The underlying order: Isomerism as a blueprint to control the behavior of sugar-based (bio)surfactants

The underlying order: Isomerism as a blueprint to control the behavior of sugar-based (bio)surfactants Adrian Sanchez-Fernandez 1 and Jia-Fei Poon 2,3 Abstract Surfactants are ubiquitous in formulated products and technologies. As one of the most important commodity chemicals, their remarkable consumption leads to the necessity of finding sustainable alternatives. Although the use of renewable sources limits the available chemical space for a “Green”production, the great variety of naturally occurring precursors, i.e., fatty acids and sugars, opens a myriad of possibilities to create biosurfactants capable of replacing the fatigued fossil-derived amphiphiles. Here, we visit the concept of isomer-directed assembly applied to sugar-based surfactants, wherein amphiphile assembly and function are fine-tuned through changes in the stereochemical and regiochemical configuration of the molecule. As such, we show how isomerism defines directional interactions and solvation, ultimately dictating the assembly of surfactants. However, a general framework to understand the structure-function relationship for these is still missing, which is key to realizing this divergent set of tools for the design of new surfactants. Addresses 1 Center for Research in Biological Chemistry and Molecular Materials (CIQUS), Department of Chemical Engineering, Universidade de Santiago de Compostela, Santiago de Compostela 15705, Spain 2 European Spallation Source, Lund 221 00, Sweden 3 Department of Food Technology, Engineering and Nutrition, Lund University, Box 124, Lund 221 00, Sweden Corresponding author: Sanchez-Fernandez, Adrian (adriansanchez. [email protected]) Current Opinion in Colloid & Interface Science 2024, 69:101768 This review comes from a themed issue on Biosurfactants (2023) Edited by Niki Baccile and Jochen Kleinen For complete overview about the section, refer Biosurfactants (2023) https://doi.org/10.1016/j.cocis.2023.101768 1359-0294/© 2023 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons. org/licenses/by/4.0/). Keywords Biosurfactants, Isomerism, Self-assembly, Rheology, Supramolecular assembly. Isomerism: a toolkit for molecular design While biological systems have evolved to develop a great functional diversity, we find that this emerges from a relatively narrow chemical space as mainly carbohydrates, lipids, and amino acids are used as building blocks [1]. Despite this apparent simplicity, chemists have pursued the rules that govern the processes of selfsustaining, highly interactive systems in order to mimic the work of Nature. Often, functional diversity or specificity is achieved using isomers, which allow to alter the behavior of biomolecules without changing the composition of the system. From the chiral nature of amino acids to the anomeric configuration of sugars, the restraints imposed by the molecular topology allow for modifying their organization, molecular recognition, and protein folding, among others [2e5]. Therefore, the use of isomerism provides an extra degree of freedom in terms of designing collective behavior, where selfassembly constitutes one of the key strategies. Nature has mastered the organization of molecules into complex and ordered arrangements that can perform highly specific biological processes. By mimicking these strategies, isomerism has also been exploited to control man-made assemblies with specific functions, such as gelation, selective compartmentalization, and scaffolding [6]. The powerfulness of hierarchical self-assembly as a key strategy to prepare complicated nanomaterials is reflected by the fact that a wide range of (bio)materials is constructed using simple building blocks. The enormous combinatorial space of peptide sequences enables the development of novel supramolecular nanostructures with specific functions, where isomerism plays an essential role [7e9]. For instance, amphiphilic peptides of identical composition but with a different amino acid sequence (constitutional isomerism) result in completely different types of nanostructures, showing the importance of directional interactions in the selfassembly [10]. In cyclic peptides, alternating sequences of peptide enantiomers (L, D) are required to planarize the unimer and enable the directional growth of 1D or 2D assemblies [11]. The behavior of amino acid-based amphiphiles at the air-water interface also show chirality-dependence, where different enantiomers (D, L, or racemic mixtures of those), leads to variations in the adsorption properties and the formation of different structural domains due to the directional hydrogen bonding [12]. The idea of directing the assembly using isomeric variations of the same molecule Available online at www.sciencedirect.com ScienceDirect Current Opinion in Colloid & Interface Science www.sciencedirect.com Current Opinion in Colloid & Interface Science 2024, 69:101768 has also been employed in non-natural molecular motifs. In carbonyl-based molecular motors, chirality defines the directionality of the interactions that allows autonomous rotation [13]. Geometric isomers of tetraphenylethenes assemble into aggregates with different structures and properties depending on the configuration of the functional side chains (cis or trans)[14]. Also, terpyridine complexes have been shown to assemble into different supramolecular architectures defined by the isomeric configuration of the molecule [15], and regioisomers of naphthalimide derivatives result in gels with different