Pyrophosphate-induced reorganization of a reporter-receptor assembly via boronate esterification; A new strategy for the turn-on fluorescent detection of multi-phosphates in aqueous solution

Article abstract of DOI:10.1039/b808027e

A new strategy for the fluorescent detection of multi-phosphates in aqueous solution is presented here. Zn^II-DPA(DPA = dipicolylamine)-appended phenylboronic acid 1?Zn forms an assembly with alizarin dye in MeOH-10 mM HEPES (1: 1 v/v) containing 10 mM NaCl at pH 7.4 at 25 °C, in which the dye binds favorably to the coordinated zinc(ii) in the Zn^II-DPA moiety. Addition of pyrophosphate (PPi) as a putative analyte causes reorganization of the complex to produce an alternative boronate ester assembly, which causes an increase in fluorescence, detectable by the naked eye. It is interesting to note that the system exhibited PPi-selectivity over other phosphates such as ATP (adenosine 5′-triphosphate), ADP (adenosine 5′-diphosphate), AMP (adenosine 5′-monophosphate) and Pi (inorganic phosphate); the competitive assay employed to determine the apparent association constants of 1?Zn with the anion analytes allows us to estimate that the binding with PPi [(1.6 ± 0.04) × 10⁶ M^-1], is 10-fold and 84-fold higher than with ATP and ADP, respectively. The sensing mechanism of 1?Zn in the presence of alizarin dye is explored using pH titrations and structural information is obtained using NMR.

Full text of DOI:10.1039/b808027e

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Pyrophosphate-induced reorganization of a reporter–receptor assembly via
boronate esterification; a new strategy for the turn-on fluorescent detection of
multi-phosphates in aqueous solution†
Aiko Nonaka,a Shoichi Horie,a Tony D. Jamesb and Yuji Kubo*c
Received 12th May 2008, Accepted 18th June 2008
First published as an Advance Article on the web 12th August 2008
DOI: 10.1039/b808027e
A new strategy for the fluorescent detection of multi-phosphates in aqueous solution is presented here.
ZnII–DPA(DPA = dipicolylamine)-appended phenylboronic acid 1·Zn forms an assembly with alizarin
dye in MeOH–10 mM HEPES (1 : 1 v/v) containing 10 mM NaCl at pH 7.4 at 25 ◦C, in which the dye
binds favorably to the coordinated zinc(II) in the ZnII–DPA moiety. Addition of pyrophosphate (PPi) as
a putative analyte causes reorganization of the complex to produce an alternative boronate ester
assembly, which causes an increase in fluorescence, detectable by the naked eye. It is interesting to note
that the system exhibited PPi-selectivity over other phosphates such as ATP (adenosine 5ꢀ-triphosphate),
ADP (adenosine 5ꢀ-diphosphate), AMP (adenosine 5ꢀ-monophosphate) and Pi (inorganic phosphate);
the competitive assay employed to determine the apparent association constants of 1·Zn with the anion
analytes allows us to estimate that the binding with PPi [(1.6 0.04) × 106 M−1], is 10-fold and 84-fold
higher than with ATP and ADP, respectively. The sensing mechanism of 1·Zn in the presence of alizarin
dye is explored using pH titrations and structural information is obtained using NMR.
The role of the phenylboronic acid segment is significant; it can
form a complex with the catechol-containing dye via boronate
Introduction
Anions are ubiquitous in biological systems1 that play significant
roles in the wide areas of biology, pharmacy, and environmental
science. The design of receptor systems for anion recognition has
therefore developed into a key area of supramolecular chemistry,2
whereby, in particular, fluorescent chemosensors have received
considerable attention due to their analytical applications.3 One
route to prepare molecular sensor systems, avoiding extensive
synthetic chemistry, is through the development of self-organized
receptor–reporter systems, obtained by linking molecular units
through reversible interactions.4 For instance, the indicator dis-
placement assay, pioneered by Anslyn and Nguyen, is a valuable
means of analyte detection.5 The study presented here has
been driven by a new approach that involves an anion-induced
reorganization of the reporter–receptor assembly. The idea is
that the binding of an anion will induce a structural change
in the system, followed by a switch in the optical properties of
the reporter. Our system incorporates a catechol-containing dye
such as alizarin (reporter) and ZnII–DPA(DPA = dipicolylamine)-
appended phenylboronic acid 1·Zn, which serves as a receptor.
