A new chiral lanthanide NMR probe for the determination of the enantiomeric purity of α-hydroxy acids and the absolute configuration of α-amino acids in water

Article abstract of DOI:10.1039/b513814k

A water-soluble, enantiopure lanthanide complex, SSS-[Ln·L ³], has been assessed as an effective chiral derivatising agent for the determination of the enantiomeric purity of α-hydroxy acids in aqueous solution. The complex displays superior ch

Full text of DOI:10.1039/b513814k

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www.rsc.org/dalton | Dalton Transactions
A new chiral lanthanide NMR probe for the determination of the
enantiomeric purity of a-hydroxy acids and the absolute configuration
of a-amino acids in water
Rachel S. Dickins* and Alessandra Badari
Received 29th September 2005, Accepted 15th February 2006
First published as an Advance Article on the web 10th March 2006
DOI: 10.1039/b513814k
A water-soluble, enantiopure lanthanide complex, SSS-[Ln·L3], has been assessed as an effective chiral
derivatising agent for the determination of the enantiomeric purity of a-hydroxy acids in aqueous
solution. The complex displays superior chemical shift non-equivalence (DDd ∼2–11 ppm) for the
diastereomeric resonances of interest compared to lanthanide shift reagents reported in the literature
(DDd <0.1 ppm, typically). 1H NMR studies have also revealed that SSS-[Ln·L3] can be used to
determine the absolute configuration of a-amino acids at physiological pH, in water. The ability of
SSS-[Ln·L3] to signal anion binding and, in particular, to distinguish between diastereomers through
optical techniques such as lanthanide luminescence and circular dichroism has also been assessed.
the examples is very small (<0.1 ppm, typically) as found in so
Introduction
many of these aqueous chiral lanthanide shift reagents. This,
The paramagnetic properties of the lanthanides have been ex-
coupled with the broadness of the observed signals, which is
ploited in NMR spectroscopy for many years to effect spectral
often associated with chemical exchange due to competing internal
simplification and resolution enhancement.1,2 The angular and
exchange processes, has meant there has been little direct appli-
distance information gleaned from the lanthanide induced shift
cation for the determination of enantiomeric purity in aqueous
has found widespread application from the determination of
solution.
solution structures of lanthanide chelates to the development
However, the resurgence of interest in the coordination chem-
of biological probes of protein and peptide structure.3,4 Chiral
istry of lanthanide complexes in aqueous solution over the past
lanthanide complexes continue to be assessed as chiral shift and
few years has led to the development of well-defined, water-soluble
derivatising agents for the NMR separation of enantiomers and
chiral systems which offer considerable scope for the design of
determination of enantiomeric purity.2,3,5 Although substantial
more effective chiral shift reagents and, where the association
developments have been reported within this field, the majority of
constant is much larger, of chiral derivatising agents. We have
studies have been performed in organic media. As a large number
been involved in the selective recognition of bioactive species
of optically active compounds are of natural origin, water is the
in aqueous solution by coordinatively unsaturated lanthanide
solvent of choice and studies in aqueous media are therefore highly
desirable. A few water soluble systems have been reported since
Reuben first introduced a ‘self-resolution’ approach to separate
the enantiomeric signals of a-hydroxy carboxylic acids using lan-
thanide ions.6 Peters et al. proposed the first genuine aqueous chi-
ral lanthanide shift reagents based on lanthanide complexes of (S)-
[(carboxymethyl)oxy]succinic acid (CMOS) which resolved the
complexes in which the bound water molecules may be readily
displaced by a variety of monodentate (e.g. fluoride, phosphate)
or chelating (e.g. lactate, carbonate) anions.10,11 The rich magnetic
and optical properties exhibited by the lanthanide ions has enabled
the anion binding to be effectively signalled through NMR (Eu,
Yb), luminescence (Eu, Tb) and chiroptical techniques (Eu, Yb).
Within these studies we have reported a first step towards the
enantiomeric nuclei of a-amino acids and carboxylates.7 Although
development of an effective chiral derivatising agent for a-hydroxy
acids in aqueous solution.12 A large lanthanide induced shift,
chemical shift non-equivalence and an apparent absence of kinetic
resolution in complex formation has been observed upon addition
of racemic lactic acid to [Yb·L1]3+. The lactate CH and CH3
resonances are clearly resolved for the (R) and (S) diastereomers
(DDd = 10 ppm) and experience a large lanthanide induced
shift; the CH resonating at +48 and +58 ppm (cf . 4.1 ppm in
the free form) and the CH3 resonating at +30 and +20 ppm
for (R) and (S)-lactate respectively (cf . 1.3 ppm in the free
form). Herein, we report the synthesis of a novel, enantiopure
lanthanide complex and assess its ability to act as an effective chiral
derivatising agent in aqueous solution, for a number of bioactive
species.
spectral resolution was sufficient to determine enantiomeric purity,
the magnitude of shift difference between enantiomers (DDd) was
small (<0.1 ppm) and displayed a strong pH dependence. This is
typical of a number of reported systems for a-hydroxy, a-amino
and carboxylic acids which are found to operate within a specific
pH range and often display no resolution beyond an optimal
pH.8 There have been reports of aqueous lanthanide shift reagents
for a-amino acids and N-acyloligopeptides operating at neutral
pH without displaying significant signal broadening.9 However,
the observed limiting chemical shift non-equivalence in each of
Department of Chemistry, University of Durham, South Road, Durham, UK
DH1 3LE. E-mail: R.S.Dickins@durham.ac.uk
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Pd(OH)2/C, acidic EtOH, RT). Complexation was achieved by
refluxing L2 in dry acetonitrile, with the appropriate lanthanide
trifluoromethanesulfonate salt. Subsequent basic hydrolysis of
[Ln·L2]3+ (0.02 M NaOH) followed by cation exchange chromatog-
raphy, yielded the neutral SSS-[Ln.L3] complexes.
