Tuning Mg(II) Selectivity: Comparative Analysis of the Photophysical Properties of Four Fluorescent Probes with an Alkynyl-Naphthalene Fluorophore

Article abstract of DOI:10.1002/chem.201800013

Four alkynyl-1-naphthyl fluorophores have been synthesised with tri- or pentadentate ligating groups suited to the binding of magnesium. Their photophysical and binding properties for magnesium, calcium and zinc ions have been assessed using absorption, e

Full text of DOI:10.1002/chem.201800013

A Journal of
Accepted Article
Title: Tuning Mg2+ selectivity: comparative analysis of the
photophysical properties of four fluorescent probes with an
alkynyl-naphthalene fluorophore
Authors: David Parker, Gareth Williams, and Edward Walter
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To be cited as: Chem. Eur. J. 10.1002/chem.201800013
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Tuning Mg2+ selectivity: comparative analysis of the
photophysical properties of four fluorescent probes
with an alkynyl–naphthalene fluorophore
Edward R. H. Walter, J. A. Gareth Williams * and David Parker *
Department of Chemistry, Durham University, South Road, Durham DH1 3LE, UK.
E-mail: david.parker@dur.ac.uk, j.a.g.williams@durham.ac.uk. Supporting information and
ORCID(s) from the author(s) for this article are available on the www under
http://dx.doi.org/10.1002/chem. xxxx
Abstract: Four alkynyl-1-naphthyl fluorophores have been synthesised with tri- or
pentadentate ligating groups suited to the binding of magnesium. Their photophysical
and binding properties for magnesium, calcium and zinc ions have been assessed
using absorption, emission and excitation spectroscopy. Each compound has a pKa
value between 6.2 and 5.1 and exhibits no significant pH response around pH 7.2.
Enhanced selectivity for Mg2+ over Ca2+ was observed with a pentadentate
phosphinate substituted system, compared to its carboxylate analogue, due to a 10-
fold lowering of affinity for the Ca2+ ion. In each case, the overall dissociation
constants for Mg2+ fall in the low mM range.
Introduction
Fluorescence spectroscopy is a powerful and extensively used tool in cell
biology, because of its high temporal resolution and sensitivity. Design strategies and
principles for the creation of fluorogenic sensors have recently been reviewed,
highlighting the need to seek systems that combine a long wavelength of excitation
with the desired affinity and selectivity profile. 1 The application of fluorescent sensing
is widespread, with a variety of fluorescent sensors providing detailed information on
2-5
6
the roles of biologically active cations
and anions in vitro and in cellulo. The use
of ratiometric probes is highly advantageous, as it allows quantitative information to
be obtained independent of environmental factors, such as uneven cell loading or
photobleaching. 7 Progress in the development of magnesium-selective fluorescent
probes, however, has been slow compared to the monitoring of other divalent
1
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Walter/Williams/ Parker
cations, such as Ca2+ and Zn2+, resulting in a very limited understanding of the
8a
specific details of magnesium homeostasis. It is worth noting that progress was made
in creating sensitive magnesium selective reagents for use in absorption spectroscopy, based
on sulfonated hydroxyl-naphthyl chromophores (λabs 604 nm) that were able to be used with
8b
small volumes of bio-fluids.
9
Magnesium is the second most abundant divalent cation in cells and is
directly involved in a number of intracellular processes, playing a fundamental role in
over 600 enzymatic reactions 10 and a key role in the stabilisation of DNA structure.11
The regulation of ‘free’ magnesium concentrations in cells is essential, with
magnesium imbalance such as the onset of hypomagnesaemia linked to a number of
cardiovascular 12, neurodegenerative 13,14 and renal 15 diseases.
The o-aminophenol-N,N,O-triacetic acid (APTRA) ligand developed by
London has been used extensively for a variety of metal ion sensing applications,
binding the ionisable ‘free’ magnesium ion with a Kd value in the low mM range.16-21
Selectivity is an issue, however, as the APTRA chelates display a much higher
affinity towards Ca2+ than Mg2+ , in every reported case (µM vs mM affinity).16-21 The
ability of APTRA indicators to act as low affinity calcium-based probes has prevented
the accurate determination of ‘free’ Mg2+ concentrations in areas where Ca2+
concentrations exceed basal levels, such as in serum and in the endo(sarco)plasmic
reticulum. The APTRA chelators were first reported over thirty years ago, yet there is
still a distinct lack of variety in ligand design for Mg2+ sensors. The novelty typically
arises from modifications to the chromophore.18-20 Bidentate β-keto acids, for
example, have been examined as alternative probes, and exhibit a higher intrinsic
22-24
selectivity towards Mg2+ over Ca2+.
The limitation of these β-keto acids arises
from the formation of mixed species in cellulo, with the probes able to bind to both
‘free’ Mg2+ and [MgATP]2–. These adducts are indistinguishable by conventional
fluorescence spectroscopic studies. 25
It is essential that greater effort be placed into the development of new
chelate ligand types for the binding of Mg2+, in order to solve this selectivity
conundrum. Here, we report the synthesis and analysis of four fluorescent probes
containing an alkynyl-naphthalene fluorophore L1–L4 (Figure 1). Levy et al have
previously demonstrated that bi-aryl systems containing an APTRA binding group
display a ratiometric response to Mg2+ and Ca2+ (L5, Figure 1). Following addition
17
of Mg2+, for example, a 30 nm hypsochromic shift in the excitation maximum was
reported.17 The development of two new phosphinate-based fluorescent indicators in
2
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particular is described, L3 and L4, as an alternative to the carboxylate-based APTRA
binding chelate used in the literature. Using an alkynyl-naphthalene fluorophore, we
have attempted to tune magnesium selectivity by changing the position of the oxygen
and nitrogen donor groups on the aromatic framework, incorporating a phosphinate
at the expense of a carboxylate group, and by modifying the ligand denticity around
the metal ion. The incorporation of the four pro-ligands into an alkynyl-naphthalene
fluorophore provided an opportunity to analyse the behaviour of the two new
phosphinate-based binding groups via fluorescence spectroscopy. It also enabled the
fluorescent systems to act as models for lanthanide(III)-based complexes created in
parallel work, to be reported elsewhere, in which pyridylalkynylaryl chromophores are
also integrated to sensitise lanthanide(III) emission efficiently. 26,27
O
N
O
O
O
O
O
O
O
O
O
O
O
N
O
O
L1
L2
O
O
O
O
N
O
NH O
O
O
P
O
P
O
O
L3
O
L4
O
O
O
O
O
O
O
N
O
N
O
O
O
O
O
O
N
O
O
L5
Mag-Fura2
O
Figure 1. Structures of L1–L4, compared to L5 , and (right) Mag-Fura-2, after London et al.17
3
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Ligand L1 features an APTRA moiety bound to a 1-naphthylalkynyl
fluorophore, and L2 is constitutionally isomeric with L1, differing in the position of the
oxygen and nitrogen donor atoms around the aromatic ring. Ligand L3 is a
phosphinate analogue of L1, containing a pentadentate APDAP binding chelate. 28
This ligand has very recently been shown to display a dramatically enhanced
selectivity for Mg2+, 28 with a 100-fold reduction in the affinity for Ca2+ compared to the
parent APTRA chelate.29 Unlike the pentadentate ligands L1–L3 that form 5-ring
chelates, the tridentate ligand L4 forms a [6,5] chelate around divalent metal ions. It
has previously been suggested that such a reduction in ligand denticity may
inherently favour the binding of the Mg2+ cation over Ca2+ and Zn2+, as the binding of
Ca2+ and Zn2+ is more enthalpically and entropically driven. 29
Thus in this work, direct comparisons will be made across the naphthalene
series to determine how the change in the position of the donor atoms, the choice of
ligating group, denticity and ring chelate size affect affinity and selectivity for Mg2+.
