The Intimate Role of Imidazole in the Stabilization ... - ACS Publications

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The Intimate Role of Imidazole in the Stabilization of Silicic Acid by a pH-Responsive, Histidine-Grafted Polyampholyte Konstantinos D. Demadis,*,† Stephan Ingmar Brückner,‡ Eike Brunner,‡ Silvia Paasch,‡ Ioanna Antonakaki,† and Mario Casolaro# †

Downloaded by UNIV OF MANITOBA on September 15, 2015 | http://pubs.acs.org Publication Date (Web): September 15, 2015 | doi: 10.1021/acs.chemmater.5b03100

Crystal Engineering, Growth and Design Laboratory, Department of Chemistry, University of Crete, Voutes Campus, Heraklion, Crete GR-71003, Greece ‡ Fachrichtung Chemie und Lebensmittelchemie, Bioanalytische Chemie, Technische Universität Dresden, 01062 Dresden, Germany # Department of Biotechnology, Chemistry and Pharmacy, Università degli Studi di Siena, Via Aldo Moro 2, I-53100 Siena, Italy ABSTRACT: A methacrylate-based polyampholyte homopolymer was synthesized starting from N-methacryloyl-L-histidine (MHist). This paper reports the inhibitory effects of poly-Nmethacryloyl-L-histidine (poly-MHist) on the in vitro silicic acid condensation. In particular, the ability of poly-MHist to retard silicic acid condensation in aqueous supersaturated solutions at three pH values, 5.5, 7.0, and 8.5, is studied. The direct role of the imidazole ring was confirmed by substantial changes in silicic acid stabilization efficiency based on the following observations: (a) the protonation degree of the imidazole ring affects stabilization. At a relatively low pH of 5.5, the imidazole is protonated and the entire polymer acquires a zwitterionic character (−COO− is also present). Inhibitory activity increases considerably. In contrast, at a high pH of 8.5, the imidazole ring is neutral and the polymer backbone is anionic due to the presence of −COO− moieties. This results in total inactivity with respect to silicic acid stabilization. (b) The use of a similar, but pH-insensitive derivative poly(N-acryloyl-Lphenylalanine) (poly-PHE), which contains a phenylalanine instead of histidine, results in total loss of silicic acid stabilization activity. Finally, poly-MHist also shows effects on colloidal silica particle morphology. Increasing the poly-MHist concentration results in a reduced size of silica particles.

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(allylamine hydrochloride),18 poly(acrylamide-co-diallyldimethylammonium chloride),18 cationically modified inulin,19 combinations of cationic/anionic assemblies,20,21 zwitterionic, phosphonate-grafted chitosan macromolecules,22,23 and neutral polymers, such as polyvinylpyrrolidone24 and polyethylene glycols.25 Our recent focus has been devoted to zwitterionic polymers, as bioinspired analogs of biopolymers that may be involved in silicic acid stabilization. Herein, we report a strategy to retard silicic acid condensation in supersaturated aqueous solutions at the pH values of 5.5, 7.0, and 8.5 by using the polyampholyte poly-Nmethacryloyl-L-histidine (poly-MHist). For comparison, we also evaluated an “analog” polymer poly(N-acryloyl-L-phenylalanine) (poly-PHE). The latter contains a phenyl residue (from phenylalanine) in the place of the imidazole ring (from histidine). Schematic structures of the polymers are shown in Figure 1. The goal of this approach was to unambiguously demonstrate the involvement of the imidazole moiety in the stabilization of silicic acid. Our interest in poly-MHist was primarily due to the following attractive features: (a) its synthesis is fairly simple and straightforward; (b) its cytotoxicity is very low; (c) it is a zwitterionic macromolecule that contains carboxylate and

