Examination of the reactivity of benzoxaboroles and related compounds with a cis -diol

Article abstract of DOI:10.1021/jo302264g

Benzoxaboroles have been emerging as an interesting and useful scaffold in drug discovery due to their apparently unique reactivity toward diols under physiological conditions. In this work, the reaction of benzoxaborole with the diol-containing, fluorescent dye Alizarin Red S is probed. Steady-state and presteady-state experiments have been conducted for the characterization of the reactions over a wide range of pH. Results indicate that Alizarin Red S reacts with both the boronic (neutral, trigonal) form as well as the boronate (anionic, tetrahedral) form of benzoxaborole in a reaction largely analogous to that previously determined for the simple phenylboronic acid. However, in certain key aspects, the reactivity of the benzoxaborole was found to differ from that of simple phenylboronic acid. The structural origin of these differences has been explored by examination of compounds related to both benzoxaborole and phenylboronic acid. These results may be applied to rational drug discovery efforts aimed at expanding the use of benzoxaboroles in medicine.

Full text of DOI:10.1021/jo302264g

Article
pubs.acs.org/joc
Examination of the Reactivity of Benzoxaboroles and Related
Compounds with a cis-Diol
†
John W. Tomsho and Stephen J. Benkovic*
Department of Chemistry, Penn State University, 414 Wartik Laboratory, University Park, Pennsylvania 16802, United States
*
S Supporting Information
ABSTRACT: Benzoxaboroles have been emerging as an
interesting and useful scaffold in drug discovery due to their
apparently unique reactivity toward diols under physiological
conditions. In this work, the reaction of benzoxaborole with
the diol-containing, fluorescent dye Alizarin Red S is probed.
Steady-state and presteady-state experiments have been
conducted for the characterization of the reactions over a
wide range of pH. Results indicate that Alizarin Red S reacts
with both the boronic (neutral, trigonal) form as well as the
boronate (anionic, tetrahedral) form of benzoxaborole in a reaction largely analogous to that previously determined for the
simple phenylboronic acid. However, in certain key aspects, the reactivity of the benzoxaborole was found to differ from that of
simple phenylboronic acid. The structural origin of these differences has been explored by examination of compounds related to
both benzoxaborole and phenylboronic acid. These results may be applied to rational drug discovery efforts aimed at expanding
the use of benzoxaboroles in medicine.
INTRODUCTION
differences arising upon reaction with boronic acids, ready
reaction with boronic acids in neutral, aqueous solution, and a
■
1
During most of the 55 years since their discovery, the
benzoxaborole heterocycle (1) has received little attention until
recently. This heterocycle consists of a phenyl ring fused with a
five-membered cyclic boronate termed an oxaborole. Indeed,
other oxaborole-containing compounds are already finding
applications in drug discovery and synthetic protocols. The
benzoxaboroles in particular have been found to possess high
water solubility and resistance to hydrolysis, excellent sugar
low pK that resembles “biological” nucleophiles. The reaction
a
and its products have been characterized across a wide range of
pH of approximately 4−9. The results of these experiments are
compared with those recently determined with the simple
18
phenylboronic acid (PBA, 4) in order to elucidate any key
2
3
mechanistic differences between the two systems. Finally,
related compounds (Figure 1) with altered heterocyclic ring
4
systems (2 and 3) or altered pK via ring substitutions (5 and
5
,6
a
(
diol) binding under physiologically relevant conditions, and
6
) have been examined to assess the cause of any differences.
7
a low pK compared with that of simple phenylboronic acid.
a
In general, it is found that benzoxaborole reacts with Alizarin
Of particular interest, the use of the benzoxaborole scaffold in
drug discovery has been on the rise.
compounds have found use as inhibitors of Leu-tRNA
Red S with a mechanism analogous to that determined for
8
,9
To date, these
18
phenylboronic acid. The reaction proceeds preferentially at
near neutral solution pH via a sequential two-step reaction in
which a bimolecular esterification is followed by an intra-
molecular cyclization resulting in a diester spiro adduct. The
unique reactivity of the benzoxaborole heterocycle arises from
the kinetic contributions of the cyclization reaction. This
enhanced cyclization appears to arise from the contributions of
the five-membered oxaborole heterocycle.
10
11
synthetases (for antifungal, antibacterial, and antitrypano-
1
2,13
somal agents.
), phosphodiesterase-4 (PDE-4, anti-inflam-
1
4
15
11
matory), β-lactamase, and several antibacterials.
In addition, this scaffold has been recently found to have
several applications in biotechnology including incorporation
into polymeric materials for sugar-responsive insulin delivery
1
6
17
and mediating the delivery of a protein toxin to the cytosol. In
general, the benzoxaborole scaffold has been selected time and
again for its enhanced reactivity or binding to target diols
whether on the cell surface or in the active site of an enzyme. In
this work, we attempt to answer the question “What properties
of this scaffold are responsible for its unique reactivity?”.
To answer this question, the reactivity of a benzoxaborole
RESULTS AND DISCUSSION
■
Steady-State Analysis. The investigation of the reaction
between 1 and ARS was begun by examining the individual
reactants as well as the products of the spontaneous reaction
that occurs in buffered aqueous solution via spectrophotometric
methods. In all cases reported in this work, Good buffers,
(1) with a model diol Alizarin Red S (ARS) in aqueous solution
has been examined in both the steady-state and presteady-state.
ARS was chosen as a model system since it has several desirable
characteristics including: fluorescence and UV/vis spectral
Received: October 24, 2012
Published: November 1, 2012
©
2012 American Chemical Society
11200
dx.doi.org/10.1021/jo302264g | J. Org. Chem. 2012, 77, 11200−11209

The Journal of Organic Chemistry
Article
Figure 1. Compound structures and designations.
versus other common buffers such as Tris or phosphate, were
chosen due to their previously determined unreactivity toward
1
8
boronic acids. Solution pH was varied from approximately 4
to 10 with buffers while extremes of pH, 1 and 13, were
achieved with dilute HCl and NaOH solutions respectively.
