Inorganic Phosphate and Nucleotides on Silica Surface

Article pubs.acs.org/JPCC

Inorganic Phosphate and Nucleotides on Silica Surface: Condensation, Dismutation, and Phosphorylation Thomas Georgelin,†,‡ Maguy Jaber,†,‡ Thomas Onfroy,†,‡ Aaron-Albert Hargrove,†,‡ France Costa-Torro,†,‡ and Jean-Francois Lambert*,†,‡ †

CNRS, UMR 7609, Laboratoire de Réactivité de Surface, Paris, France Université Pierre et Marie Curie − UPMC Paris 6, Laboratoire de Réactivité de Surface, Paris, France

‡

S Supporting Information *

ABSTRACT: We explore the reactivity of inorganic monophosphate ions (Pi) and 5′-adenosine monophosphate (AMP), adsorbed onto amorphous silica separately or together. This question has relevance for prebiotic chemistry scenarios and, more generally, for biomedical applications involving biomolecule adsorption. XRD, TGA, and 31P and 29 Si NMR results show that inorganic phosphate ions deposited on silica condense to polyphosphates at considerably lower temperatures than in bulk KH2PO4. In the same temperature range, AMP adsorbed alone undergoes dismutation reactions, yielding adenosine, ADP, and ATP; in this case, the effect of the silica surface is not obvious. When AMP and Pi are coadsorbed on silica at high loadings (5−10%), AMP dismutation and phosphorylation by Pi both occur, allowing the formation of ADP and ATP. The latter result clearly shows the ability of silica surfaces to promote the formation of molecules generally considered as “high-energy” compounds and opens the way to further research on the effect of mineral surfaces for nucleotide synthesis and ribose stabilization.

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first strategy is to use condensing agents, that is, molecules that can release free energy upon hydrolysis; if a coupling can be achieved with phosphate condensation, the overall process may be thermodynamically favorable. Diimides and potassium cyanate,6 cyanamides, and hydrogen cyanide polymers7 were tested for phosphate polymerization and cyclization, but it was concluded that such syntheses were not sufficiently robust to have been prebiotically significant.8 A possible role of mineral surfaces in prebiotic processes has been considered at least since the work of Bernal.9 This idea has been tested for several prebiotic reactions, especially the condensation of peptide bonds from monomeric amino acids,10,11 but also for phosphorylations and phosphate polymerization. Miller et al.12 have shown that, in the presence of cyanate ions, monophosphate ions from hydroxyapatite partly condense to diphosphates. Later, Yamagata et al.13 obtained ADP from AMP + inorganic monophosphate in the same way. In recent studies, condensations were observed on mineral surfaces without condensing agents. Costanzo et al.14 have demonstrated that adenosine may be phosphorylated if it is reacted for several weeks in a formamide solution containing ground phosphate minerals. No “high-energy” bonds preexisted, yet C−O−P bonds were formed. It can be suspected that

INTRODUCTION In biochemistry, ATP and other phosphorylated molecules are often called “high-energy” compounds,1 and indeed they play a fundamental role in anabolic metabolism. They would seem to pose a particular problem for prebiotic chemistry since their formation is thermodynamically uphill in aqueous solution2 and, therefore, forbidden. This is a significant question in the frame of the “RNA world” models,3 which explain later stages of evolution, but require the previous synthesis of nucleotides and thus phosphorylations.4 However, one must remember that a molecule cannot be considered as “high-energy” in an absolute way, but only with respect to some chemically possible transformation. For instance, the formation of ATP by condensation of an inorganic phosphate to ADP, giving a phosphoric anhydride (P−O−P) link ADP + Pi = ATP + H 2O (1) is endergonic in aqueous solution in standard conditions, with a ΔrG° value of about +30 kJ·mol−1, and so are the formation of a phosphoric ester link (C−O−P), or the polymerization of inorganic (mono)phosphate (P−O−P).5 All of these reactions are condensations that proceed with the release of one water molecule. According to basic thermodynamics, the corresponding equilibria are, therefore, sensitive to water activity. Abiotic phosphate condensation to yield C−O−P (i.e., phosphorylation) and/or P−O−P has been studied for almost 50 years by some of the best researchers in prebiotic chemistry, and various scenarios have been tested. A © XXXX American Chemical Society

Received: March 10, 2013 Revised: May 1, 2013

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dx.doi.org/10.1021/jp402437p | J. Phys. Chem. C XXXX, XXX, XXX−XXX

The Journal of Physical Chemistry C

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Figure 1. (a) Intensity of the main KH2PO4 XRD peak for Pi/SiO2 samples, as a function of the amount of adsorbed Pi. (b) Intensity of the main AMP XRD peak for AMP/SiO2 samples, as a function of the amount of adsorbed AMP. The open square represents sample 5%Pi-10%AMP/SiO2.

of clay silicates, which are invoked more often as a prebiotic material.

the nonaqueous nature of the solvent is crucial: the authors effectively worked in conditions of low water activity. Monophosphate has also been condensed to diphosphate in systems containing calcium phosphate precipitates and a solution of monophosphate in water/DMSO, but only when the DMSO concentration is high enough to significantly reduce the activity of water,15 and AMP may be phosphorylated to ADP.16 Many more studies have been devoted to what we may broadly call “trans-phosphorylation” reactions, in which a highenergy P−O−P or C−O−P bond is formed, but another, preexisting one is broken.15,17−22 They cannot be examined in detail here. From the preceding overview, it is apparent that reactions on mineral surfaces have a significant potential for the formation of high-energy P−O−P and C−O−P bonds. However, there is little consensus as to the fundamental mechanisms that are involved. This is because the mineral surface may influence the condensation reactions at several levels: (i) the thermodynamic level, by interacting differently with the reagents and the products, and thus modifying the free enthalpy changes of all reactions; (ii) the kinetics level, by opening new, catalytic pathways for chemical transformations and thus speeding up some reactions; this effect is often the only one acknowledged by several authors; and (iii) in addition, if the mineral is a phosphate, it may react stoichiometrically and act as a phosphate source for phosphorylations. Quite recently, knowledgeable authors concluded that “there are no known efficient prebiotic synthesis of high-energy phosphates or phosphate esters.”23 To determine if this is true, and to disentangle the different effects taking place on mineral surfaces, we have endeavored to study the reactions of monophosphate ions alone (polymerization, with P−O−P formation) and in the presence of nucleosides and nucleotides (where both C−O−P and P−O−P formations are possible). We started from precursors adsorbed on a high surface nonphosphate mineral, namely, an amorphous silica, and compared the observed reactivity with what happens in the solid state (bulk phosphates, or bulk nucleotides). We chose amorphous silica as a model solid support because earlier studies had demonstrated that it was effective in promoting peptide bond formation;24 furthermore, the silanol groups that exist on its surface make it relevant as a model for edge groups