mechanical properties [16]. Besides, supramolecular chirality can be induced to non-chiral building blocks through specific weak interactions with chiral inductors such as tartrate counterions [17]. These examples demonstrate the essential role of molecular isomerism in the modulation of supramolecular structures and hint that a wide range of possibilities exists for compositionally identical molecules. Although isomerism constitutes a powerful tool to control assembly processes, their application in surfactant development is relatively unexplored (beyond the constitutional isomers of amino acid-based surfactants [18]). For instance, tail unsaturation is often used to leverage the thermodynamic boundaries of selfassembly (a.k.a., Krafft point, T k )[19], but our understanding of its impact on structuring supramolecular assemblies compared to saturated analogs remains in its infancy. However, recent studies have shown the possibility of using what we define as “isomer-directed surfactant assembly” to control the behavior of surfactants, particularly those derived from sugars. Therefore, we aim to discuss some general aspects that define this emerging tool in surfactant design. Isomerism and biosurfactants —a world of endless possibilities Bioderived surfactants da.k.a., biosurfactants dare upsurging as sustainable alternatives to traditional surfactants [20]. Leaving apart all the fossil-derived chemicals, biosurfactants are those that can be synthesized from biomass, either through chemical or biological routes. They are more environmentally friendly than their synthetic counterparts given to the improved biodegradability, non-toxic profile, and the independency of fossil-fuel raw materials [20]. Over the years, there has been a rapidly increasing interest for biosurfactants in various fields including agriculture, cosmetic, food industry, pharmaceutical, and petroleum industry [21]. Sugar-based surfactants (alkyl polyglycosides, APGs) are synthetic, biomass-derived surfactants obtained from sugars and fatty acids, where the great variety of precursors allows potential access to many different molecular structures [22]. For instance, Koenigs-Knorr glycosylation enables the access APGs with different headgroups (degree of polymerization of the sugar unit and sugar stereochemistry) [23], in combination with a variety of headgroup-tail linkage configurations (anomerism of the O-glycosidic linkage) [24], and structures of the tail (stereoisomerism on unsaturations, regioisomers of the unsaturation position, and branching) [19]. As such, the isomerism of APGs provides a series of homologous molecules where the surfactant structure can be changed to yield a broad library of structurefunction relationships without changing the chemical composition of the system (Figure 1). Besides, this synthetic strategy allows the incorporation of nonnatural modifications and linkers to the surfactant Figure 1 Molecular structures of the possible isomeric variations of artificial APGs centered on the saturated analog (Z)-Hexadec-9-en-1-ylb -maltoside ( b -MalC16-9Z). 2Biosurfactants (2023) Current Opinion in Colloid & Interface Science 2024, 69:101768 www.sciencedirect.com [25]. Although the latter may subtly alter the chemical composition of the APGs, it also opens many possibilities in terms of functionalization (e.g., enhanced solubility or stimuli-response character) [22]. However, systematic investigations of the impact of these modifications on the assembly of these surfactants are scarce and a general understanding of the underlying phenomena is yet to be found. Bacterial biosurfactants, particularly those derived from sugars, have attracted great attention driven by the growing environmental awareness. These biosurfactants are synthesized by fungi, yeasts, and bacteria through a series of enzymatic reactions as part of the microorganism’s metabolic activity [22,26]. The theoretical biosynthesis pathway of these employs two main reactants, a sugar source (e.g., monoor disaccharides) and a hydrophobic carbon source (e.g., triglycerides or fatty acid methyl esters). A common approach involves glucose and oleic acid methyl ester, which in a yeast culture (e.g., C. Bombicola) leads to high yields of oleic acid (9-cis) sophorolipids. An important consideration is that the biosynthetic routes often yield a mixture of amphiphilic congeners with subtle variations in the chemical structure, such as the presence of lactonized forms, various degrees of acetylation, and u or u -1 headgroup-tail linkage. If required, bacterial production of biosurfactants is often followed by subsequent purification to isolate the individual components. Stereochemically, an absolute configuration is often preferred given to the stereospecific nature of the enzymatic active site. As exemplified by the production of rhamnolipid, the congeners produced via the biosynthetic route yields exclusively the (R, R)- configuration [27], while the synthetic procedures can access the four diastereomers (R, R; R, S; S, R; S, S). Other substrates and microorganisms can also be used to obtain molecular variations of the biosurfactant. For instance, the use of elaidic acid as starting material in a C. Bombicola culture leads to the formation of a sophorose-based lipid with a 9-trans unsaturated tail [28], and Ustilago maydis was used to obtain a cellobiosebased lipid [29]. Besides, microbial degradation rates of rhamnolipids is affected by the stereochemistry of the surfactant [27], which could be used to develop new purification methods for congener mixtures. Therefore, the