Solution NMR studies
The isomerism exhibited by chiral lanthanide complexes such
as [Ln·L1]3+–[Ln·L3] is well documented and understood.2 They
exhibit two independent elements of chirality denoted by the
NCCO torsion angle of the pendent arms (positive = D, negative =
K) and the NCCN torsion angle of the macrocyclic ring (positive
= dddd, negative = kkkk) which gives rise to four possible isomers
in solution. The remote chiral centre at the amide functionality
renders each of the isomers diastereomeric. Variable temperature
(295–193 K) NMR studies on lanthanide complexes of L1 confirm
the existence of two square antiprismatic isomers (Kdddd and
Dkkkk) at room temperature, which are in fast exchange on the
NMR timescale.11 In contrast, [Ln·L3] complexes appear to exist
solely as a single isomer at room temperature, as confirmed by VT
NMR, with shifts indicative of a square antiprismatic geometry.14
The partial 1H NMR spectrum of SSS-[Yb·L3] (1 mM, D2O, pD
7, 200 MHz, 295 K) is shown in Fig. 1(a). The eight distinct
resonances to high frequency (+99 to +16 ppm) can be attributed
to the four most shifted axial (᭹) and equatorial (ꢀ) protons of the
macrocyclic ring. It should be noted that the 1H NMR spectrum
of SSS-[Yb·L3] at a concentration of 10 mM revealed the presence
of many broadened peaks which disappeared upon dilution,
consistent with the formation of dimers at higher concentrations,
which was also confirmed by ESMS [1735 (10%), M2Na+].
Results and discussion
Synthesis
The synthesis of the tri-substituted ligand, L2, was attempted
by direct alkylation of tetraazacyclododecane with the appro-
priate chloroamide. However, a mixture of mono-, di- and tri-
substituted products resulted. Although the trialkylated product
was isolated via exhaustive extraction of the organic phase
with water or by column chromatography, yields were poor.
Therefore an alternative route involving the selective protection of
a single macrocyclic ring nitrogen, followed by trialkylation and
subsequent deprotection was employed (Scheme 1). Reaction of
1,4,7,10-tetraazacyclododecane-molybdenum tricarbonyl13 with
◦
benzyl bromide (dry DMF, 60 C, K2CO3), followed by decom-
plexation of the molybdenum moiety in aqueous acid, yielded
the monobenzylated macrocycle. Subsequent reaction with three
equivalents of the appropriate chloroamide (dry DMF, Ar, 60 ◦C,
K2CO3) afforded the tetra-alkylated ligand after purification
on neutral alumina. The trialkylated ligand, L2, was isolated
following reductive deprotection of the N-benzyl group (H2, 45 psi,
Recently, we have reported that the binding of anions to the co-
ordinatively unsaturated lanthanide complexes of L1 is effectively
1
signalled through H NMR as the observed lanthanide induced
shift of the macrocyclic ring protons can be directly correlated
to the nature of the donor atom in the axial position.15,16 It is
Scheme 1
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the binding anion that lie within the McConnell cone [(3cos2h −
1)r−3 dependence] will also be subjected to a lanthanide induced
shift. This can give useful information into the binding mode
of chelating anions. For example, lactate binds to [Yb·L1]3+ in
a bidentate manner, as confirmed through crystallographic and
hydration state studies.12 However, there are two possible binding
modes: the OH group may be axially disposed with the carboxylate
occupying the equatorial position or vice versa. Upon the addition
of (S)-lactate to [Yb·L1]3+ two additional singlet resonances were
evident in the high frequency range at +58 ppm, integrating to
one proton and attributed to the CH of the bound lactate, and at
+20 ppm, integrating to three protons and subsequently attributed
to the methyl resonance of the bound lactate.19 The induced
paramagnetic shift, which for Yb is largely dipolar in origin, can be
approximated to having a (3cos2h − 1)r−3 dependence and suggests
the lactate CH is closer to the principal axis of the complex than the
lactate methyl group and axial ligation therefore occurs through
the OH functionality with the carboxylate group occupying the
equatorial position (Fig. 2). This is consistent with solid state
crystallographic studies.20
Fig. 1 Partial 1H NMR (200 MHz, D2O, 295 K) of (a) SSS-[Yb·L3]
(diaqua species) showing the four axial (᭹) and four equatorial (ꢀ) protons
of the macrocyclic ring; and in the presence of (b) 10 equivalents of
(S)-lactate [(*) (S)-lactate CH3]; (c) 10 equivalents of racemic lactate [(#)
(R)-lactate CH3].
