Polyglycerol-bound phosphotriesterase enzyme model complexes for detection and hydrolysis of phosphorus species in aqueous solution

Article abstract of DOI:10.1016/j.tet.2009.03.051

Phosphotriesterase models incorporating di(2-picolyl)amino ligands supported by m-xylylene or 2-hydroxy-m-xylylene scaffolds have been tethered to the periphery of a water-soluble hyperbranched polyglycerol (PG). In aqueous solution buffered at pH 7.4, th

Full text of DOI:10.1016/j.tet.2009.03.051

Tetrahedron 65 (2009) 4298–4303
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Polyglycerol-bound phosphotriesterase enzyme model complexes for detection
and hydrolysis of phosphorus species in aqueous solution
*
Anshuman Mangalum, Rhett C. Smith
Department of Chemistry and Center for Optical Materials Science and Engineering Technologies (COMSET), Clemson University, Clemson, SC 29634, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Phosphotriesterase models incorporating di(2-picolyl)amino ligands supported by m-xylylene or 2-hy-
droxy-m-xylylene scaffolds have been tethered to the periphery of a water-soluble hyperbranched poly-
glycerol (PG). In aqueous solution buffered at pH 7.4, the polymeric complexes of Zn2þ are useful
receptors for polymeric indicator displacement assays for phosphate and pyrophosphate employing
commercial complexometric indicators. Under the same conditions, the Co3þ effectively hydrolyze
p-nitrophenylphosphate with approximately five orders of magnitude rate enhancement versus un-
catalyzed hydrolysis. These systems offer promising results as mixed-metal dual detect-decontaminate
materials for organophosphorus toxins under mild, neutral aqueous conditions.
Received 22 October 2008
Received in revised form 17 March 2009
Accepted 19 March 2009
Available online 27 March 2009
Keywords:
Hyperbranched polymer
Phosphohydrolase
Pyrophosphate sensor
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
a distance of w3.4 Å.7 One actively explored area of research is thus
to prepare small molecule bimetallic models of PTEase that can
The nerve agents of chemical warfare, as well as many widely
used commercial pesticides, are phosphoesters. Some of these
neurotoxic compounds, including G and V agents (Chart 1) are
among the most toxic synthetic materials ever isolated. The simple
structure and availability of starting materials make these agents
attractive weapons for terrorism, as exemplified by the use of sarin
(GB) in attacks on subway trains in Japan in 1995.1 Recent increases
in awareness regarding terrorist threats and the environmental
impact of pesticides highlight the desire to detect2,3 and degrade4,5
organophosphorus toxins. Phosphotriesterase (PTEase) is a bacte-
rial dizinc hydrolase enzyme capable of hydrolyzing phos-
phoesters.6 The active site of PTEase features two zinc ions held at
catalytically hydrolyze phosphoesters to biologically innocuous
phosphates. Di(2-picolylaminomethyl)xylyl scaffolded ligands of
the general form shown in Chart 2 support dizinc complexes with
M–M distances similar to that in the native PTEase active site.
Complexes of such ligands have thus been utilized in a variety of
sensing and catalytic applications under physiological conditions.8–17
An IR-dye modified variation has been applied to image phos-
phate production resulting from bacterial infection in live mice,
unequivocally demonstrating the stability of this binucleating
scaffold for extended periods in biological milieu.18 Our work
with PTEase model-based detection8 has focused on the
indicator displacement assay sensing mechanism.19 In this
Chart 1. Five of the seven globally stockpiled chemical warfare agents are simple
phosphoesters.
* Corresponding author. Tel.: þ1 864 656 6112; fax: þ1 864 656 6613.
E-mail address: rhett@clemson.edu (R.C. Smith).
Chart 2. General form of binucleating ligands whose bimetallic complexes are useful
for inorganic phosphate detection and can catalytically hydrolyze phosphoesters.