INTRODUCTION The total biogenic silica production in surface waters was estimated to be ∼240 ± 40 Tmol of “Si” per annum. Thus, marine biological systems annually process an astonishing 6.7 gigatons of “Si”.1 The fate of water-soluble “Si”, usually found in the form of silicic acid, is the biological conversion into silicon dioxide (SiO2), also known as biosilica. Environmental concentrations of dissolved “Si” in the ocean are in the range of ∼70 μM.1 Silicon dioxide cannot be synthesized at ambient conditions from such low levels of silicic acid, unless the latter increases in concentration to >∼2.5 mM at circumneutral pH. Therefore, silica-producing marine microorganisms such as diatoms must not only employ an efficient “Si” cargo system to transport “Si” intracellularly into the silica deposition vesicle (SDV2−5)6 but also need an elaborate machinery of stabilizing high concentrations of “Si” (above its maximum solubility) before silica morphogenesis occurs. Biopolymers have been identified that catalyze silicic acid condensation to form silica. For example, the condensation reaction in diatoms is enhanced by the intervention of silaffins7 or long-chain polyamines.8 Silicatein is the catalyst in sponges.9−12 However, although the role of biopolymers in silicic acid stabilization prior to biosilicification has been invoked,13,14 no such stabilizing biomacromolecule has been unambiguously identified yet. Our previous efforts in silicic acid stabilization included polyaminoamide dendrimers,15,16 polyethylenimine,17 poly© XXXX American Chemical Society

Received: August 12, 2015 Revised: September 5, 2015

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DOI: 10.1021/acs.chemmater.5b03100 Chem. Mater. XXXX, XXX, XXX−XXX

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Chemistry of Materials

monomeric (H4SiO4), dimeric (H6Si2O7), and (possibly) trimeric (H8Si3O10) silica. They also stated that measurements of [SiO2]TD (TD = total dissolved) and [SiO2]n·3 indicated that the stock solution prepared at pH 11 consisted only of molybdate-reactive silica. We adopt the definitions used by Icopini et al.32 for the different “Si” species as well as the term “molybdate-reactive”. As before,25 we define “molybdate-reactive” as the “Si” species that are responsive to the silicomolybdate test described in detail below. In accordance with the above-mentioned definition of Icopini et al., “molybdate-reactive” includes three species, namely, monomeric (monosilicic acid, H4SiO4), dimeric (disilicic acid, H6Si2O7), and possibly trimeric (H8Si3O10) silica. However, since we did not detect trimeric silica in any of our experiments (see below and our previous paper25), the term “molybdate-reactive” silica refers to a mixture of monosilicic acid (primarily) and disilicic acid, with the first being dominant under our experimental conditions. Reagents, Chemicals, and Materials. Sodium silicate Na2SiO3· 5H2O, ammonium molybdate ((NH4)6Mo7O24·4H2O), and oxalic acid (H2C2O4·2H2O) were from EM Science (Merck). Sodium hydroxide (NaOH) was from Merck; hydrochloric acid 37% was from Riedel de Haen. Acrodisc filters (0.45 μm) were from Pall-Gelman Corporation. In-house, deionized water was used for all experiments. This water was tested for molybdate-reactive silica and was found to contain negligible amounts. Poly-MHist34 and poly-PHE35 were synthesized according to published procedures. The average molecular weight of poly-MHist was found to be ∼83 kDa and of Poly-PHE was 47.6 kDa, as described in detail before.34,35 The cytotoxic effect of poly-MHist was evaluated by the cell culture of osteoblasts from mouse (MC3T3-E1). No significant cytotoxicity was observed for 2 days at a concentration up to 5 mM.34 Poly-PHE shows no toxicity as well.35 Reagents for the NMR experiments were NaOH and HCl from Sigma-Aldrich, Na2CO3 from Merck, and 29SiO2 (>99%) from CORTECNET. Preparation of Supersaturated Sodium Silicate Solutions. A solution containing silicate (500 ppm as SiO2) was prepared by dissolving 4.4 g of Na2SiO3·5H2O in 2.5 L of deionized water (a nonglass container must be used to avoid glass dissolution), followed by overnight rigorous stirring. This solution contains exclusively molybdate-reactive silica in agreement with the literature.33,36 Polymer stock solutions in water were 1% w/v (10 000 ppm). The following solutions were prepared for the silicomolybdate detection test: (a) 10 g of ammonium molybdate was dissolved in 100 mL of water and its pH was adjusted between 7 and 8 with NaOH to avoid precipitation of ammonium molybdate. (b) HCl 1 + 1 is prepared by mixing one volume of 37% HCl with an equal volume water. (c) 8.75 g of oxalic acid was dissolved in 100 mL of water. All solutions were kept in polyethylene containers (glass containers must be avoided in order to minimize SiO2 dissolution and silicate leaching into the test solutions). Silicic Acid Polycondensation Protocol. Silicic Acid Polycondensation in the Absence of Additives (“Control” Protocol). 100 mL from the 500 ppm sodium silicate stock solution (see above) was placed in a polyethylene beaker charged with a Teflon-covered magnetic stirring bar. The pH of this solution was initially ∼11.8 and is subsequently adjusted to 7.00 ± 0.1 by addition of HCl. A dilute NaOH solution is occasionally used for fine-tuning the pH. The change in the resulting volume was about 3% and was taken into account for subsequent calculations. The beaker was then covered with a plastic membrane and set aside without stirring. The solutions were checked for molybdate-reactive silica by the silicomolybdate method every hour for the first 8 h or after 24, 48, and 72 h time intervals after the onset of the pH adjustment to 7.0. There must be strict time control in measuring molybdate-reactive silica, in order to avoid conversion of higher oligomers/colloidal silica to silicic and disilicic acids. Specifically, after the ammonium molybdate and HCl solutions were added to a working sample, a period of 10 min has to pass until the solution of oxalic acid is added to the same sample. Then, another 2 min period has to follow until the final measurement. All samples (“control” and polymer-containing solutions) were treated in precisely the same way. Separate experiments were performed in which the working solutions were stirred, but no difference in molybdate-reactive silica levels was found, compared to the quiescent solutions. The