replacement of substrates and microbial strains can be used to selectively yield specific biosurfactants, potentially allowing to predesigning of the characteristics of those to suit specific applications. In the last two decades, the interest in biosurfactants has soared, and we find it challenging to summarize all the recent advances in the effect of biosurfactant structure on their behavior. Thus, we decided to focus our efforts on reviewing the latest developments in isomer-directed surfactant assembly applied to sugarbased amphiphiles. First, we will visit recent advances in the bulk assembly of biosurfactants; second, the rheological properties prompted by these assembled structures will be presented; third, the formation of higher-order structures, such as fibers, will be discussed; fourth, we will review the relevant aspects associated to interfacial organization, particularly in microemulsion stabilization; finally, we will discuss the outlook on the design of functional biosurfactants through isomerism. Packing frustration and self-assembly Above the critical micelle concentration, the spontaneous organization of surfactant molecules in solution into higher-order assemblies is driven by the solvophobic effect as an energy minimization process. For instance, micellization in aqueous solution causes the segregation of the hydrophobic tails into a low-hydrated core surrounded by a hydrophilic shell containing the surfactant headgroups [30]. The assembly of surfactants can result in nanostructures with different morphology depending on endogenous (i.e., molecular characteristics and concentration of the surfactant) and exogenous (i.e., temperature, pH, and presence of ions) properties, ranging from small, spheroidal micelles, to the formation of large one-, two-, or tree-dimensional structures. Bacterial biosurfactants are, in general terms, more structurally complex than the traditional surfactants (e.g., sodium dodecylsulfate or polyoxoethylate surfactants) due to the presence of functional groups, such as oligomeric surfactant headgroups, non-linear aliphatic tails, and ionizable moieties. This often results in a noncanonical behavior that cannot be predicted using the established theories of surfactant assembly (e.g., packing parameter) [31]. Yet, this chemical variety can be exploited to design and control self-assembly to an unprecedent extend. However, a detailed understanding of the interaction landscape is required for the design of amphiphiles with predicted function. Sugar-based surfactants constitute a valuable proxy to rationalize the behavior of bacterial sugar-based biosurfactants. Their inherent simplicity, as a glycosidic moiety linked to a linear aliphatic chain, can mimic specific interactions between unimers without the added structural complexity of bacterial biosurfactants (e.g., sophorolipids). As such, a great deal of information can be extracted about headgroup-headgroup interactions, tail packing, and hydrophobic solvation, among others. Herein, it should be noted that unimer is defined as a single surfactant molecule forming part of the assembled state, whereas monomer is used for isolated molecules in solution. Anomerization of the tail-headgroup linkage dchange of the stereo-configuration of the alkoxyl group at position 1 (see Figure 1)dis a unique strategy to finetune the surfactant properties and, thereby, the assembly of sugar-based surfactants. Early studies employing Designing surfactant isomerism to control self-assembly Sanchez-Fernandez and Poon 3 www.sciencedirect.com Current Opinion in Colloid & Interface Science 2024, 69:101768 molecular dynamic (MD) simulations of micelle formation from anomeric variations (axial, a , vs. equatorial, b ) of maltoside (Mal) surfactants with a dodecyl tail, a - and b -Mal-C12, revealed that the configuration of the maltose headgroup dictates the directionality of the hydrogen bond interactions between the sugar moieties at the micelle hydrated shell [33,34]. The orientation of b -anomer favors interactions between nearby headgroups (surfactant-surfactant) and reduces the degree of headgroup hydration compared to the a -anomer. This interplay decreases the surface area per unimer at the micelle interface and, concomitantly, modifies the molecular packing (Figure 2). As such, subtle differences in micelle morphology for the anomers of Mal-C12 were found [35]. In recent years, we have developed a library of sugarbased surfactants (Figure 1) with varied molecular architecture to understand how we can modulate intermolecular interactions to control self-assembly. Our initial studies were focused on the effect of the anomeric configuration of the alkoxyl substituents on position 1 in surfactants containing a Mal headgroup and a hexadecyl tail, a - and b -Mal-C16. This geometric variation in the molecule structure constitutes a restriction to the angular torsion of the tail relative to the headgroup without any variations in terms of chemical composition. We hypothesized that the difference in the assembled structure between the two anomers of maltoside-based amphiphiles would become more prominent with increasing the length of the aliphatic chain compared to the a - and b -Mal-C12 previously reported [35]. As such, we compared head-to-head the behavior of the two hexadecylmaltosides differing in the anomeric configuration using a combination of smallangle X-ray scattering (SAXS), small-angle neutron scattering (SANS), dynamic light scattering (DLS), and cryo-transmission electron microscopy [24,36]. Indeed, our results