the polarisability of the axial donor that ranks the second order
2
crystal field coefficient, B0 , and determines the magnitude of the
observed shift.17,18 Values of the mean shift of the four most shifted
axial protons of the macrocyclic ring for SSS-[Yb·L3] are collated
in Table 1. Hard, uncharged donors such as water give rise to the
largest shifts whereas the more polarisable donors (e.g. carbonate)
give much smaller shifts. This simple correlation has proved to be
invaluable in the determination of binding modes for a variety of
anions. For example, the phosphorylated amino acids (e.g. (S)-OP-
serine) give rise to a shift indicative of a preferred binding mode to
SSS-[Yb·L3] through the phosphate moiety rather than chelation
through the amino acid functionality (via formation of a 5-ring
chelate involving the NH2 and CO2−). Furthermore, nuclei within
Fig. 2 Ligation of lactate to the Yb centre showing the hydroxyl group in
an axial position which brings the lactate CH closer to the principal axis
compared to the lactate methyl group.
a-Hydroxy acids. The 1H NMR spectrum of SSS-[Yb·L3]
(10 mM, 200 MHz, D2O, 295 K) in the presence of a ten-fold
excess of (S)-lactate (Fig. 1b) is also consistent with bidentate
Table 1 Effect of the axial donor on the 1H NMR spectral range for SSS-[Yb·L3] (200 MHz, D2O, 295 K)
Anion
Axial donor
Shift range (ppm)
Mean shifta dHax (ppm)
(S)-OP-serine
Phosphate
Water
H2O
H2O
H2O
OH
+110 → −83
+102 → −77
+100 → −120
+99 → −56
+93 → −94
+89 → −100
+68 → −50
+65 → −43
+56 → −31
+45 → −44
82
75
70
72
68
64
43
40
36
33
(S)-Lactate
Acetate
COd−
COd−
NH2
NH2
CO−
CO−
Citrateb
(S)-Serine
(S)-Alanine
Oxalate
Carbonate
a Mean chemical shift of the four most shifted axial protons of the macrocyclic ring. b Minor species also present (>10%), due to additional possible
binding modes for the anion.
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chelation with the OH group axially disposed. An additional
singlet resonance at +31 ppm was evident, integrating to three
protons, which can be accounted for by the methyl resonance
of the bound (S)-lactate. The expected methyl doublet was not
resolved due to the broadening effect of the paramagnetic ion.
However, the exact position of the lactate CH resonance could not
be defined due to inconclusive two dimensional spectra obtained
for this system,21 although an extra singlet resonance observed in
the high frequency region of the spectrum could be accounted for.
Upon addition of 10 equivalents of racemic lactate to SSS-[Yb·L3]
a lack of enantioselectivity in adduct formation was apparent
and a 1 : 1 mixture of diastereomeric complexes was observed
2–10 ppm. As the complex displays a substantial improvement
in the chemical shift difference observed for the diastereomeric
resonances compared to reported lanthanide shift reagents (DDd <
0.1 ppm, typically) a range of related derivatives were then studied
to assess the potential of SSS-[Yb·L3] to act as an aqueous chiral
derivatising agent for a-hydroxy acids in general (Table 2). A
lack of enantioselectivity in complex formation was observed in
each case, even with more sterically demanding substrates, and
additional resonances due to the a-CH and side chain functionality
held within the McConnell cone were apparent to high frequency.
1
Due to a lack of cross peaks being observed in H–1H COSY
experiments21 the exact assignment of the bound a-hydroxy acid
resonances proved to be difficult. However, a large chemical shift
non-equivalence was apparent for the macrocyclic ring protons
in the (R) and (S) diastereomeric adducts. In particular, the
magnitude of the shift difference for the most shifted axial ring
proton in the diastereomers was of the order 2–5 ppm (Table 2).
This is a huge improvement compared to DDd values reported
1
by H NMR (Fig. 1c). The lactate methyl resonance was clearly
resolved for the (R) (+20 ppm) and (S) (+31 ppm) diastereoisomers
(DDd ∼11 ppm) and experienced a large lanthanide induced shift
(+1.3 ppm in the free form). Furthermore, the macrocyclic ring
protons for the (R) and (S) diastereomeric complexes were also
clearly resolved and displayed large DDd values ranging from
Table 2 1H NMR data for SSS-[Yb·L3] in the presence of 10 equivalents of racemic a-hydroxy acid (200 MHz, D2O, 295 K)
Ratio of (R) : (S)
diastereomers
Most shifted dHax for
(R)-diastereomer (ppm)
Most shifted dHax for
(S)-diastereomer (ppm)
a-Hydroxy acid
DDd (ppm)
1 : 1
1 : 1
1 : 1
+97 (dCH3 = +20)a
+99 (dCH3 = +30)a
2 (10)
+94
+94
+91
+96
3
2
1 : 1
1 : 1
+66
+97
+70
+92
4
5
1 : 1
+109
+114
5
a Values in parentheses indicate the chemical shift observed for the bound lactate CH3 resonance. b The resonances for the most shifted axial protons for
the (R) and (S) diastereomers were broadened and overlapped slightly, hence enantioselectivity in binding was determined using the subsequently most
shifted resonance occurring at +66 and +70 ppm for (R) and (S) mandelate respectively.
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for aqueous lanthanide shift reagents in the literature which are
generally around 1 ppm and often <0.1 ppm and enables the
determination of the enantiomeric purity of a-hydroxy acids to be
easily assessed in aqueous solution.
systematic study was therefore undertaken to determine whether
the (R) and (S) diastereomeric adducts formed upon addition of a
racemic substrate to enantiopure SSS-[Ln·L3] complexes may be
distinguished by optical methods.