The UV/vis spectra of the compounds illustrated in Figure 1
and the product of the 1:ARS reaction were taken at each pH
Figure 2. Fluorescence vs pH for ARS (●) and 1:ARS (■). Excitation
was at 440 nm while emission was monitored at 565 and 585 nm
respectively. The fluorescence at pH = 1 and 13 was similar and small
for both solution sets and was not shown for clarity. Data for 1:ARS
were fit to a two-pK equation, eq 1, and resulted in pK ’s of 4.9 and
7
a
a
studied. Utilizing the method of Tomsho, et al, pKa
8
.7.
determinations were made (the data for 1, ARS, and 1:ARS
are reported in the Supporting Information) and the results are
summarized in Table 1. Of particular note is the finding that
fold enhancement in fluorescence is observed for the 1:ARS
adduct when compared to ARS. The spectra for the 1:ARS
adduct from pH 4 - 9 exhibit unchanging wavelength maxima,
440 ± 5 nm excitation and 585 ± 5 nm emission, across pH
indicative of a single, major fluorescent species (Data not
shown). This adduct species exhibits a red shift in the emission
maxima, when compared with ARS (565 nm), consistent with
extended conjugation of the pi system. The resulting
fluorescence vs pH curve can be fit by eq 1. This equation
describes a linear reaction in which three species are
interconverting in a pH dependent manner with the middle
species solely contributing the fluorescence signal. The
Table 1. pK Values Determined by UV/Vis Spectral
a
a
Titration
compound
pKa
b
c
c
1
2
3
4
5
6
7.3 ; 7.7
b
8.4
b
8.3
b
9.1 ; 9.2
b
8.4
b
7.6
c
ARS
:ARS adduct
6.0, 11.0
4.6, 9.5, 11
resulting pK values obtained by the fitting of this data are
c
a
1
4
.9 ± 0.1 and 8.7 ± 0.1. These results may be compared with
a
The pK determinations were achieved by the method of Tomsho, et
a
those obtained from UV/vis methods and it is found that the
values obtained from the two methods are similar. Any
differences noted may be due to the fact that the fluorescent
measurements are heavily biased toward the product
contributions with their much higher signal relative to the
reactants.
7
al. which also reported the pKa of 1−3 and 6 with a lesser
concentration of DMSO in the final solution than those utilized
herein. DMSO was used to prepare high concentration stock solutions
of compounds 1−6. These data are reported here for ready
b
c
comparison. 0.5% v/v DMSO. 4% v/v DMSO.
[Fluorescent Species]
two of the observed pK ’s for the 1:ARS adduct differ
a
(
pKa2−pH)
significantly from those measured for 1 and ARS individually
while the highest (11) is an exact match for the second alcohol
deprotonation of “free” ARS. The interpretation of this finding
is that under conditions of pH ≤ 10 the reaction equilibrium
lies to the right and the product spectra are being observed,
while at high pH the reaction equilibrium lies to the left and the
spectra are reflective of the reactant.
Fluorescence characterizations were subsequently pursued
and it was found that none of the boronic acid compounds
under study exhibited significant intrinsic fluorescence. Both
ARS and the 1:ARS adduct exhibited fluorescence across a
wide range of pH as is shown in Figure 2. At the pH of
maximum fluorescence, between pH 6.5 and 7.0, a nearly 100-
[ARS] × 10
T
= 1
+ (10
)
+ 10(pKa2−pH)
(pK −pH)
·10(pKa2−pH)
a1
(1)
Equation 1 describes a model where three species are in an
equilibrium connected by two ionizations with only the middle
species contributing to the fluorescent signal. It is assumed that
the fluorescence signal is directly proportional to the
concentration of the fluorescent species, [Fluorescent Species].
The equation is placed in terms of the total concentration of
ARS, [ARS] , observed ionization constants (pK and pKa2),
T
a1
and solution pH.
The properties of the fluorescent species were further
characterized with fluorescent lifetime measurements taken
1
1201
dx.doi.org/10.1021/jo302264g | J. Org. Chem. 2012, 77, 11200−11209

The Journal of Organic Chemistry
Article
from pH 4−10. Consistent with the previous results, two
species were detected with one species accounting for 82−88%
of the fluorescent signal from pH 4−9. The major fluorescent
species’ lifetime varies from 0.49−0.64 ns while the minor
species’ lifetime was 2.49−2.05 ns as pH is varied from 4 to 10.
These data also reveal that the major fluorescent species
undergoes a change in lifetime from 0.49 ns at pH = 4 to 0.64
ns at pH 6−10, likely corresponding to the observed 1:ARS
adduct pK of 4.6−4.9. From pH 6−8, both relative fluorescent
a
contributions and lifetime measurements are unchanging. At
high pH, an explanation for the observed loss of fluorescence
may be found. Over a pH of 8−10, the lifetime of the minor
species changes from 2.36 to 2.05 ns respectively. Additionally,
the relative contribution to fluorescence of the major species
drops from 87 to 66% as pH is increased from 9 to 10. Taken
together, these data are indicative of another ionization step in
which the highly fluorescent adduct species is transitioning to a
species with much less fluorescence as a function of pH. This
finding reinforces the validity of the 1:ARS adduct pK of 8.7−
a
9.5 from the fluorescence and UV/vis spectral data, Figure 2
and Table 1, respectively.
Since useable fluorescence signal changes were noted after
the reaction between 1 and ARS in solution at near neutral pH,
steady-state titrations with fluorescence detection were
executed. Using a fixed, low concentration of ARS, 20 μM,
solutions were prepared such that the concentration of 1 was
varied from 0 to 10 mM. After a fixed incubation period, the
change in fluorescence of the solution was measured and
plotted against total [1] (Figure 3). The data were fit with eq 2
Figure 3. Steady-state fluorescent titration of ARS with 1 for the
determination of the association constant at pH 5 (red ), 6 (orange
●
■
), 7 (green ), 8 (blue ▲), and 9 (▼). [ARS] = 20 μM while [1]total
◆
was varied from 0 to 10 mM. Results of fitting to eq 2 are indicated in
Table 2.
thermodynamic binding constants. Reactions were set up at
each pH from 5 to 10 with a solution of 1 being titrated into a
solution ARS in the receiving cell. The buffer composition and
DMSO percentage were matched in both solutions to minimize
background heats of dilution. In addition, the heat of dilution of
1 was measured and used as a baseline for the reaction
measurements. Across all pH conditions examined, 1:1 binding
between 1 and ARS was found. A representative titration curve
and binding constant determination is shown in Figure 4. The
binding constants measured with this method are reported in
Table 2 and are found to closely match those determined by
the fluorescent titration method. These findings then are useful
for consideration of the overall reaction within this system for
Table 2. Comparison of the Association Constants for 1 and
ARS as Determined by Three Methods at Various pH Values
K (M−1)
Ka1 (M−1
)
K2
a
b
kinetics
a
a
11B-NMR
c
pH fluorescent titration
ITC
(k /k )
on off
5
6
7
8
9
1
690 ± 30
480 ± 20
2030 ± 80
3390 ± 420
780 ± 30
420 ± 180
1140 ± 200
1950 ± 480
840 ± 100
170 ± 25
0.40 ± 0.04
0.70 ± 0.05
0.87 ± 0.04
2580 ± 90
3190 ± 110
1140 ± 40
100 ± 10
d
ND
e
d
ND
ND
the transition from reactants to all potential products.
e
e
e
0
ND
ND
ND
0.12 ± 0.03
In our previous work with phenylboronic acid, 11B NMR
18
a
Overall K values are calculated based on the total concentration of 1
a
was found to be an extremely useful tool and provide important
information about the reaction equilibrium. This method is of
use because of its sensitivity to the changes in the configuration
about the boron atom in solution. Specifically, it allows the
independent measurement of the diester, spiro product that is
tetrahedral about the boron atom due to its distinctive chemical
shift. The structure of this product is illustrated in Figure 5 and
is labeled as the NMR Adduct since its exclusive contribution
to the reaction may be measured via this technique. The NMR
spectra thus obtained reveal two unique peaks, one from all of
the boron-containing species in rapid equilibrium (i.e., free 1
and the Fluorescent Adduct shown in Figure 5) and the other
from the slowly exchanging NMR Adduct. The data obtained
from the integration of these peaks is then used to calculate K2.
