Introduction

Though life that exists today is based on DNA genomes and protein enzymes, there is convincing evidence that hypothesizes the origin of life based on RNA (Joyce and Orgel 1999). This primordial era referred to as the “RNA world” (Gilbert 1986) hypothesizes that all modern organisms are descendants of a prebiotic self-replicating moiety, which utilized RNA as both genetic as well as catalytic material (Srivatsan 2004). This hypothesis is now an almost universally held view (Joyce 2002; Orgel 2004; Gestland et al. 2006); but could this RNA World have stood at the ultimate origin of life? This is currently still an open question. Yet while there are still substantial problems, there are now good leads for simple, spontaneous processes on the early Earth for both the synthesis of nucleotides and their concentration to oligonucleotides.

Further study on the origin of life addressed questions regarding the sources of small organic molecules that composed the first self-replicating system and the mechanism of evolution of cell-like enclosure from an abiotic supply of biomonomers (Lazcano and Miller 1999; Luisi et al. 2004). Numerous chemical and biochemical studies pertaining to the chemical evolution and origin of life, performed under simulated prebiotic conditions, have appeared owing to various pioneering work in this field (Cairns-Smith 1982; Orgel 1998; Miller 1997; Ferris 1993; Schwartz and Orgel 1995) but such aggregation of biological precursors into prebiotic biopolymers was puzzling to many. It was Bernal who proposed that adsorption of organic molecules onto mineral surface like clays would have catalytic relevance to the emergence of prototypical polymeric templates (Bernal 1951). Similarly Ferris showed that clays could catalyze activated nucleotide polymerization reactions (Ertem and Ferris 1997; Ferris et al. 1989; Ferris and Ertem 1992; Ferris and Hagan 1986; Ferris and Kamaluddin 1990). Smith studied the potential role of natural minerals on which aggregation and entrapment of nucleobases might have occurred thereby, reinforcing the potential role of natural minerals acting as scaffolds and aiding in the catalysis of reactions necessary for the evolution of early life (Smith 1998).

Like natural minerals, transition metals may have been important as catalysts for the formation of biopolymers during chemical evolution and the origin of life (Arora and Kamaluddin 2007; Arora et al. 2007; Arora and Kamaluddin 2009). Catalysts may have been important for the origins of life because they tend to direct the reaction along a few reaction pathways so that a limited array of products is obtained. Catalysts bind specific types of compounds to their surfaces and then convert them to a limited number of products. Manganese is the 10th most abundant element in the biosphere (~1014 kg of suspended and dissolved manganese found in oceans) and is second only to iron in relative terrestrial abundance of the transition metals. On average, crustal rocks contain about 0.1% by weight of Mn (Turekian and Wedepohl 1961), coordinated with oxygen, and may also exist in the bottom of seas as nodules. The existence of manganese on Mars has also been reported (Heiserman 1992). In the early stages of the Earth’s evolution, volcanoes were a major source of such elements which in turn may have been involved in adsorption and catalytic reactions of biomolecules in molecular evolution. The catalytic activity of manganese for many reactions in the presence of nucleotides, mRNA to give oligoribonucleotides is already reported (Hroacki and Hiromichi 1999). Visscher and Schwartz have concluded that the synthesis of pyrophosphate-linked oligomers from the bis-phosphoimidazolides of deoxyadenosine and deoxyguanosine, as well as from acyclic analogs of these nucleosides, is catalyzed much more effectively by Mn (II) (Visscher and Schwartz 1989).

Manganese exists in various oxidation states on Earth. The microbial oxidation of soluble Mn (II) is an important process for the formation of soluble Mn (III, IV) oxides in natural environments (Nealson et al. 1988). Abiotic oxidation of Mn (II) under aerobic condition at neutral pH proceeds only at a limited rate (Tebo et al. 1997; 2004). Microorganisms are responsible for much of the Mn-oxide as they greatly accelerate its formation rate (Nealson et al. 1988) and produce oxidation states of ≥3.4 compared to the abiotic laboratory synthesis value of 3.1 (Tebo et al. 1997).

