Ester Formation and Hydrolysis during Wet–Dry Cycles - American

Feb 4, 2014 - Nicholas V. Hud,*. ,†,§ and F. Joseph Schork. ‡,§. †. School of Chemistry and Biochemistry,. ‡. School of Chemical & Biomolecu...
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Ester Formation and Hydrolysis during Wet−Dry Cycles: Generation of Far-from-Equilibrium Polymers in a Model Prebiotic Reaction Irena Mamajanov,†,§ Patrick J. MacDonald,‡ Jingya Ying,‡ Daniel M. Duncanson,‡ Garrett R. Dowdy,‡ Chelsea A. Walker,†,§ Aaron E. Engelhart,†,§ Facundo M. Fernández,†,§ Martha A. Grover,‡,§ Nicholas V. Hud,*,†,§ and F. Joseph Schork‡,§ †

School of Chemistry and Biochemistry, ‡School of Chemical & Biomolecular Engineering, and §Center for Chemical Evolution, Georgia Institute of Technology, Atlanta, Georgia 30332, United States S Supporting Information *

ABSTRACT: Biopolymers exist within living cells as far-fromequilibrium metastable polymers. Living systems must constantly invest energy for biopolymer synthesis. In the earliest stages of life on Earth, the complex molecular machinery that contemporary life employs for the synthesis and maintenance of polymers did not exist. Thus, a major question regarding the origin of life is how the first far-from-equilibrium polymers emerged from a prebiotic “pool” of monomers. Here, we describe a proof-of-principle system, in which L-malic acid monomers form far-from-equilibrium, metastable oligoesters via repeated, cyclic changes in hydration and temperature. Such cycles would have been associated with day−night and/or seasonal cycles on the early Earth. In our model system, sample heating, which promotes water evaporation and ester bond formation, drives polymerization. Even though periodic sample rehydration and heating in the hydrated state promotes ester bond hydrolysis, successive iterations of wet−dry cycles result in polymer yields and molecular weight distributions in excess of that observed after a single drying cycle. We term this phenomenon a “polymerization ratchet”. We have quantitatively characterized the “ratchet” of our particular system. Ester bond formation rates and oligoester hydrolysis rates were determined for temperatures ranging from 60 to 95 °C. Based on these rates, a mathematical model was developed using polycondensation kinetics, from which conditions were predicted for oligoester growth. This model was verified experimentally by the demonstration that L-malic acid monomers subjected to multiple wet−dry cycles form oligoesters, which reach a steady-state concentration and mean length after several cycles. The concentration of oligoesters that persist between subsequent steady-state cycles depends on the temperature and durations of the dry and wet phases of the cycle. These results provide insights regarding the potential for very simple systems to exhibit features that would have been necessary for initiation of polymer evolution, before the emergence of genomes or enzymes.

1. INTRODUCTION Living organisms are not in thermodynamic equilibrium with their surrounding environment but are maintained in a quasisteady state by the regular input of chemical or photochemical energy. Furthermore, the biopolymers responsible for the myriad processes within each living cell are far-fromequilibrium chemical structures. The principal biopolymers (i.e., nucleic acids, polypeptides, polysaccharides) are synthesized via condensation−dehydration reactions, with the bonds linking their monomers being characterized by a positive free energy of formation in the aqueous environment of the cytoplasm. For example, the energy of the amide bond in a polypeptide backbone ranges from +2 to +4 kcal/mol in aqueous solution.1 In modern biochemistry, biopolymer synthesis from monomers is accomplished through an intricate system of energy harvesting and enzyme-controlled energy transfer.2 It stands to reason that such complicated mechanisms would not have been available in the earliest stages of chemical evolution. It has long been appreciated that many monomers of biopolymers can be produced by abiotic reactions. For example, © 2014 American Chemical Society

60 years ago, the Miller−Urey experiment demonstrated that some of the simpler biological amino acids (e.g., glycine and alanine) are readily formed in a model prebiotic atmosphere.3,4 Moreover, a number of the amino acids used in life today have been found in carbonaceous meteorites that are more than 4 billion years old.5 Likewise, other biopolymer blocks, including sugars and nucleobases, are also formed in a variety of model prebiotic reactions and found in some meteorites.6−11 Nevertheless, a plausible prebiotic reaction scheme for the coupling of monomers into biopolymers, or potential ancestral polymers (i.e., prebiopolymers), has remained elusive. The importance of low water activity to drive condensation−dehydration reactions is well appreciated, but simply drying and heating biological monomers without chemical activation has shown only modest success for the abiotic production of biopolymers. For example, attempts to produce polypeptides by the successive drying and rehydration of amino acids have typically resulted in the Received: October 31, 2013 Revised: January 24, 2014 Published: February 4, 2014 1334

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trations and lengths. This system demonstrates the generation of far-from-equilibrium polymers via a condensation−dehydration process in a model environmental cycle. Few such systems have previously been reported,12,26 and to the best of our knowledge, none have exhibited properties (e.g., forward and reverse reaction rates) that allowed the demonstration of material recycling.

conversion of less than 1% of free amino acids to dipeptides and even less to tripeptides.12,13 These low yields and the lack of longer peptides appear to be the result of the unfavorable thermodynamics of peptide bond formation1 as well as the cyclization of dipeptides into diketopiperazines.14 In light of the limited success associated with achieving peptide bond formation in simple drying reactions, we decided to investigate whether polyesters could be formed in a model prebiotic reaction that involved repeated wet−dry cycles. Previously, α-hydroxy acids have been shown to efficiently polymerize upon drying and with moderate heating. In a previous model of prebiotic polymer formation, Weber demonstrated that acid-catalyzed thermal condensation of glyceric acid at 80 °C (in the dry state) produces polymeric chains of up to 25 residues in length.15 In related reactions, Ohtani et al.16 and Kajiyama et al.17 demonstrated that dry state condensation of L-malic acid at 110−130 °C produces polymers of molecular weights up to 5 kDa that contain both α- and βlinkages (Scheme 1), with nearly complete incorporation of monomers. Similarly, industrial scale production of polylactic acid can be accomplished by direct condensation of lactic acid at moderately elevated temperature (below 200 °C) with water removal.18,19