0040-4020/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2009.03.051

A. Mangalum, R.C. Smith / Tetrahedron 65 (2009) 4298–4303
4299
Chart 3. Representation of the structure of 7–PG and 8–PG. Note that the size of PG branches varies, and this is only one possible representation demonstrating the global structure
of the PG.
strategy an optical (colorimetric or fluorescent) signal trans-
duction event is effected by displacement of an indicator from
a receptor upon exposure to an analyte. Sensitivity is attained
when the analyte binds significantly more strongly to the re-
ceptor than does the displaceable indicator. This strategy is
attractive because a variety of commercial indicators can be
utilized, providing various response wavelengths and binding
affinities with a single receptor, and the simple visual colori-
metric detection provided by this strategy is desirable for field
use. Related bioorganic model-based indicator displacement
assays for nerve agents operate via metal ion displacement from
fluorogenic ligands in response to neutral nerve agent simu-
lants20 or anionic partial hydrolysis products of G agents.21
Another of our interests is to tether PTEase models to polymeric
supports for catalytic applications. Covalent linkage or physical
encapsulation of native phosphohydrolase enzymes to polymers
and foams has already shown promise for decontamination.22,23
Covalent linkage is preferred because it prevents catalyst leaching
from the polymer, thus increasing shelf life of the material. Model
complexes should be an affordable alternative to isolated enzymes
and may be more robust components of decontamination solutions.
The polymer support we selected for this initial study is a bio-
compatible water-soluble hyberbranched polyglycerol (Chart 3).
In selecting a polymer support for a decontamination catalyst,
biocompatibility and high surface area for functionalization are ad-
vantageous. Dendrimers are promising chaperones for drug target-
ing/delivery and their high surface area and potential for supporting
cooperative effects havebeenexploited forcatalyticapplications.24–27
The often time-consuming synthesis of dendrimers, however, is the
most auspicious hindrance to their widespread application. Hyper-
branched polymers, however, can be prepared more efficiently and
retain thehigh surface area typical ofdendrimers.28–30 Biocompatible
hyperbranched polymers are thus privileged scaffolds for high ac-
tivity bioremediationand decontamination technologies. Fororgano-
phosphorus neurotoxins, polymers such as polyglycerol, which can
efficiently permeate and solubilize agents from thickened chemical
agent mixtures often used in warfare contexts (where poly(ethylene
glycol) is a preferred thickener),31 are of particular relevance in
a practical sense.4 Polyglycerol is approved in the United States and
Europe for use in pharmaceuticals,32–34 cosmetics,35,36 and even as
food additives,37,38 making it a safe candidate for protective creams.
The bioavailability of polyglycerol32,33 also suggests its potential as
a carrier support to deliver OP-decontaminating agents in pro-
phylactic medical treatment. A polymeric support also facilitates
catalyst recyclability and use in continuous membrane reactors for
water treatment.
Herein we describe the preparation of hyperbranched poly-
glycerol modified by two dinucleating PTE model ligand scaffolds.
We demonstrate that their Co(III) complexes efficiently hydrolyze
a phosphoester model compound, and that their Zn(II) complexes
are useful receptors for indicator displacement assays for phos-
phate/pyrophosphate. Both hydrolysis and detection experiments
were carried out under ambient conditions in aqueous solution
buffered at physiological pH (7.4).
2. Results and discussion
The hyperbranched PG support (Mn¼3700, PDI¼1.2) was pre-
pared via a literature protocol39 involving anionic polymerization
of glycidol from a trimethylolpropane core. In order to facilitate
attachment of PTE models, the PG was reacted with tosyl chloride
in the presence of pyridine to give tosylated polyglycerol (Tos–
PG).40 The small molecules carboxylate-derivatized binucleating
ligand moieties required for condensation with Tos–PG were
readily prepared via the sequence shown in Scheme 1. Simple SN2
reaction of PG–Tos with deprotonated 7 or 8 yields 7–PG and 8–PG,
respectively (Chart 3; the size of PG branches will vary and the
structure shown is a simplified representation). The approach se-
lected to tether enzyme model complexes to the polymers was
inspired by a reported route to hyperbranched PGs derivatized with
a,a
0-diamino-m-xylene-based pincer ligands in which highly effi-
cient complex loading was accomplished, leading to active com-
plexes for Pd-catalyzed C–C bond forming reactions.40–42 In the
current system, the efficiency of substituting 7 or 8 for tosylate
units was readily gauged by integration of well-resolved 1H NMR
resonances at w2.3 ppm (tosyl methyl group) and w8.5 ppm
(pyridyl subunit). Unfortunately, a relatively low loading efficiency
(36% and 25% of tosyl groups substituted in 7–PG and 8–PG, re-
spectively) was observed, despite repeated efforts to improve this
step. The low efficiency is presumably a result of the fairly

4300
A. Mangalum, R.C. Smith / Tetrahedron 65 (2009) 4298–4303
Scheme 1. Preparation of 7 (A) and 8 (B).