esterification and induce a change in the optical properties.6 As
part of this investigation we discovered that pyrophosphate (PPi),
which binds strongly to the ZnII–DPA, causes the reporter dye to
be expelled from the ZnII–DPA, then the reporter forms a boronate
ester with the boronic acid segment of 1·Zn. This change in the
assembly mode causes the fluorescence intensity to increase. The
phenomenon allows us to design a new type of chemosensor for
multi-phosphates. Such detection of multi-phosphates is worthy
of investigation because there is a demand for their analytical
detection in clinical applications.7 In this regard, while fluorogenic
receptors capable of sensing PPi in aqueous media are fascinating
targets, their exploration is still in its infancy, presumably due to
the fact that it is not easy to detect PPi selectively in the presence of
other kinds of phosphate such as ATP (adenosine 5ꢀ-triphosphate)
and ADP (adenosine 5ꢀ-diphosphate) in aqueous solution.8
aDepartment of Applied Chemistry, Graduate School of Science and Engi-
neering, Saitama University, 255 Shimo-ohkubo, Sakura-ku, Saitama, 338-
8570, Japan
bDepartment of Chemistry, University of Bath, Bath, UK BA2 7AY
cDepartment of Applied Chemistry, Graduate School of Urban Environmen-
tal Sciences, Tokyo Metropolitan University, 1-1 Minami-ohsawa, Hachioji,
Tokyo, 192-0397, Japan. E-mail: yujik@tmu.ac.jp; Fax: +81-42-677-3134;
Tel: +81-42-677-3134
Results and discussions
Synthesis
† Electronic supplementary information (ESI) available: Fluorescence
spectra of ARS plus 1·Zn upon adding PPi; fluorescence spectra of ARS
plus phenylboronic acid upon adding PPi; 1H,1H COSY spectrum of
alizarin plus 1·Zn with PPi; fluorescence spectra of ARS upon adding
incremental amounts of 3·Zn. See DOI: 10.1039/b808027e
We designed phenylboronic acid derivative 1·Zn as the receptor
moiety in our system; it is well-known that ZnII–DPA serves
as a suitable phosphate-binding site in aqueous solution for
developing not only artificial enzymes9 but also chemosensors.10
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The synthesis is straightforward as shown in Scheme 1. The
reductive amination of N,N-bis(2-pyridylmethyl)ethylenediaime
211 with 3-formylphenylboronic acid was carried out using
NaBH4, and the resulting DPA-appended phenylboronic acid
1 was allowed to react with Zn(NO3)2 to yield the tar-
get 1·Zn. The control 3·Zn was also synthesized from the
Boc-protected compound, N-tert-butoxycarbonyl-Nꢀ,Nꢀ-bis(2-
pyridylmethyl)ethylenediamine, 311 in a similar manner. The com-
pounds were characterized using spectroscopic methods, which
assembly in the presence of 5 equiv. of 1 under these conditions.
Next, the addition of ZnII to the solution involving ARS (50 lM)
and 1 (5 equiv.) under similar conditions induced a “turn-off”
in the fluorescence spectra (Fig. 1(b)). The quenched spectrum
with 5.3 equiv. of ZnII is almost consistent with that of ARS in
the presence of 5 equiv. of 1·Zn, suggesting that coordinated ZnII
in the ZnII–DPA species interacts with the ARS. Evidence for
the assembly formation came from ESI-MS spectroscopic data
using alizarin and 1·Zn in MeOH (m/z = 691.7635 (calcd for
[C36H34BN4O6Zn]+; 692.1894). The fluorescence properties of ARS
under several conditions in MeOH–H2O (1 : 1 v/v) were also
investigated by carrying out spectrofluorometry at varying pH
where a solution containing excess acid was titrated with standard
base (Fig. 2). ARS has almost no fluorescence over a large pH
range from 2 to 12 (Fig. 2; ●), whereas in the presence of 1·Zn
(5 equiv.) the fluorescence intensity at 586 nm increased up to pH 6,
followed by a decrease in the fluorescence intensity (Fig. 2; ꢀ).
