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β1,2-Mannans Conformationally Interesting Molecules
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2012
Polysaccharide Conformation. Part V1. Computer Model-Buildingfor Linear and Branched Pyranoglycans. Correlations with BiologicalFunction. Preliminary Assessment of Inter-Residue Forces in AqueousSolution. Further Interpretation of Optical Rotation in Terms of
ChainConformationD. A. Rees' and W. E. Scott,
Chemistry Department, The University of EdinburghJ. Chem. Soc. B, 1971, 469-479.β1,2 linked glucans and mannans are crumpled and
rigid
β1,2-Mannan a Protective EpitopePredicted to have unique conformational properties
O
HOO
O
HO
O
HO
-mannose residues atO2 and O1 will have stericinteractions
O Properties that could influence biological activity?
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2012
Oligosaccharide inhibition of mAb C3.1 binding to cell wall β-mannan
The surprizing
result of lower activity as inhibitor size increase beyond a trisaccharide
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2012
10 100 1000
0
20
40
60
80
100
inhibitor concentration [M]
% in
hibi
tion
HOO
O
HO
HOO
O
HO
OH
HO
HO
HO
HO
O
HO
R
O
n
Inhibitor IC50
mM
■ Disaccharide 44
Trisaccharide 38
Tetrasaccharide 108
■ Pentasaccharide 770
● Hexasaccharide >1000
Nitz
et al., J. Biol. Chem. 277,
3440, 2002.
Antibody Site Filled by Di-
or TrisaccharideElectronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2012
A 40 year old paradigm: The Size of an Antibody Site that Binds Polysaccharide
Antigens
Kabat E.A and H.H. Mayer, 1961, In
immunochemistry Charles C. Thomas, Springfield
Dextrans (1-6)-
glucose
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2012
800 MHz NMR Studies of a Pentasaccharide
in D2
O solution show well dispersed resonances that facilitate assignment and NOE measurements pointing to a
constrained solution conformation
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2012
Pentasaccharide: 800 MHz
O
OO
OO
OO
O
OO
H
H
H
H
H
H
HO OHHO
HO
H
H
H
H
5 mM
in D2
O, 30.0°C
Nitz
et al., J. Biol. Chem. 277,
3440, 2002.
Solution Conformation of β-1,2-Mannan Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2012
nt=1, spin lock: mix=130 ms, 6.6 kHz
Pentasaccharide: 800 MHz GTOCSY
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2012
Pentasaccharide: 800 MHz GTOCSY
Nitz
et al., J. Biol. Chem. 277,
3440, 2002.
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2012
nt=24, spin lock: mix=400 ms, 4.2 kHz
Pentasaccharide: 800 MHz TROESY
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2012
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2012
Pentasaccharide: Short Range ROEs
O
OO
OO
OO
O
OO
H
A
B
C
D
E
H
H
H
H
H
H
H
H
H
HO OHHOHO
12
2.3
2.2
2.2
n/n+1 ROEs
O
OO
OO
OO
O
OO
A
B
C
D
HO OHHO
HO
(~2.2)
E
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2012
Pentasaccharide: Long Range ROEs
O
OO
OO
OO
O
OO
H
A
B
C
D
E
H
H
H
H
H
H
H
H
H
HOHO
HOHO
12
4H
H
H2.8 3.1
H
2.7
2.7
3.1
3.2
n/n+2 ROEs n/n+3 ROEsn/n+2 ROEs
O
OO
OO
OO
O
OO
H
A
B
C
D
E
H
H
H
H
H
H
H
H
H
HO OHHOHO
12
4H
H
H
3.7
H
4.2
Nitz
et al., J. Biol. Chem. 277,
3440, 2002.
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2012
Tetrasaccharide: Long Range ROEs
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2012
Larger Oligosaccharides Clash with Antibody Site
Johnson et al., J. Biol. Chem. 287, 18078-18090, 2012.
