Enhanced selectivity for Mg2+ with a phosphinate-based chelate: APDAP: versus APTRA

Article abstract of DOI:10.1039/c7dt04698g

o-Aminophenol-N,N,O-triacetate, known as APTRA, is one of the most well-established ligands for targeting magnesium ions but, like other aminocarboxylate ligands, it binds Ca²⁺ much more strongly than Mg²⁺. The synthesis of an O-phosphinate analogue of APTRA is reported here, namely o-aminophenol-N,N-diacetate-O-methylene-methylphosphinate, referred to as APDAP. Metal binding studies monitored using UV-visible spectroscopy show that the affinity of APDAP for Ca²⁺ is reduced by over two orders of magnitude compared to APTRA, and for Zn²⁺ by over three orders of magnitude, whereas the affinity for Mg²⁺ is attenuated to a much lesser extent, by a factor of only about 7. The selectivity towards Mg²⁺ is thus substantially improved. DFT calculations support the notion that longer P-O and P-C bonds in APDAP (compared to corresponding C-O and C-C bonds in APTRA) favour a larger angle at the metal, an effect that is less unfavourable for smaller ions like Mg²⁺ than for larger ions such as Ca²⁺. Derivatives of APDAP can be anticipated that will offer improved sensing of Mg²⁺ in the presence of Ca²⁺, in the physiologically important millimolar concentration range.

Full text of DOI:10.1039/c7dt04698g

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PAPER
2
+
Enhanced selectivity for Mg with a phosphinate-
based chelate: APDAP versus APTRA†
Cite this: DOI: 10.1039/c7dt04698g
Edward R. H. Walter, Mark A. Fox,
David Parker * and J. A. Gareth Williams
*
o-Aminophenol-N,N,O-triacetate, known as APTRA, is one of the most well-established ligands for tar-
2
+
geting magnesium ions but, like other aminocarboxylate ligands, it binds Ca much more strongly than
2
+
Mg . The synthesis of an O-phosphinate analogue of APTRA is reported here, namely o-aminophenol-
N,N-diacetate-O-methylene-methylphosphinate, referred to as APDAP. Metal binding studies monitored
2
+
using UV-visible spectroscopy show that the affinity of APDAP for Ca is reduced by over two orders of
2+
magnitude compared to APTRA, and for Zn by over three orders of magnitude, whereas the affinity for
2
+
2+
Mg is attenuated to a much lesser extent, by a factor of only about 7. The selectivity towards Mg is
thus substantially improved. DFT calculations support the notion that longer P–O and P–C bonds in
APDAP (compared to corresponding C–O and C–C bonds in APTRA) favour a larger angle at the metal,
Received 12th December 2017,
Accepted 5th January 2018
2+
2+
an effect that is less unfavourable for smaller ions like Mg than for larger ions such as Ca . Derivatives
DOI: 10.1039/c7dt04698g
2+
2+
of APDAP can be anticipated that will offer improved sensing of Mg in the presence of Ca , in the
rsc.li/dalton
physiologically important millimolar concentration range.
lower-denticity cousin of BAPTA – potentially pentadentate
rather than octadentate – and features three as opposed to two
Introduction
Amino carboxylates are pre-eminent amongst ligands used to carboxylates per aminophenol unit. Available fluorescent indi-
2
+
bind to divalent group 2 metals and hard transition metal ions cators marketed for Mg are typically based on the APTRA
3
+
2+ 1,2
such as Fe and Mn . EDTA is produced on a huge scale
for sequestration and regulation of metal ions in consumer
3
products and inhibition of bacterial growth. Related amino-
phenolate-based ligands such as BAPTA {1,2-bis(O-amino-
phenoxy)-ethane-N,N,N′,N′-tetra-acetate, Fig. 1} were developed
2
+
in the 1980s by Tsien for the selective binding of Ca : the
2
+
4
octadenticity favours coordination of the large Ca ion.
A
number of fluorescent analogues of BAPTA were introduced
through the incorporation of a fluorescent aromatic in place
of, or attached to, one of the aminophenol units (e.g., Fura-2
5
–7
and Indo-1).
In conjunction with fluorescence microscopy,
they have been successfully used for the real-time monitoring
2
+
of Ca
concentrations in cell biology, physiology and
8
neurology.
2+
The smaller Mg ion, on the other hand, has received far less
attention – and much less success in terms of efforts to design
ligands that bind it with high selectivity. Perhaps the most
well-known examples to date are based on the APTRA structure
(
APTRA = o-aminophenol-N,N,O-triacetate, Fig. 1) introduced
9
by London and co-workers in the late 1980s. APTRA is a
Department of Chemistry, Durham University, Durham, DH1 3LE, UK.