2. MATERIALS AND METHODS 2.1. Materials. L-Malic acid was purchased from Sigma-Aldrich and used without further purification. Monobasic and dibasic sodium phosphates used for GPC analysis were purchased from BDH Chemicals. Barnstead Nanopure water was used for solution and eluent preparation. LC-MS grade methanol and acetonitrile were purchased from J.T. Baker Avantor Performance Materials, Inc. (Center Valley, PA). THF (HPLC grade) and sodium trifluoroacetate were purchased from Sigma-Aldrich. 2.2. Reaction Procedures. A typical polymerization reaction was conducted starting with 100 μL of 25 mM aqueous L-malic acid (pH unadjusted), which was measured to be pH 2 before and after esterformation reactions. The sample dryness, when required, was achieved by airflow delivery using a custom-built machine (described below) with reaction temperatures that ranged from 50 to 95 °C. 2.2.1. Hydrolysis Studies. A polymer sample was prepared by incubating dry L-malic acid at 85 °C for 7 days. The sample was then dialyzed using a 500 Da cutoff membrane (Float-A-Lyzer, Spectrum Laboratories) to remove any remaining monomer and dehydration side products (i.e., fumaric acid and maleic acid). During the hydrolysis studies, 25 mM (as malic acid monomer units) of aqueous polymer mixture was incubated at temperatures ranging from 70 to 95 °C with aliquots being taken at regular time intervals for analysis by gel permeation chromatography (GPC), mass spectrometry (MS), and NMR spectroscopy to determine the rate of polymer hydrolysis. 2.2.2. Environmental Cycling. Environmental cycling (temperature and water activity) was performed using a custom-built device. A BioRad MyCycler 96-well thermal cycler was used to control sample temperature during polymerization and hydrolysis cycles. In a typical experiment, the 25 mM L-malic acid solution was allowed to dry at 85 °C for 18 h, followed by a 30 min interval at room temperature, during which the samples were rehydrated, a sample for analysis was extracted, and the vials were capped. The samples then were incubated at temperatures ranging from 60 to 85 °C for 5.5 h (60.0, 61.9, 64.9, 69.4, 75.3, 80.0, 83.1, and 85.0 °C, as reported by the gradient function on the thermal cycler). Room air, dried by a Wilkerson refrigerated dryer (A01-AH-P00), was delivered at 50 °C needle inlet temperature (6% relative humidity) via a 96-needle heated gas manifold adapted from a SPE Dry 96 plate dryer (Biotage). Air delivery was controlled by a microprocessor, with intervals of 3 h on and 1 h off. 2.3. Polymer Characterization. Dry samples were dissolved in deionized water to a concentration of 25 mM (monomeric unit) prior to chromatographic analysis. Polymers were separated by size using gel permeation chromatography (GPC) on an Agilent 1260 HPLC with a Phenomenex PolySep-2000 column, preceded by a guard column. Polymers and unreacted monomers were detected and quantified based on UV absorption at 220 nm. 250 mM sodium phosphate buffer (pH = 7) was used as an eluent with 0.45 mL/min flow rate and column temperature gradient of 45−55 °C. Chromatograms were deconvoluted as a sum of Gaussians. Briefly, Igor Pro (Wavemetrics) was used to simultaneously fit the multiple peaks in each chromatogram that corresponded to the malic acid monomer, malic acid dimer, and the two intramolecular dehydration side products of malic acid (fumaric acid and maleic acid). The Gaussian curves used fit to each chromatogram were restricted to those having the same variance (FDHM), based on the assumption that molecules with similar retention times on GPC would have similar elution profiles. Gaussian curves fitted in this way to chromatogram peaks associated with the two dehydration side products were then subtracted from the raw data to yield chromatograms of only the malic acid monomer and its oligomers. The relative amount of malic acid

Scheme 1. Schematic Representation of L-Malic Acid Polyesterification Yielding α- and β-Linkages

There are reasons to expect that oligoesters have been part of biochemistry since the origin of life, and therefore, they are more than an abstract model of prebiotic polymer formation. In contemporary life, most membrane lipids are derived from triesters of glycerol, phosphate, and fatty acids. Cutin, a polyester synthesized from ω-hydroxy acids, is a main component of plant cuticle, covering all plant aerial surfaces. Furthermore, hydroxy acids are expected to have existed on the Earth for as long as amino acids, being produced in the same model prebiotic reactions that produce amino acids, and hydroxy acids are found in some meteorites.20 Finally, it has even been suggested that polyesters could have even been the ancestral precursors of polypeptides, being displaced by polypeptides over the course of chemical and biological evolution.21 Consistent with this hypothesis, the ribosome is able to catalyze polyester formation using tRNAs that are charged with α-hydroxy acids instead of α-amino acids, suggesting that the ribosome could have originally produced polyesters.22 We selected the condensation of L-malic acid (MA, Scheme 1) as a model system of prebiotic polyester formation. Malic acid is formed from pyruvate and is part of the Calvin cycle and the citric acid cycle. Malic acid is a simple, possibly prebiotic, molecule. The water solubility of poly(malic acid) (PMA) simplifies experimental protocols, quantification of yields, and modeling of hydrolysis in the solution state. Additionally, PMA has been characterized,23,24 as it is a polymer with biomedical applications.25 Here, we demonstrate that subjecting L-malic acid to repeated wet−dry conditions at varying temperatures supports the formation of oligoesters of steady-state concen1335

