UV light-induced spatial loss of sialic acid capping using a photoactivatable sialyltransferase inhibitor

Sialic acids cap glycans displayed on mammalian glycoproteins and glycolipids and mediate many glycan-receptor interactions. Sialoglycans play a role in diseases such as cancer and infections where they facilitate immune evasion and metastasis or serve as cellular receptors for viruses, respectively. Strategies that specifically interfere with cellular sialoglycan biosynthesis, such as sialic acid mimetics that act as metabolic sialyltransferase inhibitors, enable research into the diverse biological functions of sialoglycans. Sialylation inhibitors are also emerging as potential therapeutics for cancer, infection, and other diseases. However, sialoglycans serve many important biological functions and systemic inhibition of sialoglycan biosynthesis can have adverse effects. To enable local and inducible inhibition of sialylation, we have synthesized and characterized a caged sialyltransferase inhibitor that can be selectively activated with UV-light. A photolabile protecting group was conjugated to a known sialyltransferase inhibitor (P-SiaFNEtoc). This yielded a photoactivatable inhibitor, UV-SiaFNEtoc, that remained inactive in human cell cultures and was readily activated through radiation with 365 nm UV light. Direct and short radiation of a human embryonic kidney (HEK293) cell monolayer was well-tolerated and resulted in photoactivation of the inhibitor and subsequent spatial restricted synthesis of asialoglycans. The developed photocaged sialic acid mimetic holds the potential to locally hinder the synthesis of sialoglycans through focused treatment with UV light and may be applied to bypass the adverse effects related to systemic loss of sialylation.

C spectra are taken as APT experiments, in which CH / CH3 multiplicities are up and C / CH2 / solvent multiplicities are down. Reactions were monitored using TLC F254 (Merck KGaA) using UV absorption detection (254 nm) and by spraying them with potassium permanganate or 10% conc. H2SO4 in MeOH or cerium ammonium molybdate stain (Hannesian's stain) followed by charring at 300°C. Mass spectra were recorded on a JEOL AccuTOF CS JMS-T100CS (ESI) mass spectrometer. Purification by flash column chromatography was executed using automatic flash column chromatography on a Biotage Isolera Spektra One using Silicycle cartridges (Biotage, 30-100 μm, 60 Å) 4-12 g. Reactions under protective atmosphere were performed under positive Ar/N2 flow in flame-dried flasks. Reactions were performed at room temperature unless stated otherwise.

UV light exposure
LEDs emitting 312 nm, 365 nm, or 412 nm light (Thorlabs) were used as light source and DMSO-dissolved 4 was radiated in plastic test tubes at 1 cm distance from the light source. HEK293 cells growing in 96-well black/clear bottom plates (Thermo Scientific) or on coverslips were exposed to 365 nm light from the bottom (ca. 1 cm distance) for the indicated time points and at maximum or partial intensity.

Lectin staining and flow cytometry
Biotinylated SiaFind Pan-specific Lectenz (Lectenz Bio) was pre-complexed with Alexa Fluor 647 conjugated streptavidin (Thermo Fisher) for 10 minutes at 4°C in 1x PBS containing 1% bovine serum albumin (PBS-BSA) at a 1:1 ratio (μg/μg), and cells were incubated with the complexes for 1 hour at 4°C. Terminal galactose residues were detected with 1 μg/ml biotinylated-peanut agglutinin (PNA) (Vector Labs) by staining the cells for 1 hour at 4°C. After washing with PBS-BSA, the cells were incubated for 20 minutes with Alexa Fluor 647 conjugated streptavidin. The cells were washed and resuspended in PBS-BSA and 10.000 cells/sample were acquired by flow cytometry using a spectral analyzer (SA3800 SONY). Mean fluorescent intensity data were used to calculate the percentage lectin binding normalized to control and all experiments were performed 2-3 times.
Immunocytochemistry 0.7x10 5 HEK293 cells were seeded into 6-well plates containing 24 mm coverslips coated with 0.1 mg/ml poly-Llysine (Sigma) for 5 minutes. After 24 hours, cells were pulsed with 150 µM 4 or 5 for 4 hours and washed with medium followed by irradiation with 365 nm light for 30 seconds at maximum intensity. Post UV light-treatment, cells were cultured for 48 hours and fixed with 4% paraformaldehyde in 1x PBS for 10 min at room temperature. After washing with 1x PBS containing 0.1% v/v Tween-20 (PBS-T), cells were incubated in blocking buffer (1x PBS, 5% BSA) for 1 hour at RT. Coverslips were stained with 1 µg/ml biotinylated-PNA in 1x PBS containing 1% BSA for 1 hour at room temperature. Secondary staining was performed with 1 µg/ml streptavidin-Alexa Fluor 488 (Thermo Fisher) in 1x PBS with 1% BSA for 1 hour at room temperature in the dark. Nuclei were visualized by incubation with 1 µg/ml DAPI (Sigma Aldrich) in 1x PBS for 10 min at RT. Cells were mounted in fluorescent mounting medium (Dako), and images were acquired using a LS-900 confocal microscopy system (Leica Microsystems).

UV-energy measurement
Intensity of the UV-light treatment (mW/cm 2 ) was quantified using a UV power meter (Hamamatsu). The intensity was determined at 1 cm distance from the light source at max., half, and quarter capacity. Measurements were performed using no plastic (direct), through a 6-well plate bottom, a glass coverslip, and the combination. Energy was assessed over a 30 second period using average intensities.

UV-vis spectroscopy
UV-vis spectrum of 4 and 5 150 mM stock was measured using a photospectrometer (DeNovix) with a range between 190 and 840 nm over a length of 0.5 mm.

LED-NMR
A CoolLED pE-4000 was used as a light source at 365 nm and 90% intensity. The light was transmitted into the NMR sample via a fiber optic cable terminating in a finely-sanded bare-fiber tip fitted into the inner tube of a coaxial tube. Spectra were recorded on a Bruker 500 MHz AVANCE III spectrometer equipped with a Prodigy BB cryoprobe. Spectra were recorded every 20 s.