A proton shelter inspired by the sugar coating of acidophilic archaea

The acidophilic archaeons are a group of single-celled microorganisms that flourish in hot acid springs (usually pH < 3) but maintain their internal pH near neutral. Although there is a lack of direct evidence, the abundance of sugar modifications on the cell surface has been suggested to provide the acidophiles with protection against proton invasion. In this study, a hydroxyl (OH)-rich polymer brush layer was prepared to mimic the OH-rich sugar coating. Using a novel pH-sensitive dithioacetal molecule as a probe, we studied the proton-resisting property and found that a 10-nm-thick polymer layer was able to raise the pH from 1.0 to > 5.0, indicating that the densely packed OH-rich layer is a proton shelter. As strong evidence for the role of sugar coatings as proton barriers, this biomimetic study provides insight into evolutionary biology, and the results also could be expanded for the development of biocompatible anti-acid materials.

The SAM and polymer brushes were prepared on the gold electrode of a QCM chip for QCM measurements under various pH solutions. QCM is an acoustic-based sensor that detects interfacial mass losses as frequency increases (Df . 0) 15,17 . The polymer-coated chip was treated with HCl (pH 1.0) to mimic the environmental pH of acidophiles (Fig. 2b). If the H 1 from the HCl reached the Au-S bonds, one would observe frequency increases due to the partial disassembly of the SAM and attached polymer chains. If the polymer layer exerted any inhibiting effect on proton permeation, the frequency response would be weakened or silenced.
Examination of the effect of the OH-rich polymer as a proton shelter. First, the chips grafted with poly(OEGMA 475 ) were challenged with HCl. As is shown by the red line in Fig. 3a, the QCM frequency remained unresponsive to the pH 5.0 HCl but increased significantly when exposed to the pH 1.0 HCl, indicating that the H 1 from HCl penetrated the poly(OEGMA 475 ) layer and reached the Au-S bonds. As a control, poly(OEGMA 475 ) was also grafted from the acid-treated 1 SAMs and thiol initiator (vmercaptoundecyl bromoisobutyrate) SAMs (Supplementary Figs S5 and S6). The resulting chips showed no acid responses, confirming that the mass loss in Fig. 3a (red line) was driven by the acid-sensitivity of the 1 SAMs. The acid-induced partial loss of surface mass was also evidenced by atomic force microscopy ( Supplementary Fig. S7).
The poly(OEGMA 526 )-covered chips were also investigated. As shown in Fig. 3a (the black line), no significant Df was recorded when the pH value dropped from 5.0 to 1.0, indicating that the H 1 from HCl did not fully penetrate the poly(OEGMA 526 ) coating and that the local pH near the Au-S bonds was at least . 5.0. This is strong evidence that the replacement of -OCH 3 groups to -OH groups enabled the polymer coating to act as a proton shelter. Similar results were observed when OEGMA 300 (M n 5 300 g mol 21 , -OCH 3 terminated) and OEGMA 360 (M n 5 360 g mol 21 , -OH terminated) were used as another pair of -OCH 3 /-OH-presenting monomers in the SIP ( Supplementary Figs S8 and S9).
To confirm the inhibition effect of -OH groups on H 1 penetration further, we designed the following series of experiments. First, we copolymerized OEGMA 526 and OEGMA 475 at different feed ratios to tune the relative content between the -OCH 3 and -OH ends ( Supplementary Fig. S10). The result demonstrated that the pH response was completely silenced when the proportion of OEGMA 526 in the copolymer was as low as 10%; a significant proton-resistant effect was still observed even when that ratio dropped to 0.1%. Second, when the -OH ends in the 10% OEGMA 526 -containing copolymer were converted to -OCH 3 groups using MeI, the pH-responsiveness was regained, as expected ( Supplementary Fig. S11). Cyclic voltammetry (CV) experiments also indicated a higher electronic resistance of poly(OEGMA 526 ) than poly(OEGMA 475 ), consistent with protonblocking property 18  . All of these results support the notion that the OH-rich polymer brushes could act as a proton shelter.
