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Chem. Mater. 2005, 17, 2918-2923
Reactivity of Poly(anilineboronic acid) with NAD+ and NADH Bhavana A. Deore and Michael S. Freund* Department of Chemistry, UniVersity of Manitoba, Winnipeg, Manitoba, R3T 2N2, Canada ReceiVed March 24, 2005. ReVised Manuscript ReceiVed April 6, 2005
The complexation of poly(anilineboronic acid) with β-nicotinamide adenine dinucleotide (NADH) and its oxidized form, NAD+, has been investigated using electrochemical and spectroscopic techniques. At neutral pH, cyclic voltammetry of poly(anilineboronic acid) exhibits redox activity in the presence of NADH and NAD+ characteristic of self-doping. While complexation of both NADH and NAD+ with poly(anilineboronic acid) is confirmed with polarization-modulated infrared reflection absorption spectroscopic measurements, a significant difference in redox behavior is observed. 11B NMR studies suggest that the nature of species responsible for self-doping is different in the case of NAD+ and NADH, where the presence of NAD+ complex appears to facilitate the formation of the pernigraniline form of the polymer. In addition, a distinct difference in the sensitivity of open-circuit potential measurements is observed upon complexation with NADH and NAD+ as a function of the oxidation state of polymer suggesting that reduction of the polymer occurs upon complexation of NADH. The mechanism responsible for the observed behavior is supported by quartz crystal microbalance measurements.
Introduction Nucleotides, such as β-nicotinamide adenine dinucleotide (NADH) and its oxidized form (NAD+), are ubiquitous in all living systems and are required for the reactions of more than 400 oxidoreductases.1 Thus, the qualitative and quantitative analysis of the nucleotides is of great importance in a variety of fields. The complex redox behavior exhibited by the NAD+/NADH couple (Scheme 1) under physiological conditions has prompted extensive electrochemical studies of the reaction. The electrochemical oxidation of NADH has been investigated for decades as a means for quantification and creating enzyme-based electrocatalytic systems. A major complication in this field is the fact that direct oxidation of NADH at unmodified electrode surfaces proceeds at high overpotentials (>+0.5 V) and generally leads to fouling of the electrode surface as well as the formation of a mixture of oxidation products, including products of radical coupling. Attempts to develop catalytic electrode surfaces for the oxidation of NADH to enzymatically active NAD+ have concentrated on the use of redox mediator species2 and conducting polymers.3 Aromatic boronic acids are known to bind compounds containing diol moieties with high affinity through reversible ester formation (Scheme 2) where the association constant as well as the relative concentration of the neutral trigonal ester and the tetrahedral boronate ester are dependent on the pH and solvent. The reversible esterification of borate with a number of ribose-containing nucleotides and cofactors * To whom correspondence should be addressed. E-mail: michael_freund@ umanitoba.ca.
(1) (a) White, H. B. EVolution of Coenzymes and the Origin of Pyridine Nucleotides; Academic Press: New York, 1982. (b) Rawn, J. D. In Biochemistry; Patterson, N., Ed.; Carolina Biological Supply Co.: Burlington, NC, 1989. (2) For example see: Gorton, L. J. Chem. Soc., Faraday Trans. 1986, 82, 1245 and references therein. (3) Bartlett, P. N.; Simon, E. J. Am. Chem. Soc. 2003, 125, 4014 and references therein.