Results and Discussion The synthesis of ligands L1–L4 was undertaken using a
variety of alkylation and palladium-catalysed cross-coupling reactions. The
carboxylate-based ligands, L1 and L2, were synthesised from 2-aminophenol and 2-
amino-4-bromophenol respectively, as described in the supplementary information
(ESI, Schemes S1 and S2). The synthesis of the phosphinate-based ligands L3 and
L4 is described in Schemes 1 and 2.
Ligand L3 was synthesised from the ethyl ester of the parent structure
28
APDAP, 1, reported recently in a four-step synthesis from 2-nitrophenol
(Scheme 1). Selective para-bromination of 1 was achieved with [K.18-Crown-6]Br3 in
30
acetonitrile,
yielding
2
in high yield under mild conditions. The
(triisopropylsilyl)acetylene (TIPS) group was introduced via a Sonogashira cross-
coupling reaction, generating 3 in high yield, from which a one-pot alkyne de-
protection with tetrabutylammonium fluoride (TBAF) and palladium-catalysed cross-
coupling reaction gave rise to the ethyl ester of L3. Universal ester hydrolysis under
basic conditions formed L3, with ligand purity assessed by analytical reverse-phase
HPLC.
4
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O
N
OEt
O
N
OEt
O
O
N
OEt
EtO
EtO
[K.18-Crown-6]Br3,
(iPr)3Si
EtO
O
P
O
P
O
P
Pd(dppf)Cl2, CuI,
NEt3, THF
O
O
O
O
O
OEt
OEt
CH3CN
OEt
86 %
74 %
Br
2
1
Si(iPr3)
3
Br
10 %
TBAF, Pd(dppf)Cl2,
CuI, NEt3, THF
O
N
O
O
O
OEt
O
EtO
O
N
O
O
P
O
O
P
O
NaOD(aq), MeOD
quant.
OEt
L3
4
Scheme 1 Synthesis of L3
O
O
OEt
iPr2NEt, NaI, CH3CN
EtO
EtO
EtO
P
NH2
Br
Br
NH
NH
Br
O
H
I
O
I
NEt3, Pd(dppf)Cl2
O
P
OEt
48 %
68 %
Br
6
7
(iPr)3Si
77 %
Pd(dppf)Cl2, CuI,
NEt3, THF
Br
O
EtO
EtO
NH
O
NH
O
NH
O
P
O
P
O
P
TBAF, Pd(dppf)Cl2,
CuI, NEt3, THF
O
O
NaOD(aq), MeOD
OEt
OEt
quant.
19 %
Si(iPr3)
8
L4
9
Scheme 2 Synthesis of L4 from 4-bromo-2-iodoaniline.
The tridentate ligand L4 was synthesised in five-steps in moderate overall
yield (Scheme 2). Alkylation of 4-bromo-2-iodoaniline gave the mono-alkylated
intermediate 6 selectively; no traces of N,N-di-alkylation were found using a range of
conventional base and solvent combinations. The palladium-catalysed coupling of
ethyl methylphosphinate with 6 was achieved in a procedure adapted from Parker
5
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31
and co-workers.
Compound 7 formed in moderate yield, and was linked to an
alkynyl-naphthalene fluorophore in an identical procedure to L3 (Scheme 1).
Originally, it was hoped that N,N-dialkylation would give rise to the formation of a
tetradentate chelating ligand, with two carboxylate and one phosphinate oxygen
atoms and an aniline nitrogen atom available to bind to metal ions. N,N-Di-alkylation
was, however, not successful under all conditions attempted, neither from 4-bromo-2-
iodoaniline nor from compound 7, possibly due to steric inhibition of the key
substitution reaction caused by the ortho iodo or phosphinate substituents.
Photophysical studies of L1–L4
The photophysical properties of L1–L4 were examined in aqueous solution at
298K and are summarised in Table 1, comparing their behaviour with that of ligand
L5 developed by London.17 Ligands L1, L3 and L4 have large Stokes’ shifts, in the
range of 166 to 204 nm, nearly 30 nm bigger than with L5, due to the increase in
conjugation and the planarity of the alkynyl-naphthalene chromophore. Such large
Stokes’ shifts are typically found with organic fluorophores that contain an
intramolecular charge transfer (ICT) transition and arise from the stabilisation of the
excited state by lone pair conjugation involving the para aniline nitrogen atom. The
excited state of L2 in contrast, contains localised π–π* character, due to the weaker
electron donating capabilities of the para phenolic oxygen atom. A hypsochromic shift
in the absorbance, emission and excitation spectra was observed with a much
smaller Stokes’ shift of 54 nm (Table 1). The excitation wavelengths with L1,3,4 allow
the use of LEDs that operate at 365 nm, and can be pulsed to minimise the energy
absorbed by the system under examination.
Table 1. Photophysical data for L1–L5 in H2O at 298 K. Quantum yields (ꢀ) ± 20 % measured in H2O.
Ligand λabs / nm, ꢀ /103
Excitation λmax / nm a
Emission λmax / nm a
ΦH O / %
2
M-1cm-1
‘free’
358
318
350
341
325e
Mg2+ sat.
‘free’
562b
372
Mg2+ sat.
522b
372
L1
L2
L3
L4
L5
354, 14.1(3)
318, 13.8(6)
350, 10.7(2)
341, 12.9(4)
310,e n.d.
320
0.3c
1.3d
0.7c
0.8c
n.d.
318
336
550
547
341
507
507
295e
499, 358e 499, 358e
a
b
Measurements recorded in 50 mM HEPES, 100 mM KCl, pH 7.21 at 298 K; λex = 354 nm.
Fluorescence quantum yields were measured using either c Quinine sulphate (in 0.5 M H2SO4)
POPOP 33 as the standards. e Values reported by London 17, ꢀ and Φ were not determined (n.d.).
or
32
d
6
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Spectroscopic studies of ligand pKa with L1–L4
For the overwhelming majority of sensors in the literature incorporating an
APTRA binding chelate, the pKa was not determined, owing to putative instability at
low pH on prolonged standing in aqueous solution.20,29,34 No such problems were
encountered in this study, allowing the pH behaviour of L1–L4 to be studied via
fluorescence spectroscopy. In the case of L1–L3 , pKa values were calculated by
fluorescence excitation spectroscopy (ESI). A ratiometric response to pH change was
observed in acidic solutions of L1 (ESI, Figure S1) and L3 (ESI, Figure S2) following
protonation of the aniline N atom, with a non-ratiometric ‘turn-on’ observed in the
excitation spectrum of L2 (ESI, Figure S3). The pKa of the tridentate ligand L4, was
only determined by emission spectroscopy as no pronounced pH sensitivity was
seen in its excitation spectrum (ESI, Figure S4).
