Glycosylated nanomaterials: Detec on and neutralisa on of

Glycosylated nanomaterials: Detec2on and neutralisa2on of pathogenic bacteria and toxins Sarah-Jane Richards, Mathew W. Jones, Elizabeth Fullam, Gurdyal Besra and Matthew I. Gibson [email protected]
www2.warwick.ac.uk/go/gibsongroup
Background •  Polymers with pendent carbohydrate moie4es (glycopolymers) interact with lec4ns and have demonstrated binding aﬃni4es several orders of magnitude greater than a single carbohydrate.3 •  The iden4ﬁca4on and treatment of bacterial infec4ons remains a major healthcare challenge, especially to ensure appropriate applica4on of a limited spectrum of an4bio4cs.
Protein-‐carbohydrate interac4ons mediate a mul4tude of cri4cal biological recogni4on processes.1 The proteins responsible for deciphering this informa4on are termed lec4ns.2 Inhibi2on of Bacterial Toxins Role of Linker Length and Carbohydrate
•  Polymers synthesised with ~ 6 Å (short) Density
and 16 Å (long) linkers. Bacterial Toxin Binding
The cholera toxin (CTx) secreted by Vibrio cholerae binds glycosides expressed on the cell surface. Materials with high-‐aﬃnity and selec4vity for these lec4ns could ﬁnd applica4ons as an4-‐adhesive agents. PNA binding site CTx binding site Probe inﬂuence of chain length, carbohydrate density and linker length on binding inhibi4on. •  Glycopolymer library produced by t a n d e m p o s t -‐ p o l y m e r i s a 4 o n modiﬁca4ons. •  Linker length has no eﬀect on PNA inhibi4on (A). •  Longer linker has 2 – 3 fold lower MIC50 compared to shorter linker on inhibi4ng CTx (B). • 
•  Structural biology indicates CTx has deeper binding site than other galactose binding lec4ns such as Peanut agglu4nin (PNA). •  100 X more ac4ve than free galactose. •  Polymers synthesised with 10, 25 and 50 % galactose. •  By mass (A), low galactose densi4es lead to a rela4ve decrease in binding aﬃnity/inhibitory ac4vity. Tandem Post-Polymerisation Modification
‘Clickable’ units are not compa4ble with controlled radical polymerisa4on. Instead, tandem-‐post polymerisa4on modiﬁca4on are performed. •  By mole of galactose (B), 10 % and 100 % func4onalised polymers are the most ac4ve sugges4ng several features (e.g. sterics and site spanning) contribute to inhibitory ac4vity. • 
Summary
Tandem post-‐polymerisa4on modiﬁca4on allows synthesis of polymers from same chain length distribu4on. •  Longer linker has beder binding site accessibility. •  Poly(pentaﬂuorophenyl methacrylate) for easy modiﬁca4on. •  Inhibitors have to be developed for the binding site. •  β-‐D-‐galactose was ‘clicked-‐on’ to pendent alkyne moie4es. •  Results in biocompa4ble methacrylamide based (co)polymers. Jones, M.W. et. al., Polym. Chem., 2013 Jones, M. W. et. al., Chem. Sci., 2014 Richards, S-‐J.; Jones, M. W.; Hunaban, M. I.; Haddleton, D. M.; Gibson M. I. Angew. Chem Int. Ed., 2012 Detec2on of Lec2ns and Bacteria Glycogoldnanoparticles
Goldnanopar4cles have interes4ng op4cal proper4es. Red in solu4on à blue upon aggrega4on. Increased saline stability by using a Poly(ethylene glycol) linker. Carbohydrate introduced using a alkyne azide ‘click’ reac4on. •  Stable up to at least 1 M NaCl •  Lec4n Speciﬁcity – aggrega4on with ConA (mannose speciﬁc) but not with PNA (galactose speciﬁc) Synthesised glucose func4onal par4cles using a one-‐
pot method. •  Concept proved using Concanavalin A (ConA). •  Size dependent aggrega4on (B). •  Visible colour change (E) •  Tested for response to Type I ﬁmbriated E. coli. •  No colour change but increased aggrega4on noted by Absorbance with Mannose par4cles with K12 (FimH +ve). •  Tested for aggrega4on in response to FimH adhesin. •  Colour change noted with K12 (FimH +ve) and not with TOP10 (FimH –ve) un4l very high bacteria concentra4on. Colour change þ Speciﬁc þ Saline Stable ý Richards, S-‐J.; Fullam, L. C.; Besra, G. S.; Gibson M. I., J. Mater. Chem., 2014 References
1.  C. R. Bertozzi, L. L. Kiessling, Science, 2001, 291, 2357. 2.  M. Ambrosi, N. R. Cameron, B. G. Davis, Org. Biomol. Chem., 2005, 3, 1593. 3.  S. G. Spain, M. I. Gibson, N. R. Cameron, J. Polym. Sci., Part A: Polym. Chem., 2007, 45, 5031 Acknowledgements
Mad Gibson Dave Haddleton Gibson Group Mat Jones Del Besra MOAC Liz Fullam EPSRC Colour change ý Speciﬁc þ Saline Stable þ Summary
Developed a sensi4ve, rapid colourimetric bioassay for the detec4on of lec4ns and Type I ﬁmbriated bacteria. •  PEG layer increases saline stability (important for point of care diagnos4cs) but visual output is drama4cally reduced. • 

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