Elongation of Model Prebiotic Proto-Peptides by ... - ACS Publications

Article Cite This: Macromolecules XXXX, XXX, XXX−XXX

Elongation of Model Prebiotic Proto-Peptides by Continuous Monomer Feeding Sheng-Sheng Yu,†,∥ Martin D. Solano,‡,∥ Matthew K. Blanchard,†,∥ Molly T. Soper-Hopper,‡,∥ Ramanarayanan Krishnamurthy,§,∥ Facundo M. Fernández,‡,∥ Nicholas V. Hud,‡,∥ F. Joseph Schork,†,∥ and Martha A. Grover*,†,∥ †

School of Chemical & Biomolecular Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332, United States School of Chemistry & Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332, United States § Department of Chemistry, The Scripps Research Institute, La Jolla, California 92037, United States ∥ NSF/NASA Center for Chemical Evolution, United States ‡

S Supporting Information *

ABSTRACT: Mixtures of amino acids with hydroxy acids allow for the formation of peptide bonds in a plausible prebiotic scenario via ester bond formation followed by ester−amide exchange. Here, we investigate the ability of the ester-mediated reaction pathway to form even longer polymers with peptide backbones based on the specific details of the reaction protocol. Fresh monomers were fed to the polymer/monomer mixture periodically by an automated “day−night machine” that was designed to simulate wet−dry cycles that would have been common on the prebiotic Earth. Quantitative analysis of peptide bond formation in the complex oligomer mixture was enabled by a simple hydrolysis treatment. In the estermediated peptide elongation process, new monomers add to one end of the chain step-by-step without termination. The feed composition (hydroxy acids and/or amino acids) was found to determine the final oligomer distribution. Production of longer oligomers enriched in peptide bonds was more efficient when only amino acids were fed because of a smaller number of active oligomer chains. These results reveal a process for synthesizing longer depsipeptides and/or peptides that could form secondary structures, and possibly functional polymers. ization.12 Inorganic catalysts,13,14 chemical activation15,16 and alternative environments17−19 have been proposed as possible pathways to polypeptides. However, most of these reaction conditions still require high temperatures or introduction of an activating agent to produce oligopeptides. The long-standing challenge of forming polypeptides from free amino acids under potentially prebiotic conditions suggests that other plausible polymer building blocks present on the early Earth should be considered.20 Hydroxy acids are found along with amino acids in the products of the Miller−Urey reaction21,22 and in some meteorites.23,24 In our previous studies, we reported that hydroxy acids readily condense to polyester in a wet−dry cycling environment.25 When amino acids and hydroxy acids are mixed together, oligoesters are formed from hydroxy acids and are then transformed into mixed copolymers via the ester−amide exchange reaction.20 The ester-mediated pathway enables amide bond formation by lowering the activation energy barrier.26 The copolymers of hydroxy acids and amino acids, known as depsipeptides, might have functioned as proto-polymer precursors to polypeptides. Oligomeric depsipeptides of up to 14 residues in length have

1. INTRODUCTION Nearly 60 years ago, the original Miller−Urey experiment demonstrated the synthesis of simple amino acids, such as glycine, alanine, and aspartic acid, under plausible prebiotic conditions.1 Recently, products of Miller−Urey reactions were investigated using state-of-the-art analytical techniques, and more complex amino acids were also found (e.g., serine and leucine).2,3 In addition to Miller−Urey and related routes to amino acids,4 some of the building blocks of life could have also been delivered to the early Earth from extraterrestrial sources.5,6 Polypeptides can form secondary structures, such as α-helices and β-sheets, and higher order structures that can coordinate reactants and act as catalysts.7 Therefore, polypeptides may have played a key role in the origin of life on Earth, but an abiotic mechanism for amino acid polymerization has been a persistent challenge in the field of prebiotic chemistry. The simplest path to polypeptides is the polycondensation of amino acids, but this reaction is thermodynamically unfavorable in aqueous solution.8 Fox and Harada first used simple dryingheating conditions to produce copolymers of amino acids, but polymerization required relatively high temperatures, generally ranging from 150 to 200 °C.9,10 Under such conditions, even the simplest amino acid, glycine, can be decarboxylated into methylamine.11 In addition, dipeptides can cyclize to form a diketopiperazine (DKP), a side-product that inhibits polymer© XXXX American Chemical Society