We proposed that since the redox potential of the primitive Earth’s atmosphere was low and the atmosphere was less oxidized, manganese oxides of lower oxidation states were more important for selectively adsorbing and concentrating bio-molecules during chemical evolution. In the present work, we studied the interaction of ribose nucleotides namely, 5′-GMP, 5′-AMP, 5′-CMP and 5′-UMP with manganese oxides of varied Mn/O ratio (MnO, Mn3O4, Mn2O3 and MnO2). Ribose nucleotides have negatively charged phosphate groups, lone pair containing nitrogen atoms, and aromatic rings with π electron clouds. So interaction could have taken place with the negatively charged moieties of nucleotide and the positively charged surface of manganese oxides (Yao and Millero 1996; Daou et al. 2007). It was observed through a series of adsorption studies that manganese oxides of lower oxidation state are adsorbents for ribonucleotides and thus may have played a role in chemical evolution.

Experimental

Materials and Method

Manganese acetate (Merck), Ammonium oxalate (Merck), 5′- AMP (Sisco Research Laboratories, Mumbai, India), 5′-GMP (SRL), 5′-CMP (SRL) and 5′-UMP (SRL), were used as supplied. All other chemicals used were of analytical reagent grade.

Preparation of Metal Oxides

Manganese oxides were prepared from manganese oxalate through a reported method (Ahmad and Kandalam 2004). First, we synthesized manganese oxalate from two micro-emulsions (I and II) as described below. In the second step, the manganese oxalate particles were subjected to thermal decomposition to yield manganese oxides. MnO, Mn2O3 and Mn3O4 were obtained by thermal decomposition of manganese oxalate in vacuum (12 h), air (6 h), and nitrogen (8 h), respectively.

Micro-emulsion I is composed of cetyltrimethylammonium bromide (CTAB) as the surfactant, n-butanol as the co-surfactant, isooctane as the hydrocarbon phase and 0.1 M manganese acetate solution as the aqueous phase. Micro-emulsion II is comprised of the same constituents as above except for having 0.1 M ammonium oxalate instead of manganese acetate as the aqueous phase. The weight fractions of various constituents in these micro-emulsions are as follows: 16.76% CTAB, 13.90% n-butanol, 59.29% isooctane and 10.05% aqueous phase. These two micro-emulsions were mixed slowly and stirred overnight on a magnetic stirrer, and the resulting precipitate was separated from the apolar solvent and surfactant by centrifuging and washing it with 1:1 mixture of methanol and chloroform. The precipitate was dried in an oven at 120°C for 1 h. The precursors were heated under different conditions to obtain the oxides. The precursor on heating in air at 450°C for 6 h leads to the formation of Mn2O3. Under nitrogen at 500°C (8 h) Mn3O4 was obtained. MnO was obtained by decomposing manganese oxalate in a sealed quartz tube at a pressure ~10-5 torr. The sealed tube was heated slowly to 500°C at the rate of 50°C h-1. It was then annealed for 12 h and then cooled at the rate of 25°C h-1.

Manganese dioxide (MnO2) was prepared as follows: Manganese acetate (MnAc2·4H2O) and citric acid (C6H8O7·H2O) in a mol ratio 1:2 were dissolved in distilled water (500 ml) in a beaker. The solution was adjusted to pH ~6 by addition of ammonia. Then, the solution was heated to 80°C with a magnetic stirring and kept at this temperature for several hours until a wet gel was obtained. The wet gel was dried at 110°C in a drying box, and a dried gel was obtained. Then the dried gel was calcined at 380°C for 12 h in a muffle furnace. The calcined product was treated with a 2 M aqueous H2SO4 solution (50 ml) for 2 h at 80°C with magnetic stirring in order to increase the degree of oxidation of the product. After acid-treatment, the product was rinsed with distilled water and filtered, then dried at 105°C, and finally the brownish black manganese dioxide material was obtained (Xiuling et al. 2007).

Synthesized manganese oxides were ground and sieved through a 100 mesh sieve and were subjected to spectral (1600 series FTIR, Perkin-Elmer and XRD (PW1410/20, Philips Hall) and adsorption studies.

Spectral Studies

The adsorption of 5′-AMP, 5′-GMP, 5′-CMP and 5′-UMP on manganese oxides were studied by measuring absorbance at their characteristic λmax values of 259, 254, 278 and 262 nm, respectively. Absorbance was measured using a Shimadzu UV-1601 UV visible spectrophotometer. The infrared spectra of adsorbents, adsorbate, and adsorption adducts were recorded on KBr discs on a Perkin Elmer 1600 Series Fourier transform infrared spectrophotometer.