−1
b
at each pH and are expressed in units of M . Values are calculated by
dividing the observed k by koff from the kinetic progress curves at
on
c
11
each condition of pH. K values (unitless) calculated based on B-
NMR experiments as described previously. Under these conditions
2
18 d
of pH, significant peak overlap precluded accurate integration.
e
Binding under these conditions provided too low a signal to allow
for accurate measurement; ND = not determined.
to determine the binding constants and the results are
summarized in Table 2.
ΔFluorescence = [(
B] × Fluor
1/K ) + [B]
T
max
(2)
a
T
Equation 2 describes the Ka determination by two-
component fluorescent titrations; ΔFluorescence is the
The data and K calculations are shown in the Supporting
Information. Tabulated results are found in Table 2.
Inspection of these data immediately revealed that the
reaction of this system must proceed through a two-step
mechanism such as that depicted in Figure 5. In this reaction,
the first adduct formed is the result of a single dehydration step
while a second dehydration must occur to form the diester,
2
observed change in fluorescence, [B] is the total concentration
T
of boron species, and Fluormax is the maximum change in
fluorescence.
Isothermal titration calorimetry (ITC) was then used to
examine stoichiometry of binding and to determine true
1
1202
dx.doi.org/10.1021/jo302264g | J. Org. Chem. 2012, 77, 11200−11209

The Journal of Organic Chemistry
Article
found to conclusively support the existence of a ring-opened
intermediate. Additionally, the pK of a diester, ring-opened
a
adduct structure would likely be extremely low (<5) hence the
neutral, trigonal planar form would not be a significant
contributor with solution pH from 5 to 9 when the fluorescence
is most intense.
Thus far, steady-state examinations have revealed important
information about the nature of the reaction in question: (1)
UV/vis spectral analyses have elucidated the ionization
constants of the reactants and major products, (2) titrations
have characterized the thermodynamics of the overall reaction,
(
3) 11B NMR has allowed us to conclude that the reaction
proceeds in two sequential steps and to quantitate the
contribution of one of the product species, and (4) examination
of the fluorescent lifetime data combined with the application
of chemical insight has allowed us to assign the likely structure
of the major contributor to fluorescence to a structure that is
trigonal about boron with empty p orbitals and a planar
structure that provides a system with extended conjugation. Yet
there is a dearth of data that characterize the initial phase of the
reaction between 1 and ARS. For this information, we turn to
studies of the reaction in the presteady-state.
Presteady-State Analysis. Reaction kinetics were moni-
tored using a stopped-flow apparatus with fluorescence
detection. Solutions of 1 and ARS were prepared in matched
buffer solutions (buffer strength, [DMSO], etc.) from pH 4 to
9
.5. The concentration of ARS was fixed and limiting (100 μM
Figure 4. Isothermal titration calorimetry of the reaction of 1 with
final) while 1 was varied in excess (1−4 mM final). The
reaction was initiated with rapid mixing of the two solutions.
The resulting fluorescence progress curves were fit to a single
exponential and the apparent rates were plotted versus [1].
Linear fitting of these data yield the formation and dissociation
rates, via the slope (k ) and y-intercept (k ) respectively, for
ARS at pH 7. Data fits well to a one-site binding model resulting in K
a
−1
−1 −1
=
3390 ± 420 M , ΔH = −17.9 ± 0.2 kJ/mol, ΔS = 7.57 J mol K ,
and ΔG = −20.2 ± 0.2 kJ/mol.
spiro adduct. The overall binding, as determined by either
fluorescence titrations or ITC, between 1 and ARS to yield all
products reaches its maxima between pH 6 and 7. These
findings are consistent with the preferred reaction occurring
on
off
the reactions at each pH. The kon and koff data are plotted
versus pH and shown in Figure 6.
The analysis of these data was begun by the calculation of the
a1
between a singly deprotonated ARS (pK = 6.0) and the
a
K values at each pH. This was achieved by simply dividing the
value of kon by koff for each condition of pH and the results of
neutral, trigonal form of 1 (pK = 7.7). Additionally, the K data
a
2
show that at pH 7, the molar ratio of the two products depicted
in Figure 5 is nearly 1:1 yet the fluorescent lifetime experiments
tell us that one of these species is responsible for over 80% of
the observed fluorescence. The first adduct is assigned as the
species with the greater fluorescence due to the presence of a
trigonal boron species with its planar geometry and empty p-
selected conditions are recorded in Table 2. Of note is the
difference between the overall binding constant, K , and the
a
kinetic binding constant, K . This difference can be explained
a1
by the two-step sequential reaction mechanism depicted in
Figure 5. In this case, the overall K reflects the total of all
a
products formed while K and K reflect the formation of the
a1
2
19−21
orbital that act to extend conjugation.
With a nonplanar
monoester adduct and its partitioning to the diester, spiro
adduct respectively. Given this relationship, eq 3 has been
derived and used as confirmation of the proposed mechanism.
structure and a boron atom with a filled p-orbital, the diester
spiro product is assigned as the product yielding the lesser
contribution to fluorescence. The opening of the oxaborole ring
was also considered at this juncture however no evidence was
K = K + K × K
(3)
a
a1
a1
2
Figure 5. Minimal mechanism for the reaction of 1 with ARS based on experimental results from the steady state at near neutral pH’s. The
Fluorescent Adduct structure is assigned as the major source of the detected fluorescence signal while the NMR Adduct is that which can be
independently measured via its unique peak in 11B NMR. Such a mechanism accounts for the observed difference of the K ’s from kinetic (K ) and
a
a1
thermodynamic (overall K and K ) measurements via eq 4.
a
2
1
1203
dx.doi.org/10.1021/jo302264g | J. Org. Chem. 2012, 77, 11200−11209

The Journal of Organic Chemistry
Article
Figure 6. Analysis of pH profiles of kon and koff obtained from stopped-flow experiments enable partial mechanism elucidation: (A) forward rate data
k ) are fit to eq 4 (dashed, black line) and a two-pK equation (solid, blue line) in the same form as eq 1 as an approximation since an exact
(
on
a
solution for the mechanism cannot be solved, apparent pK ’s are 4.7 ± 0.1 and 7.5 ± 0.1; (B) reverse rate data (k ) are fit to eq 5.
a
off
The sequential two-step mechanism found in effect with
benzoxaborole, 1, is identical in form to that previously
determined with phenylboronic acid, 4. Other similarities
between the two systems include: UV/vis and fluorescence
spectral properties; pH profiles of fluorescence and binding,
kobs
ν
T
=
=
[
B]T
[A] [B]
18
T
⎛
+
⎞⎛
⎟⎜
+ 2
⎞
⎟
⎠
[
H ]
[H ]
k ⎜
1
B
1
+
+ 2
+
A
1
A
1
A
2
⎝
K + [H ] [H ] + ([H ]K ) + K K
⎠⎝
once the altered pK of 1 is taken into account; and the
a
+
B
⎛
⎞
⎟
⎠
k [H ] + k K
apparent preference for the reaction to occur between the
singly deprotonated ARS and the neutral, trigonal form of 1.