Calculated pKa values for L1–L4 all fall in the range of 5.2 to 6.2 (Table 2),
comparable to those reported for Mag-Fura-2, for which a pKa value of 5.0 was
21
reported by London et al.
The fluorescence of each ligand was insensitive to pH
from 6.5 to 7.5, indicating that at physiological pH the aniline nitrogen and
carboxylate / phosphinate ligating groups are fully de-protonated and available to
coordinate to metal ions. 2,21,35
Table 2. The calculated pKa values of L1–L4 in 100 mM KCl, 298 K, with the error associated to the
fitting in parenthesis. The pKa of Mag-Fura-2 is provided for comparison.
Ligand
pKa
L1
6.2(04) a
5.7(05) a
5.2(04) a
5.1(02) b
5.0 21
L2
L3
L4
Mag-Fura-2
a,b
pKa values determined by fluorescence excitation / fluorescence emission spectroscopy respectively.
Fluorescence metal ion binding studies with L1–L4
Ligand L1 In the metal-free state, L1 exhibits an excitation maximum at 358 nm.
Following addition of Mg2+, Ca2+ (Figure 2) and Zn2+ (Figure S5), a ratiometric
excitation response was observed, with a pronounced wavelength shift of 38 nm
towards the blue (Table 1, Figure 2). The excitation-based ratiometric behaviour
seen here has been reported previously for a number of different cation sensors with
2,17-21
Mg2+ and Ca2+,
including Mag-Fura-2, where a 35 nm hypsochromic shift was
7
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Walter/Williams/ Parker
21
observed in the metal-bound state. TD-DFT has been used to model the effect of
Mg2+ binding on the excited state of Mag-Fura-2, revealing that the ICT state is
perturbed following Mg2+ binding, with elimination of a charge transfer process
involving the aniline nitrogen lone pair. The excited state instead possesses localised
π–π* character in its metal-bound form, resulting in a drastic change in the spectrum
on
binding. 35
12
10
8
3
2.5
2
(C)
6
1.5
1
(A)
4
2
0.5
0
Kd = 4.5(2) mM
0
280
330
380
430
0
0.02
0.04
0.06
Concentration (M)
Wavelength (nm)
2.5
2
30
25
20
15
10
5
(B)
1.5
1
Kd = 39.6(4.9) µM
(D)
0.5
0
0
0.0004
0.0008
0
280
Concentration (M)
330
380
430
Wavelength (nm)
Figure 2. The excitation spectrum of L1 following addition of (A) Mg2+ and (B) Ca2+, λem = 530 nm, Kd
values were calculated from a 1:1 stoichiometry from the ratio of the intensity of the metal-free and
bound states against the concentration of (C) Mg2+ and (D) Ca2+, and are reported as an average of two
separate metal ion titrations, given with the experimental error in parenthesis. Ligand conc. = 5 µM in 50
mM HEPES, 100 mM KCl, pH 7.21, 298 K.
Dissociation constants of 4.5(2) mM and 39.6(5) µM were calculated following
addition of Mg2+ and Ca2+ respectively (Table 3), consistent with typical affinities
observed for APTRA analogues in the literature.17-21 Comparable values were
calculated from fluorescence emission spectral changes, with excitation at both the
isosbestic point and the wavelength maximum in the absorption spectrum (ESI).
Following excitation at the isosbestic point (λex = 330 nm), ‘turn-on’ emission
ratiometric behaviour was observed (ESI, Figure S6).
8
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Ligand L2 In contrast to the excitation- and emission-based ratiometric behaviour
reported for L1, a non-ratiometric ‘turn-on’ response was found with L2 following
addition of Mg2+, Ca2+ and Zn2+. Wavelength maxima of 318 nm and 372 nm were
observed in excitation and emission spectra respectively. A 3-, 6- and 9-fold intensity
enhancement was found following addition of Mg2+, Ca2+ (Figure 3) and Zn2 (ESI,
Figure S7), owing to the suppression of a photo-induced electron transfer (PET)
process. No hypsochromic shift was observed following metal ion binding, as the
excited state of L2 has localised π–π* character in both its ‘free’ and ‘metal-bound’
forms.
10
10
(A)
8
8
(C)
6
6
4
2
4
Kd = 1.3(1) mM
2
0
0
280
330
0
0.02
Concentration (M)
0.04
Wavelength (nm)
25
25
(B)
20
15
10
5
20
15
10
5
(D)
Kd = 32.7(3.7) µM
0
0
0
0.0002
0.0004
280
330
Concentration (M)
Wavelength (nm)
Figure 3. The excitation spectrum of L2 following addition of (A) Mg2+ and (B) Ca2+, λem = 377 nm. Kd
values were calculated from a 1:1 stoichiometry from the intensity at 318 nm against the concentration
of (C) Mg2+ and (D) Ca2+, and are reported as an average of two separate metal ion titrations, given with
the experimental error in parenthesis. Ligand conc. = 10 µM in 50 mM HEPES, 100 mM KCl, pH 7.21,
298 K.
Calculated dissociation constants for the binding of L2 are comparable to
those measured for L1. Values of 1.3(8) mM, 32.7(4) µM and 3.2(2) µM were
recorded for the binding of Mg2+, Ca2+ (Figure 3) and Zn2+ (ESI, Figure S7)
respectively. The slightly enhanced affinity observed for the binding of L2 to Mg2+ and
Ca2+ may arise from the increased donor ability of the meta aniline nitrogen atom.
9
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It is evident that despite changing the relative position of the nitrogen and
oxygen atoms on the APTRA binding framework, comparable affinity and selectivities
for Mg2+, Ca2+ and Zn2+ are found (Table 3). However, changing the position of the
donor groups does have a significant effect on the excitation and emission spectral
response. Both L1 and L2 have dissociation constants in the low mM range following
addition of Mg2+, aligning with the concentrations of ‘free’ Mg2+ in mammalian cells
9
(0.8-1.5 mM), and with the affinity and selectivity of alternative APTRA-based
sensors reported previously.17-21 In accordance with literature precedent, ligands L1
and L2 also displayed a higher affinity for Ca2+, responding in the mid-µM range.
Such an affinity profile is undesirable for Mg2+ sensing applications.36
Ligand L3
The pentadentate APDAP binding chelate was recently developed as
an alternative chelating ligand to APTRA. From initial absorbance spectroscopy
studies, it was found to display an enhanced selectivity for Mg2+, binding Ca2+ in the
low mM range.28 Described here is the first example of incorporation of the APDAP
chelate into a fluorescence system to analyse the binding of Mg2+, Ca2+ and Zn2+.