BET Surface Area

The BET surface area of the manganese oxides was estimated by using nitrogen adsorption isotherms on micromeritics ASAP 2010 (UK). Samples were activated at 473 K for 4 h prior to the measurement.

Adsorption Studies

The parameters like concentration range of adsorbate, particle size of adsorbent, contact time to attain adsorption equilibrium, quantity of adsorbent, pH and the temperature were fixed to study the adsorption of ribose nucleotides on manganese oxides. Adsorption of nucleotides on manganese oxides was studied with the use of varying concentration of nucleotides (5 × 10-5 M to 6 × 10-4 M). Twenty five milligrams each of the manganese oxides were added to 5 ml of different concentrations of aqueous solution of nucleotide. pH was adjusted by adding 0.01 M acid (HCl) or base (NaOH). Mixtures were shaken for about 2 min on Vortex shaker (Spinix) and kept at room temperature for 24 h. The mixture after 24 h was centrifuged on REMI R4C centrifuge for 35 min at 4,500 rpm. Absorbance of the supernatant was measured at the characteristic λmax of the nucleotide. The concentration of nucleotides after adsorption for 24 h was determined with the help of calibration curve made by plotting concentration vs. optical density (O. D.) of nucleotide standard solutions. The amount of nucleotide adsorbed was calculated using the absorbance of nucleotide before and after adsorption. The nucleotide- manganese adducts were washed with water, dried, and subjected to IR studies.

Field Emission Scanning Electron Microscope Studies

FE-SEM images of adsorbent, adsorbate and adduct were taken using Scanning Electron Microscope FEI Quanta 200F microscope operating at an accelerating voltage of 20 kV. Using FE-SEM, we imaged the surface morphology of the metal oxides, nucleotides and metal oxide-nucleotide adducts.

Transmission Electron Microscopy Studies

TEM images of metal oxides were recorded by Transmission Electron Microscope (FEI TECNAI G2 microscope operating at 200 kV.).

Results

Manganese oxalate dihydrate was characterized by powder X-ray diffraction. The data obtained is shown in (Fig. 1). This revealed good agreement with JCPDS (Joint Committee on Powder Diffraction Standards) (32–0647). TGA (Thermal Gravimetrical Analysis) curve of manganese oxalate shown in (Fig. 2) has two major changes occurring at 120°C (removal of two water molecules) and at 300°C (unhydrated manganese oxalate changing into their oxides) (Ahmad and Kandalam 2004). Synthesized manganese oxides were characterized by powder XRD, FE-SEM and TEM techniques. Powder XRD reveals that all four oxides viz. manganosite (MnO) (Fig. 3a) JCPDS (75–0257), hausmannite (Mn3O4) (Fig. 3b) JCPDS (80–0382), bixbyite (Mn2O3) (Fig. 3c) JCPDS (76–0150), and pyrolusite (MnO2) (Fig. 3d) JCPDS (81–2261), have good agreement with JCPDS XRD patterns. Table 1 depicts the specific surface areas of the synthetic manganese oxides which all had more or less similar values. TEM images of the manganese oxides show a spherical, nanotube and nanorod type structure with sizes ranging from 50 to 200 nm (Fig. 4a, b, c and d). FE-SEM images show the surface morphology of manganese oxides before and after adsorption as shown in Fig. 5a, b and c, (a): MnO, (b): 5′- GMP, (c): (MnO + 5′- GMP) adduct. Summarily, FE-SEM images confirm the adsorption of nucleotide on the MnO, Mn3O4 and Mn2O3 metal surface, while much less adsorption is observed on the MnO2 surface.

Fig. 1
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X- Ray diffraction patterns of manganese oxalate (Mn (C2O4).2H2O)

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Fig. 2
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TGA/DTA of manganese oxalate

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Fig. 3
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X- Ray diffraction patterns of manganese oxide (a) MnO, (b) Mn3O4, (c) Mn2O3 and (d) MnO2

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Table 1 Surface area of manganese oxides
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Fig. 4
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TEM Images of Manganese Oxides (a): MnO, (b): Mn3O4 (c): Mn2O3 and (d)MnO2

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Fig. 5
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FE-SEM images of nucleotides, manganese oxides and adduct (a): 5′-GMP, (b): MnO before adsorption, (c): MnO after adsorption