including diadenosine phosphates, S-adenosylmethionine, NAD+, NADH, adenosine triphosphate (ATP), adenosine monophosphate (AMP), and cyclic AMP have been studied using capillary electrophoresis.4 In addition, Kim et al. have studied the esterification of boric acid and borate with NAD+ and NADH using electrospray ionization mass spectrometry and 11B NMR spectroscopy. These studies demonstrate that borate binds to both cis-2,3-ribose diols on NAD+ and NADH forming borate ester.5 Similar reactions are observed with carbohydrates, vitamins, coenzymes, and ribonucleic acids containing diol moieties.6 The borate and boronic acids interaction with these polyol compounds has attracted growing interest as a basis for the construction of novel molecular recognition systems as well as platforms of diverse applications related to polyol compounds, including chromatographic and membrane separation,7-9 sensing systems,10-12 drug delivery and discovery extended by use of boradeption,13-15 and a particular cancer treatment known as (4) Ralston, N. V. C.; Hunt, C. D. Biochim. Biophys. Acta 2001, 1527, 20. (5) (a) Kim, D. H.; Marbois, B. N.; Faull, K. F.; Eckhert, C. D. J. Mass Spectrom. 2003, 38, 632. (b) Kim, D. H.; Faull, K. F.; Norris, A. J.; Eckhert, C. D. J. Mass Spectrom. 2004, 39, 743. (6) Otsuka, H.; Uchiro, E.; Koshino, H.; Okano, T.; Kataoka, K. J. Am. Chem. Soc. 2003, 125, 3493 and references therein. (7) Shinbo, T.; Nhishimura, K.; Yamaguchi, T.; Sugiura, M. J. Chem. Soc., Chem. Commun. 1986, 349. (8) Morin, G. T.; Hughes, M. P.; Paugam, M.-F.; Smith, B. D. J. Am. Chem. Soc. 1994, 116, 8895. (9) Westmark, P. R.; Gardiner, S. J.; Smith, B. D. J. Am. Chem. Soc. 1996, 118, 11093. (10) James, T. D.; Sandanayake, K. R. A. S.; Shinkai, S. Angew. Chem., Int. Ed. 1996, 35, 1910. (11) James, T. D.; Sandanayake, K. R. A. S.; Shinkai, S. Nature 1995, 374, 345. (12) James, T. D.; Sandanayake, K. R. A. S.; Iguchi, R.; Shinkai, S. J. Am. Chem. Soc. 1995, 117, 8982. (13) Pan, X. Q.; Wang, H.; Shukla, S.; Sekido, M.; Adams, D. M.; Tjarks, W.; Barth, R. F.; Lee, R. J. Bioconjugate Chem. 2002, 13, 435. (14) Stiriba, S.-E.; Frey, H.; Haag, R. Angew. Chem., Int. Ed. 2002, 41, 1329. (15) Gallop, P. M.; Paz, M. A.; Henmson, E. Science 1982, 217, 166.
10.1021/cm050647o CCC: $30.25 © 2005 American Chemical Society Published on Web 05/05/2005
ReactiVity of Poly(anilineboronic acid) with NAD+ and NADH
Chem. Mater., Vol. 17, No. 11, 2005 2919
Scheme 1. Interconversion of NAD+ (1) and NADH (2)
Scheme 2. Boronate Ester Formation
boron neutron capture therapy.16-19 In particular, sensing systems based on the optical (i.e., fluorescence, circular dichroism, and absorption) or electrochemical (i.e., voltammetry and potentiometry) response of boron-containing receptors have been explored as the signal transduction pathway of the binding event.10,20-22 Recently, we23,24 and others25,26 have developed saccharide and anion-responsive chemically modified electrodes from the electropolymerization of different boronic-acid- and boronate-substituted aromatic compounds. Our previous work demonstrates that by coupling boronic acid groups to the backbone of polyaniline, a potentiometric sensor for saccharides can be produced.23,24 The transduction mechanism in that system is the change in pKa of polyaniline that accompanies complexation and the resulting change in the electrochemical potential. Sensors produced with this approach exhibit reversible response with selectivity to various saccharides and 1,2-diols that reflect their binding constants with phenylboronic acid observed in bulk solutions. Using the same reaction we achieved the electrochemical polymerization of self-doped