Unlike its carboxylate analogue L1, ligand L3 showed no significant ratiometric
behaviour in its absorption or excitation spectra following addition of Mg2+ (ESI,
Figure S8). A hypsochromic shift was, however, observed in acidic solution (e.g. at
pH 4), suggesting that the aniline nitrogen atom may not have a prominent role in
binding to Mg2+. The small changes observed in excitation intensity on addition of
Mg2+ meant it was not feasible to generate dissociation constants accurately via this
method. Instead, Kd values were calculated from fluorescence emission spectral
changes. Following excitation at 350 nm, a wavelength maximum of 545 nm was
displayed in the ‘metal-free’ state, with an 18 nm shift to lower wavelengths and a
1.5-fold reduction in the emission intensity on saturation with Mg2+ (8.5 mM)
(Figure 4). Following addition of Ca2+ (Figure 4) and Zn2+ (ESI, Figure S9),
however, an excitation shift was observed that could be followed ratiometrically, with
a 31 nm hypsochromic shift in the metal-bound state. Such behaviour implies that the
aniline nitrogen atom binds more strongly to Ca2+ and Zn2+ than to Mg2+.
10
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14
12
10
8
13
11
9
(C)
(A)
Kd = 0.5(2) mM
6
4
2
0
7
360
460
560
660
0
0.003
0.006
0.009
Wavelength (nm)
Concentration (M)
25
20
15
10
5
1.8
1.4
1
(B)
Kd = 0.4(1) mM
(D)
0
280
0.6
330
380
430
0
0.002
0.004
0.006
Wavelength (nm)
Concentration (M)
Figure 4. The emission spectrum of L3 following addition of (A) Mg2+ (λex = 350 nm) and the excitation
spectrum following addition of (B) Ca2+ (λem = 530 nm). Kd values were calculated from a 1:1
stoichiometry from the intensity plotted against the concentration of (C) Mg2+ and (D) Ca2+, and are
reported as an average of three separate metal ion titrations, given with the experimental error in
parenthesis. Ligand conc. = 10 µM in 50 mM HEPES, 100 mM KCl, pH 7.21, 298 K.
Dissociation constants of 0.5(2) mM, 0.4(1) mM and 3.3(3) µM were
calculated respectively, assuming a 1:1 stoichiometry between Mg2+, Ca2+ and Zn2+
ions (Table 3). The ligand L3 displays increased selectivity for Mg2+ compared to the
carboxylate analogues L1 and L2. A 10-fold reduction in affinity for Ca2+ was found,
with a Kd value in the low mM range. It is likely that the increased selectivity is due to
the less acute bite angles (9o) around Mg2+ in the phosphinate chelate, as predicted
from detailed DFT studies of both [Mg(APTRA)H2O]– and [Mg(APDAP)H2O]–. It is
28
both interesting and surprising to note that L3 has a higher affinity for Mg2+ than either
L1 or L2. Its affinity falls in the desired affinity range, close to the concentration of
‘free’ Mg2+ in mammalian cells.
Ligand L4
The tridentate ligand L4, displayed a non-ratiometric response in both
its emission and excitation spectrum, following addition of Mg2+, Ca2+ and Zn2+. In the
emission spectrum, a wavelength maximum of 499 nm was found, following
excitation at 347 nm. Small spectral changes were observed after addition of high
11
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Walter/Williams/ Parker
concentrations of Mg2+ (160 mM), Ca2+ (2 mM) (Figure 5) and Zn2+ (56 µM) (ESI,
Figure S10). A Kd value of 16.5(6.6) mM was calculated for the binding of Mg2+ (ESI,
Figure S11), with the large error associated with fitting small fluorescence intensity
changes. Dissociation constants could not reliably be calculated for Ca2+ or Zn2+,
and weak binding affinity is presumed.
12
19
(A)
(C)
10
8
15
11
7
6
4
2
0
3
420
520
620
300
350
400
Wavelength (nm)
Wavelength (nm)
12
10
8
(B)
15
13
11
9
(D)
6
4
7
2
5
0
420
520
620
3
300
350
400
Wavelength (nm)
Wavelength (nm)
Figure 5. Emission spectra of L1 following addition of (A) Mg2+ (160 mM) and (B) Ca2+ (2 mM) (λex = 347
nm). Excitation spectra following addition of (C) Mg2+ and (D) Ca2+ (λem = 501 nm). Ligand concentration
= 5 µM in 50 mM HEPES, 100 mM, pH 7.2, KCl, 298K.
It has previously been suggested by Buccella and co-workers that reducing the
ligand denticity should favour the binding of Mg2+, as its binding is less enthalpically
and entropically driven than Ca2+ and Zn2+. 29 In this instance, however, it is apparent
that by reducing the number of ligating groups, 4-fold weaker binding is observed for
Mg2+ compared to that with the pentadentate ligand L1. The non-ratiometric response
and the small intensity changes in the fluorescent emission and excitation spectra
following addition of Mg2+, Ca2+ and Zn2+ make the tridentate L4 system unsuitable for
the accurate determination of the concentrations of metal ions.
12
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Table 3. The calculated dissociation constants for L1–L5 (50 mM HEPES,100 mM KCl, pH 7.2, 298 K).
Ligand
Kd (Mg2+) / mM Kd (Ca2+) / µM Kd (Zn2+) / µM
L1
L2
L3
L4
L5
4.5(2)
1.3(8)
39.6(4.9)
32.7(3.7)
1.7(1)
3.2(2)
0.5(2)
400(10)
3.3(3)
‡
‡
16.5(6.6)
1.8 17
17 17
n.d.
‡ No dissociation constants were determined from the Ca2+ and Zn2+ fluorescence titrations due to the
absence of significant spectral change. Corresponding Kd values for Mag-Fura-2 are 1.9 mM (Mg2+), 25
µM(Ca2+), 20 nM (Zn2+).
Fluorescence binding studies using L3 in competitive media
It is thought that ‘free’ Mg2+ concentrations are maintained within the narrow range of
0.7-1.1 mM in the blood plasma, comparable to the concentration of ‘free’ Mg2+ within
the majority of mammalian cells.37 Concentrations of ‘free’ Mg2+ in human serum are
often not determined due to the lack of techniques to detect ‘free’ Mg2+ ions
8
selectively. The APTRA fluorescent indicators, e.g. Mag-Fura-2, are unsuitable for
this task, due to elevated Ca2+ concentrations of around 1.1 mM in human serum. 31
The APDAP-based ligand L3 possesses many characteristics that render it potentially
suitable to detect ‘free’ Mg2+ in human serum. It has no pH interference in the
physiological range (pH 6.5-7.5) and exhibits a low mM binding affinity for Ca2+ (Kd =
0.4 mM). The binding of Mg2+ to L3 was analysed in a buffered ‘competitive medium’
containing 50 mM HEPES and concentrations of Na+ (140 mM), K+ (4 mM) and Ca2+
38
(1.1 mM) in accordance with the mean values found in human serum.