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Adsorption of nucleotides on manganese oxides was studied over a range of concentrations at neutral pH (~7.0). Adsorption isotherms were obtained by plotting the amount of nucleotide adsorbed, X e (mol/g), versus their equilibrium concentration, C e (mol L-1). Plots were made in Microsoft Excel. From the adsorption isotherm, the percent adsorption of the nucleotides on the manganese oxides was determined; this data is reported in (Table 2). Percent binding was calculated from absorbance values of nucleotides before and after adsorption at the point of the curve when saturation starts. The initial portion of the isotherms shows a linear relationship between the amount adsorbed and equilibrium concentration. At the higher concentration range, saturation phenomenon was observed (Fig. 6a, b, c and d). The adsorption data were fitted in Langmuir adsorption isotherm (Atkins and de Paul 2002) as given below.

Table 2 Percent binding of nucleotides on manganese oxides at neutral pH and Co = 40 × 10-5M
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Fig. 6
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Adsorption isotherm of nucleotides on manganese oxides: (a): 5′-GMP, (b): 5′-AMP, (c): 5′-CMP, (d): 5′-UMP

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$$ \frac{{Ce}}{{{X_e}}} = \frac{{Ce}}{{{X_m}}} + \frac{1}{{{X_m}}}{k_L} $$

Where C e is equilibrium concentration of nucleotide (mole/liter), X e is amount (in mol) of nucleotide adsorbed per gram weight of adsorbent, X m is amount of nucleotide adsorbed at saturation (mol/g), and k L is Langmuir adsorption constant (L/mol). A typical graph of C e /X e vs. Ce is a straight line (Fig. 7a, b, c and d). From the graph, X m and k L values were calculated and shown in Table 3. X m value indicates the maximum amount of nucleotide adsorbed on oxides for monolayer formation; kL value is related to the enthalpy of adsorption (Atkins and de Paul 2002). The infrared spectra of adenosine monophosphate, manganese oxide, and the manganese –nucleotide adduct are shown in Table 4.

Fig. 7
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Langmuir isotherm plots for nucleotides on manganese oxides: (a): 5′-GMP, (b): 5′-AMP, (c): 5′-CMP, (d): 5′-UMP

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Table 3 Langmuir constants (k L × 10-3) in L/mol for adsorption of nucleotides on manganese oxides (Xm × 108) mol/m2 values in bracket
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Table 4 Typical infrared spectral frequencies (cm-1) of ribonucleotides before and after absorption on manganese oxide
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Discussion

Adsorption results depicted in (Table 2) suggest that maximum adsorption of ribonucleotide occurred on the surface of manganosite (MnO), hausmannite (Mn3O4) and bixbyite (Mn2O3) while less adsorption was observed for pyrolusite (MnO2). This is further confirmed by the FE-SEM images (Fig. 5). The nanosized particles as evident from the TEM images of the oxides (Fig. 4) and more or less equal surface area of the oxides (Table 1) reveals that the surface of all oxides are conducive for adsorption of ribonucleotide.

The nature of interaction between the ribonucleotide and the oxide plays a vital role in understanding the mechanism underlying the higher adsorption affinity on certain oxides of manganese (Stumm et al. 1970; Parks and De Brum 1962; De Bruyn and Agar 1962; Bepuee and De Brvy 1968; Atkinson et al. 1967). In a manganese oxide/water system, Me-OH +2 and Me-O-1, hydroxyl groups on the solid surface are the most important sites for surface interactions. These groups can act as acids or bases, depending on the pH of the solution. In the present study, according to the literature (Kosmulski 2002; Kosmulski 2004; Kosmulski 2006; Healy et al. 1966), the zero points of charge PZPC of MnO, Mn2O3, Mn3O4 and MnO2 are >10, >10, 7.7 and 7.3 respectively. At neutral pH (pH < PZPC), the hydroxyl groups on the manganese oxide surface exist in the acidic form and the adsorbent surface is positively charged. Electrostatic interactions of positively charged surface of manganese oxides (MnO and other manganese oxides) with nucleotides may take place through negatively charged atoms in the phosphate groups, via the π electrons of the aromatic ring or rings, and/or via the lone pair of electrons on N and O atoms. Such explanations are verified by the infrared spectral studies conducted.

