. 1:
the chelate; displacement of bound waters by endogenous
anions and/or protein carboxylate residues; slowing of the
water exchange rate kex in the bound complex [32–35].
Cq
1
IS
1p
R
¼
ð1Þ
55:6 T1M þ sm
O
O
O
O
O
P
NH
R2
where C is the concentration of paramagnetic ion, q is the
number of coordinated water molecules, T1M is the
longitudinal relaxation time of the inner-sphere waters and
sm is the water exchange lifetime. According to Solomon-
Bloembergen-Morgan (SBM) theory, several other factors
further contribute towards the relaxivity, most notably sr, the
rotational correlation time and T1e, the electronic longitudinal
relaxation time. The experimental longitudinal relaxation rate
1/T1M depends on a correlation time sc that is itself a function
of the correlation times sr, sm and T1e in accordance to SBM
theory (Eq. 2) [13, 14]. At typical clinical imaging fields (20–
60 MHz) the smallest and therefore most dominant term in
Eq. 2 is sr. As sc (and therefore relaxivity) for small rapidly
tumbling complexes is greatly affected by sr, the lengthening
of this parameter by reducing molecular tumbling leads to sr
becoming comparable to both sm and T1e. Hence, attachment
of contrast agents to large, slowly tumbling proteins such as
HSA lengthens sR and enhances relaxivity.
P
O
O-
N
N
N
R1
Gd
O
N
N
N
N
Gd
O
O
O
O
O
OH2
O
O
O
O
O
O
O
O
VasovistTM
Gd.4(a-b)
a. R1 = H, R2 = Me
b. R1 = R2 = Me
The diphenylphosphinoyl moiety has been used as an
amino protecting group in peptide synthesis as the diph-
enylphosphinamide (dpp) [36]. N-dpp aziridines provide a
convenient method of introducing aminoethyl functionality
into a molecule, by ring opening of the corresponding aziri-
dine [37, 38]. This protecting group can be readily removed
under acid conditions (e.g. 95% TFA). We have been using
this methodology to introduce aminoethyl groups onto
DO3A-based ligands; however, we discovered that there is
more to this unit than simply a protecting group. It was
hypothesised that the pendant hydrophobic dpp group would
bind non-covalently to Human Serum Albumin (HSA). Such
attachment would be expected to result in the lengthening of
the rotational correlation time, sR, of the Gd(III) contrast
agent, enhancing relaxivity, r1, and MR image intensity. The
structure of the dpp moiety, containing two phenyl groups,
bears a resemblance to the pendant hydrophobic group of the
commercially available blood pool contrast agent VasovistÒ.
Due to this similarity in structure of the dpp-containing
complexes, similar HSA binding affinity was expected. We
have previously reported a strategy forrigidifyingthebinding
of hydrophobic contrast agents by incorporating a binaphthyl
moiety into a DOTA-like structure; however, this agent suf-
fered from a slow water exchange rate engendered by the
amide link to the macrocycle [39]. We hoped that complexes
such as Gd.4a/b would have faster water exchange rates,
albeit with some of the rigidity sacrificed cf. our binaphthyl-
based complexes. Herein we report the synthesis of these
complexes and study their interaction with HSA using both
luminescence (Eu) and relaxometric (Gd) methods.
1
1
1
1
¼
þ
þ
ð2Þ
sc sr sm T1e
There has been a vast amount of research into contrast
agent development towards the imaging of blood vessels
and the circulatory system [15, 16]. The utilisation of MRI
for this purpose is known as magnetic resonance angiog-
raphy (MRA) [17]. Human serum albumin (HSA) is the
most abundant protein in the circulatory system [18, 19].
The protein is made up of 585 amino acids, has a total
molecular weight of 66,400 Da and is present in human
serum at a concentration of ca. 0.67 mM [20]. Receptor
induced magnetisation enhancement (RIME) is achieved
by the incorporation of a hydrophobic moiety into the
contrast agent [21]. Conjugation of low molecular weight
Gd(III) chelates to serum proteins such as HSA through
non-covalent binding of such hydrophobic moieties not
only increases cardiovascular retention of the contrast
agent, but enhances efficiency and image intensity by
reducing the molecular tumbling of the Gd(III) chelate.
A variety of hydrophobic moieties have been incorpo-
rated into contrast agents over recent years [22–25], with the
most successful of these VasovistÒ now in clinical use [26–
31]. To varying extents, these DOTA- and DPTA-based
chelates exhibit smaller increases in r1 than theory predicts
because of three main factors that diminish the relaxivity of
the HSA-bound complexes: independent rotation around e.g.
