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. 2024 Oct 17;146(43):29621–29629. doi: 10.1021/jacs.4c10082

Abiotic Acyl Transfer Cascades Driven by Aminoacyl Phosphate Esters and Self-Assembly

Mahesh D Pol †,‡, Ralf Thomann §,∥, Yi Thomann §, Charalampos G Pappas †,‡,*
PMCID: PMC11528443  PMID: 39419499

Abstract

graphic file with name ja4c10082_0005.jpg

Biochemical acyl transfer cascades, such as those initiated by the adenylation of carboxylic acids, are central to various biological processes, including protein synthesis and fatty acid metabolism. Designing cascade reactions in aqueous media remains challenging due to the need to control multiple, sequential reactions in a single pot and manage the stability of reactive intermediates. Herein, we developed abiotic cascades using aminoacyl phosphate esters, the synthetic counterparts of biological aminoacyl adenylates, to drive sequential chemical reactions and self-assembly in a single pot. We demonstrated that the structural elements of amino acid side chains (aromatic versus aliphatic) significantly influence the reactivity and half-lives of aminoacyl phosphate esters, ranging from hours to days. This behavior, in turn, affects the number of couplings we can achieve in the network and the self-assembly propensity of activated intermediate structures. The cascades are constructed using bifunctional peptide substrates featuring side chain nucleophiles. Specifically, aromatic amino acids facilitate the formation of transient thioesters, which preorganized into spherical aggregates and further couple into chimeric assemblies composed of esters and thioesters. In contrast, aliphatic amino acids, which lack the ability to form such structures, predominantly undergo hydrolysis, bypassing further transformations after thioester formation. Additionally, in mixtures containing multiple aminoacyl phosphate esters and peptide substrates, we achieved selective product formation by following a distinct pathway that favors subsequent reactions through reactivity changes and self-assembly. By coupling chemical reactions with molecules of varying reactivity time scales, we can drive multiple reaction clocks with distinct lifetimes and self-assembly dynamics, facilitating precise temporal and structural regulation.

Introduction

Biochemical cascades1−3 form intricate networks of pathways that sustain living processes through highly selective and regulated enzymatic reactions. A prime example is the activation of carboxylic acids by adenylation, which results in the formation of acyl adenylates.4 In protein synthesis, aminoacyl adenylates (mixed phosphoric anhydrides of adenylic acid) transfer to tRNA molecules, forming ester bonds crucial for accurate and efficient peptide bond formation during translation.5 The reactivity of aminoacyl adenylates also enables them to act as acyl donors in transesterification reactions, which is essential in fatty acid metabolism involving thioesters.6 Similarly, luciferyl adenylates, formed by the catalysis of luciferin by luciferase enzymes, are key in the bioluminescence of organisms like fireflies and marine species.7 Overall, these examples highlight the way in which structure and reactivity cross-regulate both primary and secondary metabolic processes.8−10

The complexity and efficiency of biochemical cascades have inspired chemists to develop fully synthetic systems that aim to replicate these processes.11−13 However, achieving precise control and selectivity at every stage of transformation to prevent unwanted side reactions has been a significant challenge.14 To address these challenges, systems chemistry15−17 has emerged as a powerful tool, investigating the interactions and dynamics within chemical reaction networks.18−21 This approach has proven highly effective in constructing complex self-assembling cascades22−25 that involve diverse compound classes26,27 and often incorporate biocatalysts.28−32 Consequently, pathway-driven reconfigurations33−37 have been utilized to impact the properties of these reaction networks, which are highly dependent on molecular architecture,38,39 thermodynamic and kinetic parameters40−44 and assembly processes.45,46

Particularly in kinetically controlled assemblies,47 reaction cycles48−52 have been developed and further coupled with various functions such as self-assembly,53−55 catalysis,56,57 replication,58,59 and responsiveness to external stimuli.60−63 However, most of these systems operate in a single step (assembly and disassembly).64−67 The coupling of two chemical reaction cycles, in which the product of one cycle (an intermediate) serves as the substrate for a subsequent step and primarily influences the second coupling, remains rare. The reasons that remain rare are associated with the solubility and stability of activated building blocks in different environments, especially in water, which has the tendency to hydrolyze labile intermediates.

