Letter pubs.acs.org/Langmuir
Amplification of Chirality through Self-Replication of Micellar Aggregates in Water Konstantin V. Bukhryakov, Sarah Almahdali, and Valentin O. Rodionov* KAUST Catalysis Center and Division of Physical Sciences and Engineering, King Abdullah University of Science and Technology, Thuwal 23955-6900, Kingdom of Saudi Arabia S Supporting Information *
ABSTRACT: We describe a system in which the selfreplication of micellar aggregates results in a spontaneous amplification of chirality in the reaction products. In this system, amphiphiles are synthesized from two “clickable” fragments: a water-soluble “head” and a hydrophobic “tail”. Under biphasic conditions, the reaction is autocatalytic, as aggregates facilitate the transfer of hydrophobic molecules to the aqueous phase. When chiral, partially enantioenriched surfactant heads are used, a strong nonlinear induction of chirality in the reaction products is observed. Preseeding the reaction mixture with an amphiphile of one chirality results in the amplification of this product and therefore information transfer between generations of self-replicating aggregates. Because our amphiphiles are capable of catalysis, information transfer, and self-assembly into bounded structures, they present a plausible model for prenucleic acid “lipid world” entities.
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INTRODUCTION The capacity for self-replication is a fundamental property of life. To explore potential prebiotic processes, a number of artificial self-replicating systems have been devised.1−3 Among these, self-replicating micelles4,5 and vesicles6 are of particular interest7−9 as they are geometrically bounded structures not entirely dissimilar from a living cell. Self-replicating micelles can compartmentalize information and function, which is a necessary condition for evolutionary selection.10,11 The self-replication of such structures is defined differently compared to template-guided self-replication of small molecules or sequenced polymers.9 Bounded structures are considered to be self-replicating if their population can grow due to a chemical reaction that proceeds within their confines and is enabled and/or accelerated by the intrinsic properties of the structures.12 The generalized mechanism of self-replication of micellar aggregates starts with the phase transfer of reagents from the environment into the structure boundaries. Additional amphiphile molecules are then produced within the aggregates, which grow and multiply in number. The amphiphile-making reaction is accelerated within the aggregates due to solvent effects, local preconcentration, or the aggregates’ capacity for phase-transfer catalysis.13,14 Examples of self-replicating aggregates based on both bond-breaking4,12 and bond-making reactions5,15 under biphasic conditions have been described, including one instance involving chiral amphiphiles.16 Whereas examples of simple self-replication driven by micellar phase-transfer catalysis are not unusual, the capability of the self-replicating micelles for selective autocatalysis remains © 2015 American Chemical Society
to be explored. Either self-selective or crossover-selective selfreplication of bounded aggregates can provide a rudimentary mechanism for intergenerational information transfer (encoded into chemical structures). Here, we describe an example of strongly self-selective, autopoietic aggregates that grow via a bond-making copper-catalyzed azide−alkyne cycloaddition (CuAAC) reaction.17,18 Our aggregates are capable of discrimination between the two enantiomers of the starting material.
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EXPERIMENTAL METHODS
We chose to explore the behavior of the reaction between the dialkyne binaphthylphosphate 1 and heptyl azide 2 under biphasic conditions (Scheme 1), similar to the classic ethyl caprylate hydrolysis experiment reported by Luisi.4 Our choice of compound 1 was motivated by the pronounced asymmetry of the binaphthyl moiety and by the synthetic accessibility of both enantiomers. The possible products of the reaction are amphiphilic mono- and ditriazoles 3 and 4, the latter of which is a twin-tailed amphiphile loosely reminiscent of natural lipids.19 All reactions were performed in a biphasic system consisting of equal volumes of an aqueous solution of 1 (2.5 mM) and a hexane solution of 2 (2.5 mM). Copper sulfate and sodium ascorbate were added to the aqueous phase to generate CuI species and start a CuAAC reaction between 1 and 2. The solubility of heptyl azide 2 in water is negligible. Likewise, neither 1 nor the copper catalyst has any appreciable solubility in hexane. Thus, we expected the direct interphase reaction to be slow. Once micelles of amphiphile products 3 and 4 start forming, phase Received: December 25, 2014 Revised: February 10, 2015 Published: March 5, 2015 2931
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Langmuir Scheme 1. Biphasic CuAAC Reaction between Dialkyne Binaphthylphosphate 1 and Azide 2
Figure 1. Concentrations and ee’s of triazoles 3 and 4 as a function of time under various reaction conditions. Curves were generated by the leastsquares fit of data to empirical Hill-type sigmoidal functions (details in SI). Each data point is an average obtained from three independent experiments. (a) 2.5 mM dialkyne 1 and 2.5 mM azide 2; (b) 2.5 mM dialkyne 1, 2.5 mM azide 2, and 250 μM S-ditriazole 4; (c) 2.5 mM dialkyne 1, 2.5 mM azide 2, and 250 μM racemic ditriazole 4; and (d) 2.5 mM dialkyne 1, 2.5 mM azide 2, and 16.4 mM SDS. transfer of azide to the aqueous phase can be accelerated, correspondingly enhancing the reaction rate. To monitor the reactions of both enantiomers of 1 independently, we introduced an 18O isotope label into the R-1 enantiomer. The rate of 18O exchange between R-1 and water was negligible under the reaction conditions (Figure S1, SI). The degree of completion and enantiomeric composition of the reaction mixtures could be conveniently monitored by LC-MS (section 5, SI). Aliquots were taken from the reactions at regular intervals and quenched by diluting them with acetonitrile. We sought to identify nonlinear chiral induction effects in the reaction kinetics. Because such effects can manifest either as amplification or negation of the enantiomeric excess (ee), we used a mixture with a 50% ee of S-1 (i.e., 25% 18O-labeled R-1 and 75% S-1) in all of the experiments. The same rationale was behind selecting azide 2 as a limiting reagent: all reactions were performed with 1 equiv of 2, unless indicated otherwise.