Figure 1. TEM image of 8–PG (A). Insets show a set of variously sized particles (B) and
an agglomeration of smaller particles that make up larger features (C).
voluminous carboxylates lacking sufficient access to some potential
substitution sites due to crowding by the hyperbranched support.
The Zn2–8–PG metal complex was readily prepared by dissolv-
ing 8–PG with 2 equiv of Zn(ClO4)2$6H2O in 0.1 M HEPES buffer (pH
7.4). Upon dispersion in a solvent such as THF, hyperbranched
polyglycerol-supported transition metal complexes have been
shown to form micellar structures that can be fractionated by size
exclusion chromatography to produce uniformly sized nano-
structures.40 Although preparing nanostructures is not the primary
goal of the current study, we were interested in whether 7 and 8
form structures similar to those reported previously. We thus
conducted a transmission electron microscopy (TEM) study on
a sample cast from a (nonfractionated) dispersion of 8–PG in THF.
The TEM images of the Zn2–8–PG complex (i.e., Fig. 1) revealed that
irregularly shaped particles, similar in size to those previously
reported,40 were observed. The particles ranged in size from about
15 to 35 nm (Fig. 1B), and close inspection reveals that the apparent
larger features visible in Figure 1A are actually clusters of the
smaller particles (Fig. 1C). The formation of micellar or reverse
micellar nanostructures is of particular interest because catalysis
can be enhanced within micellar structures or at their surface,
depending on the substrate and solvent system employed. Catalyst
recovery could also be affected by nanofiltration, ultracentrifuga-
tion or membrane techniques for particles in this size regime. These
avenues are worth pursuing in more detail once optimized mate-
rials are accomplished.
application requiring hydrolysis of more challenging substrates,
these preliminary complexes show promise for extension to addi-
tional polymer-bound complexes. The ready water solubility of the
polyglycerol-bound derivatives and the facile separation from
a vessel of water by dialysis offer improved practical utility of the
materials versus small molecular analogues.
In order to test the viability of the PG-supported complexes to
hydrolyze phosphoesters, the hydrolysis rate of 4-nitro-
phenylphosphate (NPP) was examined in the presence of 7–PG and
8–PG Co(III) complexes. NPP is a convenient and often-used sub-
strate for such studies because the formation of its hydrolysis
product, 4-nitrophenolate, is easily followed by UV–vis spectros-
copy.43,44 By following NPP hydrolysis by absorption spectroscopy,
employing solutions with varying substrate and catalyst concen-
trations as previously reported,44 we determined the rate constants
for NPP hydrolysis in HEPES (0.1 M, pH¼7.4) with the Co(III) com-
plexes of 7–PG (4.8ꢀ10ꢁ4 sꢁ1) and 8–PG (6.2ꢀ10ꢁ4 sꢁ1). These
values indicate an approximate five orders of magnitude en-
hancement in hydrolysis rate for either of the PG-bound Co(III)
complexes versus the uncatalyzed hydrolysis rate of 2ꢀ10ꢁ9 sꢁ1 45
.
Chart 4. Structures of commercially available complexometric indicators used in the
current study. Only one protonation state/resonance structure is shown in each case.
Although higher activity would likely be necessary for practical

A. Mangalum, R.C. Smith / Tetrahedron 65 (2009) 4298–4303
4301
Table 1
Dissociation constants (Kd, in
Another interesting application of polymer-bound enzyme
m
M) for the binding of indicators to the dizinc com-
models is their ability to act as selective receptors for the detection
of biological species. In this case, the Zn(II) complexes (Zn2–7–PG
and Zn2–8–PG) were employed rather than the Co(III) complexes
used in hydrolysis studies, because Zn(II) has greater ligand lability
and thus favors reversible binding that is desirable for sensors.
