β1,2-Mannans Conformationally Interesting MoleculesPolysaccharide Conformation. Part V1. Computer...

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?


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


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


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


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


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


Pentasaccharide: 800 MHz GTOCSY

Nitz


3440, 2002.


nt=24, spin lock: mix=400 ms, 4.2 kHz

Pentasaccharide: 800 MHz TROESY


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


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


3440, 2002.


Tetrasaccharide: Long Range ROEs


Larger Oligosaccharides Clash with Antibody Site

Johnson et al., J. Biol. Chem. 287, 18078-18090, 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.


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.


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.


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.


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


A

B

C


A trisaccharide docked in the binding site of C3.1


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


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.


“A”“B”

“C”


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


24

Allowed Binding Area for (12)--Mannans Accommodates Four Residues


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


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.


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


J. Org. Chem., 70, 7381-7388, 2005.


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


J. Org. Chem., 70, 7381-7388, 2005.


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.


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


Vaccine, 30, 6263-6269, 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


Vaccine, 30, 6263-6269, 2012.


Glycopeptide Vaccine

Componentsβ-mannan trisaccharidePredicted T-cell peptides from C.albicans cell wall proteins

YGKDVKDLFDYAQELinker


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.


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.


Avoiding Dendritic Cell PrimingGlycopeptide Conjugated to Tetanus Toxoid


-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.


β1,2-Mannans Conformationally Interesting MoleculesPolysaccharide Conformation. Part V1. Computer...

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