Emission studies. In Bleaney’s theory of magnetic anisotropy25
a-Amino acids. 1H NMR studies have revealed that SSS-
[Yb·L3] binds to a-amino acids at physiological pH via a common
chelated mode. The chemical shifts observed are indicative of axial
ligation through the amine functionality11 with the carboxylate
group occupying an equatorial position (Table 1). No evidence
of competing chelation through side chain functionality was
apparent. There are very few examples of shift reagents capable
of determining the enantiomeric purity of a-amino acids at
physiological pH in aqueous media. Therefore the ability of SSS-
[Yb·L3] to act as a chiral derivatising agent for a-amino acids in
water was also assessed (Table 3). Upon addition of 10 equivalents
of racemic a-amino acid to SSS-[Yb·L3] enantioselectivity in
binding was apparent for the majority of the amino acids,
with preferential binding occurring to the (R) enantiomer. This
precludes the use of SSS-[Yb·L3] as a chiral derivatising agent for
a-amino acids. However, a large chemical shift non-equivalence
was apparent for the diastereomeric macrocyclic ring protons of
the (R) and (S) amino acid adducts, with DDd values ranging
from 4–9 ppm for the most shifted axial proton resonance.
Furthermore, the most shifted axial ring proton resonance for the
(S) diastereomer always appeared to higher frequency compared
to the (R) diastereomeric peak. Therefore SSS-[Yb·L3] may be
used in the determination of absolute configuration of a-amino
acids of unknown configuration. Indeed, although there have
been a few reports of the use of lanthanide complexes as chiral
shift reagents in aqueous media for the a-amino acids,8,9 the
assignment of the absolute configuration by such reagents is not
usually reliable. Kabuto and Sasaki have reported a consistent
correlation between absolute configuration of a-amino acids and
their shift induced by a propylenediaminetetraacetato europium
complex in aqueous solution.22 However, the observed chemical
shift differences (DDd) were very small (<0.2 ppm) and the studies
were performed at pH = 9–11.
the dipolar NMR shift of paramagnetic lanthanide complexes
2
is determined by the second order crystal field parameter, B0 .
This parameter may be measured directly from the band splitting
of the DJ = 1 transition in the europium emission spectrum.26
The europium emission spectrum also provides information on
the dipolar polarisability of the ligand donors by analysis of the
hypersensitive DJ = 2 and DJ = 4 transitions. Indeed, there have
been recent reports highlighting the sensitivity of the DJ = 2
transition to the nature of the donor in the axial position10,18 and
the ratio of the integrated emission intensities of the DJ = 2/DJ =
1 spectral band is regarded as a useful parameter in assessing
changes in the europium coordination environment.27
Upon addition of a 10-fold excess of added anion to SSS-
[Eu·L3] (2.5 mM complex in 0.1 M MOPS buffer, pH 7.4, kex
=
397 nm, 295 K) two distinct trends in the emission spectrum
were apparent (Fig. 3). The magnitude of splitting observed in
the DJ = 1 band appeared to be highly dependant upon the
nature of the axial donor (Table 4), with the more polarisable
donors giving rise to the largest band splittings. The band
splitting was found to correlate well with the most shifted axial
ring proton resonance observed in the corresponding SSS-[Yb·L3]
Luminescence and circular dichroism studies
Optical properties along with NMR spectral characteristics of
the lanthanides are determined by the nature and local symmetry
of the coordination environment.23,24 Lanthanide luminescence,
hydration state studies, circular dichroism and circularly polarised
emission have all been reported to successfully signal anion
Fig. 3 Variation in the emission spectrum of SSS-[Eu·L3] (2.5 mM
complex in 0.1 M MOPS, pH 7.4, 295 K) following the addition of a
ten-fold excess of added anion.
binding at coordinatively unsaturated lanthanide complexes.10,11
A
Table 3 1H NMR data for SSS-[Yb·L3] in the presence of 10 equivalents of racemic a-amino acid (200 MHz, D2O, 295 K, pD 7.4)
Ratio of (R) : (S)
diastereomers
Most shifted dHax for
(R)-diastereomer (ppm)
Most shifted dHax for
(S)-diastereomer (ppm)
a-Amino acid
DDd (ppm)
Alanine
Serine
Threonine
Methionine
Glutamic acid
Lysinea
1 : 1
2.2 : 1
2 : 1
1.7 : 1
2.6 : 1
1 : 1
+60
+59
+61
+64
+62
+62
+68
+65
+68
+67
+70
+68
+66
+72
5
9
6
6
6
4
4
Phenylalanine
2 : 1
a The presence of a minor species was also apparent (<7%).