E-mail: david.parker@durham.ac.uk, j.a.g.williams@durham.ac.uk
Fig. 1 The structures of selected amino carboxylate ligands: the ubiqui-
2+
tous broad-spectrum ligand EDTA, the Ca -selective ligand BAPTA, the
2+
†
Electronic supplementary information (ESI) available. See DOI: 10.1039/
APTRA ligand for Mg binding, and Mag-Fura-2, a fluorescent derivative
c7dt04698g
of APTRA.
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2
+
unit connected to, or incorporating, a fluorescent aromatic to reduce the affinity for Ca and thus enhance selectivity for
2
+
2+
reporter group, e.g., Mag-Fura-2 (Fig. 1) and related Mg over Ca . The hypothesis has been borne out by the data
6
,10
derivatives.
Yet, such ligands are not selective for Mg . Although their
described below.
2
+
2
+
Synthesis
affinity for Ca is reduced compared to BAPTA, they still retain
a higher affinity for Ca than for Mg (micromolar versus The synthesis of APDAP was achieved using the two sequences
millimolar) and indeed can be employed as Ca indicators to of reactions shown in Scheme 1. The first sequence was used
2
+
2+
2
+
respond over higher concentration ranges than the standard to prepare the phosphinate-containing alkylating agent 4.
2
+
Ca probes. Such selectivity issues apply equally to more Hydrolysis of diethyl methylphosphinate 1 with one equivalent
recently described APTRA-based systems designed to offer of water led to ethyl methylphosphinate 2. Condensation of
1
1
longer-wavelength excitation and emission. Meanwhile, the 2 with paraformaldehyde gave alcohol 3, which was sub-
use of bidentate β-keto acids, a quite different strategy explored sequently converted to its mesylate ester 4 in quantitative
2
+
12,13
for Mg binding,
suffers from other limitations, notably yield, upon treatment with mesyl chloride in the presence of
the fact that the low denticity favours the formation of mixed triethylamine as a base. Owing to the greater nucleophilicity of
ligand species in vivo, in which the coordination sphere of the amines compared to phenols, O-alkylation of o-aminophenol
metal ion may be completed by endogenous molecules such as would require prior protection of the amine. We chose instead
1
4
ATP.
to start from o-nitrophenol, 5, which was O-alkylated using 4
2
+
Nevertheless, Mg remains a highly interesting target bio- under basic conditions to give 6; completion of the reaction
1
5
medically. It is the second most abundant divalent cation in was visually evident from the loss of the bright orange colour
1
6
cells, it plays a key role in the stabilisation of DNA struc- associated with the 2-nitrophenoxide ion. Following chromato-
1
7
ture, and it is implicated in hundreds of enzymatic reac- graphic purification, reduction of the nitro group using
1
8
19
20
tions. A number of cardiovascular, neurodegenerative
and renal diseases have associated with them a mis-regu- O-alkylated aniline 7, which was then bis-N-alkylated using
lation of Mg concentrations. Greater effort is clearly required ethyl bromoacetate in the presence of base and catalytic NaI,
on the development of new classes of chelate ligand for selec- leading to 8 over a period of 4 days. Finally, hydrolysis of the
tive binding of Mg , that can be used in the presence of com- two carboxylate esters and the phosphinate ester of 8 was
petitor ions such as Ca and Zn , yet function in the physio- accomplished using sodium deuteroxide in deuterated metha-
logically important millimolar range: the concentration of nol/water mixture to give APDAP. The use of deuterated con-
free” Mg is thought to be around 1 mM in the majority of ditions allows the progress of hydrolysis to be readily moni-
hydrogen gas, catalysed by palladium on carbon, gave the
2
1
2
+
2
+
2
+
2+
2
+
“
1
31
31
mammalian cells.
tored by H and P NMR spectroscopy: the P resonance
shifts from 48.3 to 36.5 ppm on hydrolysis. The purity of the
final product was assessed by analytical HPLC using aceto-
nitrile/ammonium bicarbonate as the eluent.
Results and discussion
Strategy
Absorption spectroscopy: effect of pH
Here we describe the synthesis and metal binding properties We employed UV absorption spectroscopy as the method for
2
+
of a new ligand for Mg , namely o-aminophenol-N,N-diace- assessing the metal-binding properties of the new APDAP
tate-O-methylene-methylphosphinate, which shall be referred ligand, which allows a direct comparison to be made with
2
2
to as APDAP. The APDAP ligand is structurally similar to studies of APTRA. Evidently, fluorescence spectroscopy offers
−
APTRA, but features a phosphinate group, –OCH
place of the phenolate-bound carboxylate group, –OCH
Fig. 2). In the binding of divalent metal ions, pentadenticity require short-wavelength excitation that is impracticable for
2 2
P(Me)O , in greater sensitivity and diversity of applications, but APDAP
−
2
CO
2
itself – like APTRA – is only weakly fluorescent and would
(
can thus be maintained with formation of four 5-membered most applications. The conjugation of the APDAP ligand to a
chelate rings, but it was anticipated that the change from a fluorescent reporter unit can readily be envisaged to prepare
C–O to a P–O bond within one of the chelate rings would serve fluorescent derivatives.