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monomer and malic acid dimer in each sample was based upon the area under the best-fit Gaussian curve for each of these species. Malic acid oligomers of three monomeric units in length and longer were not fitted with individual Gaussian curves. The quantification of malic acid associated with these oligomers was determined by calculating the integrated area under the chromatogram from 17 to 21 min, less the area contributed by the monomer and dimer malic acid species. Integrated areas assigned to monomer, dimer, and longer oligomers in the 220 nm-monitored chromatograms were determined to be adequate representations of relative MA mass within each species. Specifically, the overall integrated intensity of chromatograms for samples of the same total malic acid concentration, but containing different distributions of MA between monomer and oligomers, exhibited total integrated intensities that were within experimental variation. The chromatograms of the malic acid species were then normalized by the total malic acid peak area to correct for the minor experimental variations between the GPC measurements of samples (e.g., variations in absolute volumes loaded on column). 1 H NMR spectra were recorded on a Bruker DRX500 spectrometer at 25 °C. Spectra of polymer, monomer, and other relevant compound samples in D2O were used for assignments. Reaction conversion was checked for several typical cases using polymer samples in 10% D2O at 25 °C; the water signal was suppressed using a WATERGATE pulse sequence. For MS analysis, the aqueous polymeric solution was lyophilized at −60 °C (trap temperature) at a pressure of 20 mTorr; the residue was then dissolved in THF/MeOH (v/v 50:50) containing 10−5 M sodium trifluoroacetate. MS analysis was performed using a Synapt G2 high definition mass spectrometry system (Waters Corporation, Manchester, UK), which is a hybrid quadrupole-ion mobility-orthogonal acceleration time-of-flight instrument, with typical resolving power of 20 000 m/Δm (fwhm) and mass accuracy of 9 ppm at m/z 554.2615. The instrument was operated in negative ion mode with a probe capillary voltage of 2.5 kV and a sampling cone voltage of 45 V. The source and desolvation temperatures were set to 120 and 250 °C, respectively; the nitrogen desolvation flow rate was set to 650 L h−1. The mass spectrometer was calibrated across the 50−1200 m/z range using a 0.5 mM sodium formate solution prepared in 90:10 2propanol:water v/v. Data were mass corrected during acquisition using a leucine encephalin (m/z 554.2615) reference spray (LockSpray) infused at 2 μL min−1. The scan time was set to 1 s. Data acquisition and processing were carried out using a MassLynx v4.1 and Drift Scope v2.1 (Waters Corp.) The mass spectrometer interfaces with an Acquity UPLC system fitted with a C18 (BEH 1.7 μm, 2.1 × 50 mm) column. A gradient flow of 0.300 mL min−1 using (A) H2O + 0.1% acetic acid and (B) acetonitrile was employed according to the following regimen: 0 min, 95% A; 8 min, 5% A; 9 min, 95% A; and 12 min, 95% A. The column temperature was set at 50 °C. 2.4. Modeling of Malic Acid Polymerization. A mathematical model of the malic acid polymerization under cycling conditions was developed from the models of Harshe et al. for the polycondensation of lactic acid.18 A simple condensation polymerization can be described as

of the water, but here, the volume changes significantly over time due to water evaporation and therefore cannot be neglected. The volumes of water and malic acid are assumed to be additive. The balance of water can be written as ∞

⎛ P*x ⎞ R y ,w = K y ,w ⎜ w w ⎟ = KPx w ⎝ Pt ⎠

i−1

∞

∑

zj −

j=i+1

∞

2k1zi ∑ zj − k −1W (i − 1)zi) j=1

(4)

3. RESULTS 3.1. Formation and Hydrolysis of Malic Acid Polymers at Moderate Temperatures. Prior to designing experiments for polymerization of L-malic acid (MA) by wet−dry and thermal cycling, it was necessary to determine the rate and extent of MA polymerization in single-step drying-heating reactions. For these initial experiments, 100 μL of 25 mM MA was dried and then maintained for various lengths of time at various temperatures. During the drying phase, samples were maintained at 50 °C for a period of 12 h. To facilitate drying, dehumidified, heated air (as described in Materials and Methods) was blown on samples during this stage of the reaction for a cumulative time of 8 h. Samples were then held at one of eight different elevated temperatures spanning from 70 to 95 °C for an additional 12 h. Figure 1 shows GPC analysis of the products resulting from this solid state condensation of MA. The retention time of unreacted MA monomer was determined to be 21.3 min, with MA oligomers eluting at shorter times. MA dimer and trimer elution peaks are resolved by GPC, but longer oligomers elute as a continuous, broad peak. A quantitative analysis of MA polymerization products, obtained by fitting monomer and dimer elution peaks in GPC chromatograms as the sum of Gaussians (section 2.3), revealed that the unreacted MA monomer in the 70 °C sample accounted for almost 80% of the total material, with around 20% being MA dimer, and only 0.4% being converted to trimer and higher oligomers (based on integrated area under the chromatogram, Materials and Methods). The degree of ester formation (or degree of polymerization) increased substantially with temperature, with the 95 °C sample exhibiting 52% of the total material being contained in trimer and longer oligomers, 34% as MA dimer, and less than 15% as unreacted monomer. The stacked plot of chromatograms in Figure 1 also reveals the growth of a peak with a retention time of 21.5 min that becomes substantial above 85 °C (labeled with an asterisk). This retention time, which is slightly shorter than that of the MA monomer, suggests a molecule (or molecules) of molecular weight or hydrodynamic radius that is smaller than that of the MA monomer. This peak was suspected to be fumaric and/or maleic acid, which are MA degradation products that result from the intramolecular dehydration of malic acid during heating in the dry state. Fumaric acid was previously reported as a side product in neat MA polymerization reactions carried

where Pn and Pm are polymer chains of arbitrary length n and m. Following Harshe, a balance on polymer chains of length i can be written as

dzi 1 = (k1 ∑ zi − jzj + 2k −1W dt V j=1

(3)

