(Automated) Synthesis of Well-defined Staphylococcus Aureus Wall Teichoic Acid Fragments

Abstract

Wall teichoic acids (WTAs) are important components of the cell wall of the opportunistic Gram-positive bacterium Staphylococcus aureus. WTAs are composed of repeating ribitol phosphate (RboP) residues that are decorated with d-alanine and N-acetyl-d-glucosamine (GlcNAc) modifications, in a seemingly random manner. These WTA-modifications play an important role in shaping the interactions of WTA with the host immune system. Due to the structural heterogeneity of WTAs, it is impossible to isolate pure and well-defined WTA molecules from bacterial sources. Therefore, here synthetic chemistry to assemble a broad library of WTA-fragments, incorporating all possible glycosylation modifications (α-GlcNAc at the RboP C4; β-GlcNAc at the RboP C4; β-GlcNAc at the RboP C3) described for S. aureus WTAs, is reported. DNA-type chemistry, employing ribitol phosphoramidite building blocks, protected with a dimethoxy trityl group, was used to efficiently generate a library of WTA-hexamers. Automated solid phase syntheses were used to assemble a WTA-dodecamer and glycosylated WTA-hexamer. The synthetic fragments have been fully characterized and diagnostic signals were identified to discriminate the different glycosylation patterns. The different glycosylated WTA-fragments were used to probe binding of monoclonal antibodies using WTA-functionalized magnetic beads, revealing the binding specificity of these WTA-specific antibodies and the importance of the specific location of the GlcNAc modifications on the WTA-chains.

Keywords: antibodies; automated synthesis; gram-positive bacteria; ribitol phosphate; wall teichoic acids.

Figure 1

Schematic representation of S. aureus WTA structure.

Figure 2

The library of RboP‐WTA structures, generated for this study.

Scheme 1

Assembly of building blocks. a) AllylBr, NaH, THF/DMF (7 : 1), 0 °C to rt; b) AcOH/H₂O 1 : 1, 50 °C, 300 mbar, 62 % over 2 steps; c) BnBr, NaH, THF/DMF (7 : 1), 0 °C to rt; d) 4 M HCl dioxane, 80 °C; e) NaBH₄, MeOH, 0 °C, 50 % over 3 steps; f) TBDPSCl, TEA, DCM 0 °C to rt, 95 %. g) 21, TMSOTf, DCM, rt, 92 %, 7 : 1 α/β; h) i. NaOMe, MeOH, rt, 70 % α‐anomer; ii) BnBr, NaH, THF/DMF (7 : 1), 0 °C to rt, 73 %; i) i. PMe₃, KOH, THF; ii. Ac₂O, pyridine, 24: 89 % over 2 steps; j) i. TBAF, THF, rt; ii) DMTrCl, TEA, DCM, 25: 67 %; 30: 36 %; 43: 60 %; k) i. Ir(COD)(Ph₂MeP)₂PF₆, H₂, THF, ii. I₂, sat. aq. NaHCO₃, THF, 26: 88 %; 31: 79 %; 44: 94 %; l) 2‐cyanoethyl‐N,N‐diisopropylchlorophosphoramidite, DIPEA, DCM, 13: 81 %; 14: 78 %; 15: 85 %; m) 27, TMSOTf, ACN, −40 °C to 0 °C, 28: 85 %; 41: 80 %; n) propanedithiol, pyridine, H₂O, TEA, rt; ii. Ac₂O, pyridine, 29: 59 % 2 steps; 42: 86 %; o) i. DMSO, Ac₂O; ii. NaBH₄, EtOH/H₂O, 69 % over 2 steps; iii. NAPBr, NaH, TBAI, THF; p) p‐TsOH⋅H₂O, MeOH, 75 % over 2 steps; q) i. 0.2 M NaIO₄ in H₂O, ethylene glycol, MeOH; ii. NaBH₄, MeOH; r) AllylBr, NaH, THF/DMF, 88 %; s) THF/H₂O/formic acid, 85 %; t) i. NaBH₄, MeOH; ii. TBDPSCl, TEA, DCM, 57 % over 2 steps; u) BnBr, NaH, THF/DMF; v) DDQ, DCM/H₂O; w) TBAF, THF, 62 % over 2 steps; x) TBDPSCl, TEA, DCM, 98 %.

Scheme 2

Assembly of the target WTA‐oligomers. Reagents and conditions: a) i. DCI, ACN, phosphoramidite 12, 13, 14 or 15; ii. CSO; iii. 3 % DCA in DCM. b) i. NH₃ (30‐33 % aqueous solution), dioxane; ii. Pd black, H₂, AcOH, H₂O/dioxane, 1: 87 %; 4: 96 %; 5: 55 %; 6: 16 %; 7: 68 %; 8: 78 %; 9: 88 %; 11: 70 %.

Scheme 3

Automated solid phase assembly of WTA‐fragments. Reagents and conditions: a) 3 % DCA in toluene (3 min); 12 or 15 BTT, ACN (5 min); I₂, pyr/H₂O (1 min); Ac₂O, N‐methylimidazole, 2,6‐lutidine, ACN (0.2 min); b) 25 % NH₃ (aq) (60 min); 70: 15 %; 71: 11 %; 72: 20 %; 73: 11 %; c) Pd black, H₂, dioxane H₂O, AcOH, 2: 100 %; 3: 100 %; 10: 87 %; 11: 100 %.

Figure 3

Characterization of the synthetic WTA‐fragments. A) ¹H NMR (500 MHz, D₂O) of 5, 8 and 11. B) ¹³C NMR (126 MHz, D₂O) of 5, 8 and 11. C) ³¹P NMR (202 MHz, D₂O, 10 mg/mL, pH 7) of 4, 5, 7, 8, 10 and 11.

Figure 4

Magnetic WTA‐beads for antibody binding assays. A) Workflow for the antibody binding assay: 1) Biotinylation of WTA fragments 4–11; 2) Adsorption on streptavidin coated M280 Dynabeads; 3) binding of monoclonal antibodies; 4) Alexa 488‐Protein G conjugation; 5) Readout of fluorescent beads. B–E) Binding of monoclonal antibody B) 4461, C) 4624, D) 4497 and E) 6292 to the WTA‐functionalized beads.