These
competitive binding studies were designed to provide an initial indication into how the
affinity for Mg2+ was affected following competitive cation binding, simulating whether
the APDAP chelate would respond to Mg2+ fluxes in human serum. A 26 nm
hypsochromic shift in the absorbance wavelength maximum was observed,
characteristic of partial saturation due to Ca2+ binding (Figure S12). A wavelength
maximum of 545 nm was measured, following excitation at 350 nm, and a 1.6-fold
‘turn-off’ response was found following addition of 3.5 mM Mg2+, (Figure 6).
13
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8
6
4
2
(A)
(B)
0
360
7
6
5
4
460 560
Wavelength (nm)
660
Kd = 0.11(1) mM
Figure 6. (A) Emission spectra of L3 following addition
of Mg2+ (3.5 mM). (B) Fitting of the emission intensity at
545 nm against the concentration of Mg2+ via a 1:1
binding model. (λex = 350 nm). Ligand concentration = 10
µM in 50 mM HEPES, 140 mM NaCl, 4 mM KCl,
1.1 mM CaCl2, pH 7.2, 298K.
0
0.001
0.002
0.003
Concentration (M)
A dissociation constant of 0.11(1) mM was calculated for the binding of Mg2+
in the buffered medium. Such a significant reduction in apparent affinity was
expected due to the presence of 1.1 mM CaCl2 that creates a mixture of unbound
and calcium-bound probe in solution, prior to any addition of Mg2+. Nevertheless, this
behaviour demonstrates that L3 can monitor variations in ‘free’ Mg2+ ions in a more
complex medium where concentrations of Ca2+ are above basal levels. Such an
approach is not possible with the APTRA-based sensors, because of the µM binding
affinity displayed for Ca2+.
Summary and Conclusions
Following the synthesis of four alkynyl-1-naphthalene fluorophores (L1–L4) of
differing ligand denticity, we have demonstrated that it is possible to tune Mg2+ /
Ca2+ selectivity. Each ligand possesses a pKa value in the range 5.1 to 6.2,
comparable to that of the commercial probe, Mag-Fura-2. The APTRA
analogues, L1 and L2 , were found to have comparable affinities for both Mg2+and
Ca2+ in the low mM and mid-µM range respectively, supporting literature precedent.
Changing the position of the nitrogen and oxygen donor groups, relative to the
naphthalene fluorophore, gave rise to significant differences in the absorbance,
emission and excitation spectral response to divalent cations. Ligand L1 displayed a
ratiometric response, due to the elimination of the ICT band on protonation and metal
ion binding, while a non-ratiometric ‘turn-on’ response was found for L2.
14
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By incorporating a phosphinate group into the ligand framework at the
expense of a carboxylate group, enhanced Mg2+ / Ca2+ binding selectivity is created.
The APDAP-based ligand, L3, displayed an order of magnitude reduction in affinity
for Ca2+, compared to L1 and L2 with a Kd value of 0.4(1) mM. Such affinity allowed
the binding of Mg2+ to be investigated in a ‘competitive medium’, highlighting
that L3 can be used to monitor variations in ‘free’ Mg2+ concentrations in solutions
where Ca2+ ions are present above basal levels. In the alkynyl-1-naphthyl systems
studied, the pentadentate binding chelates are preferred in order to match the
desired low mM affinity for Mg2+ binding. Reducing the ligand denticity lowered the
affinity for Mg2+; the tridentate ligand L4 displayed a 4-fold weaker binding with
respect to L1.
This comparative study highlights the advantages of using new ligand types
incorporating phosphinate donors in order to significantly change metal ion
selectivity. Further work is underway to exploit the differing selectivity profiles of
such systems, with different types of signal transduction, seeking systems combining
a ratiometric read-out and a longer a wavelength of excitation with the desired
magnesium selectivity profile. 1
Experimental
General procedures
All commercially available reagents were used as received from suppliers,
without further purification. All moisture sensitive reactions were carried out under
Schlenk-line techniques. The ethyl ester of APDAP, 1, was synthesised according to
our previous work.28 Thin-layer and column chromatography was performed on silica
1
(Merck Art 5554) and visualised under UV irradiation (254 nm). Routine H (400
MHz) and 13C (101 MHz) and 31P (162 MHz) NMR spectra were acquired on Varian
Mercury 400 NMR spectrometers. 13C NMR and 31P NMR spectra were run with
proton decoupling. Two-dimensional NMR spectra (COSY, HSQC and HSBC) were
run on Varian-600 (600 MHz) or VNMRS-700 (700 MHz) instruments. ES-MS data
was acquired on a Waters TQD mass spectrometer interfaced with an Acquity UPLC
system. Mass spectra of L1–L4 were recorded on a Waters Xevo QToF instrument in
an acetonitrile and ammonium bicarbonate buffered (25 mM) system.
15
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pKa Determination
pH measurements were recorded using a Jenway 3510 pH meter in
combination with a Jenway 924 005 pH electrode. The pH probe was calibrated
before each independent titration using buffer solutions of pH 4, 7 and 10. Samples
were prepared with a background of constant ionic strength (I = 0.1 M KCl, 298 K),
and titrated to acid. The resulting sigmoidal curve of fluorescence intensity vs. pH
was fitted by a non-linear least squares iterative analysis in Boltzmann using Origin
8.0 software. pKa values were calculated with an error associated with the fitting.
Metal ion binding studies
All divalent metal binding studies for the addition of Mg2+, Ca2+ and Zn2+ ([M2+])
were carried out in buffered solutions of 50 mM HEPES and 100 mM KCl maintained
at pH 7.2. All titrations were run in aqueous solution, with water purified by the
‘PuriteSTILLplus’ system with a conductivity of ≤0.04 µS cm-1. Stock solutions of [M2+]
were prepared containing the same concentration of the sensor used in the titration
to avoid sample dilution. Absorbance, emission and excitation spectra were run 5 min
after the addition of small aliquots of [M2+] to ensure the sample had fully equilibrated.
Dissociation constants (Kd values) for the binding of Mg2+ and Ca2+ were generated
from a 1:1 binding model obtained from a least square fitting iterative from
www.supramolecular.org. In the case of Zn2+ binding, Kd’s were calculated from a 1:1
binding model from Equation S3 and Equation S4 (See ESI for more information).
Errors associated with the Kd values were generated from two or more repetitions of
the binding experiments.
Ligand synthesis
Diethyl 2,2'-((4-bromo-2-((ethoxy(methyl) phosphoryl)methoxy) phenyl)
azanediyl)diacetate, (2)
O
N
O
O
14
11
2
12
O
O
O
P
13
9
1
10
O
3
7
8
4
6
5
Br
[K.18-crown-6]Br3 (132.3 mg, 0.24 mmol) was added to a solution of (1)
(82.5 mg, 0.21 mmol) in anhydrous acetonitrile (1.2 mL). The reaction was stirred at
room temperature for 1 h under an inert atmosphere of argon. Reaction completion
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was confirmed via ESI mass spectrometry. The solution was diluted with ethyl
acetate (10 mL) before being washed with water (10 mL) and dried over MgSO4. The
solvent was removed under reduced pressure to form a dark yellow / brown oil.