We can ascribe the observed fluorescence enhancement to the
formation of an ARS–1·Zn ensemble via boronate esterification. In
contrast, the decrease in the fluorescence intensity at pH > 6 is due
to the more favorable interaction between the catechol segment of
ARS and the coordinated ZnII in the ZnII-DPA species. The change
in the assembly mode is probably controlled by the pKa value of
ARS which is 5.5.12 On another front, the presence of PPi as a
putative analyte in the solution involving ARS and 1·Zn produced
a different pH profile when compared to the PPi-free solution
(Fig. 2; ꢁ); the fluorescence intensity increases more effectively
and reaches a maximum between pH 6 to 8. This significant
fluorescence enhancement under neutral conditions motivated us
to set up the conditions for applying the ARS–1·Zn assembly to
PPi sensing. The fluorescence titration of ARS (50 lM) with PPi in
the presence of 1·Zn (5 equiv.) was carried out in MeOH–10 mM
1
are found in the Experimental section. The H NMR spectrum
of the new compound was insightful as the methylene resonances
(3.87 ppm, s) of the DPA part of 1 were altered to AB double
doublets (4.25 ppm, d, J = 17.1 Hz; 4.38 ppm, d, J = 17.0 Hz)
upon metallation with ZnII, for example.
Scheme 1 Synthesis of 1·Zn. Reagents and conditions: (i) 3-formylphenyl-
boronic acid then NaBH4, EtOH, 12%; (ii) Zn(NO3)2·6H2O, MeOH, 89%.
Evaluation
◦
HEPES (1 : 1 v/v) containing 10 mM NaCl at pH 7.4 at 25 C;
Initially, to get an insight into the fluorescence behavior of ARS
(alizarin red S) as the reporter, stepwise titrations using the ZnII-
free ligand 1 were carried out in MeOH–10 mM HEPES (1 : 1 v/v)
containing 10 mM NaCl at pH 7.4 at 25 ◦C (Fig. 1) (HEPES = 2-
[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid). Although
ARS has almost no emission under these conditions, the flu-
orescence intensity ranging from 550 nm to 600 nm increased
upon the addition of incremental amounts of 1 (Fig. 1(a)).
This phenomenon can be explained on the basis of boronate
esterification between ARS and 1,6 the binding constant being
estimated as (3.8 0.44) × 104 M−1. One can compute from the
binding constant that 91% of ARS can be converted to the ARS–1
as expected, the fluorescence intensity at 586 nm increases upon
addition of PPi, with factors of 2.75 (250 lM of PPi) (see ESI†),
the behavior being detected by the naked-eye. The presence of
the ZnII–DPA segment is significant for the response. Indeed, the
use of ZnII–DPA-free phenylboronic acid instead of 1·Zn induced
almost no fluorescence enhancement by adding PPi under similar
conditions (ESI†).
Fig. 2 Spectrofluorimetric pH-titrations of ARS (●), ARS plus 1·Zn (ꢀ)
and ARS plus 1·Zn with PPi (ꢁ) in MeOH–H2O (1 : 1 v/v) containing
10 mM NaCl at 25 ◦C; [ARS] = 50 lM, [1·Zn] = 250 lM, [PPi] = 250 lM,
kex = 480 nm; kem = 586 nm.
Fig. 1 (a) Change in the fluorescence spectra of ARS (50 lM) upon
adding 1 in MeOH–10 mM HEPES (1 : 1 v/v) containing 10 mM NaCl at
pH 7.4 at 25 ◦C, kex = 501 nm; (b) Change in the fluorescence spectra of
ARS (50 lM) in the presence of 1 (250 lM) upon adding ZnII in MeOH–
10 mM HEPES (1 : 1 v/v) containing 10 mM NaCl at pH 7.4 at 25 ◦C,
kex = 480 nm. I0 is fluorescence intensity under the ZnII-free conditions.
