TAMEisoquin, a novel tripodal fluorescent zinc sensor with high Zn(II) affinity and Zn(II)/Cd(II) selective fluorescence response: Synthesis, coordination geometry, spectroscopy, and comparative response to biometal ions

Article abstract of DOI:10.1016/j.poly.2016.01.024

Herein we report a novel fluorescent zinc sensor 1,1,1-tris (aminomethylethane)N,N′,N″-(2-methylisoquinoline) (TAMEisoquin), [Zn(TAMEisoquin)]²⁺ complex and its structural and photophysical studies. TAMEisoquin was obtained from reduction of templated [Ni(TAMEisoquin-trisimine)]²⁺ complex to give [Ni(TAMEisoquin)]²⁺, which was subsequently decomposed with excess sodium cyanide to afford TAMEisoquin. A competition reaction between TAMEisoquin and the well-known Zn(II) chelator (N,N,N′, N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) at pH 7.2 indicated a conditional dissociation constant for [Zn(TAMEisoquin)]²⁺ of K_d = 1.4 × 10^-15 M. The affinity of TAMEisoquin for Zn(II) is indicative of a ligand design exploiting ring size, preorganization and chelate effects to potentially detect picomolar and femtomolar zinc concentrations. X-ray crystallographic analysis of [Zn(TAMEisoquin)]²⁺ showed four unique cations that preferred a distorted octahedral geometry with trigonal twist angles that range between 34(2)° and 43.1(8)°. Moreover, [Zn(TAMEisoquin)]²⁺ complex showed Zn-N bond distances slightly similar to our previously reported [Zn(TAMEpyr)]²⁺ complex, being Zn-N_heterocyclic, 2.160(1) ? and Zn-N_aliphatic, 2.178(4) ? versus 2.128(2) ? and 2.169(2) ? respectively for [Zn(TAMEpyr)]²⁺. The addition of Zn(II) to TAMEisoquin displayed 11-fold fluorescence enhancement, selective by comparison to divalent biometal ions and Cd(II) which induces only 16% of the Zn(II) response.

Full text of DOI:10.1016/j.poly.2016.01.024

Accepted Manuscript
TAMEisoquin, a novel tripodal fluorescent zinc sensor with high Zn(II) affinity
and Zn(II)/Cd(II) selective fluorescence response: Synthesis, coordination ge-
ometry, spectroscopy, and comparative response to biometal ions
Lea Nyiranshuti, Daniel P. Kennedy, Antonio G. DiPasquale, Arnold L.
Rheingold, Roy P. Planalp
PII:
S0277-5387(16)00038-3
DOI:
http://dx.doi.org/10.1016/j.poly.2016.01.024
POLY 11786
Reference:
Polyhedron
To appear in:
Received Date:
Accepted Date:
26 September 2015
20 January 2016
Please cite this article as: L. Nyiranshuti, D.P. Kennedy, A.G. DiPasquale, A.L. Rheingold, R.P. Planalp,
TAMEisoquin, a novel tripodal fluorescent zinc sensor with high Zn(II) affinity and Zn(II)/Cd(II) selective
fluorescence response: Synthesis, coordination geometry, spectroscopy, and comparative response to biometal ions,
Polyhedron (2016), doi: http://dx.doi.org/10.1016/j.poly.2016.01.024
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TAMEisoquin, a novel tripodal fluorescent zinc sensor with high Zn(II) affinity and
Zn(II)/Cd(II) selective fluorescence response: Synthesis, coordination geometry,
spectroscopy, and comparative response to biometal ions
Lea Nyiranshuti, a† Daniel P. Kennedy, a† Antonio G. DiPasquale, b Arnold L. Rheingold, b Roy P.
Planalp a*
a Department of Chemistry, University of New Hampshire, Parsons Hall, 23 Academic Way,
Durham, NH03824-3598, United States
b Department of Chemistry, University of California San Diego, La Jolla, CA92093, United States
Abstract
Herein we report a novel fluorescent zinc sensor
1,1,1-tris (aminomethylethane) N, N’, N’’-(2-
methylisoquinoline)
(TAMEisoquin),
[Zn(TAMEisoquin)]2+ complex and its structural
and photophysical studies. TAMEisoquin was
Figure 1: Synthesis of [Zn(TAMEisoquin)]2+
obtained
from
reduction
of
templated
[Ni(TAMEisoquin-trisimine)]2+ complex to give
[Ni(TAMEisoquin)]2+, which was subsequently decomposed with excess sodium cyanide to afford
TAMEisoquin. A competition reaction between TAMEisoquin and the well-known Zn(II) chelator (N, N, N’,
N’-tetrakis(2-pyridymethyl)ethylenediame (TPEN) at pH 7.2 indicated a conditional dissociation constant
for [Zn(TAMEisoquin)]2+ of Kd=1.4x10-15 M. The affinity of TAMEisoquin for Zn(II) is indicative of a ligand
design exploiting ring size, preorganization and chelate effects to potentially detect picomolar and
femtomolar zinc concentrations. X-ray crystallographic analysis of [Zn(TAMEisoquin)]2+ showed four
unique cations that preferred a distorted octahedral geometry with trigonal twist angle that range between
34(2)°and 43.1(8)°. Moreover, [Zn(TAMEisoquin)]2+ complex showed Zn-N bond distance slightly similar
to our previously reported [Zn(TAMEpyr)]2+ complex, being Zn-Nheterocyclic, 2.160 (1)Å and Zn-Naliphatic
,
2.178(4)Å versus 2.128(2) Å and 2.169(2) Å respectively for [Zn(TAMEpyr)]2+. The addition of Zn(II) to
TAMEisoquin displayed 11-fold fluorescence enhancement, selective by comparison to divalent biometal
ions and Cd(II) which induces only 16% of the Zn(II) response.