poly(anilineboronic acid) through formation of anionic boronic ester complex between 3-aminophenylboronic acid and D-fructose in the presence of fluoride.27 The complexation of boronic acid with D-fructose and subsequent formation of self-doped polymer extends the electroactivity of poly(anilineboronic acid) to neutral and alkaline media.28 Finally, this chemistry resulted in the (16) (a) Lawler, A.; Science 1995, 267, 956. (b) Flam, F. Science 1994, 265, 468. (c) Anderson, C. Science 1993, 262, 329. (17) Hawthore, M. F. Angew. Chem., Int. Ed. Engl. 1993, 32, 950. (18) Barth, R. F.; Yang, W.; Rotaru, J. H.; Moeschberger, M. L.; Joel, D. D.; Nawrocky, M. M.; Goodman, J. H.; Soloway, A. H. Cancer Res. 1997, 57, 1129. (19) Soloway, A. H.; Tjarks, W.; Barnum, B. A.; Rong, F.-G.; Barth, R. F.; Codogni, I. M.; Wilson, G. Chem. ReV. 1998, 98, 1515. (20) Eggert, H.; Frederiksen, J.; Morin, C.; Norrild, J. C. J. Org. Chem. 1999, 64, 3846. (21) Beer, P. D.; Gale, P. A. Angew. Chem., Int. Ed. 2001, 40, 486. (22) James, T. D.; Linnane, P.; Shinkai, S. Chem. Commun. 1996, 281. (23) Shoji, E.; Freund, M. S. J. Am. Chem. Soc. 2001, 123, 3383. (24) Shoji, E.; Freund, M. S. J. Am. Chem. Soc. 2002, 124, 12486. (25) (a) Nicolas, M.; Fabre, B.; Marchand, G.; Simonet, J. Eur. J. Org. Chem. 2000, 1703. (b) Nicolas, M.; Fabre, B.; Simonet, J. Chem. Commun. 1999, 1881. (c) Nicolas, M.; Fabre, B.; Simonet, J. J. Electroanal. Chem. 2001, 509, 73. (26) Pringsheim, E.; Terpetschnig, E.; Piletsky, S. A.; Wolfbeis, O. S. AdV. Mater. 1999, 11, 865. (27) Deore, B. A.; Freund, M. S. Analyst 2003, 128, 803.
Scheme 3. Proposed Redox Mechanism of PABA
chemical polymerization of a soluble, self-doped poly(anilineboronic acid).29 In the present work, we investigate the nature of the boronic acid-nucleotide (i.e., NAD+ and NADH) complexation with poly(anilineboronic acid) and its influence on the redox activity and electrochemical potential of the polymer using cyclic voltammetry, open-circuit potential, quartz crystal microbalance (QCM), 11B NMR, and polarization-modulated infrared reflection absorption spectroscopic (PM-IRRAS) measurements. Experimental Section Materials and Reagents. 3-Aminophenylboronic acid hydrochloride salt, NAD+, and NADH were purchased from Aldrich Chemical Inc. Sodium fluoride and pH 7.4 phosphate buffered saline stock solution (PBS) were purchased from Fisher Scientific. Indiumdoped tin oxide (ITO, 6 ( 2 Ω/square) glass slides were purchased form Delta Technologies, Limited. Bulk distilled water was first filtered and ion-exchanged to yield 18.3 MΩ quality water using a Milli-Q-Academic A10 system (Millipore Corporation). Electropolymerization of Poly(anilineboronic acid) (PABA). PABA was deposited electrochemically onto glassy carbon electrodes. The monomer solution was prepared using 3-aminophenylboronic acid hydrochloride salt (40 mM) and sodium fluoride (200 mM) in 0.5 M HCl. The potential was scanned between -0.1 and 1.0 V in an unstirred solution at a scan rate of 100 mV s-1 until the charge under the cathodic peak reached ∼ 0.9 mC cm-2. The proposed structure of PABA and its various oxidation states are shown in Scheme 3. For open-circuit potential measurements, the final scan was stopped at different potentials (in the range of -0.2 to 1.2) and held at that potential for 20 s. The electrode was then rinsed with water, followed by a rinse with PBS solution, and then soaked in PBS solution overnight. There was no significant change in the oxidation state of polymer after overnight equilibration in (28) Deore, B. A.; Hachey, S.; Freund, M. S Chem. Mater. 2004, 16, 1427. (29) Deore, B. A.; Yu, I.; Freund, M. S. J. Am. Chem. Soc. 2004, 126, 52.