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MATERIALS AND METHODS Materials. Potassium dihydrogenophosphate (KH2PO4; Pi), 5′-adenosine monophosphate sodium salts (AMP), 5′-adenosine diphosphate disodium salts (ADP), and 5′-adenosine triphosphate disodium salts (ATP) were purchased from Sigma-Aldrich. Aerosil 380 was provided by Evonik. It is a nonporous fumed silica with a BET surface area of 380 m2/g. Deposition Procedure. A 10 mL portion of either Pi or AMP solution of the desired concentration was added dropwise to 400 mg of silica. The mixture obtained in this way was a fluid dispersion; it was stirred for 30 min at RT and dried overnight in an oven at 70 °C. Samples containing both Pi and AMP were prepared by stepwise deposition. The required amount of Pi was deposited in a first step as described above. The sample was dried, and in a second step, AMP was deposited by dropwise addition. All samples were stored in a desiccator containing silica gel before analysis. In all cases, the natural pH of the silica dispersions was around 4. Thermogravimetric Analyses. Thermogravimetric analysis (TGA) of the samples was carried out on a TA Instruments Waters LLC, with an SDT Q600 analyzer, using a heating rate of β = 5 °C/min under a dry air flow (100 mL/min). Weight loss values presented in this paper are normalized with respect to the weight remaining at 800 °C. This is preferable to normalizing to the initial weight, as the initial hydration of fumed silica samples is highly variable. 31 P NMR. Solid-state 31P MAS NMR spectra were recorded at room temperature with a Bruker Avance 500 spectrometer with a field of 11.0 T, equipped with a 4 mm MAS probe with a spinning rate of 10 kHz. We used a simple 1-pulse sequence with a pulse length of 1.25 μs (for a π/2 pulse of about 3.75 μs), a data acquisition time of 30 ms, and a recycle delay of 5 s. The reference used to calibrate signal phosphate is Cube-CL. FT-IR Spectrometry. IR of solid samples was recorded in the transmission mode on self-supported pellets in a cell fitted with KBr windows. Samples may have two positions in the cell, in the oven, allowing in situ thermal treatments under vacuum B



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or various atmospheres, and room-temperature recording of spectra without reexposure to air. FT-IR spectra were recorded on a Bruker-Vector 22 FT-IR spectrometer equipped with a DTGS detector, having a nominal resolution of 4 cm−1, by adding 128 scans. X-ray Powder Diffraction (XRD). X-ray powder diffraction (XRD) was carried out on the final solids with a Bruker D8 Avance diffractometer using the Cu Kα radiation (wavelength λ = 1.5404 Å). XRD patterns were recorded between 3° and 70° with a step size of 0.05°.

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RESULTS XRD. Bulk KH2PO4 exhibits sharp diffraction peaks typical of a well-crystallized material. After thermal activation at 160 °C under vacuum, no change is observed in the diffractogram (Figure SI1, Supporting Information). When Pi is deposited on Aerosil 380 at high loadings, the diffraction peaks of the KH2PO4 phase may be observed in the resulting solid sample. They are present for loadings in excess of 7% KH2PO4 by weight (with respect to the SiO2 content; Figure 1a). We can surmise that Pi is adsorbed on surface sites of silica, and, therefore, unobservable by XRD, until it reaches a saturation coverage of 7 wt %, or 0.83 Pi units per nm2. When a loading higher than this value is imposed, excess Pi precipitates as bulk KH2PO4 crystals. The bulk AMP that we used as starting material for deposition was crystalline, showing the diffraction peaks of 5′-AMP·H2O, an orthorhombic phase with a = 23.0 Å, b = 9.41 Å, and c = 6.60 Å.25 Two diffraction peaks at 16° and 16.7° 2θ could not be assigned to this phase (Figure SI2, Supporting Information). Upon thermal activation at 150 °C, significant changes were observed in the diffractogram. Some of the strongest peaks of 5′-AMP disappeared, while others were weakened and broadened and several new peaks appeared. The diffractogram of heated AMP is probably best interpreted as a superposition of the peaks of 5′-AMP, crystalline 5′-ATP, hydrate and adenosine (Figure SI3, Supporting Information). When AMP is deposited on Aerosil 380, the diffraction peaks of bulk AMP are observed for weight loadings above 7 wt % (Figure 1b). This may be interpreted in the same way as for Pi deposition: AMP is adsorbed on surface sites, with a saturation coverage corresponding to 0.28 AMP molecules per nm2, and above this loading, excess AMP precipitates as bulk phase crystallites. The value of the saturation coverage found in this way seems to be at odds with previous work by Basiuk et al.26 These authors have reported a plateau for ATP adsorption isotherms from water solution on silica corresponding to the much lower value of 0.018 molecules per nm2. The saturation coverages for each adsorbate (AMP and Pi) are found to be different according to whether the other species is coadsorbed or not. Thus, in sample 5%Pi-10%AMP/SiO2, the intensity of the main AMP peak is higher by a factor 2.6 as compared with that for 10%AMP/SiO2. Similarly, the former sample shows the peaks of bulk KH2PO4, whereas 5%Pi-10% AMP/SiO2 does not (Figure 1a,b). These observations indicate that the two species adsorb (at least in part) on the same surface sites. Thermal activation of sample 5%Pi-10%AMP causes the disappearance of the AMP·H2O peaks and a significant decrease of the KH2PO4 peaks. Only the (200) peak of the KH2PO4 phase remains clearly visible (Figure 2). Thermogravimetric Analyses. The TG of bulk KH2PO4 has been discussed several times in the literature. In fact,

