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# Coacervate droplets drive organocatalyzed aqueous C-C bond formation via interfacial activation

Kevin Peyraud-Vicré, Charline Dechamps, Nicolas Martin, Valérie Desvergnes

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Kevin Peyraud-Vicré, Charline Dechamps, Nicolas Martin, Valérie Desvergnes. Coacervate droplets drive organocatalyzed aqueous C-C bond formation via interfacial activation. Journal of the American Chemical Society, 2025, 147 (41), pp.37337-37346. 10.1021/jacs.5c10731. hal-05335200

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Submitted on 28 Oct 2025

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Distributed under a Creative Commons Attribution 4.0 International License
Coacervate droplets drive organocatalyzed aqueous C-C bond formation via
interfacial activation

Kevin Peyraud-Vicré,¹,²,³ Charline Dechamps,¹,² Nicolas Martin,³,* Valérie Desvergnes¹,²,*

1. Univ. Bordeaux, CNRS, INSERM, ARNA, UMR 5320, U1212, 33000 Bordeaux, France

2. BANCO group, 2 Rue Dr Hoffmann Martinot, UFR Pharmacie, 3ème Tranche, 4ème étage, 33076 Bordeaux Cedex, France

3. Univ. Bordeaux, CNRS, Centre de Recherche Paul Pascal, UMR 5031, 115 avenue du Dr. Schweitzer, 33600 Pessac, France

* nicolas.martin@crpp.cnrs.fr; valerie.desvergnes@u-bordeaux.fr

Abstract

Compartmentalization is central to the regulation of biochemical reactions in living systems, with membraneless organelles formed by liquid-liquid phase separation (LLPS) offering dynamic environments that influence biochemical reactivity. Inspired by this principle, coacervate microdroplets have emerged as promising synthetic analogues for modulating both enzymatic and non-enzymatic reactions in water. Here, we report that coacervates can actively promote aqueous N-heterocyclic carbene (NHC) organocatalysis, enabling base-free carbon-carbon (C-C) bond formation via the Stetter reaction. Using a newly designed amphiphilic thiazolium precatalyst, we show that model polyelectrolyte coacervates not only sequester organic droplets containing the Stetter reactants, creating a unique coacervate/organic droplet interface, but also directly facilitate carbene generation. Remarkably, this activation arises from a strong electrostatic interaction between the thiazolium salt and the polyanion, which spontaneously coacervate together to form catalytically active droplets. These coacervates act as sacrificial reaction compartments, dissolving upon carbene formation while driving C-C bond formation in the absence of added base, highlighting the capacity of coacervates to restructure and respond dynamically during catalysis. Together, our results uncover a previously unrecognized mode of organocatalyst activation within LLPS-based materials, expanding the functional scope of phase-separated systems, and suggesting broader potential for leveraging multiphase systems to modulate reactivity through emergent interfacial phenomena in water.

Introduction

Compartmentalization plays a fundamental role in biological systems, enabling the spatiotemporal
organization of biochemical processes. In living cells, metabolic reactions do not occur in isolation
but are instead confined within organized microenvironments that regulate enzyme reactivity.¹
While membrane-bound organelles such as mitochondria and lysosomes provide well-defined
compartments for metabolic processes, cells also rely on a distinct form of organization based on
liquid-liquid phase separation² (LLPS). This phenomenon gives rise to membraneless organelles –
dynamic, droplet-like assemblies that regulate biochemical processes through selective
sequestration, altered local microenvironments, and interfacial effects without the need for a
surrounding membrane.³

Inspired by these biological systems, synthetic phase-separated environments such as coacervates have emerged as powerful models for studying biochemical reactivity in crowded aqueous conditions.⁴ Coacervates, formed by associative LLPS of oppositely charged polymers or biomolecules, provide aqueous environments capable of concentrating reactants and modulating enzyme reactions,⁴ ⁵ ⁶ making them promising platforms for applications in heterogeneous biocatalysis and artificial membraneless organelles.⁷ ⁸ ⁹ ¹⁰ ¹¹ Beyond enzymes, coacervates have also been shown to partition small molecules, opening perspectives for exploring non-enzymatic
reactivity and small-molecule catalysis in confined, soft microenvironments.<sup>12 13</sup> Recent studies have demonstrated that coacervates facilitate key reactions, including aldol condensation,<sup>14</sup> amide bond formation,<sup>15</sup> oligonucleotide polymerization,<sup>16</sup> imine formation,<sup>17</sup> phosphate ester hydrolysis,<sup>18</sup> or transition metal catalysis.<sup>19</sup> These findings suggest that coacervates do not merely serve as passive compartments but actively influence reaction pathways, akin to micellar catalysis,<sup>12</sup> where compartmentalized environments accelerate and guide chemical transformations. Despite these advances, most studies have focused on relatively simple reactions, leaving a significant gap in understanding how coacervates influence more complex transformations.