Herein, we design abiotic cascades driven by alterations in reactivity and self-assembly dynamics. In these systems, amino acyl phosphate esters, initially reconfigure to form thioesters. Subsequently, chimeric assemblies containing both ester and thioester functionalities within a single building block are generated. These coupled chemical reactions are triggered by the structural elements surrounding the phosphate esters, where the amino acid side chains dictate the number of couplings and the self-assembly propensity. The activated amino acids bind covalently to bifunctional peptide substrates featuring side chain nucleophiles, giving rise to further reactions or direct hydrolysis. We demonstrate that aminoacyl phosphate esters exhibit distinct reactivity patterns depending on whether they carry aliphatic or aromatic amino acid residues. This distinction impacts the stability, mechanical properties and structural arrangement of different types of activated amino acids within the cascades. Notably, we observed that when thioester formation occurs with aromatic amino acids, it leads to the fabrication of spherical aggregates, which then facilitate the construction of fibrous structures during subsequent coupling events. This process highlights the importance of thioester assembly in the initial reaction and the significance of intermolecular interactions, which were absent in phosphate esters containing aliphatic residues. Furthermore, when various aminoacyl phosphate esters compete for substrates, controlling reactivity and self-assembly enables the selective incorporation of residues into final structures that are capable of aggregation. Overall, these findings suggest that the structural elements surrounding activated amino acids can impact chemical reaction cycles in water through reactivity changes and self-assembly, without the need for complex enzymatic machinery.

Results and Discussion

Design of Acyl-Transfer Abiotic Cascades

We have previously demonstrated that the amino acid side chains in the structure of aminoacyl phosphate esters dictated the length of the oligomers that were formed during spontaneous aminolytic reactions.69 Additionally, the hydrophobicity of the amino acid side chains encoded the properties of transient assemblies upon covalent binding to substrates incorporating reactive nucleophiles.70 These examples highlighted the way in which the structural elements around activated moieties (phosphate esters, thioesters, and esters,) guided reactivity changes, supramolecular structure formation and pathway selection. Herein, we focused on applying the concept of “structured abiotic phosphates” to the design and the construction of abiotic cascades in one pot. Thus, our objective was to design systems in which the structural elements of activated amino acids could: (1) influence the reactivity of intermediates, (2) enable the coupling of chemical reactions within a single pot and (3) selectively activate pathways through self-assembly (Figure 1).

Figure 1.

Figure 1

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Schematic representation of the construction of abiotic cascades driven by the reaction between amino acyl phosphate esters (Cbz-XEP) and the dipeptide substrate Ac-CY (2) featuring cysteine and tyrosine residues. Reaction first proceeds though thioester formation, leading to the construction of chimeric structures composed of ester and thioester bonds. Aromatic amino acids within the structure of aminoacyl phosphate esters (1d, 1e) facilitate a second coupling step, where thioesters are capable of assembling into spherical aggregates. In contrast, aliphatic amino acid residues around the phosphate esters (1a, 1b and 1c) primarily lead to hydrolysis after thioester formation. Filled color spheres represent the amino acids in the structure of aminoacyl phosphate esters, while open spheres represent the amino acids in the structure of the dipeptide substrate.

To achieve these goals and satisfy the criteria above, we utilized aminoacyl phosphate esters, which effectively solubilize hydrophobic amino acid derivatives in aqueous media. We incorporated aliphatic (alanine, valine, isoleucine), aromatic natural (phenylalanine) and non-natural amino acids (naphthylalanine) to investigate how reactivity is influenced by the chemical nature of the amino acid side chains (Supporting Table 1). The stability was investigated in different forms of C-terminus activated amino acid derivatives, including phosphate esters, thioesters, and esters (Figure 2a).

Figure 2.