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Table 4 Effect of the polarisability of the axial donor on the 1H NMR shift of SSS-[Yb·L3], emission properties of SSS-[Eu·L3] and CD spectral
properties of [Yb·L3]
Anion
Most shifted dHax (ppm)
DJ = 1 splitting/104 cm−1
DJ = 2/DJ = 1 band intensity ratio
CD wavelengtha/nm
(S)-OP-serine
Phosphate
Water
(S)-Lactate
Acetate
(S)-Alanine
Oxalate
Carbonate
110
101
100
98
94
64
286
222
286
267
308
—
1.4
1.5
1.5
2.2
2.3
—
990
991
989
988
987
986
985
983
56
42
667
800
3.1
3.1
a Wavelength refers to the peak of the intense band centred around 990 nm.
ternary complexes (Fig. 4), indicating the second order crystal
field coefficient, which largely determines the pseudo-contact shift
in the paramagnetic complexes is primarily affected by the ligand
field in both Eu and Yb complexes. The intensity of the magnetic
dipole allowed DJ = 1 transition is relatively independent of the
coordination environment whereas the DJ = 2 transition, being
predominantly electric dipole in character, is highly sensitive to
the ligand field, in particular the nature and polarisability of
the axial donor. Therefore analysis of the integrated emission
intensities for the DJ = 2/DJ = 1 spectral bands for SSS-
[Eu·L3] will provide a direct measure of the polarisability of the
axial donor. The more polarisable donors (e.g. carbonate) were
found to give rise to the largest band intensity ratios (Table 4)
and a linear correlation between the observed chemical shift of
the most shifted axial ring proton in the corresponding SSS-
[Yb·L3] anion-adducts and the europium band intensity ratios
was observed. Therefore anion binding to heptadentate lanthanide
complexes is effectively signalled not only through observation of
the induced paramagnetic shift but also through characteristics in
the europium emission spectrum.
observed for each of the (R) and (S) diastereomeric adducts, the
emission studies therefore appear to be unsuitable in distinguishing
between such diastereomers.
Circular dichroism. The local coordination environment about
an ytterbium centre may be probed through near-IR CD. Ytter-
bium exhibits sensitive CD bands centred around 980 nm associ-
2
ated with the magnetic-dipole allowed 2F7/2 → F5/2 transitions.17,28
Although spectral analysis is not straightforward due to the large
number of sub-levels present, there appears to be a distinct trend
between the wavelength of the observed CD and the polarisability
of the axial donor.16
Near-IR CD spectra of SSS-[Yb·L3] (20 mM complex in 0.1 M
MOPS, pH 7.4, 295 K) in the presence of a ten-fold excess of added
anion revealed that the band centred around 990 nm moves to
shorter wavelength as the polarisability of the axial donor increases
(Fig. 5). Therefore, through observation of the form of the CD,
the nature of the coordinating anion may be assessed. Indeed,
the variation in the CD wavelength was found to correlate well
with the shift of the axial ring proton in the SSS-[Yb·L3] anion
adducts, suggesting the same axial ligand effect is manifest in each
parameter and is probably associated with the second order crystal
2
field coefficient, B0 , that determines characteristics of Eu emission
and NMR spectra (Table 4).
Fig. 4 Correlation between the most shifted axial ring proton for
SSS-[Yb·L3] and the DJ = 1 band splitting for SSS-[Eu·L3] in the presence
of a ten-fold excess of added anion.
Fig. 5 Near-IR CD spectra of SSS-[Yb·L3] (20 mM complex in 0.1 M
Emission spectra were then recorded for SSS-[Eu·L3] (2.5 mM
complex in 0.1 M MOPS buffer, pH 7.4, kex = 397 nm, 295 K) in
the presence of a ten-fold excess of a variety of (R) and (S) chiral
anions (e.g. lactate, amino acids, phosphorylated amino acids)
to deduce whether diastereomeric adducts may be distinguished
through emission studies. Identical spectral characteristics were
MOPS, pH 7.4, 295 K) in the presence of a ten-fold excess of added anion.
Near-IR CD spectra were then recorded for SSS-[Yb·L3]
(20 mM complex in 0.1 M MOPS, pH 7.4, 295 K) in the presence
of a ten-fold excess of a variety of (R) and (S) chiral anions (e.g.
a-hydroxy acids, a-amino acids, phosphorylated amino acids) in
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the hope that the differing chiral environment about the Yb centre
in the diastereomers may be effectively signalled through CD. As
in the case of the emission studies, although characteristic spectra
were obtained distinguishing between the different ternary anion
complexes, identical CD spectra were recorded for all the (R) and
(S) diastereomeric adducts.
over the appropriate drying agent. Water was purified by the
‘PuriteSTILLplus’ system.
Thin-layer chromatography was carried out on neutral alumina
plates (Merck Art 5550) and visualised under UV (254 nm)
or by staining with iodine. Preparative column chromatography
was carried out using neutral alumina (Merck Aluminium Oxide
90, activity II–III, 70–230 mesh), pre-soaked in ethyl acetate.
Cation and anion exchange chromatography were performed using
Dowex 50 W strong ion exchange resin and Dowex 1-X8(Cl),
respectively, pre-treated with hydrochloric acid (3 M).
Conclusions
The heptadentate lanthanide complexes of L3 are highly water
soluble and appear to exist as a single isomer in aqueous solution.
Such complexes are therefore highly desirable for potential use
as probes for bioactive species in aqueous solution. SSS-[Ln·L3]
complexes have been shown to act as effective NMR, luminescent
and chiroptical probes for the signalling of a variety of anions in
water. The nature of the axial donor has been shown to play a
prominent role in determining NMR, emission and CD spectral
characteristics. By observation of the lanthanide induced shift of
SSS-[Yb·L3], the DJ = 2/DJ = 1 band intensity ratio and DJ =
1 band splitting in the emission spectrum of SSS-[Eu·L3] and the
wavelength of the CD band centred around 990 nm for SSS-
[Yb·L3] the nature of the axial donor may be easily assessed.