The -OH groups must be presented in a brush form to gain the proton-shelter function, i.e., the -OH groups must be spatially confined. When a poly(OEGMA 475 )-grafted chip was treated with a pH 1.0 HCl solution containing 1 mM OEGMA 526 monomer, a Df . 0 was observed ( Supplementary Fig. S15), indicating that the free form of the -OH groups in solution did not suppress the pH response. Therefore, the surface-grafted polymer brushes were necessary for the confinement and enrichment of the -OH groups, a mimic to the conditions in glycolipids.
To assess further the effect in three-dimensional compartments, the proton-shelter was lifted up from the SAM surface by block copolymerization in which a ''proton-conductive'' layer containing exclusively poly(OEGMA 475 ) was first prepared as a spacer to set the distance between the ''proton shelter layer'' and the SAM (Fig. 3b). The chips with this two-layer architecture were subjected to acid treatment, and a prominent proton-resisting function was observed, even when the distance increased to 80 nm (24 nm of dry thickness of the poly(OEGMA 475 ) layer corresponded to 80 nm of wet thickness in HCl), proving the proton resistance effect of the -OH groups in three-dimensional space.

Discussion
In dramatic contrast to those in bacteria or eukaryotic cells, the majority of the lipids in the cell membrane of acidophilic archaeons are glycolipids or glycophospholipids that contain at least one sugar residue on the extracellular side (Fig. 1b). When the environmental pH decreased from 3.0 to 1.2, the content of glycolipids in the plasma membrane of Thermoplasma acidophilum increased and the percentage of the lipids that contained 2-4 sugar units rose from 14% to 35%, suggesting an adaptation to low pH values by extending the sugar chains on the cell surface 11 . As a systematic investigation of the  effect of -OH groups on proton resistance, our study presents strong evidence for that adaptation mechanism and pinpoints the effective site as -OH groups. The layer remained resistant to proton permeation after 1,000-fold dilution of the -OH group concentration, suggesting that there a redundancy still exists with regard to the amount of -OH groups in our experiments. Thus it is reasonable to suppose that a layer of a few sugar units (1-2 nm in thickness) is capable of exerting moderate effects on acid defense (Fig. 1c).
The concentration of -OH groups inside the poly(OEGMA 526 ) brushes was estimated to be 0.5 M (see Supplementary Information for the detailed calculation), which is on the similar order of magnitude with that on the cell surfaces of acidophilic archaeons, thus justifying our simulation 19 .
Our study proves the strong proton-resistance of surface confined -OH groups and also paves the way for the investigation of the underlying mechanism. The proton-resistance properties of other groups, such as -NH 2 and epoxy groups, could be studied by changing the monomers, the direction of our ongoing experiments. The results, combined with a theoretical analysis, are expected to reveal the mechanisms.
The bioinspired poly(OEGMA 526 ) membrane possesses a strong proton-sheltering effect that has been quantitatively defined as  raising the pH from 1.0 to . 5.0. Therefore, our biomimetic study may be expanded for the development of biocompatible anti-acid strategies, such as tooth protection and drug-carriers that withstand gastric acid digestion 20 . Our conclusion also provides information for the design of novel paints that offer protection to vehicles, buildings and infrastructures against acid rain corrosion, an area for which realistic solutions are still lacking. Furthermore, as the first organosulfur compound that has been found to form an acid-sensitive Au-S bond, compound 1 could be used to study the nature of Au-S bonds.