Figure 2

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(a) Chemical structure of aminoacyl phosphate esters (1x), thioester products (1x-2) in the first reaction and diester products in the second reaction. (b) Hydrolysis profile of 10 mM 1a–1e in 0.6 M borate buffer pH 9.1, analyzed using UPLC. (c, d) Time-dependent thioester and diester formation from the reaction between 10 mM Cbz-XEP (X = A, V, I, F, Nal) and 10 mM 2 in 0.6 M borate buffer pH 9.1. (e) Time-dependent thioester and diester product formation of the reaction between 10 mM 1d and 10 mM 2 in 0.6 M borate buffer pH 9.1. 1d-2 is shown with a different color compared to (c) to avoid confusion. Storage and loss modulus as a function of time for the systems containing (f) 10 mM 1d with 10 mM 2 and (g) 10 mM 1a, 1d, 1e with 10 mM 2. Solid squares represent the storage modulus (G′) and the open squares the loss modulus (G″). Thioester (1d-2) and diester (1d–2–1d) products presented in (e) are the same as those presented in (c, d). They are included here for direct comparison with the rheology data. Similarly, in (g), the result of the mechanical properties of 1d with 2 is reproduced from (f) for comparison. In all graphs, error bars represent the standard deviation of three independent experiments.

As a peptide substrate, we selected a short dipeptide sequence featuring cysteine and tyrosine residues, appended at the N-terminus with an acetyl group (Ac-CY) (Supporting Table 2). This bifunctional substrate allows us to study the competition between thioester and ester formation, as well as the self-assembly propensity of the different intermediates formed in the cascades.

Differential Reactivity Patterns from Aminoacyl Phosphate Esters

Building up from our previous observations on the reactivity of aminoacyl phosphate esters featuring an alanine residue Cbz-AEP (1a),70 we sought to further investigate the hydrolysis trends by modifying the amino acid side chains with longer alkyl chains. Thus, we synthesized Cbz-VEP (1b) and Cbz-IEP (1c), using valine (V) and isoleucine (I) residues, respectively. We followed the same synthetic protocol for coupling and isolating the aminoacyl phosphate esters as described earlier.70 Time-dependent Ultra Performance Liquid Chromatography (UPLC) experiments showed a significant increase in the stability of 1b and 1c compared to 1a in 0.6 M borate buffer at pH 9.1. Specifically, 1b exhibited a half-life of approximately 29 h, while 1c demonstrated an even longer half-life of 58 h (Figure 2b and Supporting Table 3). In contrast, the half-life of 1a was found to be around 3 h. These results indicate that the introduction of bulky aliphatic side chains might enhance the stability of aminoacyl phosphate esters due to steric hindrance, which could protect the acyl phosphate bond from hydrolysis.68 Moreover, the half-lives of Cbz-FEP (1d) and Cbz-NalEP (1e) where found to be around 1.5 h. This indicates a potential inductive effect from the aromatic amino acids, making the acyl carbon more electrophilic and, therefore, more susceptible to nucleophilic attack.

Next, we introduced the dipeptide substrate Ac-CY (2) into the systems, which contains nucleophilic groups from cysteine and tyrosine residues, facilitating the formation of thioester and ester bonds in tandem. The higher nucleophilicity of thiols enabled the initial formation of a thioester. Subsequently, the hydroxyl group of tyrosine facilitated the formation of ester bonds by performing a nucleophilic attack on the carbonyl group of the thioester (Figure 2a). We prepared samples by mixing various amino acyl phosphate esters (10 mM) with 10 mM 2 in 0.6 M borate buffer at pH 9.1. Specifically, in the cases of 1d and 1e, thioester formation occurred rapidly within 15 min, reaching a maximum conversion of around 8 mM. Notably, for 1b and 1c, maximum conversion occurred within 6 and 7 h, respectively (Figure 2c). Despite the differences in the kinetics of thioester formation, relatively high yields were obtained in all samples, indicating that the amino acid side chains have a minimal effect on the yield of product formation during the first reaction. Subsequently, further intermolecular attack by the free hydroxyl group of tyrosine led to the formation of a diester, the second product of the reaction. The construction of this transient product (diester) in the second reaction varied significantly among the different samples. The highest yield was observed with 1e, reaching a maximum of 4.1 mM after 48 h, while 1d produced 3 mM after 36 h. Among the aminoacyl phosphate esters containing aliphatic amino acids, 1a produced 0.36 mM of the diester after 2 h, whereas 1b and 1c generated 1 mM after 120 h and 0.7 mM after 360 h, respectively (Figure 2d,e). These observations highlight that the number of couplings in the cascade can be affected by chemical design (amino acid side chains). The reactivity and structural elements (aromatic versus aliphatic amino acid side chains) around the aminoacyl phosphate esters play a crucial role in determining the preferred pathway between second reaction or direct hydrolysis.