Carbon-carbon (C-C) bond formation is a cornerstone in both biological metabolism and synthetic chemistry. Enzymes catalyze C-C bond formation in aqueous environments with remarkable efficiency,<sup>20</sup> yet synthetic methodologies often rely on organometallic reagents in organic solvents. Developing aqueous-phase strategies for C-C bond formation using simple catalysts is thus a major challenge at the interface of synthetic and biological chemistry,<sup>21</sup> not only for expanding sustainable catalysis but also for mimicking the efficiency of enzymatic processes in water. In this context, N-heterocyclic carbenes (NHCs) have emerged as powerful organocatalysts for C-C bond formation.<sup>22 23</sup><sup>24</sup> Originally described by Breslow as key intermediates in biochemical pathways,<sup>25 26</sup> NHCs and their azolium precursors are structurally related to the coenzyme thiamine diphosphate and mimic key aspects of its reactivity. Despite their susceptibility to hydrolysis, NHC-organocatalyzed benzoin condensation was the first reaction of its kind successfully performed in water.<sup>27 28 29</sup> The analogous Stetter reaction, which provides efficient and atom-economical C-C bond formation through reverse polarity (umpolung),<sup>30 31 32 33 34</sup> has also been recently demonstrated in water between an aldehyde and a Michael acceptor.<sup>35</sup> Given the low water solubility of organic reactants, this reaction involved phase-separated organic droplets suspended in the aqueous medium, suggesting activation at the organic/water interface.<sup>35</sup> Such an organic/aqueous phase separation highlights the potential of water not just as a solvent but also as a dynamic medium capable of concentrating organic reactants and facilitating interfacial chemical reactivity.<sup>36</sup>

Here, we expand this concept by investigating the role of coacervates in aqueous NHC organocatalysis. We first design a new amphiphilic thiazolium salt precatalyst tailored for aqueous reactivity and explore how model polycation/polyanion coacervates favor, guide, and activate C-C bond formation via the Stetter reaction. Unlike previous studies where reactants partition within coacervates and react in the coacervate matrix, we find that highly hydrophobic reactants instead phase-separate into organic droplets. Rather than being isolated from the aqueous medium, these droplets are spontaneously sequestered within coacervates, forming a unique organic/coacervate interface (Figure 1). Strikingly, we demonstrate that coacervates surrounding the organic droplets promote the generation of the thiazolium-derived carbene specie in situ without the need for an additional base. We attribute this activation to a strong interaction between the polyanion involved in coacervate formation and the thiazolium salt. Notably, we find that this interaction also induces the coacervation of the thiazolium itself with the polyanion, producing new organocatalytically-active coacervates that independently promote C-C bond formation.

Overall, our findings expand the functional role of coacervates, demonstrating that they act not only as passive compartments but as active modulators of interfacial reactivity and catalysis. By showing that aqueous phase-separated environments can both sequester organic droplets and facilitate chemical activation, we introduce a new paradigm in coacervate-based catalysis. More broadly, our study underscores the potential of multi-phase systems to create dynamic reaction environments that guide chemical reactivity through emergent interfacial phenomena, offering new avenues for aqueous organocatalysis, interfacial chemistry and molecular self-organization.
<figure><img src="image_3.png" /><figcaption>**Figure 1.** Schematic representation of coacervate-driven interfacial activation of NHC-catalyzed C-C bond formation.</figcaption></figure>

## Results

### Coacervates promote and guide C-C bond formation via organocatalyzed Stetter reaction

The development of efficient organocatalytic systems in aqueous environments remains a major challenge³⁷, particularly for C-C bond-forming reactions. In this study, we designed the water-compatible thiazolium salt **1** inspired by the catalytic properties of thiamine diphosphate, a natural enzyme cofactor derived from vitamin B1 (Figures 2a,b). This thiazolium salt was highly soluble in water (up to 100 mM) and was engineered with amphiphilic properties to promote localization at water/organic interfaces, potentially improving reaction dynamics while mitigating hydrolytic degradation, an issue commonly affecting azolium salts and NHC-based organocatalysts in aqueous media.³⁸ ³⁹ ⁴⁰ ⁴¹ ⁴² The amphiphilic nature of **1** was confirmed by micelle formation assays using pyrene fluorescence in water, giving a critical micelle concentration of 0.65 ± 0.03 mM (Figure S1).