C–C bonds in the link between the hydrophobic group and
Experimental section
Reagents are commercially available and were used with-
out further purification. Human serum albumin (HSA),
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J Incl Phenom Macrocycl Chem (2011) 71:435–444
437
Hydration state, q, determination
product number A-1653 (fraction V powder 96–99% albu-
min, containing fatty acids) was purchased from Sigma-
Aldrich. Dry solvents were obtained using an Innovative
Technology inc. PureSolv solvent purification system.
1H NMR spectra were recorded using a Bruker 300 or
400 MHz spectrometer and referenced to residual solvent
peaks. All chemical shifts are recorded in ppm. Accurate
mass ESMS were recorded on a Finnigan MAT 900 XLT
high resolution double focusing mass spectrometer with
tandem ion trap (polyethylenimine reference compound) at
the EPSRC National MS Service at Swansea, UK. Elec-
tronic absorption spectra were recorded on a Shimadzu
UV180 spectrometer. IR measurements were carried out
using a Perkin-Elmer Spectrum One FTIR spectrometer.
Concentrations of the complexes used in the studies were
determined by ICP-MS using an Agilent Technologies
7500ce inductively coupled plasma mass spectrometer.
Lifetimes were measured by direct (395 nm) and indirect
(kex = 270 nm) excitation of the sample with a short 40 ms
pulse of light (500 pulses per point) followed by monitoring
the integrated intensity of light (DJ = 2) emitted during a
fixed gate time of 0.1 ms, at a delay time later. Delay times
were set at 0.1 ms intervals, covering 4 or more lifetimes.
Excitation and emission slits were set to 5 nm. The data is
applied to the standard first order decay (Eq. 6), minimized
in terms of k by iterative least-squares fitting operation using
Microsoft Excel, where Iobs is the observed intensity, I0 is the
initial intensity, and t is the time (ms). The calculated k
values obtained in H2O and D2O are then applied to Eq. 7 to
calculate the hydration state, q [41].
Iobs ¼ Ioeꢀkt þ offset
ð6Þ
ð7Þ
q ¼ 1:2½ðkH O ꢀ kD OÞ ꢀ 0:25ꢂ
2
2
1H relaxation data
HSA binding studies
Observed longitudinal water proton relaxation times (T1obs
)
were measured on a Stelar Spinmaster spectrometer (Stelar,
Mede (PV) Italy) operating at 20 MHz, by means of the
standard inversion-recovery technique (16 experiments, 2
scans). A typical 90° pulse width was 3.5 ls and the
reproducibility of the T1obs data was 0.5%. The para-
magnetic water proton relaxation rate, R1p and relaxivity,
r1p were determined following Eqs. 3–5, where 0.38 is the
diamagnetic contribution of the bulk water molecules [40].
Complex concentrations were determined via mineraliza-
tion with nitric acid or by ICP-MS.
For both luminescence and relaxivity studies, aqueous
solutions were buffered to pH 7.4 using phosphate buffered
saline solution (10 mM PBS, 150 mM NaCl). Titrations
were carried out at fixed complex concentration (0.1 mM)
with varied [HSA] at 25 °C. The Eu(III) complexes were
excited indirectly, via the dpp chromophore, at kex
*270 nm and the variations in intensity of the DJ = 2
band recorded. The concentrations of the Eu(III) solutions
was determined by ICP-MS while the exact concentration
of HSA was determined by electronic absorption spec-
troscopy. The equilibrium Eq. 8 was assumed, where K is
the affinity constant and [Ln] represents the Ln(III) com-
plex. The concentration of HSA-bound complex, [LnHSA],
is determined from Eq. 9, where [HSA]T and [Ln]T are the
total concentrations of HSA and the Ln(III) complex [23].
1
R1obs
¼
ð3Þ
T1obs
R1p ¼ R1obs ꢀ 0:38
r1p ¼ R1p=½Gdꢂ
ð4Þ
ð5Þ
Luminescence emission spectra were recorded using a Jo-
bin-Yvon Horiba FluoroMax-P spectrometer (using Data-
K
½HSAꢂ þ ½Lnꢂ ꢀ ½LnHSAꢂ
ð8Þ
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ꢀ
ꢁ
ꢀ
ꢁ
2
½HSAꢂT þ½LnꢂT þ1=K ꢀ
½HSAꢂT þ½LnꢂT þ1=K ꢀ4½HSAꢂT ½LnꢂT
½LnHSAꢂ ¼
ð9Þ
2
Max for Windows v2.2). Samples were held in a
10 9 10 nm or 10 9 2 nm quartz Hellma cuvette and a
cutoff filter (550 nm) was used to avoid second-order dif-
fraction effects. Eu(III) excitation was indirect
(kex = 270 nm).