minority enantiomer R-1 had a visible induction period (Figure S3a, SI), no steplike speed-up could be observed for the reaction of the major S enantiomer. Furthermore, the initial rate of the reaction increased for both enantiomers with the increase in concentration of azide 2 in the organic phase (Figure S5a-b, SI). Therefore, although 1 is practically insoluble in hexane, it is capable of acting as a phase transfer catalyst in its own right. Thus, the transfer of azide 2 to the aqueous phase is enabled, and the 1 → 3 CuAAC reaction takes place. We did not observe any significant reaction speed-up for the major enantiomer concomitant with the formation of 3, which suggests that monotriazole 3 is not a significantly better phase transfer catalyst compared to 1. The cause of the enhancement of the ee of monotriazole 3 was revealed after we performed LC-MS analyses of aqueous and methanolic solutions of the 50% S-enantioenriched RS-1 mixture. Both samples were filtered through 0.2 μm PVDF membrane filters. For the methanol solution, the observed ee was the expected 50%. The ee of the filtered aqueous sample,
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RESULTS AND DISCUSSION We discovered that ee of triazole products 3 and especially 4 is enhanced in the initial stages of the reactions compared to that of starting material 1 (Figure 1a). Whereas the reaction of 2932
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Figure 2. Proposed mechanism of chiral amplification. (a) Formation of M1 aggregates through a sequential 1 → 3 → 4 process. (b) Autocatalytic amplification of S-4 in M2 aggregates.
incorporation of dialkyne 1 into M2 aggregates is stereoselective, with a preference for the same chirality. Because M2 species are negatively charged, this transfer of 1, which also bears negative charge, is likely a relatively slow process and one that is strongly affected by the concentration of 1. A reaction starting with a sufficiently high initial concentration of 1 will be accelerated by M2 due to high local concentrations of both 1 and 2 in the aggregates. Because the concentration of dialkyne 1 in the aggregates is likely lower than the concentration of neutral, hydrophobic 2, ditriazole 4 will be predominantly produced. As the reaction progresses and 1 is depleted, it is no longer transferred to M2, and the accelerated phase is over. The M1 aggregates that consist primarily of 1 and 3 are not as efficient at the phase transfer of azide 2. Thus, the local concentration of 2 within M1s is low, and there is no preference for the formation of ditriazole 4. On the other hand, we expect the M1s to be more disordered compared to the M2s, which makes for an easier and faster incorporation of additional dialkyne 1. Thus, monotriazole is the product that is preferentially produced in the initial stages of the reaction within M1s. This sequential 1 → 3 → 4 reaction predominates in the reactions not preseeded with 4. To test this hypothesis, we performed a reaction in the presence of sodium dodecyl sulfate (SDS), a common anionic surfactant (Figures 1d and S4b, SI). We expected SDS micelles, just like aggregates of the M2 type, to be efficient at transferring hydrophobic azide 2 to the aqueous phase but slow at exchanging with the anionic “head” 1. The crucial difference between SDS and 4 is the strong asymmetry of the latter. Thus, we anticipated that SDS would accelerate the reaction for both S-1 and R-1 and favor the production of ditriazole 4 in the initial stages of both reactions. This is exactly what we observed. Ditriazole products 4, both R- and S-, were the major constituents of the reaction mixture after azide was exhausted, even though the reaction was performed with only 1 equiv of azide. The ee of both products 3 and 4 was amplified relative to that of 1 but not to the same extent as it was in the reactions preseeded with 4 or in the nonseeded reaction. This indicates that some residual long-range order remains in SDS micelles containing 1, 3, and 4, but the self-organization of the binaphthol “heads” is disrupted. Direct insight into the process of aggregation of 3 and 4 was gained from cryo-TEM images of their aqueous solutions
however, was enriched to ca. 95%. This indicates that racemic 1 forms aggregates that are poorly soluble in water and are removed from the reaction in its initial stages. The simple consideration of the differences in solubility between racemic and enantiopure 1 cannot explain the complex evolution of the enantiomeric composition of the reaction mixtures or the sigmoidal kinetics of the conversion of the minor R-1 enantiomer. The evolution of ee’s of monotriazole 3 and ditriazole 4 products were markedly different throughout the reactions (Figures 1a and S3a, SI; green markers). We hypothesized that ditriazole 4 might be a more efficient phasetransfer catalyst than either monotriazole 3 or starting material 1. To test this assumption, we set up a reaction seeded with 10 mol % (250 μM) S-4 (Figures 1b and S3b, SI). The effect we observed was both significant and unexpected. The reaction was strongly accelerated, reaching ∼30% conversion of azide 2 in the first 30 min, compared to just ∼3% for the reaction with no added 4 (Figure S6a,b, SI). Remarkably, the observed acceleration was due to the rapid self-amplification of