With this catalytic system in place, we investigated the Stetter reaction using benzaldehyde (1.2 equivalents) and chalcone (1 equivalent) as highly reactive model aldehyde and Michael acceptor, respectively, in the presence of **1** as the precatalyst (0.3 equivalents) and DBU (0.3 equivalents) as an organic base (Figure 2c). Under conventional conditions in organic solvents, the reaction proceeds via in situ generation of the active carbene through deprotonation of the thiazolium salt – a critical step for catalyst formation –, followed by nucleophilic addition to benzaldehyde to form a nucleophilic acyl anion equivalent, known as the “Breslow intermediate”⁴³ ⁴⁴ (Figure 2d). This intermediate subsequently undergoes a Michael addition to chalcone, providing 1,4-dicarbonyl compounds. A competing reaction produces benzoin as a side product, formed when benzaldehyde, the most electrophilic species, undergoes condensation with the Breslow intermediate.⁴⁵ ⁴⁶ ⁴⁷ Unlike previously reported conditions, which generally employ high reactant concentrations (>1 M), our study used significantly lower concentrations (50 mM benzaldehyde, 42.5 mM chalcone, 12.5 mM thiazolium **1**, 12.5 mM DBU), enabling us to probe reactivity under dilute aqueous conditions, which we assumed to be more compatible with coacervate stability. The reaction was performed at 75°C for 2 hours under magnetic stirring, yielding the expected 1,4-diketone and benzoin (Figure 2c). Product structures were confirmed by NMR spectroscopy (Figure S2). The conversion was quantified by recovering unreacted chalcone while the selectivity (1,4-diketone:benzoin ratio) was determined by ¹H NMR spectroscopy of the crude (Figure S3).

Specifically, we evaluated the reaction conversion and selectivity in different media, including THF – a conventional organic solvent for the Stetter reaction –, water, and a suspension of model coacervate droplets. To prepare the coacervate droplets, we mixed equimolar charge amounts of poly(diallyldimethylammonium chloride) (PDDA, M<sub>w</sub> = 8,300 g.mol<sup>-1</sup>, ~ 50 monomers) and poly(sodium acrylate) (PAA, M<sub>w</sub> = 5,100 g.mol<sup>-1</sup>, ~ 50 monomers) at 300 mM final monomer concentration at pH 8 (Figure 2e). This mixture formed a turbid suspension containing micrometer-
sized coacervate droplets (Figure 2f), which remained stable upon heating at 75°C (Figure S4). To assess the impact of coacervate droplets on the reaction, we used this suspension as the reaction medium in place of THF or water. Results revealed a moderate conversion in THF (25 ± 5%, Figure 2g), with a diketone:benzoin selectivity ratio of 65:35 (Figures 2h,i and Figure S3). Performing the reaction in water led to a striking improvement, with a conversion increased to 69 ± 8% (Figure 2g), and a selectivity shifted to 85:15 (Figures 2h,i and Figure S3). This enhancement is particularly notable given that commercial thiazolium salts generally perform poorly in aqueous media, where they are often reported to suffer from hydrolysis and thus reduced catalytic efficiency. Strikingly, a substantially higher conversion (89 ± 9%, Figure 2g) was observed in the presence of coacervate droplets compared to pure water, along with a slight increase in selectivity (92:8) in favor of the Stetter 1,4-diketone product (Figures 2h,i and Figure S3). This suggests that coacervate droplets not only enhance reaction efficiency but also guide selectivity by favoring the Stetter product over benzoin. These findings demonstrate that the Stetter reaction can be efficiently performed in aqueous environments with a properly designed organocatalyst. More importantly, coacervate droplets do not merely act as passive compartments but actively modulate reactivity and selectivity.
<figure><img src="image_4.png" /><figcaption>**Figure 2.** a, Chemical structure of thiamine diphosphate (the thiazolium core is highlighted in purple). b, Scheme of the two-step synthesis of the amphiphilic thiazolium salt 1. c, Scheme of the model Stetter reaction used in this study. d, Scheme of the umpolung reactivity of NHC-organocatalyzed Stetter reaction. e, Chemical structure of the polyelectrolytes used to assemble coacervate droplets. f, Optical microscopy image of PDDA/PAA coacervate microdroplets produced at 300 mM monomer concentration and at 1:1 charge ratio. Scale bar, 10 µm. Inset shows a picture of the turbid suspension of coacervate droplets. g, Conversion of the Stetter reaction in different media in the presence of 0.3 equivalents of DBU. Data is shown as mean ± s.d. (n = 3). h, Examples of ¹H NMR spectra used to determine the selectivity of the Stetter reaction (1,4-diketone:benzoin ratio) of the crude from different media. Values correspond to integration of the peaks. i, Selectivity of the Stetter reaction in different media in the presence of DBU. Data is shown as mean ± s.d. (n = 3).</figcaption></figure>