Received: July 23, 2017 Revised: November 18, 2017

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DOI: 10.1021/acs.macromol.7b01569 Macromolecules XXXX, XXX, XXX−XXX

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Macromolecules been identified in model prebiotic reactions: their backbones are still a mix of ester and amide bonds, and their abundance is relatively low. Depsipeptides enriched in amino acids are of special interest because they are more likely to possess structural properties and functions leading to catalytic activity (similar to polypeptides). In the ester-mediated elongation reaction, each amino acid is added stepwise to the C-terminus of oligomers, without termination, as shown in Scheme 1. Direct amide bond

the analysis of the g/G oligomer mixture, a mixture of depsipeptides composed of various sequences of glycolic acid and glycine residues, by combining HPLC−UV/MS techniques with a hydrolysis treatment to reduce product complexity. We then compare the product distribution observed between two different feeding protocols (addition of glycolic acid and glycine versus addition of glycine alone), evaluating their impact on the oligomer distribution and their potential to produce longer chain peptides.

Scheme 1. Major Reaction Path of Depsipeptide Elongation via Esterification and the Ester-Amide Exchange Reactiona

2. EXPERIMENTAL METHODS 2.1. Materials. Glycolic acid, glycine, glycine anhydride, diglycine, triglycine, methyl glycolate, formic acid, 2,5-dihydroxybenzoic acid, and phosphoric acid were purchased from Sigma-Aldrich. Methanol, HPLC grade water and acetonitrile were purchased from EMD Millipore. 99% of 1,1,3,3-tetramethylguanidine was purchased from Alfa-Aesar. 2.2. Day−Night Machine. The cycling experiment with feeding was conducted by a custom-configured instrument, shown in Figure S3, which is the combination of a Bio-Rad MyCycler 96 well thermocycler and a Beckman Biomek 3000 Automation Workstation. A sample solution of 100 μL containing 100 mM of glycolic acid and 100 mM of glycine was heated to 95 °C within polypropylene tubes in the 96 well thermal cycler. Every 24 h, a pipet controlled by the Biomek 3000 workstation rehydrated the dry samples with 100 μL of an aqueous solution containing fresh monomer, typically 20 mM each of glycolic acid and glycine, or only 20 mM of glycine. 2.3. Oligomer Characterization. Before characterization, each sample was dissolved in 200 μL of water, sealed in a vial, and then incubated at 95 °C for 48 h to hydrolyze ester linkages. The pH of these solutions was approximately 2.8 due to the presence of glycolic acid. Next, the aqueous solutions were diluted 2-fold with acetonitrile to 50% v/v acetonitrile/water. Samples were analyzed with an Agilent 1260 HPLC coupled to an Agilent 6130 single quadrupole mass spectrometer and an inline Agilent UV absorbance detector (210 nm). Oligomers were separated using a SeQuant ZIC-HILIC column (150 × 2.1 mm, 3.5 μm particle size, 100 Å). The flow rate was 0.2 mL min−1 and the column temperature was held at 40 °C. Injection volume is 5 μL. The mobile phase was (A) water with 0.5% v/v formic acid and (B) acetonitrile. The gradient method started with 5% A and then ramped to 50% A over 25 min. The mobile phase composition was held at 50% A for 5 min and then returned to 5% A in 1 miniute. For MS analysis, all data were obtained with negative-ion mode electrospray ionization with a capillary voltage of 2.0 kV. Log2-ratio analysis was performed using MZmine 2 software and plotted in OriginPro 9.0. Tandem MS experiments were performed on a Waters quadrupole/ time-of-flight (qTOF) Xevo G2 mass spectrometer. Samples were diluted to 0.039 mg mL−1 of starting monomers by addition of water and directly infused into the mass spectrometer by electrospray ionization in negative-ion mode. The capillary voltage was set at 2.0 kV, the sampling cone voltage was 30 V, the extraction cone voltage was 3.0 V, and the source temperature was 90 °C. The low mass resolution was 7 and high mass resolution was 15. The desolvation gas flow was 600 L h−1 and the desolvation temperature was 250 °C. Collision energy for fragmentation was in the range 15−25 eV (labframe). 2.4. Synthesis of Standard Compounds. For the synthesis of the 1g1G dimer standard (HO-g-G-CO2H), 2 mmol of methyl glycolate and 2 mmol of glycine were mixed in 500 μL of 1,1,3,3tetramethylguanidine and 100 μL of methanol for methanolysis. The solution was stirred for 8 h at 100 °C. The crude products were first purified by passage through an anion exchange column packed with 25 g of QAE Sephadex A-25 resin (GE Healthcare) with a pH 5 ammonium formate elution buffer running a gradient from 50 to 500 mM. Products were then passed through a cation exchange column packed with 15 g of Dowex 50WX8 hydrogen form (Sigma-Aldrich) charged with 0.1% formic acid solution. Final isolation of product was

a

Elongation gradually leads to longer oligomers and enrichment in peptide bonds, until free amino acids are depleted.

