ZinCast-1: A photochemically active chelator for Zn2+

Article abstract of DOI:10.1039/b913605c

Two strategies were applied to the synthesis of ZinCast-1, a nitrobenzhydrol-based caged complex that upon photolysis exhibits a nearly 400-fold difference in binding for Zn²⁺. The Royal Society of Chemistry 2009.

Full text of DOI:10.1039/b913605c

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ZinCast-1: a photochemically active chelator for Zn2+w
Celina Gwizdala, Daniel P. Kennedy and Shawn C. Burdette*
Received (in Austin, TX, USA) 9th July 2009, Accepted 22nd September 2009
First published as an Advance Article on the web 14th October 2009
DOI: 10.1039/b913605c
Two strategies were applied to the synthesis of ZinCast-1,
a nitrobenzhydrol-based caged complex that upon photolysis
substitution reaction with 3,4-dimethoxybenzoic acid was used
to assemble the backbone of the cage. After installation of the
nitro group, 5 was reduced with sodium borohydride to
provide ZinCast-1 in 16% overall yield. In contrast to our
previous report,7 the benzophenone was reduced cleanly to the
corresponding benzhydrol in reasonable yield (33%) when the
temperature was maintained at 70 1C and excess borohydride
was quenched when the reaction was complete.
exhibits a nearly 400-fold difference in binding for Zn2+
.
Caged compounds are valuable tools for delineating signaling
pathways of small molecules and metal ions.1 While caged
Ca2+ has been utilized extensively,2,3 similar complexes are
not available for other metal ions except Cu2+ 4
. We are
interested in Zn2+ homeostasis and signaling, so we are
developing new caged complexes. We have reported on a
complex that releases Zn2+ upon cleavage of a ligand back-
bone.5 The reduction of chelate effects produces a drastic
increase in free Zn2+; however, generating modest changes
in metal binding equilibra can provide insight into different
processes.
The second pathway utilizes a Pd-catalyzed boronylation
and an aryl-aldehyde cross coupling reaction. The unique
reagent {[K(18-crown-6)]ICl2}n, an analogue of the corres-
ponding tribromide,12 was used to access the aryl iodide. This
streamlines our synthetic route by eliminating the halogen
exchange step.7 The Pd-catalyzed reaction of
6 with
bis(picanolato) ester provided 7 for the aryl-aldehyde coupling
using tris(1-naphthyl)phosphine (P(1-NAP)3).13 As shown
previously,7 conversion to the boronic acid is not necessary
since the boronate ester is coupled efficiently (52% yield).
While more work is needed to fully reveal the scope of this
reaction, the overall yield of 26% of ZinCast-1 from 3 suggests
this will be a versatile method for making cages.
Modulating metal–ligand interactions photochemically
provides an uncaging mechanism that changes the free metal
ion binding equilibria moderately.6 Recently we reported on
two new methods for preparing nitrobenzhydrol-based metal
ion cages using an aza-crown ether substrate.7 The optimal
preparative method utilized Pd-catalyzed coupling reactions,
but since the model ligand has a low affinity for Pd, we wanted
to demonstrate the efficacy of our method with ligands
designed to bind d-block metals like Zn2+. We also wanted
to ascertain how this uncaging strategy would modulate Zn2+
concentrations in aqueous solution.
The photochemistry of ZinCast-1 was evaluated by irradiating
a 25 mM solution of the free ligand in buffer (20% DMSO,
50 mM HEPES, 100 mM KCl, pH 7) at 350 nm for 3 h with a
150 W source. By monitoring the absorption of the ZinCast-1
photoproduct ZinUnc-1 (2, lmax = 349, e = 33 300 MÀ1 cmÀ1
;
Fig. 2, top) and measuring the intensity of the source by iron
oxalate actinometry,14 a quantum yield of 0.7 Æ 0.1% was
calculated for the conversion. The nomenclature ZinUnc
refers to the uncaged version of the ZinCast chelator. The
low quantum yield may be attributed to energetically low-lying
charge transfer (CT) states in the dimethoxynitrobenzene15,16
ZinCast-1 (1) incorporates an aryl-dipicolylamino (DPA)
ligand with a nitrobenzhydrol caging group. A DPA motif was
chosen since similar ligands are components of numerous
Zn2+ sensors.8–10 The shorthand name ZinCast comes from
the ability of the ligand to cast off zinc upon photolysis.
