Orthogonal photoswitching of heterobivalent azobenzene glycoclusters: the effect of glycoligand orientation in bacterial adhesion

Abstract

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.

Keywords: FimH; azobenzene glycoconjugates; carbohydrate recognition; docking; orthogonal photoswitching.

Figure 1

Cartoon of the photoswitchable glycoconjugates investigated in this account. The previously described heterobivalent azobenzene glycocluster 6βGlc3αMan 1 [24] is shown as structural formula and as the corresponding symbolic representation; the other structural formulas are derived accordingly: the homobivalent analog of 1, 6αMan3αMan 2 and the individual structural components of 1 and 2, the monovalent glycoazobenzene-functionalized mannosides 6βGlc 3, 6αMan 4, and 3αMan 5. Note that the ortho-fluorinated S-azobenzene units (ABF₄) can be isomerized orthogonally to the O-azobenzene (AB) photoswitch. (Orthogonal) photoswitching alters the relative spatial orientation of the two sugar units. Glucose (Glc) moieties are colored in blue and mannose (Man) in green according to the symbol nomenclature for carbohydrates (SNFG) [–30].

Scheme 1

Synthesis of the homobivalent azobenzene glycocluster 6αMan3αMan 2. Reagents and conditions: a) BF₃∙Et₂O, HSAc, dry CH₂Cl₂, −10 °C to rt, 18 h, 97%; b) (i) Na₂CO₃, MeOH, rt, 3 h; (ii) Xantphos-Pd-G3, Et₃N, dry THF, −78 °C, 1.5 h, 31% over two steps; c) Xantphos-Pd-G3, Et₃N, dry THF, rt, 3 h, 59%; d) NaOMe, CH₂Cl₂/MeOH 1:2, rt, 2 h, 96%.

Scheme 2

Synthesis of the antennas 6βGlc 3 and 3αMan 4 (A), and 6αMan 5 (B). Reagents and conditions: a) DTT, Et₃N, dry DMA, rt, 1 d; b) Xantphos-Pd-G3, Et₃N, dry THF, −10 °C to rt, 20 h, 63% (16, over two steps), 73% (17, over two steps); c) NaOMe, CH₂Cl₂/MeOH 1:2, rt, 3 h, 77% (3), quant. (4, 4 h), quant. (5); DTT: 1,4-dithio-ᴅ-threitol; DMA: dimethylacetamide.

Figure 2

A: Wavelength-selective photoswitching of the α-ᴅ-mannopyranosyloxy-AB and -ABF₄ antennas comprised in the homobivalent glycocluster 2. The PSS values after irradiation with 365, 435, and 520 nm light, respectively, are shown (cf. Table 1). B: Wavelength-selective photoswitching of the azobenzene mannosides 3, 4, and 5 after irradiation with 365, 435, and 520 nm light (cf. Table 1). C: The thermal relaxation of 2 from Z_ABZ_ABF4 to E_ABE_ABF4 is described by the rate constants k₁–k₄ (top) and thermal relaxation of Z-3, Z-4, and Z-5 is described by k₅, k₆, and k₇ (bottom).

Figure 3

Comparison of the inhibitory potencies of 1, 2, 4, and 5 in the different isomeric states. The depicted RIP(MeMan) values are relative to the reference inhibitor MeMan tested on the same plate. Error bars of the inhibition of E. coli (GFP-PKL1162) adhesion to mannan reflect standard deviations (cf. Supporting Information File 1, Table S6).

Figure 4

Three-dimensional representation of the superimposed most stable ligand–protein complexes from IFD for E- and Z-6αMan 4 (A: E in red, Z in yellow) and E- and Z-3αMan 5 (B: E in turquoise, Z in magenta), as well as of the EE, ZZ, EZ, and ZE isomers of glycocluster 6βGlc3αMan 1 (C) and of 6αMan3αMan 2 (D). The protein FimH (1UWF) is depicted as ribbon diagram and the ligands are displayed as stick models (glycoclusters 1 and 2: EE: blue; ZZ: violet; EZ: green; ZE: red). Superposition of the isomers shows the similarity of the binding of the terminal mannoside antenna within the FimH CRD and the different orientations of the “rest” of the molecule at the periphery of the CRD (the scaffold mannoside and, for 1 and 2, the second antenna).