As the polarisability of this axial donor increases the observed
paramagnetic NMR shift decreased, a larger DJ = 2/DJ = 1
band intensity ratio and a larger DJ = 1 splitting (cm−1) in the Eu
emission spectra was observed and the CD band centred around
990 nm moved to shorter wavelength.
Upon the addition of (R) and (S) chiral anions to SSS-
[Ln·L3] no apparent changes in the form of the Eu emission or
Yb near-IR CD spectra were observed. Therefore the signalling
of diastereomeric adducts through optical techniques is not
appropriate for such systems. However, the SSS-[Yb·L3] diastere-
omeric ternary adducts formed upon the addition of racemic a-
hydroxy acids, a-amino acids and phosphorylated amino acids
were easily distinguishable by 1H NMR and exhibited large
chemical shift non-equivalences. In fact, an apparent absence of
kinetic resolution in complex formation between SSS-[Yb·L3] and
racemic a-hydroxy acids was observed and augurs well for the
development of such systems as aqueous chiral derivatising agents.
Furthermore, the chemical shift non-equivalence (DDd ∼2–5 ppm)
and large lanthanide induced shifts exhibited by this unique chiral
derivatising agent are far superior compared to values reported
in the literature for aqueous lanthanide shift reagents which are
generally <0.1 ppm. Enantioselectivity in complex formation was
observed with SSS-[Yb·L3] in the presence of racemic a-amino
acids, which precludes its use as a chiral derivatising agent for such
species. However, the (S)-amino acid adducts always experienced
a greater lanthanide induced shift compared to the (R)-adducts,
therefore such complexes may be effectively used to determine
the absolute configuration of a-amino acids in aqueous media at
physiological pH.
Infrared spectra were recorded on a Graseby-Specac “Golden
Gate” Diamond ATR accessory spectrometer. Melting points were
recorded using a Ko¨fler block and are uncorrected.
1H and 13C NMR spectra were recorded on a Varian Mercury-
200 (1H at 199.975 MHz, 13C at 50.289 MHz), Varian Unity 300
(1H at 299.908 MHz, 13C at 75.412 MHz) or a Varian Inova
500 (1H at 499.7 MHz, 13C at 125.67 MHz) spectrometer. Two-
dimensional spectra were carried out on a Varian Inova 500
spectrometer. Spectra were referenced internally relative to tert-
butanol for paramagnetic complexes or to the residual protio-
solvent resonances which are reported relative to TMS. All
chemical shifts are given in ppm and coupling constants are in
Hz. 1H NMR spectra for the anion ternary adducts were obtained
by adding an excess (∼10 equivalents) of the anion, as a solid, to
a solution of the complex (10 mM in D2O). The pD was carefully
adjusted to 7.5 ( 0.4) using sodium deuteroxide.29
Electrospray mass spectra were recorded on a VG Platform II
(Fisons instrument), operating in positive or negative ion mode
as stated, with methanol as the carrier solvent. Accurate mass
spectra were recorded at the EPSRC Mass Spectrometry Service
at the University of Wales, Swansea.
Corrected emission spectra were recorded on a S.A. Fluorolog
3, operating with DataMax software using quartz fluorescence
cuvettes of path length 1 cm, a 420 nm cutoff filter and an excitation
wavelength of 397 nm (Eu). Eu3+ spectra were recorded between
570 nm and 730 nm, at 0.5 nm increments with 1 s integration
time. Excitation and emission slits were 10 and 1 nm respectively.
Data analysis was performed using an iterative least-squares fitting
procedure, assuming a 1 : 1 binding stoichiometry, operating in
Microsoft Excel. Statistical errors were estimated to be <10%,
and experimental errors were within 20%. Emission spectra in the
presence of added anions were obtained by adding an excess of
the anion (10 equivalents), as a solid, to a solution of the complex
(0.50 ml, 2.5 mM) prepared in MOPS (0.1 M, pH 7.4) buffer.
Near-IR CD spectra were recorded in four successive scans
using a Jasco J-810 spectropolarimeter at 295 K using a 1 cm path
length and slits of 60 lm. To the complex solution (20 mM in
0.1 M MOPS buffer, pH 7.4) was added an excess (>10 equiv.)
of the anion as a solid. Spectra were recorded immediately and
showed no change in form 24 h later.
Ligand synthesis
Experimental
2-Chloro-N-[(S)[1-methoxycarbonyl-3-methyl] butyl] ethana-
mide. (S)-Leucinemethylester hydrochloride (3 g, 16.5 mmol)
was dissolved in dry diethyl ether (100 ml) under argon and
triethylamine (5.52 ml, 39.6 mmol) was added. The solution was
cooled to −20 ◦C (acetone, CO2 (s) bath) and chloroacetyl chloride
(1.58 ml, 19.8 mmol) in dry diethyl ether (60 ml) was added
General procedures and characterization techniques
Reactions requiring anhydrous or inert conditions were carried
out using Schlenk-line techniques under an atmosphere of dry
argon. Anhydrous solvents when required were freshly distilled
3094 | Dalton Trans., 2006, 3088–3096
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dropwise, maintaining the temperature at −20 ◦C. The reaction
mixture was allowed to warm to room temperature and stirred
for 1 h. The resulting white precipitate was dissolved in water (60
ml) and the organic layer washed with hydrochloric acid (0.1 M,
80 ml) and water (3 × 80 ml), dried (K2CO3) and the solvent
was removed in vacuo to give a brown oil (3.21 g, 88%); mmax
(film)/cm−1 3304 (NH), 1745 (COO), 1680 (CONH); dH (200 MHz,
CDCl3) 6.90 (1H, br s, NH), 4.68–4.56 (1H, br m, CHNH), 4.04
(2H, s, ClCH2CO), 3.72 (3H, s, OCH3), 1.69–1.50 (3H, br m,
CDCl3) 173.8 (CO2Me), 173.4 (CO2Me), 171.6 (CONH), 171.5
(CONH), 59.6 (br, NCH2CO), 53.8–52.3 (br, ring-CH2, OCH3),
50.7 (CHNH), 47 (ring-CH2), 41.3 (CH2CH), 25.1 (CH2CH),
23.0 (CH3), 21.1 (CH3); m/z (ES+) 384 (100%, MCa2+), 728 (15%,
MH+), 766 (5%, MK+) (Found: MH+, 728.4878. C35H66N7O9
requires MH+, 728.4922).