Reaction medium in aqueous conditions exhibits phase separation and hierarchical organization
To rationalize the observed enhancement of the Stetter reaction in water and in the presence of
coacervates compared to THF, we investigated the organization of the reaction medium by confocal
fluorescence microscopy. A clear and homogeneous reaction solution was observed in THF, with a
slight color change from yellow to pale yellow upon addition of DBU, attributed to thiazolium
deprotonation and the formation of the Breslow intermediate (Figure 3a).²⁵ Confocal fluorescence
microscopy of this sample, after addition of the hydrophobic fluorescent dye Nile Red (0.75 µM) to
visualize organic environments, revealed that Nile Red fluorescence distributed uniformly throughout
the sample (Figures 3d,g). In contrast, the reaction in water led the formation of a turbid suspension
that turned light brown upon DBU addition (Figure 3b), and that contained organic microdroplets
observed by confocal microscopy (Figure 3e,h). This latter observation suggests that two key factors
contribute to improved reactivity in water compared to THF. (1) Phase separation and local
concentration effects: the formation of organic droplets implies that hydrophobic reactants undergo
spontaneous phase separation in water, leading to a higher local concentration within these
droplets, likely enhancing reactivity. The estimated volume fraction of the organic phase was ~2
vol%, corresponding to an approximate local reactant concentration of ~2M, which is significantly
higher than the global concentration (~40 mM). To further probe this effect, we conducted the
reaction at higher reactant concentrations (~1.7M). Phase separation still occurred in water, but the
difference in reactivity between THF and water diminished at this concentration (Figure S5). This
suggests that the reactivity enhancement observed at lower concentrations in water is primarily due
to the high effective molarity within organic droplets. (2) Interfacial effects: The amphiphilic nature
of the precatalyst ensures its localization at the water/organic interface. Beyond simple
accumulation, the interfacial environment may influence key catalytic steps, potentially stabilizing
intermediates or altering transition state dynamics, possibly through hydrogen bonding with water as
described for “on water” chemical mechanisms.³⁶

The reaction medium in the presence of coacervate droplets also appeared as a turbid suspension and became brown upon addition of DBU (Figure 3c), similarly to water. Coacervates were doped with fluorescein-amine-functionalized PAA (fluo-PAA, 3 mM) to visualize them by confocal fluorescence microscopy. Images of the turbid suspension revealed a complex, hierarchical organization, where organic droplets containing the reactants and Nile Red still formed, coexisting as a distinct phase (Figure 3f). Notably, these droplets were spontaneously sequestered within coacervates, likely driven by differences in surface tension between the organic and coacervate phases ($\gamma_{org} = 4.8 \pm 0.6 \text{ mN.m}^{-1} \gg \gamma_{coa} = 0.035 \pm 0.005 \text{ mN.m}^{-1}$, Figure S6 4) and total interfacial energy minimization. Additionally, most of the green fluorescence was observed around organic droplets, pointing to the accumulation of fluo-PAA at their interface, forming a PAA-enriched interfacial region (Figure 3f,i). Line intensity analysis further confirmed this enrichment at the coacervate/organic boundary, while also revealing weaker but detectable signal inside the organic droplet (Figure S7). This minor partitioning may reflect partial complexation of PAA with thiazolium cations, analogous to ion-pair extraction or phase-transfer catalysis phenomena. The predominant interfacial This local accumulation is tentatively attributed to attractive electrostatic interactions between negatively charged PAA and positively charged thiazolium 1, which likely localizes at the organic droplets' interface due to its amphiphilic nature. Consistently, control experiments in the absence of thiazolium show no PAA accumulation around the organic droplets (Figure S8), confirming that shell formation is specifically driven by electrostatic interactions with the precatalyst. This multi-layered structural organization could influence reaction dynamics, with the polyanion potentially stabilizing key intermediates or altering activation barriers.

