Synthesis of Peptides from Glycine on Anatases with Different Crystal Facets

Abstract

Semiconductor minerals are widely present on the surface of Earth, but their roles in the process of peptide formation from amino acids are less studied, especially the role of different crystal facets in the origin of life. In this research, High Performance Liquid Chromatography (HPLC), thermogravimetric analysis (TA/DTA), Nuclear Magnetic Resonance (NMR) and simulation calculations were used to study the condensation of glycine on the surface of anatase with (001) crystal facets and ordinary anatase as well as the reaction mechanism. Combined with TA/DTA and heating experiments (80–130 °C), it was found that anatase with (001) crystal facets and ordinary anatase could both catalyze the condensation of glycine to form corresponding oligopeptides (mainly DKP, Gly₂ and Gly₃). Anatase with (001) crystal facets shows better catalytic effect, which can reduce the condensation temperature of glycine to 90 °C. With the increase in temperature, the condensation efficiency of anatase with (001) crystal facets for Gly₂ is relatively higher, and the maximum yield is about 0.20 mg/m². The condensation efficiency of ordinary anatase for Gly₃ is relatively higher, and the maximum yield is about 0.28 mg/m². The results of FTIR and simulation calculation show that the electron density of the carboxyl group changes after glycine is adsorbed on the surface of anatase, which is easily subject to the nucleophilic attack of amino groups to promote the condensation reaction. These results can provide reference for the research of condensation of small biomolecules on semiconductor mineral surfaces in the origin of life.

1. Introduction

The origin and evolution of early life on Earth has always been one of the major scientific frontier issues [1]. Although a large number of studies have been carried out on the origin of life, and the results are constantly emerging, the origin of life has not been completely solved in the field of life sciences. Among the many events that led to the emergence of life, the formation of the first biopolymer by condensation of monomeric molecules (for example, by condensation of amino acids to form peptides) is a crucial process, and the polymerization of amino acids (AAs) to peptides has attracted significant attention for a long time due to its importance in various fields [2]. Bernal [3] proposed that the mineral surface may play an important role in this step because amino acids can establish strong interactions with the mineral surface, be concentrated on it and even be activated for condensation reactions, eventually leading to the formation of the first oligopeptide. Understanding the interaction between amino acids and mineral surfaces is very important for better understanding the surface interaction of biological components and the role of mineral surfaces in the origin of life on Earth [4,5].

Since Bernal [6] and Goldschmidt [7] proposed the conclusion that “minerals are an important material basis in the origin of life”, a large number of scholars have put forward general principles and assumptions in terms of mineral-assisted biosynthesis, and clay minerals are the most common in the specific mineral groups that are cited [3]. Many scholars have used the interaction between clay minerals and amino acids to explore the evolution of early biological macromolecules, but the mechanism is still not fully explained [8]. Hu et al. [3] used orthoclase to study the reaction mechanism of amino acid condensation to generate peptide bonds based on density functional theory, analyzed the role of metal ions, surface of orthoclase and amino acid side chains, and concluded that metal ions could catalyze the polymerization of amino acid molecules to generate peptide bonds, amino acid side chains can affect the formation of peptide bonds and the catalytic properties of different ionic systems are significantly different. Zhong et al. [9] found that glycine on anatase surfaces can undergo dehydration condensation reactions in the range of 80–200 °C and that oligopeptides such as DKP, Gly₂ and Gly₃ are formed in the range of 80–120 °C, among which DKP is the main product of condensation under heating conditions. In the paper of Wu [10], the salt-induced peptide formation (SIPF) reaction in the amino acid polymerization simulation system was discovered in 1988 [11] based on the theoretical study of electrolyte solutions and complexes. This reaction provides a new perspective for the formation of peptide bonds in the primitive earth environment [10]. Chaudhuri et al. [12] analyzed the peptide formation of various amino acids and related mechanisms using first-principles calculations. A recent study by Chien and Yu [13] showed that the effective way to form amino acid-rich oligomers is ester-mediated peptide formation. Martra et al. [14] demonstrated by in situ spectroscopy that continuous addition of vapor-phase glycine monomers on the surface of silica (and TiO₂) will result in the formation of oligopeptides of up to 16 units. Combined with spectral measurements and quantum chemical simulations, Si–OH on SiO₂ surfaces can directly or indirectly participate in peptide formation as a key site; in addition, some scholars have pointed out that Lewis and Brønsted sites on the mineral surface play a catalytic role, but the presence of proton transfer promoter molecules (such as H₂O, NH₃ and Gly) provides a more significant kinetic energy barrier reduction [15,16]. El Samrout et al. [2] used thermogravimetric analysis (TA/DTA), XRD and other methods to further characterize the continuous steps of poly-Gly formation on the surface of silica and also studied the mechanism of polypeptide chain growth and secondary structure formation in the vapor deposition of glycine on the surface of silica [17].