Concentrations were substituted for Eu(III) emission
intensity (Eq. 10), where Icalc is the calculated Eu(III)
emission intensity (to be compared with the experimentally
observed intensity), IEu is the intensity of the Eu(III)
complex when in the absence of HSA (i.e. at the start of the
123

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J Incl Phenom Macrocycl Chem (2011) 71:435–444
titration) and IEuHSA is the intensity of the Eu(III) complex
when entirely bound to HSA (i.e. at the end of the
titration).
solution was stirred under nitrogen for 72 h at 85 °C (3a)
or 120 °C (3b). After cooling, the solution was filtered
through Celite and the solvent removed under reduced
pressure. The product was purified by silica column chro-
matography with solvent gradient elution from 100% DCM
to 8% MeOH, 1% ammonia and 91% DCM.
ꢀ
ꢁ
1
Icalc
¼
½EuꢂT IEu þ ðIEuHSA ꢀ IEuÞ½EuHSAꢂ
ð10Þ
½Euꢂ
Equations 9 and 10 were combined to give Eq. 11, which
was minimized in terms of K. Data analysis was performed
using an iterative least-squares fitting procedure, assuming
a 1:1 binding stoichiometry, operating in Microsoft Excel.
rac-1-[20-(Diphenylphosphinylamino)-propyl]-4,7,10-
tris(methoxycarbonylmethyl)-1,4,7,10-
tetraazacyclododecane, 3a
ꢀ
ꢁ
!
IEu ꢃ½EuꢂT
ðIEuHSA ꢀIEuÞ
þ
1
2 (0.311 g, 0.78 mmol), K2CO3 (0.11 g, 0.79 mmol) and
1a (0.21 g, 0.82 mmol). 3a (0.121 g, 23%) was obtained as
a yellow oil. Rf = 0.60 (10% MeOH, 1% NH3, 90%
DCM); dH (300 MHz; CDCl3) 1.1 (3 H, d, CH3) 1.9–4.4
(25 H, br, NCH2CH2N (16 H), OCCH2N (6 H),
NCH2CHCH3 (2 H), CH2CHCH3 (1 H)), 3.7 (9 H, 29 s,
CO2CH3) 7.4 (6 H, m, ArH), 7.9 (4 H, m, ArH); dC
(75 MHz; CDCl3) 23.5 (CHCH3), 43.4 (CH2CHCH3), 48.4,
50.4, 51.4, 53.5 (ring CH2), 51.7, 52.0, 52.5 (CO2CH3),
55.0, 55.3, 55.9 (OCCH2N), 66.2 (NCH2CH2N), 129.8
(ArC), 132.1 (ArC), 133.3 (ArC), 134.4 (ArC), 172.0
(CO2), 173.5 (CO2), 174.3 (CO2); dP (121 MHz; CDCl3)
24.1; m/z (HR-FAB?) [M ? H]? calcd for C32H49N5O7P;
646.33696, found; 646.33703; kmax (DCM)/nm 269; mmax
cm-1 3352 (br), 2951, 2825 (CH), 1733 (C=O), 1667,
1438, 1308, 1196 (P=O), 1112.
Icalc
¼
½EuꢂT
ꢀ
ꢁ
0
@
1
A
!
½HSAꢂT þ½EuꢂT þ1=K ꢀ
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ꢃ
=2
ꢀ
ꢁ
2
½HSAꢂT þ½EuꢂT þ1=K ꢀ4½HSAꢂT ½EuꢂT
ð11Þ
Relaxometric data analysis of the Gd–dpp complexes was
carried out in a similar manner to that of the Eu(III)
analogues, using an iterative least-squares fitting
procedure, assuming
a
1:1 binding stoichiometry,
operating in Microcal Origin. In order to define the
concentration of the HSA-bound Gd species in terms of
its relaxation rate, R1calc, Eq. 12 was used, which was
combined with Eq. 9 to give Eq. 13, where R1Gd is the
observed longitudinal relaxation rate of the Gd(III)
complex in the absence of HSA (i.e. at the start of the
titration) and R1GdHSA is the observed longitudinal
relaxation rate of the Gd(III) when entirely bound to
HSA (i.e. at the end limiting asymptotic value of the
titration) and [HSA]T and [Gd]T are the total concentrations
of HSA and the Gd(III) complex.