Together, these findings suggest that both phase separation in water and hierarchical structuring
in coacervate environments play a role in enhancing reactivity and guiding selectivity in the Stetter
reaction.
<figure><img src="image_5.png" /><figcaption>**Figure 3.** a-c, Photographs of the reaction media in THF (a), water (b) and the suspension of coacervate droplets (c) before and after addition of DBU. The solution remains clear in THF, but appears turbid in water and under coacervate conditions. Upon addition of DBU, a brown color appears in water and coacervate samples, attributed to the formation of Breslow intermediate enaminol. d-f, Confocal fluorescence microscopy images of the reaction media containing all reagents (including DBU) in THF (d), water (e) and the coacervate suspension (f), doped with Nile Red (0.75 µM, magenta). In the coacervate condition, fluorescently labeled PAA (fluo-PAA, 3 mM) was also added, and the transmitted light image was overlaid to help visualize PDDA/PAA coacervates, which appeared only faintly fluorescent. Scale bars, 50 µm. g-i, Schematic representation of the reaction media in THF (g), water (h) and coacervate droplets' suspension (i).</figcaption></figure>

## Coacervate droplets as active organocatalytic phases

Acetate counterions have been reported to enable in situ carbene formation in imidazolium-based ionic liquids.<sup>48 49</sup> Given that PAA, a polyacetate, accumulates at the organic droplet interface, we thus investigated whether coacervates could actively promote the Stetter reaction in the absence of an external base. To test this, we conducted the Stetter reaction without DBU. As expected, no reaction occurred in THF or in water (Figure 4a), which confirmed that the base was critical to activate the thiazolium salt. In contrast, in the presence of coacervates, the reaction proceeded very efficiently, with conversion (89 ± 3%) and selectivity (93:7 diketone:benzoin ratio) comparable to those obtained in the presence of DBU (Figures 4a,b and Figure S9). This striking result indicates that the coacervate phase itself enables in situ carbene generation and catalysis without requiring an added base. To further confirm that free thiazolium micelles in the continuous dilute phase do not contribute significantly to the reactivity, we performed the reaction using only the bulk coacervate phase
separated from the supernatant after centrifugation. Identical conversion and selectivity were observed (Figure S10), indicating that the catalytic activation arises from the coacervate phase.

To pinpoint the specific component responsible for this activation, we performed control experiments using PAA and PDDA separately. PAA, but not PDDA, promoted the Stetter reaction in the absence of DBU, resulting in comparable conversion (89 ± 1%) and selectivity (92:8 diketone:benzoin ratio) to that observed under coacervate conditions (Figures 4a,b and Figure S5). These results highlight polyacrylate species as key contributors to carbene formation. Interestingly, the reaction proceeded with similar kinetics in the presence of either DBU or PAA, with conversion and selectivity plateauing after 2 hours – though with higher final values in the case of PAA (Figures 4c,d and Figure S11). This comparable kinetic profile suggests that PAA does not accelerate the reaction rate but instead alters the reaction environment in a way that may favor carbene formation, leading to higher overall conversion and selectivity. While this may involve a shift in the reaction equilibrium, other mechanisms, such as stabilization of reaction intermediates or modulation of local acid-base properties, cannot be ruled out. Temperature-dependent studies further revealed that, with PAA, both conversion and selectivity declined significantly below 75°C, whereas the reaction catalyzed by DBU was relatively insensitive to temperature (Figures 4e,f and Figure S12). This temperature-dependence observation suggests that PAA-mediated activation may rely on temperature-sensitive acid-base equilibria or that increased thermal energy is required to facilitate carbene formation in the presence of PAA. At first glance, direct deprotonation of the thiazolium precatalyst by PAA seems unlikely given its relatively low pKa (~6 from ref. ⁵⁰), which makes it a much weaker base than DBU. However, elevated temperature could transiently enhance the basicity of carboxylate groups and improve their accessibility,⁵¹ potentially facilitating carbene formation and contributing to the observed temperature dependence of conversion and selectivity. Furthermore, within coacervates, PAA forms polyelectrolyte complexes with PDDA, neutralizing carboxylate anions and thus reducing their availability for deprotonation, making the observed activation by PAA in these coacervates even more surprising. Notably, comparison of conversion and selectivity between PDDA/PAA coacervates and PAA/thiazolium systems (Figure 4a,b) shows comparable reactivity in both cases, indicating that PAA can activate the thiazolium precatalyst even when associated with PDDA.