Complex synthesis
Yb·L2. Ytterbium(III) triflate (0.44 g, 0.72 mmol) in dry
acetonitrile (1 ml) was added to a solution of L2 (0.512 g, 0.72
mmol) in dry acetonitrile (4 ml) and the mixture was heated to
reflux under argon overnight. The reaction solution was dropped
onto stirring diethyl ether (50 ml) and the resulting yellow
precipitate was collected by centrifugation and filtration. The
solid was redissolved in the minimum amount of acetonitrile and
the precipitation procedure repeated once more. A yellow solid
resulted (0.70 g, 75%), mp 142–144 ◦C; mmax (solid)/cm−1 1742
(COO), 1627 (CONH); dH (200 MHz, CD3OD) partial assignment
119.5 (1H, br s, ring-Hax), 76.4 (1H, br s, ring-Hax), 67.8 (1H,
br, ring-Hax), 53.9 (1H, br, ring-Hax), 43.0 (1H, br s, ring-Heq),
35.8 (1H, br s, ring-Heq), 31.9 (1H, br s, ring-Heq), 27.0 (1H, br,
ring-Heq), 12.1, −17.5, −19.1, −29.7, −33.8, −36.3, −41.1, −42.8,
−48.5, −78.1, −91.0; m/z (ES+) 450 (100%, M2+) (Found: M2+,
450.2109. C35H64N7O9Yb requires M2+ 450.2077).
3
CH2CH), 0.92 (6H, d, J = 5.8, CH3); dC (50.29 MHz, CDCl3)
172.8 (CO2Me), 166.6 (CONH), 52.2 (OCH3), 51.0 (CHNH),
42.4 (ClCH2CO), 40.7 (CH2CH), 24.7 (CH2CH), 22.7 (CH3), 21.6
(CH3); m/z (ES+) 244 (100%, MNa+) (Found: MNa+, 244.0739.
C9H16NO3NaCl requires MNa+, 244.0716).
1-Benzyl-4,7,10-tris-[(S)-((1-methoxycarbonyl-3-methyl)butyl)-
carbamoylmethyl]-1,4,7,10-tetraazacyclododecane. 2-Chloro-N-
[(S)[1-methoxycarbonyl-3-methyl] butyl] ethanamide (1.33 g,
6.00 mmol) in dry N,N-dimethylformamide (5 ml) was added to
a stirred solution of 1-benzyl-1,4,7,10-tetraazacyclododecane13
(0.526 g, 2.00 mmol) and fine mesh anhydrous potassium
carbonate (0.830 g, 6.00 mmol) in dry N,N-dimethylformamide
(25 ml). The reaction mixture was heated at 60 ◦C under an argon
atmosphere for 36 h. The solvent was distilled off under vacuum
and the resulting brown oil was extracted into dichloromethane
(40 ml), washed with purite water (3 × 40 ml), brine (40 ml),
dried (K2CO3) and concentrated to dryness. The mixture was
purified by alumina column chromatography (gradient elution
from dichloromethane to 0.5% methanol–dichloromethane) and
the product was isolated as a brown oil (0.821 g, 50%); Rf (Al2O3;
10% CH3OH–CH2Cl2; I2 and UV detection) 0.38; mmax (film)/cm−1
1741 (COO), 1659 (CONH); dH (200 MHz, CDCl3) 8.0–7.8 (3H,
br s, NHCO), 7.3–7.1 (5H, m, Ar), 4.5 (3H, m, CHNH), 3.70–2.40
(33H, br m, ring-CH2, CH2CO, OCH3, CH2Ph), 1.58–1.40 (9H,
br m, CH2CH, CH2CH), 0.87 (18H, m, CH3); dC (50.29 MHz,
CDCl3) 173.4 (CO2Me), 173.2 (CO2Me), 171.3 (CONH), 170.9
(CONH), 138.0 (ipso-Ar), 129.0, 128.5, 127.4 (C–Ar), 59.9–59.0
(br, NCH2Ph, NCH2CO), 53.4–52.3 (br, ring-CH2, OCH3), 50.5
(CHNH), 41.3 (CH2CH), 25.1 (CH2CH), 23.0 (CH3), 22.1 (CH3);
m/z (ES+) 429 (100%, MCa2+), 840 (30%, MNa+), 440 (20%,
MK+Na+) (Found: (MHK)2+, 428.7514. C42H72N7O9K requires
(MHK)2+, 428.7542).
