Glycosylated Nanomaterials: Neutralisa3on and Detec3on of Bacteria and Toxins Sarah-Jane Richards &
Matthew I. Gibson
Department of Chemistry, University of Warwick, UK
S-J.Richards@warwick.ac.uk
www.warwick.ac.uk/go/gibsongroup
Mul3GlycoNano 2014 – 26th June Protein-Carbohydrate Interactions
Cell signalling
Fertilisation
Cellular adhesion of
•  Viruses
•  Bacteria
•  Bacterial toxins
Inflammation
You Houng, Nat. Mater., 2010, 9, 485 Gamblin, Chem. Rev, 2009, 109, 131 Miura, JPOLA, 2007, 45, 5031 Imberty, Chem. Eur. J., 2008, 14, 7490 Spain, Polym. Chem., 2011, 2, 60 Turnbull, B., Rev. Mol. Biotech., 2002, 90, 231 Affinity
Why Materials?
Predicted linear
response
Number of Binding Epitopes
Kiessling, Angew, Chem., 2006, 45, 22348 Alexander et al, JACS, 2007, 129, 11014 Mammam, J. Med. Chem., 1995, 38, 4179 Spain et al. Polym. Chem., 2011, 2, 60 Applications: Anti-adhesion Therapy
Interactions can be inhibited at
nM of glycopolymers
Influenza inhibition in mice
Sharon, N. Biochimica et Biophysica Acta, 2006, 527–537
Hidari et al. Glycobiology 2008, 18, 779
Glycopolymers by Post-Polymerisation Modification
Jones, M. W., Richards, S-­‐J., Haddleton, D. M. Gibson, M. I.; Polym. Chem., 2013, Richards, S-­‐J., Jones, M. W., Hunabun, M. I., Haddleton, D. M., Gibson, M. I.; Angew. Chem., 2012 HO
HO
O
HO
HO
HO
HN
n
O
O
NaN3
N3
HO
Cu(I)Ligand
O
HO
OH
HN
O
n
O
O
Cu(I)Ligand
OH
O
N3
N
N
N
OH
OH
OH
O
O
OH
HO
HO
O
O
HO
HO
O
HO
O
O
N
N
N
Jones, M. W., L. OXen, Richards, S-­‐J., Lowery, R., Phillips, D. J. Haddleton, D. M. Gibson, M. I.; Chem. Sci., 2014 HN
O
F
n
O
n
O
F
HN
HN
F
HN
n
O
x
O HN
y
O
n
O
N
N
N
R
F
F
OH
-  Easy to make 50 gram
scale
-  1 column/distillation
-  Compatible with RAFT/
ATRP
-  Quantitative
functionalisation with
non-hindered amines
-  Density control
Theato, P.; J. Pol. Sci. A., 2008, 46, 6677
-  Sequentially
modified
polymer libraries
Selective Binding of Cholera-Toxin
Enzymatic domain
Induces toxic effect
Carbohydrate
binding domain
Binds to epithelial cells
to promote cell uptake
Anti-adhesion therapy does not target
bacteria, so less evolutionary stress
Galectins – at least 13
β-D Galactose
Sigma-Aldrich – 8 Galactose-’specific’ lectins
How do we engineer a high-affinity binder for
cholera toxin, without total synthesis of
complex carbohydrates?
GM-1 ganglioside
Glycan Accessibility as a Tool for Lectin Specificity
Peanut Agglutinin
Cholera Toxin
w
w
Asn 14
w
Asp 80
Asn 90
Gly 213
Ser 211
Asp 83
Lys 91
w
Glu 51 Asn 127
w
Gln 56
w
w
w
Gly 104
w
w
Trp 88
Ile 58
His 13
Gly 33
Gln 61
w
w
Glu 129
w
Leu 212
Glu 11
Ile 101
Leu 31
16 Å
Asn 41
6.3 Å
Kiick et al.; Macromol. 2007, 40, 7103 and Biomac., 2006, 7 483
Glycopolymer Library
O
O
O
O
F
i)
O
F
F
F
O
+
H2N
HN
NH2
m
m = 0 or 2
m
Br
n
O
F
x
O
HN
dw/d(LogM)
HN
OH
O
HO
N3
OH
O
OH
HO
HO
x = 10, 25, 50, 100
y = 90, 75, 50, 0
N
-1
-1
Br
y
O
x
O
HN
HO
n = 25, 50, or 100
2.0
HO
O
2.5
O
ii)
Br
y
O
m
O
N
N
O
HO
-1
Mn - 7800 g.mol
Mn - 6100 g.mol
Mn - 7250 gmol
PDi - 1.19
PDi - 1.24
PDi - 1.32
1.5
1.0
0.5
0.0
1000
10000
-1
Mw (gmol )
1000
10000
1000
Mw (gmol-1)
Polymer
GP1
GP2
GP3
GP4
GP5
GP6
GP7
GP8
GP9
DP[a]
18
33
70
18
33
70
33
33
33
10000
Mw (gmol-1)
Linker[b]
Short
Short
Short
Long
Long
Long
Long
Long
Long
Density[c]
100
100
100
100
100
100
50
25
10
Mw/Mn[d]
1.29
1.27
1.26
1.32
1.28
1.27
1.23
1.21
1.20
OH
OH
Peanut Agglutinin
Polymer length
Polymer length