1. Introduction
Zinc is the second most abundant element in the body with very diverse biological
functions such as structural, enzymatic, catalytic, charge neutralization and signaling.1 Although
zinc proteins and metallothioneins regulate the concentration of zinc in the body, the disruption
of zinc homeostasis is associated with various diseases such as diabetes,2 Alzheimer’s,3 and
prostate cancer.4,5This is critical because concentration of free zinc in the body generally vary
between 1 nM and 1 mM6,7 with particular high concentration in the brain, eyes, pancreas and
spermatozoa.7 For instance, numerous metalloneurochemistry studies showed that free zinc
causes detrimental effects to central nervous system.8-11 Therefore, the detection of zinc in living
organisms, environment, and food samples is important to understand functions of free zinc in
the cellular environment and its effects on human health.
In recent years, a great number of fluorescent zinc sensors have become available to
satisfy the criteria of metal ion readout, rapid response, selectivity, and biological stability.12-14
However, one of the challenges associated with zinc sensors is their ability to induce similar
fluorescence response for Zn(II) and Cd(II)15,16 since they are both divalent d10 metal ions, which
possess similar coordination and photophysical properties. According to the U.S Environment
Protection Agency (EPA), cadmium is listed as one of the 126 priority pollutants with a half-life
between 15 and 20 years in humans.17-19 Similar to zinc, excessive exposure to cadmium is
linked with various health effects such as kidney dysfunction, pulmonary cancer, and lung
diseases.18,19 It is therefore important to discriminate these two metal ions in environmental and
biological studies, such that we are studying effects of chelate ring size and metal-ion binding
pocket size upon preference for Zn(II) relative to Cd(II).
In the past, we have developed the
chemistry of two tripodal chelator frameworks,
20
cis,cis-1,3,5-triaminocylohexane (TACH) and
21
1,1,1-tris(aminomethyl)ethane (TAME)
by
attaching suitable pendant groups to provide
tripodal hexadentate chelators (Scheme 1). We
are mainly interested in hexadentate tripodal
ligands because of their size selective nature,
formation of metal complexes with defined
configurations, and high binding affinity due to
six coordinating nitrogen atoms on the ligand.
Scheme 1: The TACH and TAME frameworks and
their 2-methylpyridyl derivatives serving as basis for
the Zn sensor TAMEisoquin described herein.
2

Our previous studies with hexadentate tripodal chelators such as tachpyr showed marked size
selectivity attributed to the metal-binding cavity size. For example, the tachpyr complexes of
Cd(II), Hg(II) or In(III) were weakly bound and distorted relative to Zn(II) or Ga(III)
complexes.22,23 In the present article we explore the chemistry of TAME framework by using
isoquinoline as coordinating fluorophore. We use isoquinoline because our previous results
showed that substituents ortho to the heterocyclic nitrogen create substantial steric hindrance
24
upon metal complexation. Therefore, using isoquinoline rather than quinoline prevents steric
congestion at the C3-apexof the complex. TAMEisoquin sensor operates through photoinduced
electron transfer (PET) mechanism whereby Zn(II) binding prevents fluorescence quenching,
and thus enhances emission intensity. In this paper, we report synthesis of a novel indicator,
TAMEisoquin (Scheme 2), its structural and photophysical properties relative to Zn(II) sensing,
and the selectivity of response to Zn(II) relative to Cd(II) and other divalent biometal ions.
Scheme 2. A. Preparation of isoquinoline-1-carboxaldehyde: i. SOCl2, MeOH, reflux,
16 h. ii. a)0.5 equiv LiAlH4, THF, -78°, 15 min. b)AcOH. B. Preparation of
TAMEisoquin: i) 3.0 equiv 1-isoquinolinecarboxaldehyde, Ni(ClO4)2·6H2O, H2O, EtOH,
12 h; ii) NaBH4, H2O, EtOH, 25 °C, 12 h; iii) NaCN(aq), CH2Cl2 extraction and
evaporation
3

