Dipartimento di Chimica Organica e Industriale

Anna Bernardi Carbohydrate Mimics: Design, Synthesis, and Biological Activity

IASOC 2006Ischia, September 16 - 21, 2006

Organic Chemistry from Synthesis to the Interfaces of Life Sciences

Glycoconjugate and cell surfacerecognitionfrom Science 291, 2357 (2001)

www/sciencemag.org/Science Supplemental Material -- Carbohydrates Web Resources.htm

Electron micrograph of the glycocalixat the surface of a red blood cell

The glycocalix

Functions resulting from glycan-lectininteractions

Directing trafficking of glycoconjugates(intra- and extra-cellular)

Mediating and modulating cell adhesion(cell-cell, cell-matrix)

Mediating and modulating signalling(intra- and extra-cellular)

Sugar/protein interactions as targetDevelopment of probes, diagnostics and drugs

Sear, P.; Wong, C. H.; Angew. Chem. Int. Ed. 1999, 38, 2300

Cell surface carbohydrate-protein interactions play important roles in cell recognition processes.

Cell-cell interaction ( inflammation, fertilization)

Cell-virus interaction (e.g. flu virus)

Cell-molecules interaction (e.g. hormones, toxin)

Bacterial adhesion and biofilm formation (E.coli, H. pylori)

Carbohydrate - Protein interactions are involved in the early stages of many cell communication events, hence they are interesting targets for drug development

Oligosaccharide mimics could be made:metabolically stablemore bioavailableeasier to synthesizehigh affinity ligands ?

Oligosaccharides’drawbacks as drugs

metabolically unstable

low bioavailabilitydifficult to synthesizelow affinity ligands

Biotin – avidin complex

Oligosaccharide – MBP complex

small epitopes on complex scaffolds

REPLACE THE SCAFFOLDKEEP EPITOPE ORIENTATION

REPLACE THE SCAFFOLDKEEP EPITOPE ORIENTATION

HOO

OHO2C

O

OO O

HO

OHNHAc

OHOH

HO

O

OHOH

AcHN

sialyl Lewisx

HO

OMeOH

OH

HOO

OHO2C

OOH

O

OH

Ernst's mimic

HO

OMeOH

OH

Cy

IC50 (E) 80 µM800 µM

H. C. Kolb, B. Ernst Chem. Eur. J. 1997, 3, 1571 The sialyl Lewisx case

OO

OO

OH

XHO

O

HOO

OHO

HOW DO WE REPLACE AXIALLY SUBSTITUTED RESIDUES ?HOW DO WE REPLACE AXIALLY SUBSTITUTED RESIDUES ?

2 Manα1-X=OH 3,4-GalX= NHAc 3,4-GalNAc

DCCHD A Group of Conformationally Stable Cyclohexanediols

DCCHD A Group of Conformationally Stable Cyclohexanediols

OR

OR

CO 2RCO 2R

RO

RO

RO CO 2R

CO 2R

OR

CO 2RCO 2R

OR

ROCO 2R

CO 2RRO

RO

RO CO 2R

CO 2R

RO

RO CO 2R

CO 2R

CO 2R

CO 2R

ROOR

CO 2R

CO 2R

ROOR

CO 2R

CO 2R

RO

Rel. En.(kcal/mol)

0.0 + 4.1

0.0

0.0

+ 2.7

+ 3.7

Rel. En.(kcal/mol)

Rel. En.(kcal/mol)

MM3*

Bernardi, Arosio, Manzoni, Micheli, Pasquarello, Seneci J.Org.Chem. 66, 6209 (2001)

COOH

COOH

DCCHD: A Class of Pseudo-Monosaccharide Scaffolds

DCCHD: A Class of Pseudo-Monosaccharide Scaffolds

HO

HO CO2RCO2R

HOHO

CO2RCO2R

HO

OH

CO2RCO2R

(S,S)-Cyclohex-4-en-1,2-dicarboxylic acid

Bernardi, Arosio, Dellavecchia, Micheli Tetrahedron:Asymmetry 40, 3403 (1999)Bernardi, Arosio, Manzoni, Micheli, Pasquarello, Seneci J.Org.Chem. 66, 6209 (2001)

OsO4

1. MCPBA2. H2O

60% overall yieldNo intermediatepurificationBoth enantiomersavailable

H

H

O

O

O

CO2H

CO2HH

H

HOO

HOO

OHO The 1,2-disusbtituted α Mannose Case:

the oligomannose oligosaccharideThe 1,2-disusbtituted α Mannose Case:

the oligomannose oligosaccharide

Manα1-2Manα16Manα13 6 Manβ1- 4GlcNAcβ1 - 4GlcNAc3

Manα1-2Manα1-2Manα1

Manα1-2Manα1

Man9GlcNAc2, the least processedN-glycan from mature proteins

Man9GlcNAc2, the least processedN-glycan from mature proteins

OHOHO

OHOH

OOHO

HO

OR

OH

The Manα1-2Man fragment is recognizedby DC-SIGN, the dendritic cell receptor that

recognizes HIV-gp120 and other pathogens.