Purification by silica gel column chromatography (gradient 100 % hexane to 100 %
ethyl acetate) formed the title compound as a colourless oil. (101 mg, 82 %). 1H NMR
(700 MHz, CDCl3) 7.05 (1 H, dd, J 9, 2, H4), 7.00 (1 H, d, J 2, H6), 6.81 (1 H, d, J 9,
H3), 4.28 – 4.06 (12 H, m, H7, H9, H11, H13), 1.64 (3 H, d, J 15, H8), 1.33 (3 H, t, J 7,
H10), 1.23 (6 H, t, J 7, H14); 31P NMR (283 MHz, CDCl3) + 47.2; 13C NMR (176 MHz,
CDCl3) 170.6 (s, C12), 151.4 (d, J 12, C1), 138.6 (s, C2), 125.5 (s, C4), 122.0 (s, C3),
117.7 (s, C6), 114.7 (s, C5), 65.1 (d, J 110, C7), 61.0 (d, J 7, C9), 60.8 (s, C11 or C13),
53.2 (s, C11 or C13), 16.5 (d, J 6, C10), 14.1 (s, C14), 12.5 (d, J 98, C8); ESI-LRMS
[C18H2879BrNO7P]+ (+) m/z 480.1; ESI-HRMS calcd for [C18H2879BrNO7P]+ 480.0787
found 480.0778.
Ethyl 2‐({2‐[(dimethyl phosphoryl)methoxy]‐4‐{2‐[tris(propan‐2‐yl)silyl]ethynyl}
phenyl}(2‐ethoxy‐2‐oxoethyl)amino)acetate, (3)
12
14
O
O
13
7
11
O
N
O
P
9
2
O
O
3
4
O
10
1
8
6
5
15
16
Si
17
18
Compound (2) (273.9 mg, 0.57 mmol) was dissolved in anhydrous THF (1.8
mL) and the solution was degassed via three freeze-pump-thaw cycles.
(Triisopropylsilyl)acetylene (0.64 mL, 2.85 mmol), triethylamine (0.56 mL,
4.02 mmol), Pd(dppf)Cl2 (56.7 mg, 0.08 mmol) and copper iodide (22.0 mg,
0.1 mmol) were added, and the solution was degassed via a further three freeze-
pump-thaw cycles. The solution was stirred at 65 oC for 6 d under an inert
atmosphere of argon, reaction completion being determined by ESI mass
spectrometry. The solvent was removed under reduced pressure to form a dark
brown residue. Purification by silica gel column chromatography (gradient 100 %
CH2Cl2 to 95 % CH2Cl2 / 5 % MeOH) formed the title compound as a pale brown oil
1
(270.9 mg, 86 %). Rf (silica, 95 % CH2Cl2 / 5 % MeOH) = 0.54; H NMR (600 MHz,
CDCl3) 7.08 (1 H, dd, J 9, 2, H4), 6.97 (1 H, d, J 2, H6), 6.81 (1 H, d, J 9, H3), 4.31 –
17
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4.07 (12 H, m, H7, H9, H11 and H13), 1.65 (3 H, d, J 15, H8), 1.36 (3 H, t, J 7, H10), 1.25
(6 H, t, J 7, H14), 1.13 (3 H, br m, H17), 1.11 (18 H, s, H18); 31P NMR (243 MHz,
CDCl3) + 47.5; 13C NMR (151 MHz, CDCl3) 170.7 (s, C12), 149.9 (s, C1), 139.8 (s, C2),
126.8 (s, C4), 119.9 (s, C3), 117.5 (s, C6), 117.4 (s, C5), 106.6 (s, C15), 89.8 (s, C16),
65.3 (d, J 111, C7), 60.9 (d, J 7, C9), 60.8 (s, C11 or C13), 53.3 (s, C11 or C13), 18.7 (s,
C18), 16.5 (d, J 6, C10), 14.2 (s, C14), 12.6 (d, J 98, C8) 11.3 (s, C17); ESI-LRMS
[C29H49NO7PSi]+ (+) m/z 582.3; ESI-HRMS calcd for [C29H49NO7PSi]+ 582.3007 found
582.3016.
Diethyl 2,2'-((2-((ethoxy(methyl)phosphoryl)methoxy)-4-(naphthalen-1-
ylethynyl)phenyl)azanediyl)diacetate, (4)
O
O
11
6
13
O
14
N
O
P
12
9
1
2
O
O
O
10
7
3
8
5
4
15
16
17'
17
21'
18'
19'
18
19
21
20'
20
Compound (3) (252.5 mg, 0.434 mmol) and 1-bromonaphthalene (58 µL, 0.41
mmol) were dissolved in anhydrous THF (1.8 mL) and the solution was degassed by
three freeze-pump-thaw cycles. Triethylamine (1.2 mL), Pd(dppf)Cl2 (43.9 mg, 0.06
mmol) and copper iodide (61.2 mg, 0.32 mmol) were then added and the solution
was degassed once more. Tetrabutylammonium fluoride (TBAF, 1 M solution in THF,
0.6 mL, 0.6 mmol) was then added before the brown solution was degassed with
three final freeze-pump-thaw cycles. The reaction mixture was stirred at 65 oC for 19
h under an inert atmosphere of argon, before the solvent was removed under
reduced pressure to form a dark brown / black residue. Purification by column
chromatography (gradient 100 % hexane to 100 % ethyl acetate) formed the title
compound as a pale brown oil (24.1 mg, 10 %). 1H NMR (700 MHz, CDCl3) 8.39 (1 H,
d, J 8, H20’), 7.85 (1 H, d, J 9, H17’), 7.82 (1 H, d, J 8, H20), 7.72 (1 H, dd, J 7, 2, H18),
7.60 – 7.57 (1 H, m, H19’), 7.53 – 7.51 (1 H, m, H18’), 7.46 – 7.43 (1 H, m, H19), 7.25 (1
H, dd, J 6, 2, H5), 7.15 (1 H, d, J 2, H3), 6.90 (1 H, d, J 8, H6), 4.36 – 4.09 (14 H, m,
H7, H9, H11 and H13), 1.68 (3 H, d, J 15, H8), 1.37 (3 H, t, J 7, H10), 1.26 (6 H, t, J 7,
H14); 31P NMR (101 MHz, CDCl3) + 53.8; 13C NMR (176 MHz, CDCl3) 170.7 (s, C12),
150.1 (s, C2), 139.8 (s, C1), 133.2 (s, C17), 130.2 (s, C18), 128.3 (s, C17’), 128.6 (s,
18
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C20), 126.7 (s, C19’) 126.5 (s, C5), 126.4 (s, C21’), 126.3 (s, C20’), 126.2 (s, C18’), 125.3
(s, C19), 120.9 (s, C21), 120.0 (s, C6) 117.1 (d, J 7, C3) 94.0 (s, C15), 87.0 (s, C16), 64.6
(s, C7, C9, C11 or H13), 65.2 (s, C7, C9, C11 or H13), 60.9 (s, C7, C9, C11 or H13), 53.3 (s,
C7, C9, C11 or H13), 16.6 (s, C10), 14.2 (s, C14), 12.6 (d, J 97, C8); ESI-LRMS
[C30H35NO7P]+ (+) m/z 552.1; ESI-HRMS calcd for [C30H35NO7P]+ 552.2153 found
552.2151.