2. Experimental
2.1. General Considerations
All the materials listed below were of research grade or a spectro-grade in the highest
purity available and were generally used without further purification except diethyl ether, which
was distilled from sodium benzophenone. Anhydrous methanol, acetonitrile, and the salts
Zn(ClO4)2.6H2O,
Cd(NO3)2.4H2O,
Ni(ClO4)2·6H2O,
Co(BF4)2.6H2O,
Fe(ClO4)2.6H2O,
Mn(ClO4)2.6H2O, and AgNO3 were purchased from Aldrich. The salts NaCl, KCl, and CaCl2
were purchased from EM science. Anhydrous grade EtOH was purchased from Pharmco.
1,1,1-Tris(aminomethyl)ethane was synthesized as we previously reported.21 (N,N,N’N’-
Tetrakis-(2-pyridylmethyl)(ethylenediamine) “TPEN” was from TCI. DMSO-d6, CDCl3 and DMF-
d6 were obtained from Cambridge Isotope Laboratories. The UV-visible spectra were measured
using Varian Cary 50 Bio spectrometer with 3 cm3 quartz cuvettes (1 cm path-length). The
fluorescence spectra were recorded using a Cary Eclipse fluorescence spectrophotometer with
3 cm3 quartz cuvettes (1 cm path-length). 1H and 13C NMR were obtained using a Varian
Mercury 400 MHz NMR instrument and chemical shifts are reported in ppm relative to the
deuterated solvents used. Elemental analysis was performed by Atlantic Microlabs (Atlanta,
Georgia). Drying was accomplished under a stream of nitrogen or under reduced pressure (ca.
10-2 Torr) with a standard Schlenk line.
2.2. Synthesis of compounds
2.2.1. Methyl 1-isoquinolinecarboxylate
To a suspension of 1-isoquinoline carboxylic acid (3.00 g, 17.3 mmol) in MeOH (30 mL)
was added SOCl2 (6.18 g, 3.79 mL, 52.0 mmol) via pipet. The resulting pale yellow solution
was refluxed under a stream of nitrogen for 16hr. The solvent was then removed under vacuum
affording pale yellow solids. The residue was dissolved into H2O (20 mL), neutralized with
excess Na2CO3, and extracted with CH2Cl2 (4x20 mL). The organic extracts were combined,
dried over Na2SO4, filtered, and the solvent removed by vacuum leaving behind a yellow oil.
The oil was distilled under reduced pressure affording methyl 1-quinolinecarboxylate as a
colorless oil (1.59 g, 49%, bp=144-145°C, 700 mTorr). 1H NMR (CDCl3): 8.84 ppm (1H, d, J=8.8
Hz); 8.63 ppm (1H, d, 5.6 Hz); 7.883 ppm (1H, d, J=8.0 Hz); 7.83 ppm (1H, d, J=5.6 Hz); 7.71
ppm (2H, m); 4.10 ppm (3H, s). 13C NMR (CDCl3): 166.35, 148.24, 141.65, 137.01, 130.65,
128.89, 127.20, 126.97, 126.46, 124.40, 53.06 ppm. IR (liquid film): 3055, 2951, 2849, 1720 cm-
1
.

2.2.2. 1-Isoquinolinecarboxaldehyde.
A solution of methyl 1-isoquinolinecarboxylate (1.00 g, 5.34 mmol) in dry THF (30mL)
was cooled to -70°C in a dry ice/MeOH bath. To that solution was added a suspension of
LiAlH4 (0.10 g, 2.67 mmol) in THF (10 mL). The resulting brown reaction mixture was stirred for
15min followed by a quench with glacial acetic acid (1.3 mL). Once the mixture warmed to RT
the aluminum salts were precipitated with a few drops 1N NaOH. The heterogeneous mixture
was filtered, and the solvent was removed under vacuum. The residue was dissolved into a
small portion of CH2Cl2 and chromatographed on silica (1:1hexanes:EtOAc , Rf=0.79) affording
1
1-isoquinolinecarboxaldehyde (0.28g, 33%) as a crystalline white solid. H NMR (CDCl3): 10.40
ppm (1H, s); 9.33 ppm (1H, m); 8.76 ppm (1H, d, J=5.6 Hz); 7.91 ppm (2H, m); 7.77 ppm (2H,
m). 13C NMR (CDCl3): 195.63, 149.79, 142.41, 136.85, 130.74, 130.01, 126.91, 126.31, 125.70,
125.45 ppm. IR (KBr pellet): 3033, 2844, 1703 cm-1.
2.2.3. [Ni (TAMEisoquin-trisimine)] (ClO4)2·MeCN
To a solution of TAME (30.9 mg, 264 mmol) in EtOH (2.3 mL) was added Ni
(ClO4)2·6H2O (93.6 mg, 256 mmol) dissolved in H2O (4.0 mL). The resulting pale blue solution
was titrated with 1-isoquinolinecarboxaldehyde (121 mg, 770 mmol) dissolved in EtOH (3.0 mL).
A finely divided orange powder precipitated from solution immediately. The heterogeneous
reaction mixture was allowed to stir at RT for 16hr. The solids were filtered on a glass frit,
washed with portions of Et2O (3x10mL), and dried under vacuum. The crude trisimine complex
was recrystallized from 1:1 MeOH: MeCN (v/v) via Et2O diffusion as large, lustrous orange
needles (137 mg, 62%). IR (KBr pellet): 3256, 2964, 2933, 1623, 1589, 1458, 1322, 1093 cm-1.
(Found: C, 53.06; H, 3.99; N 11.54. C35H30N6NiCl2O8·C2H3N requires: C, 53.33; H, 3.99; N,
11.77 %.). Crystals suitable for X-ray crystallography were obtained by slow Et2O diffusion into
a 1:1 MeCN: MeOH (v/v) solution of the complex.
2.2.4. TAMEisoquin
To a suspension of [Ni (TAMEisoquin)] (ClO4)2·2MeCN (50 mg, 63 mmol) in H2O (10 mL)
was added NaBH4 (151 mg, 4.00 mmol). The resulting mixture was sonicated and then shaken
to afford copious amounts of a pale pink precipitate. The mix was stirred at RT for 3hr. The
reaction was then quenched with the addition of conc. HClO4 (pH 6). The mixture was
centrifuged and the supernatant was decanted away from the solid plug. The solids were
washed with Et2O (2x20 mL) and then dried under vacuum. The resulting pale pink triamine

