Automated Oligosaccharide Synthesis

Peter H. SeebergerPeter H. Seeberger

Biopolymers: OverviewBiopolymers: Overview

Replication

O B

XOPO

O B

O

O

O-O

XTranslation

HC C N

HCH C

RNH

O

R

O

ProteinsNucleic Acids

HO O O OHO

NHAc

O OHO

NHAc

OH

O

HO OHO

HO

OH OHHO OHO

O

HO

O

O

Transcription

Oligosaccharides - Glycoconjugates

Glycosyltransferases

GenomicsGenomics ProteomicsProteomics GlycomicsGlycomics

Biopolymer Interactions

Nucleic Acids Proteins

Nucleic Acid - Protein Interactions

Protein - ProteinInteractions

Nucleic Acid - Nucleic AcidInteractions

Carbohydrates

Carbohydrate - Protein Interactions

Carbohydrate - Nucleic AcidInteractions

Carbohydrate - CarbohydrateInteractions

Comment by Cell Biology Correspondent

“…This is perhaps the greatest tour de force organic and biochemists have yet achieved. Like NASA with its Apollo program, Khorana’s group has shown it can be done, and both feats may never be repeated…”

Nature 1973, 241, 33.

Science 2001, 291, 1523

The Automated Oligosaccharide SynthesizerThe Automated Oligosaccharide Synthesizer