In order to confirm the selective coupling from the −SH group of the cysteine residue, we isolated the thioester peak (1x–2) from all the reactions between 1x and 2 using flash column chromatography. 1H and 13C NMR analysis confirmed that the coupling exclusively occurred due to nucleophilic attack by the thiol group rather than the hydroxyl group of tyrosine. Additionally, from the same reaction mixtures, we isolated the diester products (1a–2–1a and 1d–2–1d) and confirmed their structure using UPLC, UPLC-MS, 1H and 13C NMR spectroscopy. The UPLC-MS analysis and kinetic profiles of all systems involving various aminoacyl phosphate esters (XEPs) with 2 are available in the Supporting Information (Supporting Figures S1–S11).

Given that the second coupling occurred with a higher yield in the presence of aromatic amino acids, we investigated the concentration effects of aminoacyl phosphate esters in the cascade. We prepared samples of 1d with 2 at different concentrations. Diester product formation was less efficient at lower concentrations, yielding 21% at 1 mM and 48% at 2.5 mM. However, yields increased significantly at higher concentrations, reaching 58% at 5 mM and 64% at 10 mM (Supporting Figure S12). In order to drive more coupling toward the second reaction, we tested 20 mM aminoacyl phosphate esters (1d and 1e) with 10 mM of 2. In both cases, this resulted in a notable enhancement in diester yields (Supporting Figure S13). The increased yields at higher concentrations suggest an assembly event and a critical aggregation concentration required to efficiently promote the second coupling in the cascade. To further support these findings and highlight the importance of hydrophobic interactions for the second reaction, we prepared samples of 1d and 1e with 2 in a cosolvent environment containing 20% acetonitrile (ACN). The yields of thioester formation (first reaction) were similar with and without the organic solvent. However, the yields of diester products were significantly lower, producing only 0.75 mM and 1.2 mM for 1d and 1e, respectively (Supporting Figure S14). These results indicate that self-assembly facilitates the coupling between the two reactions, and diminishing aromatic interactions minimizes the second coupling. We furthermore conducted refueling experiments on the library containing 1d and 2 following the complete hydrolysis of the diester product. First, we regenerated approximately 8.5 mM of peptide 2 from its disulfide form using the reducing agent TCEP (tris(2-carboxyethyl)phosphine). Upon the subsequent addition of 1d, we successfully reformed both the thioester (7 mM) and diester (2.6 mM), with minimal yield loss compared to the initial reaction (Supporting Figures S15 and S16). In samples containing aliphatic aminoacyl phosphate esters, after thioester formation, we observed side reactions involving deacetylation products (<0.7 mM) (Supporting Figures S17–S19).

Effect of Self-Assembly on Cascade Formation

To further support the formation of aggregates, we performed continuous turbidity measurements for the reactions of Cbz-XEPs with 2 over a 24-h period, monitoring the absorbance at 600 nm. These experiments confirmed a high aggregation propensity in case of 1d and 1e. In contrast, aminoacyl phosphate esters incorporating aliphatic amino acid side chains remained soluble throughout the entire 24-h process (Supporting Figure S20). Using rheology, we found that the stiffness of the samples containing 1d and 1e was gradually enhanced, accompanied by the formation of hydrogels (G′ > G″). Following the reaction of 1d with 2 over time, we noticed that the stiffness was reduced and the system behaved as solution. This behavior is attributed to the hydrolysis of the final transient ester products. The reaction of 1a with 2 behaved like a liquid (G″ > G′) from the early stages of the reaction and remained unchanged until complete hydrolysis (Figure 2f,g). The higher G′ value observed for the 1d with 2 system indicates the formation of a hydrogel, despite its comparable turbidity to the aliphatic systems. This observation can be attributed to the transparent nature of the hydrogel, which forms a more homogeneous network with fewer large aggregates, resulting in reduced light scattering. In contrast, the 1e with 2 system shows increased turbidity due to larger aggregates, leading to an opaque gel. Supporting Table S4 highlights the macroscopic transitions observed for the different systems. The structural organization at different stages of the reactions was monitored using cryo and negatively stained Transmission Electron Microscopy (TEM) experiments. TEM experiments revealed distinct morphological differences between the assemblies formed by aminoacyl phosphate esters with aromatic residues and those with aliphatic residues.