The absorption spectrum of APDAP in aqueous solution at
pH = 8.5 shows one main band centred at 254 nm (ε = 4480
−
1
−1
M
cm ) accompanied by a distinct shoulder at 285 nm. The
variation of the spectrum with pH was monitored over the pH
range 4.5–8.5. Acidification leads to a decrease in the absor-
bance across all wavelengths, particularly the main band at
2
54 nm: the spectrum under acidic conditions features a
broad band with a rather ill-defined maximum around 260 nm
Fig. 3). Such changes are consistent with those expected
(
upon protonation of the electron-donating amino group.
The sigmoidal fit of the absorbance at 254 nm against pH gave
Fig. 2 The structure of the new phosphinate ligand APDAP shown
alongside the tricarboxylate APTRA.
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Scheme 1 Synthesis of (a) the alkylating agent 4 and (b) APDAP from o-nitrophenol.
a ground-state pK of 6.03(8) (Fig. 3, inset). This suggests that,
a
over the normal physiological pH range (pH 6.5–7.5), the
amine will be largely unprotonated and therefore available for
metal ion binding, together with the less basic carboxylate and
phosphinate oxygen atoms. It is encouraging that there is little
change in the absorbance with pH over the above physiological
range: metal ion probes that are targeted for use in biomedical
areas should ideally not be affected by pH under the prevailing
conditions, otherwise an observed response cannot be unequivo-
cally attributed to metal ion binding but rather may arise from
variation in pH. Similarly, competitive protonation leading to
decomplexation of the target metal ion once bound needs to
2
+
be avoided. A Mg -saturated solution of APDAP showed very
Fig. 3 Absorption spectrum of APDAP in aqueous solution at varying little variation with pH in its UV-visible spectrum (Fig. 3,
pH values between 8.5 and 4.5; concentration = 50 μM, in the presence
of 100 mM KCl, T = 298 ± 3 K. The inset shows the sigmoidal fit (red
inset).
In the case of APTRA, the pK has not been accurately deter-
mined in the majority of its derivatives, owing to it being
a
line) to the absorbance at 254 nm against pH (black diamond data
points). The blue circles in the inset show the change in the absorption
2+
somewhat unstable in solution through decarboxylation and
at 254 nm with pH after saturation with Mg (100 mM), with a trendline
in black.
2
3
oxidation reactions; a pK value of 5.5 was determined for
a
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2
4
one APTRA derivative by absorption spectroscopy.
Table 1 Comparison of the metal binding affinities of APDAP and
APTRA , obtained via absorption spectroscopy in aqueous solution
a,b
Magnesium-bound APTRA has similarly been found to be
largely pH-insensitive.
APDAP
APTRA
2
+
Absorption spectroscopy: effect of divalent metal ions Mg ,
Ca and Zn
pK
a
6.03(8)
79(5)
12.7(8) mM
9.30(5) × 10
1.08(5) mM
6(1) × 10
17(4) μM
5.5
K (Mg )/M−1
2
+
560(30)
1.79 mM
1.02(7) × 10
2
+
2+
2+
K
d
(Mg
)
2
+
−1
2
5
K (Ca )/M
The effect of increasing concentrations of the above metal ions
on the absorption spectrum was investigated in aqueous solu-
tion, buffered to pH = 7.21 using HEPES, at a temperature of Kd (Zn2
2
+
K
d
(Ca
)
9.80 μM 7
2+
−1
4
K (Zn )/M
7(1) × 10
+
)
14 nM
2
98 ± 1 K. The evolution of the spectra, together with the
a
b
pK
a
value of APTRA is from ref. 24. Metal-binding data for APTRA
corresponding fits to estimate binding constants, are shown in
Fig. 4. The standard deviations indicated were obtained from
three independent sets of measurements in each case. The
binding affinities of APDAP for each metal ion are compared
with those previously reported for APTRA in Table 1.
For each of the three metal ions investigated, it can be seen
that the absorbance of the main bands progressively decreased
with increasing concentration of the metal. However, the
change in the spectral profile is rather different. In the case of
Ca and Zn , there is a large decrease in absorbance of the
main band at 254 nm and of the long-wavelength shoulder,
whilst a new band starts to emerge at about 273 nm (with an
additional high-energy band at about 237 nm for Ca ). In con-
trast, the spectral profile does not change substantially upon
are those of Buccella and co-workers, ref. 22.
2+
2+
2+
addition of Mg . The changes with Ca and Zn are more
similar to the effect of protonation (Fig. 3) than those induced
by Mg . One may tentatively conclude, therefore, that the
amine plays a much less significant role in the binding of
Mg than Ca or Zn : it is possible that the APDAP binds
Mg in a quasi-tetradentate O4 coordination mode, without
much participation of the nitrogen lone pair, but for the larger
Ca ion and for the more polarisable Zn ion it is penta-
2+
2+
2+
2+
2+
2
+
2+
2+
2+
dentate O N (Fig. 5).