Photoelectrons generated by semiconductor minerals under various conditions play various roles in the evolution of life on earth, such as synthesizing substances, protecting cells and providing energy [18]. Nowadays, the study of crystal facets is a hot topic in the field of semiconductor minerals. Amino acids, as the constituent units of proteins, contain multiple functional groups such as –COOH, –NH₂, –SH, etc. These functional groups can selectively bind to different crystal facets, thus affecting the exposure of crystal facets [19]. Crystal facets exposure can better increase the catalytic effect of semiconductor minerals. Anatases are widely studied as models of semiconductor minerals. In general, the ordinary anatase is composed of a (101) crystal face with a higher surface energy, while a (001) crystal face with a higher reactivity is almost non-existent due to its low surface energy. Liu et al. [8] studied the adsorption and mechanism of glycine on anatase with exposed (001) and (101) facets. Pantaleone et al. [20] studied the interaction of 11 amino acids with the TiO₂ (101) anatase surface by means of PBE-D2* periodic simulations, both from a static and dynamic point of view. However, most studies have focused on the adsorption of amino acids on (110) crystal facets anatases [21,22,23,24], the mechanism and rate of amino acid polymerization and the self-assembly of the resulting peptides [17], and there are few studies on the comparative analysis of amino acids on the mineral surface with different crystal facets. In addition, the conversion process of biological small molecules studied in a large number of studies is basically carried out under dry, wet, dry–wet circulation and other conditions. In addition to direct heat energy, the energy intake ignores the role of a large amount of light energy. So, in this research, under the condition of simulating the existence of a large amount of thermal energy and light energy in the early earth environment, glycine small molecules were used as the research object to study the condensation and synthesis of peptides on the surface of anatases with different crystal facets under ordinary equilibrium conditions. The role of semiconductor minerals in the formation of early biological macromolecules was discussed, and the catalytic condensation efficiency of two anatases were analyzed.

2. Materials and Methods

2.1. Materials

Anatase with (001) crystal facets samples were provided from Environmental Microbiology Lab of Southwest University of Science and Technology, and its synthesis and characterization method were carried out according to Liu et al. [8]. The ordinary anatase samples were purchased from Shijiazhuang Yunpo Chemical Co., Ltd. (Shijiazhuang, China) Gly₂, Gly₃ and DKP were purchased from Kelun Pharmaceutical (Chengdu, China), Beijing Aoboxing (Beijing, China) and Kelon Chemical (Chengdu, China), respectively.

2.2. Thermal Analysis Experiments

An amount of 25 mg of anatase with (001) crystal facets or ordinary anatases was put into a 5 mL centrifuge tube, 3 mL of prepared 0.09 mol/L glycine solution was added and the mixture was treated for 24 h in shaking incubator (25 °C, 150 rpm). The drying solution was taken out and placed in the oven at about 50 °C. After drying, the samples were taken out and subjected to thermogravimetric experiments in nitrogen and air atmospheres, respectively. The flow rate was 100 mL/min, and the sample dosage was 16–18 mg. Before the experiment, the carrier gas was purged to remove some physical water. The TG spectra were analyzed by TA instrument universal Analysis 2000 software.