Here Pw* and Pt are respectively the vapor pressure of water and the total pressure, and xw is the mole fraction of water in the liquid phase. The mass transfer coefficient is Ky,w, and KP also incorporates the pressure terms. The water fraction in the gas phase is assumed to be zero because it is negligible relative to the vapor pressure of water at the polymerization temperatures. All simulations and calculations were performed in MATLAB, using the patternsearch function for error minimization and the ode15s function for integration of eqs 2 and 3.

(1)

k −1

∞

where the rate of water removal by evaporation is

k1

Pn + Pm HooI Pn + m + H 2O

∞

dW = k1 ∑ zi ∑ zj − k −1W ∑ (i − 1)zi − R v , w dt i=1 j=1 i=1

(2)

where zi is the number of moles of Pi in the system, W is the number of moles of water, and V is the system volume. Harshe formulated the equations in terms of concentrations, by neglecting any volume change 1336

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in the estimation of kinetic constants for MA polymerization or in subsequent experiments discussed below. While useful for quantification, it is not possible to resolve malic acid oligomers by GPC beyond trimers. Negative mode ESI-MS analysis was therefore used to identify higher-order oligomers. This analysis was performed on a sample prepared by solid-state polymerization of malic acid at 85 °C, which was subsequently dialyzed against water using a 500 Da cutoff membrane in order to decrease the monomer signal. The highresolution mass spectrum of this sample is shown in Figure 2.

Figure 2. Mass spectrometric analysis of malic acid oligomers produced by heating malic acid (MA) in the dry state. Negative mode electrospray ionization (ESI) mass spectra of the poly-MA (PMA) sample prepared by incubation of solid MA at 85 °C for 72 h, followed by dialysis using 500 Da cutoff membrane to minimize the malic acid signal, which reveals MA oligomers of up to at least 10 monomeric units. All labeled species correspond to [M−H]− ions.

Figure 1. GPC analysis of PMA formed by solid state condensation as a function of temperature. The monomer, dimer, and trimer peaks are labeled on the 85 °C chromatogram because these peaks are most easily identified for reactions at this temperature; reactions carried out at lower temperatures show minimal dimer formation and reactions carried out at higher temperatures show increasing amounts of dehydration side products (i.e., fumaric acid and maleic acid; marked with an asterisk). Samples were maintained at the temperatures indicated on each chromatogram for 12 h, after first being dried from a 25 mM solution at 50 °C for a period of 12 h.

The signals observed in the mass spectrum are consistent with MA oligomers of up to at least 10 monomeric units in length. Assignments to oligomeric species were made, in all cases, with mass accuracies better than 10 ppm. It is possible that branched MA oligomers are also generated during solid-state polymerization, as each MA has two carboxylic acid groups. Such oligoesters would not be resolved by GPC or distinguished by mass spectrometry from linear structures. However, in the present study, our primary focus was to find reaction cycles that promote the formation of oligomers of three or more MA monomeric units, which would include linear or branched oligomers. Thus, determination of structural heterogeneity was not pursued as part of this study. 3.2. Estimation of Kinetic Rate Constants. To estimate the polymerization rate constant k1, a monomer solution of 25 mM was heated under cyclic temperature conditions in an open vial, over a period of 4 days. During the first 12 h of each day, the temperature was held at fixed temperatures ranging from 70 to 95 °C, and during the second 12 h, the temperature was lowered to 50 °C. A significant amount of water was generated during the condensation polymerization, and thus hydrolysis and water evaporation were both included in the estimation of the polymerization rate constant. Hydrolysis rates for poly-MA (PMA) were determined by measuring the change in product distribution (by GPC) of dilute aqueous solutions of preformed PMA heated at various temperatures between 70 and 95 °C (Figure S3). Prior to heating, the PMA stock solution was dialyzed against water using a 500 Da cutoff membrane to reduce the amount of MA monomer in the stock solution of preformed PMA. During heating, the vials containing the PMA solutions were sealed to prevent evaporation. Control experiments in which MA

out at 110−130 °C and as the main product for reactions carried out at 150 °C or higher.17 The 21.5 min peak was confirmed as MA dehydration products by the coinjection of fumaric acid and maleic acid authentic standards with products of MA polymerization reactions. Furthermore, assignment of this chromatographic peak is also supported by NMR and MS analysis of the same samples that contain 1H resonances and accurate masses, respectively, which indicate the presence of fumaric and/or maleic acid (Supporting Information, Figure S1). 1 H NMR spectra of products from a malic acid polymerization reaction indicate that the amount of fumaric acid and maleic acid produced during a single drying cycle does not exceed 1.4% (Figure S2). However, the extinction coefficients of fumaric acid and maleic acid at 220 nm are 75 and 137 times greater, respectively, than that of MA (Table S1), which explains why such minor side products are detected in UVmonitored chromatograms (Figure 1). Fortunately, fumaric acid and maleic acid are chromatographically separated from the polymeric products of MA. Furthermore, hydrolysis of malic acid polymers confirmed that only minimal amounts of maleic and/or fumaric acid are incorporated into MA polymers (Figures S1B). Thus, these side products were not considered 1337