Therefore, an analogous reaction mechanism is proposed.
It is seen that ARS may exist as any of three species, from the
fully protonated A to the monodeprotonated A to the fully
2
3
+
1
+
⎜
⎝
B
1
K + [H ]
A
+
⎛
⎜
⎞
⎟
K [H ]
1
+
2
+
A
1
A
A
2
⎝ [H ] + ([H ]K ) + (K K ) ⎠
0
1
1
(4)
deprotonated A . These ionization steps are connected by the
2
equilibria K1A and K respectively. B and B , connected by the
A
Equation 4 describes the formation of the three monoester,
adduct species, P , P , and P , from reactions between the
various ionization states of ARS and 1. pK , pK , and pK
are fixed to 6.0, 11.0, and 7.7 respectively based on the results
of the UV/vis determination of the pK ’s of 1 and ARS.
2
1
2
B
equilibrium K , represent the neutral, trigonal and anionic,
1
2
3
1
A
A
2
B
1
tetrahedral forms of 1, respectively. The reactions between the
various ionization states of ARS and 1 may then proceed via
three parallel, single-step reaction paths to form intermediate
1
a
products P , P , and P . Each of these intermediate products
1
2
3
+ 2
+
P
P
1
P
v
P]T
(k [H ] ) + (k [H ]K ) + (k K K )
−
1
−2
1
−3
2
represent a differing ionization state of the monoester adduct of
:ARS and they exist in rapid equilibrium with the others as
kobs = [
=
+ 2
+
P
P
P
1
[H ]
+
([H ]
K1 + K1 K2
)
(
)
P
P
2
dictated by solution pH and K1 and K . These products may
(5)
then undergo a second, intramolecular attack to form the cyclic,
Equation 5 describes the dissociation of the three species, P ,
1
diester product P that contains an anionic, tetrahedral boron
P
4
P , and P , that are connected by two ionization steps, K and
2
3
1
atom.
K2 respectively. pK1 and pK2P are fixed to 4.6 and 9.5,
P
P
With an essentially identical format of the mechanistic
respectively, based on the results of the UV/vis determination
proposal for the reaction between 1 and ARS as was
of the pK ’s of 1:ARS adduct.
18
a
determined for the reaction with 4, the previously determined
rate equations for k , eq 4, and k , eq 5, versus pH were
An inconsistency was noted when eq 4 was applied to the kon
vs pH data graphed in Figure 6A (dashed, black line). A
reasonable fit was found for the right half (pH 6.5−9.5) of the
graph where the contributions of the middle (A + B ) and
on
off
evaluated for compatibility with this system. The equations
were modified to reflect the revised numbering of the steps
1
1
used in Figure 7 and the pK values found for this system in
a
bottom (A + B ) arms of the mechanism are controlling and
1 2
B
independent measurements from the steady-state for 1 (pK ),
ARS (pK , pK ), and the 1:ARS complex (pK , pK ). As
1
the ionization of 1 is evident. At pH < 6.5, significant deviations
between the data and the fit predicted by eq 4 were observed.
In essence, eq 4 predicts a change in reaction rate, from the
A
A
P
1
P
1
2
2
can be seen in Figure 6 B, eq 5 provides an excellent fit to the
koff vs pH data. The k , k , and k values obtained from this
upper path (A + B ) to the middle path (A + B ), controlled
−1
−2
−3
0
1
1
1
A
fitting are recorded in Figure 7.
by the ionization of A to A as represented by pK . These data
0 1 1
1
1204
dx.doi.org/10.1021/jo302264g | J. Org. Chem. 2012, 77, 11200−11209

The Journal of Organic Chemistry
Article
Figure 7. Proposed mechanism for the spontaneous reaction between 1 and ARS in aqueous solution as pH is varied from acidic (top) through near
neutral (middle) to basic (bottom). Due to an inability to explicitly solve the necessary rate equations, values for k and k were estimated. *Values
1
3
were determined in independent experiments.
indicate that the reaction is proceeding at low pH in a manner
Subsequently, the second step of the reaction, the intra-
molecular attack and closure to form the diester spiro adduct
P , was investigated in greater detail. The largest observed K ,
A
that is inconsistent with the independently determined pK1
=
6.0. In order to assess the extent of the deviation, the data are fit
4
2
with a two-pK equation in the same form as eq 1 (solid, blue
from pH = 7 data, was assigned as the equilibrium constant for
a
line). It is found that the right arm’s apparent pK is 7.5 ± 0.1, a
formation of P4 from P . Thermodynamic reaction cycle
a
2
calculations using this data and pK1 and pK2P allowed
P
good match for the pK of 1, and that the left arm’s pK is 4.7 ±
a
a
0
.1. This pK is strikingly similar to that found for the ionization
determination of the equilibria that exist between P → P
a
1
4
of the products of the reaction under acidic conditions (P to P
and P → P respectively. Examination of the kinetics of the
3 4
reverse reaction were enabled by synthetic preparation of P4
under anhydrous conditions in acetonitrile. A freshly prepared
1
2
P
via pK ).
1
With this finding, it is proposed that the observed deviation
of the data from eq 4 may be explained by contributions from
the product side of the reaction, that is, from pK1 and K2.
solution of P in acetonitrile was rapidly mixed with a 25-fold v/
4
P
v excess of aqueous buffer at pH 5−9 utilizing a stopped-flow
reaction apparatus. Progress curves were monitored by
fluorescence detection and fit to double exponential equations.
For this fitting, the slow rate was fixed to the koff value for that
pH obtained in Figure 6B. The rates thus obtained for the
Attempts to derive equations for these models with the
mechanism illustrated in Figure 7 proved exceedingly complex
so the question was examined by modeling the reaction with
the program KinTek Global Kinetic Explorer v.3.0. Indeed, it
was found that the observed reaction progress curves, at pH 5
and 7, could be accurately simulated with a sequential two-step
model given the experimentally determined rates of reaction
decomposition of P are listed in Table 3. These data, when
4
11
combined with the K data obtained by B NMR experiments,
2
also enable the calculation of the forward reaction rates via the
(Supporting Information). Given a level of confidence in the
relationship K = k /k .