2,2'-((2-((Hydroxy(methyl)phosphoryl)methoxy)-4-(naphthalen-1-ylethynyl)
phenyl)azanediyl)diacetic acid, L3
O
OH
9
6
HO
10
N
O
P
1
2
O
O
OH
7
3
8
5
4
11
12
13'
16'
13
17'
14'
15'
14
15
17
16
The ethyl ester of L3 (4) (11 mg, 0.02 mmol) was dissolved in CD3OD (2 mL)
containing NaOD (0.4 M in D2O, 0.5 mL). The pale yellow solution was stirred under
1
argon at room temperature for 48 h. Ester hydrolysis was confirmed by H NMR
spectrometry and ESI-LRMS. The solution was lyophilised to form the title compound
as a pale brown solid in quantitative conversion (7 mg). QToF-LRMS [C24H21NO7P]–
(–) m/z 466.1; ESI-HRMS calcd for [C24H21NO7P]– (–) 466.1056 found 466.1044.
Reverse phase HPLC (0 % - 100 % - 0% CH3CN in ammonium bicarbonate buffer
(25 mM) tR = 7.1 min; ꢀ! ! = 0.7 %.
!
Ethyl 2-((4-bromo-2-iodophenyl)amino)acetate, (6)
7
8
O
9
10
NH
1
4
O
I
6
2
5
3
Br
N,N-Diisopropylethylamine (0.96 g, 1.3 mL, 7.50 mmol) and ethyl
bromoacetate (600 μL, 5.40 mmol) were added to a solution of 4-bromo-2-iodoaniline
(641.4 mg, 2.15 mmol) and sodium iodide (130.2 mg, 2.87 mmol) in anhydrous
o
acetonitrile (3.8 mL). The reaction was heated at 85 C for 24 h under an inert
19
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atmosphere of nitrogen, before additional N,N-diisopropylethylamine (1.2 mL, 6.89
mmol) and ethyl bromoacetate (470μL, 4.24 mmol) were added. The reaction mixture
o
was stirred for a further 40 h at 85 C. After cooling to room temperature the brown
reaction mixture was diluted with ethyl acetate (15 mL), filtered through celite, and
washed with water (10 mL) and brine (10 mL). Organic extracts were combined and
dried over MgSO4, and the solvent was removed under reduced pressure to form a
brown residue. Purification by silica gel column chromatography (gradient 100 %
hexane to 80 % hexane / 20 % ethyl acetate) formed the title compound as an off-
white solid (565 mg, 68 %). Rf (80 % hexane / 20 % ethyl acetate) = 0.72; m.p. 77 -
o
1
78 C; H (700 MHz, CDCl3) 7.77 (1 H, d, J 2, H3), 7.29 (1 H, dd, J 9, 2, H5), 6.29 (1
H, d, J 9, H6), 4.86 (1 H, br t, J 5, -NH), 4.26 (2 H, q, J 7, H9), 3.89 (2 H, d, J 5, H7),
1.30 (3 H, t, J 7, H10); 13C (176 MHz, CDCl3) 170.0 (C8), 145.4 (C1), 140.6 (C3), 132.1
(C5), 111.4 (C4), 109.6 (C6), 85.3 (C2), 61.6 (C7), 46.0 (C9), 14.1 (C10); ESI-LRMS
[C10H11NI79Br]+ (+) m/z 384.1; ESI-HRMS calcd for [C10H11NI79Br]+ 383.9096 found,
383.9099.
Ethyl 2-((4-bromo-2-(ethoxy(methyl)phosphoryl)phenyl)amino) acetate, (7)
7
8
O
9
10
NH
O
P
12
1
4
O
O
6
13
2
11
5
3
Br
Ethyl 2-((4-bromo-2-iodophenyl)amino)acetate, (6) (390.2 mg, 1.02 mmol),
ethyl methyl phosphinate (as its EtOH adduct, 200.8 mg, 1.30 mmol) and
triethylamine (0.58 mL, 4.16 mmol) were added to dry toluene (degassed by three
freeze-pump-thaw cycles). The solution was degassed by a further three cycles
before the addition of Pd(dppf)Cl2.CH2Cl2 (19.2 mg, 0.024 mmol). The reaction
o
mixture was stirred at 118 C under an inert atmosphere of argon for 22 h. After
cooling to room temperature the solvent was removed under reduced pressure to
form a dark brown residue. Purification by silica gel column chromatography
(gradient 100 % hexane to 1:1 ethyl acetate / hexane) formed the title compound as
1
a yellow oil (137 mg, 39 %). Rf (silica, 1:1 ethyl acetate / hexane) = 0.52; H (600
MHz, CDCl3) 7.50 (1 H, br s, NH), 7.43 (1 H, dd, J 12, 2, H3), 7.38 (1 H, dd, J 8, 2,
H5), 6.38 (1 H, dd, J 8, 6, H6), 4.21 (2 H, q, J 7, H9), 4.13 (2 H, m, H12), 3.89 (2 H, br
s, H7), 1.66 (3 H, t, J 15, H11), 1.32 (3 H, t, J 7, H13), 1.25 (3 H, t, J 8, H10); 13C (151
MHz, CDCl3) 170.1 (s, C8), 150.1 (d, J 7, C1), 136.5 (d, J 2, C5), 134.8 (d, J 10, C3),
20
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114.1 (d, J 122, C2), 113.7 (s, C), 112.9 (d, J 10, C6), 108.0 (d, J 15, C4), 61.3 (s, C9),
60.9 (d, J 7, C12), 45.1 (s, C7), 16.4 (d, J 7, C13), 16.3 (d, J 106, C11), 14.2 (s, C10); 31
P
(243 MHz, CDCl3) + 45.6; ESI-LRMS [C13H1979BrNO4P]+ (+) m/z 364.5; ESI-HRMS
calcd for [C13H1979BrNO4P]+ 364.0313 found, 364.0320.
Ethyl 2-((2-(ethoxy(methyl)phosphoryl)-4-((triisopropylsilyl)ethynyl)phenyl)
amino)acetate, (8)
10
12
O
13
NH
O
P
11
8
1
2
O
6
O
9
3
7
5
4
14
15
16
Si
17
Compound (7) (757.0 mg, 2.09 mmol) was dissolved in anhydrous THF (7
mL) and the solution was degassed via three freeze-pump-thaw cycles.