1
The binding profile was investigated based on H NMR data
whereby alizarin was employed in place of ARS because the H3
signal (∇) of alizarin is considered to be diagnostic for boronate
ester formation;6c Fig. 3 shows how the aromatic protons of each
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together, a plausible mechanism for the PPi-induced fluorescence
enhancement in the assembly is illustrated in Scheme 2. ARS
binds to 1·Zn efficiently to form the ARS–1·Zn assembly, which
is equilibrating between the boronate ester form (A) and the
catechol–ZnII form (B). Under neutral conditions, form (B) is more
favoured than form (A) and suppresses the fluorescence arising
from ARS. When PPi is added into the solution, PPi favorably
binds to the (A) form, producing a shift in the equilibrium towards
(A). The ditopic binding of PPi to (A) affords the ternary complex,
PPi–ARS–1·Zn, accompanied by a fluorescence enhancement.
Fig. 3 1H NMR spectra in CD3OD–D2O (9 : 1 v/v) (400 MHz) at 23 ◦C.
[alizarin] = 2.4 mM, [1·Zn] = 2.4 mM; (a) alizarin, (b) 1·Zn, (c) alizarin
plus 1·Zn, (d) alizarin plus 1·Zn with PPi. The spectrum of (d) was obtained
after (solid (PPi)–liquid (2.4 mM of alizarin and 1·Zn in CD3OD–D2O (9 :
1 v/v))) two-phase extraction in view of the low solubility of PPi under
NMR detectable conditions.
Scheme 2 A plausible mechanism for the PPi-induced fluorescence
enhancement.
component reflect the PPi-induced organization in the assembly.
No perturbation of the chemical shifts for alizarin and 1·Zn was
observed when they are mixed (Fig. 3(a) and (b) vs. Fig. 3(c)).
However, when PPi was added to the mixture the chemical shifts
changed dramatically and became complicated (Fig. 3(d)): an
additional 1H,1H COSY measurement (ESI†) helped us to assign
the signals; the resonances arising from alizarin were clearly
distinguishable signals in the presence of PPi. These observations
can be explained on the basis of boronate ester formation whereby
the PO–B (sp3) dative bond would be partially subject to solvolysis
in protic media.13 The entirely up-field shift of the resonances here
fully supports the formation of a boronate ester;6c for example, the
H∇ signal was significantly shifted from 7.22 (d, J = 8.3 Hz) to 6.75
(d, J = 8.0 Hz) and 6.81 ppm (brd). In contrast, we notice that the
a-protons of the pyridine ring (H8) were down-field shifted from
8.78 (d, J = 4.6 Hz) to 9.02 ppm and somewhat broadened upon
addition of PPi, indicating that PPi binds to the coordinated ZnII
in the ZnII-DPA species.8c These insights allow us to postulate that
the PPi is ditopically bound within the alizarin–1·Zn assembly,
both to the ZnII and the boronate ester segment. In 11B NMR
measurements, an upfield shift (Dd = 15.82 ppm) of the boron
signal was observed upon the addition of PPi in a MeOH–D2O (9 :
1 v/v) solution of 1·Zn (2.4 mM) and alizarin (2.4 mM). However,
we also detected in the spectra signals assignable to ZnII-free DPA;
The anion selectivity of the ARS–1·Zn system was then exam-
ined using PPi, ATP, ADP, AMP (adenosine 5ꢀ-monophosphate),
2−
−
Pi (HPO4 mainly equilibrates with H2PO4 under the neutral
conditions) and other biologically as well as chemically important
anions, in MeOH–10 mM HEPES (1 : 1 v/v) containing 10 mM
NaCl at pH 7.4 at 25 ◦C. As shown in Fig. 4, when titrations with
PPi were performed a significant enhancement in the fluorescence
intensity was obtained. Almost similar but less effective responses
were obtained in the case of ATP and ADP addition, whereas
AMP as well as Pi induced no response in the fluorescence spectra.
