Photoswitchable precision glycooligomers and their lectin binding

Summary The synthesis of photoswitchable glycooligomers is presented by applying solid-phase polymer synthesis and functional building blocks. The obtained glycoligands are monodisperse and present azobenzene moieties as well as sugar ligands at defined positions within the oligomeric backbone and side chains, respectively. We show that the combination of molecular precision together with the photoswitchable properties of the azobenzene unit allows for the photosensitive control of glycoligand binding to protein receptors. These stimuli-sensitive glycoligands promote the understanding of multivalent binding and will be further developed as novel biosensors.


Photochemical isomerization
UV-visible absorption spectra were recorded using quartz cuvettes on a Cary 50 spectrophotometer equipped with a Peltier-thermostated cell holder (precision of 0.1 °C). Irradiation experiments were performed using a LOT-Oriel 1000 W medium-pressure Xenon lamp equipped with cut-off filters (λ max = 360 nm with FWHM = 43 nm for the E → Z isomerization, and λ > 400 nm for the Z → E isomerization).

S5
Thermal Z → E isomerization: Figure S3: UV-vis absorption spectra of E-Azo-Gal(1,3)-3 in buffer solution, its PSS mixture upon irradiation at 360 nm, and the time-evolution spectra of the PSS mixture at 25 °C over 45 hours (the curves were recorded with 3 h intervals).

SPR PA-IL inhibition/competition studies:
A sensor chip SA coated with streptavidin was conditioned with three consecutive injections of 100 µl 1 M NaCl and 50 mM NaOH in a flow rate of 100 µL/min. Then, biotinylated -D-galactose-PAA (0.1 nM in HBS-EP buffer) was immobilized on flow cell 2 with HBS-EP buffer in a flow rate of 5 µL/min. The characteristic sigmoidal shape (see Figure S4) was fitted with the Hill equation to obtain the IC 50 values k: where Start is the value at 100% binding and equals 100, End is the value where no binding is observed anymore and equals 0, x corresponds to the concentration and n describes the cooperativity [4]. IC 50 values describe the inhibitory potential of a ligand for a specific receptor which is no direct measure for the affinity of a ligand but can be related to the affinity of the ligand in a competitive experiment e.g. as we have performed with PA-IL and against -D-galactose.

Computational modelling of Azo-Gal(1,3)-3 and Azo-Gal(1,3,5)-5
The structural models of Azo-Gal(1,3)-3 and Azo-Gal(1,3,5)-5 in E-and in all-Z-configurations of the connecting azobenzene group have been represented in a crystal structure of Pseudomonas aeruginosa Lectin LecA, complexed with 1-methyl 3-indolyl--D-galactopyranoside at 1.45 Å resolution. The PA-IL protein structure has been inferred from the Protein Data Bank (PDB code 4ljh). Since this is a very recent and well resolved crystal structure co-crystallized with galactopyranoside analogues, the divalent and the trivalent linker could be modeled to bridge two binding sites at once on the tetrameric protein with a grafting from approach: the galactose ligands were extended by a few atoms belonging to the linker backbone until the rest of the backbone could be matched up and the final structure was then obtained by energy minimization, employing 1,000 steps of steepest descent followed by 10,000 steps of conjugate gradient minimization in implicit solvent, using the Amber12 [5]. Force field parameters were taken from the general Amber force field (GAFF) [6], amended by parameters to adequately represent the Azo-moieties. [7] Additionally, in the supporting information we show that the force field parametrisation is agreeing very well with quantum mechanically derived structures at the B3LYP/6-31G(d,p) level of theory using Gaussian03.
[http://www.gaussian.com/g_misc/g03/citation_g03.htm] The pictures have been rendered using VMD. This indicates that close to the overall minimum (with all torsions in their minimum energy (trans-) states) the agreement between MM and QM is easier to achieve, as expected. However, also the gross conformational properties agree reasonably well for the higher energy state represented by structures in a). Table S1: Selected Azo-Gal-(1,3,5)- 5 showing the versatility of the polymer structure upon UVirradiation. All configurations shown have been generated from the low energy configuration of the all-trans state (azo-moieties and all other bonds) A2 by reorienting the side chains either up (denoted u in the chart below) or down (d) with respect to the reference structure A2 which has the side chains in an u-u-u configuration. We then isomerize the linker from trans to cis for each azo-group. In the models shown, this involved a rigid rotation around the central N=N bond, followed by a short minimization to relax the structure, leading to the conformations shown in column 1. Note that the molecule is not planar; in A2 and D2, the leftmost galactose residue is approximately pointing in and out of the plane of the paper. Only structures are shown that can potentially bind to PA-IL by bridging two binding sites, other possible conformations such as u-d-u have been excluded.