Polyvalent mannobiosides have potential as

antiviral compounds.

The Manα1-2Man fragment is recognizedby DC-SIGN, the dendritic cell receptor that

recognizes HIV-gp120 and other pathogens.

Polyvalent mannobiosides have potential as

antiviral compounds.

MC/EM

-240

-180

-120

-60

0

60

-60 0 60 120 180 240Phi

Psi

MC/SD, 10 ns

-240

-180

-120

-60

0

60

-60 0 60 120 180 240Phi

Psi

S

E

S E

OHOHO

OHOH

OOHO

HO

OMe

OH

α−(1,2)-Mannobiosideα−(1,2)-Mannobioside

AMBER* GB/SA waterMacroModel 5.5

AMBER* GB/SA waterMacroModel 5.5

X-Ray: both presentNMR: both must be present to satisfy NOE constraints

Phi: O5-C1-O1-C2’Psi: C1-O1-C2’-C1’

∆E S-E= 6 kJ/mol

Dwek et al Chem Rev. 2002, 102, 371-386

1’

3’1

1

HOO

HOO

OHO

1’

3’

55

Pseudo-1,2-MannobiosidePseudo-1,2-Mannobioside

MC/SD 10 ns

-240

-180

-120

-60

0

60

-60 0 60 120 180 240Phi

Psi

AMBER* , GB/SA waterAMBER* , GB/SA water

MeOOC

MeOOC

1. TMSOTf, -20°COAcO

AcO

OAc

OTCA

CO2MeCO2Me

HO

O

+ 2. MeONa/MeOH

2. Cu(OTf)2, AllOH

92%

1. MCPBA

OAcOHO

HO

OHOH

O

OR

RO2CRO2C

Manα1,2-Manα mimic

Eur. J. Org. Chem. 2004,5119

The NOE data closely parallel thosefound for the natural disaccharide and are in agreement with the two limitconformations predictedThe compound is more stable thanmannobioside to mannosidase-catalyzed hydrolysis

The NOE data closely parallel thosefound for the natural disaccharide and are in agreement with the two limitconformations predictedThe compound is more stable thanmannobioside to mannosidase-catalyzed hydrolysisES

HOO

HOO

OHO

Tetrameric, type II transmembrane protein (C-lectin), expressed by immature dendritic

cellsBinds to a broad spectrum of pathogens (including HIV, Ebola, Dengue, Leishmania

etc) mainly by targeting highly mannosylated glycoproteinsSome pathogens, including HIV, use DC-SIGN to infiltrate the immune systemDC-SIGN is currently regarded as a new target in infectious diseases

DC-SIGN Dendritic Cell-Specific ICAM-3 Grabbing Nonintegrin

The interaction is multivalentand Ca dependent

Mannose-based multivalentinhibitors of DC-SIGN havebeen reported (J. Rojo et al)

At mM concentration ps-1,2-mannobioside inhibitsDC-SIGN mediated viralentry in an Ebola model

HOO

HOO

OHO

MeOOC

MeOOC

1. Aniline, InBr3 CO2MeCO2Me

HN

OTBDMS

O2. TBDMSCl

ClCH2COClCO2Me

CO2Me

N

TBDMSO

Cl

O

CO2MeCO2Me

N

TBDMSO

O

Pd(OAc)2

PtBu2

N O

Ar

1. ArCHO, piperidineHO

CO2Me

CO2Me

Buchwald et al JACS 2003, xxxx

80%

40%

2. TBAF

Synthesis of potential kinase inhibitors

N O

Ar

OHm

Indolidinones are known kinase inhibitors

N-substitution with polyhydroxylated rings known to improve activity

Marco Re

Collaboration with Pierfausto Seneci - Unimi

HOO

HOO

OHO

O O OO

OO

-O2C

O

OO OR

HO

OHHO

HO

HO OH

OHOH

OHOH

HOAcHN

HOOH NHAc

OHOHGM1

III

N

IIIIV

R= Ceramide

GM1: Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glcβ1-OR

GM2: GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glcβ1-OR

GM3 Neu5Acα2-3Galβ1-4Glcβ1-OR

GM4: Neu5Acα2-3Galβ1-OR

IV IN IIIII

The 3,4 disusbtituted Galactose Case:

Monosialo Gangliosides

The 3,4 disusbtituted Galactose Case:

Monosialo GangliosidesGangliosides are important constituents of biological membranes, particularly in the nervous system, where they participate in various recognition processes.Ganglioside GM1 is also the specific membrane receptor of Cholera Toxin (CT).

Gangliosides are important constituents of biological membranes, particularly in the nervous system, where they participate in various recognition processes.Ganglioside GM1 is also the specific membrane receptor of Cholera Toxin (CT).

OO

OO

OH

OH

O O OO

OO

HO2CO

OO O- ceramide

HO

OHHO

HO

HO OH

OHOH

OHOH

HO

HOOHAcNH

OHOH

AcHN

B5PENTAMER

ASUBUNIT

The cholera toxin

Mimicking the GM1-osMimicking the GM1-osO O O

OO

OHO2C

O

OO OH

HO

OHHO

HO

HO OH

OHOH

OHOH

HO

HOOH NHAc

OHOH

AcHN

O O OO

OHO2C

O

HO

OHHO

HO

HO OH

OHOH

HO

OH NHAc

AcHN

COORCOOR

GM1-os

ps-GM1

J. Am. Chem. Soc. 121, 2032 (1999)

GM1-os and ps-GM1 havethe same structure and the same affinity for CT

GM1-os and ps-GM1 havethe same structure and the same affinity for CT

O O O

HO2C

H

OHHO

HO

HO OH

OH AcHN O

O CO2RCO2R

R

ps-GM1

HO2C

HPh

HO2C

Me H

HO2C

HCy

KD (µM)

190

1010

45

Polyvalency improves the affinity to the nM range(Org. Biomol. Chem. 2004, 2113; J. Am. Chem. Soc. 2005,127, 3660 )

NMR studies have allowed rational design of the mimics(Org. Biomol. Chem. 2004,1, 785; Chem. Eur. J. 2002, 4597; Chem. Eur. J. 2004, 4395)

OO

OO

OH

OH

OSP

HN

HNSP

O

SCAFFOLD

psGM1

Linker

SP

OR

O O

RO

Spacer

R = Me, Et

O

OO O

H2N OO

O NH2

NH2H2N Linker

MeO2C

O

O

NH

O

NHO

O

O

NHBoc

NHBoc

O

O

NHBoc

NHBoc

Collaborazione conUniversità di Utrecht

SCAFFOLD O O OO

COOHCOOHCOOHHOOC

Collaborazione conUniversità di Parma

OO O

O

OBnO2C

HMe

OAcAcO

AcOOAc OAc

OAcNHAc

COOHCOOMepsGM1-(R)lat

190 µM

Org. Biomol. Chem. 2004, 2113 J. Am. Chem. Soc. 2005,127, 3660

POLYVALENT psGM1 LIGANDSPOLYVALENT psGM1 LIGANDSPOLYVALENT psGM1 LIGANDSOO

OO

OH

OH

NHO

H

OO

OOOH

OH

OH OH

OHOH

NHAcCOOMe

O

Me

NH

O O

NH

O3

O

O

Me

OO

OOOHOH

OHOH

OH OH

NHAcCOOMe

O

HNH

O NH

O

O O

NH

NH

NH

O

OO

O

O

O

HOOC

HOOC3 3

3

Divalent Calixarene: Fluorescence titrationsDivalentDivalent CalixareneCalixarene: : FluorescenceFluorescence titrationstitrations

In this assay, the divalentcalixarene appears more activethan the natural ligand o-GM1

In this assay, the divalentcalixarene appears more activethan the natural ligand o-GM1

IC50= 48 nMβ = 3950

0 .1 µ M C T

[L ] n M0 2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 0 1 2 0 0 1 4 0 0