revealed striking differences between the assembly behavior of these two compositionally identical surfactants. The structure of the micelles was profoundly affected by the change in the anomeric configuration of the surfactant. The equatorial configuration of b -Mal-C16, forms 1D, semiflexible micelles da.k.a., worm-like micelles (WLMs) dwith contour lengths around 860 nm, while the axial a -MalC16 forms shorter cylindrical micelles with lengths around 50 nm, both in the dilute regime and at the same concentration (10 mM). Also, differences were found in the flexibility of the assemblies; a -Mal-C16 forms stiffer micelles, with persistence lengths around 45 nm, than those of the saturated analog, with persistence length of ca. 30 nm. Besides, temperatureand concentrationdependent transitions occur for a -Mal-C16, which undergo an ellipsoid-to-cylinder transition with increasing either of those. In contrast, b -Mal-C16 micelles retain similar structural features across the temperature (10e95  C) and concentration (1e10 mM) ranges. The combination of scattering methods allowed a detailed determination of the variations in the internal organization and hydration of the unimers. These results confirmed that the packing of these two surfactants is different. The size of the micelle cross-section of the a -Mal-C16 micelles is around 15 % smaller than the b - Mal-C16 micelles (6.0 nm) [36]. In addition, the corefinement of SAXS and SANS data indicates that the headgroup region of the a -anomer is more strongly hydrated, which results in a larger area per at the micelle interface from a packing perspective. In fact, the radius of the micelle core, which is 9 % smaller for the a -MalC16, suggests that the orientation of the headgroup governs the packing of the entire amphiphile. These changes result in an increase of the spontaneous micellar curvature and subsequently promote the formation of shorter assemblies for the a -Mal-C16. In contrast, the b - analog forms much longer micelles owing to the reduced area per unimer associated to the decrease in the hydration of the headgroup region. Our observations were in a good agreement with the molecular perspective given by Kanduc et al. on their MD characterization of b -Mal-C12 surfactant in water [34]. The change in the anomeric configuration of the headgroup leads to a redistribution of the H-bond network. The b -analog forms densely packed molecular layers driven by the strong H-bonding between neighboring disaccharides, which is accompanied by a reduction in the H-bonding available for water-headgroup interactions. In addition, our micellization results suggest that the a -configuration completely changes the interaction landscape in the headgroup region by redistributing such a H-bond network and favoring Figure 2 Differences between the assembled structures of a - and b -Mal-C16 in water. Reproduced with permission from the study by Abel et al. [33]. Copyright 2023 by American Chemical Society. 4Biosurfactants (2023) Current Opinion in Colloid & Interface Science 2024, 69:101768 www.sciencedirect.com hydration. Therefore, this variation drives the morphological change observed in the micellar phase and demonstrates an important point: molecular organization can be directed by controlling the anomerism of the building blocks. Aiming to exploit this concept of controlling the assembly through variations in the surfactant structure, we then turned to explore the unsaturation of the aliphatic tail as a design motif. We synthesized b -MalC16-9Z, an analogous amphiphile to the b -Mal-C16 where a double bond with a cis configuration was introduced at 9-position [19]. As expected, a significant reduction of the Krafft temperature (T k =<5  C for b - Mal-C16-9Z, T k =27  C for b -Mal-C16) was observed, allowing the surfactant to remain soluble in the whole liquid range of water and extending the boundaries of self-association compared to the saturated analog. The structural characterization by SANS revealed that this surfactant forms a temperature-resilient micellar phase composed of WLMs. Their contour length was slightly larger than that of the saturated analog (920 nm) but great differences were found in terms of persistence length, strongly related to the micelle flexibility. b - Mal-C16-9Z was found to form much more flexible assemblies with persistence lengths around 20 nm compared to the stiffer b -Mal-C16 assemblies (vide supra). This could be attributed to the inherent mobility of unsaturated tails compared to saturated analogs [37], conferring a more conformationally flexible micelle core. This observation was corroborated by means of neutron spin echo (NSE), where the characterization of the dynamics revealed that the unsaturated surfactant forms a more dynamic assembly in the nanoscopic scale [38]. Interestingly, this flexibility translates in a more dynamically arrested system in terms of mesoscopic entanglement due to the higher chances of potential contact points between more flexible WLMs (vide infra). Following a similar systematic principle, Tabor et al. performed an investigation on the behavior of amphiphiles with a sugar-oligo(ethylene oxide) hybrid headgroup linked to an aliphatic tail through an O-glycosidic bond in equatorial configuration ( b ). They synthesized a wide range of alkyl-oligo(ethylene glycol)-glycosides with subtle structural variations [25]. Notably, they explored the influence of the headgroup using different sugars with different diastereomeric configuration: monosaccharides dglucose (Glc), galactose (Gal), and mannose (Man) dand