1-[20-(Diphenylphosphinylamino)-20-(methyl)-propyl]-
4,7,10-tris(methoxycarbonylmethyl)-1,4,7,10-
tetraazacyclododecane, 3b
2 (0.305 g, 0.79 mmol), 1b (0.217 g, 0.80 mmol) and
K2CO3 (0.112 g, 0.81 mmol). 3b (0.102 g, 19%) was
obtained as a yellow oil. Rf = 0.70 (10% MeOH, 1% NH3,
90% DCM); dH (300 MHz; CDCl3) 1.2 (6 H, br s,
CH2(CH3)2), 2.2–3.5 (24 H, br, NCH2CH2N (16 H),
OCCH2N (6 H), NCH2C(CH3)2 (2 H)), 3.6, 3.6, 3.7 (9 H, 39
s, OCH3), 7.4 (6 H, m, ArH) 7.8 (4 H, m, ArH); dC (75 MHz;
CDCl3) 29.0 (C(CH3)2), 38.4 (C(CH3)2), 51.0 (NCH2CH2N),
54.2, 54.8 (OCCH2N), 69.3 (NCH2C(CH3)2), 127.4 (ArC),
130.5 (ArC), 172.4, 172.8 (CO2); dP (121 MHz; CDCl3) 19.4
(s); m/z (HR-FAB?) [M ? H]? calcd for C33H51N5O7P;
660.35261, found; 660.35262; kmax (DCM)/nm 269; mmax
cm-1 3370 (br), 2926, 2852 (CH), 1732 (C=O), 1655, 1378,
1438, 1215 (P=O), 1110.
ꢀ
ꢁ
1
R1calc
¼
½GdꢂT R1Gd þ ðR1GdHSA ꢀ R1GdÞ½GdHSAꢂ
½GdꢂT
ð12Þ
ꢀ
ꢁ
!
IGd ꢃ ½GdꢂT
þ
Þ
1
R1calc
¼
0
@
½GdꢂT
ðR1GdHSA ꢀ R1Gd
ꢀ
ꢁ
1
A
!
½HSAꢂT þ½GdꢂT þ1=K ꢀ
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ꢃ
=2
ꢀ
ꢁ
2
½HSAꢂT þ½GdꢂT þ1=K ꢀ4½HSAꢂT ½GdꢂT
ð13Þ
Aziridines 1a/b [36] and 1,4,7-tris(methoxycarboxymeth-
yl)-1,4,7,10-tetraazacyclododecane 2 [43] were prepared
according to literature procedures.
General methyl ester hydrolysis
General dpp ring opening synthesis
Following literature methods [5], the methyl ester protected
macrocycles were dissolved in 1 M LiOH (5 mL) and
heated at 80 °C for 24 h. The resulting solution was loaded
The appropriate aziridine (1a/b) and 2 were dissolved in
dry MeCN (3a) or DMF (3b). On addition of K2CO3 the
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J Incl Phenom Macrocycl Chem (2011) 71:435–444
439
onto a Dowex 50W strong acid-cation exchange column
(H? form), washed with water and eluted with 12%
ammonia solution. The solvent was then removed under
reduced pressure and the residue taken up in water and
lyophilized to give the product.
lowered to pH 5.5 and lyophilised. The product was
extracted from the salt residue with 20% dry MeOH/DCM
solution which was then removed under reduced pressure.
The residue was taken up in water and lyophilised to give
the Ln(dpp) complex.