Figure 2. XRD of 5%Pi-10%AMP after drying at 70 °C and after thermal activation at 160 °C under vacuum.

changes in the physical properties of this compound in the 200−340 °C temperature range were once assigned by specialists of solid-state physics to a “high-temperature phase transition”, before it was realized that this transition actually consists of a dehydration accompanied by phosphate condensation.27 Our thermogram (Figure 3, trace a) is in agreement with previously published data.27,28 It shows a complicated weight

Figure 3. DTG traces of (a) bulk KH2PO4 (intensity ×1/5; this trace is offset for better legibility), (b) the Aerosil 380 silica support, (c) 5% Pi/SiO2, and (d) 10%Pi/SiO2.

loss profile associated with an endothermic effect between 200° and 340°. The total weight loss in this region (13.3% of the initial weight) corresponds to complete dehydration to a final formula of KPO3 (theoretical weight loss, 13.2% of the initial weight). More specifically, the DTG trace in Figure 3 exhibits at least three maxima at 260, 278, and 303 °C and a shoulder at around 225 °C. The first and the last peak are clearly endothermic, as shown by DTA (data not shown). Other authors have observed three weight loss steps, better resolved than in our sample,27,28b maybe because of a better crystal C



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maxima. The presence of bulk AMP·H2O in this sample prior to TG is confirmed by XRD measurements (Figure 2), and therefore, it is not surprising to observe the signature of this phase in the thermogram. However, its thermal transformations do show a slight influence of the other components of the solid phase (i.e., the silica support and/or the inorganic phosphates). Finally, we also recorded the thermogram of a physical mixture of KH2PO4 and AMP·H2O. The DTG trace could be interpreted as a mere superposition of the two components of the mixture, without any interference between them. 31 P- NMR. Bulk Inorganic Phosphate, and Inorganic Phosphate on Silica. According to the literature data,29 monophosphate (also called orthophosphate) in solution has a pH-dependent shift. The 31P signal lies at +0.3 ppm in the predominance range of (H2PO4)− and shifts to higher δ values upon pH increase, to reach +3.0 ppm in the predominant range of (HPO4)2− (a +2.7 ppm downfield shift). We observe the signal of solid KH2PO4 at +4.1 ppm (a +3.8 ppm downfield shift with respect to (H2PO4)− in solution). Obviously, intramolecular interactions, such as H-bonding, that are present in the crystalline phase exert an influence on the chemical shift at least as important as the degree of protonation. In sample 5%Pi/SiO2 (Figure 5, spectrum b), the main signal is narrow, indicating solution-like mobility, and centered at

quality. There is now agreement that at least partial phosphate polymerization occurs, but the details of the process are still unknown, the peaks being attributed to “multiple overlapping polymerization events”. In contrast, samples 5%Pi/SiO2 and 10%Pi/SiO2 only show one detectable, endothermic weight loss event at 185 °C (Figure 3, traces c and d), a significantly lower temperature than in bulk KH2PO4. The typical DTG pattern of bulk KH2PO4 does not seem to be present, despite the fact that KH2PO4 crystals are observed by XRD in the higher loading sample. The sharp DTG peak around 185 °C may be quantified. When corrected for the blank, the corresponding weight loss is considerably lower than the theoretical value calculated for quantitative condensation of all phosphate P-OH groups. Therefore, TG data suggest that an intermediate degree of condensation, between the diphosphate and the polymetaphosphate, is reached in a single step. The TG of bulk AMP (Figure SI4, Supporting Information) shows three thermal events. The first one at 128 °C is endothermic and corresponds to a 5.20% weight loss, very close to one water molecule per AMP (calculated: −5.18%). It is attributable to the loss of the hydration water in the initial phase AMP·H2O. A second, sharp exothermic event at 188 °C corresponds to a 15.00% weight loss. It is followed by more diffuse, almost athermic events. Between 65 and 400 °C, the total weight loss is around −30%, indicating considerable pyrolytic degradation of the AMP molecule. When AMP is deposited on silica alone (10%AMP/SiO2, Figure 4, trace b), TG shows a progressive, rather featureless

Figure 5. 31P NMR of (a) bulk KH2PO4, (b) 5%Pi/SiO2, and (c) 5% Pi/SiO2 after thermal activation at 160 °C under vacuum.

+1.15 ppm, closer to the aqueous phase signal than to the bulk solid. It accounts for 53% of the total 31P intensity. Deconvolution shows that a broader signal is also present at +4.1 ppm (25.3% of total 31P). One last signal at −8.8 ppm accounts for 21.5% of total 31P. The reasons for the existence of a narrow component will be considered in the Discussion. The broader peak at +4.1 ppm corresponds to a minor amount of solid KH2PO4 crystals. Finally, the peak at −8.8 ppm indicates that a significant amount of phosphate has dimerized to diphosphate (see below; higher polymers are not present because they would give a signal in the −12 to −25 ppm range as well). This presumably happens during the drying step at 70 °C. After thermal activation of this sample at 160 °C under vacuum, one observes (Figure 5, spectrum c) three maxima at

Figure 4. DTG of (a) blank (Aerosil 380), (b) 10%AMP/SiO2, and (c) 5%Pi-10%AMP/SiO2.

weight loss. The sharp events of bulk AMP are not observed. Between 120 and 400 °C, the total blank-corrected weight loss represents 34% of the initial mass of AMP, a figure similar to that observed for bulk AMP. When AMP is deposited on silica together with Pi (Figure 4, trace c), sharp endothermic weight loss events are apparent, at 114, 190, and 204 °C. The peak positions and general appearance of the DTG trace are reminiscent of bulk AMP, except that the event around 200° now clearly has two separate D



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Table 1. 31P NMR Signals of 5%Pi/SiO2 Compared to Bulk KH2PO4a δA (ppm) − Q0 bulk Pi bulk Pi-160 °C (spectrum not shown) 5%Pi/SiO2 5%Pi/SiO2-160 °C a

+4.1 +4.1 +1.1 +4.1 +4.1 +1.5

(100%) (100%) (53.2%) (25.3%) (14.3%) (5.8%)

δB (ppm) − Q1

δC (ppm) − Q2

−8.8 (21.5%) −8.3 (45.9%)

−19.3 (33.9%)

Chemical shifts (ppm) in the three ranges defined in the text are listed together with the relative contribution of each signal to the total intensity.