To explore the unexpected activation by PAA, we asked whether it might directly be involved in ionic interactions with the thiazolium precatalyst. To test this, we mixed the two components under reaction-relevant conditions (12.5 mM thiazolium 1, 300 mM PAA), in the absence of PDDA or reactants. Surprisingly, this mixture spontaneously formed coacervates (Figure 4g,e), despite its unconventional non-stoichiometric charge ratio, indicating a strong interaction between PAA and the thiazolium salt. These droplets displayed hallmark coacervate behavior: a broad size distribution in the micron range (Figure S13a) and liquid-like dynamics evidenced by fusion events and rapid fluorescence recovery after photobleaching (FRAP) (Figure S13b,c). Turbidity and optical microscopy-based phase diagram analysis further revealed robust coacervate formation in excess PAA, whereas near charge stoichiometry primarily yielded aggregates (Figure S13d,e). Coacervation was driven by a combination of electrostatic attraction between the anionic polymer and cationic precatalyst, as well as amphiphilic aggregation of 1 (Figure S13f,g). These findings suggest that PAA may facilitate thiazolium deprotonation not simply through conventional acid-base chemistry, but via a unique microenvironment created through coacervate formation. Such compartmentalization may stabilize charged intermediates, lower local dielectric constant, or promote proton abstraction through specific ion-pairing interactions.

Together, these observations suggest that coacervates formed between PAA and the thiazolium salt represent a new class of organocatalytically active materials, distinct from the more inert PDDA/PAA system, and likely central to the observed additional base-free activation.
<figure><img src="image_6.png" /><figcaption>**Figure 4.** a,b, Conversion (a) and selectivity (b) of the Stetter reaction in different media without DBU. Data is shown as mean ± s.d. (n = 3). c,d, Evolution of the conversion (c) and selectivity (d) of the Stetter reaction over time for the reaction performed in pure water (with (w/) DBU) or in an aqueous solution of PAA (without (w/o) DBU). Note that the x-axis is not a linear scale. The vertical dotted line shows the condition typically used (2h of reaction). e,f, Evolution of the conversion (e) and selectivity (f) of the Stetter reaction at decreasing temperatures for the reaction performed in pure water (with (w/) DBU) or in an aqueous solution of PAA (without (w/o) DBU). Note that the x-axis is not a linear scale. The vertical dotted line shows the condition typically used (75°C). Data in c-f is shown as mean ± s.d. (n = 2). g, Scheme and optical microscopy image showing the formation of coacervate microdroplets by mixing thiazolium 1 (12.5 mM) and PAA (300 mM). Scale bar, 10 µm. Inset shows a picture of the turbid suspension of PAA/thiazolium coacervate droplets.</figcaption></figure>

Restructuring of reaction medium during coacervate-activated C-C bond formation

Having identified spontaneous coacervation between PAA and the thiazolium precatalyst, we next sought to monitor their temporal evolution upon heating at 75°C in the absence of reactants. Given that the coacervate interior was more hydrophobic than surrounding aqueous medium, with a relative polarity close to that of methanol (Figure S14), they sequestered Nile Red, forming homogeneous droplets where fluorescently labeled PAA and Nile Red co-localized (Figure 5a, t = 0). Strikingly, the PAA/thiazolium coacervates progressively dissolved over time at 75°C, coinciding with the emergence of organic droplets containing only Nile Red (Figure 5a). Concurrently, a low-density, brown organic phase became visibly distinct in the reaction tube (Figure 5a, t = 120 min, inset, and Figure S15). Although the precise nature of this organic phase could not be conclusively identified by NMR and mass spectrometry, its formation likely arises from degradation products of the carbene
generated by PAA. The disappearance of the coacervate phase may be linked to thiazolium deprotonation: upon neutralization of the thiazolium salt, the NHC no longer interacts with PAA, leading to coacervate dissolution (Figure 5a). This provides strong indirect evidence that PAA facilitates thiazolium deprotonation and carbene generation in situ.