complex was dissolved into MeOH (3 mL). To the solution was added excess NaCN (19.1 mg,
391 mmol) dissolved in H2O (1 mL). The mixture was shaken vigorously and then extracted
with CH2Cl2 (3x3 mL). The organic fractions were combined, dried over Na2SO4, decanted, and
the solvent was removed under vacuum to afford TAMEisoquin as a pale yellow oil (34.0 mg,
1
99%). H NMR(CDCl3): 8.39 ppm (3H, d, J=6.0 Hz); 8.21 ppm (3H, d, J=8.8 Hz); 7.77 ppm (3H,
d, J=8.0 Hz); 7.60 ppm (3H, m); 7.50 ppm (3H, d, J=6.0 Hz); 7.46 ppm (3H, m); 4.36 ppm (6H,
s); 2.77 ppm (6H, s); 2.53 ppm (3H, br.s); 1.02 ppm (3H, s). 13C NMR (CDCl3): 159.54, 141.85,
136.31, 130.01, 127.35, 127.15, 127.06, 125.38, 120.06, 57.09, 54.04, 39.22, 21.95 ppm. The
ligand was also prepared as a hydrochloride salt by dissolving the free amine into EtOH and
adding conc. HCl dropwise. The resulting white precipitate was isolated, washed with Et2O, and
dried under vacuum. (Found: C, 54.31; H, 5.74; N, 10.86. C35H36N6·6HCl·H2O requires: C,
54.07; H, 5.70; N, 10.81%).
2.2.5. [Zn (TAMEisoquin)] (ClO4)2.2H2O.1/2MeCN
To a solution of TAMEisoquin (28.3mg, 52.3 mmol) in MeOH (3mL) was added
Zn (ClO4)2·6H2O (19.5mg, 52.3 mmol) dissolved in MeOH (1mL). The mixture was allowed to
stand for 5min, and then Et2O (20mL) was added affording copious amounts of a white
precipitate. The heterogeneous mixture was centrifuged and the supernatant was decanted.
The solid plug was washed with Et2O and then dried under vacuum affording the desired
1
complex as a white powder (38.4 mg, 91%). H NMR (DMSO-d6): 8.44 ppm (3H, d, J=8.8 Hz);
8.15 ppm (3H, d, J=8.0 Hz); 8.01 ppm (3H, t, J=6.8 Hz); 7.95 ppm (3H, d, J=6.0 Hz); 7.89 ppm
(3H, t, J=7.2Hz); 7.75 ppm (3H, d, J=6.0 Hz); 4.83 ppm (3H, m); 4.75 ppm (3H, m); 4.58 ppm
(3H, m); 3.28 ppm (3H, m); 3.05 ppm (3H, m); 0.76 ppm (3H, s). IR (KBr): 3280, 3070, 2920,
1627, 1598, 1101 cm-1. (Found: C, 50.83; H, 4.59; N, 10.10. C35H36N6ZnCl2O8.2H2O.1/2MeCN
requires: C, 50.19; H, 4.85; N, 10.57%). Crystals suitable for X-ray analysis were obtained via
Et2O diffusion into a 1:1 MeOH: MeCN (v/v) solution of the complex.
2.2.6. [Cd (TAMEisoquin)] (NO3)2.2H2O
To a solution of TAMEisoquin (43.7mg, 80.8 mmol) in MeOH (1.5 mL) was added
Cd (NO3)2·4H2O (22.9 mg, 74.2 mmol) dissolved MeOH (1.5 mL). The mixture was allowed to
stand for 5min, and then Et2O (20 mL) was added affording copious amounts of a white
precipitate. The heterogeneous mixture was centrifuged and the supernatant was decanted.
The solid plug was washed with Et2O and then dried under vacuum affording the desired
complex as a white powder (55.9 mg, 96%). Microcrystals were obtained via Et2O diffusion into
6