formation is also possible but we have found it to be less efficient than the ester-mediated pathway.26 It is possible that new building blocks formed on other parts of early Earth, including in the atmosphere, were transported and mixed with existing depsipeptides, enabling continued oligomer extension. Therefore, we investigated whether continuously “feeding” with fresh monomers could allow for the formation of long depsipeptides enriched in peptide bonds. The objective of this work is to identify model prebiotic scenarios that enable the continuous growth of oligomers with a peptide-like backbone. The copolymerization of glycolic acid (g) and glycine (G) was chosen as a model reaction because of the relatively high abundance of those monomers in the products of model prebiotic reactions, and because of their observed high reactivity in dry-phase reactions than other amino acids (Figure S1 and S2).20 In previous studies we have used tandem mass spectrometry (MS) to obtain sequence information from the depsipeptides mixture, but it is difficult to quantify peptide fragments by tandem MS experiments due to the lack of appropriate standards. In this study, we first present B


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Macromolecules achieved by flowing products through a Teledyne Combiflash-Rf+ flash chromatography system using a RediSep C18aq 150 g Gold column and a UV−vis detector. Identification of the desired product was verified with an Agilent 6130 single quadrupole mass spectrometer using a 2.0 kV ESI capillary voltage. The expected [M − H]− value is 132.03 Da for the 1g1G dimer. Species with m/z values equal to 132.1 and 265.0 Da (a common [2M − H]− ESI adduct) were observed. In a similar procedure, diglycine was combined with methyl glycolate in the initial reaction for the synthesis of the 1g2G trimer (HO-g-G-GCO2H). For the trimer, the expected [M − H]− is 189.05 Da. Signals of 189.0 and 379.0 Da ([2M − H]−) were detected.

Previously, we used a reverse-phase column to separate lactic acid/valine oligomer mixtures.26 However, the same method was not successful in the current study due to the low hydrophobicity of the g/G depsipeptides. Hao et al. used hydrophilic interaction chromatography (HILIC) to separate similar mixtures containing glycine, diglycine and triglycine from the Maillard reaction.27 On the basis of their procedure, we successfully used a HILIC column to separate the g/G depsipeptides. The quality of this separation is demonstrated by our LC−MS results shown in Figures S4−S7, including mass spectra from negative-ion and positive-ion modes. The identification of each species is also supported by a high resolution MS analysis as in Figure S8. For the sample without hydrolysis treatment, the mass spectrum of each peak revealed many oligomers with distinct m/z values that coeluted from the HPLC column. For example, the 1g2G trimer, the 3g3G hexamer, and the 4g4G octamer were all found to elute at 10 min (Figure S4b). Because of this extensive peak overlap, it was difficult to measure the concentrations of individual oligomer species by their MS or UV signals. HPLC peak overlap was alleviated by heating the samples in the solution state at 95 °C for 48 h, which hydrolyzed the ester bonds and reduced the number of oligomer species. The corresponding total ion chromatogram showed more resolved peaks, compared to the sample without ester hydrolysis treatment (Figure S6a). Signals for oligomers containing multiple glycolic acid units disappeared after the 48-h hydrolysis reaction. The mass spectrum of each LC peak for the posthydrolysis samples contained only one dominant species. The effect of the hydrolysis treatment was further visualized through two-dimensional log2-ratio plots (Figure 2). This approach is commonly applied in metabolomics to illustrate the difference between two sets of LC−MS results.28 The symbol color of each species indicates the ratio of peak areas in the MS chromatograms before and after hydrolysis:

3. RESULTS 3.1. Quantitative Analysis of the g/G Depsipeptides. To reduce the complexity of the oligomer mixtures produced in our reactions, and to reveal the abundance of those oligomers with consecutive amino acids (i.e., consecutive amide linkage in the backbone), we relied on the higher thermal stability of amide bonds relative to ester bonds. The overall procedure is shown schematically in Figure 1. Each sample was maintained

Figure 1. Experimental procedure of feeding experiments. The oligomer mixture was synthesized by feeding fresh monomers once during each wet−dry cycle. The complexity of the oligomer product mixture was reduced by the hydrolysis of ester linkages. Orange sunbursts indicate the positions of ester linkages in the two example depsipeptides.