+4.1 ppm (14%), −8.2 ppm (45.0%), and −19.3 ppm (33.9%), and deconvolution reveals a fourth component at about +1.5 ppm (5.8%). The component at +1.5 ppm lies in the same range as the predominant peak before thermal activation, but is now much broader and corresponds to a nonmobile species, probably dispersed monophosphate ions interacting with silica surface groups (i.e., adsorbed monophosphates). In this sample, most or all of the physisorbed water has been desorbed, and the remaining monophosphates are forced to interact with silanols. In both bulk KH2PO4 and adsorbed monophosphate, the environment of the P atom consists of four terminal oxygens. This type of environment can be denoted as O4P, or Q0. The −8.2 ppm peak falls in the chemical shift range of either the diphosphate ion or terminal phosphate groups in polyphosphates, according to solution NMR studies.29b In these compounds, the phosphorus environment is O3P(OP). It has one other P atom as a second neighbor, and this environment can be called Q1. The −19.3 ppm could correspond to the middle groups of linear polyphosphates, and/or to cyclopolyphosphates. The corresponding phosphorus environment is O2P(OP)2, with two P neighbors, that is, a Q2 phosphorus. In solution, the P nucleus of the middle group in the linear triphosphate resonates at −20.0 to −22.0 ppm depending on the protonation state.29b In the solid state,30a,b signals around −18.0 to −20.0 ppm were assigned to middle groups. Note that there is a strong dependence of the terminal groups peak position on the nature of the compensating cation. In the case of linear polyphosphates longer than the tetraphosphate, one would expect several chemically nonequivalent phosphate groups with slightly different chemical shifts. In our sample, no clear structure was observed in the broad peak at −19.3 ppm. For the sake of easy discussion in the present paper, we will distinguish three chemical shift ranges: range A (δ > 0 ppm), monomeric phosphates (Q0); range B (0 to −12 ppm), terminal phosphates in polyphosphates (Q1); and range C (−12 to −25 ppm), middle phosphates in polyphosphates (Q2) (see Table 1). In summary, the thermal activation treatment has caused phosphates in Pi/SiO2 to polymerize to a large extent, along Piads + Piads = P−Piads + H 2Ogas

conclusion is in agreement with previously mentioned TG results. The above assignments in terms of phosphate polymerization are in good agreement with the literature, but an alternative interpretation could be proposed. The appearance of signals in the B and C ranges upon thermal treatment could be interpreted instead as grafting of the monophosphates on surface silanol groups, yielding the environments O3P(OSi) (for single grafting) and O2P(OSi)2 (for digrafting). These environments may alternatively be called Q3(1 Si) and Q2(2 Si). Distinguishing them from the all-phosphorus Q1 and Q2 mentioned above is not easy because of the lack of generally accepted reference compounds. Some studies of silicasupported phosphate catalysts have observed 31P signals in ranges B and C and assigned them to Q1(1 Si) and Q2(2 Si); for example, Maki et al.32 propose that Q1(1 Si) resonates at −8.3 ppm and Q2(2 Si) at −24.4 ppm. To check this possibility, we recorded the {29Si−1H} CPMAS and 1-pulse 29Si NMR spectra of the same samples. CPMAS spectra are shown in Figure 6, and 1-pulse spectra in

Figure 6. {1H−29Si} CP-MAS spectra of (a) starting silica (Aerosil 380), (b) 5%Pi/SiO2 dried at 70 °C and rehydrated, and (c) the same, heated at 160 °C under vacuum.

(2)

Figure SI5 (Supporting Information). In both the starting silica and 5%Pi/SiO2, three signals are apparent at around −90, −100, and −110 ppm, corresponding to the Q2, Q3, and Q4 environments of silicon, that is, (OH)2Si(OSi)2, (OH)Si(OSi)3, and Si(OSi)4, respectively.33 Q2 and Q3 are surface species bearing one and two silanols, which enhances their signals when detected with proton CP, while the Q4 are bulk species. No new peaks appear upon phosphate deposition and activation. A slight variation is recorded in the relative intensity of the Q2 component after Pi deposition and drying at 70 °C (Figure SI5, Supporting Information), but further thermal

with similar reactions for longer polyphosphates. These reactions produce one water molecule per P−O−P bond formed and, therefore, become thermodynamically favorable upon drying. This is also the case for the formation of peptide bonds between adsorbed amino acids on the same support.31 Phosphate polymerization is not complete in our conditions since (i) some isolated monophosphates remain, and (ii) even when restricting the discussion to polyphosphates, terminal groups (range B in 31P NMR) represent a larger fraction than middle groups, which indicates rather short chains. This E



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treatment at 160 °C does not cause any measurable change in the 29Si spectra. Indeed, if grafting occurred, for example, on the terminal silanol of a Q3 group (OH)Si(OSi)3, this would result in its transformation to Q4(1P), that is, Si(OSi)3(OP), and according to data on similar systems, this type of silicon should resonate about 2 ppm upfield from “regular” Q4, that is, Si(OSi)4. Thus, the relative intensity in the Q3 region should decrease, with the appearance of a corresponding signal slightly downfield of Q4. This should easily be observable since, in 5% Pi/SiO2, the P/silanol molar ratio is about 0.25; in a study of phosphorus grafting from PCl5, Zheng et al. were able to clearly observe that kind of effect even for a P/silanol ratio of 0.08.34 Therefore, the negative result is a strong indication that there is no grafting in our conditions. Thus, the spectacular changes in phosphorus environments caused by heating to 150−200 °C do not affect the silicon environment, and therefore, it is unlikely that they should be caused by grafting. More will be said on this point in the general discussion. Bulk Nucleotides. Bulk AMP has a single 31P peak at −1.35 ppm (Figure SI6, spectrum a, Supporting Information and Table 2), whereas ADP has a broad signal that can be resolved

