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

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.

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.

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

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.

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.

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 = 33300 M�1 cm�1;

Fig. 2, top) and measuring the intensity of the source by iron

oxalate actinometry,14 a quantum yield of 0.7 � 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

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 � 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 mMconcentrations, DMSO co-solvent was required to maintain

solubility of the apo ligand. Upon addition of Zn2+ the

absorption of ZinCast-1 (lmax = 262 nm, e = 5620 M�1 cm�1)

eroded, and the binding was fit to a 1 : 1 isotherm with a

14.3 mM Kd. ZinUnc-1 absorption also diminished upon the

Department of Chemistry, University of Connecticut, Storrs,CT 06269-3060, USA. E-mail: shawn.burdette@uconn.eduw Electronic supplementary information (ESI) available: Experimentalprocedures, additional spectroscopic data and NMR spectra for newcompounds. See DOI: 10.1039/b913605c

This journal is �c The Royal Society of Chemistry 2009 Chem. Commun., 2009, 6967–6969 | 6967

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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

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

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+.

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 mMZinUnc-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.

6968 | Chem. Commun., 2009, 6967–6969 This journal is �c The Royal Society of Chemistry 2009

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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

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+

from the photolyzed ZinCast-1. Inset: changes in normalized

integrated emission.

This journal is �c The Royal Society of Chemistry 2009 Chem. Commun., 2009, 6967–6969 | 6969

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