Building on our work with related dizinc complexes,8 we tested the
polymer-bound analogues as macromolecular receptors in in-
dicator displacement assays (IDAs) for inorganic phosphate de-
tection. The first step in this direction was to determine how
Zn2–8–PG complexes influence the absorption spectra of com-
mercially available complexometric indicators upon binding, and
whether the indicators are successfully displaced by various ana-
lytes. A large color change between the complex-bound and free
(analyte-displaced) indicator states is desired because this would
facilitate detection of small quantities of analyte.
plexes of the listed ligands, determined from absorption spectroscopic data (in 0.1 M
HEPES buffer at pH 7.4). Structures and abbreviations for indicators are provided in
Chart 4, and absorption spectra are provided in Supplementary data
Dissociation constants (Kd, mM)
Indicator
8–PG
7–PG
9
10
ARS
MB9
MX
PAR
PV
60
42
29
350
48
63
280
5.1
21
NAa
83
NAa
640
28
3.4
110
68
18
160
59
98
480
30
2.9
ZC
a
Spectroscopic data could not be adequately fit using this model.
These changes in binding constant are accompanied by concomi-
tant changes in how effectively inorganic phosphate anions dis-
place the indicators to produce colorimetric responses. Though
m-xylylene-supported bis(di(2-picolylamine)) dizinc complexes
are useful phosphate (Pi) sensors,9–16 there is some evidence that
pyrophosphate (PPi) can bind more strongly, at least in some
cases.8,46 Furthermore, selectivity can be acquired simply by judi-
cious selection of the indicator to be displaced by the analyte (i.e.,
by selecting an indicator with an appropriate Kd).46 The polymer-
bound analogues behave similarly: when the dizinc complex is the
receptor, any of the indicators shown in Chart 4 are selective for
inorganic phosphates (Pi or PPi) over all other common anions
screened. No notable change in absorbance was observed in re-
sponse to Fꢁ, Clꢁ, Brꢁ, Iꢁ, AcOꢁ or HSOꢁ4 (all in 0.1 M HEPES,
pH¼7.4). An even greater specificity can be attained when the most
weakly binding indicators are employed. Specifically, we found that
PPi can be selectively detected versus Pi at the same concentration
when MX or PV is used as the indicator (The response using MX is
shown in Fig. 2; other absorption spectra are shown in Supple-
mentary data.). It is noteworthy that the best receptor–indicator
pairs for the polyglycerol differ from those for small molecule an-
alogues,8 emphasizing the importance of independent Kd mea-
surements and indicator displacement trials in the course of
polymer-bound sensor development.
On the basis of our previous study,8 we selected six dyes (Chart
4) as indicators capable of potentially large color changes. As an
example of how dramatically spectral properties can change upon
complexation, data from a titration of MX with Zn2–8–PG and
subsequent displacement of the indicator by addition of pyro-
phosphate are shown in Figure 2 (similar data for the remaining
dyes are provided in Supplementary data). Data from absorption
spectroscopic titrations were used to extract dissociation constants
(Kd) for the indicators (Table 1). Table 1 also provides Kd values for
the indicators with dizinc complexes of nonpolymer-bound ligands
9 and 10 (Chart 2) for comparison. Because the polymer-bound
variations are in significantly altered environments in terms of both
polarity and sterics, it is not surprising that there is a marked
change in the dissociation constants for indicators bound to Zn2–7–
PG or Zn2–8–PG complexes versus the small molecule analogues.
+Zn2-8-PG
1
0.8
+Zn2-8-PG
0.6
0.4
0.2
0
3. Conclusions
We have prepared artificial enzyme modified, water-soluble
hyperbranched polyglycerols. The Co(III) complexes of these mate-
rials catalyze the hydrolysis of a phosphoester model substrate in
neutral aqueous buffer, with a rate enhancement of five orders of
magnitude versus the uncatalyzed reaction. Zinc(II) complexes of PG-
bound enzyme models are highly selective receptors for inorganic
phosphates over other common anions. There are significant differ-
ences in binding constants between the polyglycerol-supported en-
zyme models and their small molecule analogues. Caution should
thus be exercised when attempting to extend data from small mol-
ecule model systems to polymer-bound analogues, especially in IDA
studies where relative binding strengths dictate the selectivity of the
receptor–indicator pairs for a given analyte. Additional polymer
supported PTEase model-derivatized hyperbranched polymers are
currently being prepared for optimization in our laboratory to im-
prove upon the strategies reported herein.