4
2
+
Inspection of the binding constant data in Table 1 reveals
some intriguing differences between APDAP and APTRA. The
binding constant of APDAP for Mg is 79(5) M which is
reduced by a factor of approximately 7 compared to that of
APTRA. In contrast, the affinity for Ca is lowered by a factor
of 110. Similarly, the affinity for Zn ions plummets by a
factor of >10 . Clearly, then, the selectivity for Ca or Zn
over Mg is reduced on going from the carboxylate to the
phosphinate, as desired. Although the new ligand does not
show the required selectivity profile for Mg , competition
from Ca and Zn will clearly be lowered. Pentadentate
ligands showing sensitivity in the millimolar range for both
2
+
−1
2
2
2+
2
+
3
2+
2+
2
+
2
+
2
+
2+
2
+
2+
Mg and Ca have not previously been reported. The biden-
tate β-keto acids mentioned earlier do show such a range, but
do not offer the high denticities necessary to avoid formation
of mixed ligand species.
APDAP versus APTRA: a theoretical evaluation
For possible insight into the differing effects of the change
2
+
2+
from carboxylate to phosphinate on Ca and Mg binding, a
Fig. 4 Absorption spectra (left) and binding curves with associated fits
2+
2+
2+
in red (right) for the addition of (A) Mg , (B) Ca , and (C) Zn to
APDAP in aqueous solution. The metal chloride salts were used in each
case; [APDAP] = 50 μM, [HEPES] = 50 mM, [KCl] = 100 mM, pH = 7.21,
T = 298 ± 3 K. The reported binding constants are an average from three
separate metal ion titrations, with the standard deviation in parenthesis.
Fig. 5 Plausible coordination modes for APDAP with divalent metal
2+
ions: the tetradentate mode (B) may be closer to that adopted by Mg
,
2+
2+
with the pentadentate (A) for Ca and Zn
.
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2 2
series of DFT calculations was carried out for complexes of the [Mg(APTRA)(H O)] . Comparing [Zn(APTRA)(H
O)]− and
−
−
two ligands. Details of the functionals and basis sets employed [Zn(APDAP)(H O)] , on the other hand, shows little difference
2
are given in the Experimental section. Crystal structures of the between them in M–L bond lengths. The Mg–L lengths are
2
+
2+
Mg and Zn complexes of APTRA have been reported by mostly shorter than corresponding Zn–L lengths, for both
Buccella and co-workers, in which the metal ions are O N- APTRA and APDAP. The shorter Mg–N compared to Zn–N
4
coordinated by the pentadentate ligand, with the sixth coordi- bond length that is calculated may appear somewhat inconsist-
nation site occupied by a bridging carboxylate from a neigh- ent with the conclusions from UV-visible spectroscopy, sum-
2
2
bouring complex. For simplicity in the calculations, and as a marised in Fig. 5, that suggest a stronger interaction of the
2
+
2+
more likely parallel of behaviour in solution, the DFT calcu- nitrogen lone pair with Zn than with Mg . However, the UV-
lations were run with a water molecule occupying the sixth visible data are obtained in aqueous solution, where an H
site. The optimised structures obtained in this way for the molecule or an OH group could occupy the sixth coordination
2
O
2
+
2+
2+
Mg , Ca and Zn complexes are shown in Fig. S1 of the site in place of the amine nitrogen atom. The DFT calculations
ESI.† There is a good agreement between the calculated M–L do not allow for this possibility, being based on the solid-state
bond lengths and those determined crystallographically by structure. Some caution must therefore be exercised in using
2
+
2+
Buccella and co-workers for the Mg and Zn complexes of the theoretical data, with regard to the spectroscopic changes.
APTRA (a comparison is provided in Table S1†). This offers Table 3 summarises the bite angles around the metal for
confidence in the approach, in the absence of crystal struc- the four complexes. Here again, it is evident that [Mg(APDAP)
−
2
tures of the APDAP complexes. Ideally, the calculation of rela- (H O)] differs from the other three: the bite angles are largest
tive metal binding energies would be helpful, but such a task for this complex. Such a trend is consistent with longer P–C
is hampered by the likely increase in coordination number of and P–O bonds compared to corresponding C–C and C–O
2
+
Ca beyond 6, probably up to 8, through coordination of bonds (Table S2†), but the effect is clearly more significant for
2
+
2+
additional water molecules. Multiple anions of the form the Mg complex than for the Zn complex (e.g., ArO–M–O
−
[
Ca(APTRA)(H O) ] are likely in solution. We focused instead angles of 83.9° and 78.6° respectively).
2 n
on differences in key bond lengths and angles in the structures
2
+
2+
Discussion: the effect of structural changes in the chelate ring
on metal ion selectivity
of the Mg compared to the Zn complexes, which are both
expected to be 6-coordinate and which feature metal ions of
very similar ionic radius.