Blood Group Determinants and Tumor Associated Antigens

O

OPivPivOBnO

OOBnO

OBnBnOO

TCAHN

BnO

O OOPivO

OBnBnOOO

BnO OPivO

OBn

OO

PivOOPivOBn

HO

O

PivOOPivOBn

OOBnO

OBnBnOO

TCAHN

BnO

O OOPivO

OBnBnOOO

BnO OPiv

OBn

OHO

HO

Lex

O

PivOOPivOBn

OOO

OBnBnOO

TCAHN

BnO

O OOPivO

OBnBnOOO

BnO OPiv

OBn

PivOO

HO

O

OPivPivOBnO

OOBnO

OBnBnOO

TCAHN

BnO

OO

O

PivOOPivOBn

LeyLex

Ley

OFmocOBnO

OPiv

OBn

O PO

(OBu)2

OFmocOPivO

OBnBnOOP

O(OBu)2

OOBn

LevOFmocO

TCAHNO P

O(OBu)2

O

PivOOPivOBn

O PO

(OBu)2

OBnO OBn

OFmocBnO O P (OBu)2

O

Angew. Chem. Int. Ed. 2004, 43, 602

ancora

OBnO

BnOOPiv

OBn

OOBnO

OBnO

O

OBnOBnO

OBnO

OBnOBnO

OAcBnO

Leishmania Cap - 9 h

O

PivOOPivOBn

OOBnO

OBnBnOO

TCAHN

BnOO OO

PivO

OBnBnOOO

BnO OPiv

OBn

OHO

Lex - 11.5 hO

OPivPivOBnO

OOBnO

OBnBnOO

TCAHN

BnOO OO

PivO

OBnBnOOO

BnO OPiv

OBn

O

PivOOPivOBn

OO

Ley - 14 h

Ley-Lex - 23 h

OPivOO

OBnBnOO

NHTCA

BnOO OO

PivO

OBnBnOOO

BnO OPiv

OBn

OPivOOPiv

OBn

OOOBnO

OBnBnOONHTCA

BnOO O

OPivOOPiv

OBn OOPivPivO

BnO

O

OOBnO

NHAc

OBnOO

BnONHAc

OBnOBnO

O

OBn

OBnOBnO

OAc

O

BnO

OBnOBnO

OAcBnO

O

N-Linked Core Region - 9 hOO

AcO

OBnBnOOO

BnO OPivOOO

AcO

OBnBnOBnO O

BnO PivO

MeOO

Proteoglycan Linkage Region - 9 h

BnO OBnO PivO

BnO OBnO PivO

OBnO OO

PivO

O

BnO OBnO BnO

OBn

OBnO OO

PivOBnO O

BnO BnO

OBnOLev

O

n = 3

Phytoalexin Elicitor - 16 h

OBnO

BnO

BnO

O

OBnO

BnO

BnOO

O

OBnO

BnO

BnO

O

OBnO

BnO

BnO

O

OBnO

BnO

BnO

O

OBnO

BnO

BnO

O

OBnO

BnO

BnO

O

OBnO

BnO

BnO

O

OBnO

BnO

BnO

O

OBnO

BnO

BnO

OAc

Mannose Oligomer - 23 h

O

O

OAcBnO

BnO

O

O

BnOBnO

O

O

BnOBnO

n = 3

Rhamnose Pentamer10 h

OOOBn

OPivORBnO

OO OBn

PivOBnO

OBnO OBn

BnOBnO

Gb3 - 6.5 h

Automated Synthesis of Complex Structures

Automation of Difficult GlycosylationsAutomation of Difficult Glycosylations

β-Mannosylation

α-Galactosylation

α-Sialylation

OBnO

OBnO

OPiv

O

OBnO

BnOOPiv

O

OBnBnO

OBnO

O

O

OBnBnO

ONHTCA

O

OBnBnO

BnOO

O OBn

PivOOPiv

Globo-H Hexasaccharide

OBzO

OBn

O

OBz

OOTrocHN

AcO

AcO

OAc

AcO OO

OBnBnOCOOMe

OAc

GM3 Trisaccharide

OO

BnOBnO

BnO

O

O

BnOBnO

BnO

O

OOBn

HOO

OPh

OOBn

O

OO

OPivO

BnO

BnOO

O

OOBn

BnO

OO

PhPh

Globo-H Series of Tumor-Associated AntigensGlobo-H Series of Tumor-Associated Antigens

OHOOHO

OH

O

OHO

HOOH

O

OHHO

OOH

OCeramide

O

OHHO

ONHAc

O

OHHO

HOO

O OH

HOOH

Globo-H

OHOOHO

OH

O

OHO

HOOH

O

OHHO

OOH

OCeramide

O

OHHO

ONHAc

O

OHHO

HOOH

Gb5 (SSEA-3)

OHOOHO

OH

O

OHO

HOOH

O

OHHO

HOOH

OCeramide

Gb3

Globo-H: RetrosynthesisGlobo-H: Retrosynthesis

O

OPiv

OBn

OBnO

OO

OPiv

OBnO

BnO

O

BnO

OBnBnO

OO

NHTCA

OBnBnO

OO

O

OBnBnO

BnO

O

PivOOPivOBn

O

OPiv

OBn

OBnO

OO

OPiv

OBnO

BnO

O

BnO

OBnBnO

HO

O

OPiv

OBn

FmocOBnO

O

OPiv

OBnFmocO

BnO

O

BnO

OBnBnO

FmocO

O

TCAHN

OBnBnO

FmocO

O

OFmoc

OBnBnO

BnO

O

PivOOPivOBn

O OP

O

OBu

OBu

O P

O

OBu

OBu

O P

O

OBu

OBu

O P

O

OBu

OBu

NPh

CF3

O

NPh

CF3

Globo-H Hexasaccharide

Gb3 Trisaccharide

Werz, D. B.; Castagner, B.; Seeberger, P. H. J. Am. Chem. Soc. 2007, 129, 2770

Gb3 synthesisGb3 synthesis

OHO

TMSOTfDCM-15¼C;Pip., DMF

TMSOTfDCM-15¼C;Pip., DMF

TMSOTfDCM-30¼C;Pip., DMF;ethyleneGrubbs' cat.