1
a 1:1 MeNO2: MeCN (v/v) solution of the complex. H NMR(DMSO-d6): 8.69 ppm (3H, d, J=6.0
Hz); 8.43 ppm (3H, d, J=8.4 Hz); 8.14 ppm (3H, d, J=8.0 Hz); 8.05 ppm (3H, d, J=6.0 Hz); 7.96
ppm (3H, t, J=7.6 Hz); 7.84 ppm (3H, t, J=7.2 Hz); 4.72 ppm (6H, s); 4.55 ppm (3H, m); 3.24
ppm (6H, m); 0.89 ppm (3H, s). IR (KBr pellet): 3219, 3065, 2919, 2869, 1625, 1381, 1346,
1312 cm-1. (Found: C, 51.83; H, 4.64; N, 13.93. C35H36N6CdN2O6 .2H2O requires: C, 51.70; H,
4.96; N, 13.78%).
2.2.7. Photophysical studies
Stock solutions of the ligand “TAMEisoquin” and metal ions were prepared in
1:1 DMSO: H2O (v/v) and 100% water respectively. The stock solution of the ligand was used to
make 14 µM in 0.1 M HEPES buffer pH 7.2 and titrated with various metal ions aliquots. The
samples used for photophysical studies contained 0.4% (v/v) DMF/H2O except for quantum
yield determination where 1:1 DMF: H2O was used. All spectral measurements were carried out
in a 3cm3 cuvette with a 1 cm path-length. The fluorescence spectra were conducted with an
excitation wavelength (λex) of 321 nm.
Recrystallized [Zn (TAMEisoquin)](ClO4)2 was used to prepare serial dilutions of the
complex and its quantum yield of [Zn(TAMEisoquin)]2+ was determined by comparing with a
quinine sulfate standard25 measured in 1N H2SO4. The formula used to calculate the quantum
yield
of
the
Zn(II)
complex
is:
2










Gradx
Gradst
η
η
x




Φ = Φ
x
st
2


st
The subscripts ‘x’ and ‘st’ refer to the experimental sample and the standard, respectively, and
‘Grad’ refers to the slope of a least-squares straight line through a plot of integrated
fluorescence intensity vs. absorbance.26 The refractive indices, ɳ, were neglected in the
computation and may introduce a small amount of error in the quantum yield reported herein.
2.2.8. Binding constant studies
In order to further investigate the affinity of Zn(II) for TAMEisoquin, we carried out a
ligand exchange reaction with the strongly binding chelator TPEN (N, N, N’, N’-Tetrakis- (2-
pyridylmethyl)-ethylenediamine. Stock solutions of [Zn(TAMEisoquin)]2+ and TPEN were
prepared in 0.1 M HEPES buffered at pH 7.2 with 0.1 M KNO3 as ionic strength adjuster. A 14
µM solution of [Zn(TAMEisoquin)]2+ with 1.0 equiv of TPEN was prepared in the HEPES buffer
pH 7.2. A decrease in the emission intensity at 350 nm was monitored over the course of three
7