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2012
Mapping the Carbohydrate Epitopeof Candida albicans Protective Antibody C3.1
Functional Group ReplacementSequential hydroxyl group substitution
HO H
HO CH3O
Nycholat
and Bundle
Carbohydr. Res., 344, 555-569, 2009.Bundle, et al., ACS Chem. Biol., 7, 1754-1763, 2012.
Nikrad, Beierbeck
and Lemieux. A Novel Procedure for the Detection of the Intermolecular Hydrogen Bonds Present in a Protein-Oligosaccharide Complex, Can. J. Chem., 70, 241-253, 1992.
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2012
OOMeO
O
MeO
Ph OH
2.) L-Selectride, THF, -78 oC
1.) Me2SO, Ac2O
OOMe
OH
OO
HO
Ph1.) Bu2SnO, PhCH3;2.) MeI, DMF;
OOMe
OH
OO
MeO
Ph
71%
80%
OOMe
OPMB
OO
O
Ph
SN
NO
OMeOPMB
OOPhBu3SnH, AIBN,
PhCH3, reflux
OOMe
OH
OO
HO
PhO
OMeOPMB
OO
HO
Ph TCDI, PhCH3Bu4NHSO4, 5% aq. NaOH
OOMeO
OPh OHO
OMeOH
OOPh
2.) L-Selectride, THF, -78 oC
1.) Me2SO, Ac2O
DDQ, CH2Cl2/H2O
30%
64%
78%86%
83% (2 steps)
PMBCl
Synthesis of 3-O-Methyl β-Mannopyranoside
Synthesis of 3-O-Deoxy β-Mannopyranoside
Nycholat
and Bundle Carbohydr. Res., 344, 555-569, 2009.
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2012
OOMeO
O
MeO
Ph
OO
OAc
BnOBnO
BnO
OOMeO
OPh
OO
OAc
BnOBnO
BnO
MeONa, MeOHquant. 88%
65% 72%2) L-Selectride, THF, -78 oC
1) Me2SO, Ac2O
75% 74%
OOMeHO
MeO
OOHO
HO
HO OH
HOO
OMeHO
OOHO
HO
HO OH
HO
H2, Pd/C, MeOH, CH2Cl2
OO
OAc
BnOBnO
BnO
NH
CCl3
59% 97%O
OMeOO
MeO
Ph OHO
OMeOOPh OH
TMSOTf, CH2Cl2, 0 oC
Synthesis of the 3-deoxy and 3-O-methyl -Mannobiosides
Nycholat
and Bundle Carbohydr. Res., 344, 555-569, 2009.
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2012
Functional Group Replacement
DeoxyRelative potency
O-MethylRelative potency
3-OH inactive inactive4-OH inactive inactive6-OH 238 382′-OH ND 943′-OH 26 504′-OH inactive 4.66′-OH 7.3 5.3
OCH3OHO
HO
OH
OOHO
HO
OHOH
2'
4
Relative potency = 100
3
6'
Inhibition by Modified Disaccharide Glycosides
Bundle, et al., ACS Chem. Biol., 7, 1754-1763, 2012.
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2012
O-3
O-4
O-4'O-1
O-2''
Key Polar Contacts (red)
A
B
C
Bundle, et al., ACS Chem. Biol., 7, 1754-1763, 2012.
Site of chain extension
Site of attachment to phosphomannan side chains via phosphodiester or a glycosidic
bond
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2012
A
B
C
Johnson et al., J. Biol. Chem. 287, 18078-18090, 2012.
A trisaccharide docked in the binding site of C3.1
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2012
Which β-Mannan is the Protective Epitope?