The thermal analysis experiments of two types of anatases were carried out by using the SDT Q600 synchronous thermal analysis instrument from American TA instruments, and the TG spectra were analyzed by TA instrument universal Analysis 2000 software.

2.3. Heating Experiment of Glycine on the Surface of Anatases

The anatase with (001) crystal facets or ordinary anatase (25 mg) was put into a 5 mL centrifuge tube, 3 mL of prepared 0.09 mol/L glycine solution was added to the centrifuge tube, and the mixture was treated for 24 h in a shaking incubator (25 °C, 150 rpm). The samples were taken out and dried at about 50 °C in the oven. After drying, they were taken out and heated at 80 °C, 90 °C, 100 °C, 110 °C, 120 °C and 130 °C for 2 h, respectively. The glycine adsorbed on the surface and its peptide products were eluted by adding 3 mL 0.01 mol/L CaCl₂ solution to the centrifuge tube, and then the supernatant was centrifuged and filtered with 0.45 μm filter membrane for HPLC determination. Three groups of parallel experiments were set up.

2.4. Illumination Experiment for Condensation of Glycine on the Surface of Anatases

The pre-treatment process is the same as above. The xenon was placed in a xenon lamp climate-resistant test chamber for illumination (60 °C, 670 W/m²), and the dry–wet cycle was maintained during illumination. The samples were taken at 24 h, 48 h, 72 h, 96 h, 120 h and 144 h for HPLC determination.

2.5. HPLC Analysis

Preparation of mobile phase: 0.941 g of sodium hexane sulfonate was dissolved in a certain volume of ultrapure water, and the pH was adjusted to 2.5 with H₃PO₄ to 500 mL. The mobile phase was filtered with a 0.45 μm filter membrane, and ultrasonic exhaust was performed before use. Test conditions: flow rate of 0.6 mL/min, sodium hexane sulfonate:acetonitrile = 95:5, temperature of 30 °C, absorption wavelength of 200 nm and injection volume of 50 μL. The production of oligopeptides was determined by standard curve method.

2.6. H-NMR Analysis

Hydrogen NMR spectroscopy analysis was carried out on an NMR spectrometer, and a pre-saturation technology composed of “noesyprld” pulse sequences was adopted to achieve complete inhibition of the water peak signal [25]. The oligopeptide products obtained by heating glycine on two types of anatases at 130 °C were collected and concentrated. After the liquid products were dried, the solid was dissolved in deuterium water and inhaled into the dried nuclear magnetic tube for testing. Test conditions: the measured frequency was 600 MHz, scanned 64 times, PW = 30 °C and RD = 5.0 s.

The ULTRASHIELD 600 PLUS nuclear magnetic resonance spectrometer from Bruker was used to analyze the condensation products of glycine on two types of anatases.

2.7. FTIR Analysis

The product was mixed with 100 mg dry potassium bromide, ground and pressed into pieces for FTIR determination. The scanning range was 400–4000 cm⁻¹.

An iCE3000 Fourier Transform Infrared Spectrometer (FTIR Spectrometer) from Perkin Elmer Co., Ltd. (Shanghai, China). was used for FTIR analysis.

2.8. Simulated Caculation

The simulation calculation was carried out according to Liu et al. [8].

3. Results and Discussion

3.1. Effect of Temperature on the Condensation of Glycine on the Surface of Anatases with Different Crystal Facets

Figure 1 shows the TA/DTA curves and DSC curves of blank glycine in nitrogen and air. In the absence of anatase, glycine undergoes three stages of weightlessness during continuous heating in an air atmosphere, ranging from around 200−300 °C and around 550 °C (Figure 1a). In the nitrogen atmosphere, the weightlessness peak exists between 200–300 °C, while the weightlessness peak around 550 °C disappears. In the study of the pyrolysis process of amino acids, Hao et al. [26] found that the weight loss of glycine can be divided into several stages, in which the pyrolysis between 200 °C and 300 °C can produce the intermediate product DKP, which gradually cleaves into CO, HCN and other substances. Based on this, we can infer that there are two weightlessness peaks at 200–300 °C in Figure 1a, and the first one is the weightlessness caused by the dehydration condensation of glycine itself to DKP, and the second one is the weightlessness caused by the decomposition of glycine into other small molecules (H₂O, CO, NH₃, etc.). The disappearance of the peak at about 550 °C in nitrogen atmosphere indicates that this stage is the oxidative decomposition process of the residual material after the decomposition of glycine. Figure 1b shows the endothermic and exothermic process of the substance change in each weightlessness stage of the reaction. It can be seen from the figure that the condensation and decomposition of glycine is an exothermic process at 200–300 °C, and the weightlessness at about 550 °C is an endothermic process.