OBnO

FmocOBnO

OPivO

POBuOBu

O

O

OBnFmocO

BnOOPiv

OP

OBu

OOBu

O

OBnBnO

FmocOOBn

O CCl3

OBnO

OBnO

OPiv

O

OBnO

BnOOPiv

O

OBnBnO

HOBnO

O

Gb346% isolated yield

NH

Crude HPLC

Globo-H SynthesisGlobo-H Synthesis

Crude HPLC

OBnO

OBnO

OPiv

O

OBnO

BnOOPiv

O

OBnBnO

OBnO

O

OO

OBnBnO

HOTCAHN

cat. TMSOTfDCM, -10¼C25 min;Pip., DMF

O

OBnBnO

BnOOFmoc

O

cat. TMSOTfDCM, -10¼C25 min;Grubbs' cat.ethylene

NPh

CF3O

PivOOPiv

OBn

O

NPh

CF3

OBnO

OBnO

OPiv

O

OBnO

BnOOPiv

O

OBnBnO

OBnO

O

O

OBnBnO

ONHTCA

O

OBnBnO

BnOO

O OBn

PivOOPiv

Globo-H Hexasaccharide

Purified Globo-HPurified Globo-H

OBnO

OBnO

OPiv

O

OBnO

BnOOPiv

O

OBnBnO

OBnO

O

O

OBnBnO

ONHTCA

O

OBnBnO

BnOO

O OBn

PivOOPiv

Globo-H Hexasaccharide

30% overall yield after column chromatography

Werz, D. B.; Castagner, B.; Seeberger, P. H. J. Am. Chem. Soc. 2007, 129, 2770

ββ-Mannosylation-Mannosylation

Kim, K. S.; Kim, J. H., Lee, Y. J.; Lee Y. J.; Park, J. J. Am. Chem. Soc. 2001, 123, 8477-8481

-No pre-activation necessary

-Compatible with linker olefin

-No acceptor by-product formation

OOO

Ph

BnO

OBn

O

CO2H

Tf2O (1 eq.)DTBMP (2 eq.)DCM

OOO

Ph

BnO

OBn

OTf

+

OO

OHO

OOOO

OPh

BnO

OBn

Elongation of the C3 PositionElongation of the C3 Position

OPivO

BnO

BnOAcO

O

NH

CCl3O

OBn

BnO

OCB

OO

Ph

OOBn

O

OO

OPivO

BnO

BnOO

O

OOBn

BnO

OO

PhPh

OHO

TMSOTfToluene;NaOMe, MeOHDCM

Tf2O, DTBMP;TBAF, THF;

O

OCB

OBn

OO

OPh

TIPSO

Tf2O, DTBMPEthyleneGrubbs' Cat.

60% isolated yield (over 6 steps)

Codée, J. D. C.; Kröck, L.; Castagner, B.; Seeberger, P. H. in preparation

Sialic Acid Disaccharide Building BlockSialic Acid Disaccharide Building Block

Hanashima, S.; Castagner, B.; Esposito, D., Nokami, T.; Seeberger, P. H. Org. Lett. 2007, 9, 1777.

OTrocHN

AcO

AcO

OP(OBn)2

COOMe

OAc

AcO + TMSOTf (0.15 eq.)OHO

OBnBnO

OO

OBnBnOOTrocHN

AcO

AcO

COOMe

OAc

AcO

OTrocHN

AcO

AcO

OAc

AcO OO

OBnBnOCOOMe

OAc O CF3

NPh

86% isolated yield (α προδυχτ)11:1 (α/β)

ΕτΧΝ, ΜΣ 4 , −78 …Χ

1. ΠηΙ(ΟΑχ)2, ΒΦ3⟨ΟΕτ2, ∆ΧΜ; Αχ2Ο, πψριδινε2. Ν2Η4⟨ΑχΟΗ, ∆ΜΦ3. ΧΦ3Χ(ΝΠη)Χλ, ΧσΧΟ3, ∆ΧΜ

