Communications
buffer over a 24 h time period, while 5 showed > 50%
hydrolysis (Figure S6). Although the use of carbonate and
carbamate ester linkages in self-immolative systems are more
common (due to the additional thermodynamic driving force
from the release of CO2 in the cascade reaction),[21,22] our
findings suggest that the carbonate ester linkage was not
optimal because of the low aqueous stability observed for 5.
In addition, we found that incorporation of the carbonate
ester linkage was synthetically more challenging and less
reliable (i.e. when comparing the synthesis of 4 versus 5),
which further discouraged its use in a metalloprotein proin-
hibitor approach. Indeed, despite numerous attempts, we
were unable to achieve a satisfactory synthesis for the
carbonate ester analog of compound 3.
After establishing a strategy for the addition of H2O2
activated protecting groups to the appropriate ZBGs, the full-
length inhibitors 1,2-HOPO-2 and PY-2 were protected with
4-bromomethylphenyl boronic acid pinacol ester in the
presence of K2CO3 in DMF to yield compounds 1 and 2,
respectively. Activation of 1 and 2 by H2O2 to release 1,2-
HOPO-2 and PY-2 was confirmed by absorption spectrosco-
py (Figure S7 and S8). The spectral changes observed for 1
and 2 (obtained under the same reaction conditions as those
used for compounds 3 and 4) suggest that the cleavage
kinetics for the proinhibitors are comparable to the ZBGs.
The IC50 values of the proinhibitors 1 and 2 against MMP-9
were found to be greater than 1 mm, representing a > 100
fold-increase than the active inhibitor (Table 1). When 1 and 2
Scheme 2. Structures of proinhibitors 1 and 2 and their active inhib-
itors 1,2-HOPO-2 and PY-2, respectively, and the protected ZBGs 3–5.
The ROS-triggered self-immolative protecting group can
be attached to the MMPi by using either an ether (3, 4) or
carbonate ester (5) linkage at the hydroxy group of the ZBG
(Scheme 2). To determine which linker strategy provided the
best overall approach, both the cleavage kinetics and solution
stability of protected ZBGs 3–5 were examined (see Support-
ing Information). The ability of these compounds to be
activated by H2O2 was evaluated by using electronic spec-
troscopy. A sample of each compound in HEPES buffer
(50 mm, pH 7.5) was activated with an excess (18 equiv)[12–15]
of H2O2 and the change in absorbance was monitored over
time. In all cases, the spectra of the protected ZBG
compounds decreased over time while the spectra of the
free ZBG appeared, demonstrating the expected cleavage
reaction (Supporting Information, Figure S1–S3). To confirm
that the boronic ester moiety was necessary for H2O2
cleavage, the ZBGs were prepared with benzyl protecting
groups without the boronic ester. For these compounds, no
change in absorbance was observed over time in the presence
of H2O2 (Figure S4). Additionally, the selectivity of the
boronic ester towards H2O2 was confirmed by examining
cleavage in the presence of KO2 and catalase (Figure S5). As
expected,[12,20] the superoxide anion was unable to activate the
protected ZBGs.
The rates of conversion of compounds 3–5 to their
respective activated ZBGs were then determined by mon-
itoring the change in absorption using pseudo-first order
reaction conditions with an excess of H2O2. The calculated
rate constants indicated that the carbonate ester linkage in
compound 5 provided the fastest conversion with a rate
constant of 6.7mÀ1 sÀ1, while rate constants of 4.0mÀ1 sÀ1 and
2.9mÀ1 sÀ1 were found for compounds 3 and 4, respectively
(see Supporting Information). Upon examination of the
solution stability of these compounds, 3 and 4 were stable in
Table 1: IC50 values of proinhibitors and inhibitors against MMP-9 and
MMP-12 as measured using a fluorescence based assay. Data are the
average of two experiments.
Pro-
inhib-
itor
IC50
Inhibitor
IC50
Enzyme
1
1
2
2
>1 mm[a]
1,2-HOPO-2 6.1(Æ0.2) mm
MMP-9
MMP-12
MMP-9
17.8(Æ1.1) mm
1,2-HOPO-2 0.053(Æ0.01) mm
>1 mm[b]
PY-2
9.8(Æ0.7) mm
12.9(Æ0.03) mm PY-2
0.035(Æ0.003) mm MMP-12
[a] 46% inhibition at 1 mm. [b] 27% inhibition at 1 mm.
were tested against MMP-12, their IC50 values were found to
be in the micromolar range (Table 1), which was again > 100-
fold less effective than their activated counterparts. Both sets
of experiments show that when the ZBG of the inhibitor is
protected, the ability of the compounds to inhibit MMPs is
severely attenuated.
Having established that proinhibitors 1 and 2 could be
effectively protected and activated in the presence of H2O2,
the ability of these compounds to inhibit MMPs after
activation was evaluated. Using a fluorescence-based assay,
compounds 1 and 2 were tested with MMP-9 and MMP-12 in
the presence of H2O2 at concentrations close to their reported
IC50 values.[11] MMP-9 is considered a high-value MMP target
in the context of ischemia-reperfusion injury associated with
stroke.[7] The percent inhibition of proinhibitors 1 and 2 were
evaluated after one hour of activation with and without H2O2.
As expected, when there is no hydrogen peroxide present,
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2010, 49, 6795 –6797