Figure 2 shows the TA/DTA curves (a,b) and DSC curves (c,d) of two types of anatases adsorbing glycine in air and nitrogen atmosphere, respectively. There are four and three weightlessness stages in air atmosphere (Figure 2c) and nitrogen atmosphere (Figure 2d), respectively. Compared with the weightlessness analysis of blank glycine, it can be concluded that the first weightlessness stage of glycine adsorbed on two types of anatases is the same in the process of heating glycine from 40 °C to 600 °C, whether in nitrogen or in air atmosphere. Therefore, it is speculated that the weightlessness process is the dehydration condensation of glycine on the surface of anatases, which is an exothermic process. This result is similar to the results of Meng et al. [27] and corresponds to the small weightlessness peak of blank glycine between 200–300 °C in Figure 1. It is inferred that a large amount of glycine oligopeptides may be formed, and compared with the blank glycine, it is observed from the TA/DTA curve that the condensation temperature was advanced to about 120 °C in the presence of anatases, indicating that anatases had the effect of reducing the dehydration condensation temperature of glycine. In the presence of semiconductor mineral anatases, the dehydration condensation of glycine small molecules is more inclined to the formation of glycine-related oligopeptides, while the blank glycine is mainly the formation of other inorganic small molecules at high temperature. In the air atmosphere, comparing the initial temperature of dehydration condensation of glycine on two types of anatases, the temperature required for glycine condensation on the surface of anatase with (001) crystal facets is lower than that of ordinary anatases, indicating that the reaction on anatase with (001) crystal facets is easier than that on ordinary anatases.

3.2. Glycine Condensation Products Catalyzed on the Surface of Anatases with Different Crystal Facets

Figure 3a shows the HPLC spectra of glycine cyclic dipeptide (DKP), glycine (Gly), glycine linear dipeptide (Gly₂) and glycine tripeptide (Gly₃). Figure 3b shows the HPLC spectra of blank glycine was heated at 130 °C. The result shows that small molecules of glycine cannot be condensed to form oligopeptides at the highest temperature of 130 °C in the absence of semiconductor mineral anatases.

Figure 4a–f show the HPLC spectra of heating product after small molecules of glycine are adsorbed on two types of anatase and heated at 80 °C, 90 °C, 100 °C, 110 °C, 120 °C and 130 °C, respectively. It can be seen in Figure 4 that only a small amount of glycine dipeptide molecules are formed when at 80 °C. When the temperature rose to 90 °C, small molecules DKP and Gly₃ began to appear in the products for the (001) crystal facets group, while the amount of production was relatively small, but only Gly₂ was produced for the ordinary anatase group [28]. When heating at higher temperatures, the content of several small-molecule substances generated on the two anatases gradually increased. When the temperature reached 130 °C, the peak of small-molecule Gly₃ began to split, and it seemed that the peak of a new substance appeared. Through the analysis of the above phenomena, it can be concluded that both anatases can catalyze the dehydration and condensation of glycine small molecules adsorbed on the surface to form corresponding short peptide small molecules under the heating conditions. In addition, within a certain temperature range, with the increase in energy supply, the content of several small peptides formed by condensation also increases [29]. When the temperature reached 130 °C, the Gly₃ peak began to split, possibly because longer peptide chains were formed.