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monomer was kept in solution at 25 mM for 8 days at temperatures from 70 to 95 °C showed no evidence of polymer formation. Thus, the forward polymerization reaction was assumed to be negligible in these solution-phase hydrolysis experiments. Using our experimental data, the polymerization and hydrolysis rate constants were then estimated using eqs 2−4. Based on the observation that the water in a 25 mM MA solution evaporates in 15 min, a mass transfer coefficient of KP = 0.022 mol/h was estimated using eqs 3 and 4. To estimate the kinetic coefficients at each temperature, the sum-squared error between the model prediction and the experimental measurements was minimized. At each time point, the relative intensities of the monomer, dimer, and the combination of all larger species were calculated from the GPC measurements and were compared to the predictions from the model, assuming that each monomer (free or in polymer) contributes equally to the GPC-measured intensity (Materials and Methods). A comparison of the experimental data with the optimized kinetic values is shown in Figure S4. For both the polymerization and hydrolysis rate constants, the experimentally derived values display a linear behavior between ln(k) and 1/T (Figure S5), providing forward and reverse rate constants by the equations k1 = (3.0 × 1015) exp(−1.5 × 104/T) and k−1 = (1.9 × 108) exp(−8.6 × 103/T). The units on the rate constants are M−1 h−1, with T in K. The R2 value for the polymerization reaction fit is 0.965, and for the hydrolysis reaction R2 is 0.999. The data for the polymerization reaction has a noticeable curvature (Figure S4A), indicating a more complicated reaction than the secondorder bimolecular model. As one possible source of additional complexity, this reaction is expected to be sensitive to the viscosity of the reaction milieu, since chain ends must come in close proximity to react. As the system dries, milieu viscosity will increase, reducing the effective value of the rate “constant” k1. Furthermore, the amount of water remaining in the nominally dry state will depend on drying temperature. In contrast, the hydrolysis reaction is expected to be more ideal, since the solutions will have the same water activity for all temperatures, and viscosity should be less dependent on temperature for the hydrated samples. 3.3. Prediction of a Polymerization Ratchet by Malic Acid in Wet−Dry Cycles. The polycondensation kinetics model described in section 2.4 was used to predict the timedependent oligomer and water compositions under cyclic conditions. The cycle consisted of hot, dry phases lasting 18 h, 30 min ambient periods, and cold, wet phases lasting 5.5 h, according to the 24 h cycle described in section 2.2.2. In particular, the temperature during the hot/dry phase (Td) was held constant at 85 °C, while the temperature during the cool/ wet phase (Tw) was varied between 60 and 85 °C. The temperature-dependent values of the rate constants were calculated using the fits presented in the previous section. As shown in Figure 3A, the mass fraction of MA present as free monomer is predicted to decrease during each dry phase and increase during each wet phase. Overall, less free monomer is present for lower values of Tw, since hydrolysis rates decrease with temperature. Correspondingly, the plot in Figure 3C shows opposite trends for MA trimers and longer MA oligomersproduction of these species is promoted during the dry phases, with loss due to ester hydrolysis during the wet phases. However, a crossover behavior can be seen for the dimer concentration during each dry phase after the initial dry

Figure 3. Model prediction of the system composition under a cyclic environment: (A) monomer concentration; (B) dimer concentration; (C) trimer-and-longer concentration. All concentrations are normalized to represent the fraction of monomer in each species. The temperature during the wet phase (Tw) is denoted by curve color, with red for 85 °C and blue for 60 °C (intermediate Tw are provided in Materials and Methods). The dry temperature is 85 °C for all cases. Dashed vertical line marks the end of the first dry subcycle, whereas solid line indicates the end of the first wet subcycle.

phase (Figure 3B). For high Tw, net production of dimers is observed during the dry phase, as monomers form dimers, while at low Tw, dimers are lost during the dry phase due to their incorporation into longer MA oligomers. 3.4. Experimental Demonstration of a MA Polymerization Ratchet. MA polymerization experiments driven by repeated wet−dry cycles were performed using a home-built environmental cycling machine (section 2.2.2). Briefly, 25 mM solutions of MA were subjected to multiple cycles in which samples were dried at 85 °C for 18 h under the flow of dehumidified air (dry phase), then rehydrated at room temperature, and incubated in sealed test tubes at temperatures ranging from 60 to 85 °C for 5.5 h (wet phase). Samples were collected at the end of dry and wet phase and analyzed by GPC. The normalized distributions of MA between monomer, dimer, and longer oligomers, as derived from GPC analysis, are plotted in Figure 4 as a function of cycle. As expected, MA monomer fractions decrease during the dry phases and increase during the wet phases, whereas the fractions of trimer and longer oligomers exhibit the opposite behavior. The dimer 1338

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of oligomers being maintained between cycles and with fewer cycles being necessary to reach steady state levels. At the highest temperature used during the wet phase (85 °C), trimer and longer oligomers formed during the dry phase were almost completely hydrolyzed back to MA monomers and dimers after each wet phase. Examination of MA distributions for individual samples further emphasizes the difference between MA samples that are subjected to various temperatures during the wet phase of each cycle. The MA product profiles for the final (i.e., eighth) dry and wet phases, as derived from GPC chromatograms, are shown in Figure 5 for the samples with the two extreme wet-

Figure 5. Deconvolved GPC chromatograms of representative samples after their eighth dry and eighth wet phases, for cycles with the wet phase temperatures (Tw) of 60 and 85 °C. Black lines are the chromatograms with the degradation products removed. Red lines represent the monomer and green the dimer; both are based on Gaussian fits. Blue lines are the residuals used for calculation of trimerand-longer products. Panel A corresponds to the lowest wet phase temperature (Tw of 60 °C) after the eighth dry cycle, and panel B corresponds to the same sample after the subsequent dry cycle. Panel C corresponds to the highest wet phase temperature (Tw of 85 °C) after the eighth dry phase, and panel D corresponds to the same sample after the subsequent wet cycle. All vertical axes are normalized with respect to the total integrated intensity of the black traces. The total area integrations were performed between retention times of 18 and 22 min. The arrows indicate the retention time cut offs at which 1, 5, and 25% fraction of the total area eluted.

