Chemoenzymatic Preparation of a Campylobacter jejuni Lipid-Linked Heptasaccharide on an Azide-Linked Polyisoprenoid

Complex poly- and oligosaccharides on the surface of bacteria provide a unique fingerprint to different strains of pathogenic and symbiotic microbes that could be exploited for therapeutics or sensors selective for specific glycans. To discover reagents that can selectively interact with specific bacterial glycans, a system for both the chemoenzymatic preparation and immobilization of these materials would be ideal. Bacterial glycans are typically synthesized in nature on the C55 polyisoprenoid bactoprenyl (or undecaprenyl) phosphate. However, this long-chain isoprenoid can be difficult to work with in vitro. Here, we describe the addition of a chemically functional benzylazide tag to polyisoprenoids. We have found that both the organic-soluble and water-soluble benzylazide isoprenoid can serve as a substrate for the well-characterized system responsible for Campylobacter jejuniN-linked heptasaccharide assembly. Using the organic-soluble analogue, we demonstrate the use of an N-acetyl-glucosamine epimerase that can be used to lower the cost of glycan assembly, and using the water-soluble analogue, we demonstrate the immobilization of the C. jejuni heptasaccharide on magnetic beads. These conjugated beads are then shown to interact with soybean agglutinin, a lectin known to interact with N-acetyl-galactosamine in the C. jejuni heptasaccharide. The methods provided could be used for a wide variety of applications including the discovery of new glycan-interacting partners.

Synthesis of (Z, E)-8-N-m-benzyl azido-amino-3,7-dimethyl-2,6 octadiene monophosphate (11)..........12 Figure S1. Azide isoprenoid products were reacted with DBCO-TAMRA for fluorescence monitoring after respective enzymatic reactions. Benzylazide GPP, BPP, and BP were conjugated to DBCO-TAMRA and product was monitored by HPLC. Two isomers of TAMRA are apparent in the bottom chromatogram with AzGPP and expected as these are present in commercial sources of DBCO-TAMRA. TAMRA isomers (* and **) in BPP and BP appear to co-elute as each peak represents a different size isoprenoid and peak broadening typical of these products does not allow for resolution observed with the GPP. Samples were blanked with a water injection and the chromatograms were offset along the y-axis by 80 fluorescent units for each sample. Isoprenoid starting material identity was confirmed by LC-MS

Figure S2. HPLC analysis of UDP-diNAcBac formation with sequential addition of PglF, E, and D.
Arrows indicate the elution of respective products. All reactions were performed as single pot reactions from UDP-GlcNAc with successive enzymes. Samples were blanked with a water injection and the chromatograms were offset along the y-axis by 50 absorbance units for each sample.

Figure S3. SDS-PAGE, Ponceau Staining, and Western blot of proteins used in this work.
SDS-PAGE gels were stained with coomassie. Nitrocellulose blots were stained first in ponceau to confirm successful transfer. Western blots were treated with primary mouse anti-His (1:10,000 dilution) followed by anti-mouse conjugated HRP (1:20,000 dilution). Staining occurred with a chemiluminscent substrate and an exposure time of 30s. All Pgl expressions were carried out in BL21-Star cells and PglI cultures contained 3% ethanol.    Synthesis of neryl acetate (7) Nerol (5 g, 32.4 mmoles), pyridine (10 mL) and acetic anhydride (10 mL) were added to a round bottom flask. The reaction was allowed to stir at room temperature overnight and then diluted with ether. The crude product was washed with saturated sodium bicarbonate (2x), water, then brine. The organic layer was dried with MgSO 4 and the solvent removed by rotary evaporation. The resulting oil was used directly for subsequent allylic oxidation without further purification. 1

Synthesis of 8-N-m-benzyl alcohol-amino-3,7-dimethyl-2,6 octadien-1-ol (3 or 9)
A round bottom flask was flame-dried under argon. Once cooled, 50 mL CH 2 Cl 2 , 1.3 g of 3-aminobenzyl alcohol (10.56 mmols) and 2.0 g (9.5 mmols) of the aldehyde (2 or 8) were added followed by 0.75 mL glacial acetic acid (13.11 mmols) and 3.0 g of Na(OAc) 3 BH (14.15 mmols). The mixture was left overnight and extracted with chloroform the following day. The product was purified using 30% EtOAc/Hexanes to isolate the desired product. A contaminate with an R f value similar to the desired compound was co-purified but did not appear to impact downstream synthesis.

Synthesis of (E, E,)-8-N-m-benzyl azido-amino-3,7-dimethyl-2,6 octadiene diphosphate (5)
The benzylazide geranyl alcohol (4) was brominated followed by subsequent diphosphorylation. 8-N-m-benzyl alcohol-amino-3,7-dimethyl-2,6 octadien-1-ol (333 μmol) was added to a flame dried flask under argon as 1 mL of a 100 mg/mL solution in methylene chloride. From a 1 M solution, 0.17 mL of PBr 3 in Cl 3 CH (170 µmol) was then added to the flask, without further addition of solvent. Bromination occurred instantaneously. Without further S12 purification, 1.8 mL of tris tetra n-butyl ammonium diphosphate in acetonitrile (0.5 mg/mL) was added and the reaction (999 µmol) was left at room temperature for 2h. The solvent was removed under reduced pressure without heat. The resulting viscous solution was resuspended in minimal 25 mM ammonium bicarbonate (generally less than 0.5 mL). The crude reaction was then placed directly on NH 4 + charged cation exchange resin and eluted with the same buffer. The compound was then frozen and lyophilized prior to purification by HPLC. 1  To a flame dried flask, 1 mL of a 100 mg/mL solution in CH 2 Cl 2 of (10) (333 μmol) and excess trichloroacetonitrile (217 µL, 2.2 mmol) was added under argon. Solid tetra-nbutylammonium dihyrogen phosphate (226 mg, 6.7 mmol) was then added, which was prepared from the lyophilized product after titrating phosphoric acid. 3 The mixture was stirred for 10 min and the solvent removed. The viscous solution was resuspended in an equilibrated mixture of THF (2 mL) with 25% (v/v) ammonium hydroxide (0.2 mL) and stirred for 30 min. Next, 5 mL of a toluene:methanol (1:1) was added for an additional 20 min. The resulting precipitate was removed by filtration and the solvent removed. The crude reaction was resuspended in minimal 25 mM ammonium bicarbonate with 10% isopropanol (generally less than 0.5 mL) and placed directly on NH 4 + charged cation exchange resin and eluted. The compound was then frozen and lyophilized prior to purification by HPLC. Purified fractions were dried under vacuum. 1