2 4 −4
model, the rates k , k , and k are assigned the values given in
At this juncture, the similarities and differences of the
reactions of benzoxaborole, 1, and simple phenylboronic acid,
1
2
3
Figure 7. The value of k is the result of fitting eq 5 to the data
2
18
from pH 6.0 to 9.5. This fit also yields a value for k that is
4, with ARS as a model diol are examined in order to assess
any inherent benefit arising from the utilization of the
benzoxaborole scaffold. The similarities include optimal binding
at near neutral pH and a shared mechanistic scheme with the
preferred route of reaction occurring between the singly
deprotonated ARS (A ) and the boronic form of 1 (B ).
3
essentially zero. Given that all other steps in this lower reaction
−1
−1
cycle are known, k is calculated to be 0.3 M
s
via
3
thermodynamic closure. The value for k is approximated to be
1
−
1 −1
20 M
s
via comparison of observed rates in Figure 6A and
thermodynamic evaluation of the upper reaction cycle.
1
1
1
1205
dx.doi.org/10.1021/jo302264g | J. Org. Chem. 2012, 77, 11200−11209

The Journal of Organic Chemistry
Article
a
path (2550 ± 95 and 2250 ± 250 M−1 s−1, respectively). This
type of simple reactivity was not observed for the 1:ARS system
Table 3. Cyclization Step Reaction Rates vs pH
rate (s−1)
where the contributions of both Ka1 and K needed to be
2
pH
k4
k−4
accounted for (Table 2 and eq 3) to accurately replicate the
binding of the overall reaction. In essence, it is believed that the
5
6
7
8
9
.0
.0
.0
.0
.0
0.26
0.39
0.20
ND
ND
0.65 ± 0.07
0.56 ± 0.09
0.23 ± 0.02
0.18 ± 0.01
0.99 ± 0.11
relatively greater contribution of the diester adduct, P , to the
4
overall reaction is responsible for the observed enhanced
reactivity of the benzoxaborole scaffold.
This assertation was then tested by examining the reactions
between ARS and several other compounds that are structurally
related to both 1 and 4, Figure 1. Compounds were selected to
examine the roles that the oxaborole heterocycle (benzoxabor-
in, 2, and 3,3-gem-dimethyl-benzoxaborole, 3) and compound
a
The rate of the reverse reaction, k , is obtained from observation of
the rates of decomposition of synthetically prepared P when rapidly
mixed with aqueous buffer of various pH. The forward rate, k , is
calculated using the relationship K = k /k . ND = not determined
−4
4
4
2
4
−4
due to unavailability of K measurements.
2
pK (4-trifluoromethyl-phenylboronic acid, 5, and 5-methyl-
a
benzoxaborole, 6) might play in the reactivity of the
Differences can be found in both the steady-state and
presteady-state. In the steady-state, there are the expected
benzoxaborole scaffold. These compounds’ pK ’s were
a
independently determined and are listed in Table 1. The
reactions’ kinetics were examined in the presteady-state after
rapid mixing with a stopped-flow apparatus with an
experimental setup nearly identical to that used above. The
major difference in this case is that reaction progress was
monitored via time-resolved, UV/vis spectral collection with
photo diode array detection rather than fluorescence. This
detection method was chosen to enhance the likelihood of
reliably detecting contributions of the cyclic, diester product,
P4, that has been determined to have a much smaller
fluorescence response than the intermediate, monoester
product, P2.
differences in binding vs pH due to the differing pK of 1 and 4,
a
Table 1. The optimal pH of binding for 1:ARS is found to be
between 6 and 7 while that for 4:ARS was determined to be 7−
8
. At pH = 7, the overall association constant is about 25%
−1
greater for 1:ARS vs 4:ARS, 3200 and 2550 M , respectively.
Additionally, it is noted that the K for 1:ARS is greater at every
2
pH for which the data is available, Figure 8.
Separate solutions of ARS and the compounds 1−6 were
prepared in matched aqueous buffer solutions at pH 6 and 8. In
all cases, the final concentrations of ARS and 1−6 were 0.1 and
4
1
mM respectively after mixing. Data were collected for up to
00 s after mixing and the spectra from 350 to 700 nm were
examined. Singular value decomposition (SVD) analysis was
used to extract the number of detectable species, including
intermediates, contributing to the observed reaction spectra.
The resulting SVD basis vectors have been included in the
Supporting Information.
In the case of a simple one-step reaction in which a spectral
change is observed, two species corresponding to the reactant
and product should be observed. Three species will be observed
in the case of a two-step reaction in which the reactant,
intermediate, and product have differing spectra. The results of
these analyses on the reactions between 1−6 and ARS are
summarized in Table 4. It can be seen that only compounds 1
and 6 exhibit three species in the reaction indicative of
significant contributions to the reaction from intermediate and
Figure 8. Comparison of the calculated K values for the 1:ARS (red)
2
18
and 4:ARS (blue) reactions at pH 5−10. At all pHs for which data
are available, the formation of the full-ester, spiro adduct by the
benzoxaborole is preferred over the phenylboronic acid.
Table 4. Number of Species Present in the Reaction between
ARS and Various Boron Compounds as Determined by SVD
Comparison of the reactions in the presteady-state and the
subsequent detailed reaction mechanisms illuminate the
potential source of the increased reactivity/binding observed
for the benzoxaborole scaffold. Examination of the individual
rates given in Figure 7 with those determined for 4 do not show
any striking differences, as might be expected for comparisons
between two molecules with such similar structures. What is
seen, however, is that the cyclization reaction for the formation
a
Analysis of Time-resolved Reaction Spectra
boron compound
pH
1
2
3
4
5
6
6
8
3
3
2
2
2
2
2
2
2
2
3
3
a
Time-resolved UV/vis spectra (350-700 nm) were captured for the
of P from 1 occurs at a slightly faster rate and that this product
4
pre-steady state reactions of boron compounds 1−6 with ARS at pH 6
and 8. Singular value decomposition analysis revealed that only the
compounds with an unsubstituted benzoxaborole heterocycle posses
the signature, two-step reaction profile. SVD basis vectors for these
designations are included in the Supporting Information.
is more stable, as reflected in the greater K values (Figure 8),
2
than is observed for the reaction with 4. For the 4:ARS
reaction, it was seen that the overall binding was dictated by Ka1
with overall reaction K at pH 7 equal to Ka1 for the middle
a
1
1206
dx.doi.org/10.1021/jo302264g | J. Org. Chem. 2012, 77, 11200−11209

The Journal of Organic Chemistry
Article
product species. The simplest interpretation is that the rate of
formation of the corresponding full ester, spiro adduct for 1 and
overall reaction course. Indeed, it is proposed that this
kinetically competitive cyclization step is the source of the
enhanced reactivity of the benzoxaborole heterocycle.