Specifically, cryo-EM analysis of reactions between 1d and 1e with 2 revealed the formation of spherical aggregates during thioester formation (first reaction), while no structure was visualized for 1b (Figure 3a).

Figure 3.

Figure 3

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(a) From left to right: Cryo-EM images of reactions involving 10 mM 1b, 1d and 1e each with 10 mM 2, taken after 1 h for 1b and after 1 min for 1d and 1e. To capture only the thioester structure, samples were analyzed by cryo-EM at the specified times, ensuring the presence of thioester but not diester. (b) Time-dependent TEM images of the reaction between 10 mM 1d and 10 mM 2. (c) TEM images of reactions involving 10 mM Cbz-XEP (1a, 1c, 1e) and 10 mM 2 after 24 h. All samples were prepared in 0.6 M borate buffer at pH 9.1.

Subsequent reactions of 1d and 1e with 2, showed a transformation into a dense fibrillar network, primarily consisting of the diester product. A progressive shortening of the fibers was noticed, which was associated with the hydrolysis of the diester product. Fibrillar assemblies of the diester products were also visualized in a cosolvent environment containing 20% acetonitrile (Supporting Figures S21 and S22). Furthermore, time-dependent TEM analysis showed that 1a did not form any distinct assemblies when combined with 2 throughout the entire process (Figure 3c). However, fibrillar structures were observed for the diester products of 1b and 1c indicating that these amino acids can form assemblies even at low concentrations of diester products (Supporting Figure S23). Overall, the formation of thioesters from aminoacyl phosphate esters and the dipeptide substrate (2) is favored in our system due to the higher nucleophilicity of thiols compared to hydroxyl groups, particularly under the experimental pH conditions. The conversion between esters and thioesters typically requires catalysts such as transition metals or enzymes, along with harsh conditions like elevated temperatures and high salt concentrations in organic solvents.71 Instead, the aggregation of thioesters plays a critical role in stabilizing these intermediates by creating a microenvironment that favors the persistence of thioesters over esters. This aggregation further drives the reaction pathway toward intermolecular thioester–ester couplings by bringing reactive species into close proximity, facilitating interactions that would otherwise be rare in solution. The proximity effect lowers the activation barrier for acyl transfer, promoting the formation of complex chimeric structures rather than simpler esters, which would likely be favored under thermodynamic control. These findings highlight the crucial role of aggregation in both stabilizing reactive intermediates and promoting specific intermolecular reactions within the system.

