ISSN 00360244, Russian Journal of Physical Chemistry A, 2010, Vol. 84, No. 3, pp. 409–414. © Pleiades Publishing, Ltd., 2010. Original Russian Text © G.A. Bektenova, 2010, published in Zhurnal Fizicheskoi Khimii, 2010, Vol. 84, No. 3, pp. 475–481.
PHYSICAL CHEMISTRY OF SOLUTIONS
Ionization Constants of Boronic Acids and Their Complexation with Diols G. A. Bektenova Bekturov Institute of Chemical Sciences, Ministry of Education and Science of the Republic of Kazakhstan, Almaty, 050010 Kazakhstan Received February 2, 2009
Abstract—Ionization constants of a number of boronic acids were determined spectrophotometrically in aqueous solutions. The effect of different substituents on their acid properties is considered. The strongest acids are shown to be phenylboronic acid derivatives containing a nitro group. A model study of the interac tion between boronic acids and polyvinyl alcohol depending on pH is analyzed to reveal the optimum condi tions for the formation of a stable boronatediol complex. DOI: 10.1134/S003602441003012X
INTRODUCTION Despite a number of advantages, the covalent immobilization of enzymes through functional groups of a protein matrix often results in changes in its con formation and a loss of the catalytic activity of the enzymes. For glycoprotein enzymes, it is possible to perform covalent immobilization through the polysac charide part of the enzymes. Bifunctional phenylbo ronic acid (PBA) derivatives can be used here as spac ers (spatial bridges between the enzyme and a carrier) for the covalent immobilization of glycoproteins. In this case, the native enzyme conformation is best pre served, and a loss of catalytic activity is less probable. Interaction with an enzyme proceeds through complexation between the polysaccharide part of the enzyme and the dihydroxyboryl groups of the spacer (a PBA derivative) [1–5]. The formation of this com plex and its stability depend on the acidic properties of the boronic acids. Since boronic acids are relatively weak acids (for PBA, рК = 8.86 [6]), the formation of a stable boronate–diol complex takes place in an alka line medium (at pH ~ 8 [1]). This does not correspond to the pH optimum of the maximum activity of a series of enzymes, including glucose oxidase (pHopt 5.5–6.0) [7]). In preparing new spacers based on PBA, we felt it to be important that we shift the pH optimum of the formation of the boronate–diol complexes to a more acidic range by increasing the ionization constants of the boronic acids as compared to those of PBA and the 3aminophenylboronic acid (APBA) used as a spacer [1]. We therefore synthesized a series of aromatic boric acid derivatives [8] with consideration given to the effect of different substituents on the strength of the boronic acids and determined their ionization con stants. The ionization constants of ~30 boronic acids were previously determined in [6, 9, 10], including
some compounds of interest to us (e.g., PBA, p and mtolylboric acid (TBA), and p and mcarboxy PBA). In the abovementioned works, the ionization con stants of boronic acids were found by the potentiomet ric method in water–alcohol solutions because the solubility of these compounds is limited in water. This is no obstacle to a more sensitive spectrophotometric determination of the ionization constants of arylboric acids, which is very convenient in finding the рКа of poorly soluble substances [11]. This method was also chosen because we were very interested in acidity characteristics obtained in water, since enzymes are immobilized with the help of boronic acids in aqueous solutions. EXPERIMENTAL In the work, phenylboronic acid derivatives were used as test objects [8]. The individuality and structure of the obtained compounds were confirmed by liquid chromatographic and spectral methods. Using a universal buffer system (a mixture of 0.04 M solution of boric, phosphoric, and acetic acids and 0.2 M NaOH solution), a series of solutions with different pH values from 4.0 to 10.0 with steps of 0.2 pH units were prepared for each boronic acid. All reagents used to prepare the buffer solutions were chemically pure. The pH range depended on the expected рКа value of the boronic acid under study; the extreme pH values of this range differed from the рКа by not less than 2 pH units. The pH values of the boronic acid solutions were measured with an accu racy of ±0.03 pH units. The absorption spectra of solutions were measured in 1 cm quartz cuvettes against a buffer solution with a corresponding pH value. The analytical wavelength for each absorption maximum of a boronic acid solu
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Δε/ε0 (a)
5
0.6 4
(b)
2
0.4
3
5
0.4
1 6
0.2
2
0.2 4
1
3
5
7
9
2
0.4
5
7
(c)
9 (d)
0.4
1
2 4
0.2
1
0.2
3
5
7
9
5
7
9 pH
Fig. 1. pH dependences of relative changes in the optical density of boronic acid solutions before and after interaction with poly vinyl alcohol: (a) PBA 1, 4TBA 2, 3TBA 3, 3NO24TBA 4, 3NO24BrPBA 5, ωBr4TBA 6; (b) 4СООНPBA 1, 3СООНPBA 2, 2NO24СООНPBA 3, 3СООН5NO2PBA 4, benzoic acid 5; (c) 4СООСН3PBA 1, 3СООСН3PBA 2, 2NO24СООСН3PBA 3, 3COOCH35NO2PBA 4; (d) 3NH2PBA 1, 3CONHC6H4NH25NO2PBA 2.