It is well established that larger metal ions will tend to suffer a
From the calculated bond lengths collated in Table 2, it can larger reduction in complex stability with increasing chelate
1
,25
2+
be seen that, for APTRA, the M–L bond lengths are very similar ring size than smaller cations.
Larger cations, such as Ca ,
for the Mg and Zn complexes. In contrast, for APDAP, there will favour the formation of 5-membered chelate rings over
2
+
2+
2
+
2+
are significant differences between the Mg and Zn com- 6-membered, with an ideal bite angle of 69°. Smaller cations
plexes for most of the M–L bonds. In general, M–L bond are more amenable to the formation of 6-membered chelates.
−
lengths are shorter in [Mg(APDAP)(H
2
O)] compared to A comparison between the bite angle in the O-carboxylate and
2+
2+
2
Table 2 Calculated M–L bond lengths for the Mg and Zn complexes of APTRA and APDAP, bound in a pentadentate manner, with one H O
molecule completing the coordination sphere
Bond length/Å
[Mg(APDAP)]−
[Mg(APTRA)]−
[Zn(APDAP)]−
[Zn(APTRA)]−
M–N
M–OAr
M–O(CO
M–O(PO
M–O(CO
M–O(CO
2.233
2.088
n/a
1.966
2.056
1.971
2.093
2.336
2.161
2.037
n/a
2.033
2.028
2.135
2.366
2.350
n/a
1.991
2.022
2.020
2.302
2.382
2.251
2.037
n/a
2.019
2.013
2.258
2
CH
MeCH
2
O)
2
2
O)
2
CH
CH
2
N)
N)
2
2
2
M–O(H O)
2+
2+
2
Table 3 Calculated L^M^L bite angles for the Mg and Zn complexes of APTRA and APDAP, bound in a pentadentate manner, with one H O
molecule completing the coordination sphere
Angle/°
[Mg(APDAP)]−
[Mg(APTRA)]−
[Zn(APDAP)]−
[Zn(APTRA)]−
ArO–M–N
78.4
79.8
80.1
83.9
73.9
77.3
77.1
75.2
71.2
78.9
78.0
78.6
72.1
78.1
78.2
74.6
−
N–M–O
N–M–O
−
−
a
ArO–M–O
a
O− = phosphinate oxygen for APDAP, O-carboxylate for APTRA.
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unit. Future functionalization of APDAP with fluorescent
groups may be anticipated to offer access to new sensors for
2
+
Mg with improved selectivity.
Experimental
General
Commercially available reagents were used as received from
suppliers without further purification. Solvents used were lab-
oratory grade; anhydrous solvents were dried over the appropri-
ate drying agent. All moisture-sensitive reactions were carried
out by Schlenk-line techniques, under an inert atmosphere of
Fig. 6 Calculated ArO–M–O− bite angles in the Mg complexes of (A)
APTRA and (B) APDAP, showing the shorter M–O lengths with APDAP
compared to APTRA and the longer P–O (versus C–O in APTRA) and
2+
2+
P–C (versus C–C in APTRA). The blue circle represents the Mg ion.
Structures (C) and (D) illustrate typical 5- and 6-membered chelates
formed with ligands such as ethylenediamine and trimethylenediamine either argon or nitrogen. For air sensitive reactions, solvents
respectively; the black circle represents a generic metal ion with ideal
bite angles as described by Martell and Hancock.
were degassed using the freeze–pump–thaw cycle method.
1
Water was purified by the ‘PuriteSTILLplus’ system with a con-
ductivity of <0.04 µS cm− . Thin layer chromatography was per-
1
formed on neutral alumina (Merck Art 5550) or silica (Merck
Art 5554) and visualized under UV irradiation (254 nm) or by
ively, is shown in Fig. 6. A schematic of 5-membered and staining with either iodine or potassium permanganate.
phosphinate chelating units in APTRA and APDAP, respect-
6
-membered chelates is also included for comparison. The Column chromatography was carried out either manually
using neutral alumina or silica (Merck Silica Gel,
lengths found for [Mg(APDAP)(H O)] suggest that the phos- 230–400 mesh) or using a Teledyne Combi-flash instrument
increase in the ArO–M–O bite angle and the shorter M–L bond
−
2
f
phinate-based APDAP ligand sets up a structure on binding to equipped with RediSep R silica cartridges to perform auto-
2
+
1
13
1
mated elution. H (400 MHz) and C{ H} (101 MHz) and
Mg that is somewhat more like a 6-membered chelate. In
-membered chelates, the ideal bite angle is 109.5°, signifi-
3
1
1
6
P{ H} (162 MHz) NMR spectra were acquired on Bruker
cantly larger than the value of 69° for a 5-membered chelate. Avance or Varian Mercury 400 NMR spectrometers. Electrospray
−
ionisation mass spectra were acquired on a Waters TQD mass
The larger ring chelate formed in [Mg(APDAP)(H
2
O)] may
2
+
2+
thus explain why the relative affinity for Mg versus Ca is spectrometer interfaced with an Acquity UPLC system.