Figure 1. Schematic structures of the polymers poly-MHist and polyPHE in their ionic forms. The imidazole (blue) and phenyl (red) functionalities are highlighted appropriately.

imidazolium moieties (the latter is particularly thoughtprovoking as histidine has been identified to play a key role in biosilicification in sponges);26−28 (d) the carboxylate and amide groups present in the backbone of poly-MHist may assist in detecting possible polymer entrapment within the silica matrix during silicification (this was possible in previous research with a number of polymeric inhibitors);15,16,20−23 (e) Poly-MHist also possesses an amide functionality (that actually links histidine to the polymethacrylate backbone) that may be important in silicic acid stabilization. It was reported by our group that the neutral polymers polyethyloxazoline (PEOX)29 and polyvinylpyrrolidone (PVP)24 are efficient silicic acid stabilizers. The approach undertaken in this work is based on the following principles: (1) utilization of water-soluble sodium silicate (Na2SiO3·5H2O) as the silica synthon (in contrast to TEOS used in several in vitro studies) and (b) utilization of a modified methacrylate polymer in which histidine groups have been chemically introduced by design. Poly-MHist is a polymer possessing both cationic (−imidazole+) and anionic charge (−COO−). (c) The pH of silicic acid condensation is circumneutral. This is the pH region where silicic acid condensation is the fastest. (d) The initial silicic acid concentration in our experiments is 500 ppm (expressed as ppm of SiO2), corresponding to ∼8.33 mM. It should be noted that, although the intracellular silica pool can be as high as 450 to 700 nM/cell,30 the actual level seems to range from less than 1 mM to about 20 mM (equivalent to a solution of ∼1% w/v SiO2) as recalculated from the silica content and the biovolume for more than 70 species that have been compared for their silica content.31 (e) The focus presently is additive-induced retardation/inhibition of condensation of silicic acid but also examination of the morphological features of precipitated silica nanoparticles that form after (occasionally unavoidable) silica formation occurs.

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EXPERIMENTAL SECTION

Definitions and Si Species Nomenclature. Icopini et al.32 summarized and presented the arguments of Iler33 and others and stated that the silicomolybdate method used in their work was able to detect different low molecular weight silica polymers, including B


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Chemistry of Materials 29

Si NMR Spectroscopy. 29Si NMR experiments were performed at an operating resonance frequency of 59.63 MHz using a Bruker Avance 300 spectrometer with commercial 10 mm HR probes (number of scans 120, 60 s repetition time). During the signal acquisition, Waltz16 1H decoupling was applied. The chemical shift was referenced relative to tetramethylsilane (TMS) using a standard with 1% TMS and 10% CDCl3 in acetone-d6. The time needed for one experiment was ∼2 h.