S-4 in the initial moments of the reaction. Close to 13% of S-1 was converted to S-4 in the first 5 min (Figure S6b, SI). At the same time, the concentration of monotriazole intermediate S-3 remained low, indicating that the S-3 → S-4 step must be extremely fast in the reaction preseeded with S-4. This kinetic behavior is impossible for a simple sequential reaction. The concentration of S-4 stabilized at ∼0.5 mM after 30 min and resumed a slow growth after 8 h (Figure S3b, SI). The rate of conversion of minority enantiomer R-1 was unaffected by the addition of S-4. The reaction seeded with 10 mol % of racemic 4 was also accelerated, albeit not to the same degree as the one preseeded with enantiopure S-4 (Figures 1c and S4a, SI). As in the enantiopure-seeded reaction, the major S-4 enantiomer was amplified. Unexpectedly, there was no amplification of minority R-4, suggesting that a threshold minimum concentration of dialkyne 1 is necessary for the amplification mechanism to operate. This observation, as well as the growth-plateau kinetics for the formation of the majority enantiomer S-4, led us to propose a mechanism for the autocatalytic amplification of 4 (Figure 2). Ditriazole product 4 forms predominantly homochiral aggregates M2, which are competent at catalyzing the phase transfer of azide 2 to the aqueous phase. We assume that the 2933
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OUTLOOK The focused efforts of chemists to synthesize nucleotides and nucleotide-like molecules under plausible prebiotic conditions have met with limited success. This suggests that the spontaneous emergence of template replicators with the degree of complexity necessary for life is unlikely. The observations described here may be particularly relevant to the lipid world theory of the origins of life20 as well as the emerging field of systems chemistry.21,22 Our amphiphiles assemble into structures capable of both function (phase-transfer catalysis) and information transfer (as chirality) between generations. This provides support for the idea that lipidlike, catalytically competent molecules could have played a dominant role in the prebiotic era, before the emergence of more specialized information-bearing structures such as RNA.
(Figures 3 and S11 and S12, SI). Both enantiopure 3 and 4 assemble into ellipsoidal aggregates with an average diameter of
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ASSOCIATED CONTENT
S Supporting Information *
Experimental details of organic synthesis; characterization (1H and 13C NMR and fluorescence spectra, HRMS data); extended conversion plots; and a list of abbreviations. This material is available free of charge via the Internet at http://pubs.acs.org.
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Figure 3. Cryo-TEM images of aggregates of 3 and 4 in water. (a) S-3, ellipsoids; (b) S-4, ellipsoids; (c) RS-3, network; and (d) RS-4, tubule and toroids.
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Tel: +966-128084592.
approximately 20 nm (Figure 3a,b). In 100 μM or more concentrated solutions of S-4, a small number of thin (1 to 2 nm) nanowires could be seen. Finally, we observed twinned/ joined micellar aggregates of S-4 (Figure S11b, SI). These structures could be seen in most samples, irrespective of the concentration of S-4. Assemblies similar to these could be intermediates in the self-replication of S-4 aggregates. The morphologies exhibited by the racemic compounds were significantly more diverse. Racemic 3 assembled into networklike extended structures with roughly spherical nodes connected by thinner isthmuses (Figures 3c and S11c, SI). For racemic ditriazole 4, we observed four major aggregate types: ellipsoids, flat sheets, tubules, and toroids (Figures 3d and S11d, SI). The ellipsoids were prevalent, and their average size was somewhat smaller than that for enantiopure S-4. We further explored the aggregation behavior of 3 and 4 by obtaining their fluorescence emission spectra for a range of concentrations (section 6, SI). The spectra of enantiopure compounds were different from the spectra of corresponding racemates. Furthermore, the change in fluorescence behavior with concentration was distinct for every compound surveyed (Figures S8 and S10, SI). This agrees well with the cryo-TEM observations, which suggest a unique mode of self-assembly for each of the triazole products. Our cryo-TEM and fluorescence studies support the existence of long-range order in the assemblies of both enantiopure and racemic 3 and 4. Furthermore, the propensity of enantiopure 4 to aggregate into highly monotonous ellipsoidal structures (section 7 and Figure S12, SI) suggests that in the initial, autocatalytic stage of the reactions the number of aggregates increases as the reactions progress. Whereas the formation of new aggregates “from scratch” is a possibility, this hypothesis does not readily explain the autocatalysis phenomena or the unusual 1 → 4 kinetic behavior. Therefore, self-replication of the aggregates through growth and partitioning is a more likely operational mechanism.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We are grateful to Dr. Rachid Sougrat for his assistance with cryo-TEM and to Prof. Jean Fréchet for helpful discussions. REFERENCES
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