Table 1 shows the standard curve of corresponding oligopeptides measured by HPLC; the R² values are all greater than 0.99, which have a good linear relationship and can be used to calculate the content of products. Figure 5a–c show the changes in the products under different temperature conditions. In this research, only Gly₂ was generated on the surface of two types of anatases at 80 °C. As the temperature increased to 90 °C, a small amount of DKP and Gly₃ appeared on anatases with (001) crystal facets, while only Gly₂ existed under the action of ordinary anatases. After 100 °C, under the action of two types of anatases, glycine condensation products DKP, Gly₂ and Gly₃ began to be produced in large quantities. The maximum amount of Gly₂ generated under the catalysis can reach about 0.12 mg/m²; Gly₃ is about 0.13 mg/m².

The above results showed that the two anatases had different condensation efficiency for related oligopeptides in the condensation process of glycine. Anatase with (001) crystal facets had better condensation efficiency on Gly₂, and ordinary anatase had better condensation efficiency for Gly₃. Compared with ordinary anatases, anatases with (001) crystal facets show better catalytic effect at relatively low temperature, which is consistent with the results of thermal analysis.

3.3. Analysis of Glycine Condensation Products Catalyzed on the Surface of Anatases with Different Crystal Facets under Light Irradiation

Figure 6 shows the production of related peptides after 24 h, 48 h, 72 h, 96 h, 120 h and 144 h for the anatase with (001) crystal facets group at 60 °C under light and dark conditions. On the surface of anatase with (001) crystal facets, with the extension of illumination time, the condensation products of glycine small molecule are DKP, Gly₃ and Gly₂, successively, and only Gly₂ and Gly₃ are generated under dark conditions.

Figure 7 shows the peptides synthesized from glycine on the surface of ordinary anatase under the same experimental conditions, and trace DKP is formed under dark conditions, which is consistent with the results on anatase with (001) crystal facets. It shows that under the condition of light as energy, both anatases have the effect of catalyzing the condensation of glycine to form oligopeptides.

Figure 8 indicates the production amount of related oligopeptides catalyzed by two types of anatase under light and dark conditions measured by HPLC. Figure 8a shows the formation of DKP. Under dark conditions, no DKP was formed on anatase with (001) crystal facets, and only a small amount of DKP was formed under the action of ordinary anatase. With the extension of dark treatment time, the content did not change, which may be formed during the pre-treatment process. Under the illumination condition, the content of DKP increased significantly, which formed from catalytic condensation of two types of anatases. Under the action of ordinary anatase, it reached the maximum after 72 h, about 0.12 mg/m². Under the action of anatase with (001) crystal facets, it reached the maximum after 120 h, about 0.10 mg/m². Under the action of two types of anatases, the production of DKP decreased after reaching the maximum. Figure 8b shows the amount of Gly₂ produced under different light and dark conditions. The content of Gly₂ increased with the extension of treatment time, and the amount of Gly₂ under light conditions was significantly higher than that under dark conditions. After reaching the maximum value, the Gly₂ under light conditions decreased significantly, and the decrease in dark conditions was not obvious.

Figure 8c shows the production of Gly₃, which is similar to that of DKP. The production of Gly₃ is lower under dark conditions and does not change significantly with time. The amount of Gly₃ production reached a maximum of about 0.04 mg/m² after 120 h of illumination under the catalysis of two anatases, and the amount of light production also decreased after reaching the maximum. In summary, under light conditions, both anatases have the effect of catalyzing the condensation of glycine to form corresponding oligopeptides. Compared with heating, using light as an energy source, the catalytic condensation yield is lower; under light conditions, after the production of each oligopeptide reached the maximum, the phenomenon of reduced production occurred when the irradiation continued. As model semiconductor minerals, anatases have typical photocatalytic properties. Electrons and holes can be generated under irradiation conditions, which may produce reduction and oxidation effects, respectively. The dehydration and condensation of glycine catalyzed by anatases is not a redox reaction. Combined with the analysis of the amount of oligopeptides products under heating and irradiation conditions, it can be deduced that light and heat are the same energies in the catalytic condensation of glycine catalyzed by anatases, which can provide energy and reduce the activation energy. Karina et al. [30] showed that at about 70 °C, the semiconductor mineral FeS₂ had a certain degradation effect on glycine-related oligopeptides. The decrease in oligopeptide production in this research is speculated to be related to this factor.