2920 Chem. Mater., Vol. 17, No. 11, 2005
Figure 1. Cyclic voltammograms of PABA-modified gold electrode in phosphate buffer at pH 7.4 in the presence of NADH and NAD+. Concentration NADH + NAD+: (a) 0, (b) 10 + 0, (c) 7.5 + 2.5, (d) 5 + 5, (e) 2.5 + 7.5, and (f) 0 + 10 mM. Scan rate 100 mV/s.
PBS. For PM-IRRAS characterization, films were deposited on indium-doped tin oxide coated glass in a similar manner. Characterization. Cyclic voltammetric and potentiometric measurements were performed using a CH Instrument CHI-660 workstation controlled by a PC. A three-electrode cell was used, which consisted of a gold (3.0-mm diameter) working electrode, a platinum coil auxiliary electrode, and an Ag/AgCl reference electrode. Open-circuit potential measurements were performed using a PABA-modified gold electrode as a working electrode and Ag/AgCl reference electrode. Quartz crystal microbalance experiments were performed with a CH Instrument model 400 series QCM integrated with potentiostat. Measurements were carried out in a single-compartment, three-electrode cell using a commercial gold, vapor-deposited on a 8 MHz AT-cut quartz crystal as working electrode (International Crystal Manufacturing, Co., Inc.). The calibrated response was 2.05 ng Hz-1. PM-IRRAS spectra were recorded at a resolution of 8 cm-1 using Nexus 870 spectrometer (Thermo Nicolet Corporation). A grazing angle of 67° was used to collect 300 interferograms for each spectrum. 11B NMR studies were carried out using a Bruker AMX 500 NMR spectrometer. The samples were prepared using 10% D2O in pH 7.4 PBS. Chemical shifts were determined relative to borontrifluoride etherate as a reference.
Results and Discussion Cyclic Voltammetry. The redox properties of PABA thin films in the presence of NADH and NAD+ at various concentrations were studied in pH 7.4 PBS. As is the case with polyaniline,30 PABA thin-films cycled in pH 7.4 PBS lose their redox activity due to deprotonation and loss of dopant as shown in Figure 1a. However, in the presence of nucleotides (i.e., NAD+ and/or NADH) PABA films become redox active (Figure 1b-f) due to complexation of boronic acid with cis 2,3-ribose diols and subsequent formation of self-doped polymer. CVs of PABA thin films show a single redox couple in the presence of NADH (Epa 0.05 and Epc -0.10 V). In contrast, a second redox couple is observed in the presence of NAD+ at Epa 0.34 and Epc 0.18 V. With increasing the relative concentration of NAD+ in the mixture of NADH and NAD+, a gradual evolution between the two voltammetric responses is observed with an increase in the overall magnitude of peak current. The peak current increases with NAD+ concentration up to 20 mM. In the presence of NAD+, the CVs exhibit redox behaviors very similar to (30) (a) Ray, A.; Richter, A. F.; MacDiarmid, A. G.; Epstein, A. J. Synth. Met. 1989, 29, E151. (b) MacDiarmid, A. G.; Yang, L. S.; Huang, W. S.; Humphrey, B. D. Synth. Met. 1987, 18, 393.
Deore and Freund
Figure 2. 11B NMR spectra of 5 mM 3-aminophenylboronic acid in the presence of 20 mM NADH (a) and NAD+ (b) at pH 7.4 PBS.