Figure 4. Population versus time profiles in the cycling environment: (A) monomer concentration; (B) dimer concentration; (C) trimerand-longer concentration. All concentrations are normalized to represent the fraction of monomer in each species. The temperature during the wet phase (Tw) is denoted by curve color, with red for 85 °C and blue for 60 °C (intermediate Tw values are provided in Materials and Methods). The dry temperature is 85 °C for all cases. Samples are taken at the end of each dry and wet period; the lines connecting the symbols are used to guide the eye. The experimental data points for Tw of 60 and 85 °C are represented as ×’s and circles, respectively.

fraction exhibits a more varied behavior, as it increases or decreases during the wet period, depending on temperature. Overlaying theoretical and experimental results illustrates the excellent qualitative agreement, and even reasonable quantitative agreement, between model predictions and actual data (Figure S7). The ratchet mechanism exhibited by MA polymerization during repeated wet−dry cycles is most apparent from the change in the fraction of trimer and longer MA oligomers for the sample that experiences the wet phase at 60 °C (the 60 °C wet series). Following the fraction of MA oligomers present in this sample at the end of each dry phase shows a clear increase in the fraction of MA in longer oligomers that begins at 30% after the first dry phase and increases to a steady-state value of 53% after six cycles. Between dry phases, the fraction of these longer oligomers drops to only 10% after the first wet phase, but it eventually reaches a steady state level of 32% after seven cycles. The behavior of samples subjected to wet phases at higher temperatures is similar, except with decreasing amounts

phase temperatures (Tw) of 60 and 85 °C. Figures 5A and 5C correspond to the chromatograms of the samples collected after the eighth dry phase. The product distribution in these samples is similar as indicated by the close retention times at which 1%, 5%, and 25% of the total material elute. At the end of the eighth wet phase, the sample from the 60 °C-wet series (Figure 5B) still contains MA oligomers three and more units long, whereas the sample from the 85 °C-wet series (Figure 5D) is almost completely hydrolyzed to monomers and dimers. Survival of the trimer and longer oligomers in the wet phases of the 60 °Cwet series allows for their elongation during the dry phases. A comparison of the 60 °C-wet series products after the eighth dry phase (Figure 5A) with the 85 °C-wet series products after the same dry phase (Figure 5C) reveals an oligomer distribution that is similar, but noticeably weighted 1339

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toward the longer species (i.e., shorter retention times) in the 60 °C-wet series. Based on the retention times measured for MA oligomers, the chromatograms in Figures 5A and 5C reveal malic acid oligomers of at least 8 monomeric units in length to be present after the eighth dry phase for the 60 °C-wet series and 85 °C-wet series. The existence of oligoesters of at least this length is supported by the fact that mass spec analysis of reaction mixtures that exhibit MA oligomer GPC retention times exclusively >19.5 min contain MA oligomers that are at least 10 mers (e.g., Figure 2). The chromatogram shown for the 60 °C-wet series after its eighth wet phase (Figure 5B) still shows that about 1% of MA oligomers persist through the wet phase with a length of 8 or more monomeric units. Thus, a MA oligomer length of at least 8 monomeric units is supported once steady state kinetics have been reached for the 60 °C-wet series, whereas the maximum length of oligomers persisting through the wet phases of the 85 °C-wet series appears to be limited to trimers (Figure 5D). 3.5. Predicting Existence of Poly-MA Polymers over a Wider Rage of Cycle Durations. Given the good agreement observed between our predictive model and experimental data with MA polymerization in wet−dry cycles, our model was used to predict conditions that would support far-fromequilibrium MA polymers over a greater range of parameters. For these predictions, the duration of the wet phase, the wetphase temperature, and the dry-phase temperature were varied, while the total cycle length remained fixed at 24 h. In particular, this parameter space was explored for conditions that would lead to sustained MA oligomer existence. We quantify this concept of oligomer existence as the fraction of oligomers that are trimers or longer, after the end of eight complete cycles. Since each cycle ends with a wet phase, during which significant depolymerization may occur, these predictions are focused on those conditions that will lead to the steady state persistence of polymers through the wet phase. Figure 6A shows the minimum duration of the dry phase of a cycle required to achieve 1% of MA in trimer-and-longer products after the end of eight cycles. As expected, for higher dry-phase temperatures and lower wet-phase temperatures (upper left corner), we predict that MA oligomers can be sustained with shorter lengths of the dry phase. Over the range of temperatures considered (60−85 °C), the required dryphase duration spans across a wide range. When the dry-phase time is 24 h (i.e., no wet phase), the condition of 1% oligomer existence is satisfied for all temperature combinations investigated, even those in the lower right region of Figure 6A (i.e., when the wet-phase temperature is higher than the dryphase temperature). When the dry-phase temperature is at the maximum considered (85 °C) and the wet-phase temperature is minimum (60 °C), the amount of monomer in trimer or longer is still 2.5% after a dry period of 3 h, with a wet period of 21 h. However, when the dry-phase temperature is minimum (60 °C) and the wet-phase temperature is maximum (85 °C), only 0.1% of monomer is in trimers or longer after a dry period of 21 h and a wet period of 3 h. Thus, there is a wide range of potential cyclic environmental conditions where water can be episodically present (as would be the case on the prebiotic earth), but that does not necessarily prevent MA oligomers from being generated and sustained. A larger threshold value is analyzed in Figure 6B, where conditions are sought that produce MA trimer-and-longer oligomers in an abundance of at least 5%. In this case, compared to Figure 6A, the contours are shifted toward the