6, relative to all other compounds, is competitive with the rate
of intermediate formation so that three species, rather than two,
are observed.
Finally, a stopped-flow method that obtains time-resolved
UV/vis spectra of the reaction was used for identification of
readily formed reaction intermediates. This method allows the
rapid comparison of the reactivity of several structurally related
compounds. These experiments compared the effects of
modifications to the benzoxaborole heterocycle and compound
pKa (from distal aromatic ring substitutions) on the
compounds’ reactivities. Based on these results, it appears
that the five-membered oxaborole ring itself is a major
contributor to the unique reactivity observed with the
benzoxaborole scaffold. It is proposed that the reactivity arises
from the geometry about the neutral, trigonal planar boron
atom when it is constrained in a five-membered ring. This ring
strain is alleviated when the boron atom forms an anionic,
tetrahedral species by either hydroxlyation (conjugate base
CONCLUSIONS
■
It has been observed that the benzoxaborole scaffold has been
selected time and again for its enhanced reactivity or binding to
target diols, whether on the cell surface or in the active site of
an enzyme. Thus far, the rationale for its use seems to have
arisen simply from phenomenological observations of its
effectiveness when compared to other, similar compounds
such as phenylboronic acid. In this work, we address the
question “What properties of this scaffold are responsible for its
unique reactivity?” by determining the mechanism of the
reaction between benzoxaborole (1) and a model diol, ARS.
Additionally, the reactivity of 1 is compared and contrasted
with other structurally related compounds (2−6) in an attempt
to pinpoint the structural source of these properties.
formation) or diesterification (i.e., formation of the spiro P4
species). These results may now provide a basis for further
improvements to this scaffold and a rationale for the expanded
use of this cyclic boronate in applications ranging from drug
discovery to synthetic protocols.
Experiments in the steady-state were executed to determine
basic properties of benzoxaborole and the products formed in
the reaction with ARS for comparison to those previously
determined in the reaction between ARS and phenylboronic
acid (4). As could be expected in the comparison of two
compounds with such close structures, many similarities were
found. These included general spectral (UV/vis, fluorescence)
properties, responses to pH, and fluorescence lifetime measure-
ments indicating two species contributing to the fluorescent
signal. The major differences included a tighter binding
observed for 1 with the maximum occurring at a lower solution
pH. Also, greater formation of the diester, spiro adduct was
observed at all pH conditions examined when compared with
the previously determined data for 4. Combined, these data
confirm that the reaction is proceeding in two sequential steps.
Presteady-state analyses utilizing stopped-flow fluorescence
spectrometry were used to elucidate a detailed mechanism for
the reaction (Figure 7). This mechanism was found to parallel
that determined for the reaction between 4 and ARS in our
previous work. The preferred reaction occurs at near neutral
pH between the singly deprotonated ARS and neutral, trigonal
EXPERIMENTAL SECTION
■
Common solvents and reagents were obtained from commercial
sources and were of the highest available purity. 2-(Hydroxymethyl)-
benzene boronic acid cyclic monoester (benzoxaborole, 1) was
purchased from Lancaster Synthesis, Inc. and purified by silica gel
column chromatography with 25% ethyl acetate in hexanes. 2-(2-
hydroxyethyl)benzene boronic acid cyclic monoester (benzoxaborin,
2
) and 3,3-gem-dimethyl-benzoxaborole (3) were provided by
Scynexis, Inc. 5-Methyl-benzoxaborole (6) was provided by Anacor
Pharmaceuticals, Inc. ARS and all buffer solutions were prepared with
double-deionized water in triple-rinsed, acid-washed glassware. The
ARS stock solution was 10 mM and was protected from light. All
aqueous solutions were filtered through 0.45 μm Supor syringe filters
from Pall Life Sciences. Stock solutions of 1−6 were 200 mM in
DMSO. All solutions were stored at 4 °C in polypropylene tubes.
Unless otherwise noted, all data were plotted and analyzed using
KaleidaGraph v3.5 by Synergy Software.
Buffers were prepared as 500 mM stock solutions and adjusted to
final pH as indicated with either HCl or NaOH: Glycine (pH = 3.0
and 3.5), Acetic acid-Sodium Acetate (pH = 4.0, 4.5 and 5.0), MES
1
(A and B respectively) to form the monoester adduct, P ,
1
1
2
before intramolecular ring closure to the diester spiro adduct,
P4. The reaction is much less favorable at either lower or higher
solution pH. A key difference was noted in this system when
(
pH = 5.5, 6.0, and 6.5), HEPES (pH = 7.0, 7.5, and 8.0), CHES (pH
= 8.6, 9.0, 9.5, and 10.0), and CAPS (pH = 10.5 and 11.0). Final
solution pH was determined by preparing mock solutions (5 mL,
lacking only compounds) and measuring pH with a pH meter
calibrated against aqueous buffer solutions using a combination
electrode without correction for liquid junction potentials.
the kinetic Ka1 (k /k ) was compared with the steady-state
on off
overall reaction K . In the reaction between 4 and ARS, K was
a
a1
essentially identical to the overall K indicative of a system
a
UV/Visible Spectra Collection. Solutions were placed into 1 mL,
cm path length quartz cuvettes and spectral scans (1 nm resolution)
where the reaction is controlled by the initial association
reaction. In the 1:ARS system, the contributions of both Ka1
1
were taken at room temperature. Scans were taken in the following
regions: 240−340 nm for 1−6; 240−700 nm for ARS; 350−700 nm
for 1:ARS. Solutions consisted of 1 mM 1, 100 μM ARS, or 8 mM 1
and 100 μM ARS, respectively. All solutions contained either 0.5% or
4% v/v DMSO and any one of 100 mM HCl, 100 mM NaOH, or 50
mM buffer. Ionization constants were derived from these data by the
and K needed to be taken into account to reproduce the
2
observed overall reaction K (eq 3).
a
Additionally, another difference between the systems was
noted when the rate equations determined for 4:ARS were
applied to the benzoxaborole system. While the equation that
describes the reverse reaction rates as a function of pH (eq 5)
revealed an excellent fit to the koff data, the forward reaction
rate equation (eq 4) fit the kon data poorly at pH < 6 (Figure
7
method of Tomsho, et al.
Fluorescence Spectra Collection. Measurements were collected
at room temperature. Emission scans were taken from 460 to 800 nm
with an excitation wavelength of 440 nm. Excitation scans were then
taken from 300 to 550 nm with an emission wavelength equal to the
wavelength of maximum fluorescence determined in the emission scan.
All slit widths were set to 2 nm. Solutions consisted of 20 μM ARS, 4%
v/v DMSO, 0 or 2 mM 1, and any one of 100 mM HCl, 100 mM
NaOH, or 50 mM buffer.