One should further note that the two nonequivalent crystallographic positions in ATP show strong splittings both in the O2P(OP)(OC) and the O2P(OP)2 ranges, which are attributable to different foldings of the phosphate chain. These effects are of the same order of magnitude as the differences in second neighbors. Finally, after thermal activation of bulk AMP, a strong signal appears in the O2P(OP)(OC) range, and a weaker one in the O2P(OP)2 range. Both are very broad. The most likely interpretation is the appearance of both ADP and ATP. The formation of these two more phosphorylated nucleotides from AMP probably implies a dismutation reaction also yielding adenine (see general discussion). AMP Adsorption and Thermal Activation. After AMP adsorption on silica and drying at 70 °C (5%AMP/Si), the main signal is observed at +0.45 ppm (73.2% of total 31P) and a second one appears with a chemical shift around −9.95 ppm (15.1%) (Figure 7, spectrum b). The latter signal could

Table 2. Chemical Shifts (ppm) for Bulk AMP, Bulk ADP, and Bulk ATP

bulk AMP bulk ADP bulk ATP

δ Q1

δ Q1 (1C)

δ Q2

δ Q2 (1C)

O3P(OP)

O3P(OC)

O2P(OP)2

O2P(OP)(OC)

−18.9 −21.85

−9.35 −9.25 −14.3

−1.35 −7.4 −7.35

into two components at −7.4 and −9.35 ppm (Figure SI6, spectrum b, Supporting Information). In comparison to solution NMR results,35 the signal at −7.4 ppm corresponds to the β position of ADP, that is, to a O3P(OP), or a Q1 environment, whereas the signal at −9.35 ppm corresponds to the α position, that is, to a O2P(OP)(OC), or a Q2 (1C) environment. The broad signal at about +1 ppm remains unexplained. Aging of the commercial ADP sample results (over several years) in the appearance of additional shoulders of the signals of hydrated ATP (see below), indicating a very slow dismutation under room conditions. Bulk ATP (Figure SI6, spectrum c, Supporting Information) has five signals at −7.35, −9.25, −14.3, −18.9, and −21.85 ppm, not including a small peak at +0.65 ppm, which may be due to a small amount of monophosphate from hydrolysis. According to the literature,36 the signal at −7.35 ppm corresponds to the γ position of ATP (in a di- or trihydrate phase), which is a Q1 environment. The two signals at −9.25 and −14.3 ppm correspond to the α phosphate or Q2 (1C) environments of two ATPs in nonequivalent crystallographic positions. In the same way, the signals at −18.9 and −21.85 ppm correspond to two nonequivalent β phosphates, that is, Q2 or O2P(OP)2. If we classify the signals observed for the three nucleotides according to the chemical environment of P nuclei, there are two main ranges: −1 to −8 ppm, Q1 environment, with O3P(OP) around −7.5 ppm and O3P(OC) strongly downfield (−1.35 ppm); and −8 to −22 ppm, Q2 environment, subdivided into O2P(OP)2 (−16 to −25 ppm) and O2P(OP)(OC) or Q2 (1C) (−8 to −16 ppm).

Figure 7. 31P NMR of (a) bulk AMP, (b) adsorbed AMP (5%AMP/ Si), and (c) adsorbed AMP (5%AMP/Si) heated at 150 °C under air flow.

encompass the contributions of O3P(OP) and O2P(OP)(OC) in ADP. A narrow component is also apparent at +0.45 ppm (8.8%), corresponding to a solution-like species, probably Pi. Thermal activation at 160 °C leads to a strong increase of the signal at about −10 ppm, which now accounts for about 40% of the total intensity, and the appearance of a smaller one at around −22 ppm. In fact, the spectrum is very similar to that of thermally activated bulk AMP (except for its lower resolution due to the lower amount of phosphorus in the sample; Figure 7, spectrum c). This is despite the fact that sample 5%AMP/ SiO2 did not originally contain bulk AMP, but only adsorbed molecules (cf. discussion of the XRD results). For comparison, we also recorded the spectra of both ADP and ATP deposited on silica (Figure SI7, Supporting Information). ATP on silica shows a very similar spectrum to that of bulk ATP, probably indicating that the predominant species consists of crystalline ATP hydrates precipitated independently of the silica surface. A sharp peak at 1.1 ppm could correspond to a minor amount of hydrolysis giving monophosphate ions. This peak is more intense for ADP on silica, which would mean a more important hydrolysis. Indeed, in this case, a broad component is also apparent around 0 ppm, F