weeks (T=25°C). A 14 µM solution of [Zn(TAMEisoquin)]2+ without the competing ligand was
also monitored as a control for photobleaching.
3. Results and Discussion
3.1. Synthesis of TAMEisoquin and its Ni(II), Zn(II) and Cd(II) complexes
To prepare TAMEisoquin, we rely on the Ni-templated condensation of isoquinoline
aldehyde analogous to the previously reported synthesis of TAMEpyr (N,N',N''-tris(2-
pyridylmethyl)-tame) from TAME and pyridine-2-carboxaldehyde.21 The required aldehyde is
prepared from 1-isoquinolinecarboxylic acid via reduction of its methyl ester (Scheme 2a). The
reaction of TAME, Ni(ClO4)2.6H2O and 3.0 equiv of 1-isoquinolinecarboxaldehyde gave the
orange complex [Ni(TAMEisoquin-trisimine)]2+ in high yields. The ligand is prepared by treating
[Ni(TAMEisoquin-trisimine)]2+ with excess NaBH4 and liberating TAMEisoquin from the metal
center via 6.0 equiv of NaCN (Scheme 2b). TAMEisoquin ligand is also prepared as a
hydrochloride salt by treating an ethanolic solution of the free ligand with concentrated HCl and
isolating the resulting white powder under vacuum. Zn(II) and Cd(II) TAMEisoquin complexes
are isolated by reactions of the corresponding metal salts and the ligand in MeOH with Et2O
cosolvent to improve precipitation and yields. A 1:1 M:L composition of all metal complexes was
verified by elemental analysis.
3.2. Structural studies
Crystallographic studies reveal the coordination sphere of the TAMEisoquin ligand and
particularly the fit of Zn(II) within the TAME-heterocycle framework. To assess the flexibility of
TAMEisoquin, an X-ray study of [Zn(TAMEisoquin)](ClO4)2.1/2MeCN (Figure 2) was carried out
which showed four unique [Zn(TAMEisoquin)]2+ cations in the asymmetric unit, labeled A,B,C,
and D respectively. All four cations can be described as distorted octahedral complexes with
twist angles that range between 34(2)º and 43.1(8)º, as viewed along the C3 symmetry axis.
The three C–N bonds of TAME restrict ligand twisting such that a structure between trigonal
prismatic (α=0º) and octahedral (α=60º) is obtained.27 The average distance Zn-Naliphatic is
slightly longer than the average Zn-Nheterocyclic, at 2.178(4) vs. 2.16(1) Å for cation A. This
behavior agrees with our previously published structure of [Zn(TAMEpyr)]2+ where the distance
of Zn-Naliphatic and Zn-Nheterocyclic were 2.169(2) and 2.128(2) Å respectively.21 Analogous to the
[Fe(tptMetame)]2+ complex reported by Al-Obaidi28 (where tptMetame = the pyridylmethyl
TAME-based chelator, 1,1,1-tris{[N-(2-pyridylmethyl)-N-methylamino]-methyl}ethane), each
8

cation has six chelate rings and three stereogenic nitrogen atoms, potentially giving rise to a
large number of isomers. The conformation of the three six membered chelate rings with
reference to the C3 axis in molecule A is δcap. The absolute configuration of the cation in
molecule A as defined by the arrangement of the five membered chelate rings is assigned as ∆.
The chiral aliphatic nitrogen donors are designated S,S,S. Lastly, the conformation of the five
membered chelate rings is slightly puckered and has been assigned as λλλ. Thus
stereochemistry for molecule A follows as ∆ δcap λλλ (SSS), and molecules B-D are similarly
assigned (Table 1).
In summary, [Zn(TAMEisoquin)]2+ (ClO4)2 assumes a number of
conformational modes in the solid state, which are attributed to the flexibility of TAME
framework.
We attempted synthesis of [Cd(TAMEisoquin)]2+; unfortunately crystals suitable for X-ray
crystallographic analysis were not isolated. We were able to perform computational studies on
[Cd(TAMEisoquin)]2+ complex (Figure S9) which revealed that the Cd complex is more
distorted than the Zn complex. The computational data showed that average bond length for
Cd-N(amine) (2.43Å) is longer than the average bond length for Cd-N (heterocyclic) (2.36 Å).
Known CdN6 complexes have crystallographically measured bonds that vary between ca. 2.3-
2.4Å which support the validity of the computational results.29 However, comparison between Zn
and Cd complexes shows that the Cd complex is more distorted. For example, the largest
deviation from 180⁰ for the Cd complex was observed in N5-Cd-N3 (152.70⁰) which is
significantly higher compared to the largest deviation for Zn complex, N5-Zn-N3 (160.84⁰).
9

Figure 2: ORTEP plot (50% probability) for [Zn(TAMEisoquin](ClO4)2.1/2MeCN. Coun
solvents and hydrogens were omitted for clarity. Color code: C, gray; Zn, Red; N, blue. Sele
lengths (Å): Zn1B-N1B, 2.173(4); Zn1B-N2B, 2.164(5), Zn1B-N3B, 2.142(5); Zn1B-N4B,
Zn1B-N5B, 2.168(6) and Zn1B-N6B, 2.168(5)
Complex
Zn-N
Zn-N (amine)
Average bond
length (Å)
Twist angle
Stereochemical
configuration
(heterocyclic)
Average bond
length (Å)
2.160(1)
α (°)
Cation A
Cation B
Cation C
Cation D
2.178(4)
2.164(6)
2.158(1)
2.163(1)
34(2)
38(2)
43.1(8)
40(3)
∆δCAPλλλ(SSS)
∆λCAPδδδ(RRR)
∆λCAPλλλ(SSS)
∆λCAPλλλ(SSS)
2.160(1)
2.150(1)
2.154(1)
Table 1: Selected structural data of four cations of [ZnTAMEisoquin] 2+: Average bond lengths (Å), twist angles(°), and
stereochemical configurations are reported.
3.3. Photophysical studies
10