rac-1-[20-(Diphenylphosphinylamino)propyl]-4,7,10-
tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane, 4a
Europium(III) rac-1-[20-
(diphenylphosphinylamino)propyl]-4,7,10-
tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane,
Eu.4a
3a (0.121 g, 0.19 mmol). 4a (0.075 g, 58%) was obtained
as a white hygroscopic powder. dH (300 MHz; D2O) 1.9
(3H, s, CH3), 1.2–4.0 (25 H, br, NCH2CH2N (16 H),
OCCH2N (6 H), NCH2CHCH3 (2 H), CH2CHCH3 (1 H)),
7.6 (6 H, m, ArH), 7.9 (4 H, m, ArH); dC (75 MHz; D2O)
24.7 (CHCH3), 46.0 (CHCH3), 50.9, 51.3, 51.7, 52.1, 53.0,
53.7 (NCH2CH2N), 58.3, 59.2 (OCCH2N), 62.4
(NCH2CHCH3), 131.5 (ArC), 134.3 (ArC), 135.4 (ArC),
174.0, 178.7, 180.7 (CO2); dP (121 MHz; D2O) 28.1;
m/z (HR-FAB?) [M ? H]? calcd for C29H43N5O7P;
604.29001, found; 604.29002; kmax (H2O)/nm 269; mmax
cm-1 3335 (OH), 1583 (C=O), 1410, 1175 (P=O), 1094.24,
996, 862.
EuCl3ꢁ6H2O (15.4 mg, 42.0 lmol) and 4a (20.8 mg,
35.0 lmol). Eu.4a (25.7 mg, 99%) was obtained as a white
hygroscopic powder. dH (400 MHz; D2O; 4 °C) selected
resonances: 25.7 (1H, s, NCHHCH2N axial), 33.0 (1H, s,
NCHHCH2N axial), 34.7 (1H, s, NCHHCH2N axial), 34.9
(1H, s, NCHHCH2N axial), typical of a square-anti pris-
matic (SAP) geometry about Eu, the remaining resonances
span -17.4 to 7.65 ppm; m/z (HR-ESMS?) [M ? H]?
calcd for C29H40N5O7PEu; 754.1872, found; 754.1876
(
153Eu); kmax (H2O)/nm 273; mmax cm-1 3368, 2984, 1594
(C=O), 1437, 1396, 1083 (P–O).
1-[20-(Diphenylphosphinylamino)-20-(methyl)-propyl]-
4,7,10-tris(carboxymethyl)-1,4,7,10-
tetraazacyclododecane, 4b
Europium(III) 1-[20-(diphenylphosphinylamino)-20-
(methyl)-propyl]-4,7,10-tris(carboxy methyl)-1,4,7,10-
tetraazacyclododecane, Eu.4b
3b (0.102 g, 0.16 mmol). 4b (0.079 g, 71%) was obtained
as a white hygroscopic powder. dH (300 MHz; D2O) 1.5 (6
H, s, CH3), 2.5–3.7 (24 H, br, NCH2CH2N (16 H),
OCCH2N (6 H), NCH2C(CH3)2 (2 H)) 7.5 (6 H, m, ArH),
7.7 (4 H, m, ArH); dC (100 MHz; D2O) 21.6 (CH3), 42.8
(C(CH3)2), 48.8, 49.6, 51.8, 55.7, 56.5 (NCH2CH2N), 58.8,
61.2 (OCCH2N), 66.6 (NCH2C(CH3)2), 128.5 (ArC), 131.4
(ArC), 132.9 (ArC), 137.9 (ArC), 171.0, 178.1, 180.2
(CO2); dP (121 MHz; D2O) 26.4; m/z (ESMS?) 618
[M ? H]?; kmax (H2O)/nm 270; mmax cm-1 2970, 1738
(C=O), 1586, 1379, 1203 (P=O), 1088.
EuCl3ꢁ6H2O (11.2 mg, 31.0 lmol) and 4b (14.4 mg,
23.0 lmol). Eu.4b (8.1 mg, 46%) was obtained as a white
hygroscopic powder; dH (400 MHz; D2O) 24.8 (1H, s,
NCHHCH2N axial), 26.2 (1H, s, NCHHCH2N axial), 33.6
(1H, s, NCHHCH2N axial), 35.6 (1H, s, NCHHCH2N
axial), typical of a square-anti prismatic geometry (SAP)
about Eu, the remaining resonances span -20.4 to
9.1 ppm; m/z (HR-ESMS?) [M ? H]? calcd for
C30H42N5O7PEu; 768.2029, found; 768.2026 (153Eu); kmax
(H2O)/nm 270; mmax cm-1 3369, 2868, 1590 (C=O), 1389,
1085 (P–O).