δI

0

2

4

6

8

1 0

o -G M 1d iv a le n t c a lix

J. Am. Chem. Soc. 2005,127, 3660

OO

OO

OH

OH

OOligosaccaride-O

OHNH

NHAc

OH

O NH

O

NH

O

peptide

β-N-Linked

peptide

In N-linked glycopeptides and glycoproteins the oligosaccharides are βlinked to the Asn side chain of a Asn-Xaa-Thr/Ser consensus sequenceKunz, H. et al Chem. Rev. 2000, 100, 4495-4537

OO

HOAcNH

OHα N-linked

HN

O HN

O

NH

O

peptide

peptide

Oligosaccaride

α-N-linked glycosylamides and glycopeptides are likely to be metabolically stable analoguesα-N-linked glycosylamides and glycopeptides are likely to be metabolically stable analogues

Their chemical properties and biological activity are virtuallyunexplored:

•one natural α-N-linked glycopeptide(nephritogenoside) isolated from natural sources (Shibata et al J.Biol.Chem.1998, 263, 12843)

• one synthetic α-N-linked chitobiosylpeptide: the configuration of the anomeric carbon controls the conformation of the peptide (Imperiali et alJ. Am. Chem. Soc. 2004, 127, 8421)

Their chemical properties and biological activity are virtuallyunexplored:

•one natural α-N-linked glycopeptide(nephritogenoside) isolated from natural sources (Shibata et al J.Biol.Chem.1998, 263, 12843)

• one synthetic α-N-linked chitobiosylpeptide: the configuration of the anomeric carbon controls the conformation of the peptide (Imperiali et alJ. Am. Chem. Soc. 2004, 127, 8421)

α-N-glycosylamides, a new class of glycoconjugatesα-N-glycosylamides, a new class of glycoconjugates

The stereoselective synthesis of α glycosylamides is not trivial

OHO

HOOH

OH

OH

NH3, NH4Cl0°C

OHO

HOOH

OH

NH2

OHO

HOOH

OH

NH2

H2ORNH2 + RI

3P O

+ RI3P ORNHCORII

1) RIICOX

2) H2OR N PRI

3

R''CO X

R = PyranoseR = Pyranose

N3R R N PRI3+ RI

3P

Staudinger reduction/acylation can be used to avoid amineanomerization

iminofosforane

AcOAcO

N3

AcO

OAcO

AcOAcO

NHAcOAc

OAcO

AcOAcO

NHCOCF3

AcO

OAcO

a. PMe3b. Ac2O

b. (CF3CO)2Oa. PMe3

Resa = 80%

Resa = 51%

Kovács, L.; Ösz, E.; Domokos, V.; Holzer, W.; Györgydeák, Z. Tetrahedron 2001, 57, 4609-4621.

100% β

100% α

However, the iminofosforane is also prone to isomerization

O

NRO

PR'3

ONRO PR'3

kacRIICOX

ON

RO PR'3

COR''

x

keq keq

Selectivity dependson the kac/keq ratio

Selectivity dependson the kac/keq ratio

O

NRO

PR'3

O

N3

ROPR'3

RIICOX

O

N

RO

PR'3R''OC

x

kac

O

N3OAc

AcOAcO

OAcO

NOAcO

AcO

OAc

Me

PPh3

ON

OAcAcO

AcO

OAc

O

NAcO

AcO

OAc

N2

O

Me

OPPh3

PPh3

ON

OAcAcO

AcO

OAc

PPh3

O=PPh3

RCOSPy

Cu2+

O

NHCORAcOAcO

AcO

OAc

Damkaci, F.; DeShong, P. J. Am. Chem. Soc. 2003, 125, 4408

DeShong Isoxazoline Method

PPh2O

O

ROBnO

BnO

N3BnO

OBn

+

OBnO

BnOHN

BnO

OBn

O

R

α

OHO

HOHN

OH

OH

O

R

Bianchi A., Bernardi A. Tetrahedron Letters 2004, 2231; J. Org. Chem. 2006, 4565

The Staudinger ligation: a general method for the synthesis of α-glycosylamides

The Staudinger ligation: a general method for the synthesis of α-glycosylamides

OBnOBnO

OBn

BnON

Ph2PR

OOO

R

O

OBnOBnO

OBn

BnON

PPh2

The intramolecular acyltransfer competes more efficiently with the anomerization process

The intramolecular acyltransfer competes more efficiently with the anomerization process

H2O

O

N3BnO

BnOBnO

OBn

O

NHCOCH3BnO

BnOBnO

OBn

1.2 eq

y = 77%

O

N3BnO

BnO

BnO OBn

O

NHCOCH3BnO

BnO

BnO OBn

1.2 eqy = 73%

Staudinger ligation: acetylation of α-glycosyl azidesStaudinger ligation: acetylation of α-glycosyl azides

