Pentavalent Sialic Acid Conjugates Block Coxsackievirus A24 Variant and Human Adenovirus Type 37–Viruses That Cause Highly Contagious Eye Infections

Coxsackievirus A24 variant (CVA24v) and human adenovirus 37 (HAdV-37) are leading causative agents of the severe and highly contagious ocular infections acute hemorrhagic conjunctivitis and epidemic keratoconjunctivitis, respectively. Currently, neither vaccines nor antiviral agents are available for treating these diseases, which affect millions of individuals worldwide. CVA24v and HAdV-37 utilize sialic acid as attachment receptors facilitating entry into host cells. Previously, we and others have shown that derivatives based on sialic acid are effective in preventing HAdV-37 binding and infection of cells. Here, we designed and synthesized novel pentavalent sialic acid conjugates and studied their inhibitory effect against CVA24v and HAdV-37 binding and infection of human corneal epithelial cells. The pentavalent conjugates are the first reported inhibitors of CVA24v infection and proved efficient in blocking HAdV-37 binding. Taken together, the pentavalent conjugates presented here form a basis for the development of general inhibitors of these highly contagious ocular pathogens.

5 tetrafluoropropoxy)phosphazine in 90:10 CH3CN/H2O. Semi-preparative high performance liquid chromatography (HPLC) was performed on a Gilson system HPLC, using a YMC-Actus Triart C18, 12 nm, S-5 μm, 250 × 20.0 mm with a flow rate 20 mL.min -1 , detection at 214 nm and eluent system A: aqueous 0.005% formic acid, and B: CH3CN 0.005% formic acid. Column chromatography was performed on silica gel (Merck, 60 Å, 70-230 mesh ASTM). Thin layer chromatography (TLC) were performed on Silica gel 60 F 254 (Merck) with detection under ultraviolet (UV) light and/or development with 5% H2SO4 in EtOH and heat. Automated flash column chromatography was performed using a Biotage® Isolera One system and purchased pre-packed silica gel cartridges (Biotage® SNAP Cartridge, KP-Sil). Freeze drying was performed by freezing the diluted CH3CN/water solutions in dry ice-acetone bath and then employing a Scanvac CoolSafe freeze dryer connected to an Edwards 28 rotary vane oil pump. Organic solvents were dried using a Glass Contour Solvent Systems (SG Water USA) except CH3CN (freshly distilled from CaH2) and MeOH that were dried over molecular sieves 3 Å.
All commercial reagents were used as received. All target compounds were ≥95% pure according to HPLC UV-traces. Statistics were calculated using GraphPad Prism 7 (GraphPad Software, Inc, La Jolla, CA). Microwave reactions were performed using a Biotage® Initiator microwave synthesizer; temperatures were monitored by an internal IR probe; stirring was mediated magnetically and the reaction were carried out in sealed vessels.

General procedure for sialidation -Method A
An oven dried round bottom flask was charged with magnetic stirring bar, activated molecular sieves (4 Å, 9.0 g), thiophenyl donor (1.71 mmol, 1.0 eq), azidoalcohol (4.60 eq) and silver trifluoromethanesulfonate (AgOTf, 2.0 eq). The flask was closed with rubber septa and placed under vaccum in the dark for 16 h. Under dark conditions the flask was transferred to nitrogen atmosphere and at rt was added freshly distilled CH3CN (45 mL) and anhydrous CH2Cl2 (30 mL). The mixture was allowed to stir at rt for 30 min before being cooled to -74 °C degrees. In a separate oven dried v-shaped round bottom flask was added IBr (1.40 eq) and anhydrous CH2Cl2 (2.4 mL, final concentration of 1 M) under nitrogen atmosphere. After the IBr was completely dissolved the solution was injected all at once into the stirring solution at -74 °C. The reaction was allowed to perform under dark conditions for 5.5 h at -74 °C. Diisopropylethylamine (DIPEA, 6.0 eq) was then added and the reaction allowed to perform for an additional 30 min before warming to rt. The solution was subsequently filtered through a celite plug and concentrated under reduced pressure. The resulting mixture was pre-purified by automated flash chromatography (ethylacetate (EtOAc)/acetone gradients) before purification on preparative HPLC (CH3CN/H2O 20-80% gradient 30 minutes) affording protected sialosides in pure alpha anomeric form.
The protected sialosides (0.37 mmol, 1.0 eq) were subsequently dissolved in CH3OH (44.7 mL) and NaOCH3 (4.5 eq) was added in portion to reach a final concentration of 0.03 M (significantly more concentrated solutions result in breakdown of the sialoside). The reaction was allowed to stir overnight at rt under nitrogen atmosphere before neutralizing (pH 7-8) the mixture with pre-washed Dowex 50x8 H + -Form. The mixture was concentrated under reduced pressure, re-dissolved in minimal amount of CH3OH and purified on preparative HPLC (gradient: 5% à 20% CH3CN/H2O in 20 min) affording the sialosides 11 and 12. See chemical synthesis for specific yields and analytical data. 7 mixture dissolved in water (50 mL) and titrated with aqueous NaOH (1 M) until pH >12. The resulting solution was extracted with CH2Cl2 (four times) and the combined organic layers were washed with NaOH (0.5 M, one time), brine (two times), and water (one time). The organic phase was dried over Na2SO4, filtered and concentrated under reduced pressure affording a highly viscous light-yellow oil in quantitative yield.