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be contributions from both polyphosphates and polyphosphorylated adenosine (see above). The only exception is thermally activated 10%Pi-10%AMP/SiO2, where several components may be clearly identified, allowing a more precise chemical attribution. The results of spectrum deconvolutions are summarized in Table 3. In samples, where inorganic phosphate was present in low amounts (1%Pi-5%AMP/SiO2 and 1%Pi-10%AMP/SiO2), a narrow signal was observed with the same features as the one previously assigned to monophosphate in supported aqueous solution (Figure 5 and corresponding discussion), together with a broad component that could correspond to adsorbed AMP (Figure 7). A broad signal in range B indicates that P− O−P bonds have already been formed, as either ADP or diphosphate. The deconvolutions indicate that, with respect to the amounts initially introduced in the impregnation solution, intensity is mostly missing from the AMP peak; therefore, the event that has taken place upon drying is probably AMP dismutation rather than its phosphorylation by inorganic phosphates, or condensation of the latter to diphosphates. Further thermal activation at 160 °C has no clear effect for 1% Pi-5%AMP/SiO2, but results in more advanced modifications for 1%Pi-10%AMP/SiO2, where Q2 signals are now apparent in range C (i.e., P−O−P−O−P links are present). For the samples with higher Pi (5%Pi-10%AMP/SiO2 and 10%Pi-10%AMP/SiO2), the percentages of inorganic phosphates after drying are very close to the nominal values (compare columns 2 and 3 of Table 3), meaning that they are still essentially unreacted at this stage. In 5%Pi-10%AMP/SiO2, they are distributed between solution-like and bulk (precipitated KH2PO4), whereas in 10%Pi-10%AMP/SiO2, they are mostly in bulk form. In 5%Pi-10%AMP/SiO2, about one-fourth of the expected intensity is missing from the AMP signal, and a corresponding amount is present in the B range, most probably due to ADP. For these samples too, heating at 160 °C causes an increase of the signals in range B and the appearance of signals in range C. It is interesting to see at the expense of what other signals they are formed. Considering first 5%Pi-10%AMP/SiO2, the increase of the signal in the B and C ranges upon heating is +30.5% of the total intensity. The decrease in the monophosphate signals is −24.1%, and the decrease of the AMP signal is −6.3%.

and could correspond to the other expected product of the hydrolysis reaction, namely, AMP. AMP Deposition on Pi/SiO2 and Thermal Activation. In the next step, we studied samples containing both Pi and AMP on silica (Figure 8). Most samples show signals in ranges A, B, and

Figure 8. 31P NMR of 1%Pi-5%AMP/SiO2, before (a) and after (b) thermal activation at 160 °C under vacuum; 1%Pi-10%AMP/SiO2, before (c) and after (d) thermal activation in the same conditions; 5% Pi-10%AMP/SiO2, before (e) and after (f) thermal activation in the same conditions; and 10%Pi-10%AMP/SiO2, before (g) and after (h) thermal activation in the same conditions.

C. At least two and sometimes three separate signals can be identified in the deconvolution of range A. In contrast, the rather low resolution does not allow identifying separate signals in either range B or range C; in each of these ranges, there may

Table 3. Results of 31P NMR Peak Deconvolutions for the Spectra Presented in Figure 8a Pi (%)

Pi (%) initially deposited

AMP (%)

AMP (%) initially deposited

1%Pi-5%AMP/SiO2 70 °C 1%Pi-5%AMP/SiO2 160 °C 1%Pi-10%AMP/SiO2 70 °C 1%Pi-10%AMP/SiO2 160 °C 5%Pi-10%AMP/SiO2 70 °C 5%Pi-10%AMP/SiO2 160 °C 10%Pi-10%AMP/SiO2 70 °C 10%Pi-10%AMP/SiO2 160 °C

25.3 44.4

35.1 35.1

45.4 24.1

64.9 64.9

27.3 31.5

n.d. n.d.

20.1

21.2

61.3

78.8

10.8

n.d.

12.4

21.2

40.1

78.8

34.6

12.8

55.5

56.9

33.0

43.1

11.5

n.d.

31.4

56.9

26.7

43.1

22.2 (total) (PPi): 5.2

14.6

74.9

71.8

25.1

28.2

n.d.

n.d.

37.3

71.8

3.0

28.2

23.3 (total) (PPi): 6.1

38.9 (PPi):14.1

a

range B Q1 and Q2 (1C) (%)

range C Q2 in ATP and polyphosphates (%)

samples

Abbreviations: n.d. = not detected. PPi = signals attributable to polyphosphates. G



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For 10%Pi-10%AMP/SiO2, the corresponding figures are an increase in the B and C ranges, +61.2%; a decrease in the monophosphate signals, −39.6%; and a decrease of the AMP signal, −21.5%. In the latter two samples, whatever happens between 70 and 160 °C affects both the inorganic phosphate and the AMP. If we compare these two samples, we see that the amount of AMP destroyed by the 160 °C treatment is higher when there is more inorganic phosphate in the sample. Besides, in these two cases, the decrease of the Pi signal is higher than the increase of the polyphosphate (PPi) signals. It appears then that, at least in 10%Pi-10%AMP/SiO2, the two deposited molecules do not react separately, but together; namely, the AMP is phosphorylated by the inorganic phosphate. In both 5%Pi-10%AMP/SiO2 and 10%Pi-10%AMP/SiO2, we estimate that about one-half of the reacted monophosphate has phosphorylated AMP. IR Spectroscopy. Vibrational spectroscopies should be able to provide information on the formation of the phosphoric anhydride and phosphoester bonds. Raman spectroscopy could be a technique of choice to evidence these functional groups. Unfortunately, it was not applicable to our samples due to strong fluorescence. 5%AMP/SiO2 was observed in situ by FT-IR under vacuum as a function of temperature. The full spectrum is provided in the Supporting Information (Figure SI8) and is of course dominated by the bands of SiO2 (see ref 37 for a discussion of the IR bands of silica). It must be noted that, while the OH stretching band of isolated silanols is clearly apparent at 3730 cm−1, no well-defined band can be attributed to PO−H stretching (this mode was observed at 3662 cm−1 in the work by Busca et al. on H3PO4/SiO2).38 Some bands of adsorbed organic molecules are observable in the window of transparency. Their evolution is displayed in Figure 9, with difference spectra in the inset. Five vibrational bands are observed in the 1400−1750 cm−1 range. The band at 1637 cm−1 is too narrow to be assigned to adsorbed water, and its evolution upon thermal activation is not compatible with such an assignment. Besides, water is not

present in high amounts according to the general appearance of the νOH stretching region. Molecules containing the adenine cycle do have an IR band at about 1630 cm−1 (δNH2 + νCN + νCC).39 Besides, it has been reported that adenine protonation causes the disappearance of this band, which is replaced by another one at higher frequency (1670 cm−1 in the case of ATP).39c We tentatively assign the band at 1637 cm−1 to a molecule containing the unprotonated adenine ring, and the band at 1680 cm−1 to the corresponding protonated form. This means that, in the unactivated sample, AMP is distributed between its protonated and unprotonated forms, an observation that is not unexpected since the pH of the deposition solution was close to the pKa value. The main change that is observed upon thermal treatment is then a decrease of the unprotonated form, and an increase of the protonated one. This observation is compatible with the postulated dismutation reaction. One of the products of the dismutation, the (unphosphorylated) adenosine, is significantly less acidic than AMP, and therefore, the contribution of the protonated adenine cycles should increase if it is formed.