tion was selected separately for each compound. The analytical wavelength varied from 218 to 238 nm. The Bouguer–Lambert–Beer law was observed in the used concentration region of boronic acids (0.6–l.0 × 10–4 M). The рКа values were calculated by one of the fol lowing equations [11]: di – d if di > dm, then рКа = pH + log , (1) d – dm if
di < dm,
then
dm – d рКа = pH + log , d – di
(2)
where di, dm, and d are the optical density of the ion, the neutral molecule, and their mixture respectively at the analytical wavelength. For compounds containing a second functional carboxyl group along with the dihydroxyboryl group, two ionization constants were found. In order to increase the statistical reliability of the results, the рКа values were determined 4–6 times for each boronic acid. The determination error did not exceed ±0.02 pH. The interaction of polyvinyl alcohol (PVA) modi fied by cyclohexanone with boronic acids was studied in 0.1 M sodium phosphate buffer solutions. For each boronic acid, a series of solutions with different pHs
from 4.5 to 9.0 were prepared with steps of 0.5 pH units. The concentration of boronic acids was 0.6– 1.0 × 10–5 M. Equal excess weight portions of PVA were placed into the solutions. The obtained suspen sion was stirred for 10 h at room temperature. The time in which the system reached equilibrium state was determined ahead of time and was 6–8 h. The amount of bound boronic acid was found spectrophotometri cally by the difference in optical densities of the boronic acid solution before and after interaction at the analytical wavelength corresponding to the absorption maximum of the solution. Buffer solutions with similar pH values were used as comparison solu tions in measuring the absorption spectrum in the UV region. Based on the results of our analysis, the pH depen dences of relative changes in the solution optical den sity (proportional to the concentration) were plotted (Fig. 1). RESULTS AND DISCUSSION Table 1 lists the results of spectrophotometric titra tion for the whole series of synthesized PBA deriva tives. We were most interested in bifunctional boronic
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Table 1. Ionization constants of boronic acids in 0.1 M universal buffer solution at 298 K No.
Ка
рКа
PBA derivatives СООН
В(ОН)2
СООН
В(ОН)2
1
PBA*
8.90
1.26 × 10–9
2
3NH2PBA*
9.15
7.08 × 10–10
3
4TBA
9.26
5.50 × 10–10
4
4СООНPBA
5
4СООСН3PBA
7.69
2.04 × 10–8
6
3TBA
9.00
1.00 × 10–9
7
3СООНPBA
8
3СООСН3PBA
9
2NO24CООНPBA
3.95
8.56
4.04
1.12 × 10–4
9.12 × 10–5
8.56 8.79
2.75 × 10–9 1.15 × 10–8
7.94 3.42
2.75 × 10–9
3.72 × 10–4
1.62 × 10–9 4.35 × 10–9
10
2NO24CООСН3PBA
8.36
11
3СООН5NO2PBA
12
3СООСН35NO2PBA
6.59
2.57 × 10–7
13
3СОNHC6H4NH25NO2PBA
6.45
3.55 × 10–7
14
3NO2TBA
7.70
2.00 × 10–8
15
ϖBr4TBA
9.49
3.24 × 10–10
16
3NO24BrPBA*
5.52
3.06 × 10–6
3.43
6.85
3.80 × 10–4
1.41 × 10–7
Note: Values pKa (COOH) = 3.95, 4.04, 3.42, and 3.43 for compounds 4, 7, 9, and 11 respectively. Asterisks mark compounds obtained at the Institute of Organic Chemistry, Ural Division, Russian Academy of Sciences.
acids (BAs) that could be used as spacers in the immo bilization of enzymes.