Next, we added the reactants (benzaldehyde and chalcone) to the PAA/thiazolium coacervates to observe how the reaction medium evolved during catalysis. Despite their hydrophobic interior, the reactants still formed a distinct organic phase, producing a hierarchical structure with organic droplets embedded within PAA/thiazolium coacervates (Figure 5b, *t* = 0). As the reaction progressed, the green-fluorescent coacervate layer gradually disappeared (Figure 5b), reinforcing the idea that this phase plays a sacrificial role in carbene formation. The reaction products, which are highly hydrophobic, likely localized within the organic droplets that persisted at the end of the reaction. This transformation was accompanied by the gradual appearance of a pink coloration throughout the sample (Figure 5b, insets at *t* = 0 and *t* = 120 min, and Figure S15), providing a qualitative visual cue that the reaction had occurred. Notably, this color change was not observed in experiments conducted in water in the absence of both PAA and DBU (Figure S15), reinforcing its association with catalytic activity within the coacervate phase.

Finally, we revisited our initial observation of a PAA-rich layer surrounding organic droplets within PDDA/PAA coacervates. Our findings now suggest that these structures correspond to PAA/thiazolium coacervates surrounding organic droplets, embedded within PDDA/PAA coacervates, forming a hierarchical multi-phase organization (Figure 5c, *t* = 0). FRAP experiments confirmed that the PAA/thiazolium shell remain liquid-like, with recovery dynamics comparable to that of PAA/thiazolium coacervates in isolation (Figure S16). During the reaction, the sacrificial PDDA/thiazolium layer was progressively consumed due to thiazolium deprotonation by PAA, enabling carbene formation (Figure 5c and Figure S17). However, unlike PAA alone, the overall integrity of the coacervate phase was preserved due to structural stabilization by PDDA, eventually yielding organic droplets containing the reaction products within PDDA/PAA coacervate droplets (Figure 5c, *t* = 120 min). A similar pink coloration developed during the reaction (Figure 5c, insets at *t* = 0 and *t* = 120 min, and Figure S15), consistent with the reaction proceeding under base-free conditions within the coacervate environment.

Together, these results demonstrate that coacervates are not mere passive reaction compartments. Rather, they actively participate in catalysis by enabling carbene generation in the absence of an external base. The observed restructuring of the coacervate phase during the reaction, where PAA/thiazolium coacervates act as sacrificial droplets, underscores the dynamic role of these microenvironments in catalysis. This highlights a novel function of coacervate microenvironments as self-activated organocatalytic phases, offering a promising strategy for tuning catalytic activity in aqueous media.
<figure><img src="image_7.png" /><figcaption>**Figure 5.** **a-c**, Confocal fluorescence microscopy images (i) and schematic representation (ii) of the different coacervate systems during the reaction at 75°C: PAA/thiazolium coacervates without benzaldehyde nor chalcone (a), and PAA/thiazolium coacervates (b) and PDDA/PAA coacervates with thiazolium (c) in the presence of benzaldehyde and chalcone. Nile Red (0.75 µM) and fluo-PAA (3 mM) were added to visualize hydrophobic environments (magenta) and coacervate phases (green), respectively. The dotted white circles in c highlight the faintly fluorescent PDDA/PAA coacervates (see also transmitted light channel in **Figure S17**). Scale bars, 10 µm. Insets at *t* = 0 and *t* = 120 min show photographs of the reaction media without added dyes. A clear pink color of the reaction media is observed after 2h in the presence of reactants, which indirectly indicates that the reaction occurred.</figcaption></figure>
Discussion

Our study demonstrates that coacervate-based systems facilitate C-C bond formation in aqueous environments without the need for external organic bases or solvents. This finding builds upon previous research, such as Breslow's pioneering work on NHC catalysis with enzyme-mimetic polycations,²⁹ which required DMSO as a co-solvent to achieve efficient reaction. In contrast, our system relies solely on aqueous LLPS between polyanionic species like PAA, and an amphiphilic thiazolium salts, to create microenvironments that promote carbene reactivity.

A striking feature of our system is the presence of discrete organic droplets, containing hydrophobic reactants and products, embedded within coacervates. This hierarchical organization contrasts with traditional models, in which reactants are solubilized within the coacervate phase. Instead, our systems create unique coacervate/organic phase interfaces, which resonates with recent work describing oil-in-coacervate-in-water droplets formed during an autocatalytic reaction network.⁵² Together, these studies underscore the broader potential of multiphase coacervate/oil systems to regulate chemical reactivity through emergent interfacial phenomena.

