Angewandte
Chemie
erode as a new peak appears at 10.1 ppm, corresponding to an
aldehyde a-hydrogen of 2 (Figure S27). Furthermore, bulk
photolysis provided 2 as the major product. The identity of
this product was confirmed by TLC, NMR, FTIR, LC-MS and
GC-MS analysis.
We employed established protocols using 4-(2-pyridyl-
azo)resorcinol (PAR) to quantify metal binding properties of
NTAdeCage since the Zn2+ complex lacks a spectroscopic
signature.[17] The PAR signal changes fit
a
1:1
Zn2+:NTAdeCage binding model (Figure S9 and S10). Meas-
urements in triplicate reveal a Kd of 0.10 Æ 0.01 pm (40 mm
HEPES, 100 mm KCl, pH 7.0). This value is comparable to
that of unmodified NTA measured under the same conditions
with PAR (Kd = 3.7 Æ 0.4 pm) and the values measured by
potentiometric titration (logK = 10.7, Kd = 19.9 pm).[31] The
IDA photoproduct has log K of 7.2 for Zn2+ at pH 7 (Kd =
63 nm),[31] so the DKd (Kd IDA/Kd NTAdeCage) for this
photocaging system is nearly 630000. The DKd value provides
a convenient means to compare the metal ion releasing
properties of different photocages semi-quantitatively. The
DKd of NTAdeCage is smaller than those measured for Cleav
photocages, but is significantly higher than any Cast photo-
cage measured to date.[7]
We also used a metal ion displacement assay to evaluate
the impact of the other biologically relevant divalent cations
on the photocaged complex. By titrating [Zn(NTAdeCage)]À
with Ca2+ or Mg2+ in the presence of PAR, the appearance of
the [Zn(PAR)2] can be used to ascertain the concentration at
which interference becomes problematic. While no displace-
ment was observed from [Zn(NTAdeCage)]À when titrating
with Mg2+, at approximately 100 mm, a 10-fold excess with
respect to [Zn(NTAdeCage)]À in the selectivity experiment,
Ca2+ displaces most of the bound Zn2+ yielding [Ca(NTAde-
Cage)]À (Figure S13); however, at typical resting intracellular
[Ca2+] (ca. 100 nm), the displacement is negligible. Provided
Ca2+ do not exceed normal basal levels, [Zn(NTAdeCage)]À
should deliver Zn2+ as desired upon photolysis.
Both the buffering capacity NTAdeCage prior to irradi-
ation and the magnitude of the DKd after photolysis indicate
the photocage should be suitable for biological applications
where resting free Zn2+ concentrations are in the nm range. To
demonstrate metal ion release, [Zn(NTAdeCage)]À was
photolyzed and ZTRS,[32] a fluorescent Zn2+ sensor (Kd =
5.7 nm) was used to monitor increases in available Zn2+
(Figure S11 and S12). Aliquots from a bulk [Zn(NTAde-
Cage)]À solution being photolyzed were analyzed owing to
ZTRS photobleaching that occurred when the sensor was
exposed to the light source for uncaging. An increase in
fluorescence intensity at 512 nm, which is consistent with
emission of [Zn(ZTRS)]2+, in aliquots from the photolyzed
[Zn(NTAdeCage)]À solutions treated with ZTRS, demon-
strates that while higher affinity NTAdeCage chelator
effectively competes with ZTRS for Zn2+, the IDA photo-
product does not. The composite spectroscopic investigations
and proof-of-concept Zn2+ releasing assays demonstrate the
validity of the proposed uncaging mechanism.
Figure 3. Photolysis of [Zn(NTAdeCage)]À (80 mm) under simulated
physiological conditions (40 mm HEPES, 100 mm KCl, pH 7.5). Irradi-
ation at 365 nm (LED, 3 WcmÀ2) leads to the erosion of the absorption
band at 274 nm associated with the photocaged complex and the
concurrent formation of a band at 230 nm characteristic of photo-
product 2. Inset: change in absorbance at 230 nm with respect to
photolysis time in seconds.
photoproduct appeared in the LC trace in both experiments,
the m/z of photoproduct could not be detected by the coupled
MS instrument, so the photolyzed sample was subjected to
additional GCMS analysis. The mass of the photoproduct
(m/z 151) and the fragmentation pattern (m/z 105.0, 77.1 and
51.0) match the theoretical and actual 3-nitrobenzaldehyde
(2) spectrum. By monitoring the disappearance of NTAde-
Cage or the [Zn(NTAdeCage)]À complex, under these
experimental conditions the quantum yields (Fphotolysis) were
found to be 29 Æ 2% and 27 Æ 5% respectively.
In addition to the expected photoproducts (Figure 1),
several minor photoproducts observed in the LCMS indicate
the formation of a-amino radicals and possible coupling
between nitro compounds and 2; however, Zn2+-binding
appears to suppress these alternate photolysis pathways
yielding cleaner conversion to 2 (Figure S3 and S4). A similar
product distribution is observed when the photolysis is carried
out in slightly basic water (pH 7.5) in the absence of buffer
(Figure S1 and S2). Under non-aqueous conditions, such as in
methanol, the relative amount of these minor photoproducts
increases. Future detailed mechanistic studies will help
identify ligand design factors that limit radical branching
pathways and understand the uncaging mechanism in greater
detail.
Multiple spectroscopic analyses of the photoreaction also
confirm the LC/GCMS observations. The UV/Vis spectrum of
80 mm [Zn(NTAdeCage)]À (40 mm HEPES, 100 mm KCl,
pH 7.5) contains an absorption band at 274 nm and a minor
feature at 320 nm. During photolysis (lex = 365 nm), the
intensity of the 274 nm band decreases and a new peak
forms at 230 nm with an isosbestic point at 254 nm that
corresponds to the absorption of 2. The increase in absorb-
ance at 320 nm is attributed to the tail of the nitrobenzalde-
hyde peak and the formation of the minor photoproducts
observed in the LCMS. Complete photolysis of NTAdeCage
and the [Zn(NTAdeCage)]À complex occur within 6 min
(Figure 3). When monitoring the photolysis by 1H NMR
spectroscopy, the aliphatic protons initially sharpen and then
The feasibility for using this photocage in biological
systems was demonstrated by measuring Zn2+ transport
following the heterologous expression of the human (h) zinc
Angew. Chem. Int. Ed. 2015, 54, 13027 –13031
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