OH B OH
As is well known [12], the strength of acids increases considerably in the order COOH < Br < Cl < F < NO2 under the action of electronegative substitu ents. It has been shown that the introduction of a nitro group raises the PBA ionization constant by almost an order of magnitude, the largest effect being from the nitro group at the orthoposition. Our previous quantum chemical calculations of the protonation energy E H+ , kJ/mol, of the B(OH)2 group in model boroncontaining systems [13] indicate that, depending on the nature of the substituent, it decreases in the order (HO)3SiOB(OH)2 > H3CB(OH)3 > HOB(OH)2 1394 1379 1376 > FB(OH)2 > O2NB(OH)2 , 1342 1184 which correlates with the corresponding increase in acidity. A similar dependence is also maintained for E H+ when passing to a series of phenylboronic acid mole cules: RUSSIAN JOURNAL OF PHYSICAL CHEMISTRY A
O C , HO
1344
OH B OH
,
1300
O N O
OH B OH 1269
Thus, the quantum chemical calculations also indicate an increase in the acidity of the –В(ОН)2 group if –COOH and –NO2 substituents are present in the PBA aromatic ring. The introduction of an electron acceptor carboxyl group into the PBA molecule should increase the ion ization constant of the dihydroxyboryl group due to a shift of the electron density in the benzene ring and an increase in excess charge δ+ on the boron atom. In the region of dihydroxyboryl group ionization (pH > 6), however, the carboxyl group (as follows from its рКа values for different compounds) is completely ionized (рКа1) and boronic acid exists in the form of carboxy late ion PBAСОО–. This obviously suppresses the ionization of compounds via the –В(ОН)2 group (рКа2). For p and mcarboxy derivatives of PBA we therefore obtained lower values of the second ioniza Vol. 84
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tion constant than one might have expected. To prove this, we studied the ionization of the above com pounds’ methyl esters. As can be seen from Table 1, the protection of the carboxyl group ensures the inde pendence of –В(ОН)2 group ionization; hence, the acidity of the latter increases (in methyl esters of p and mcarboxy PBA) by almost an order of magni tude.
hydrogen (structure (1)) or boron (structure (2)) by cycling the dihydroxyboryl group
As expected, the acidic properties of boronic acids containing a nitro group are considerably enhanced. Introduction of the nitro group into the pTBA mole cule led to an increase in the ionization constant by two orders of magnitude (compounds 3 and 14, Table 1). The effect of the nitro group remarkably reduces even the effect of the ionized –СООН group, and the acid ity of 3СООН5NO2PBA approaches the values of its methyl ester. The introduction of nitro groups with other electron acceptor substituents is especially effec tive (compounds 13, 16).
Our data from PMR spectral studies also testifies to the presence of interaction between the –NO2 and –В(ОН)2 groups in this compound. Due to this inter action, the electron density on the boron atom increases and the acidic properties of the dihydroxy boryl group are reduced. The second ionization con stant of 2NO24СООНPBA (Ка2 = 1.62 × 10–9) is even somewhat lower than the ionization constant of initial pСООНPBA (Ка2 = 2.75 × 10–9). The effect of electron donor substituents is mani fested, as expected, in a decrease in the ionization constants of boronic acids. A comparison (Table 2) of PBA ionization constants previously determined by the potentiometric method in water [9] and our data obtained by the spectrophotometric method shows that the results are very similar. However, the ioniza tion constants found in aqueous and water–alcohol [6, 9] solutions at 298 K, as shown in Table 2, are sub stantially different, indicating the importance of sol vent in ionization. It should be noted that the spectrophotometric method is convenient, quite accurate, and repeatable in determining the ionization constants of boronic acids. Seven compounds out of the 16 studied are bifunctional and can be used as spacers to immobilize glycoproteins through diol groups. By comparing the new data on the ionization constants and the best val ues of the enzymes’ pH optima, it is possible to select a spacer for any glycoprotein and to perform immobi lization with minimal loss of catalytic activity. We also considered the most favorable complex ation conditions. For this purpose, a BA–diol system was analyzed under static conditions where it was pos sible to study a stationary equilibrium state not com plicated by kinetic factors. We used PVA as the diol. Though this polymer is a 1,3polydiol, its use as a model compound in our studies is allowable because the mechanisms of BA complexation with 1,2 and 1,3diols are identical [15]. The usability of PVA is also supported by it being a polymer with a regular struc ture containing equal hydroxyl groups bonded to sec ondary carbon atoms [16]. Reactions with PVA there fore proceed with greater regularity and yield more definite results than, e.g., reactions with cellulose con taining OH groups bonded to primary and secondary carbon atoms and demonstrating different reactivity. We previously considered a mathematical model of complexation corresponding to the formation of the simplest boronate–diol complex, where two OH groups of BA interact with the hydroxyls of one diol
With regard to the effect of different substituents and their position in the boronic acid molecule, we may assume that the strength of the bifunctional PBA derivatives should increase in the order COOH NH2 < B(OH)2
COOH <
<
B(OH)2
(I)
(II)
B(OH)2
B(OH)2 (III)
(IV)
Br O2N
COOH <
<
B(OH)2 (V)
COOH
NO2 <
NO2 B(OH)2
B(OH)2
(VI)
(VII)
(PBA (II) is included in this series for generality). This assumption is completely confirmed by the experimental data; the sole exception is compound (VI). Its anomalously low acidity is likely explained by interaction between the nitro and dihydroxyboryl groups. Different types of this interaction are consid ered in the literature. In [14] for instance, the proba bility of a hydrogen bond forming due to a nitrogen lone pair and a vacant boron orbital is discussed. How ever, this type of the interaction requires the presence of electron donor groups at the nitrogen atom, which persuaded us to accept the standpoint of the authors of [10], who considered the possibility of forming a hydrogen bond between nitro group oxygen and
O N B
O
N+ (1)
OH
OH
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Table 2. Comparative data on the ionization constants of some boronic acids determined by potentiometric (I) and spec trophotometric (II) methods in different solvents at 298 K Compound PBA pTBA mTBA pСООНPBA
Ка
Solvent water 25% EtOH water 25% EtOH water 25% EtOH water 25% EtOH
mСООНPBA
water 25% EtOH
I
II
1.37 × 10–9 1.97 × 10–10 – 1.00 × 10–10 – 1.40 × 10–10 – – 3.06 × 10–5 (Ка1) 1.90 × 10–10 (Ка2) – – 2.22 × 10–5 (Ка1) 1.12 × 10–10 (Ка2)
1.26 × 10–9 – 5.50 × 10–10 – 1.00 × 10–9 – 1.12 × 10–4 (Ка1) 2.75 × 10–9 (Ка2)
molecule. The expression for the optimum pH value at which a stable boronate–diol complex forms was obtained: рНоpt = (1/2)рКа – (1/2)рКD + 7.