OHO
HOHO
OHO
HOHO O
OHO
HO
HO
OHO
O
P
O
O OOHO
HO
OH
O
Reducing Epitope
Non-Reducing Epitope
OHO
HOHO
OHO
HOHO O
OHO
HO
HO
OHO
OHO
HOHO
O
OO
HOHO
HO
O
Non-Reducing Epitope
Reducing Epitope
A
B
C
A
B
C
Phosphodiester linked Glycosidically linked
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2012
HOO
O
HO
HOO
O
HO
OH
HO
HO
OH
O
HO
R
OH
HOO
O
R
HOO
O
HO
OH
HO
HO
HO
HO
O
HO
O
R = HR = OMe
Freezing Epitope Frame Shifting
Costello and Bundle, Carbohydr. Res., 357, 7-15, 2012.Bundle, et al., ACS Chem. Biol., 7, 1754-1763, 2012.
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2012
“A”“B”
“C”
Johnson et al., J. Biol. Chem. 287, 18078-18090, 2012.
The Limits of Frame Shifting: Tetrasaccharide or larger oligosaccharide are unable to frame shift without steric
clash with the protein or significant oligosaccharide conformational changes
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2012
24
Allowed Binding Area for (12)--Mannans Accommodates Four Residues
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2012
Synthesis, Conjugation and Evaluationof a Trisaccharide Conjugate Vaccine
Wu, X.; Bundle, D. R.
J. Org. Chem., 70, 7381-7388, 2005.
Tetanus ToxoidOHO
HO
HOO
O
OHOHO
HOO
OHOHO
HO OH
O
O
SNH
10-15
βMan βMan βMan Tetanus ToxoidLinker •
Gives high antibody titres in rabbits
•
Reduces fungal load in live challenge experiments
• Mediocre response in mice
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2012
OBnOBnO
BnO OHOAll
OBnOBnO
BnO
OAcO
NH
CCl3TMSOTf, CH2Cl2
OBnOBnO
BnO
O
OAll
OBnOBnO
BnO
OAcOBnO
BnO
BnOO
OAll
OBnOBnO
BnO HO
OBnOBnO
BnOO
OAll
OBnOBnO
BnO
OBnOBnO
BnO
OAcO
OBnOBnO
BnOO
OAll
OBnOBnO
BnO
OBnOBnO
BnO OH
O
(1) NaOMe/MeOH
93%
(2) DMSO/Ac2O(3) L-selectride, THF 88%
81%
+
(1) NaOMe/MeOH(2) DMSO/Ac2O(3) L-selectride, THF 77%
TMSOTf, CH2Cl2
OBnOBnO
BnO
OAc
O
NH
CCl3
Kochetkov, N. K et al. Carbohydr. Res. 33, C5, 1974.Ekborg, G.; Lönngren, J.; Svensson, S. Acta Chem. Scand. B29, 1031, 1975.Mathew, F.; Mach, M.; Hazen, K. C.; Fraser-Reid, B. Synlett, 9, 1319, 2003.
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2012
OHOHO
HOO
O(CH2)3S(CH2)2NH2
OHOHO
HO OH
OHOHO
HOO
O(CH2)3S(CH2)2NH2
OHOHO
HOO
OHOHO
HO OH
OHOHO
HO O
O(CH2)3S(CH2)2NH2
OHOHO
HOO
OHOHO
HOO
OHOHO
HO OH
(a) 2-aminoethanethiol hyrochloride, MeOH/CH2Cl2, hv 365 nm(b) Na/NH3, t-BuOH, THF
80%
OO
OBn
Wu, X.; Bundle, D. R.
J. Org. Chem., 70, 7381-7388, 2005.
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2012
OHOHO
HOO
O
OHOHO
HOO
OHOHO
HO OH
O
ONO2
O
O
O2N
DMF
SNH2
OHOHO
HOO
O
OHOHO
HOO
OHOHO
HO OH
O
O NO2
O
SNH
+
aqueous bufferTetanus Toxoid
Tetanus ToxoidOHO
HO
HOO
O
OHOHO
HOO
OHOHO
HO OH
O
O
SNH
10-15
Wu, X.; Bundle, D. R.