To explore the effect of peptide sequence on the two distinct chemical reactions, we modified the initial sequence 2 by introducing an anionic (aspartic acid, D), an aliphatic (valine, V) and a positively charged (arginine, R) amino acid residue at the C-terminus. Our aim was to evaluate the impact of these C-terminal modifications on the yields of thioester and diester products, considering both kinetic and assembly effects derived from the chemical structure of the tripeptide substrates. We used 1d and 1e due to their high reactivity and propensity to form assemblies upon transferring the aromatic amino acids into the new activated forms (thioesters and esters). From the reactions of 1d and 1e with Ac-CYD (3), we observed lower yields for both thioester and diester products compared to 2. Specifically, the reaction of 1d with 3 produced 7.4 mM of thioester after 30 min and 1.5 mM of diester after 12 h, while 1e with 3 yielded 6.4 mM after 30 min and 3.7 mM after 6 h, respectively. When replacing 3 with Ac-CYV (4), we observed higher yields for the products of both reactions, which were similar to those produced using 2. Additionally, when Ac-CYR (5) was used, the reactions of 1d yielded 8.0 mM of thioester after 30 min and 1.9 mM of diester after 36 h. In comparison, 1e produced 7.3 mM of thioester after 30 min and 3.2 mM of diester after 48 h (Supporting Figures S24–S38). TEM analysis provided insights into the structural reconfigurations over time. Upon mixing 1e with 3, an initial nondefined assembly was observed during thioester formation, which later transitioned into a dense fibrillar network following the second coupling and high yield of diester formation. Similarly, when 1e was mixed with 5, a nondefined structure was also observed during thioester formation, but short fibrillar assemblies appeared after the formation of the diester. In the tripeptide sequence involving valine residues, fibers and ribbon-like assemblies were observed (Supporting Figure S39). These results suggest that incorporating anionic or cationic residues at the C-terminus of the tripeptide substrates influences product yields in both reactions, by affecting the self-assembly propensity of the intermediates formed.

Selective Coupling in Abiotic Cascade Networks

In order to investigate the competition between phosphate esters and substrates in cascade formation, we conducted experiments using various mixtures. The mixtures included combinations of 1d/1b with 2, 1e/1b with 2 and 1d/1e with 2, combining two activated amino acids with one peptide substrate (Figure 4a upper panel). The selection of 1b and 1e was based on their differing reactivity and propensity to form the second coupling product. Additionally, we aimed to explore whether coassembly effects between the activated amino acids could influence the efficiency of thioester and diester formation, particularly for aliphatic amino acid residues, which typically showed lower coupling yields. Notably, in the mixture containing 1e/1b and 2, in 5 min we observed the formation of 3.9 mM of thioester from 1e, which is significantly higher compared to the 0.4 mM formed from 1b after 72 h. The thioester from 1e further converted into 3.5 mM of its diester product after 24 h (Figure 4b). Similarly, in the 1d/1b mixture with 2, 1d produced 3.8 mM of thioester after 5 min, which then converted to 1.9 mM of the diester product within 24 h. In the mixture containing 1d/1e with 2, we observed similar thioester concentrations for both 1d and 1e. After 12 h, 1d yielded 0.4 mM of its diester product, while 1e produced 1.8 mM of diester. Additionally, we detected the formation of a mixed diester from both 1d and 1e, with a concentration of 1.5 mM (Supporting Figures S40–S46). TEM analysis of these libraries demonstrated that fibrillar assemblies formed over time, consistent with the structures observed in the individual component libraries (Supporting Figure S47).

Figure 4.

Figure 4

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(a) Schematic representation of different species formed in mixtures containing two aminoacyl phosphate esters (1b, 1e) and the bifunctional dipeptide substrate (2) at the upper panel, as well as one aminoacyl phosphate ester (1e) and two bifunctional tripeptide substrates (3, 4), at the lower panel leading to the selective formation of diester products. To avoid confusion, we used different color coding for valine in the tripeptide substrate (4) distinct from that used for the valine in the aminoacyl phosphate ester (1b). Time-dependent formation of thioester and diester products in samples containing a mixture of (b) 10 mM 1b, 10 mM 1e, and 5 mM 2 in 0.6 M borate buffer at pH 9.1, and (c) 10 mM 1e, 10 mM 3 and 10 mM 4 in 0.6 M borate buffer at pH 9.1. Error bars represent the standard deviation of three independent experiments.

These findings demonstrate distinct behaviors for mixtures of aminoacyl phosphate esters with aliphatic and aromatic residues. The aliphatic amino acids, such as 1b, displayed slower reaction kinetics and lower yields in forming the transient products. In contrast, aromatic amino acids, such as 1e, showed higher reactivity and a greater propensity for self-assembly. Notably, libraries containing aromatic amino acids (1d, 1e) exhibited a clear preference for the formation of the 1e diester, emphasizing its stronger self-assembly behavior. The formation of mixed diesters in the 1d/1e mixtures highlights the role of coassembly, where interactions between the aromatic residues facilitate the integration of both into a single structure. Overall, these results show that both the reactivity of the aminoacyl phosphate esters and their self-assembly behaviors play crucial roles in determining the cascade outcomes, with aromatic residues showing greater efficiency in forming the transient products.