(3)
The derived expression not only confirms our assump tion about the dependence of the рНоpt value on the Table 3. Dependence of the pH optimum of interaction be tween PVA and boronic acids on B(OH)2 group acidity No.
рКа
рНopt
1 2 3 4 5 6 7 8 9 10 11 12 13 14 16
8.90 9.15 9.26 8.56 7.69 9.00 8.56 7.56 8.79 8.36 6.85 6.59 6.45 7.70 5.52
7.70 8.20 7.90 7.70 6.35 8.40 6.95 6.35 7.65 5.95 5.20 5.20 6.00 6.35 4.36
Note: For benzoic acid pKa = 4.12; numbers of compounds are given in Table 1. RUSSIAN JOURNAL OF PHYSICAL CHEMISTRY A
Ka ( H2 O ) K a ( EtOH ) 6.4–6.9 5.5 7.1
3.7 14.5 9.12 × 10–5 (Ка1) 2.75 × 10–9 (Ка2) – –
4.1 24.5
ionization constant of boronic acid and diol, but also indicates a shift to a more acidic range relative to the рКа of boronic acid. The complexation of boronic acids with diol was studied experimentally under static conditions using a model of the BA–PVA system crosslinked by cyclo hexanone. A measure of complex stability was the rel ative change in a solution’s optical density before and after interaction. The obtained dependences are almost uniform curves with maxima in a more acidic range than the рКа of the corresponding acid. Figure 1 and Table 3 present the results of studies. CONCLUSIONS Thus, based on the performed study (which basi cally corresponds to the proposed model), it is possible to quite accurately select the optimum conditions for the immobilization of glycoproteins using PBA deriv atives as spacers. For glucose oxidase (pH optimum, 5.5–6.0), stronger boronic acids are most suitable as spacers for performing immobilization in an acidic medium. In order to immobilize acetylcholinesterase (pH optimum, 7.5–8.0), which is also a glycoprotein with a ~3% carbohydrate content, the use of weaker boronic acids in an alkaline medium is most effective. The obtained data could be useful in the development of sensitive elements of biosensor devices and various molecular recognition systems based on polymers and boronic acids [3–5]. Vol. 84
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9. D. L. Yabroff, J. E. K. Branch, and B. Bettmann, J. Am. Chem. Soc. 56, 1850 (1934). 10. B. Bettmann, G. E. K. Branch, and D. L. Yabroff, J. Am. Chem. Soc. 56, 1865 (1934). 11. A. Albert and E. Sergeant, Ionization Constants of Acids and Bases (Methuen, London, 1962; Nauka, Moscow, Leningrad, 1964). 12. W. Gerard, The Organic Chemistry of Boron (Academic, London, 1961; Chemistry, Moscow, 1966). 13. G. A. Bektenova, E. E. Dil’mukhambetov, and A. S. Tulegenov, Vestn. KazNU, Ser. Khim., No. 1 (45), 409 (2007). 14. M. Lauer, H. Bohnke, R. Grottelen, Chem. Ber. 118, 246 (1985). 15. A. E. Gren’ and V. V. Kuznetsov, Chemistry of Cyclic Boric Acid Esters (Nauk. Dumka, Kiev, 1988) [in Rus sian]. 16. S. N. Ushakov, Polyvinyl Alcohol and Its Derivatives (Nauka, Moscow, Leningrad, 1960), Vol. 1 [in Rus sian].
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