■

DISCUSSION The basic question we wanted to address was whether deposition on the silica surface promotes the formation of P−O−P and/or C−O−P bonds, eventually after moderate thermal activation. Different answers were obtained for the three systems under study and will now be recapitulated. The Pi/SiO2 System. In the 5%Pi/SiO2 sample dried at 70 °C and reequilibrated at room temperature and humidity, the predominance of a solution-like signal probably means that the majority of the phosphate ions belong to a “supported aqueous phase”. Indeed, fumed silicas retain large amounts of water at room humidity, and hydrophilic species solvated by this water may have solution-like mobility. In this sample, the amount of physisorbed water is only about 8.25% by weight (from TG), which would correspond to one monolayer of water based on the surface area of 380 m2/g. However, the adsorption of water on silica is initially not monolayer, but patchy, and NMR work on amino acid adsorption has shown that a high mobility of adsorbed molecules may be achieved in comparable conditions.40 Quantification of the narrow signal indicates that the supported solution would have a high effective concentration, namely, 4.4 mol·L−1. These high effective concentrations may not be sufficient to favor diphosphate formation. If we use the equilibrium constants provided in 41, the equilibrium diphosphate concentration should still be only in the range of one per thousand. There are two ways to explain this discrepancy. Either the diphosphates were formed during the drying stage, prior to rehydration at RT, and are not yet fully equilibrated in the presence of the supported solution, or they are much less soluble than the monophosphates, which would provide a driving force for their formation. Anyway, the fact that drying at 70 °C is sufficient to transform a fraction of Pi to pyrophosphate is coherent with observations made for Pi adsorbed on the calcium phosphate surface.16 In this work, thermodynamic measurements have confirmed that the free enthalpy of pyrophosphate formation is slightly negative after adsorption if anhydrous conditions are maintained. In TG heating conditions, 5%Pi/SiO2 undergoes a clear-cut thermal event at 180 °C that results in a strong alteration of the phosphorus environments. We have already discussed the

Figure 9. In situ FT-IR spectra of 5%AMP/SiO2 heated to 100 °C (a), 125 °C (b), and 150 °C (c) under dry N2 flow. The arrows indicate the intensity increase or decrease upon increasing treatment temperatures. H



Article

question of whether this is grafting (Si−O−P bond formation) or polymerization (P−O−P bond formation), and concluded in favor of P−O−P bonds. There are quite a few studies in the heterogeneous catalysts literature that reach divergent conclusions on the occurrence of phosphate grafting on silica.32,38,42,43 To cut a long story short, it seems that grafting on silanols is favored when fully protonated H3PO4 is deposited on silica, but not when the precursor is (H2PO4)−. If we consider only the results obtained in the present study, it is obvious that the transformation occurring between 150 and 200 °C changes the phosphorus environment without changing the silicon environment. The question remains of why, in CP-MAS spectra, there is a slight decrease of the relative Q2 contribution upon drying at 70 °C (but not upon heating to higher temperatures). Since there is no corresponding alteration in P environments, this is probably an effect on the efficiency of 1H−29Si polarization transfer, rather than on the number of Q2. Pending further investigation, this explanation would be compatible with Hbonding of the silanol groups to the phosphates, as proposed before.44 The surface−phosphate interaction mechanism may differ according to whether the precursor is (H2PO4−) or H3PO4; that is, surface acidity plays an important role. The picture that emerges is as follows. After drying at 70 °C and rehydration, the phosphates in Pi/SiO2 are present as highly concentrated monophosphates in a supported aqueous solution, together with minor KH2PO4 and diphosphates. After heating up to 160° (in vacuo) or 180−200° (in flowing air for TGA experiments), extensive polymerization takes place. Here, the key is the low activity of water during the heating stage, as was remarked by Holm and Baltscheffsky45 for a completely different phosphate polymerization scenario in hydrothermal conditions. However, polymerization does not go all the way to the metaphosphates, producing di- and linear oligophosphates instead. In summary, a catalytic effect is evident, as supported phosphates condense at a significantly lower temperature than bulk ones, but the final outcome of the reaction is different between both cases. Therefore, there is also a thermodynamic effect of the adsorption to preferentially stabilize some products of the polymerization reaction. These conclusions hold for a “subsaturation” sample where most of the phosphate ions interact with the silica surface. Therefore, it is not surprising that these ions can be activated by the surface and that condensation reactions can be catalyzed. However, according to Sabatier’s principle, a too strong interaction with the surface would result in trapping of reagents rather than catalysis. This is what would happen if the phosphate groups were grafted and immobilized on the silica surface. Therefore, some care was taken to exclude this possibility in interpretation of the results. The situation is reminiscent of peptide formation catalyzed by the same surface.31 The AMP/SiO2 System. Silica-supported AMP is chemically modified by moderate thermal activation, but the same is already true for bulk AMP. 31P MAS NMR and XRD show that bulk AMP heated at 160 °C is partly transformed to ADP and ATP. This is most likely due to dismutation reactions formally written as 2AMP = ADP + Adenosine

respectively. From the enthalpic point of view, these reactions do not involve either hydrolysis (with consumption of one water molecule) or condensation (with water elimination); rather, C−O−P bonds are exchanged for P−O−P bonds. From the entropic point of view, this “scrambling” of phosphate groups should be favored. In fact, in aqueous solution, it can be calculated that reaction 3′ is slightly favored: 2AMPaq = ADPaq + adenosineaq