6). These data, however, revealed an apparent pKa that
depended on the ionization of the intermediate product (P →
1
P2) rather than on the ionization of any of the reactants. This
result was taken as further evidence for the significant
contribution of the intramolecular cyclization step in the
1
1207
dx.doi.org/10.1021/jo302264g | J. Org. Chem. 2012, 77, 11200−11209

The Journal of Organic Chemistry
Article
Fluorescence Lifetime (TCSPC) Determination. Data were
collected at room temperature with a NanoLED-495 excitation source
1 MHz refresh rate) while the emission wavelength was set to 590 nm
20 nm bandpass). Data were collected with Data Station v. 2.5 and
The ARS solution additionally contained 0.02−0.20 mM ARS while
the boron compound solutions contained 2 mM 1; in both cases, the
final concentrations of both ARS and 1 are halved upon mixing for
reaction initiation. Reaction progress curves were analyzed using the
Applied Photophysics Pro-Data Viewer v. 4.2.0 for fitting to a single
exponential curve to obtain apparent rate data.
(2). Reverse Reaction Kinetic Determinations. The stopped-flow
instrumental setup was as detailed for the forward reaction kinetic
determinations above except the instrument was configured for
asymmetric mixing with a ratio of 1:25 and data collection up to 60 s.
The 1:ARS full-ester adduct solution in acetonitrile is freshly prepared
(
(
analysis was done with DAS6 software. Solutions consisted of 20 μM
ARS, 2 mM 1, 4% v/v DMSO, and 50 mM buffer (pH 4−10). Ludox
CL colloidal silica solutions were used as light scattering standards.
Steady State Fluorescence Titrations. Measurements were
collected at 25.0 °C with excitation at 460 nm and emission at 590 nm
utilizing 5 nm slit widths. The Ka of the 1:ARS complex was
determined by two-component fluorescent titrations by the method of
22
Springsteen and Wang. The K values were determined by fitting the
as described above. Buffer solutions, 50 mM in ddH O, of the desired
2
a
data to eq 2. These samples contained 50 mM buffer, 5% v/v DMSO,
pH (5−9) are prepared. Mixing for reaction initiation results in a 25-
fold dilution of the 1:ARS full-ester adduct solution into buffer
resulting in 4% v/v acetonitrile final. Reaction progress curves were
analyzed using the Applied Photophysics Pro-Data Viewer v. 4.2.0 for
fitting to a double exponential curve to obtain apparent rate data.
(3). UV/vis Kinetic Determinations with Photo Diode Array
Detection. The stopped-flow instrumental setup was as detailed for
the forward reaction kinetic determinations above except the
instrument was configured for photo diode array detection of the
UV/vis spectra. The light source utilized was a xenon short arc lamp
with the diffraction grating of the monochromator taken out of line
and 14 nm slits. Path length was 1 cm and blanks were taken on mock
reaction solutions lacking only boron compounds and ARS. For each
pH examined, two solution sets were prepared such that all contained
2
0 μM ARS, and 0−10 mM 1 in ddH O. An ARS solution and
2
solutions of all other components except for ARS were prepared and
equilibrated at 25.0 °C in separate vessels. Upon ARS solution
addition, the complete reaction solution was mixed well and
maintained in the dark at 25.0 °C for 3 min prior to the measurement
of fluorescence.
Isothermal Titration Calorimetry. A solution consisting of 0.4
mM ARS was titrated with 8 mM 1, both solutions also contained 50
mM HEPES at pH 7 and 4% v/v DMSO in ddH O. A blank titration
2
of 1 into buffer lacking ARS was done to correct for the heat of
dilution of the solution of 1. The cell temperature was held to 25.0 °C,
injection volume was 10 μL, and injection duration and spacing were
24 and 240 s respectively. After correction for heat of dilution of 1, the
5
0 mM buffer and 4% v/v DMSO in ddH O. The ARS solution
data were fit to a one-site binding model using MicroCal Origin 5.0 for
ITC software package. From these data, the association constant and
thermodynamic parameters of the reaction were determined.
2
additionally contained 0.20 mM ARS while the boron compound
solutions contained 8 mM 1−6; in both cases the final concentrations
of both the ARS and boron compounds are halved upon mixing for
reaction initiation. Time resolved reaction spectra from 350 to 700 nm
were analyzed using the Applied Photophysics ProK Global Analysis v.
11
B-NMR. Spectra were collected at 96.21 MHz using a 4.9 μs 90°
pulse, 488 ms FID acquisition time, and a 1 s acquisition delay. The
sweep width was set to 87.2 ppm and the temperature to 25.0 °C. All
chemical shifts were referenced to an external standard of BF (Et O)
1
.0.8 software for SVD analysis and fitting to either one- or two-step
3
2
reaction mechanism models.
at 0.0 ppm. Samples were prepared in 5% v/v DMSO and 10% v/v
D O in ddH O as the lock solvent and were placed into quartz NMR
2
2
ASSOCIATED CONTENT
Supporting Information
tubes. Each sample consisted of 50 mM buffer, 10 mM boron
compound 1, and 7.5 mM ARS. Two thousand scans were taken for
each sample and the data were then processed using SpinWorks v2.5.5.
A minimum of three independent determinations were made at each
pH examined from 5 to 10.
■
*
S
Spectral data for the pK determinations of 1, ARS, and the
a
1
1
:ARS complex; NMR and MS spectra for the synthesis of the
:ARS diester adduct in acetonitrile; B NMR integration data
11
Preparation of the 1:ARS Full-ester Adduct (P ). Using
4
and K calculation; KinTek modeling results; SVD basis vectors
2
standard procedures to exclude moisture, Alizarin Red S (173 mg,
for 1−6 at pH 6 and 8. This material is available free of charge
via the Internet at http://pubs.acs.org.
0.5 mmol), benzoxaborole (74 mg, 0.5 mmol), and anhydrous
potassium carbonate (173 mg, 1.25 mmol) are sealed in a round-
bottomed flask under an argon atmosphere. To this is added 10 mL of
acetonitrile (dried by stirring over anhydrous potassium carbonate
under argon) and the resulting suspension is stirred vigorously for ∼18
h at room temperature in the dark. Stirring is stopped and the
suspension is allowed to settle. Using dry needles, syringes, and syringe
filters, the supernatant was removed and filtered through a 0.45 μm
nylon syringe filter taking care to minimize solution exposure to air.
AUTHOR INFORMATION
Corresponding Author
Tel: 814-865-2882. Fax: 814-865-2973. E-mail: sjb1@psu.edu.
Present Address
Department of Chemistry and Biochemistry, University of the
Sciences in Philadelphia, 600 South 43rd St., Philadelphia, PA
19104.
■
*
†
1
The solution was protected from light and used immediately. H NMR
(
7
CD CN) δ: 8.25 (m, 2H), 8.02 (s, 1H), 7.92 (m, 2H), 7.77 (d, 1H*),
3
11
.45 (m, 3H*), 6.78 (br s, 1H*), 5.06 (s, 2H*). B-NMR (CD CN)
3
Notes
δ: 32.3 (1.00), 16.3 (0.020), 8.2 (0.025); MS ESI- m/z (relative
intensity) [M] 217.0 (50.3%), [M + K] 237.5 (13.5%), [ARS]
3
−2
−2
−
The authors declare no competing financial interest.