disaccharides dmaltose, lactose (Lac), and cellobiose (Cel). The results reveal major differences between the surfactants under the same concentration and temperature conditions. b -Glc-EO3C18-9Z and b -Gal-EO3-C18-9Z form WLMs, where the Gal-based micelles are slightly longer than those of the Glc-based surfactant. In stark contrast, b -Man-EO3C18-9Z shows a lipid-like behavior resulting in the formation of bilayers, i.e., lamellae and vesicles. As the only difference in the system is the structural configuration of the hydroxyl groups, changes in the assemblies must be ruled by the headgroup. Despite the complexity of the aggregation behavior of the monosaccharide-based surfactants, their assembly can again be rationalized through the underlying interactions between the sugar units. The orientation of the hydroxyl groups has been shown to define the hydration behavior and clustering of monosaccharides [39]. The axial configuration of the groups confers a more hydrophobic character to the sugar, leading to a redistribution of the hydrogen bond network around the molecule and favoring sugar-sugar interactions. This subtle variation potentially extends to the headgroup moiety and is the cause of the major structural differences in the assemblies. Changing to a disaccharide-based headgroup also prompts the formation of different structures. The larger, more hydrophilic headgroups result in an increase in the spontaneous curvature of the aggregate, forming shorter micelles. Also, differences were found for the studied diastereomers. The b -Lac-EO3-C18-9Z forms shorter ellipsoidal structures than the b -Mal-EO3-C189Z analog. A more striking result was observed in the case of the b -Cel-EO3-C18-9Z due to the extremely low solubility compared to the other compositionally identical surfactants. Again, the variations must arise from the specific interaction landscape of the carbohydrates. The Mal headgroup consists of two glucose units linked in an axial configuration, a -(1 /4), while Cel presents an equatorial configuration, b -(1 /4). Comparison can be drawn to the polymeric forms of these sugars, starch and cellulose, respectively. While starch (poly-Mal) is soluble in water, cellulose (poly-Cel) is very insoluble due to the strong H-bonding between the sugar units in equatorial configuration, which causes a very stable crystalline packing [40]. In the case of the surfactants, the differences in headgroup topology must control the balance between intra- (headgroup-headgroup) and inter- (water-headgroup) H-bonding. Clearly, Celconfiguration creates a strongly cohesive headgroupheadgroup H-bonding network, largely neglecting hydration effects that results in an insoluble aggregate. The Mal-surfactant balances both populations of Hbonding, resulting in the formation of elongated micelles with (possibly) a relatively low headgroup hydration degree. On the other side of the scale, the Lacsurfactant allows a higher degree of hydration, resulting in a bulkier effective headgroup size and the formation of shorter (ellipsoidal) micelles. Thus, the structural characteristics of the sugar units are key at defining the interaction landscape and packing of the surfactant unimers. The effect of tail unsaturation was also studied for these hybrid surfactants. Structural investigations confirmed Designing surfactant isomerism to control self-assembly Sanchez-Fernandez and Poon 5 www.sciencedirect.com Current Opinion in Colloid & Interface Science 2024, 69:101768 that the tail-saturated b -Glc-EO3-C18 undergoes selfassembly to flexible cylinder, but only at a temperature above its Krafft point, i.e., 48  C[41]. In contrast, WLM formation of the unsaturated b -Glc-EO3-C18-9Z occurred readily at 25  C, presumably due to a synergistic effect of both the ethylene glycol group and tailunsaturation. Both contributions drastically lower the Krafft point of the amphiphile than the saturated analog and to b -Glc-C18 (no EO linker), i.e., >100  C. Notably, differences were also in the structural features of the assemblies, where both b -Glc-EO3-C18 and b -GlcEO3-C18-9Z form WLMs of similar contour length (ca. 120 nm at 3.2 mM), but micelles from the unsaturated analog are much more flexible (persistence length of 17 nm at 3.2 mM) than those of the saturated surfactant (persistence length of 32 nm at 3.2 mM). Again, tail unsaturation provides leverage to reduce the Krafft temperature and introduces a design point to control micelle morphology. In the case of biosurfactants, only a handful of systematic comparisons on the effect of isomerism on their assembly have been performed. This could be attributed to the enzymatic routes used for the synthesis that often yield to congener mixtures [31,42], requiring further purification to isolate the single components. In addition, the structural complexity of these bioamphiphiles, associated to the presence of multiple functional side chains (vide supra),means thatonesinglesurfactantcanprovideahighlycomplex, broad spectrum of supramolecular structures (micelles, vesicles, lamellae, and so on) under different conditions [32]. To date, only a few groups have focused their efforts on characterizing the pure surfactants. The behavior of structurally related deacetylated sophorolipids, containing a sophorose (Sop) headgroup, constitutes a good starting point. The self-assembly of tail saturated and cis-unsaturated C18-sophorolipids, b - Sop-C18-COOH, and b -Sop-C18-9Z-COOH, respectively, has been investigated by Baccile et al. at basic pH (ca. 11) [43]. It must be noted that the terminal carboxylic