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.
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INTRODUCTION During most of the 55 years since their discovery,1 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 discovery2 and synthetic protocols.3 The benzoxaboroles in particular have been found to possess high water solubility and resistance to hydrolysis,4 excellent sugar (diol) binding under physiologically relevant conditions,5,6 and a low pKa compared with that of simple phenylboronic acid.7 Of particular interest, the use of the benzoxaborole scaffold in drug discovery has been on the rise.8,9 To date, these compounds have found use as inhibitors of Leu-tRNA synthetases (for antifungal,10 antibacterial,11 and antitrypanosomal agents.12,13), phosphodiesterase-4 (PDE-4, anti-inflammatory),14 β-lactamase,15 and several antibacterials.11 In addition, this scaffold has been recently found to have several applications in biotechnology including incorporation into polymeric materials for sugar-responsive insulin delivery16 and mediating the delivery of a protein toxin to the cytosol.17 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 (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 © 2012 American Chemical Society
differences arising upon reaction with boronic acids, ready reaction with boronic acids in neutral, aqueous solution, and a low pKa that resembles “biological” nucleophiles. The reaction 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 phenylboronic acid (PBA, 4)18 in order to elucidate any key mechanistic differences between the two systems. Finally, related compounds (Figure 1) with altered heterocyclic ring systems (2 and 3) or altered pKa via ring substitutions (5 and 6) have been examined to assess the cause of any differences. In general, it is found that benzoxaborole reacts with Alizarin Red S with a mechanism analogous to that determined for phenylboronic acid.18 The reaction proceeds preferentially at near neutral solution pH via a sequential two-step reaction in which a bimolecular esterification is followed by an intramolecular 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.
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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, Received: October 24, 2012 Published: November 1, 2012 11200
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Figure 1. Compound structures and designations.
versus other common buffers such as Tris or phosphate, were chosen due to their previously determined unreactivity toward boronic acids.18 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 studied. Utilizing the method of Tomsho, et al, 7 pK a 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
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-pKa equation, eq 1, and resulted in pKa’s of 4.9 and 8.7.
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 resulting pKa values obtained by the fitting of this data are 4.9 ± 0.1 and 8.7 ± 0.1. These results may be compared with 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.
Table 1. pKa Values Determined by UV/Vis Spectral Titrationa compound
pKa
1 2 3 4 5 6 ARS 1:ARS adduct
7.3b; 7.7c 8.4b 8.3b 9.1b; 9.2c 8.4b 7.6b 6.0, 11.0c 4.6, 9.5, 11c
a
The pKa determinations were achieved by the method of Tomsho, et al.7 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 comparison. b0.5% v/v DMSO. c4% v/v DMSO.
[Fluorescent Species]
two of the observed pKa’s for the 1:ARS adduct differ 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]T × 10(pKa2 − pH) 1 + 10(pKa2 − pH) + (10(pKa1− pH)·10(pKa2 − pH))
(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]T, observed ionization constants (pKa1 and pKa2), and solution pH. The properties of the fluorescent species were further characterized with fluorescent lifetime measurements taken 11201
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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 pKa of 4.6−4.9. From pH 6−8, both relative fluorescent 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 pKa of 8.7− 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 the transition from reactants to all potential products. In our previous work with phenylboronic acid,18 11B NMR 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. The data and K2 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,
Table 2. Comparison of the Association Constants for 1 and ARS as Determined by Three Methods at Various pH Values Ka (M−1)
Ka1 (M−1)
K2
b
pH
fluorescent titrationa
ITCa
kinetics (kon/koff)
5 6 7 8 9 10
690 ± 30 2580 ± 90 3190 ± 110 1140 ± 40 100 ± 10 NDe
480 ± 20 2030 ± 80 3390 ± 420 780 ± 30 NDe NDe
420 ± 180 1140 ± 200 1950 ± 480 840 ± 100 170 ± 25 NDe
B-NMRc
11
0.40 ± 0.70 ± 0.87 ± NDd NDd 0.12 ±
0.04 0.05 0.04
0.03
a
Overall Ka values are calculated based on the total concentration of 1 at each pH and are expressed in units of M−1. bValues are calculated by dividing the observed kon by koff from the kinetic progress curves at each condition of pH. cK2 values (unitless) calculated based on 11BNMR experiments as described previously.18 dUnder these conditions 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]T × Fluormax (1/K a) + [B]T
(2)
Equation 2 describes the Ka determination by twocomponent fluorescent titrations; ΔFluorescence is the observed change in fluorescence, [B]T is the total concentration 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 11202
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found to conclusively support the existence of a ring-opened intermediate. Additionally, the pKa of a diester, ring-opened adduct structure would likely be extremely low (