Gadolinium(III) rac-1-[20-
General complex synthesis
(diphenylphosphinylamino)propyl]-4,7,10-
tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane,
Gd.4a
Two separate solutions of the appropriate pro-ligand and
LnCl3ꢁ6H2O (slight excess to ensure all ligand used in
complexation) in water (5 mL) were prepared and were
each adjusted to pH 7 by addition of NaOH. On mixing, the
pH fell to *5 and the solution was adjusted to pH 6 using
NaOH. After heating at 90 °C for 2 h, the pH was raised to
10 and the solution was filtered through a Celite plug to
remove any precipitated Ln(OH)3. The solution was then
GdCl3ꢁ6H2O (10.7 mg, 29.0 lmol) and 4a (12.0 mg,
20.0 lmol). Gd.4a (15.0 mg, 99%.) was obtained as a
white hygroscopic powder. m/z (HR-ESMS?) [M ? H]?
calcd for C29H40N5O7PGd; 756.1886, found; 756.1885
(
155Gd); kmax (H2O)/nm 270; mmax cm-1 2819, 1587 (C=O),
1426, 1330, 1109 (P–O).
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J Incl Phenom Macrocycl Chem (2011) 71:435–444
Gadolinium(III) 1-[20-(diphenylphosphinylamino)-20-
(methyl)-propyl]-4,7,10-tris(carbo-xymethyl)-1,4,7,10-
tetraazacyclododecane, Gd.4b
GdCl3ꢁ6H2O (19.5 mg, 52.5 lmol) and 4b (21.7 mg,
35.0 lmol). Gd.4b (17.3 mg, 64%) was obtained as a
white hygroscopic powder. m/z HR-ESMS ? [M ? H]?
calcd for C30H42N5O7PGd; 773.2057, found; 773.2060
(
158Gd); kmax (H2O)/nm 277; mmax cm-1 2628, 1575 (C=O),
1436, 1402, 1187, 1128, 1054 (P–O).
Results and discussion
The proligands 4a and 4b were synthesised via ring
opening of the aziridine precursors (1a/b) with 2 followed
by base hydrolysis of the methyl esters. Complexation was
achieved in water using the appropriate LnCl3 salt. Com-
plex formation was verified by ESMS (and 1H NMR
spectroscopy of the Eu(III) analogues) (Scheme 1).
Fig. 1 Emission spectra of Eu.4a (0.1 mM, pH 7.4) in the absence
(solid line) and presence of HSA (dashed line 0.16 mM, dot-dash line
0.32 mM, dotted line 1.6 mM)
transitions, i.e. from the 5D0 excited state to the 7FJ ground
state of Eu(III) where J = 0, 1, 2, 3, 4. The spectrum was
obtained by indirect excitation via the dpp chromophore
(kex = 270 nm); this sensitised emission is much more
intense than that obtained via direct excitation. As O–H
oscillators can quench the excited state of Eu(III) com-
plexes [41, 42], the hydration state of Eu.4a/b could be
Luminescence studies
The luminescence emission spectrum of Eu.4a at pH 7.4
(Fig. 1—solid line) is typical in shape and form of Eu(III)
complexes (the Eu.4b spectrum, not shown, is near iden-
tical). The peaks correspond to the DJ = 0 to DJ = 4
O
O
OMe
OMe
P
O
NH
K2CO3
CH3CN or DMF
N
N
HN
N
N
N
R2
R1
O
P
N
MeO
O
MeO
O
N
N
R1
R2
MeO
MeO
O
O
1(a-b)
2
3(a-b)
a. R1 = H, R2 = Me
b. R1 = R2 = Me
1M LiOH
O
O
O
N
OH
P
P
O
O
NH
R2
NH
R2
LnCl3.6H20
H2O, pH 6
N
N
N
N
R1
R1
Ln
O
O
HO
N
N
N
O
O
HO
4(a-b)
O
O
Ln.4(a-b)
Ln = Gd, Eu
Scheme 1 Synthesis of proligands and complexes
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J Incl Phenom Macrocycl Chem (2011) 71:435–444
441
determined by exploiting the differing quenching ability of
O–H cf. O-D oscillators. The difference in rate constants
(measured in H2O and D2O) for depopulation of the excited
state, enable q, the hydration state (number of inner-sphere
waters) to be determined for the Eu(III) complexes
(Eq. 7). These values can be reliably extrapolated to
the analogous Gd(III) complexes [2]. At pH 7.4 both
Eu.4a (kH O ¼ 2:14 msꢀ1; kD O ¼ 0:64 msꢀ1) and Eu.4b
2
2
(kH O ¼ 2:67 msꢀ1; kD O ¼ 1:18 msꢀ1) gave a value of
2
2
q = 1.5. Fractional q values such as this are indicative of
the presence of a q = 2 species in equilibrium with a q = 1
species. A high value of q should be reflected in a high
relaxivity for the corresponding Gd(III) complexes.