30
25
25
MIC50 (µM Galactose)
MIC50 (µM Galactose)
Cholera Toxin
20
15
10
5
0
GP1
GP2
GP3
Short Linker
GP4
GP5
GP6
Long Linker
20
15
10
5
0
GP1
GP2
GP3
Short Linker
GP4
GP5
GP6
Long Linker
Richards, S-­‐J., Jones, M. W., Hunabun, M. I., Haddleton, D. M., Gibson, M. I.; Angew. Chem., 2012, 51, 7812 Gou, Y., Richards, S-­‐J., Haddleton, D. M. Gibson, M. I.; Polym. Chem., 2012, 3, 1634-­‐1640 OXen, L., Richards, S-­‐J., Fullam, E., Besra, G. S. Gibson, M. I.; J. Mater. Chem. B, 2013, 1, 2665-­‐2672 Jones, M. W., Richards, S-­‐J., Haddleton, D. M. Gibson, M. I.; Polym. Chem., 2013, 4, 717-­‐723 Jones, M. W., L. OXen, Richards, S-­‐J., Lowery, R., Phillips, D. J. Haddleton, D. M. Gibson, M. I.; Chem. Sci., 2014, 5, 1611-­‐1616 Applications: Detection
-
-
-
-
-
Direct
Label-free Easy
Sensitive Quantifiable in real-time
Potential Sensor for Ricin
HO
HO
HO
1.4
O
S
O
S
1.0
0.8
[ConA] 0.6
0.4
HO
HO
HO
OH
OH
Absorbance
O
S
HO
HO
HO
HO
HO
HO
O
SH
HO
HO
HO
OH
OH
HAuCl 4/Reflux
O
S
OH
1.2
0.2
450
500
550
600
650
700
1.0
(Normalised)
Abs700 (Normalised)
AuNP 1
AuNP 2
AuNP 3
Abs
700
0.5
0.0
0.1
1
log[ConA] (nM)
10
100
1.0
1.0
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2
0.0
0.0
0
5
10
15
20
25
Time (Minutes)
Richards, S-­‐J., Fullam, E., Besra, G. S. Gibson, M. I.; J. Mater. Chem. B, 2014, 2, 1490-­‐1498 Normalised Diameter
Wavelength (nm)
1.6
1.2
1.2
1.0
0.8
0.6
0.4
0.2
450
1.0
[FimH-­‐ bacteria] 1.4
Absorbance
Absorbance
1.4
Abs700 (Normalised)
1.6
[FimH+ bacteria] 1.0
Colour Change þ
Fast þ
Specific þ
Wavelength (nm)
Wavelength (nm)
Saline Stability ý 0.8
0.6
0.4
500
550
600
650
700
0.2
450
500
550
600
650
700
K12
Top10
K-12
+ FimH
0.5
TOP10
- FimH
OD = 0.25
0.0
1E-3
0.01
log(OD)
0.1
1
OD 0.005
[Bacteria] Richards, S-­‐J., Fullam, E., Besra, G. S. Gibson, M. I.; J. Mater. Chem. B, 2014, 2, 1490-­‐1498 5
O
O
O
O
(OH) 5
(OH) 5
O
O
SH
1.8
0.08
1.6
O
O
O
)5
(OH
[ConA]
1.4
(O
H)
Colour Change ý
Fast ý
Specific þ
Saline Stability Cu(I)Br/DMSO/TBTA
O
n
5
O
O
N
N N
5
O
(OH
)
(OH) 5
HO
O
O
PEG func3onalised Abs700
Absorbance
(OH
)
O
40 nm
OH
SH
n
O
O
O
O
O
O
HO
5
O
OH
"-Gal
)5
or
(O
H)
O
HO
HO
!-Man
N3
OH
(OH
HO
1.2
0.06
1.0
0.8
0.6
0.4
0.04
0.2
0
450
50
500
100
550
150600
200650 250 700
Time (min)
Wavelength
(nm)
Richards, S-­‐J., Fullam, E., Besra, G. S. Gibson, M. I.; J. Mater. Chem. B, 2014, 2, 1490-­‐1498 OH
HN
F
F
O
F
F
F
O
O
S
n
OH
HO
S
10
HO
HN
OH
S
O
HO
H
N
O
O
SH
n
OH
HO
HO
HN
OH
O
HO
H
N
O
O
S
n
Colour Change þ Fast þ
Specific þ
Saline Stability þ Richards, S-­‐J., Fullam, E., Besra, G. S. Gibson, M. I., In Prepara3on Summary
•  Tandem Post-Polymerisation Modification
•  Multivalent inhibitors that have good affinity AND
specificity
•  Glycogoldnanoparticles
•  Optimised polymer coating for
lectin detection
Acknowledgements
Gibson Group Members
Matthew
Gibson
Dave Haddleton
Del Besra
Elizabeth Fullam
Mathew Jones
-
-
-
-
-
-
-
-
Gemma-Louise Davies
Yunhua Chen
Catherine Cooper
Robert Deller
Caroline Biggs
Daniel Phillips
Tom Congdon
Lucienne Otten
-
Daniel Mitchell
Richard Lowery
Ben Martyn
Lewis Blackman
Sang-Ho Won
Laura Wilkins
Robert Keogh
Glycosylated Nanomaterials: Detec3on and Neutralisa3on of Bacteria and Toxins Sarah-Jane Richards & Matthew I.
Gibson
Department of Chemistry, University of Warwick, UK
S-J.Richards@warwick.ac.uk
www.warwick.ac.uk/go/gibsongroup
Mul3GlycoNano 2014 – 26th June