J. Org. Chem., 70, 7381-7388, 2005.
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2012
Rhodamine labelled second antibody
Rabbit Antibody raised against trisaccharide tetanus toxoid glycoconjugate
Candida albicans
Trisaccharide Vaccine Induces Antibodies that bind to Native Antigen on Candida Cells
Lipinski, T., et al.,
Vaccine, 30, 6263-
6269, 2012.
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2012
Rabbit Challenge
Protection experiments in neutropenic rabbits
Rabbits immunized twice with oligosaccharide-TT conjugates, rendered neutropenic and then challenged with live C. albicans.
ImmunizationCyclophosphamide
Challenge with live CandidaDay 0 Day 21 Day 28 Day 34
Termination
Lipinski, T., et al.,
Vaccine, 30, 6263-6269, 2012
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2012
Candida counts in New Zealand While Rabbits Immunized with Trimannose-TT and TT, and Challenged with C. albicans (3X103
cfu)
Statistical analysis by Generalized Estimating Equation: significant reduction of Candida counts in kidney and liver no statistically significant effect for spleen and lungs.
Control / Vaccinated ratio of Candida in examined organs: Kidney: 9.3, p=0.016; Liver: 193.6, p=0.035; Spleen: 1.3, p=0.8; Lungs: 20.4, p=0.99. p-statistical significance value
Lipinski, T., et al.,
Vaccine, 30, 6263-6269, 2012.
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2012
Glycopeptide Vaccine
Componentsβ-mannan trisaccharidePredicted T-cell peptides from C.albicans cell wall proteins
YGKDVKDLFDYAQELinker
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2012
C. albicans Glycopeptide Vaccine
(with Jim Cutler)
NH
O
O
R
O
OO
HN
O
OHOHO
O
HO
O
OHOHO
O
HO
OHOHO
HOOH
SNH
OO
O
O
3
4
O OO
OO
ON
O
O O TrtSCOOH
O
SO
OOOHOHO
O
HO
O
OHOHO
O
HO
OHOHO
HOOH
SNH
HS
O
OHOHO
O
HO
O
OHOHO
O
HO
OHOHO
HOOH
SNH2
II
1
TentaGel Rresin
13x Solid-phasepeptide synthesis
Conjugation
peptide
1. Tris · HCl buffer, 0.05 MpH 8.9, 4h
2. purification by RP-HPLCYield: 64 - 81 %
2
borate buffer
Carrier peptide fromsurface proteinof C. albicans
Dziadek; Jacques; Bundle. Chem. Eur. J.,
14, 5908-5917, 2008.
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2012
Fungal Challenge
DC cells cultured and pulsed with glycopetide conjugate injected into Balb/c mice
day 9 day 14
Second injection of antigen pulsed DC
Mice immunized with glyoconjuagte in CFA
day 28
Challenge with C. albicans
day 42
-(Man)3
-Hwp1 -(Man)3
-QGETEEALIQKRSY
-(Man)3
-Eno1 -(Man)3
-DSRGNPTVEVDFTT
-(Man)3
-Pgk1 -(Man)3
-VPLDGKTITNNQRI
-(Man)3
-Gap1 -(Man)3
-NRSPSTGEQKSSGI
-(Man)3
-Fba -(Man)3
-YGKDVKDLFDYAQE
-(Man)3
-Met6 -(Man)3
-PRIGGQRELKKITE
Hong, Dziadek, Bundle, Cutler PNAS 105,13526-13531,
2008.
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2012
Avoiding Dendritic Cell PrimingGlycopeptide Conjugated to Tetanus Toxoid
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2012
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2012
-MannanTrisaccharide
T cell peptide Tetanus Toxoid
Triethylene glycollinker
Triethylene glycollinker
3 + ALUM
3 + MPL
MPL
ALUM
Buffer alone
3 without adjuvant
• 100% protection of mice
• Adjuvant not required
Xin
et al., PLoS
One 7, e35106, 2012.
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2012