ZinCast-1 uncaging involves a nitro group abstracting a
benzylic hydrogen atom, which converts the alcohol into a
ketone (Fig. 1). After uncaging, the nitrogen lone pair is
partially delocalized onto the keto-oxygen atom. DFT
calculations at the B3LYP/6-31G level of theory with
Gaussian 0311 suggests a >50% decrease in electron density
on the aniline nitrogen atom of the benzophenone. The less
electron rich nitrogen results in a weaker interaction with
Zn2+, which shifts the equilibrium between the bound and
unbound form of the chelator. The desired ligand was syn-
thesized by two different methods using N-phenyl-di(2-picolyl)-
amine (3) as the starting material (Scheme 1). In the first, a
polyphosphoric acid (PPA) promoted electrophilic aromatic
or aniline fragments. ZinUnc-1 was synthesized on
a
preparative scale to allow the spectroscopic and metal binding
properties to be measured without interference from other
species. In analogous experiments
a quantum yield of
1.0 Æ 0.2% was calculated for [Zn(ZinCast-1)]2+. The
increased quantum yield of the metal species is consistent with
other nitrobenzhydrol-based caged complexes,6,7 which we
hypothesize results from shifting of an anilino CT state away
from the absorption of uncaging.
The Zn2+ binding affinity of both ZinCast-1 and ZinUnc-1
was measured in the 4 : 1 aqueous buffer–DMSO solution to
predict the ability of the cages to increase free Zn2+. While
[Zn(ZinCast-1)]2+ is soluble in aqueous solution at high mM
concentrations, DMSO co-solvent was required to maintain
solubility of the apo ligand. Upon addition of Zn2+ the
Department of Chemistry, University of Connecticut, Storrs,
CT 06269-3060, USA. E-mail: shawn.burdette@uconn.edu
w Electronic supplementary information (ESI) available: Experimental
procedures, additional spectroscopic data and NMR spectra for new
compounds. See DOI: 10.1039/b913605c
absorption of ZinCast-1 (lmax = 262 nm, e = 5620 MÀ1 cmÀ1
)
eroded, and the binding was fit to a 1 : 1 isotherm with a
14.3 mM Kd. ZinUnc-1 absorption also diminished upon the
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Scheme 1 Synthesis of ZinCast-1.
Fig. 1 Uncaging action of ZinCast-1. Conversion of the ligand from
a benzhydrol to a benzophenone decreases the electron density on the
aniline nitrogen atom, which lowers the ligand’s affinity for Zn2+
.
addition of Zn2+, but the dissociation constant of the 1 : 1
complex decreased by nearly 400-fold to 5.5 mM (Fig. 2,
bottom). The change in binding affinity is consistent with the
theoretical calculations predicting a reduction in electron
density on the aniline nitrogen atom, which would result in a
weaker metal–nitrogen bond. A weakly bound (log K12 = 1.45)
2 : 1 L : M species was observed by NMR titration in DMSO
at 10 mM ZinCast-1; however, these low affinity complexes
will not be present at the concentrations used in the spectro-
photometric experiments.
Since aryl-DPA ligands have been used as Cd2+ and Cu2+
chelators,17,18 the binding properties of ZinCast-1 with these
metal ions was investigated. The 55-fold decrease in binding
strength for Cd2+ from 425 mM to 22.6 mM for caged and
uncaged ZinCast-1, respectively, is similar to the magnitude of
change observed with Zn2+; however, the decreased stability
of the complexes suggests that aryl-DPA does not accommodate
the coordination requirements of Cd2+ as well as it does for its
smaller congener. Very little change in binding strength was
measured with Cu2+ for ZinCast-1 (Kd = 4.5 mM) and
ZinUnc-1 (Kd = 1.6 mM). While the increased stability of
the Cu2+ complexes follows the predictions of the Irving Williams
series,19 neither size nor Lewis acidity arguments explain the
minute affinity change. With the exception of this current
study and one previous report, nitrobenzhydrol-derived caged
Fig. 2 Changes in absorption of 25 mM [Zn(ZinCast-1)]2+ upon
irradiation at 350 nm by a 150 W source (top). Titration of 25 mM
ZinUnc-1 with ZnCl2 (bottom). Inset 1 : 1 isotherm of the absolute
value of the absorption changes at 355 nm. Error bars represent
deviations over 3 trials.