⎛ peak area after hydrolysis ⎞ log 2‐ratio = log 2⎜ ⎟ ⎝ peak area before hydrolysis ⎠

at 95 °C for 48 h to hydrolyze all ester linkages. We anticipated some amide bonds might be hydrolyzed as well, but to a far lesser extent (see section 3.3). Overall, this procedure allowed us to reduce chromatographic overlap and to focus on oligomers containing the more stable amide bonds.

(1)

Although the peak areas in the MS chromatograms are usually not used for quantitation without standards, this approach provides a way to semiquantitatively compare two LC−MS experiments. For example, the pentamer series (Figure 2, inset) ranges from 3g2G to 1g4G. For the same chain length,

Figure 2. Log2-ratio analysis of LC−MS data before and after hydrolysis treatment. The inset focuses on the details of the pentamer series. The sample was prepared by drying a mixture of glycolic acid (100 mM) and glycine (100 mM) at 95 °C for 16 days. A 20 mM solution of glycolic acid and glycine was added to the dry mixture every 24 h. C


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Figure 3. Tandem MS sequencing analysis of depsipeptides in the hydrolyzed g/G reaction product mixture. Sample was prepared by drying a mixture of glycolic acid (100 mM) and glycine (100 mM) at 95 °C for 16 days. A 20 mM solution of glycine was added to the dry mixture every 24 h. Besides the main fragment ion series from α-cleavages, signals from β-cleavages (#) and CO2 losses (&)30 were also observed.

oligomers containing more glycolic acid units tended to elute earlier. Those oligomers containing multiple glycolic acid residues coeluted with other oligomers as well. After the hydrolysis treatment, the peak areas of glycolic acid-rich oligomers decreased and those of the 1gnG depsipeptides series increased. Since the log2-ratio values of those species containing multiple glycolic acid units were mostly below −4 (less than 6% of their initial peak area), we concluded that the hydrolysis treatment had removed nearly all ester bonds. According to our previous findings,20 the remaining 1gnG series should have only one dominant sequence: 1g-(G)n, in which all units are linked by amide bonds with the single glycolic acid residue located at the N-termini. This interpretation is further supported by Figure 3. In addition to glycolic acid N-capped depsipeptides, we also found pure glycine peptides (nG) without any glycolic acid units. These pure peptides could be the result of either glycine polycondensation or hydrolysis of amide bonds in depsipeptides. Because we detected those oligomers before the hydrolysis treatment, it is likely that some were produced by glycine polycondensation. We were unable to directly estimate the amounts of nG in the sample without the hydrolysis treatment due to intense peak overlap, but the increase of nG peptide peak area suggests that some homo-G peptides were released from depsipeptides during the hydrolysis treatment. The possible causes of glycine polycondensation and the effect of peptide bond hydrolysis are discussed below. Because of its significance in limiting peptide growth, we attempted to detect the cyclic dimer (diketopiperazine, DKP) in the reaction mixtures. Because of its low ionization efficiency, no distinct signal could be detected in the negative-ion mode mass chromatograms. Parts a and b of Figure S9 show the UV and positive-ion mode total ion chromatogram for the expected DKP. Although no clear signal was observed by MS in the total ion trace, the DKP retention time was confirmed by injecting a DKP standard and comparison with the UV detector traces. As shown in Figure S9, parts c and d, the mass spectra for the DKP