Methods 2-Bromo-2-methyl-propionic acid 4-[bis-(11-hydroxy-undecylsulfanyl)-methyl]phenyl ester (1). The dithioacetal initiator (1) was synthesized using the following procedure: (1) Synthesis of 4-formylphenyl -2-bromo-2-methylpropanoate 21 : Hydroxybenzaldehyde (0.61 g, 5 mmol), triethylamine (0.75 mL, 1.033 mmol), and dry dichloromethane (25 mL) were added to a 50-mL round-bottom flask with a stir bar. The mixture was cooled to 0uC, followed by the dropwise addition of ice-cold bromoisobutyryl bromide (0.67 mL, 5.39 mmol). After stirring at 0uC for 1 h, the reaction was continued for another 12-16 h at room temperature. Water (20 mL) and dichloromethane (10 mL) were added to the mixture for a two-phase extraction. The aqueous phase was further extracted with dichloromethane (2 3 30 mL), and the organic phase was concentrated by rotary evaporation to remove the dichloromethane. The resulting crude extract was dissolved in dichloromethane (40 mL), washed with a saturated sodium bicarbonate solution (3 3 40 mL), and dried over MgSO 4 . The removal of the dichloromethane resulted in a yellowish oil, which was passed through a column (silica gel, neutral, with petroleum ether:ethyl acetate 5 20:1) and then vacuum dried overnight. The final product (4-formylphenyl -2bromo-2-methylpropanoate) was a white solid, obtained in high purity and with a high yield (0.2168 g, 80% yield). 1  (2) Synthesis of dithioacetal initiator 22 : Mercaptoundecanol (0.48 g, 2 mmol): 4formylphenyl -2-bromo-2-methylpropanoate (0.271 g, 1 mmol) and toluene (10 mL) were added to a 50 mL round-bottom flask with a stir bar. The mixture was refluxed to 110uC, followed by the addition of p-toluenesulfonic acid (0.009 mL, 0.05 mmol). After stirring at 110uC for 3 h, the reaction was stopped by the addition of triethylamine (3 mL). The mixture was concentrated by rotary evaporation to remove the toluene. The removal of the toluene resulted in an orange oil, which was passed through a column (silica gel, neutral, with petroleum ether: ethyl acetate5 2:1 with 2% triethylamine as eluent) and then vacuum dried overnight. The final product was a white solid, obtained in high purity and with a high yield (1.4040 g, 93.1% yield). 1  SAMs. The QCM chips were incubated in 1 mM ethanol solution of 1 for 18 hours. The reaction was executed at ambient temperature, under nitrogen atmosphere protection and protected from light.
SIP. The SIP of OEGMA from the SAMs of 1 as the initiators was performed as reported previously 15 using bipyridine as a ligand and water: ethanol 5 1: 1 as a solvent, with a molar ratio of OEGMA/CuBr 2 /bipyridine/CuBr/ 5 400/1/30/10 (i.e., 10/0.025/0.75/0.25 mM). The reaction was terminated by MilliQ-water (pH 6.2), and the chips were then rinsed thoroughly with MilliQ-water and ethanol to remove any salt particles and dried under a nitrogen flow. The dry thicknesses of the surfacetethered polymer brushes were determined by Ellipsometry.
QCM measurements. All of the QCM measurements were conducted with a relative humidity controlled below 25%. The operation temperature was set at 25uC. The chips (AT cut, 5 MHz; HZDW, Hangzhou, China) were placed in a home-built QCM with control software purchased from Resonant Probes GmbH (Goslar, Germany). For the liquid-phase measurements, the QCM was operated in a flow-through mode at a speed of 80 mL min 21 . Milli-Q water (pH 5 6.2) with a resistivity of 18.2 MV cm 21 was used for the solution preparation.
AFM. The AFM images of the samples in their dry state were taken in the tapping mode (Digital Instruments, Santa Barbara). The polymer-coated QCM chips were imaged before and after washing with a dilute acid solution.
Ellipsometry. The dry film thickness was measured using an M-2000V spectroscopic ellipsometer (J. A. Woollam Co., Inc.) at angles of 65u, 70uand 75uand wavelengths from 400 nm to 800 nm. The ellipsometric data were fitted for thickness using materialspecific models (Cauchy layer model) from a vendor-supplied software, with fixed (A n , B n ) values (1.46, 0.01). Each datum was an average of three measurements.