polyaniline31 and PABA24 in acidic solution with exception that the two sets of redox couples are more closely spaced and shifted to lower potentials. These results are similar to the redox behavior of self-doped PABA in the presence of D-fructose as a function of pH of electrolyte solution.28 The redox chemistry of self-doped PABA is clearly quite different from that of previously reported forms of self-doped polyaniline such as sulfonated polyaniline, likely due to the pHdependent nature of the boronic acid group of PABA.28 In the presence of NAD+, the two redox couples represent the conversion of the nonconducting, reduced form into the conducting intermediate oxidized form (leucoemeraldine to emeraldine) and subsequent conversion to the nonconducting fully oxidized form (emeraldine to pernigraniline).31 However, in the case of NADH (Figure 1b), the absence of a second redox couple suggests that the further oxidation of emeraldine to pernigraniline form of the PABA is not energetically favorable. The difference in the redox behavior of PABA in the presence of NAD+ and NADH likely stems from a difference in the structure of boronic acid-nucleotide complexes, which are known to form in solution.4,5 The fact that two distinct redox behaviors are observed indicates that neither electron transfer between the polymer and the nucleotide nor exchange between nucleotide complexed and in solution occurs on the time scale of the voltammetric experiment. To investigate the possible nature of the complex of PABA with the oxidized (NAD+) and reduced (NADH) nucleotides, 11 B NMR of monomer solutions were carried out in the presence of the nucleotides (see Figure 2). A monomer solution was prepared by mixing the 5 mM 3-aminophenylboronic acid and 20 mM nucleotides in pH 7.4 PBS with 10% D2O. In the presence of NADH, a single resonance is observed with a chemical shift at 28.8 ppm (Figure 2a), which indicates that the major boronic acid species exists in the neutral trigonal form32 as illustrated in Chart 1 (I or II). However, in the presence of NAD+ (Figure 2b), an additional resonance signal is observed approximately 20 ppm upfield from the trigonal boronic acid signal, indicative of the formation of a tetrahedral anionic boronic acid32 as illustrated in Chart 1 (III). The presence of two peaks indicates the slow exchange between the two forms on the 11B NMR time (31) (a) Huang, W. S.; Humphrey, B. D.; MacDiarmid, A. G. J. Chem. Soc., Faraday Trans. 1986, 82, 2385. (b) Sariciftci, N. S.; Kuzmany, H.; Neugebauer, H.; Neckel, A. J. Chem. Phys. 1990, 92, 4530. (32) For a recent review on boronic acid-diol chemistry see Springsteen, G.; Wang, B. Tetrahedron 2002, 58, 5291.
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Chart 1. Proposed Structures of Boronate Esters with NADH (I and II) and NAD+ (III)
scale.5 These observed structural differences are likely due to the electrostatic stabilization of the tetrahedral anionic boron provided by the positively charged nicotinamide group in NAD+.5 While not conclusive regarding the structure in the polymer, the 11B NMR results indicate that, upon complexation with nucleotides, the nature of the boronate esters responsible for self-doping of PABA is different. In the presence of NAD+, self-doping can result from two sources, including the anionic tetrahedral boronate ester as well as the nucleotide phosphate. The anionic tetrahedral boronic acid-NAD+ adduct apparently supports the further oxidation of self-doped PABA (emeraldine to pernigraniline) due to the close proximity of the charged species to the polyaniline backbone. In contrast, the neutral trigonal boronic acidNADH adduct only provides the anionic phosphates on the nucleotide which are further removed. Due to the relatively large separation of the charged phosphate group in the nucleotide from the cationic amine in the polymer backbone, oxidation from the emeraldine to the pernigraniline form of polyaniline is less energetically favorable. PM-IRRAS. Voltammetric and NMR results indicate that PABA is capable of forming covalent bonds with the diol functionalities of nucleotides resulting in the generation of cyclic esters. PM-IRRAS is an ideal tool for monitoring this reaction within polymer thin films. Figure 3 shows the formation of boronate esters by the complexation of NAD+ and NADH with PABA. Figure 3a shows the main vibrations of polyaniline at 1603, 1510, and 1160 cm-1 and correspond to quinoid, benzenoid, and C-N stretching ring modes, respectively.33 Vibrations characteristic of aromatic boronic acids observed at 986 and 1115 cm-1 correspond to B-OH bending modes, while the vibrations attributed to asymmetric B-O stretching and B-F stretching mode lie at 1330 and 888 cm-1, respectively.33,34 Following the reaction of PABA thin films with the nucleotides in pH 7.4 PBS, the disap(33) Epstein, A. J.; McCall, R. P.; Ginder, J. M.; MacDiarmid, A. G. In Spectroscopy of AdVanced Materials; John Wiley & Sons: New York, 1991.