from both the reaction mixture and the standard compound at that retention time in positive-ion mode did show the expected DKP [M + H]+ signals. DKP could be produced via the cyclization of diglycine, or the hydrolysis of longer peptides.29 Since pure glycine peptides are observed, it is not surprising that DKP is detected. Note that the ester-mediated reaction can also produce cyclized 1g1G dimer (diketomorphalino), but it can be converted back to linear dimer readily through the hydrolysis of the ester bond. Tandem MS experiments confirmed the sequences of the oligomers in hydrolyzed samples (Figure 3). All detected depsipeptides with 1gnG composition had one dominant sequence: one glycolic acid unit on the “N-terminus,” followed by multiple glycine units, consistent with the proposed ester− amide exchange mechanism. The longest depsipeptide with precursor ion signal sufficiently high for a tandem MS experiment was 1g9G, as the intensities of longer depsipeptides were too low for tandem MS analysis with adequate signal-tonoise ratio. The UV extinction coefficient of each oligomer was determined by injecting standard compounds in known concentrations. While the glycine monomer, DKP, and short peptides are commercially available, hydroxy acid−amino acid dimers and trimers are not. The 1g1G and 1g2G standards were synthesized as described in Experimental Methods and characterized by LC−MS (Figure S10 and S11). The UV responses of 1g1G, 1g2G, and DKP are shown in Figure S12. Although the chromatographic peaks of these three species did not have ideal Gaussian profiles, their UV responses were linear with R2 values better than 0.99. Therefore, their UV response factors were used directly for quantitation. The standards of 1G, 2G, and 3G with different concentrations were used to obtain the UV response factors of pure peptides (Figure S13). To estimate the extinction coefficients of longer oligomers, the extinction coefficients of amide bonds and carboxylic acid groups were calculated from the results of short oligomers, similar to the approach used by Codari et al.31 Assuming the D


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Figure 4. Instantaneous yield and conversion for different feeding approaches based on HPLC−UV analysis. (a and b) Yields of 1gnG (black), nG (cyan), and DKP (dark blue) over time. (c and d) Instantaneous conversion of unreacted glycine monomer (red) and the total yield of products (blue), the sum of 1gnG, nG, and DKP. Experiments shown in parts a and c were fed with glycolic acid and glycine, and experiments shown in parts b and d were fed with only glycine. All samples were hydrolyzed at 95 °C for 48 h prior to analysis.

monomers. If all glycine monomers were converted into oligomers, the profiles of total oligomer yield and conversion should be within experimental error. Parts a and b of Figure 4 show the instantaneous yields of three different products, 1gnG, nG, and DKP, for the two feeding compositions. In both cases, the major product was the 1gnG oligomers, with yields typically being three to four times greater than those of the nG pure amino acid series. This result confirmed that even under the high temperature of 95 °C, when both ester−amide exchange and direct amide bond formation can proceed (see below), the ester-mediated pathway was the favored reaction. Parts c and d of Figure 4 show the percent conversion of glycine monomers and the total yield of products (i.e., the sum of 1gnG, nG, and DKP). For both feeding compositions, the total yield of oligomers and the conversion of glycine follow each other closely, indicating our quantitation method accounts for most of the products formed in the reaction and that most of the glycine monomer consumed is incorporated into oligomers. Also, for both feeding compositions, the instantaneous yield and conversion generally remained stable over time. This result suggests the ester-mediated reaction is efficient during the 20 days reaction. However, in the case of glycineonly feeding (Figure 4d), it appears that the rate of glycine incorporation into oligomers may be slowing toward the end of the study, possibly due to a reduction in the availability of the hydroxy acids needed for the ester−amide exchange, or the formation of water-insoluble products. This decrease in the rate of conversion was not observed in Figure 4c when glycolic acid was also added at each glycine feeding. Besides the hydrolysis of depsipeptides, nG pure peptides might be produced directly (without the ester-amid exchange

extinction coefficients of the chromophores in the longer oligomers are as same as those in the short oligomers, we calculated the UV extinction coefficients of 1gnG and nG oligomer series based on the numbers of amide and carboxylic acid groups. The full list of UV response factors (1/extinction coefficient) used in this work is provided in Table S2. 3.2. Efficiency of the g/G Copolymerization with Different Feeding Compositions. Two different feeding compositions were studied for the purposes of peptide formation; feeding with equal amounts of glycolic acid and glycine (1:1), or feeding with glycine only (0:1). In both sets of experiments, the reactions started with a 1:1 mixture of glycolic acid and glycine. The instantaneous yields of different products and the conversion of glycine monomer were evaluated by the following parameters: ⎛ ∑∞ xPx(t ) ⎞ ⎟⎟ × 100% instantaneous yield = ⎜⎜ x = 1 ⎝ P1G(t0) + F1Gt ⎠

(2)