Figure 1 shows that there is a marked change in the
intensity of the luminescence on addition of HSA. The
spectra shown for 0.16, 0.32 and 1.6 mM HSA show a
decrease in Eu(III) emission intensity on binding to HSA.
In the presence of the protein, the diphenylphosphinamide
moiety binds to hydrophobic sites in HSA and the Eu(III)
luminescence is quenched (no Eu(III) emission is seen at
1.6 mM HSA). The intense emission in the absence of
HSA is a result of energy transfer from the dpp triplet
excited state to the 5Do excited state of Eu(III). The
decrease in sensitization (or quenching) observed upon
addition of HSA is likely to originate from an energy
transfer mechanism, most likely involving the solitary
tryptophan residue in the protein found in subdomain IIA
(site I), a typical site for binding of MRI contrast agents
[26–31]. Addition of HSA also causes a change in hydra-
tion state of the Eu(III) complexes; under physiological
conditions in the presence of HSA (0.1 mM Eu.4a/b,
Fig. 2 Plot of DJ = 2 Eu(III) emission intensity versus [HSA] for
Eu.4a (left) and Eu.4b (right) showing the experimental data (solid
circles) and the calculated data (line).*0.2 mM Eu.4(a–b), 0.1 M
NaCl, pH 7.4 (10 mM PBS), 25 °C
The binding constants obtained by this method for
Eu.4a (27,000 M-1 12%) and Eu.4b (32,000 M-1
14%) are similar and show a fairly high affinity of these
complexes for HSA, indeed slightly higher than that
of the benchmark HSA-binding contrast agent VasovistÒ
(*11,000 M-1) [29, 45]. These values suggest that at
imaging concentrations (0.1 mM Eu.4a/b, 0.67 mM HSA)
the complexes would be [90% bound to HSA (at 298 K).
The values for the two complexes are too similar (within
the errors of this method of determination) for any con-
clusions to be drawn with respect to the slightly higher
value for Eu.4b.
0.67 mM HSA, pH 7.4) both Eu.4a (kH O ¼ 1:34
2
msꢀ1; kD O ¼ 0:75 msꢀ1) and Eu.4b (kH O ¼ 1:83 msꢀ1
;
2
2
kD O ¼ 1:05 msꢀ1) show a significant reduction in inner-
Relaxivity studies
2
sphere hydration (q = 0.4 and q = 0.6 respectively). Pre-
dominantly q = 2 complexes (such as Eu.4a/b in the
absence of HSA) are susceptible to displacement of inner-
sphere waters by competitive binding of endogenous serum
anions such as carbonate or protein carboxylic acid resi-
dues [44]. This decrease in emission intensity on increasing
HSA concentrations can be used to determine binding
constants for Eu.4a/b to HSA. Eu(III) emission spectra
were recorded (0.1 mM complex) with varying concen-
trations of HSA. The complexes were excited indirectly,
via the chromophore, at kex*270 nm and the variations in
intensity of the DJ = 2 band (615 nm) were plotted vs.
[HSA] (Fig. 2). The titrations of Eu.4a and Eu.4b were
carried out at pH 7.4 (phosphate buffered saline). The
decrease in emission intensity as HSA concentration is
increased, fits to a 1:1 binding isotherm; for related com-
plexes it is common to observe only one strong affinity site
on HSA [22–31].
The relaxivities (298 K, 20 MHz) of the Gd(III) complexes
were recorded at pH 7.4: Gd.4a r1 = 7.6 mM-1 -1
s
and
. These values are higher
than typical q = 1 complexes such as [GdDOTA]-
(r1 = 4.1 mM-1 -1
) [46] and indicative of a predomi-
Gd.4b. r1 = 7.3 mM-1 -1
s
s
nantly q = 2 species (in equilibrium with a q = 1 species);
this is consistent with the hydration state data obtained for
the Eu(III) analogues. In the presence of HSA (0.1 mM
Gd.4a/b, 0.67 mM HSA) the relaxivities were: Gd.4a
r1 = 11.7 mM-1 -1 and Gd.4b. r1 = 16.0 mM-1 -1
s .
s
These increased relaxivities are disappointing given the
apparently high binding affinities these complexes have for
HSA; however, given the luminescence data on the Eu(III)
analogues these lower than hoped for values are to be
expected. It was noted that the q-values of Eu.4a and
Eu.4b were both significantly lowered on binding to HSA
(from q = 1.5 to q = 0.4 and 0.6 for Eu.4a and Eu.4b
123

442
J Incl Phenom Macrocycl Chem (2011) 71:435–444
again showing a relatively high affinity for HSA. At
imaging concentrations (0.1 mM Gd.4a/b, 0.67 mM HSA)
the complexes would be [90% bound to HSA (at 298 K).