3.4. Identification of Peptides Condensed from Glycine though NMR

Figure 9 shows the NMR analysis of glycine condensation products on the surface of two types of anatases. Since the concentration of the product after the condensation reaction at 130 °C is still low, the spectrum was locally amplified. Figure 9 show the hydrogen spectrum of the product after the reaction of glycine anatase with (001) crystal facets and ordinary anatase, respectively.The more obvious chemical shifts in the spectrum are 3.51 ppm, 3.63 ppm and 3.75 ppm, and the peaks are attributed to the hydrogen in glycine α-CH₂. Because of the N–H and O–H in the NMR belong to active hydrogen, proton substitution is easy to occur in hydrogen spectroscopy with D₂O as solvent [31], which is not easy to be detected.

Through the local amplification diagram 1–3 in Figure 9, we can see that there are many low concentration peaks. The chemical shift of the hydrogen absorption peak on the –NH₂ group is between 1 ppm–3 ppm, there is proton exchange of active hydrogen, and the peak shape of the H peak on the –NH₂ group presents a wide peak or double and triple splitting peak state [32]; in addition, a broad peak appeared between 8.4 ppm and 8.5 ppm. According to the literature analysis, this peak may belong to the oligopeptide peptide bond –NHCO–hydrogen [33,34]. A large number of peaks with chemical shifts between 3.8 ppm and 4.2 ppm can be seen in the local amplification diagram 2 of Figure 9, which are basically attributed to the hydrogen of –CH₂– at different positions in the oligopeptide chain [35,36]. In summary, the appearance of amide group H and a large number of –CH₂– group H except glycine in the NMR hydrogen spectrum of the condensation product further indicates that both anatases have the ability to catalyze the condensation of glycine to peptides under certain energy conditions. The peak shape and chemical shift of the partial position peak of H in the –CH₂– of the glycine condensation products on the two anatases surfaces are different, which may be caused by the different concentration of the condensation products.

3.5. Analysis of Peptides Condensed from Glycine though FTIR

Table 2 shows the affiliation and displacement of pure glycine-related groups measured under the same conditions: the medium–strong peak at 3171.3 cm⁻¹ is the anti-symmetric stretching vibration peak of NH₃⁺, its symmetric stretching vibration is at 1521.7 cm⁻¹, and the CH₂ group has an anti-symmetric stretching vibration with small absorption at 2917.3 cm⁻¹. The strong sharp absorption peak at 1613.3cm⁻¹ is the antisymmetric stretching vibration of COO– [37]. Figure 10 shows FTIR spectra of glycine condensation products on the surface of two types of anatases under different temperature conditions. The strong and wide absorption peak at 3400 cm⁻¹ is the asymmetric stretching vibration of glycine N–H bonds in the sample [38]; however, the absorption peak of the NH₃⁺ group moves towards the direction of high wavenumber after heating; that is, the blue shift phenomenon is generated. The strong absorption peak of antisymmetric stretching vibration of the COO– group at 1613.3 cm⁻¹ also moves to higher wavenumber direction after heating on two types of anatases, while the bending vibration generated by the NH₃⁺ group at 1521.7 cm⁻¹ moves to the direction of low wavenumber and generates a red shift. The absorption peak of glycine at 2917.2 cm⁻¹ and the absorption peak at 1333.1 cm⁻¹ are less affected by heating. It is the fingerprint region of the glycine molecule in the range of wavenumber from 400 cm⁻¹ to 800 cm⁻¹, and the characteristic group Ti–O of anatase is also in this region. As shown in Figure 10, the strong and broad absorption peaks generated by the characteristic stretching vibration of anatases at around 400 cm⁻¹–700 cm⁻¹ cover up some of the glycine absorption peaks. Meanwhile, only Ti–O–Ti groups can be absorbed in the two characteristic absorption peaks of ordinary anatases in this range after heating [39]. The Ti–O–Ti groups of the two anatases produced few changes during heating, which may be affected by the glycine absorption. In conclusion, two types of anatases catalyze the condensation of glycine to related oligopeptides, which mainly affect the glycine amino and carboxyl groups. Under the catalysis of anatases, glycine can reduce the activation energy of the reaction so that the peptide’s synthesis from glycine can occur.