⎛ ⎞ P1G(t ) instantaneous conversion = ⎜1 − ⎟ × 100% P1G(t0) + F1Gt ⎠ ⎝ (3)

where Px(t) is the amount of oligomer (1gnG or nG) containing x glycine units at time point t. The summation of xPx(t) is the total amount of glycine units in the oligomers. F1G is the feeding rate of fresh glycine monomer and P1G is the amount of unreacted glycine monomer at a given time point. The denominators in both equations give the total amount of glycine units in the mixture. eq 2 gives the yields of different oligomers, 1gnG, nG, and DKP, at a given time point. The result of eq 3 is the instantaneous conversion of glycine E


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were performed in MATLAB with the patternsearch function for minimization and the ode15s function for integration of differential equations. The experimental data and simulation with optimized rate constants are shown in Figure S16. At 95 °C, kh is 6.03 × 10−5 L mol−1 h−1 and kc is 1.75 × 10−3 h−1. Previously determined ester hydrolysis rate constants at 95 °C are 3.07 × 10−3 for polylactic acid26 and 1.35 × 10−2 L mol−1 h−1 for polymalic acid.25 Thus, as expected, the amide bonds are considerably more stable than ester bonds under similar hydrolysis conditions. In this experiment of 3 days hydrolysis, 40% of the 1g2G trimer was consumed. In our day−night cycling experiment, water evaporated in the first 30 min. Therefore, up to 0.25% of amide bond might be hydrolyzed in each cycle. We expect the potential effect of amide bond hydrolysis on the oligomer distribution in the cycling experiments is minimal. However, it is expected to be significant during the hydrolysis procedure, in which the depsipeptides are maintained in a heated and hydrated state for 2 days. 3.4. Oligomer Distribution under Different Feeding Compositions. We further evaluated the effect of feeding composition by comparing the distribution of 1gnG depsipeptides. The oligomer distribution after 2 days of hydrolysis at 95 °C was measured by HPLC−UV (Figure S17). Because the hydrolysis treatment also cleaves amide bonds to some extent, the experimental distribution might not precisely reflect the actual distribution. In particular, the abundance of long oligomers may be underestimated due to the greater probability for an amide bond to be hydrolyzed. To estimate the initial distribution of the 1gnG series without amide bond hydrolysis, we solved eqs 4 to 7 backward in time, using the experimental data as the initial condition and assuming all amide bonds have the same hydrolysis rate. The estimated distributions between the two experiments are shown in Figure 5. As expected, the reconstructed distribution indicates that more long chain oligomers were produced than actually observed in the experimental distribution. When both glycolic acid and glycine were used for sample feeding, the distribution of oligomers remains unchanged over time. Although the abundance of longer oligomers gradually increased, the dominant species appeared to be the 1g2G trimer containing only two amide bonds. In the second case, when the samples were fed only glycine, the initial distribution was similar to the first case, but it shifted to longer chain length as time passed. The dominant species changed from 1g2G to 1g4G by the end of our study. The difference in oligomer distribution between the two feeding compositions arises from the number of active chains in the mixture. When feeding with glycolic acid, more oligomer chains can be initiated over the course of the study. The increasing number of active chains may consume free glycine more rapidly, but not necessarily contribute to the elongation of existing chains. Much of the free amino acids would then be consumed by making and extending many short oligomers, instead of extending a smaller number of longer oligomers. When no additional glycolic acid is added during sample feeding, free glycine is primarily consumed to elongate the smaller number of active chains formed during the first few cycles. By combining the step-growth polymerization and the ester−amide exchange reaction, the ester-mediated pathway leads to a similar chain-extension behavior as chain-growth living polymerization,33 in which no termination step is present to deactivate oligomer chains. The average molecular weight is