The nature of the interaction between ribonucleotides and the manganese oxides is mainly electrostatic, which was further elucidated by means of infrared absorption study which showed shifting of particular metal oxide-phosphate peaks (Daou et al. 2007; Antelo et al. 2005; Chitrakar et al. 2006; Li and Stanforth 2000). Electrostatic interaction of phosphate groups with positively charged groups and hydroxyl sites at the surface of magnetite was postulated by Daou et al. Antelo et al. similarly stressed the importance of pH and ionic strength while explaining the higher adsorption of phosphate to goethite in acidic medium. Higher adsorption of phosphate by goethite at acidic pH was also explained by Chitrakar et al. and Li and Stanforth in lines of electrostatic forces of attraction as the primary mode of interaction. The shift in wavelengths of characteristic frequencies of ribonucleotides in nucleotide-manganese adducts (Table 4) indicates interactions between the ribonucleotides and manganese. It appears that the binding may also be mediated by either N-1 or N-7 for 5′GMP and 5′AMP, and N-3 for 5′CMP and 5′UMP. The adsorption is presumably related to the involvement of N-1, N-3 and N-7 of the base residues, lone pair of nitrogen atom, and to the dissociation of the two available protons of the phosphate groups. A shift towards lower wavelength of N-H bending from 1,647 cm-1 to 1,629 cm-1 shows the involvement of the NH2 groups present on the base of 5′GMP ribonucleotides in the interaction of ribonucleotides with manganese. A strong band at 1,084 cm-1 in 5′GMP corresponding to C-O-P vibration has been shifted to 1,102 cm-1. Thus interaction likely takes place through amino and phosphate groups of ribonucleotides with positively charged manganese oxide surfaces (Arora and Kamaluddin 2009).

The presence of significant change in the typical infrared frequencies of N-H and C-O-P suggests that the adsorption of ribonucleotides is a surface phenomenon occurring on the surface of metal oxides. Ribonucleotides interact through their purine or pyrimidines residue and phosphate group with the positively charged surface of the mineral. Greater adsorption of 5′-GMP and 5′-AMP may be due to the higher number of π electrons in their aromatic ring system.

Manganese oxides with different oxidation states like 2, 3 and 4 exist (Bish and Post 1988; Burns et al. 1985; De Guzman et al. 1993; Shen et al. 1992) with decreasing average volume per Mn ion (A3) in each form of the oxide (Li and Stanforth 2000). The prebiotic condition existing in the bottom of sea was essentially reducing (Wachtershauser 1988). This is suitable for the adsorption of ribonucleotides on the surface of the Mn (II) and Mn (III) which changed slowly to the more stable state of manganese oxide, Mn (IV) with the increasingly oxidizing nature of the primitive Earth’s atmosphere (Jortner 2006). The oxic conditions prevailing at bottom of seas were clarified by thallium isotope studies carried out by Nielson (Nielsen et al. 2006) and further reported by Sleep and Bird (Sleep and Bird 2008).

At neutral pH the adsorption of 5′-GMP was the greatest of all the ribonucleotides with all of the oxides studied (Table 2). In general, MnO and Mn3O4 oxides of manganese were found to have an extremely high affinity toward ribonucleotides as the extent of surface covered \( \left( {\theta = kLCe/1 + kLCe} \right) \) was determined to be about maximum (~100%). Purine nucleotides (5′GMP and 5′AMP), which have one more ring and a lone pair of electrons at the N-7 position, are adsorbed more strongly on manganese oxides than are the pyrimidine nucleotides (5′CMP and 5′UMP).

Conclusion

All of the manganese oxides synthesized from manganese oxalate were nanosized, their surface morphology was irregular and porous, and all had more or less similarly high specific surface areas. All these observations are favorable and conducive to higher adsorption of ribose nucleotides. Our studies also revealed that nucleotides have a greater binding affinity on lower oxidation state manganese oxides (MnO and Mn3O4). Adsorption was higher for 5′-GMP and 5′-AMP compared to 5′-CMP and 5′-UMP. We conclude that electrostatic interactions are likely the main contributing factors for the observed adsorption on manganese (II) and (III) oxides. The higher electrostatic interaction between the positive metal-oxide surface and the negative part of the ribonucleotide could have been conducive for adsorption and preservation of these biomolecules as well as for promoting further polymerization reactions.