α only

OAc

PPh2

OAc

PPh2

Bianchi, A.; Bernardi, A. Tetrahedron Lett. 2004 45, 10, 2231-2234

β only

O

N3BnO

BnOBnO

OBn

2) PFP-OAc

ONHCOCH3

BnO

BnOBnO

OBn1) PMe3 y = 77%

α only

>97:35685:1565-CH=C(CH3)2

93:765---(CH2)3COOMe

α/β ratioYield (%)α/β ratioYield (%)R =

--100:077-CH3

>97:37684:1640-CH(CH3)2

95:58183:1770-CH2CH(CH3)2

93:78384:1660-(CH2)3CH3

Method B (DMF)Method A (CHCl3, hν)

O

N3BnO

BnOBnO

OBn

PPh2

OCOR

O

NHCORBnO

BnOBnO

OBn

+

B nO O

NB nO

B nO

O B n

NN

PPh 2R O C O

B nO O

NB nO

BnO

O B n

NN

PPh 2R O C O

P Ph 2

O R

O

BnO O

NB nO

B nO

O B n

Ph 2P

O

R O C

BnO O

N 3B nO

B nO

O Bn

PP h2O H

OH 2O

B nO O

NB nO

B nO

O B n

NN

Ph 2P

R O C O

N 2

BnOO

N H C O RBnO

B nO

O Bn

B nOO

NB nO

B nO

O B n

P h2P

O

C O R

+

EZ

triazad iene

im inophosphorane (aza-y lide)

A. Bianchi, A. Russo, A. Bernardi Tetrahedron: Asymmetry 2005, 16, 381-386

Mechanism of the Staudinger ligation of glycosyl azides

solvent α/β ratio Yield (%)Toluene 86:14 75*1:3 DMA:Tol 75:25 66

+

OPPh2

O NHCbz

O

OMe O

NHBnOBnO

BnO

OBn

O

COOMe

NHCbz

O

N3BnO

BnOBnO

OBn

O

N3BnO

BnO

BnO OBnPPh2

O

O Me

MeO

HNBnOBnO

BnO OBn

O Me

Me

α : β 98 : 2

70%

H2 (4 atm), Pd-C

DMA / AcOH / H2O85 : 10 : 5

O

NHBnO

BnOBnO

OBn

O

COOMe

NHCbz

O

NHHO

HOHO

OH

O

COOMe

NH2

DeprotectionDeprotection

O

N3HO

HOHO

OH

PPh2

OCOR

O

NHCORHO

HOHO

OH

+ DMA, 2% DMPU

NHCOR

H

HH OH

OH HO

HHO

HO+

..or no protection..or no protection

98:256 -(CH2)3COOMe

98:220

> 98:250-(CH2)3CH3

α/β ratioYield (%)R =

> 98:240

O

N3HO

HOHO

OH

PPh2

OCOR

O

NHCORHO

HOHO

OH

+ DMA/DMPU

Filippo Nisic

-H2C OMe

O

NHCbz

-H 2 CO M e

O

N H C b z

O

NHCOCH3OH

H2

HO

H3

H4

HO

H5

H1

OH

O

NHCOCH3OH

H2

HO

H3

OH

H4

H5

H1

OH

s

s

s s

ss

s

m

mm

w

w w

O

NHCOCH3

H3C

H3

OHOH

H5

H1

OH

The α-glycosylamides examined so far conserve the usual chairconformation of the pyranose ring

The α-glycosylamides examined so far conserve the usual chairconformation of the pyranose ring

4C11C4

Good mimicry of the sugar !

Università di Milano

Daniela ArosioSilvia MariAldo BianchiGiancarlo TerraneoDonatella InvernizziČrtomir PodlipnikGilles MarcouHelena PosteriPierangelo MereghettiAndrea RussoFilippo NisicMarco ReSara Sattin

Donatella PotenzaLaura BelvisiPierfausto Seneci

MadridJesus Jiménez-BarberoJavier Caňada

MIURCNR – ISTMCISIEuropean Network Glycidic ScaffoldsGSK - Verona

€

SevillaJavier RojoJosé ReinaPedro Nieto

CSIC

IBS – GrenobleFranck FieschiGeorges Tabarani

Università di ParmaRocco UngaroAlessandro CasnatiFrancesco SansoneMarco Fontanella