Consequently, ARS–1·Zn shows a selective response towards
phosphates in the following order; PPi > ATP > ADP > AMP >
Pi. The addition of carboxylates as putative oxoanions other than
phosphates induced either no (for AcO−) or a low response (for
ꢀꢀ
ꢀꢀ
8.15 (d, J = 4.8 Hz, H8 ), 7.52 (td, J = 7.7, 1.7 Hz, H6 ), 7.10 (d, J =
ꢀꢀ
ꢀꢀ
7.9 Hz, H5 ) and 7.06 ppm (dd, J = 7.5, 5.1 Hz, H7 ). The singlet
peak at 3.77 ppm is also assignable to the ZnII-free NCH2Pyr.
One can envisage that the PPi-associated alizarin–1·Zn assembly
is susceptible to solvolysis to afford solvent-inserted alizarin-1 and
Zn2P2O7. Buffer-free conditions during the NMR measurements
may facilitate decomposition of the ZnII–DPA segment. Taken
Fig. 4 Plots of fluorescence intensity at 586 nm of ARS (50 lM) and
5 equiv. of 1·Zn as a function of anion concentration.
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citrate). Also one can detect no fluorescence enhancement under
84, compared to ATP and ADP, respectively. This selectivity is
presumably due to the total anionic density of the P–O involved
in the interaction between ARS-1·Zn and multi-phosphorylated
species.8e
−
these conditions using monovalent (HCO3−, F−, Br−, I−, NO3
,
ClO4 and N3−) as well as divalent (SO42−) anions. Subsequently
we tried to determine the association constants from the titration
data. However, the titration curve obtained for the addition of PPi
(0–500 lM) displayed a somewhat sigmoidal progression because
of multiple equilibria as well as an excess amount of 1·Zn (also
capable of binding to the anion) making it impossible for simple
nonlinear curve fitting to reproduce the observed progression.
Thus, although the anion-triggered fluorescence enhancement is
not due to the regular indicator displacement assay, we decided
to determine the apparent binding constant of PPi to the receptor
1·Zn (Ka) in line with the assumption based on the equilibrium
from eqn (1)
−
Conclusions
We synthesized the ZnII–DPA-appended phenylboronic acid 1·Zn,
which interacts with ARS in aqueous solution to form an ARS–
1·Zn assembly with a low fluorescence intensity under neutral
conditions. The binding of PPi could induce a structural variation
in the assembly, followed by a switch in its optical properties.
Although the preliminary results represented here require an
improvement in the responsive ability toward target anions, our
proposed system has conceptual novelty with regard to the sensing
principle for the detection of multi-phosphates in aqueous media.
One merit of the system is that a judicious choice of catechol-
containing dye as the indicator, as well as a change in the metal
ion coordinated to the DPA, would allow us to tune not only
spectral but also selectivity features as required. Also, given the
rich chemistry of boronic acids, variation of the receptor moiety
is possible. Further elaborated systems based on this concept are
now underway.
ARS–1·Zn + G ꢂ G–1·Zn + ARS*
(1)
where ARS–1·Zn is regarded as form B in Scheme 2, G is the
guest anion, and G–1·Zn and ARS* represent a G-coordinated
ZnII–DPA entity and a boronate esterified ARS, respectively. The
mass balance equation and the equilibrium constants (KI and Ka)
were used to define P and Q.14 For this approach, the binding
constant of ARS to ZnII–DPA in the form B could be estimated as
KI; we carried out the fluorescent titration of ARS upon adding
incremental amounts of phenylboronic acid-free ZnII-DPA, 3·Zn,
being estimated for KI as 5.4 × 104 M−1 (ESI†).
Experimental
P = [1·Zn]t − 1/(QKI) − [ARS*]t/(Q + 1)
NMR spectra were taken on Bruker DRX-400 or DPX-400 (1H:
400 MHz; 13C: 100.7 MHz; 11B: 128 MHz) spectrometers. Chemical
shifts (d) are reported downfield from the initial standard Me4Si.