with ΔrG° = −20 kJ/mol.46 The corresponding thermodynamic data for the solid phase are lacking; in contrast to condensation reactions, such as phosphate polymerization, the effect of working in dry conditions on the reaction equilibrium is not straightforwardly assessed. It is not surprising altogether that dismutation should occur in the bulk, but we cannot tell if the reaction reaches equilibrium. When supported on silica (in conditions where no bulk crystallites are present), AMP dismutation reactions occur as well. In fact, from 31P NMR, the results of thermally activating bulk AMP on the one hand, and well-dispersed AMP/SiO2 on the other hand, are very similar. It is not impossible that these reactions are catalyzed by the silica surface, since a small amount of ADP appears to form already at 70 °C. Since they are already rather facile in the bulk, establishing the limits of the catalytic effect would require more precise work. One of the necessary products of the dismutation reaction, adenosine, cannot of course be evidenced by 31P NMR, but its formation has been confirmed by other techniques, namely, in situ IR and XRD. Overall, the occurrence of dismutation is, therefore, firmly established. The Pi-AMP/SiO2 System. XRD shows that inorganic phosphates and AMP are in competition for surface adsorption sites during the initial deposition and drying steps. For each component, the surface saturates at lower loadings when the other component is present (even though they have been introduced in a two-step procedure). Thermal activation of mixed Pi-AMP/SiO2 systems leads to rather variable effects depending on the proportion of each constituent. At low phosphate loadings, AMP dismutation is still observed, but it is probably inhibited to a degree as compared with phosphate-free AMP/SiO2. This would be understandable if the dismutation of supported AMP involved interaction with silica surface groups, a possibility that we left open in the discussion of the AMP/SiO2 systems. It would then be understandable that the presence of phosphates competing for the adsorption sites could deactivate these groups by preventing them from interacting with AMP. At the highest phosphate loadings (i.e., in 5%Pi-10%AMP/ SiO2), in opposition, thermal reactivity seems to be enhanced. Both linear polyphosphates and polyphosphorylated nucleotides (ADP and ATP) are formed by thermal activation. Quantification of the signals indicates that a net phosphorylation of AMP by inorganic phosphates is achieved. This conclusion is rather unescapable based on our data, but it raises some puzzling questions: − Phosphorylation is observed only at high inorganic phosphate loadings, when bulk KH2PO4 is present in the samples. It would seem logical to conclude that bulk KH2PO4 is involved in the phosphorylation reaction. But then, where does phosphorylation occur? Is it a solidstate reaction at the boundary between KH2PO4 and

(3)

and 3AMP = ATP + 2Adenosine

(3′)

(4) I



Article

energy in standard, aqueous phase biochemistry. Of course, we do not advocate a simplistic model in which full-fledged ATP molecules emerged in a single step on mineral surfaces, but this type of reactivity has been invoked as a constituent in more sophisticated scenarios.51 In order for them to rest on a firm basis, it is important to carry out such studies of simple model systems.

silica particles containing adsorbed AMP or at the boundary between KH2PO4 and AMP bulk crystallites? The latter possibility does not seem to be favored according to our results on physical mixtures of the two solids, where crystallites of each species appear to react independently of each other. − What is the mechanism for AMP phosphorylation to ADP (and ATP)? Does the phosphate group on the AMP molecule condense with an inorganic phosphate? Or do the AMP molecules first dismutate into adenosine and ADP (ATP), the adenosine reacting in turn with inorganic phosphates? These questions will be the object of future investigations. At the present time, one may offer a temporary answer to the question that motivated our investigations: phosphorylation of biomolecules by monophosphate ions can indeed occur in scenarios involving mineral surfaces in the dry state, but the phenomena that are taking place are more complicated than one would have expected. A similarity with biochemistry can be underlined. Extensive investigation of ATP synthase, including the Nobel-prize winning works of Boyer and Walker,47 has shown that this enzyme works by building an environment (the “tight” catalytic site) where water elimination from ADP + Pi is thermodynamically favored and ATP actually becomes the “low-energy” compound. Later on, free enthalpy is used by the enzyme to expel the ATP molecule into the aqueous solution, an environment where it is a “high-energy” molecule. In our scenario, drying the mineral surface with adsorbed ADP also temporarily provides an environment where ATP is the “lowenergy” molecule, allowing its synthesis by condensation. Later on, it may be released to the solution by desorption. Thus, if we speculate that a take-over occurred for ATP synthesis during the early steps of prebiotic evolution, from mineral surfaces to the world of biochemistry, some basic operational principles were preserved during this transition.

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■

ASSOCIATED CONTENT

S Supporting Information *

X-ray diffractograms, TG traces, and NMR spectra as mentioned in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Telephone: (+) 3341275519. Fax: (+)33144276033. Address: Laboratoire de Réactivité de Surface, Case courrier 178, UPMC, 3 Rue Galilée, 94200 Ivry-sur-Seine, France. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS A.-A.H. acknowledges a grant from the REU program between the University of Florida and UPMC − Paris. REFERENCES

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CONCLUSION

Silica surfaces are able to catalyze monophosphate condensation to polyphosphates upon moderate heating. Condensation reactions of inorganic anions in the presence of mineral surfaces have been observed in several different systems. For instance, condensation of (mono)silicates to polysilicates was observed on ferrihydrite by Swedlund et al.,48 and recently, the same reactivity was evidenced and studied in depth on titania surfaces.49 In those studies, polymerization already occurs in the presence of an aqueous phase at room temperature, which does not seem to be the case on silica. Indeed, it must be underlined that different mineral surfaces may exhibit very diverse reactivities; in particular, such minerals as iron oxyhydroxides50 or clays should be studied in the same conditions. Probably more original is the demonstration that inorganic monophosphates also have the ability to directly phosphorylate AMP in moderate conditions. The reaction mechanism has not yet been unraveled and probably involves, as one of its steps, AMP dismutation, which has been observed independently in the absence of phosphates. Even though the specifics are not fully understood, it appears that silica surfaces are perfectly able, upon moderate thermal activation, to induce the formation of both P−O−P and C−O− P bonds, which are involved in the storage and release of free J



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Inorganic Phosphate and Nucleotides on Silica Surface

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