76% οϖε ρ 4 στεπσ

Solution Phase Sialyl Lewis X SynthesisSolution Phase Sialyl Lewis X Synthesis

OTrocHN

AcO

AcO

OAc

AcOO

O

OBnBnOCOOMe

OAc

OBzO

OBz

OBn

O NHAllocO

OBz

O

OBnBnOO

LevONHTCA

OBnOO

OBzO

OBz

OBn

O NHAllocO

OBz

O

OBnBnOO

LevONHTCA

OBnOHO

N2H4áH2OAcOH, pyridine

allyl alcohol95%

OAcHN

HO

HO

OH

HOO

O

OHHOCOOH

OH

OHO

OH

OH

O NH2

O

OH

O

OHHOO

ONHAc

OHOO

O OBnOC(NPh)CF3

OAcOAc

O OHOH

OH

O OBnOAc

OAc

OTrocHN

AcO

AcO

OAc

AcOO

O

OBnBnOCOOMe

OAc

OBzO

OBz

OBn

O NHRO

OBz

O

OBnBnOO

ONHTCA

OBnOO

Yb(OTf)3MS 4 , CH2Cl2dioxane, Et2O

CH2Cl2-15 ¼C, 93%

88%

OTrocHN

AcO

AcO

OAc

AcO OO

OBnBnOCOOMe

OAc O CF3

NPh

+ TMSOTf

(1.5 eq.)

6

6

66

Sialyl Lewis X

Time Allocation During Oligosaccharide SynthesisTime Allocation During Oligosaccharide Synthesis

Building Block Synthesis

Oligosaccharide Assembly

Depro-tection

Solution Solution PhasePhase

Building Block Synthesis

Depro-tection

Automated Solid PhaseAutomated Solid Phase (currently)(currently)

weeks - months weeks - months days

weeks - months 1 day days

Building Block Synthesis

Automated Solid Phase Automated Solid Phase (soon)(soon)

Days - weeks 1 day hours

Synthetic Carbohydrate AntigensSynthetic Carbohydrate Antigens

Development of Vaccine Candidates Against Development of Vaccine Candidates Against Parasites, Bacteria and CancerParasites, Bacteria and Cancer

Carbohydrate Vaccine Development PathCarbohydrate Vaccine Development Path

Conjugate and formulate

M. tuberculosis

Identify unique antigen

Clinical Development

Synthesize antigen

Avian fluBacterial antigens

Test Immunogenicity

Cancer antigens

Preclinical Development

ancora Malaria

Challenge animal model

Leishmania, HIV

B. anthracis

An Anti-Toxin Malaria VaccineAn Anti-Toxin Malaria Vaccine

QuickTime and aªPhoto - JPEG decompressor

are needed to see this picture.

Clinical and Anti-parasite Immunity to Malaria

• many protein-based vaccines explored

• carbohydrate-based vaccines very successful against other diseases

Malaria Statistics (1994 WHO Estimate) • 40% of world population at risk• 5% infected (300 million people)• 100 million clinical cases • 2-3 million deaths (1% of cases fatal (predominantly children < 5 years)

The Plasmodium falciparum Life Cycle

OOHO +H3N

OH

O

OO

OHOH

OH

OHOHO

OHO

OHOHO

OOH

OHOHO

OO

OHOHO

HOHO

P-O

O

P

O-

O(CH2)2NH3+

O

OOHO +H3N

OH

O

OO

OHOH

OH

OHOHO

OHO

OHOHO

OOH

OHOHO

OO

OHOHO

HOHO

P-O

O

P

O-

O(CH2)2NH3+

O

1) Substance isolated from P. falciparum - structure postulated

2) Synthesis of structure to confirm assignment

3) Use synthetic molecule as anti-toxin vaccine candidate

Nature, 2002, 418, 785

18961896 Golgi Postulates Malaria Toxin Golgi Postulates Malaria Toxin

2002 2002 Toxin IdentifiedToxin Identified

An Anti-Toxin Malaria VaccineAn Anti-Toxin Malaria Vaccine

Glycosyl Phosphatidyl Inositol (GPI): Structure

>GPI-proteins

>Free GPIs

GPI-proteins

GPI anchor

OOHO

H2N

OH

OH

OO

OHOH

OH

OHOHO

OHO

OHOHO

OOH

OHOHO

OH

O

POHO

O

PO

OH

O HN

OROR'

