Thisbe K. Lindhorst

Optoglycomics describes an emerging research field in which photoswitchable glycoconjugates are employed to probe and control carbohydrate–protein interactions with light. In most cases, azobenzene serves as the photoswitchable chromophore, which can be reversibly isomerized between its E and Z state, thereby substantially altering the spatial presentation of conjugated glycans. This light-induced conformational change can modulate the affinity of glycan ligands for their receptors and the inhibitory properties of glycosidase and glycosyltransferase inhibitors. Furthermore, glycosylated surfaces, including the cellular glycocalyx, as well as other glycosylated supramolecular assemblies, can be manipulated with the high spatial and temporal precision afforded by light. Although optoglycomics is still in its infancy, the remarkable success of photopharmacology suggests that it has the potential to become a powerful tool for investigating the still not conclusively understood complexity of glycobiology and to inspire new diagnostic and therapeutic strategies for glycan-associated diseases.

Glycoclusters are important molecular tools for studying carbohydrate–protein interactions. It is advantageous to derive glycoclusters from carbohydrate-based scaffolds. Modulating the chair conformation of the scaffolding monosaccharide between ⁴C₁ and ¹C₄ enables the variation in the spatial organisation of the resulting glycoclusters, allowing the consequences for carbohydrate recognition in biological systems to be studied. Recently, we introduced a xylose derivative capable of adopting two complementary chair conformations, which was conjugated to glycoligands via Suzuki–Miyaura cross-coupling to afford homobivalent glycoclusters. Here, we expand this approach by further exploring the potential of xylose as a conformationally ‘switchable’ carbohydrate scaffold. Two additional features were introduced, (i) alkyne groups to facilitate conjugation reactions through click chemistry, and (ii) orthogonal protection to enable the synthesis of heterobivalent glycoclusters in addition to homobivalent analogues. This strategy enabled the synthesis of a focused library of homo- and heterobilvalent glycoclusters, which were subjected to preliminary biological evaluation. Notably, inversion of the chair conformation of the xyloside scaffold exerted a significant effect on the potency of the respective compounds as inhibitors of mannose-specific bacterial adhesion.

Over recent decades, the glycocalyx, an extracellular organelle composed of a multitude of glycolipids, glycoproteins, proteoglycans, and glycoRNA, has gained considerable interest in cellular biology. While research in this field has revealed its tremendous importance in ever more aspects of physiological and pathological cellular processes, many of the principles that govern the role of the glycocalyx in these processes on a molecular level are still unknown. In order to unravel the fundamental laws underlying glycocalyx function, new technologies are required that enable the distinction between individual subprocesses within the intricate environment of the glycocalyx. Here, we establish an experimental platform to investigate the dynamics of the glycocalyx at the nanometer and microsecond length and time scales in a bottom-up fashion. We synthesized defined oligosaccharides and installed them on supported lipid bilayers. This way, synthetic glycolipids were assembled to glycocalyx model systems with tunable properties. By investigating these tunable model systems with interferometric scattering (iSCAT) microscopy, we gain access to the required spatiotemporal resolution. We found a strong correlation between the molecular structure of several investigated model glycans and global dynamics of the system. Our findings are corroborated by atomistic molecular dynamics simulations and coarse-grained Brownian dynamics simulations. Our results provide the first direct experimental evidence on the relationship between glycan structure, organization, and dynamics, offering a robust and versatile basis for a quantitative understanding of glycocalyx biology and physics at the molecular level.

Functional glycomimetics is suited to study the parameters of carbohydrate recognition that forms the basis of glycobiology. It is particularly attractive when a glycoligand allows for the investigation of two different states, such as varying distance between multiple glycoligands. Here, a xylopyranoside was employed as a scaffold for the presentation of two mannoside units which are ligands of the bacterial lectin FimH. The chair conformation of the central xyloside can be switched between a ⁴C₁ and a ¹C₄ conformation whereby the two conjugated mannoside ligands are flipped from a di-equatorial into a di-axial position. Concomitantly, the distance between the two glycoligands changes and, as a consequence, so does the biological activity of the respective bivalent glycocluster, as shown in adhesion–inhibition assays with live bacteria. Molecular modeling was employed to correlate the inter-ligand distance with the structure of the formed glycocluster–FimH complex. Our study suggests that conformational switches can be employed and further advanced as smart molecular tools to study structural boundary conditions of carbohydrate recognition in a bottom-up approach.