For 11B NMR 15% BF3–Et2O was employed as the external
standard. Electrospray ionization mass spectra were obtained on
a Mariner System 5231 spectrometer whereas a Jeffermine D2000
was employed for the calibration. Fast atom bombardment (FAB)
mass spectra were obtained on a JEOL JMS-DX 303 double
focusing spectrometer where m-nitrobenzyl alcohol was used as a
matrix. Elemental analyses were obtained on an EISON EA1108
or ThermoFinigan Flash EA1112. Fluorescence and absorption
spectra were measured using a JASCO FP-6300 and Shimadzu
UV-3100PC spectrophotometer, respectively. Reagents used for
the synthesis were commercially available and used as supplied.
ARS and alizarin were recrystallized from EtOH. Dry EtOH
was prepared according to standard procedures. The MeOH and
H2O for spectroscopic measurements were purchased as analytical
grade and used as received.
Q = [ARS*]/[ARS − 1·Zn]
Q = (I − I0)/(Ilim − I)
Q is termed the indicator ratio, and can be obtained using the
fluorescence intensity of ARS when coordinated to ZnII in the form
B (I0) and when existing as the esterified boronate (Ilim). Fig. 5
shows the relationship between Gt/P vs. Q, where in the case of
PPi it is hard to obtain a linear relationship between them. In
particular, at Q < 2.5 ([PPi]t = 167 lM, 3.34 equiv.), the deviation
became large. This result could be explained on the basis of an
excess amount of 1·Zn compared to that of ARS in the solution;
PPi could bind more efficiently to free 1·Zn than to ARS-bound
1·Zn. The linearity was consequently obtained ranging from 167
to 300 lM of [PPi]t, which allows us to elucidate the apparent
association constant Ka by dividing KI by the slope of the plot
(Fig. 5). Three individual measurements afforded (1.6 0.04) ×
106 M−1 as the Ka value with PPi. Similar analyses were carried
out for other phosphates (ATP and ADP), as shown in Fig. 5, to
elucidate the Ka with ATP and ADP as (1.6 0.28) × 105 M−1
and (1.9
0.19) × 104 M−1, respectively. It is noteworthy that
N-3-Dihydroxyborylbenzyl-Nꢀ,Nꢀ-bis(2-pyridylmethyl)
ethylenediamine 1
1·Zn exhibited selective binding to PPi by a factor of 10 and
Under an Ar atmosphere, N,N-bis(2-pyridylmethyl)ethylene-
diamine (4.60 g, 19.0 mmol) and 3-formylphenylboronic acid
(2.85 g, 19.0 mmol) were dissolved in dry EtOH (300 mL) that had
been degassed by three freeze–pump–thaw cycles. The solution,
˚
containing molecular sieves 3 A (10 g), was stirred overnight at
room temperature. After checking the progress of the reaction by
TLC, a dry EtOH solution (80 mL) of NaBH4 (1.44 g, 38.1 mmol)
was added to the solution and the mixture was further stirred
at room temperature for 1 h. After filtration the solution was
evaporated in vacuo. The residue was partitioned between AcOEt
(300 mL) and H2O (300 mL) and the water phase was extracted
Fig. 5 A competitive titration algorithm to determine the association
constants of 1·Zn with the multi-phosphates; PPi (●), ATP (ꢂ), ADP (ꢀ).
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with CH2Cl2 (50 mL × 10). The organic phase was dried with dry
Na2SO4 and filtered. The material obtained was chromatographed
on silica gel (Wakogel C-300) using a gradient of MeOH (0–100%
(v/v)) in CH2Cl2 as an eluent, and then washed with AcOEt and
Et2O. In this way, 843.9 mg of 1 was obtained in 12% yield.