3.6. Mechanism Analysis of Glycine Condensation on the Surface of Anatases with Different Crystal Facets through Simulated Calculation

Figure 11 shows the variation of electron cloud density of glycine absorbed on two types of anatases. Glycine (zamphoteric ion, dissociated molecules M1 and M2) forms a bridging structure with two Ti atoms connected by oxygen on the surface of two anatases. The blue area near the Ti atom on the surface of the carboxyl carbon atom and anatases represents the loss of electrons, while the red area near the carboxyl oxygen atom represents the gain of electrons. It can be seen from Figure 11 that the electron cloud density of Ti atoms connected with the carboxyl terminal decreases after glycine is adsorbed to (001) crystal facets and (101) crystal facets, while the electron cloud density of the oxygen atom at the carboxyl terminal increases and the electron cloud density of carboxyl carbon atoms decreases. The simulation results correspond to the vibration changes of the glycine carboxyl terminal and Ti–O under adsorption and heating conditions measured through FTIR. The change in electron cloud density of the carboxyl terminal caused by adsorption results in vulnerability to nucleophilic attack by other glycine amino acids under certain energy conditions, and nucleophilic reactions occur [40,41]. Specifically, as shown in Figure 12, the two anatases activate glycine through adsorption and other processes, reduce the activation energy of the condensation reaction, and finally promote the formation of peptide bonds and the extension of peptide chains [42]. In addition, the comparison of electron cloud density changes adsorbed by glycine on the surface of the two types of anatases shows that the adsorption sites of glycine on the (001) crystal facets and (101) crystal facets are different and that the electron cloud density changes are different after adsorption, which suggests that anatase with (001) crystal facets has more catalytic activity [43]. The dominant crystal facets of anatase with (001) crystal facets and ordinary anatases are (001) crystal facets and (101) crystal facets, respectively, and there are differences in the atomic or electronic structures between the two crystal facets. Many scholars believe that the more catalytic effect of (001) crystal facets is due to more Ti−5c atoms existing on its surface [44,45]. Barcaro et al. [46] point out the catalytic effect on polymerization depends mainly on the relative position of the adsorbed molecules. So, the different condensation efficiency of these two types of anatases on Gly₂ and Gly₃ under heating conditions may also be caused by different adsorption sites. Schmidt et al. [47] showed that the adsorption of amino acids on the surface of TiO₂ in aqueous solution preferentially occurred in acidic solution and showed specific adsorption with Ti atoms on the surface, and the main groups involved in the adsorption process were amino and carboxyl groups common to all amino acids. Barcaro et al. [46] point out the motion of the Gly droplet towards the interface during the equilibration dynamics was slower than the adsorption of water, The interface became partially hydroxylated and solvated by water molecules hydrogen- bonded to the out-of-plane oxygen atoms or physisorbed on Ti atoms. With only a few surface sites available for direct contact, amino-acid binding must compete with surface–solvent interactions, and with this exact agreement with the experimental results, it is nearly impossible to simulate an efficient polymerization process without performing several restricted molecular dynamics stages. Due to the necessity of considering solvation effects, the model is constructed as close as possible to the real reaction system in the subsequent simulation calculation.

4. Conclusions

The anatase with (001) crystal facets and ordinary anatase can catalyze the condensation of glycine to form the corresponding oligopeptide under the condition of providing a certain amount of energy, and the catalytic condensation of anatases with (001) crystal facets is stronger. After different forms of glycine are adsorbed on the surface of anatases by bridge structures through carboxyl groups, the electron cloud density of two carboxyl oxygen atoms on glycine carboxyl groups increases. At the same time, the electron cloud density of carboxyl carbon atoms and Ti atoms on the surface of connected anatases decreases. The change in electron cloud density of carboxyl groups caused by adsorption makes amino acid molecules subject to nucleophilic attack by other free or adsorbed amino acid groups and finally condensate to form peptide bonds.