−
−
18.9 (100%), [M] 434.9 (90.2), [M + K] 473.0 (14.7%); λmax
ACKNOWLEDGMENTS
(
CH CN) 460 nm. *these protons are attributed to 1 and are labeled
■
3
according to the number present in the product molecule for clarity;
however an approximately 17-fold excess is observed in the reaction
due to the limited solubility of ARS.
We thank Dr. C. Tony Liu for critical review of the manuscript;
Anacor Pharmaceuticals, Inc. of Palo Alto, CA, for providing a
sample of compound 6; and Scynexis, Inc. of Research Triangle
Park, NC, for providing samples of compounds 2 and 3.
Presteady State Kinetics. (1). Forward Reaction Kinetic
Determinations with Fluorescence Detection. Reaction progress
was monitored utilizing a stopped-flow reaction analyzer with
fluorescence detection. The excitation wavelength was 450 nm with
a 4.65 nm slit width, emission was monitored with the use of an
OG530 nm long wave pass filter, temperature was controlled at 25.0 ±
REFERENCES
(1) Torssell, K. Ark. Kemi 1957, 10, 507−511.
(2) Li, X.; Zhang, Y.-K.; Liu, Y.; Ding, C. Z.; Li, Q.; Zhou, Y.;
Plattner, J. J.; Baker, S. J.; Qian, X.; Fan, D.; Liao, L.; Ni, Z.-J.; White,
G. V.; Mordaunt, J. E.; Lazarides, L. X.; Slater, M. J.; Jarvest, R. L.;
Thommes, P.; Ellis, M.; Edge, C. M.; Hubbard, J. A.; Somers, D.;
■
0
.1 °C, and reaction progress was followed for up to 100 s. For each
pH and ARS concentration examined, two solution sets were prepared
such that all contained 50 mM buffer and 4% v/v DMSO in ddH O.
2
1
1208
dx.doi.org/10.1021/jo302264g | J. Org. Chem. 2012, 77, 11200−11209

The Journal of Organic Chemistry
Article
Rowland, P.; Nassau, P.; McDowell, B.; Skarzynski, T. J.; Kazmierski,
W. M.; Grimes, R. M.; Wright, L. L.; Smith, G. K.; Zou, W.; Wright, J.;
Pennicott, L. E. Bioorg. Med. Chem. Lett. 2010, 20, 3550−3556.
(
3) Dixon, D. D.; Lockner, J. W.; Zhou, Q.; Baran, P. S. J. Am. Chem.
Soc. 2012, 134, 8432−8435.
4) Snyder, H. R.; Reedy, A. J.; Lennarz, W. J. J. Am. Chem. Soc. 1958,
0, 835−838.
5) Dowlut, M.; Hall, D. G. J. Am. Chem. Soc. 2006, 128, 4226−4227.
6) Berube, M.; Dowlut, M.; Hall, D. G. J. Org. Chem. 2008, 73,
471−6479.
7) Tomsho, J. W.; Pal, A.; Hall, D. G.; Benkovic, S. J. ACS Med.
Chem. Lett. 2012, 3, 48−52.
8) Adamczyk-Wozniak, A.; Cyran
Sporzynski, A. J. Organomet. Chem. 2009, 694, 3533−3541.
9) Baker, S. J.; Tomsho, J. W.; Benkovic, S. J. Chem. Soc. Rev. 2011,
0, 4279−4285.
10) Rock, F. L.; Mao, W.; Yaremchuk, A.; Tukalo, M.; Crepin, T.;
(
8
(
(
́
́
6
(
̇
́
ski, M. K.; Zubrowska, A.;
(
́
́
(
4
(
Zhou, H.; Zhang, Y. K.; Hernandez, V.; Akama, T.; Baker, S. J.;
Plattner, J. J.; Shapiro, L.; Martinis, S. A.; Benkovic, S. J.; Cusack, S.;
Alley, M. R. K. Science 2007, 316, 1759−1761.
(
11) Obrecht, D.; Bernardini, G.; Dale, G.; Dembowsky, K. In Annual
Reports in Medicinal Chemistry; Elsevier Inc.: New York, 2011; Vol. 46,
pp 245−262.
(
12) Ding, D.; Meng, Q.; Gao, G.; Zhao, Y.; Wang, Q.; Nare, B.;
Jacobs, R.; Rock, F.; Alley, M. R. K.; Plattner, J. J.; Chen, G.; Li, D.;
Zhou, H. J. Med. Chem. 2011, 54, 1276−1287.
(
13) Qiao, Z.; Wang, Q.; Zhang, F.; Wang, Z.; Bowling, T.; Nare, B.;
Jacobs, R. T.; Zhang, J.; Ding, D.; Liu, Y.; Zhou, H. J. Med. Chem.
012, 55, 3553−3557.
14) Akama, T.; Baker, S. J.; Zhang, Y.-K.; Hernandez, V.; Zhou, H.;
2
(
Sanders, V.; Freund, Y.; Kimura, R.; Maples, K. R.; Plattner, J. J. Bioorg.
Med. Chem. Lett. 2009, 19, 2129−2132.
(
15) Xia, Y.; Cao, K.; Zhou, Y.; Alley, M. R. K.; Rock, F.; Mohan, M.;
Meewan, M.; Baker, S. J.; Lux, S.; Ding, C. Z.; Jia, G.; Kully, M.;
Plattner, J. J. Bioorg. Med. Chem. Lett. 2011, 21, 2533−2536.
(
16) Kim, H.; Kang, Y. J.; Kang, S.; Kim, K. T. J. Am. Chem. Soc.
2
012, 134, 4030−4033.
17) Ellis, G. A.; Palte, M. J.; Raines, R. T. J. Am. Chem. Soc. 2012,
34, 3631−3634.
18) Tomsho, J. W.; Benkovic, S. J. J. Org. Chem. 2012, 77, 2098−
106.
19) Huebner, A.; Qu, Z. W.; Englert, U.; Bolte, M.; Lerner, H. W.;
Holthausen, M. C.; Wagner, M. J. J. Am. Chem. Soc. 2011, 133, 4596−
609.
20) DiCesare, N.; Lakowicz, J. R. J. Phys. Chem. A 2001, 105, 6834−
840.
21) Yoon, J.; Czarnik, A. W. J. Am. Chem. Soc. 1992, 114, 5874−
875.
22) Springsteen, G. Tetrahedron 2002, 58, 5291−5300.
(
1
(
2
(
4
(
6
(
5
(
1
1209
dx.doi.org/10.1021/jo302264g | J. Org. Chem. 2012, 77, 11200−11209