One of the key findings of our work is that thiazolium-rich coacervates are not merely passive compartments, but actively participate in C-C bond formation by promoting in situ thiazolium deprotonation and subsequent carbene formation at organic droplet interfaces. This capability establishes a new paradigm in coacervate-enabled organocatalysis. Importantly, beyond their relevance to modern biology, coacervates have also been hypothesized to play roles in early chemical evolution by providing localized environments conducive to reaction, making them plausible protocell models.⁵³ ⁵⁴ ⁵⁵ ⁵⁶ Although our system is not prebiotic in a strict sense, it offers a compelling model for exploring how rudimentary catalytic processes might have emerged in soft, compartmentalized assemblies. In particular, the use of thiazolium catalysts, structurally inspired by thiamine diphosphate, echoes ancient biochemical motifs. Thiamine diphosphate, which contains a reactive thiazole ring,⁵⁷ plays a central role in cellular metabolism, including the Wood-Ljungdahl (acetyl-CoA) pathway, one of the oldest known anabolic carbon fixation routes.⁵⁸ The potential availability of thiazole-like structures in early Earth conditions raises the possibility that such cofactors could have mediated proto-metabolic chemistry prior to the emergence of protein enzymes.⁵⁹ This has led to the hypothesis that nucleotide-derived coenzymes may be evolutionary relics of early metabolism.⁶⁰ ⁶¹ ⁶²

Focusing more directly on the role of compartments, Spruijt and colleagues have proposed that coacervates might have preceded enzymes as “catalytic generalists,” performing primitive catalytic functions before the emergence of protein-based catalysts.¹³ In their hypothesis, coacervates need not have acted as fully-fledged protocells that compartmentalized all steps of proto-metabolism, but could have played a more distributed role, facilitating specific reactions in the prebiotic milieu. Our results align with this perspective: the catalytic activity arises not from a uniform sequestration of reactants within the coacervate matrix, but rather from the formation of localized interfaces between coacervates and embedded organic droplets. This spatial heterogeneity suggests that early soft compartments may have supported chemical reactivity in a decentralized fashion, offering microenvironments that selectively stabilized certain reactions over others. Crucially, our system achieves high selectivity for the desired diketone product, addressing a key challenge in prebiotic chemistry: avoiding non-selective reactivity and by-product formation in complex, multicomponent mixtures. Selective catalysis is essential for the emergence of biologically relevant molecules and pathways, and our results suggest that coacervate-based systems could offer a route to that selectivity under fully aqueous conditions.

Overall, our findings suggest that coacervate-based systems may offer a versatile platform for designing selective, water-compatible organocatalytic systems, supporting the broader vision that soft, compartmentalized systems can bridge the gap between modern bioinspired catalysis and plausible prebiotic chemical organization. To move beyond speculation, further research should explore how interfacial dynamics, molecular architecture, and phase behavior influence reactivity
and selectivity, particularly when incorporating components and reactions with greater prebiotic relevance.

## Conclusion

In summary, this study highlights a previously unreported activation of NHC organocatalyzed reaction with liquid-liquid phase separated (LLPS) systems in aqueous media. We demonstrated that coacervates can actively mediate aqueous NHC organocatalysis, enabling base-free C-C bond formation via the Stetter reaction. This reactivity is driven by strong electrostatic interactions between a newly designed amphiphilic thiazolium precatalyst and a polyanion, giving rise to catalytically active coacervates. These coacervates spontaneously sequester hydrophobic reactants into organic droplets, forming reactive coacervate/organic interfaces. At these interfaces, carbene generation occurs without external base, leading to efficient catalysis in water. Remarkably, the coacervates function as sacrificial compartments, dynamically disassembling during the reaction. Overall, our findings reveal a new mode of organocatalyst activation within LLPS-based systems. This work positions coacervates as active, tunable platforms for promoting complex chemical transformations in water, and highlights how interfacial phenomena in multiphase systems can be harnessed for catalysis.
More broadly, our study advances the concept of coacervates as dynamic chemical microreactors, opening new directions for designing responsive, phase-separated materials for aqueous synthetic chemistry.

## Author contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

## Acknowledgments

This study and K.P.V. received financial support from the French government in the framework of the University of Bordeaux's France 2030 program/RRI "Frontiers of Life". V.D., N.M. and C.D. acknowledge the French National Research Agency (ANR-23-CE07-0025-02) for funding. N.M. also acknowledges funding from Région Nouvelle-Aquitaine (AAPR 2020-2019-8330510). We thank Etienne Ducrot for help with confocal microscope studies and Jean-Paul Chapel for help with interfacial tension measurements. We thank the CNRS and the INSERM for financial support. HRMS were performed at IECB, Bordeaux.

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