Spectroscopic study of products from reaction of Zn(II) with TAMEisoquin is consistent
with weakened π bonding in isoquinoline upon coordination and a 1:1 stoichiometry of Zn(II)-
TAMEisoquin interaction. TAMEisoquin in aqueous DMF exhibits four maxima at 322nm (logε1 =
3.96 0.01), 311nm (logε2 = 3.91 0.01), 283 nm (logε3 = 4.00 0.01), and 272 nm (logε4 =
4.16 0.01) (Figure 3). When TAMEisoquin is exposed to 1.0 equiv of Zn(II) ion there is a slight
red shift of the four maxima with a concomitant enhancement of intensity at 325 nm and 313 nm
(logε1’ = 4.05 0.01 and logε2’ = 4.00 0.01 respectively) and attenuation of intensity at 284 nm
and 273 nm (logε3’ = 3.99 0.01 and logε4’ = 4.19 0.01 respectively. There is no further change
when TAMEisoquin is exposed to >1.0 equivalent of metal ion, supporting 1:1 M:L stoichiometry
as confirmed by Job’s plot analysis (Figure 4)
Figure 3: UV-Vis absorbance spectra of 56 µM TAMEisoquin in 0.1M HEPES buffer, pH 7.2. The black line
represents TAMEisoquin alone and the red line represents TAMEisoquin and 1 equivalent Zn(II).
11

Figure 4: Job’s plot analysis for fluorescence intensity of [ZnTAMEisoquin]2+ in 0.1 M HEPES buffer, pH 7.2 at
350 nm. Fluorescence intensity at 0 molar ratio of Zn(II) is due to background fluorescence of TAMEisoquin.
12

Figure 5: Quantum yield determination of [Zn(TAMEisoquin)]2+ in DMF:Water (1:1).
To determine emission efficiency, we measured the quantum yield of
[Zn(TAMEisoquin)]2+ by using quinine sulfate as a standard (1N H2SO4, Φf=0.54).29 A quantum
yield of 0.0077 is obtained for [Zn(TAMEisoquin)]2+ with a linear emission intensity up to a
concentration of ca. 20 mM (log K = 6.32 0.02 where F=K·[Zn(TAMEisoquin)]2+) (Figure 5).
TAMEisoquin shows nearly no fluorescence when excited with 321 nm light while the
addition of an equimolar amount of Zn(II) causes an 11-fold enhancement of emission intensity
(λem=350nm) (Figure 6). The emission intensities are detected at 350 nm and 480 nm
respectively. The Zn(II)-cation binds to both the secondary amines and the heterocyclic
nitrogens of TAMEisoquin preventing the PET-mediated quenching of the isoquinoline groups.
The sensor is then turned-on and the resulting luminescent response amplified via the
chelation-enhanced fluorescence (CHEF) mechanism.30,31 The emission intensity at 480nm is
due to excimer interaction between two isoquinoline molecules.32 Consequently, the decrease
in emission intensity at 480 nm is due to the breaking up of isoquinoline excimers upon
[Zn(TAMEisoquin)]2+ formation.
13

Figure 6: Fluorescence spectra of 14 µM TAMEisoquin (λex=321 nm) in the presence of an
increasing concentration of Zn(II) in 0.1 M HEPES buffer pH 7.2.
3.4. Binding constant determination
The binding affinity of Zn(II) to TAMEisoquin was determined by using a ligand exchange
method at pH 7.2 and ionic strength µ = 0.1 M. TPEN (N,N,N’,N’-Tetrakis-(2-pyridylmethyl)-
ethylenediamine), a well-known zinc chelator, was added to a solution of [ZnTAMEisoquin] 2+
and a decrease of approximately 50% of the original emission intensity remained constant after
1 week (Figure 7). From this data, a conditional dissociation constant Kd of 1.4 x 10-15 M was
calculated. The reaction was repeated with 2.0 eq of competing TPEN with the same outcome
(not shown). Thus, although timeframe of response is an issue for Zn(II) sensing in a situation of
changing Zn(II) concentration, the initial uptake of Zn(II) is quite rapid as shown by Job’s plot
studies (Figure 4).
14

Figure 7: Binding constant of [ZnTAMEisoquin]2+ in 0.1 M HEPES buffer, pH 7.2, at 350 nm. TPEN is used as a
competing ligand and equilibrium is reached after one week.
3.5. Metal-binding preference
A screen of comparative response of Zn(II) to other mono- and divalent metal ions
indicates the preference of TAMEisoquin toward Zn(II). The responses of TAMEisoquin to the
metal ions alone were first determined (Figure 8, black bars). TAMEisoquin is used as a
reference and the low fluorescence responses to dicationic metal ions of the first d-series, Mn(II)
to Cu(II), are expected based on their quenching behavior. The weak responses of Na(I), K(I),
and Ca(II) may be attributed to their very weak interactions with TAMEisoquin in accordance
with hard-soft acid-base theory and therefore an inability to engage the CHEF mechanism.
Although probes based on the PET/CHEF mechanism readily detect Cd(II), TAMEisoquin only
induces a small enhancement (16% of the Zn(II) response) of emission intensity at 350 nm. In
comparison, the emission intensity of cadmium complexes of N,N,N',N'-tetrakis(8-
quinolylmethyl)ethylenediamine (TQEN), and the 1– or 3–isoquinolyl analogues are
approximately 60% and <15% respectively of their corresponding zinc complexes.15,33 In order
to determine relative preferences of TAMEisoquin for Zn(II) against these metal ions, we
introduced 1.0 equiv of Zn(II) to the solution containing TAMEisoquin and selected metal ions
15