reaction) in the acidic environment provided by glycolic acid. The pH of the initial solution used in our experiment is about 2.8. Rodriguez-Garcia et al. have reported the condensation of glycine in an acidic condition.32 To further investigate this effect, the pH of the solution containing only glycine was lowered to 2.8 by phosphoric acid, and then was subjected to the same 95 °C wet−dry cycles. Glycine peptides and DKP indeed formed after a few cycles (Figure S14), with the yields of nG peptides and DKP being similar to those in experiments that contained glycolic acid. The dominant species was glycine trimer, with oligomers up to 7 amide linkages observed after 12 days of reaction (Figure S15). Therefore, it was concluded that hydroxy acids enable amide bond formation not only via the ester−amide exchange reaction pathway, but also via direct polycondensation in an acidic environment. However, the majority of peptide bond formation (over 75%) occurred via the ester−amide exchange reaction. 3.3. Extent of Amide Bond Hydrolysis. Although the hydrolysis treatment was designed to remove all ester bonds and thereby reduce the complexity of the product mixtures, a fraction of the amide bonds may be cleaved as well. To estimate the loss of amide bonds, we monitored the hydrolysis rate of the 1g2G standard under the same condition used for ester bond hydrolysis (48 h at 95 °C, pH 2.8). This treatment of 1g2G produced all expected products, i.e., 1g1G, 2G, 1g, 1G, and DKP. The experimental results are shown in Figure S16, along with the prediction from our kinetic model. In our previous work, we established a model for the copolymerization and hydrolysis of depsipeptides.26 Here, a simplified version was used to study the kinetics of amide bond hydrolysis. POi represents oligomers with hydroxy termini and PNi with amine termini. Subscript i indicates the number of amide bonds. For example, PO2 is the 1g2G trimer and PN1 is the 2G dimer. The hydrolysis of POi is represented by eq 4. The first term is the loss of oligomers due to the amide bond hydrolysis. The second term represents the generation of oligomers from the hydrolysis of longer oligomers. The hydrolysis of PNi (pure glycine peptides) is represented by eq 5, which is similar to the equation for POi ; however, eq 5 also includes a third term for the hydrolysis from longer glycine peptides. Because the formation of DKP is observed in our experiment, we also include the cyclization of diglycine in eqs 6 and 7. Pc is the amount of DKP, which we assume to only be generated from the cyclization of the glycine dimer (PN1 ). Therefore, the rate equation for PN1 has one additional term for cyclization. ∞

dPiO = −ikhWPiO + khW ∑ POj dt j=i+1

(4)

∞

∞

dPiN = −ikhWPiN + khW ∑ POj + 2khW ∑ PkN dt j=i+1 k=i+1

(5)

dPc = kcP1N dt

(6) ∞

∞

dP1N = −khWP1N + khW ∑ POj + 2khW ∑ PkN − kcP1N dt j=2 k=2 (7)

The rate constants for amide bond hydrolysis (kh) and diglycine cyclization (kc) were determined by fitting the data of 1g2G hydrolysis with eqs 4−7. All simulations and calculations F


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Figure 5. Estimated chain length distribution of 1gnG before the hydrolysis treatment. The distribution was predicted from eqs 4−7, based on the HPLC−UV measurement of the hydrolyzed samples. The y-axis is the number of glycine units in each oligomer.

Figure 6. (a) Number of oligomer chains in 1gnG. (b) Average number of consecutive amide bonds in each oligomer distribution. The results of the two feeding compositions are represented by red (1:1) and blue (0:1). Lines with light and strong colors represent the experimentally measured distribution and the model-estimated distribution prior to hydrolysis, respectively.

determined by the ratio between monomers and initiators, and the active chains have the ability to connect new monomers sequentially. We also studied an unfed experiment, starting with the same amounts of glycolic acid and glycine (Figure S18). As we expected, without the new monomers for chain elongation, fewer oligomers were formed and the oligomer distribution remained unchanged. The behavior of the ester-mediated amide bond formation reaction is confirmed by the data shown in Figure 6a. In the glycolic acid and glycine (1:1) feeding experiment, the number of chains indeed increased linearly over time, suggesting that adding glycolic acid initiates new chains. When no additional glycolic acid is used for feeding, the total chain number remained constant after the first 8 cycles. We further calculated the average number of amide bonds in each distribution and summarized the results in Figure 6b. The estimated distributions have higher values than the experimental distribution as expected. When feeding with both glycolic acid and glycine, the average chain length stayed around 2.5. In contrast, feeding with only glycine gave a more efficient growth of average chain length, presumably because fewer new chains can nucleate without the addition of glycolic acid, favoring elongation of existing chains. In the glycine only (0:1) feeding experiment, the final chain number was only 56% of the initial amount of glycolic acid. This fewer number of chains compared to the number of potential chain initiators might be the result of glycolic acid evaporation, similar to what we observed when using lactic acid.26 The evaporation rate of glycolic acid was monitored in a closed reactor with the procedure described in our previous work.26 At the same drying temperature (85 °C), the evaporation of glycolic acid appeared to be much slower than that of lactic acid (Figure S19), which may be one of the

reasons that glycolic acid is more efficient than lactic acid in catalyzing depsipeptide formation. Compared to lactic acid, more glycolic acid would be retained under the same conditions, and continue to promote peptide bond formation through ester−amide exchange. The longest oligomer observed by UV detection was 1g8G. MS analysis with electrospray ionization (ESI) enabled the detection of even longer oligomers, such as 1g13G (Figure 7),