300
350
400
450
500
550
Wavelength (nm)
600
650
700
MX + 1.2 Zn2-8-PG
1
0.8
0.6
0.4
0.2
0
Murexide (MX)
MX + 1.2 Zn2-8-PG
then 1.2 PPi
300
350
400
450 550
Wavelength (nm)
500
600
650
700
Figure 2. Upper chart: changes in absorption spectra of murexide (5.0ꢀ10ꢁ5 M) as up
to 1 equiv of Zn2–8–PG is added to the solution. The first trace is murexide alone, and
each subsequent trace represents addition of 0.1 equiv of Zn2–8–PG. Lower chart:
absorption spectra for murexide alone (blue trace), the same solution immediately
after addition of 1.2 equiv Zn2–8–PG (black trace), and subsequently 1.2 equiv pyro-
phosphate (PPi, red trace). All solutions were in 0.1 M HEPES buffer at a pH of 7.4.
Spectra are corrected for dilution factors.
4. Experimental
4.1. Materials and characterization methods
All reagents were obtained from Aldrich Chemical Co., TCI
America, Alfa Aesar, Mallinckrodt, or MP Biomedicals, LLC and used

4302
A. Mangalum, R.C. Smith / Tetrahedron 65 (2009) 4298–4303
as received. Hyperbranched polyglycerol39 and tosylated poly-
glycerol40 were prepared as previously described. NMR spectra
were recorded on a Bruker Avance 300 spectrometer and chemical
m/z): calcd for C34H34N6O3 574.2692; found 574.2700. This material
was hydrolyzed to form the acid without further purification.
shifts are reported in parts per million (
internally referenced to tetramethylsilane (
d
ppm). Proton NMR was
0.0) or residual solvent
4.2.4. 3,5-Bis[(bis(2-pyridylmethyl)amino)methyl]-4-hydroxy-
benzoic acid (7)
d
signal; 13C NMR chemical shifts were reported relative to the re-
sidual solvent peak. TEM imaging was performed using a Hitachi H-
7600 variable kV TEM with acceleration voltage of 120 kV. The
sample was prepared on formwar coated copper grids by drop-
casting, and samples were dried in an oven overnight. Compounds
4 and 5 were prepared as previously reported.47
Compound 2 (0.2 g, 0.3 mmol) was dissolved in 10 mL of ethanol
and w1.2 g of NaOH was added into the reaction mixture. After
addition, solution was refluxed overnight. Volatiles were removed
by rotary evaporation and water was added into the residue and
solution was extracted with dichloromethane (3ꢀ30 mL). The or-
ganic layer was dried over Na2SO4, filtered, and volatiles were re-
moved under reduced pressure (0.09 g, 26.30%). 1H NMR (300 MHz,
4.2. Synthesis
CDCl3):
d
¼3.85 (s, 4H, C–CH2), 3.95 (s, 8H, N–CH2), 7.15 (m, 4H,
pyridyl), 7.52 (d, 4H, J¼7.8 Hz, pryidyl), 7.65 (m, 4H, pryidyl), 8.10 (s,
4.2.1. Methyl 3,5-bis[(bis(2-pyridylmethyl)amino)methyl]-
benzoate (6)
2H, aromatic), 8.55 (d, 4H, J¼4.2 Hz, pryidyl). 13C NMR (75.4 MHz,
CDCl3):
d
¼59.3, 63.0, 121.1, 122.7, 123.0, 124.3, 131.0, 137.1, 149.1,
A solution of 5 (1.0 g, 3.1 mmol) in 5 mL of anhydrous THF was
stirred at 0 ꢂC under inert nitrogen atmosphere. In another vial,
a solution of triethylamine (1.3 g, 12.9 mmol) and DPA (1.3 g,
6.5 mmol) was prepared and then added dropwise to the solution
of 5. The reaction was allowed to warm at room temperature after
complete addition and the reaction mixture was stirred for 3 days.