In this research, we elucidate the mechanism of peptide condensation from glycine on the surface of two types of anatases, which can provide a scientific reference for the role of semiconductor minerals in the formation process of biological macromolecules in research on the origin of life.

Author Contributions

Conceptualization, J.C.; methodology, X.Z. and J.C.; software, H.W. (Haiyan Wu) and Z.L.; validation, X.N. and M.L.; investigation, L.Z.; resources, H.W. (Haiyan Wu); writing—original draft preparation, J.C. and X.Z.; writing—review and editing, J.C.; visualization, H.W. (Hongfu Wei); supervision, W.H. and M.L. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Conflicts of Interest

The authors declare no conflict of interest.

Footnotes

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Figures and Tables

Figure 1. TA/DTA/DSC spectra of blank glycine in two kinds of atmosphere. (a) TA/DTA spectra of blank glycine in two kinds of atmosphere. (b) DSC spectra of blank glycine in two kinds of atmosphere.

Figure 2. TA/DTA/DSC spectra of glycine absorbed on two types of anatases in two kinds of atmosphere. (a) TA/DTA spectra in air atmosphere. (b) DSC spectra in air atmosphere. (c) TA/DTA spectra in nitrogen atmosphere. (d) DSC spectra in nitrogen atmosphere.

Figure 3. HPLC spectra of glycine condensation products catalyzed on the surface of anatases. (a) HPLC spectra of glycine and oligopeptides. (b) HPLC spectra of glycine heated at 130 °C (insert: HPLC spectra of oligopeptides at 130 °C).

Figure 4. HPLC spectra of heating product after glycine absorbed on two kinds of anatases under heating conditions. (Note: (a) 80 °C. (b) 90 °C. (c) 100 °C (d) 110 °C. (e) 120 °C. (f) 130 °C.).

Figure 5. Glycine oligopeptide products catalyzed by two types of anatases under different temperature conditions. (Note: (a) DKP production amount. (b) Gly2 production amount. (c) Gly3 production amount.).

Figure 6. HPLC spectra of glycine oligopeptide products catalyzed by anatase with (001) crystal facets under light and dark condition (60 °C). (Note: (a) 24 h. (b) 48 h. (c) 72 h. (d) 96 h. (e) 120 h. (f) 144 h.).

Figure 7. HPLC spectra of glycine oligopeptide products catalyzed by ordinary anatase under light and dark condition (60 °C). (Note: (a) 24 h. (b) 48 h. (c) 72 h. (d) 96 h. (e) 120 h. (f) 144 h.).

Figure 8. The amount of glycine oligopeptide products catalyzed by two types of anatases at different times under light and dark conditions (60 °C). (Note: (a) DKP production amount. (b) Gly2 production amount. (c) Gly3 production amount.).

Figure 9. NMR analysis of glycine condensation products on the surface of two types of anatases. (a) NMR analysis of glycine condensation products on the surface of anatase with (001) crystal facets. (b) NMR analysis of glycine condensation products on the surface of ordinary anatase.

Figure 10. FTIR spectra of glycine condensation products on the surface of two types of anatases under different temperature conditions.

Figure 11. The density difference of glycine absorbed on two types of anatases with different crystal facets.

Figure 12. Thermal polymerization process of glycine on the surface of two types of anatases.

Table 1

Standard curve of corresponding oligopeptide.

Production	Standard Curve	R2
DKP	y = 252.20x + 0.3087	0.9987
Gly2	y = 117.01x + 0.0692	0.9992
Gly3	y = 220.07x + 0.10329	0.9958

Table 2

The shift of pure glycine-related group.

Type of Vibration	Absorption Peak Displacement (cm−1)
υas (NH3+)	3171.3
υas (CH2)	2917.3
υas (COO–)	1613.3
δs (NH3+)	1521.7
ρw (CH2)	1333.1

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