(Figure 8, red bars). TAMEisoquin detects Zn(II) in the presence of one equivalent of Na(I),
K(I), Ca(II) and Mn(II). The addition of Zn(II) to a solution containing 1.0 equiv of Fe(II), Cu(II),
Co(II) or Ni(II) showed slight fluorescence enhancement, which can be ascribed to a small
amount of displacement of these fluorescence–quenching d-metal ions by Zn(II). Moreover, with
reference to TPEN, the relative affinity of Zn(II) for TAMEisoquin against these metal ions is as
expected. Thus, the analysis of the fluorescence spectra (Figure 8) revealed that the order of
binding affinity of TAMEisoquin to 3d-metal ions agrees with Irving-Williams series
(Mn<Fe<Co<Cu>Zn).34 The low emission intensity for the Cd(II)–TAMEisoquin interaction
Figure 8: Preference of TAMEisoquin (TIQ) for Zn(II) over selected divalent metal ions in 0.1 M
HEPES buffer at pH 7.2. The black bars represent the emission of TAMEisoquin in the presence of
1eq of divalent metal ions of interest. The red bars represent the emission when 1 eq of Zn(II) is
added to each solution. The response I is normalized in reference to the free emission of the free
ligand “TIQ” (I0).
indicates that TAMEisoquin will detect Zn(II) with greater sensitivity than Cd(II).
Binding preference of TAMEisoquin for Zn(II) over Cd(II) is not definitively established in
the present studies, however the response of [Cd(II)(TAMEisoquin)]2+ challenged by Zn2+(aq)
indicates that Cd(II) is partially displaced. This result in concert with the computational study of
[Cd(II)(TAMEisoquin)]2+ indicating considerable distortions in CdN6 coordination sphere,
suggests that the poor fit of Cd(II) in TAMEisoquin leads to a decrease in the stability of
16

[Cd(II)(TAMEisoquin)]2+ relative to complexes without the binding-pocket constraints of
TAMEisoquin. It has been noted in the case of ligands that are sterically hindered near the
metal coordination site, such as 2-methyl-1,10-phenanthroline, that the formation constants for
Cd(II) are slightly larger than for Zn(II) (log β2 (Cd(II)) = 9.68 vs. log β2 (Zn(II)) = 9.35).35 The
present results suggest the formation constants of Zn(II) and Cd(II) for TAMEisoquin are on par,
but further study is necessary to verify this and to elucidate factors that will improve Zn/Cd
selectivity.
4. Conclusion
In this study, a novel zinc fluorescent sensor, TAMEisoquin was successfully
synthesized. The X-ray crystallographic analysis of [Zn(TAMEisoquin)]2+ reveals four unique
cations attributed to the flexibility of TAME framework. All four cations in the asymmetric unit
show a distorted octahedral geometry. Photophysical and metal ion competition studies
demonstrate Zn-binding stoichiometry of 1:1 and preference of Zn(II) over selected bioavailable
metal ions. The hexadentate, preorganized design of TAMEisoquin confers a high stability for
Zn(II) upon the sensor. The comparison of Zn(II) and Cd(II) responses confirms that
TAMEisoquin exhibits a stronger fluorescence response to Zn(II) while a computational study
showed that Cd(II) complex is more distorted than the Zn(II) complex but size selectivity for
Zn(II) over Cd(II) is not yet definitely shown. Further studies will be directed to improving the
aqueous solubility of the sensor and further modifying the ligand to improve preferences of
Zn(II) relative to Cd(II).
Acknowledgment
We acknowledge the National Science Foundation (CHE-1012897) for financial support. We
also thank Dr. Sarah Joiner for assistance with the computational studies of the Cd complex.
Appendix A. Supplementary data
CCDC
No
1416945
contains
the
supplementary
crystallographic
data
for
[Zn(TAMEisoquin)](ClO4)2. These data can be obtained free of charge via
http://www.ccdc.cam.ac.uk/conts/retrieving.html, or from the Cambridge Crystallographic Data
17

Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223-336-033; or email:
deposit@ccdc.can.ac.uk
ASSOCIATED CONTENT
Supporting information
1H NMR, 13C NMR and single crystal X-ray data
AUTHOR INFORMATION
Corresponding Author
E-mail: roy.planalp@unh.edu
Phone: +1 603 862 2471
† These authorscontributed equally.
Notes
The authors declare no competing for financial interest
18

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Abstract
Figure 1: Synthesis of [Zn (TAMEisoquin)] 2+
21