increased compared to APTRA, in line with the conclusions
Optical spectroscopy; determination of pK and metal ion
affinities
a
of landmark studies by Hancock and Martell 30 years ago on
1
5
- versus 6-membered chelates in general. With regard to the
specific case of phosphinates, a similar increase in affinity for UV-Vis absorption spectra were measured using a Uvikon XS
smaller metal ions has been observed in phosphinate-substi- spectrometer operating with LabPower software. The sample
2
+
3+
tuted azamacrocycles, in binding studies with Cu , Ga and was held in a quartz cuvette with a path length of 1 cm.
3
+ 26
Fe .
Spectra were recorded against pure solvent in an optically
matched cuvette. pH measurements were recorded using a
Jenway 3510 pH meter in combination with a Jenway
9
24 005 pH electrode. The pH probe was calibrated before each
Concluding remarks
independent titration using commercially available buffer
In summary, the new, pentadentate APDAP ligand has been solutions of pH 4, 7 and 10. Samples were prepared with a
synthesised in a four-step sequence starting from 2-nitro- background of constant ionic strength (I = 0.1 M KCl, 298 K).
2
+
phenol. Although it displays a weaker affinity towards Mg
Aqueous solutions were titrated to acid using 0.025 M, 0.05 M
than APTRA, the phosphinate-based ligand has a dramatically and 0.1 M concentrations of HCl(aq). The resulting sigmoidal
2
+
2+
lower affinity for Ca . The K
d
values for Ca are in the milli- curve of either absorbance or fluorescence intensity vs. pH was
molar range compared to the mid-micromolar values typical of fitted by a non-linear least squares iterative analysis by
2
+
APTRA and its derivatives. The affinity for Mg remains of an Boltzmann using Origin 8.0 software.
order of magnitude that renders APDAP potentially suited to All metal binding studies were carried out in buffered solu-
biomedical applications, given that [Mg ] is of the order of tions of 50 mM HEPES and 100 mM KCl maintained at pH 7.2.
2
+
2
+
1
mM in most mammalian cells. Moreover, the ligand offers Stock solutions of [M ] contained the same concentration of
high denticity, which is likely to ensure that the formation of the ligand in the cuvette, to avoid sample dilution over the
2
+
2+
undesirable mixed ligand species with Mg , as occurs with course of the titration. Small aliquots of [M ] were added in
β-keto acids for example, is impeded. The change in relative each instance, with the absorbance spectrum recorded
2
+
2+
binding affinities of Mg and Ca , compared to the all- 5 minutes after each addition to ensure that the sample had
carboxylate APTRA, is probably linked to the larger chelate equilibrated. Dissociation constants (K values) were calculated
ring associated with the incorporation of the phosphinate from the experimental data as described in the ESI.†
d
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Paper
Computations
(Ethoxy(methyl)phosphoryl)methyl methanesulfonate, 4.
All calculations were carried out with the Gaussian 09
2
7
package. The geometries were optimised at the hybrid-DFT
2
8
B3LYP functional with no symmetry constraints using the
2
9
6
-311++G(d,p) basis set for all atoms. The Gaussian default
3
0
polarisation continuum model (IEFPCM) was applied to all Methylsulfonyl chloride (0.65 mL, 8.4 mmol) was added to an
calculations using water as solvent. Frequency calculations ice cold solution of 3 (808.8 mg, 5.95 mmol) and triethylamine
on these optimised geometries revealed no imaginary (1.25 mL, 8.96 mmol) in anhydrous THF (5.7 mL). The solu-
frequencies.
tion was warmed to room temperature and stirred for an
additional 1 h under an inert atmosphere of argon. After 1 h,
THF was removed under reduced pressure, the dark orange
residue was re-dissolved in CH Cl (20 mL) and washed with
Synthetic procedures and characterisation data
Ethyl methylphosphinate, 2.
2
2
4
brine (15 mL). The organic extracts were dried over MgSO ,
and the solvent was removed under reduced pressure to form
an orange residue. Purification by silica gel column chromato-
2 2 2 2
graphy (gradient 100% CH Cl to 95% CH Cl /5% MeOH)
formed the title compound as a yellow oil (1.03 g, 82%).