The triethylammonium bromide was filtered off and volatiles were
158.9, 160.3, 167.8. HRMS (FAB, m/z): calcd for C33H33N6O3
561.2614; found 561.2623.
4.2.5. Compound 8–PG
A solution of 8 (23 mg, 0.04 mmol) and PG–Tos (100 mg) in 5 mL
of N,N-dimethylformamide (DMF) was stirred for 2 days at 70 ꢂC.
Salts were removed by filtration and volatiles were removed under
reduced pressure. The remaining residue was triturated with tol-
uene (3ꢀ10 mL), yielding an orange solid (50 mg). 1H NMR
removed under reduced pressure, leaving behind
a red oil.
Dichloromethane (50 mL) was added and solution was washed
with saturated Na2CO3 (aq) (3ꢀ50 mL). The organic layer was col-
lected and dried over Na2SO4, then volatiles were removed under
reduced pressure, yielding a viscous red oil (1.3 g, 72.0%). 1H NMR
(300 MHz, DMSO-d6):
d¼2.29 (s, 9H, Tos–CH3), 3.34 (br, 31H, poly-
ether), 3.63 (s, 4H, C–CH2), 3.70 (s, 8H, N–CH2), 7.10 (m, 6H,
2ꢀaromatic H–tosyl), 7.26 (m, 4H, pyridyl), 7.48 (dd, 6H, 1J¼8.1 Hz,
2J¼1.8 Hz, 2H, tosyl aromatic), 7.58 (d, 4H, J¼7.8 Hz, pyridyl), 7.75
(m, 6H, PTE aromatic and pyridyl), 8.49 (dd, 4H, 1J¼2.4 Hz,
2J¼0.9 Hz, pyridyl). 13C NMR (75.4 MHz, DMSO-d6):¼21.3, 21.5,
58.2, 59.6, 122.6, 122.7, 125.8, 126.0, 128.5, 128.7, 128.9, 129.4, 137.0,
138.0, 146.3, 149.3, 159.7.
(300 MHz, CDCl3):
d
¼3.74 (s, 4H, C–CH2), 3.82 (s, 8H, N–CH2), 3.93
(s, 3H, OCH3), 7.15 (br, 4H, pyridyl), 7.6 (m, 8H, pryidyl), 7.75 (s, 1H,
aromatic), 7.95 (d, 2H, J¼1.5, aromatic), 8.52 (m, 4H, pryidyl). 13C
NMR (75.4 MHz, CDCl3):
d
¼58.2, 60.1, 67.9, 122.0, 122.8, 128.8,
130.2, 133.8, 136.5, 139.7, 148.9, 159.5, 167.2. HRMS (FAB, m/z): calcd
for C34H34N6O2 558.2743; found 558.2749. This material was hy-
drolyzed to form the acid without further purification.
4.2.6. Compound 7–PG
A solution of 7 (60 mg, 0.1 mmol) and PG–Tos (260 mg) in 5 mL
DMF was stirred for 2 days at 70 ꢂC. After cooling to room tem-
perature, a precipitate was removed by filtration and DMF was
removed under reduced pressure. The residue was triturated with
toluene (3ꢀ10 mL), yielding a yellow solid (90 mg). 1H NMR
4.2.2. 3,5-Bis[(bis(2-pyridylmethyl)amino)methyl]-benzoic acid (8)
Compound 6 (1.3 g, 2.2 mmol) was dissolved in 60 mL of ethanol
and w1.2 g of NaOH was added into the reaction mixture. After
addition, the solution was refluxed overnight. Volatiles were re-
moved by rotary evaporation, then water was added to the residue
and extracted with dichloromethane (3ꢀ30 mL). The organic layer
was dried over Na2SO4, and volatiles were removed under reduced
pressure to give a yellow solid (0.09 g, 7.00%). 1H NMR (300 MHz,
(300 MHz, DMSO-d6):
d
¼2.29 (s, 12H, Tos–CH3), 3.34–3.83 (52H, PG
overlapping C–CH2 and N–CH2), 7.10 (m, 8H), 7.35 (m, 4H, pyridyl),
7.48 (m, 11H, 2ꢀaromatic H–tosyl), 7.67–7.82 (m, 9H), 8.49 (br, 4H,
pyridyl). 13C NMR (75.4 MHz, DMSO-d6):
122.4, 122.7, 123.1, 125.0, 128.5, 137.2, 138.0, 146.3, 149.2, 149.2,
159.0.