Additionally, we conducted experiments by mixing 1d or 1e with two tripeptide substrates, 3 and 4 in a single pot. Our aim was to investigate the way in which the tripeptide sequences influence the selective formation of products in the cascade, when they are incorporated into the activated amino acids in both reactions (Figure 4a lower panel). Upon mixing 1e with 3 and 4, we observed the formation of 3.3 mM thioester with 3 after 5 min and 5.4 mM with 4 after 30 min. In the second reaction, the diester product primarily formed from 4, reaching a maximum yield of 2.8 mM after 384 h. In contrast, the second coupling product with 3 resulted in a significantly lower yield of only 0.3 mM after 6 h (Figure 4c). Similarly, in the reaction of 1d with 3 and 4, we observed a comparable trend, with diester product formation being significantly more pronounced with peptide 4 (Supporting Figures S48–S53). These findings highlight the important role of specific amino acids within tripeptide substrates in influencing efficiency and selectivity within the cascades. Notably, our approach demonstrates the ability to incorporate peptide sequences containing nucleophiles, irrespective of their length. This capability has been effectively observed in both dipeptides and tripeptides, which guided cascade formation. Detailed reaction pathways for the mixtures containing three components are available in the Supporting Information (Supporting Figures S54 and S55).

Conclusions

In this work, we focused on constructing abiotic cascades by influencing the reactivity and self-assembly dynamics of acyl transfer reactions initiated by aminoacyl phosphate esters. We demonstrated that the structural elements surrounding phosphate esters can modulate their reactivity and half-lives, ranging from hours for aromatic amino acids to days for aliphatic residues. This variability in turn dictates reaction pathways and influences the propensity for self-assembly when the structural elements (amino acid side chains) are transferred into other activated forms, such as esters and thioesters. Aminoacyl phosphate esters containing aromatic residues facilitated a two-step coupling process, initially forming thioesters capable of assembling into spherical aggregates. This process enabled the fabrication of chimeric assemblies incorporating both ester and thioester bonds within a single structure. Conversely, activated amino acids with aliphatic residues predominantly underwent hydrolysis after thioester formation. Furthermore, we achieved selective product formation in mixtures containing aminoacyl phosphate esters and peptide substrates by leveraging self-assembly as a selection mechanism. By promoting or inhibiting specific pathways based on structural elements and assembly dynamics, we enhanced both the efficiency and specificity of the reactions. Our approach demonstrates how the interplay between assembly and reactivity enables the coupling of chemical reactions, driving complex transformations. In future, we will focus on further understanding how minimal changes in the structure of amino acid side chains can further affect reactivity and influence nonequilibrium cascade formation. Coupling chemical reaction cycles through reactivity changes and self-assembly, along with the potential incorporation of chemical shunts,19 presents a novel opportunity to direct orthogonal functions within chemical reaction networks. Chemical shunts, driven by environmental factors or structural modifications, can serve as selective pathways that redirect intermediates. This allows reaction networks to operate with internal clocks, offering a new level of temporal regulation over complex chemical processes.

Acknowledgments

We thank Christoph Warth for analytical support. We gratefully acknowledge Lenard Saile for fruitful discussions and insightful comments. The graphical abstract was created using BioRender.com/h71f504.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.4c10082.

  • Materials and Methods description and additional UPLC chromatograms, NMR data, LC-MS analyses, kinetic profiles, reaction pathways, tables with chemical structures, transmission electron microscopy images, and turbidity measurements (PDF)

The work was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy-EXC-2193/1-390951807 and the European Union (ERC-2023-StG grant, PhosphotoSupraChem, 101117240).

The authors declare no competing financial interest.

Supplementary Material

ja4c10082_si_001.pdf (5.3MB, pdf)

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Associated Data

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Supplementary Materials

ja4c10082_si_001.pdf (5.3MB, pdf)

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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