pearance of the 888 cm-1 band is observed corresponding to the removal of fluoride associated with the boronic acid during polymerization. As seen in Figure 3b and c, after complexation with NAD+ and NADH, the disappearance of vibration at 986 cm-1 and increase in the intensity of 1330 cm-1 vibration is observed. This is consistent with the loss
Figure 3. PM-IRRAS spectra of PABA film (a) reacted with 10 µM (b) NAD+ and (c) NADH in pH 7.4 PBS.
of the free B-OH group which occurs concomitantly with an increase in asymmetric B-O bond formation, consistent with the formation of boronate ester. In Figure 3b and c, the new vibrations at 1080 and 1470 cm-1 are attributed to ribose and adenine moiety, respectively.35 The vibrations at 1218 (NAD+) and 1245 (NADH) and above 1603 cm-1 are attributed to nicotinamide moiety.35 The presence of these vibrations confirm the complexation of nucleotides with PABA. After complexation with NAD+ (Figure 3b), the high relative intensity ratio of quinoid to benzenoid ring modes suggests that the PABA is in the oxidized form. These results (34) Socrates, G. Infrared Characteristic Group Frequencies; John Wiley & Sons: New York, 1994. (35) Yue, K. T.; Martin, C. L.; Chen, D.; Nelson, P.; Sloan, D. L.; Callender, R. Biochemistry 1986, 25, 4941.
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Figure 4. Sensitivity of PABA to NADH (A) and NAD+ (B) in pH 7.4 PBS. PABA film was oxidized at 1.0 V (b) and reduced at -0.1 V (O) for 20 s.
suggest that complexation with NAD+ results in self-doping through ester formation, in agreement with CV and 11B NMR results. In contrast, following exposure to NADH (Figure 3c), a red shift in the peaks corresponding to the quinoid and benzenoid ring modes as well as a decrease in their relative intensity ratio is observed. The decrease in the ratio of relative intensities of quinoid to benzenoid ring modes indicates that the polymer is reduced in the process. The reduction of PABA likely occurs due to hydride transfer from NADH to one of the nitrogens in the polyaniline backbone, consistent with previous reports.3 As indicated in the voltammetric study, this redox process appears slow. Open-Circuit Potential Measurements. The clear difference in voltammetric behavior as a function of the redox state of the nucleotide suggests that it may be possible to manipulate the complexation reaction by altering the oxidation state of the polymer. To explore the sensitivity of PABA films toward the redox state of the nucleotide, open-circuit potential measurements were carried out as a function of oxidation state of the polymer. Since complexation changes the nature of the boronic acid substituent and in turn the electrochemical potential of polyaniline, we can monitor the binding event by measuring changes in the electrochemical potential of polymer. To change the oxidation state of polymer, PABA thin films were held at different potentials in the range of -0.2 to 1.2 V in 0.5 M HCl. The electrode was then rinsed with water, followed by a rinse with PBS solution, and then soaked in PBS solution overnight to allow the electrochemical potential to stabilize. A significant difference in the open-circuit potential sensitivity of PABA is observed upon complexation with nucleotides. A positive shift in electrochemical potential is observed for NAD+ and a negative shift is observed for NADH as shown in Figure 4. The positive shift in the potential of oxidized PABA for NAD+ is a result of complexation, similar to that observed for saccharides using PABA,23,24 although the sensitivity is much higher in the case of NAD+.
Deore and Freund
This is expected due to the higher binding constant associated with cyclopentane diol as well as the two sites available in the nucleotide. Under these conditions, the electrochemical potential is sensitive to the change in the pKa of the PABA as a result of the formation of an anionic boronic acid-diol complexation. However, in the case of NADH, the negative shift in the potential is likely a result of net effect of pKa change and complexation which is dominated by reduction of the polymer,3 consistent with PM-IRRAS results. No significant difference in the sensitivity is observed for NAD+ as a function of the oxidation state of the polymer (within the range of 0.2 to 1.0 V). However, films exposed to potentials