d
¼21.2, 54.5, 59.3, 122.3,
CDCl3):
d
¼3.32 (s, 4H, C–CH2), 3.52 (s, 8H, N–CH2), 6.84 (t, 4H, J¼6.0,
pyridyl), 7.19–7.38 (m, 8H, pryidyl), 7.79 (s, 2H, aromatic), 8.38 (d,
4H, J¼4.5, pryidyl). 13C NMR (75.4 MHz, CDCl3):
¼58.9, 59.9, 121.9,
d
122.9, 129.3, 131.0, 136.4, 137.4, 138.3, 149.0, 159.2, 173.8. HRMS
4.3. Preparation of metal complexes
(FAB, m/z): calcd for C33H33N6O2 545.2665; found 545.2674.
4.3.1. For Co2–7–PG and Co2–8–PG
4.2.3. Methyl 3,5-bis[(bis(2-pyridylmethyl)amino)methyl]-4-
hydroxybenzoate (2)
A solution of 7–PG or 8–PG containing 1 mmol of ligand unit
was dissolved in 9.8 mL of methanol and 0.2 mL of 0.1 mmol
CoSO4$7H2O was added into it. Solution was stirred overnight.
Further methanol was removed under reduced pressure. HEPES
buffer (10 mL, 0.1 M) was added into it in order to make 1 mmol
Co2–7–PG and Co2–8–PG solution, which is further used for hy-
drolysis titrations.
Compound 1 (1.0 g, 6.6 mmol) was taken in a round bottom
flask. DPA (3.9 g, 19.6 mmol), paraformaldehyde (0.6 g, 20.0 mmol),
and 40 mL toluene were then added. After addition, the reaction
mixture was refluxed overnight. After cooling to room temperature,
toluene was removed under reduced pressure and the residue was
dissolved in CH2Cl2. The resultant solution was washed with satu-
rated Na2CO3 (4ꢀ50 mL). The organics were dried over Na2SO4 and
then volatiles were removed by rotary evaporation. The desired
product was obtained as a viscous red oil (2.6 g, 69.0%). 1H NMR
4.3.2. For Zn2–7–PG and Zn2–8–PG
A solution of 7–PG or 8–PG containing 1.5 mmol of ligand unit
and 3 mmol of Zn(ClO4)2$6H2O was dissolved in 10 mL of 0.1 M
HEPES buffer to make 1.5 mmol Zn2–7–PG and Zn2–8–PG solution
which was further used for TEM experiment and the indicator
displacement assay. For TEM experiments, Zn2–8–PG solution was
removed under reduced pressure and solid residue was dispersed
in the THF.
(300 MHz, CDCl3):
d
¼3.85 (s, 4H, C–CH2), 3.90 (s, 11H, N–CH2 and
OCH3), 7.15 (m, 4H, pyridyl), 7.50 (d, 4H, J¼7.8 Hz, pryidyl), 7.65 (m,
4H, pryidyl), 7.95 (s, 2H, aromatic), 8.55 (dd, 1J¼6.0 Hz, 2J¼0.9 Hz,
4H, pryidyl). 13C NMR (75.4 MHz, CDCl3):
d
¼51.7, 54.5, 59.7, 120.1,
122.0, 122.9, 124.4, 131.1, 136.6, 148.8, 159.1, 160.7, 167.3. HRMS (FAB,

A. Mangalum, R.C. Smith / Tetrahedron 65 (2009) 4298–4303
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Absorption spectra were recorded on a Varian Cary 50 Bio UV–vis
spectrophotometer. Samples for all absorbance spectra used 0.1 M N-
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of 1 cm. Indicator displacement assay protocols,8 kinetic analyses,44
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brand method)48 have been described previously. All absorption
spectra for titrations and Benesi–Hildebrand plots used are provided
in Supplementary data.
Acknowledgements
We thank the Clemson University Department of Chemistry and
Center for Optical Materials Science and Engineering Technologies
for support, and Amar Kumbhar for assistance with TEM.
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