Figure 6. Minimum duration of the dry phase required for (A) 1% and (B) 10% of monomers to be incorporated in trimer-and-longer oligomers after eight cycles. All cycle times were 24 h overall, with the dry phase carried out at temperature Td (vertical axis) and incubation in the wet phase at temperature Tw (horizontal axis). Contours are shown for dry phase durations.

upper-left corner, indicating that higher dry-phase temperatures and/or lower wet-phase temperatures are needed for the steady state level of 5% or more of MA in trimer-and-longer species. Additionally, this plot illustrates that for lower values of the dryphase temperature (i.e., 61 °C or lower) it is not possible to achieve 10% in trimer-and-longer species, for any wet-phase temperature of 60 °C or higher (lower gray area of plot in Figure 6B).

4. DISCUSSION Our results show that it is possible to synthesize far-fromequilibrium oligomers from unactivated monomers by simply subjecting these starting materials to multiple wet−dry cycles. In particular, our experiments have shown that oligoesters formed by malic acid (MA) during a dry phase at 85 °C of 18 h duration can persist through a subsequent wet phase of 5.5 h, with the concentration and mean length of oligoester depending on the wet-phase temperature (from 60 °C to at least 85 °C). The MA oligomers of samples in the wet phases of each cycle are far-from-equilibrium molecules for all wet-phase temperatures, as MA monomers are thermodynamically favored over MA oligoesters in aqueous solution. When compared to previously reported attempts to achieve peptide bond formation in model prebiotic reactions with unactivated amino acids, oligoester formation is much more accessible. As noted above, the drying and heating of unactivated amino acids produces essentially only dipeptides and in low yields.12,13 Even with the addition of amino acid activating agents, peptide formation in model prebiotic drying reactions is still limited to very short oligomers.27 Taken together, the results presented here and those of past studies support the hypothesis that 1340

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in backbone structure and variability that would not result from other hydroxy acids that possess only one carboxylic acid group. As mentioned in the description of our experimental system (Materials and Methods), samples were prepared from the free acid form of malic acid, without adjustment of pH. These samples had an initial pH of 2, which did not change appreciably after wet−dry cycling. Thus, in our experiments, ester bond formation and hydrolysis are dominated by acid catalysis. It has been argued that the early Earth’s atmosphere would have had a considerably higher partial pressure of CO2 than today,32 which could have caused the early oceans and pools of fresh water on land to be slightly acidic due to dissolved carbonic acid. However, it is very unlikely that the prebiotic environment would have been as acidic as the samples investigated in this study. We recognize that with increasing pH there will be a reduction in the rate of acid-catalyzed ester formation and an increase in the rate of base-catalyzed hydrolysis of ester linkages. As previously noted by Weber, drying−heating reactions of glyceric acid carried out at neutral pH were about 100-fold slower in ester formation and about an order of magnitude faster in the rate of hydrolysis compared to acidic conditions.15 The malic acid system has been presented as a first step in the demonstration of polymer ratcheting that can be driven by periodic wet−dry cycles. Increased pH of the sample during either phase would be expected to increase the number of cycles required to reach the steady-state level of oligoester concentration and length. We note that acidcatalyzed reactions, including ester formation,33,34 peptide bond formation,13 acetal bond formation,35 and glycosidic bond formation and hydrolysis,36,37 can be catalyzed by general acid catalysis, such as by ammonium ions or hydrated and partially dehydrated divalent cations. Such catalysts are prebiotically plausible and would have been concentrated during drying (when the polymerization reaction is also favored). Thus, another dimension that needs to be explored in order to create a truly plausibly prebiotic scenario for the formation of far-from-equilibrium polymers by a simple environmental cycle is the interplay between pH and inorganic catalysts. Increasing the pH during polymerization would also increase the rate of transesterification. Wolfenden et al. concluded that rate of glycerol-β-monoacetate intramolecular transesterification was 6000 times faster than hydrolysis, with the rate of transesterification being on the order of 0.01 s−1 at pH 6 and 37 °C.38 Thus, we would anticipate that a considerable fraction of oligoesters formed at higher pH would undergo transesterification, which could lead to the formation of cyclic products that depend on the length and flexibility of the oligoester. We note that ionic species with m/z values consistent with the formation of cyclic oligoesters are observed in our mass spectra (Figure 2), and cyclic oligoester macrocycles exist in life today.39 It is therefore reasonable to propose that some functional oligoesters could have been cyclic. Additionally, transesterification-based recombination has been proposed as a mechanism by which nucleic acids (which are phospho-diesters) were rearranged in sequence as part of the prebiotic search for functional sequences.40 The simple process of mixing, or shuffling, the sequences of pre-existing oligoesters through transesterification could have been an important process as well.