General procedure for amide coupling and azide formation
To a round-bottom flask equipped with a magnetic stirring bar was added distilled water (36 mL), TBDPS protected aminoalcohol (32.29 mmol, 1.0 eq) and Na2CO3 (67.80 mmol, 2.10 eq). The mixture was cooled to 0 °C and under vigorous stirring a solution of acid chloride (38.75 mmol, 1.20 eq) in 1,4dioxane (36 mL) was dropwise added. After addition was complete, the mixture was allowed to warm to rt and stirred for 5-16 h. At this point the reactions were either: a) continued by addition of NaN3 (129.16 mmol, 4 eq) and heated to 50 °C, or b) filtered through a plug of celite, concentrated under reduced pressure to remove 1,4-dioxane, extracted with CH2Cl2 (three times), washed with water (two times), dried over Na2SO4, filtered and concentrated under reduced pressure affording a viscous crude oil. The crude oil (19.72, 1.0 eq) was dissolved in dimethylformamide (DMF, 41.5 mL) under nitrogen atmosphere and NaN3 (59.16 mmol, 3 eq) added in portion. The reactions were monitored by LC-MS until completion (24-30 h) and solvent removed under reduced pressure. The resulting crude was purified on automated flash chromatography (5% à 50% EtOAc in n-heptane) yielding a viscous oil.
See chemical synthesis for specific yields.

General procedure for removal of TBDPS protecting group
An oven-dried round bottom flask equipped with a magnetic stirring bar was charged with TBDPS protected azido alcohol (5.46 mmol, 1.0 eq), flushed with N2, and an. CH2Cl2 (11.2 mL) added. To the stirring mixture was added either: a) tetrabutylammoniumfluoride (TBAF, 1 M in THF, 6.0 mL, 1.2 eq), or b) trifluoroacetic acid (TFA, 26.7 eq). The reactions were stirred at rt for 16 h and monitored with TLC.
To mixtures indicating incomplete conversion after 16 h was added additional TBAF (1 M in THF, 1.0 eq) and stirring proceeded for 2 h before the reaction mixtures were concentrated under reduced pressure resulting in a crude oil which was purified by automated flash chromatography (CH3OH/CH2Cl2 gradients). See chemical synthesis for specific yields.

Docking and Calculations
The length of the spacers connecting the central core fragments 19 (alpha) and 20 (beta) to the five sialic acid residues was investigated computationally. Two different orientations of the chair conformations of 19 and 20 were considered in the investigation. The calculations showed that 26 and 28 containing spacers with 13 main chain atoms were off sufficient length to not cause strain on the spacer atoms; the average bond angles over the carbon atoms was at an optimal of 109 and 113°, which is in agreement with the OPLS_2005 force field for these kinds of angles. Shortening the spacers to contain 11 main chain atoms, as in 40 did not change the C-C-C bond angles. A shorter spacer as in 48 with a total length of eight main chain atoms resulted in significant strain as manifested by an average C-C-C bond angle of 123.3°. In addition, the shorter spacer of 48 also resulted in steric clashes with amino acids of CVA24v.

15
Supplementary Tables   Table S1. Table of spacer structures and angles over carbons in the designed pentavalent sialic acid conjugates. Values for the highest resolution shell are given in parentheses.

Chemical synthesis
Synthesis of PEG spacer 3
The product was purified on preparative HPLC (
Azide formation was performed after semi-purification (filtration and extraction
Isolated in 58% over two steps. Azide formation was performed after semi-purification (filtration and extraction
However, azide formation was performed directly with the addition of NaN3 without any semi-purification.

5-azido-N-(5-((tert-butyldiphenylsilyl)oxy)pentyl)pentanamide
Compound was synthesized according to the general procedure for TBDPS protection, amide coupling and azide formation. This resulted in a mixture which was used directly in the TBDPS deprotection

Synthesis and analytical data of azido sialosides 11-18
Azido sialosides were synthesized according to general procedure for sialidation described in the material and methods section, except for azido sialoside 18 (see below). Azido sialoside 11 and 12 were synthesized according to "Method A", while azido sialosides 12-17 were synthesized according to

2-nonylopyranosyl))-onate (15)
Synthesized according to "Method B", 34% after two steps. 18 was synthesized according to general method B, with minor modifications. In the sialidation step 4bromo-1-butanol (1.20 eq) was used as the acceptor, yielding an acetyl protected aliphatic bromo sialoside after purification (CH2Cl2/CH3OH 10:0.2 à 10:1) as a mixture of anomers, in addition to elimination product. The mixture (711 mg, 1.135 mmol) was subsequently dissolved in dimethylsulfoxide (DMSO, 32 mL) and treated with NaN3 (6.0 eq) followed by tetra-n-butylammonium iodide (2.0 eq). The reaction was allowed to stir under nitrogen atmosphere for 22 h. The mixture was diluted in CH2Cl2, washed with water, HCl (1 M), and brine. The organic layer was dried with Na2SO4, filtered and concentrated under reduced pressure affording crude product, which was used without additional

1,2,3,4,6-penta-O-propargyl -b-D-glucoryranoside (20)
A round bottom flask was charged with a magnetic stirring bar and commercial 2-propynyl-tetra-Oacetyl-β-glucopyranoside (1.29 mmol, 1.0 eq) and CH3OH (105 mL) was added. To the stirring solution was added NaOCH3 (4.40 eq) in portion. The mixture was stirred for 4 h before neutralization with Amberlite IR 120 (H-Form) to pH ≈7. CH3OH was removed under reduced pressure to afford a white solid. The white solid was dissolved in anhydrous DMF (19.9 mL) and cooled to 0 °C, to this mixture was added NaH (60% in mineral oil, 7.60 eq) in portion. The mixture was stirred for 45 minutes at 0 °C, and subsequently propargyl bromide (6.0 eq) was added. The mixture was allowed to warm to rt and