complexes have only been screened for Ca2+ and Mg2+
affinity.2,7 The Cu2+ binding behavior warrants additional
investigation; however, if free Cu2+ was present in a biological
assay, photolysis of ZinCast-1 would not change its concentration
significantly, which gives the caged complex a modicum of
selectivity for Zn2+
.
To demonstrate the ability of the new caged complex to
release Zn2+, [Zn(ZinCast-1)]2+ was photolyzed in the
presence of the fluorescent sensor ZP1B, which has a Kd of
13 mM for Zn2+ (Fig. 3).20 Upon irradiation, the emission of
the sensor increases as ZinCast-1 is converted into ZinUnc-1.
Nearly complete restoration of emission and post-photolysis
absorption measurements suggest minimal photobleaching of
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investigations. The current study provides a roadmap for
preparing new cages and guiding investigations of photo-
physical properties of nitrobenzyl derivatives.
This work was supported by the University of Connecticut.
The authors thank B. Wong and Prof. S. J. Lippard for the
generous donation of ZP1B.
Notes and references
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Fig. 3 Fluorescence response of ZP1B upon uncaging of ZinCast-1.
The emission intensity of a solution of 5 mM ZP1B (50 mM HEPES,
100 mM KCl, 20% DMSO, pH 7) was recorded before and after the
addition of 40 mM ZnCl2. The emission was integrated between 480
and 620 nm and normalized to the maximum response. Subsequent
addition of 25 mM ZinCast-1 partially reduced the emission of the
ZP1B complex, which has a Kd of 13 mM for Zn2+. Irradiation of the
solution in an Applied Photophysics photoreactor (two 4 W lamps,
lex = 350 nm) resulted in a complete photolysis of ZinCast-1 within
50 minutes. This behavior is consistent with ZP1B acquiring Zn2+
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ZP1B occurs. While this method illustrates light-induced
changes in Zn2+ binding equilibria, the low intensity source
(two 4 W lamps) precludes photolysis at a rate relevant to
biological applications. Flash photolysis is the preferred
uncaging technique since it provides high intensity high-speed
light bursts that are capable of photolyzing cages efficiently.21
When ZinCast complexes with binding and photochemical
properties suitable for studying Zn2+ biology become available,
detailed flash photolysis studies will be conducted.
The induced changes in free Zn2+ concentrations upon
photolysis of [Zn(ZinCast-1)]2+ can be simulated using HySS.22
An equimolar mixture of ZinCast-1 and Zn2+ (50 mM each)
results in 21 mM of free Zn2+ at equilibrium, a concentration
above the activation (r1 mM) of many extracellular Zn2+
receptors; however by using a 10-fold excess of ZinCast-1 (50 mM)
to Zn2+ (5 mM), free Zn2+ concentrations can be maintained
at 1.3 mM. If flash photolysis converts 50% of the cage to
ZinUnc-1, equilibrium free Zn2+ would increase to 33 mM and
2.1 mM for each scenario, respectively. Although these
calculations do not account for endogenous Zn2+ or biologi-
cal chelators, the modeling indicates that if the Zn2+ affinity of
future ZinCast derivatives is increased, ZinCast complexes are
capable of modulating free Zn2+ under physiological conditions.
In conclusion, we have reported on two synthetic routes for
accessing ZinCast-1, a new caged complex for Zn2+. ZinCast-1
has a mM affinity for Zn2+ that decreases over 2 orders of
magnitude after photolysis. Future investigations will focus on
optimizing the quantum efficiency of uncaging, and tuning
the Zn2+ binding properties of cages for specific biological
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Chem. Commun., 2009, 6967–6969 | 6969