1H NMR (400 MHz, CD3OD) d 8.25 (d, J = 4.5 Hz, 2H),
7.64 (td, J = 7.7, 1.7 Hz, 2H), 7.62 (d, J = 7.1 Hz, 1H), 7.58 (s,
1H), 7.28 (t, J = 7.4 Hz, 1H), 7.17–7.23 (m, 5H), 4.11 (s, 2H),
3.87 (s, 4H), 3.25 (t, J = 5.4 Hz, 2H), 3.04 (t, J = 5.5 Hz, 2H);
13C NMR (100.7 MHz, 50 mM in CD3OD) d 159.9, 149.8, 138.6,
135.3, 135.1, 131.4, 128.3, 127.2, 124.9, 123.8, 60.6, 53.0, 52.7,
46.4; ESI MS: m/z 377 ([M + H]+); elemental analysis: anal. calcd
for C21H25BN4O2·0.3 H2O: C 66.09; H 6.76; N 14.68%, found: C
65.96; H 6.64; N 14.28%.
36.60, 28.67; ESI MS: m/z 468 ([M − NO3]+); elemental analysis:
anal. calcd for C19H26N6O8Zn: C 42.91; H 4.93; N 15.80%, found:
C 43.00; H 4.85; N 15.65%.
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◦
THF, washed with Et2O, and dried under heat at 40 C. In this
way, 687.9 mg of 1·Zn was obtained in 89% yield.
1H NMR (400 MHz, CD3OD) d 8.74 (d, J = 4.6 Hz, 2H), 8.13
(app. t, J = 7.7 Hz, 2H), 7.66 (t, J = 6.4 Hz, 2H), 7.61 (d, J =
7.9 Hz, 2H), 7.54 (s, 1H), 7.50 (d, J = 6.7 Hz, 1H), 7.33 (d, J =
7.4 Hz, 1H), 7.29–7.22 (m, 1H), 4.38 (d, J = 17.0 Hz, 2H), 4.25
(d, J = 17.1 Hz, 2H), 4.11 (brs, 1H), 3.49 (brs, 1H), 2.94 (brs,
2H), 2.63 (s, 2H); 13C NMR (100.7 MHz, CD3OD) d 157.1, 149.7,
142.6, 136.3, 136.0, 135.6, 134.9, 134.4, 132.4, 131.8, 129.1, 126.2,
125.7, 59.1, 54.2, 53.4, 44.6; ESI MS: m/z 219 ([M − 2(NO3)]2+;
elemental analysis: anal. calcd for C21H25BN6O8Zn·0.5 H2O·0.5
MeOH: C 42.70; H 4.78; N 14.23%, found: C 42.50; H 4.42; N
14.11%.
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N-tert-Butoxycarbonyl-Nꢀ,Nꢀ-bis(2-pyridylmethyl)
ethylenediamine zinc (II) complex 3·Zn
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N -tert-Butoxycarbonyl-Nꢀ ,Nꢀ -bis(2-pyridylmethyl)ethylenedi-
amine (500 mg, 1.46 mmol) and Zn(NO3)2·6H2O (297.5 mg,
1.46 mmol) were dissolved in MeOH (50 mL). The resulting
mixture was stirred for 1 h at room temperature. After removal
of the solvent in vacuo the resulting residue was recrystallized with
MeOH. In this way, 6.26 g of 3·Zn was obtained in 44% yield.
1H NMR (400 MHz, CD3OD) d 8.71 (d, J = 4.8 Hz, 2H), 8.19
(td, J = 7.7, 1.6 Hz, 2H), 7.68–7.73 (m, 4H), 4.51 (d, J = 16.2 Hz,
2H), 4.19 (d, J = 16.2 Hz, 2H), (t, J = 6.7 Hz, 2H), 2.76 (t,
J = 6.6 Hz, 2H), 1.38 (s, 9H); 13C NMR (100.7 MHz, CD3OD) d
158.52, 156.42, 149.42, 142.94, 126.58, 126.36, 81.05, 58.11, 53.99,
14 K. A. Connors, Binding Constants, The Measurement of Molecular
Complex Stability, John Wiley & Sons, New York, 1987.
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The Royal Society of Chemistry 2008
Org. Biomol. Chem., 2008, 6, 3621–3625 | 3625
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