(Triisopropylsilyl)acetylene (2.4 mL, 10.7 mmol), triethylamine (2.2 mL, 15.8 mmol),
Pd(dppf)Cl2 (157.3 mg, 0.21 mmol) and copper iodide (116.3 mg, 0.61 mmol) were
added, and the solution was degassed via a further three freeze-pump-thaw cycles.
o
The solution was stirred at 65 C for 3 d under an inert atmosphere of argon.
Reaction completion was determined by ESI mass spectrometry. The solvent was
removed under reduced pressure to form a dark brown residue. Purification by silica
gel column chromatography (gradient 100 % hexane to 100 % ethyl acetate 4:1)
formed the title compound as a pale brown oil which slowly solidified to a pale brown
solid overnight (747 mg, 77 %). m.p = 82 oC – 83 oC; 1H NMR (600 MHz, CDCl3) 7.71
(1 H, br s, NH), 7.47 (1 H, dd, J 14, 2, H3), 7.42 (1 H, dd, J 9, 2, H5), 6.40 (1 H, dd, J
9, 6, H6), 4.21 (2 H, q, J 7, H12), 4.16 – 3.87 (4 H, m, H8 and H10), 1.68 (3 H, d, J 15,
H7), 1.32 (3 H, t, J 7, H9), 1.26 (3 H, t, J 7, H13), 1.10 (21 H, s, H16 and H17); 31P NMR
(243 MHz, CDCl3) + 46.6; 13C NMR (151 MHz, CDCl3) 170.1 (s, C11), 150.8 (d, J 7,
C1), 137.6 (d, J 2, C5), 136.5 (d, J 10, C3), 113.4 (d, J 123, C2), 111.3 (d, J 14, C4),
110.7 (d, J 10, C6), 61.3 (s, C12),106.8 (s, C14), 88.5 (s, C15) 60.9 (d, J, 6, C8), 45.0 (s,
C10), 18.7 (s, C17), 16.5 (d, J 105, C7), 16.3 (d, J 7, C9), 14.2 (s, C13), 11.3 (s, C16);
ESI-LRMS [C24H41NO4PSi]+ (+) m/z 466.2; ESI-HRMS calcd for [C24H41NO4PSi]+
466.2543 found 466.2535.
21
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10.1002/chem.201800013
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Walter/Williams/ Parker
Ethyl 2-((2-(ethoxy(methyl)phosphoryl)-4-(naphthalen-1-ylethynyl)phenyl)
amino) acetate, (9)
10
12
O
13
NH
O
P
11
8
1
2
O
6
O
9
3
7
5
4
14
15
16
16
20'
17'
18'
17
18
20
19'
19
Compound (8) (109.7 mg, 0.24 mmol) and 1-bromonaphthalene (31 µL, 0.22
mmol) were dissolved in anhydrous THF (1 mL) and the solution was degassed by
three freeze-pump-thaw cycles. Triethylamine (0.7 mL), Pd(dppf)Cl2 (40.6 mg, 0.06
mmol) and copper iodide (42.1 mg, 0.22 mmol) were then added and the solution
was degassed once more. Tetrabutylammonium fluoride (TBAF, 1 M solution in THF,
0.35 mL, 0.35 mmol) was then added before the brown solution was degassed with
three final freeze-pump-thaw cycles. The reaction mixture was stirred at 65 oC for 18
h under an inert atmosphere of argon, before the solvent was removed under
reduced pressure to form a dark brown / black residue. Purification by column
chromatography (gradient 100 % hexane to 100 % ethyl acetate) formed the title
1
compound as a pale orange oil (78 mg, 20 %). H NMR (700 MHz, CDCl3) 8.40 (1 H,
d, J 8, H20’), 7.85- 7.83 (2 H, m, H17’ and NH), 7.80 (1 H, d, J 8, H20), 7.71 (1 H, dd, J
7, 1, H18), 7.65 (1 H, dd, J 15, 2, H3), 7.60 – 7.56 (2 H, m, H5 and H19’), 7.52 – 7.50 (1
H, m, H18’), 7.44 – 7.42 (1 H, m, H19), 6.51 (1 H, dd, J 9, 6, H6), 4.24 (2 H, q, J 7, H12),
4.19 – 3.93 (4 H, m, H10 and H8), 1.73 (3 H, d, J 15, H7), 1.35 (3 H, t, J 7, H9), 1.28 (3
H, t, J 7, H13); 13C NMR (176 MHz, CDCl3) 170.1 (s, C11), 151.0 (d, J 6, C1), 137.1 (d,
J 2, C5), 136.2 (d, J 10, C3), 133.2 (d, J 14, C2), 130.0 (s, C18), 128.3 (s, C17’ and C20),
126.6 (s, C19’), 126.4 (s, C18’), 126.2 (s, C20’), 125.3 (s, C19), 121.2 (s, C21), 112.5 (s,
C17’ or C21’), 111.7 (s, C17’ or C21’), 111.1 (s, C4), 110.0 (d, J 10, H6), 94.1 (s, C14),
86.1 (s, C15), 61.4 (s, C12), 60.9 (d, J 6, C8), 45.1 (s, C10), 16.44 (d, J 106, C7), 16.4
(d, J 7, C9), 16.1 (s, C13); 31P NMR (283 MHz, CDCl3) + 46.7; ESI-LRMS
[C25H27NO7P]+ (+) m/z 436.4; ESI-HRMS calcd for [C25H27NO7P]+ 436.1678 found
436.1672.
22
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10.1002/chem.201800013
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ChemEurJ 2018
Walter/Williams/ Parker
2-((2-(Hydroxy(methyl)phosphoryl)-4-(naphthalen-1-ylethynyl) phenyl)amino)
acetic acid, L4
7
8
HO
NH
O
P
1
4
O
2
OH
6
5
3
9
10
11
11’
14'
15'
12
13
12’
13'
15
14
The ethyl ester of L4 (9) (46.0 mg, 0.10 mmol) was dissolved in CD3OD (1.8
mL) containing NaOD (0.4 M in D2O, 0.2 mL). The pale yellow solution was stirred
under argon at room temperature for 4 d. Ester hydrolysis was confirmed by 1H NMR
spectrometry, and ESI-LRMS. The solution was then lyophilised to form the title
compound as a pale brown solid, in quantitative conversion (30 mg). QToF-LRMS
[C21H17NO4P]– (-) m/z 466.1; ESI-HRMS calcd for [C21H17NO4P]– (-) 378.0905 found
1
378.0895. H NMR (400 MHz, D2O) 8.19 (1 H, d, J 8), 7.69 – 7.60 (3 H, m), 7.48 –
7.47 (1 H, d, J 7), 7.43 (1 H, t, J 7), 7.36 – 7.21 (4 H, m), 6.27 (1 H, m), 1.32 (3 H, d,
J 14); 31P (176 MHz, D2O) + 31.7; Reverse phase HPLC (0 % - 100 % - 0% CH3CN in
ammonium bicarbonate buffer (25 mM)), tR = 6.6 min; ꢀ! != 0.8 %.
!
Acknowledgements We thank EPSRC (EP/ L01212X/1) for generous support.
Key Words Magnesium; fluorescence; selectivity; ligand; calcium
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