Water (264 µL, 14.7 mmol) was added to diethyl methyl-
phosphinate 1 (2 g, 14.7 mmol) at room temperature. The col-
ourless solution was stirred vigorously for 18 h under an inert
atmosphere of argon. Reaction completion was determined by
1
2
H (400 MHz, CDCl ) 4.46–4.34 (2 H, m, H ), 4.25–4.11 (2 H,
3
4
1
3
m, H ), 3.15 (3 H, s, H ), 1.63 (3 H, d, J 16, H ), 1.39 (3 H, t, J 8,
H ); P{ H} (162 MHz, CDCl ) + 43.7; C (101 MHz, CDCl )
3.0 (d, J 106, C ), 61.6 (d, J 7, C ), 37.8 (s, C ), 16.5 (d, J 6, C ),
5 13 5
2.6 (d, J 100, C ). ESI-LRMS [C H O PS] (+) m/z 217.3;
5
31
1
13
3
3
5
2
4
1
6
1
3
1
P NMR spectroscopy. The title compound formed with one
3
+
equivalent of ethanol as a by-product and was used in situ in
+
ESI-HRMS calculated for [C H O PS] 217.0300 found,
5
13 5
1
subsequent steps without any additional purification. H NMR
217.0321.
1
(
400 MHz, CDCl ) 7.25 (1 H, dq, J 538, 2, H ), 4.17–3.16 (2 H,
3
Ethyl methyl((2-nitrophenoxy)methyl)phosphinate, 6.
3
2
4
13
1
m, H ), 1.48 (3 H, dd, J 16, 4, H ), 1.24 (3 H, t, J 4, H ); C{ H}
) 62.4 (d, J 24, C ), 16.2 (d, J 24, C ), 14.6
d, J 97, C ); P{ H} NMR (176 MHz, CDCl ) + 33.4; ESI-LRMS
3
4
NMR (101 MHz, CDCl
(
[
3
2
31
1
3
+
+
3 9 2 3 9 2
C H PO ] (+) m/z 109.0; ESI-HRMS calculated for [C H PO ]
1
09.0419 found, 109.0414.
Ethyl (hydroxymethyl)(methyl)phosphinate, 3.
Anhydrous potassium carbonate (302.9 mg, 2.19 mmol) was
added to a solution of 2-nitrophenol (98.9 mg, 0.71 mmol) and
4
(228.2 mg, 1.05 mmol) in anhydrous acetonitrile (1.2 mL).
The reaction mixture was heated at 85 °C for 48 h under an
inert atmosphere of argon. The pale yellow precipitate was
removed by filtration and the solvent was removed under
reduced pressure to form a yellow residue. Purification by
The procedure adopted was based on one developed previously
for related mesylated phosphinate esters. Ethyl methyl phos-
phinate 2 (14.7 mmol) was heated at 85 °C in anhydrous THF
3
1
(5 mL) for 30 min. Paraformaldehyde (707.9 mg, 23.60 mmol)
silica gel column chromatography (gradient 100% CH
2 2
Cl to
was then added. The reaction mixture was heated at 75 °C
9
5% CH Cl /5% MeOH) formed the title product as a pale
2
2
3
1
under an inert atmosphere of argon and monitored by
P
1
yellow oil (115.3 mg, 63%). H NMR (600 MHz, CDCl
3
) 7.88–7
NMR. After 5 h the colourless solution was cooled to room
temperature before the THF was removed under reduced
pressure. The residue was then dissolved in CH Cl (20 mL)
8
5 (1 H, m) 7.58–7.54 (1 H, m), 7.13–7.09 (2 H, m), 4.39–4.26
9
7
8
(
4 H, m, H and H ), 1.69 (3 H, d, J 15, H ), 1.33 (3 H, t,
2
2
10 13
1
1
J 7, H ); C{ H} (151 MHz, CDCl
3
,) 151.8 (d, J 13, C ), 140.1
before the inorganic salts were removed by filtration. The
solvent was removed under reduced pressure to form a colour-
less oil. Purification by silica gel chromatography (gradient
2
3
(
C ), 134.4 (CH), 125.8 (CH), 121.9 (CH), 114.7 (C ), 61.4 (d, J 7,
9
7
9
8
31
1
C ), 65.2 (d, J 111, C ), 16.5 (C ), 12.2 (C ); P{ H} NMR
+
(
242 MHz, CDCl
3
) + 48.5; ESI-LRMS [C10
60.1; ESI-HRMS calculated for [C10 14NO
60.0699.
Ethyl ((2-aminophenoxy)methyl)(methyl)phosphinate, 7.
H
14NO
5
P] (+) m/z
from 100% CH
title compound as a pale yellow oil (747 mg, 37%). R
2
Cl
2
to 90% CH
2
Cl
2
/10% MeOH) formed the
= 0.20
+
2
2
H
5
P] 260.0701 found
f
1
(silica, 95% DCM; 5% MeOH, visualisation with KMnO ); H
4
3
1
3
NMR (400 MHz, CDCl ) 4.13 (2 H, m, H ), 3.85 (2 H, m, H ),
2
4
3
.07 (1 H, br s, OH), 1.54 (3 H, d, J 12, H ), 1.35 (3 H, J 4, H );
P{ H} NMR (176 MHz, CDCl ) + 52.0; C NMR (101 MHz,
3
1
1
13
3
3
1
4
3
CDCl ) 61.1 (d, J 7, C ), 59.8 (d, J 110, C ), 17.7 (d, J 6, C ), 16.6
2
79
+
(
d, J 91, C ); ESI-LRMS [C
6
H
7
BrN
2
] (+) m/z 139.0; ESI-HRMS
7
9
+
calculated for [C H BrN ] 139.0456 found, 139.0449.