Eu·L2. The title compound was prepared following a method
similar to Yb·L2, using europium(III) triflate (0.37 g, 0.62 mmol)
in dry acetonitrile (3 ml) and L2 (0.450 g, 0.62 mmol) in dry
acetonitrile (3 ml). A yellow solid resulted (0.713 g, 87%), mp >
178 ◦C (decomp.); mmax (solid)/cm−1 1742 (COO), 1627 (CONH);
dH (200 MHz, CD3OD) partial assignment 24.8 (1H, br s, ring-
Hax), 16.0 (1H, br s, ring-Hax), 13.5 (1H, br s, ring-Hax), 9.28
(1H, br s, ring-Heq), −1.66, −4.83, −6.40, −7.33, −9.43, −11.6,
−13.6, −14.9, −19.0; m/z (ES+) 440 (100%, M2+), 514 (40%,
[M3+ + (CF3SO3−)]2+) (Found: [M3+ + (CF3SO3−)2]+, 1178.3163.
C37H65N7O15F6S2Eu requires [M3+ + (CF3SO3−)2]+ 1178.3097).
Yb·L3. Yb·L2 (0.25 g, 0.18 mmol) was dissolved in the
minimum amount of methanol and treated with an aqueous
sodium hydroxide solution (0.02 M, 37 ml). The solution was
brought to pH 6, reduced to small volume and loaded onto a
cationic exchange column (Dowex, 50 WH+), eluting with water
and then aqueous ammonia solution (6%). The ammonia layer
was dried giving the product as a yellow solid (0.080 g, 50%), mp
> 250 ◦C; mmax (solid)/cm−1 1625 (br, CONH, COO); dH (200 MHz,
CD3OD) partial assignment 105.6 (1H, br s, ring-Hax), 99.1 (1H,
br s, ring-Hax), 79.0 (1H, br s, ring-Hax), 75.0 (1H, br s, ring-Hax),
43.4 (1H, br s, ring-Heq), 27.0 (3H, br s, ring-Heq), 21.7, 18.7, 10.4,
8.15, −17.0, −22.8, −24.3, −33.0, −36.3, −46.7, −51.9, −54.3,
−58.5, −61.7, −121.3; m/z (ES+) after addition of CF3COOH:
429 (100%, [M + 2H+]2+) (Found: MH+, 857.3681. C32H57N7O9Yb
requires MH+ 857.3607).
1,4,7-Tris-[(S)-((1-methoxycarbonyl-3-methyl)butyl)carbamoyl-
methyl]-1,4,7,10-tetraazacyclododecane L2. A hydrochloric acid
solution (1 M, 1 ml) and a catalytic amount of palladium
hydroxide on carbon were added to 1-benzyl-4,7,10-tris-[(S)-
((1-methoxycarbonyl-3-methyl)butyl)carbamoylmethyl]-1,4,7,10-
tetraazacyclododecane (1.07 g, 1.31 mmol) in ethanol (45 ml)
and the mixture was treated with hydrogen (45 psi) at room
temperature for 48 h. The reaction mixture was filtered through
Celite and the solvent was removed under vacuum. The residue
was taken into dichloromethane (30 ml) and washed with a
solution of sodium bicarbonate (30 ml) and brine (1 × 30 ml),
dried (K2CO3) and concentrated to dryness to give the product
as a yellow-orange glassy solid (0.77 g, 81%), mp 120–122 ◦C;
mmax (solid)/cm−1 1739 (COO), 1665 (CONH); dH (200 MHz,
CDCl3) 8.0–7.84.(3H, br s, NHCO), 80–2.55 (35H, br m, ring-
CH2, CH2CO, OCH3, ring-NH, CHNH), 2.00–1.20 (9H, br m,
CH2CH, CH2CH), 1.00–0.87 (18H, br, CH3); dC (50.29 MHz,
Eu·L3. The title compound was prepared following a method
similar to that for Yb·L3, using Eu·L2 (0.500 g, 0.37 mmol),
dissolved in the minimum amount of methanol and an aqueous
sodium hydroxide solution (0.02 M, 75 ml). A yellow solid resulted
(0.028 g, 50%), mp > 250 ◦C; mmax (solid)/cm−1 1625 (br, CONH,
COO); dH (200 MHz, CD3OD) partial assignment 25.7 (1H, br s,
ring-Hax), 23.4 (1H, br s, ring-Hax), 18.0 (1H, br s, ring-Hax), 17.6
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(1H, br s, ring-Hax), 11.03, −1.12, −2.34, −2.78, −3.32, −5.13,
−5.37, −6.89, −7.28, −8.84, −9.33, −11.1, −12.8, −14.6, −15.43;
m/z (ES+) after addition of CF3COOH: 418 (100%, [M + 2H+]2+)
(Found: MH+, 836.3474 C32H57N7O9Eu requires MH+ 836.3430).
11 R. S. Dickins, A. S. Batsanov, D. Parker, H. Puschmann, S. Salamano
and J. A. K. Howard, Dalton Trans., 2004, 70.
12 R. S. Dickins, C. S. Love and H. Puschmann, Chem. Commun., 2001,
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13 K. P. Pulukkody, T. J. Norman, D. Parker, L. Royle and C. J. Broan,
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Acknowledgements
We thank EPSRC and the Royal Society for a Dorothy Hodgkin
Fellowship (RSD) for support.
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3096 | Dalton Trans., 2006, 3088–3096
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