This study focuses on the kinetics of light-induced mesophase transitions in lyotropic liquid crystals containing a mixture of phospholipids and azobenzene amphiphiles. Lipid membranes organize in a wide range of morphologies, directly influencing their functionality and the efficiency of associated components such as proteins. Transitions between mesophases occur naturally during membrane fusion and can also be triggered by multiple factors, such as pH, salinity, temperature and light. Employing light to isomerize artificial photoswitchable lipids in mixed model membranes containing 1,2-dipalmitoylphosphatidylcholine or 1,2-didecanoylphosphatidylcholine revealed light-induced structural changes including mesophase transitions from a lamellar to a cubic Pn3m phase. Performing time-resolved small-angle X-ray scattering measurements, the kinetics of the change in membrane repeat distance and the transition from a lamellar to a bicontinuous cubic phase could be captured on the timescale of tens of seconds. The results demonstrate new possibilities for investigating intermediate states during mesophase transitions that are important to understand membrane fusion, and they highlight the potential of photoswitchable lipids for designing bespoke drug delivery systems.

Unexpected but insightful: the affinity of trisaccharides for the mannose-specific bacterial lectin FimH depends on the mode of scaffolding and on the anomeric group (azide versus propargyl).

Functional glycomimetics are suited to study the parameters of carbohydrate recognition that forms the basis of glycobiology. It is particularly attractive when a glycoligand allows for the investigation of two different states, such as varying distance between multiple glycoligands. Here, a xylopyranoside has been employed as a scaffold for the presentation of two mannoside units which are ligands of the bacterial lectin FimH. The chair conformation of the central xyloside can be switched between a 4C1 and a 1C4 conformation whereby the two conjugated mannoside ligands are flipped from a di-equatorial into a di-axial position. Concomitantly the distance between the two glycoligands changes and as a consequence also the biological activity of the respective bivalent glycocluster, as shown in adhesion-inhibition assays with live bacteria. Molecular modeling was employed to correlate the inter-ligand distance with the structure of the formed glycocluster-FimH complex. Our study suggests that conformational switches can be employed and further advanced as smart molecular tools to study structural boundary conditions of carbohydrate recognition in a bottom-up approach.

Over recent decades, the glycocalyx, an extracellular organelle comprised of a multitude of glycolipids, glycoproteins, proteoglycans and glycoRNA, has gained considerable interest in cellular biology. While research in this field has revealed its tremendous importance in evermore aspects of physiological and pathological cellular processes, many of the principles that govern the role of the glycocalyx in these processes on a molecular level are still unknown. In order to unravel the fundamental laws underlying glycocalyx function, new technologies are required that enable the distinction between individual subprocesses within the intricate environment of the glycocalyx. Here, we establish an experimental platform to investigate the dynamics of the glycocalyx at the nanometer and microsecond length and time scales in a bottom-up fashion. We synthesized defined model glycans and installed them on supported lipid bilayers, assembling glycocalyx model systems with tunable properties. By investigating these tunable model systems with interferometric scattering (iSCAT) microscopy, we gain access to the required spatiotemporal resolution. We found a strong correlation between the molecular structure of several investigated model glycans and global dynamics of the system. Our findings are corroborated by atomistic and coarsegrained molecular dynamics simulations. Our results provide the first direct experimental evidence on the relationship between glycan structure, organization, and dynamics, offering a robust and versatile basis for a quantitative understanding of glycocalyx biology and physics at the molecular level.

Carbohydrate recognition is fundamental to a plethora of cellular processes and hence the elucidation of the structural determinants of the recognition process is a prerequisite for understanding and manipulating carbohydrate–protein interactions, such as in the inhibition of carbohydrate-specific bacterial adhesion. For receptor binding, glycoligands have to be properly oriented in three-dimensional space and additionally, secondary interactions exerted by multivalent glycoligands have an effect on affinity. A recently introduced orthogonally photoswitchable heterobivalent azobenzene Glc/Man glycocluster was utilized to examine these aspects of carbohydrate recognition in a bacterial adhesion–inhibition assay. The measured results were systematically contextualized employing new reference compounds such as the respective homobivalent Man/Man glycocluster. An in-depth study comprising the analysis of the photochromic properties and the potential as inhibitors of bacterial adhesion of the synthetic glycophotoswitches in their different isomeric states led to new insights into the role of ligand orientation in carbohydrate recognition. The experimental results were underpinned by molecular modeling.