measurements of molybdate-reactive silica were run on a HACH 890 spectrophotometer from the Hach Co., Loveland, CO, U.S.A. Silicic Acid Polycondensation in the Presence of Polymers. 100 mL portions of the 500 ppm sodium silicate stock solution (see above) were placed in polyethylene containers and charged with Teflon− covered magnetic stir bars. In each container, different volumes of polymer solutions (from the prepared 10 000 ppm stock solutions) were added to achieve a desirable polymer concentration. These ranged from 20 to 40, 60, 80, and 100 ppm, and the added volumes were 200, 400, 600, 800, and 1000 μL. After that, the same procedure as the “control” protocol was followed. Determination of Molybdate-Reactive Silica. Frequently, there is a misconception about the use of the terms “silica”, “silicate”, and “silicic acid”. In this paper, the term “silica” indicates the product of silicic acid polycondensation. The term “silicate” indicates all the forms of the species Si(OH)4 at various deprotonation states Si(OH)4−xx− with x = 0, 1, 2, 3, 4; x = 4 means that silicic acid is fully deprotonated which exists at pH regions well above 12.37 Note that mono- and disilicic acid rapidly interconvert and they both contribute to molybdate-reactive silica. Molybdate-reactive silica was quantified using the wellestablished silicomolybdate spectrophotometric method. For our studies, we used a modification of (several) published procedures38−43 (5% precision) as follows: A 2 mL aliquot from the working solution is filtered through a 0.45 μm syringe filter and then diluted to 25 mL in a special cylindrical cell (the Hach Co.) of 1 cm path length. Then, 1 mL of ammonium molybdate stock solution and 0.5 mL of HCl (1:1 dilution of the concentrated solution) are added to the sample cell; the solution is mixed well by shaking and finally left undisturbed for 10 min. After that, 1 mL of oxalic acid stock solution is added and thoroughly mixed again. The solution is set aside for 2 min. After the second time period, the photometer is set to “zero absorbance” using a sample of water plus all chemicals used for the silicomolybdate test except for silicic acid (“blank”). Finally, the sample absorbance is measured (at 420 nm) and is expressed as “ppm of SiO2”. The detectable concentration range for this specific protocol is 6−75 ppm (expressed as SiO2). In order to calculate the concentration in the original solution, an appropriate dilution factor is applied. The silicomolybdate method is based on the principle that ammonium molybdate reacts only with “reactive” silica (see definitions above) and any phosphate present at low pH (about 1.2) and yields heteropoly acids, yellow in color. Oxalic acid is added to destroy the molybdophosphoric acid (in case phosphate is present in the water) leaving the silicomolybdate complex intact and thus eliminating any color interference from phosphates. It should be emphasized that molybdate reacts only with the monomeric, dimeric, and possibly trimeric forms as stated above but is totally unreactive to colloidal silica particles, if the measurements are performed with strict time control (see above). This was verified experimentally in our laboratory. Within the strict time control of the above-described measurements, only mono- and disilicic acids are reactive. Reagents, Materials, and Procedures for NMR Studies. Preparation of the Isotope Labeled Sodium Metasilicate. 29SiO2 was melted with sodium carbonate forming Na229SiO3 in a solid state reaction.44 Afterward, the resulting solid was pestled to a fine powder. Preparation of the NMR Samples. 3.6 mg of Na229SiO3 was placed in a Falcon Tube and dissolved in 2 mL of nanopure H2O and 1 mL of D2O. A volume of 36 μL of the respective 10 000 ppm polymer stock solution was added to the samples except for the “control” samples. To ensure that all sodium metasilicate was completely dissolved, the solution was treated for 3 min in an ultrasonic bath. Intending to minimize the errors of the final sample volume necessary to adjust the pH value, the pH value was adjusted once by using a 2.0 M hydrochloric acid and a 2.5 M sodium hydroxide solution. Afterward, the solution was filled up with nanopure water to 3.6 mL by the difference of 600 μL and the applied polymer stock solution plus the solution volume of hydrochloric acid/sodium hydroxide needed to adjust the pH. The pH then was adjusted once more by a volume less than one percent of the sample volume. After transferring the solution into a 10 mm NMR tube, the thus prepared NMR sample was inserted into the magnet and the measurements were started immediately.