Conclusion
Eu(III) and Gd(III) complexes bearing pendant diph-
enylphosphinate residues have been prepared which dis-
play a hydration state q = 1.5 at pH 7.4, reflected in the
relatively high relaxivities that were observed for Gd.4a/b.
The hydrophobicity of the dpp moiety enables non-cova-
lent binding to HSA. This binding event can be monitored
using luminescence (Eu) and relaxometry (Gd) giving
relatively high binding affinities, whereby [90% of the
agents would be non-covalently bound to HSA under
physiologically relevant concentrations of complex and
HSA. Unfortunately the expected relaxivity gains were
disappointing; this can be attributed primarily to dis-
placement of inner-sphere waters on binding to the protein.
This lowering of hydration state was shown by luminescent
lifetime measurements on the Eu(III) analogues.
Fig. 3 Plot of the observed longitudinal relaxation rate, R1obs, versus
[HSA] for Gd.4a (left) and Gd.4b (right) showing the experimental
data (solid circles) and the calculated data (line).*0.2 mM Gd.4(a–
b), 0.1 M NaCl, pH 7.4 (10 mM PBS), 20 MHz, 25 °C
respectively). This displacement of inner-sphere waters by,
presumably, carboxylate residues within the HSA binding
site is a common problem for neutral q = 2 complexes
[44]. VasovistÒ (q = 1) has r1 *35 mM-1 -1
under
s
As the binding affinities of the complexes was shown to
be favourably high, we are seeking to maintain this high
affinity whilst suppressing the reduction in hydration state
(caused by endogenous anion binding) by incorporating
negatively charged substituents in the alpha position of the
carboxylate arms [8, 9]. We are also investigating further,
the binding of Ln.4a. The compound synthesised in this
report was a racemic mixture of the R and S enantiomer (at
CH2CH(CH3)NHP). Although in previous work using
binaphthyl-derived DOTA-based complexes we saw no
discrimination between R and S binaphthyl units, it is
possible that the R and S-enantiomers of Ln.4a have dif-
ferent binding affinites (e.g. the S-enantiomer of EovistÒ
has been shown to bind to HSA with a greater affinity than
the R-enantiomer) [48].
similar conditions (298 K, pH 7.4, 20 MHz) [47]. When
MRI contrast agents bind to large slowly tumbling proteins
such as HSA, relaxivity is enhanced; however, lower than
expected relaxivities are often a result of several effects:
lowering of hydration state by competitive binding of
endogenous anions, rotational freedom within the bound
agent (i.e. about C–C bonds, thus the motion of the contrast
agent is not effectively coupled to the motion of the pro-
tein), and slowing of the water exchange rate. A combi-
nation of these factors (particularly the first two) is likely to
explain the lower than hoped for relaxivities of HSA-bound
Gd.4a/b. It is interesting to note that a higher relaxivity
was observed for HSA-bound Gd.4b. cf. Gd.4a
(r1 = 16.0 mM-1 -1 cf. r1 = 11.7 mM-1 -1
s ); this may be
s
tentatively assigned to the fact that there is additional steric
bulk in Gd.4b (two methyl groups in the link to the
phosphinamide) leading to less free rotation of the bound
complex.
Acknowledgments We thank the EPSRC National Mass Spec-
trometry Service Centre, Swansea for high resolution ESMS.
Relaxivity versus [HSA] titrations were carried out for
Gd.4a/b in the same manner as for the Eu(III) complexes,
the difference being that relaxation rate measurements
were used instead of luminescent emission. As expected,
the observed relaxation rates increased upon addition of
HSA. This is because of the increased rotational correlation
time, sR, of the protein bound complex (Fig. 3).
The data analysis was carried out in a similar manner to
that of the Eu(III) analogues. The affinity constants
calculated from the relaxivity data are, as expected,
similar to those calculated from luminescence: Gd.4a
(21,000 M-1 15%) and Gd.4b (26,000 M-1 15%),
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