Figure 7. Longest oligomers observed by ESI−MS from the 0:1 feeding experiment. The sample was prepared by drying a mixture of glycolic acid (100 mM) and glycine (100 mM) at 95 °C for 16 days. A 20 mM solution of glycine was added to the dry mixture every 24 h. Diluted samples were analyzed by direct injection into a Waters Xevo G2 mass spectrometer. G


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Fresh monomers can be added to the C-termini of oligomers continuously. Adding more glycine eventually leads to longer oligomers compared to our previously reported studies with a closed system of hydroxy acids and amino acids. Faster growth of average chain length was found when only glycine was fed to the dry mixture every cycle. Oligomers containing up to 13 repeated glycine residues have been identified by MS, longer than is needed to form secondary structures. These results provide insights on which chemical and environmental scenarios should be further investigated as the possible origins of biomolecules.

but their UV responses were too low to obtain quantitative measurements. Moreover, we found insoluble products in the 0:1 experiment after 16 days of reaction. Oligoglycines longer than five residues are known to be insoluble in water.34−36 Therefore, the abundance of long oligomers measured by HPLC−UV analysis might be lower than actual values because it only detects products that can be dissolved in water. We attempted to detect longer and possibly insoluble oligomers by MALDI−MS analysis. As shown in Figure S20, the MALDI− MS analysis did not provide further evidence of longer peptides.

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4. DISCUSSION In this study, we compared the impact of two feeding compositions on the growth of peptides produced by the hydroxy acid-catalyzed ester−amide exchange reaction. We focused on the polymerization of glycine, mainly because of its higher reactivity and its high abundance in model prebiotic reactions, compared to other amino acids. In principle, the method described in this work can be used to polymerize other amino acids. Other simple amino acids, like alanine and valine, are more likely to form regular secondary structures that would also be of interest,37 but longer reaction times will likely be necessary due to their lower reactivity and lower solubility compared to glycine. Future work will investigate if specific feeding protocols can lead to long chain depsipeptides that form regular secondary structures, as has been shown recently for amino acids activated by carbonyl sulfide chemistry.37 The evaporation of hydroxy acids gradually changes the ratio of the two building blocks over time and leads to peptideenriched oligomers, as confirmed by our previous study of lactic acid/alanine copolymerization.20 On the other hand, if the hydroxy acids can be recycled back to the dry oligomer mixture through a return (such as a raining process), this would lead to depsipeptides with short consecutive peptide fragments. For example, from our feeding experiment that regularly adds hydroxy acids by simulating a raining process, the most abundant species appear to be the 1g2G heterotrimer and 1g3G heterotetramer. In addition, our results provide insights on the possible product distribution from prebiotic mixtures. In the Miller− Urey type experiment, the ratio between amino acids and their hydroxy acid counterparts depends on the pH and the concentration of NH3 in the reaction medium.38,39 High pH in the electric discharge experiment leads to higher amino acid/ hydroxy acid ratio. If those mixtures were to undergo repeated wet−dry cycles, it should produce depsipeptides enriched in amino acids. On the other hand, electric discharge experiments with low pH results in low amino acid/hydroxy acid ratio and thus forms short hydroxy acid-amino acid fragments. Those hetero-oligomers can still be polymerized through reversible esterification and hydrolysis reactions, and potentially lead to depsipeptides with secondary structures and/or catalytic capabilities.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.7b01569. Reaction of alanine and valine, LC−UV/MS analysis, data for the hydrolysis of the 1g2G oligomer, analysis of standard compounds, UV response curves, evaporation rate of hydroxy acid, yield of glycine peptides in a cycling experiment without glycolic acid, and MALDI−MS analysis (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected] (M.A.G.). ORCID

Ramanarayanan Krishnamurthy: 0000-0001-5238-610X Facundo M. Fernández: 0000-0002-0302-2534 Martha A. Grover: 0000-0002-7036-776X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank David Bostwick for this assistance on HPLC and MALDI−MS experiments, and David Fialho for advice on standard synthesis. This work was jointly supported by the NSF and the NASA Astrobiology Program, under the NSF Center for Chemical Evolution, Grant CHE-1504217.

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REFERENCES

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