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RESULTS Effect of Poly-MHist on Molybdate-Reactive Silica Stabilization at pH Values 5.5, 7.0, and 8.5. “Long-Term” Experiments (3 Days). Poly-MHist is a zwitterionic polymer that possesses protonated imidazole (cationic) and carboxylate (anionic) groups. The protonation constants were determined to be 2.00 for the carboxylic acid and 7.53 for the imidazole groups.34 At pH ∼ 7, poly-MHist may exist in two forms (in equilibrium). The first has the imidazole ring protonated (∼77%), resulting in a zwitterionic form (also taking into account the negative carboxylate group) of zero total charge. The second has a neutral imidazole ring (∼23%), resulting in a negatively charged backbone. In both tautomeric forms, the carboxyl moiety is deprotonated. Initially, poly-MHist was tested for its ability to stabilize molybdate-reactive silica and, thus, inhibit colloidal silica formation in “long-term” (3-day duration) experiments at various concentrations. Molybdate-reactive silica was quantified by the silicomolybdate method.38,43 Figure 2 shows the results

Figure 2. Stabilization of molybdate-reactive silica at pH 7.0, in the presence of poly-MHist (20−100 ppm) during the course of 3 days.

during 3-day silicification experiments at poly-MHist levels of 20, 40, 60, 80, and 100 ppm at pH 7. It becomes apparent that poly-MHist exerts only a minor stabilization effect (∼20 ppm silicic acid above the control) for 80 and 100 ppm concentrations. Silicification experiments were also carried out at pH 5.5 where, notably, silicic acid condensation is much slower. At pH 5.5, the imidazole ring should be fully protonated, and polyMHist should be zwitter-ionic with charges balanced. Silicic acid stabilization experiments (Figure 3) showed that the effect is marginal for the first 24 h, with only ∼50 ppm silicic acid being stabilized above the “control”, at poly-MHist concentrations of 80 and 100 ppm. However, after 48 h, molybdate-reactive silica levels increase to ∼90 and 118 ppm (for 80 and 100 ppm poly-MHist C


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Chemistry of Materials

Figure 5. Stabilization of molybdate-reactive silica at pH 7.0 in the presence of poly-MHist (10, 20, 40, 60, 80, and 100 ppm) during the course of 8 h. Inset: comparative graph of the 8th hour measurement.



concentration of 100 ppm, poly-MHist is able to quantitatively stabilize all initial (500 ppm) molybdate-reactive silica. The stabilization capability of poly-MHist at pH 5.5 is shown in Figure 6. Although the stabilization of molybdate-reactive

concentrations, respectively) above the control values. Finally, poly-MHist at a level of 100 ppm is able to additionally stabilize 157 ppm molybdate-reactive silica. These results strongly indicate that the protonated imidazole ring is playing a role in the stabilization process. Finally, stabilization experiments were performed at pH 8.5, where the imidazole ring is expected to be nonprotonated and neutral. Interestingly (see Figure 4), the stabilizing ability of

Figure 6. Stabilization of molybdate-reactive silica at pH 5.5 in the presence of poly-MHist (10, 40, 80, and 100 ppm) during the course of 8 h. Inset: comparative graph of the 8th hour measurement.

silica seems to be only 85 ppm above the control (at 100 ppm polymer concentration and after 8 h), overall, the inhibition performance is excellent. For example, at only 40 ppm polymer concentration, the total stabilized molybdate-reactive silica is ∼400 ppm. The increase in polymer concentration induced additional silicic acid stabilization. Again, this is most likely an event due to the protonation of the imidazole ring. Finally, the effect of poly-MHist on the stabilization of molybdate-reactive silica was studied at pH 8.5, where the imidazole ring is neutral (nonprotonated). The results are shown in Figure 7. Furthermore, there appears to be a concentration dependence of stabilizing efficiency. On the basis of the data shown in Figures 5, 6, and 7, a clear trend is apparent; molybdate-reactive silica stabilization efficiency increases proportionally with increasing poly-MHist concentration. This is more clearly demonstrated in Figure 8, where molybdate-reactive silica levels (at 4 h condensation time) were plotted against poly-MHist concentration. This phenomenon is reminiscent of threshold inhibition of several inorganic sparingly soluble salts, where such a concentration-dependent relationship is expected. This phenomenon is reminiscent of threshold inhibition of several


poly-MHist is completely lost, and silicic acid levels are identical to those of the “control”. On the basis of these results, it appears that protonation of the imidazole ring has a profound effect on the inhibition of silica condensation. “Short-Term” Experiments (8 h). The above results taken together reveal an additional important point, that any substantial inhibitory activity takes place at timescales

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