group of these surfactants (opposite to the headgroup) should be fully deprotonated at this pH, thus conferring the surfactant a bolaform character, i.e., two hydrophilic moieties linked by an aliphatic chain. The characterization in the dilute regime (3 mM) using SAXS revealed that these surfactants assemble into core-shell spheroidal micelles (Figure 3). The analysis of the data shows that the size of the micelle cross section is around 2.7 nm, a relatively small size compared to that of the synthetic b -Mal-C16 micelles. This was attributed to the ability of the surfactant to expose both hydrophilic moieties to water at opposite sides of the micelle, requiring only one molecule across the micelle to form a hydrophobic pocket surrounded by a hydrophilic shell. The unsaturation of b -Sop-C18-9Z-COOH results in a subtle decrease in the size of the micelle cross section compared to b -Sop-C18-COOH. Besides, the length of b -Sop-C18-9Z-COOH micelles (parametrized as aspect ratio) was found to be larger than that of b -Sop-C18-COOH. The parallelism in the trends observed with tail unsaturation between these biosurfactants and the b -Mal-C16 and b -Mal-C16-9Z micelles suggests that tail packing affects the overall micelle size, possibly attributed to the higher conformational flexibility of the unsaturated tail, resulting in the formation of more asymmetric assemblies. Figure 3 SAXS data, structural parameters, and schematic models of assembled sophorolipids and glucolipids at pH 11. Reproduced from the study by Baccile et al. [43]. Copyright 2023 American Chemical Society. SAXS, small-angle X-ray scattering. 6Biosurfactants (2023) Current Opinion in Colloid & Interface Science 2024, 69:101768 www.sciencedirect.com Another comparison on the effect of the tail can be drawn to the behavior of the trans-unsaturated amphiphile, b -Sop-C18-9E-COOH. This surfactant is constituted by a linear C18 aliphatic chain, as b -SopC18-COOH, but it contains a trans unsaturation in position 9. Indeed, b -Sop-C18-9E-COOH forms a clear solution upon mixing with water in alkaline conditions (pH w11), suggesting the formation of an isotropic, nanoscopic colloidal phase [28]. Although the SAXS data also confirm the formation of ellipsoidal assemblies with a prolate distribution of mass, an interesting observation is the 2-fold longer (ca. 6.4 nm) crosssection than b -Sop-C18-COOH and b -Sop-C18-9ZCOOHmicelles.Thistrendisalsoobservedinthe radius of gyration of the micelle, which is ca. 3.3 nm b - Sop-C18-9E-COOH and 1.7 nm for b -Sop-C18-9ZCOOH, confirming a different unimer packing. It should be noted that the comparisons in terms of internal micelle structure should be taken with caution, as the selection of a different mathematical models for the fit (e.g., prolate or oblate) or the use of different model constraints could lead to miscomparisons. However, the radius of gyration, as it is determined from the low-q regime of the SAXS data, depends less on the distribution of mass of the assembly. Thus, it can be confirmed that an overall change in micelle shape is observed in the case of b -Sop-C18-9E-COOH compared to the saturated and cis analogs. Although the effect of variations in the unsaturation and isomerism in the aliphatic tail of sophorolipids has been studied, much less effort has been made to understand the effect of the headgroup isomerism. Indeed, no systematic investigations were found that directly compared this effect, to the best of our knowledge. However, some results from different studies can be related, at least qualitatively, to decipher the influence of headgroup configuration. The structurally similar cellobioselipids provide a valuable comparison on the effect of headgroup configuration on the self-assembly. For instance, the behavior of the surfactant b -CelC16-COOH can be compared to that of the sophorolipid analog b -Sop-C16-COOH. It should be noted that, while the Cel-headgroup is linked through an O-glycosidic bond in the terminal carbon of the tail, the predominant congener of the sophorolipid presents the O-glycosidic bond at the subterminal position. In addition, the bacterial synthesis of the cellobiose lipids yields to a heterogeneous mixture of congeners, where the dominant species is b -Cel-C16-COOH [29]. As expected, major differences are observed in the behavior of the b -Cel-C16-COOH compared to the sophorosimilars. The b -Cel-C16-COOH seems to form amorphous aggregates at high pH, which transition toward the formation of crystalline fibers in the micrometric scale at neutral and acidic pH (vide infra). This observation agrees with the studies on synthetic amphiphiles based on cellobiose and can be attributed to the dominant tendency of the system to form supramolecular crystals due to the strong van der Waals and directional hydrogen bond interactions between the unimers [25]. Changes in the packing parameter of surfactants are also achieved through the variation of the apparent size of the headgroup [30]. This strategy can be employed in sugar-based biosurfactants by changing the number of sugar units in the saccharide. Glucolipids are structurally similar to sophorolipids but differ in that the former contains a monosaccharide headgroup in the diastereomer form of glucose. Again, one of the most relevant aspects of these surfactants is their bolaform structure in alkaline conditions, where the terminal carboxylic group shall be deprotonated. Indeed, b -Glc-C16COOH and b -Glc-C16-9Z-COOH at pH 11 have been shown to form small, spheroidal (coffee-bean) micelles co-existing with larger