6
7
2
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Compound 6 (118.1 mg, 0.46 mmol) was dissolved in ethanol
2,2′-((2-((Hydroxy(methyl)phosphoryl)methoxy)phenyl)aza-
(20 mL). Palladium on charcoal (10 wt%, 51.6 mg, 0.03 mmol) nediyl) diacetic acid, APDAP.
was added and the reaction mixture was stirred at room temp-
erature for 2 h under an atmosphere of hydrogen. After
this time, the reaction mixture was filtered through a
Celite plug. The solvent was removed under reduced pressure
to form a pale yellow oil (104 mg, 96%). Compound 7 was used
in subsequent steps without any additional purification.
1
3
4
5
3
H NMR (600 MHz, CDCl ) 6.86–6.82 (2 H, m, H , H , H or
The tris-ester 8 (82.6 mg, 0.21 mmol) was dissolved in CD OD
3
6
3
4
5
6
H ), 6.74–6.70 (2 H, m, H , H , H or H ), 4.29–4.07 (4 H, m,
(
2
4 mL) and NaOD (0.4 M in D O, 1.4 mL). The pale yellow solu-
7
9
8
10
H and H ), 1.64 (3 H, d, J 14.6, H ), 1.34 (3 H, t, J 7, H );
tion was stirred under an inert atmosphere of argon at room
temperature for 20 h. Hydrolysis of the ethyl esters was moni-
3
1
1
13
1
P{ H} NMR (162 MHz, CDCl
3
) + 47.4; C{ H} (151 MHz,
1
2
3
4
5
6
CDCl
1
C
1
3
) 146.3 (d, J 11.9, C ), 136.4 (C ), 122.8 (C , C , C or C ),
1
31
tored by H NMR spectrometry, P NMR spectrometry and
ESI-LRMS. The solution was lyophilized to form the title com-
3
4
5
6
3
4
5
6
3
4
18.5 (C , C , C or C ), 115.7 (C , C , C or C ), 112.2 (C , C ,
or C ), 64.6 (d, J 111, C ), 61.0 (d, J 7, C ), 16.6 (s, C ),
5
6
7
9
10
pound as an off-white solid in almost quantitative yield
8
+
2.6 (d, J 98, C ); ESI-LRMS [C10
H
16NO
3
+
P] (+) m/z 230.6;
1
(
62 mg, 93%). H NMR (600 MHz, D
2
O) 6.95 (1 H, dd, J 1.8, 8,
ESI-HRMS calculated for [C H NO P] 230.0946 found
6
4
5
3
1
0
16
3
H ), 6.83 (2 H, m, H and H ), 6.78 (1 H, br m, H ), 3.93 (2 H,
d, J 9, H ), 3.71 (4 H, s, H ), 1.29 (3 H, d, J 14, H ); P{ H}
NMR (283 MHz, D O) + 36.54; C{ H} NMR (176 MHz, D O)
2 2
79.1 (C ), 150.6 (C ) 139.4 (C ), 121.6 (C or C ), 118.5 (C or
C ), 118.1 (C ), 113.4 (C ), 56.5 (C ), 14.2 (d, J 95, C ); reverse
phase HPLC (0%–100%–0% CH CN in ammonium bicarbon-
2
30.0949.
Diethyl 2,2′-((2-((ethoxy(methyl)phosphoryl)methoxy)phenyl)
7
9
8
31
1
13
1
azanediyl) diacetate, 8.
10
1
2
4
5
4
1
5
3
6
9
8
3
ate buffer (25 mM), t = 1.2 min).
R
Conflicts of interest
N,N-Diisopropylamine (410 μL, 2.36 mmol) and ethyl bromo-
acetate (158 μL, 1.42 mmol) were added to a solution of 7
There are no conflicts to declare.
(107.5 mg, 0.46 mmol) and sodium iodide (125.6 mg,
0
.97 mmol) in anhydrous acetonitrile. The reaction was heated
for 2 d at 85 °C under an inert atmosphere of argon before the Acknowledgements
addition of further N,N-diisopropylamine (120 μL, 0.69 mmol)
We thank Durham University and EPSRC for support.
and ethyl bromoacetate (50 μL, 0.45 mmol). The reaction
mixture was stirred at 85 °C for a further 2 d, before being
cooled and diluted with ethyl acetate (10 mL). Inorganic impu-
rities were removed by filtration before the organic filtrate was
washed with water (10 mL) and brine (10 mL). Organic extracts
References
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4
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3
4
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6
3
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3
ate buffer (25 mM), t = 9.6 min).
R
Dalton Trans.
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This journal is © The Royal Society of Chemistry 2018
Dalton Trans.