oligoesters (or even polyesters) could have appeared on Earth at the same time as polypeptidesif not earlier. Moreover, the reduced stability to hydrolysis of esters relative to amides could have allowed polyesters to undergo sequence evolution more readily than polypeptides, before the advent of complex cellular machinery (discussed below). Our theoretical models of MA oligoester formation and hydrolysis driven by wet−dry cycles indicate a considerable range of temporal and temperature phase space could support the persistence of far-from-equilibrium oligoesters. While we have only modeled up to eight wet−dry cycles, corresponding to the maximum number of cycles investigated in our experiments, it is easy to imagine that far-from-equilibrium polymers on the prebiotic Earth were the result of many wet− dry cycles under conditions that might have taken years to reach steady state. While experimental models of monomers subjected to hundreds, or even thousands, of wet−dry cycles may be necessary to demonstrate the true nature of far-fromequilibrum polymers that could have existed on the early Earth, such experiments are not currently practical. Nevertheless, the possibility of a polymerization ratchet that relies upon very small forward polymerization rates, and correspondingly slower hydrolysis rates, is very attractive as a model for the origin of the first biopolymers. Such a system could have allowed the emergence of functional polymers under conditions that minimize the degradation or irreversible reaction of monomers. For example, as illustrated in the present study with MA, intramolecular dehydration of the hydroxy acid at higher temperatures might have interfered with the long-term steady state production and recycling of polyesters by reducing the concentration of hydroxy acid monomers. While the highly labile nature of soluble oligoesters could be perceived as a disadvantage for proto-biopolymers, the ability for a prebiotic system to repeatedly cycle building blocks between the monomeric and polymeric states would have been essential for exploring polymers with different sequences in order to find functional polymers that advanced their own survival, either by adopting stable secondary structures (thereby reducing their rate of spontaneous hydrolysis)28 or by catalyzing beneficial reactions (e.g., catalyzing monomer formation).29 The true advantage of reversible polymerization, from the standpoint of chemical evolution,30,31 would be best demonstrated from a starting pool of mixed monomers that allow for the production of polymers with a diversity of monomer compositions and sequences. In the present study, we have only examined ester formation by malic acid, restricting our system to oligoesters that are homogeneous in composition and sequence. Nevertheless, these studies were necessary to demonstrate that hydroxy acids and oligoesters can function respectively as monomers and oligomers from which far-from-equilibrium polymers can be created and maintained by a model environmental cycle. To demonstrate the evolution and selection of oligoesters based on functional selection will require a starting pool of mixed hydroxy acids. Such studies will require more sophisticated analytical approaches due to the greater complexity inherent to a mixed-sequence system. Additionally, the dicarboxylic acid nature of malic acid may facilitate oligoester formation, and therefore polymerization reactions with other hydroxy acids may not proceed as readily as reported here for malic acid. On the other hand, as noted in the Introduction, malic acid produces both α- and β-linkages in thermally produced oligoesters, which also presents a level of complexity 1341

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5. SUMMARY AND CONCLUSIONS Polymer formation by the drying and heating of monomers has long been hypothesized as an early and important step in the origin of life, before the advent of enzymes. However, model prebiotic reactions have typically utilized chemically activated monomers or have been carried out in solutions containing condensing agents. In particular, peptides have been formed by the drying of amino acids in the presence of dehydrating reagents, such as water-soluble carbodiimide41 or pyrophosphate.42 Model prebiotic studies of de novo oligonucleotide formation have most often used monomers with phosphate groups activated by imidazole or related molecules.43,44 The use of condensing agents introduces additional complexity to a chemical system and limits the ability for the system to recycle monomers without a mechanism for monomer reactivation, which renders such systems, in our opinion, less prebiotically plausible. Previously reported possible prebiotic dry-state polymerization reactions performed without activating reagents proved to be of limited success. Examples of such reports include the synthesis of peptides12,13 and of nucleic acid analogues,35 both of which resulted in low yields (about 1%) of oligomers and chain lengths mostly limited to dimers and trimers. Successful dry-state polymerization of monomers still presents challenges to prebiotic polymer formation and evolution. The dry state would prevent the manifestation of biopolymer properties, such as folding into a functional form, ligand binding, and catalysis. However, dissolution of preformed polymers in water to achieve functionality does not address their susceptibility toward hydrolysis. Thus, an important demonstration of the current study is that a system of regular wet−dry cycles, starting with unactivated monomers, can result in the formation of far-from-equilibrium polymers. Although the achievement of far-from-equilibrium polymers by such a process has long been speculated as an important prebiotic mechanism for the formation of biopolymers (or proto-biopolymers), this current study represents, to the best of our knowledge, the first experimental demonstration of the creation of a steady state of oligomers that continuously recycle with the monomeric state and which was initiated from a pool of unactivated monomers. We have termed the process by which a steady state of coexistence of oligomers and monomers is driven wet−dry cycles a “polymerization ratchet” to emphasize the fact that, despite regular hydrolysis of oligomers during the wet phase of each cycle, oligomers subjected to multiple cycles grow to concentrations and mean lengths that are greater than those produced in a single dry phase. Given the simplicity of such a system that exhibits properties believed to be important for the origin and early evolution of the first proto-biopolymers, we propose that polymerization ratchets were important mechanisms in the emergence of the first functional biopolymers. In terms of possible prebiotic environments that would have driven polymerization ratchets, wet−dry cycles could have existed on arid coastal areas that are even common at the present time. Limitations of the described model include the experimental setup that deviates from the prebiotically plausible pH and temperature ranges and the synthesis of a product that is not a contemporary biopolymer. Nevertheless, our model enables demonstration on a laboratory time scale of dehydration condensation of polymers in aqueous solution with monomer recycling.

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ASSOCIATED CONTENT

S Supporting Information *

NMR spectra of reaction products, extinction coefficients, chromatograms of PMA subjected to hydrolysis, plots of data used to determine rate constants, comparisons of experimental data, and kinetic models. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (N.V.H.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Drs. Manshui Zhou and David Gaul for assistance with MS analysis. This work was jointly supported by the NSF and the NASA Astrobiology Program, under the NSF Center for Chemical Evolution, CHE-1004570, and the NASA Astrobiology Institute.

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REFERENCES

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