assemblies (Figure 3)[43]. Characteristically, these bolaamphiphiles assemble in the second dimension when lowering the pH due to the protonation of the carboxylate moiety, with b -Glc-C16COOH forming infinite bilayers while b -Glc-C16-9ZCOOH assembles into vesicles. It must be kept in mind that b -Glc-C16-COOH possesses a high temperature boundary for the assembly (Krafft temperature), associated to the high melting point of the saturated tail (T m =69  C), which is reduced upon inclusion of tail unsaturation (T m =13  C). For b -Glc-C16-9Z-COOH, the packing of unsaturated tails is expected to be more mobile than the saturated counterpart, contributing to reduce the bending energy of the membrane. This effect causes the membrane to bend and form vesicles, in contrast to the stiffer lamellar assemblies of the saturated analog. This parallels the widely accepted behavior of lipid membranes based on saturated and unsaturated amphiphiles, where variations in the degree of unsaturation in the lipid tails leads to the modulation of the bending energy of the assembled bilayers [44]. In pursue of gaining function generality, the missing piece was the formation of long WLMs, as these are known to confer gel-properties to the system. The most relevant systematic investigations, in our opinion, were recently published by Poirier et al. [45,46]. In the pH range from 7 to 9, deprotonation of b -Glc-C16-9ZCOOH promotes the micellar phase to transition toward the formation of 1D fibers and WLMs upon the addition of divalent and trivalent salts (e.g., Mn 2þ ,Ca 2þ ). Although they did not explore the behavior of other biosurfactants with varied isomerism, studies on sugarbased surfactants indicate that change in the molecular structure could lead to the modulation of the structure of the assemblies [25]. For instance, previous investigations had shown that b -Glc-C16-9Z-COOH undergoes a unidimensional growth upon Na þ addition. This was allowed, at least in part, by the conformational flexibility of the unsaturated tail [43]. In contrast, the Designing surfactant isomerism to control self-assembly Sanchez-Fernandez and Poon 7 www.sciencedirect.com Current Opinion in Colloid & Interface Science 2024, 69:101768 more rigid tail of the saturated analog b -Glc-C16COOH does not allow the spontaneous curvature required for the 1D assembly even in the presence of salts. Another class of biosurfactants with potential structural tuneability is rhamnolipids. The protocol of bacterial synthesis for these surfactants yields to a mixture of congeners (vide supra), thus, limiting the investigation of different structural varieties of the isolated surfactants. Synthetic approaches to obtain enantiomerically pure rhamnolipids have been developed, allowing to extract structure-function relationships for these amphiphiles [47,48]. Unfortunately, no structural investigations have been made of the assemblies formed by different stereoisomers, to the best of our knowledge. However, the variety of intraand inter-molecular interactions for these surfactants could open many possibilities in terms of surfactant design [49,50]. Predictable and unpredictable mechanical responses A key aspect of self-assembled structures is their ability to generate function compared to the unfunctional isolated monomers. One of the most recurrent aspects is the modification of the rheological response of the system, where an entangled colloidal network develops different mechanical responses to those of the pure liquid, such as high viscosity, shear-thinning, and viscoelastic properties [51,52]. These properties can be exploited in a wide range of consumer goods, pharmaceutical products, and industrial processes. Reaching the required rheological properties for each application becomes essential and, for that, the structure-function relationship of the amphiphiles must be properly understood, both in terms of the chemistry of the surfactant and exogenous conditions. The concept of gelby-design can be achieved through molecular assembly in the first dimension [53]. In particular, surfactants have been exploited to yield viscoelastic responses by forming an entangled network of elongated, semiflexible cylindrical nanostructures. Self-assembly to WLMs is dictated by a relatively low spontaneous curvature or in terms of critical packing parameter, 1/3 <p<1/2 [30]. Unlike polymers, the selfassembly into WLMs is a dynamic process, implying that the supramolecular nanostructure constantly undergo continuous scission and recombination. With a typical contour length in the range between 10 2 and 10 3 nm, not only are WLM systems highly viscous owing to the inhibited free rotation in the entangled regime but also flexible due to the relatively short persistent length d the length of local rigid domains is between 10 1 and 10 2 nm [51]. Due to alignment of the WLM in parallel to the direction of the flow promoted by mechanical shearing, these systems often exhibit shear-thinning rheological behavior promoted by a decrease in the number of entanglement points [52]. This viscoelastic behavior implies that the material exhibits elastic properties when the force is applied under a short time but frictional losses when the force is applied for a long time. The viscoelastic property of elongated WLM can typically be described using the Maxwellian model due to the significantly slower reptation time ( t rep ) than breaking time ( t b ). Compared to numerous ionic surfactant systems, there are far less non-ionic surfactants that undergo WLM formation and can be used as rheological modifiers. Polyoxyethylene sorbitan monooleate and polyoxyethylene cholesteryl ether are two archetype WLMforming non-ionic surfactants, but they require additives such as dodecanoyl-N-methylethanol-amide, monolaurin, and polyoxyethylene dodecyl ether, respectively [54,55]. However, multicomponent WLM systems involve more complex formulation schemes. In contrast, sugar-based surfactants have shown the ability to assemble into WLMs and confer specific rheological responses to the system without requiring other components (Figure 4a) [24]. Consequently, they have attracted increasing attention owing to the additive-free formulation along with improved biocompatibility, biodegradability, and independency of fossil-based raw materials [20,56]. In addition, the tailorable character of the molecules leads to a great variety of possibilities without varying the chemical composition of the system (as long as it involves only isomeric variations). We recently demonstrated a vast difference in the rheological profile of a -Mal-C16 and b -Mal-C16: not only is the viscosity of a -Mal-C16 is several orders of magnitude lower than its b -analog but also does it show a Newtonian rheological profile whereas the latter exhibits shear-thinning rheological profile [24,36,57]. SANS, SAXS, and rheo-SANS were used to study the origin of these difference and elucidate the structureefunctional response of these surfactants. Our results confirmed the shear-thinning properties of the b - Mal-C16 WLMs to be associated to micellar entanglement, whereas the a -analog shows a low likelihood of entanglement due to the shorter and stiffer structure of the assemblies. Disruption of the entangled micellar network upon applying stress above the critical shear rate is reflected by a decrease of the viscosity, resulting from the shear-induced domain alignment of the b -MalC16 micelles. Thus, the rheology of the system changed dramatically with varied anomerism, and importantly, we set the first design rules to modulate the macroscopic response of sugar-based surfactant systems based on isomerism. A typical approach to tailor the assembly mechanism of these surfactants is the introduction of structural variations in the surfactant molecules. Although the length of the aliphatic tail often correlates to the rheology of 8Biosurfactants (2023) Current Opinion in Colloid & Interface Science 2024, 69:101768 www.sciencedirect.com the system (longer tails, higher viscosities), there is a rather narrow window for such strategy to be practically useful in the design of surfactants aimed for roomtemperature applications. For example, shortening the alkyl chain of a viscous solution of b -Mal-C14 by two carbon-atoms ( b -Mal-C12) renders the rheology to low viscosity Newtonian fluids [35,58]. In contrast, two extra carbon-atom ( b -Mal-C16) results in viscoelastic gels [36] coupled with Krafft point above room temperature, which limits application prospects. We have already seen that the incorporation of double bonds to the aliphatic tails can leverage this limitation with b -Mal-C16-9Z, resulting in the formation of WLMs at room temperature [19]. The question is: how this change in the surfactant structure affects the rheological response of the system? We explored the linear and oscillatory rheology behavior of b -Mal-C16-9Z above the overlap concentration at 25  C. The assemblies of the tail-unsaturated analog displayed shear-thinning character coupled with viscoelastic properties (Figure 4b). This response is similar to that of the b -Mal-C16 at 50  C but differs in that the unsaturated analog exhibits a higher zero-shear viscosity than its saturated counterpart (150 vs 86 Pas at 100 mM, respectively) [19,24]. Interestingly, despite both showing a similar relaxation time, the viscoelastic properties of b -Mal-C16-9Z were better fitted in the Maxwell model than b -Mal-C16, which implies a faster breakage of the micelle (lower scission energy) for the unsaturated analog. Also, the unsaturated surfactant solutions show a different flow behavior than the saturated counterparts. As shown by tensile strength texture analysis, the b -Mal-C16-9Z prompts a stringy texture to the solution when a solid probe was pulled apart from the surface of the sample, which was not observed for b -Mal-C16. These differences in the rheological behavior and extensional flow were connected to the faster mesoscopic dynamics detected by means of NSE and DLS. The more conformationally flexible packing of the unsaturated tails leads to faster segmental diffusion than the more tightly packed saturated analog. Therefore, the introduction of a cis-unsaturation also contributes to tuning the mechanical response of the system. Our working hypothesis was based on the fact that WLMs of similar contour length but differences in the segmental flexibility define the degree of entanglement of the network. For instance, the boundary condition of 1D stiff rods (uncooked spaghetti) would provide much less entanglement possibilities than 1D semiflexible rods (cooked spaghetti); a more Figure 4 Structure-property relationship of sugar-based surfactants on rheology. (a) Linear and oscillatory rheology of b -MalC16 at 50 mM ( ◊ ), 100 mM (,), and 200 mM (B). (b) Linear and oscillatory rheology of b -Mal-C16-9Z at 100 mM. (c) Linear and oscillatory rheology of Glc-EO3-C18-9Z at various concentrations. (d) Metalogels derived from the assembly of Glc-C18-9Z-COOH with different cations. Adapted from Refs. [19,24,25,45]. Copyright 2022 by Elsevier Inc. 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