Protein

Glycosylation

EtN-Phosphorylation

Acylation

ONHR

R'OH Ceramide

Glycosyl Phosphatidyl Inositol (GPI): Occurrence

Exoenzymes e.g. alkaline phosphatase,

Adhesion molecules e.g Neural cell adhesion molecules

Complement regulatory proteins e.g. DAF, CD59

Protozoa surface antigens e.g. SAG1, MSP1

Yeast T. brucei P. falciparum T . gondii Human

High copy (10-20 Million per cell) Low copy

Semi-Automated Assembly of the GPI Glycan

J. Am. Chem. Soc. 2002, 124, 13434.

OBnOBnO

OBnO

OBnOBnO

OBnO

OBnOBnO

OTIPSO

OBnOBnO

OBnBnO

R

OBnOBnO

OBnAcO

O

NHCl3C

OBnOBnO

OAcBnO

O

NHCl3C

OBnOBnO

OAcTIPSO

O

NHCl3C

OBnOBnO

OBnBnO

O

NHCl3C

OHO

1. 0.5 equiv TMSOTf

2. 10 equiv NaOMe

Grubbs' catalyst

Ethylene

1. 0.5 equiv TMSOTf

2. 10 equiv NaOMe

1. 0.5 equiv TMSOTf

2. 10 equiv NaOMe

1. 0.5 equiv TMSOTf

44% overall yield9 hours

0.1 equiv TMSOTf

1. NBS, CH3CN/H2O (67%)

2. DBU, Cl3CCN (75%)

CH2Cl2, 4 MS

OHOBnO N3

OBn

O

OO

OBnOBn

OBn

R =

NHCCl3 R =

89%

OBnOBnO

OBnO

OBnOBnO

OBnO

OBnOBnO

OTIPSO

OBnOBnO

OBnBnO

OOBnO N3

OBn

O

OO

OBnOBn

OBn

Synthesis of a Malaria Vaccine CandidateSynthesis of a Malaria Vaccine Candidate

Nature, 2002, 418, 785

OOHO H3N

OH

OO

OOH

OHOH

OHOHO

OHO

OHOHO

OOH

OHOHO

OO

OHOHO

OHOH

POO

O

PO O KLH-glycan

NH

NHS N

O

O

KLH

OOHO H3N

OH

OO

OOH

OHOH

OHOHO

OHO

OHOHO

OOH

OHOHO

OO

OHOHO

OHOH

POO

O

PO O

NH3

SNH1. pH 8.0

2. maleimide-activated KLH

H3NO

O

SH

maleimide-activated KLHH3N

OO

S NO

O

KLH

KLH-cysteine

OBnOBnO

OBnO

OBnOBnO

OBnO

OBnOBnO

OTIPSO

OBnOBnO

OBnBnO

OOBnO N3

OBn

ORRO

OOBn

OBnOBn

NPO NHCbzO CN

C(CH3)2

OH

CSA, HOCH2CH2OH

(81%)

OBnOBnO

OBnO

OBnOBnO

OBnO

OBnOBnO

OTIPSO

OBnOBnO

OBnBnO

OOBnO N3

OBn

ORRO

OOBn

OBnOBn

Cl2P(O)OMe, Pyr88%

OOBnO N3

OBn

OO

O OBnOBnOBn

OBnOBnO

OBnO

OBnOBnO

OBnO

OBnOBnO

OOR

OBnOBnO

OBnBnO

PO O

1. DBU, CH2Cl2; 2. Na, NH3, THF

(75%, 2 steps)

1. Tetrazole, CH3CN;

2. t-BuOOH, CH3CN (84%, 2 steps)

R = TIPS

R = H

R =

TBAF, THF 61%

PO NHCbzO CN

O

Survival andparasitaemia

Cerebral Histology

Systemic Pathology

Vaccines vs Vaccines vs ControlsControls

Does an Anti-GPI Response Protect Does an Anti-GPI Response Protect from Malaria Mortality?from Malaria Mortality?

QuickTime and aªPhoto - JPEG decompressor

are needed to see this picture.

Tools for Epitope Mapping & Biosynthesis InvestigationsTools for Epitope Mapping & Biosynthesis Investigations

OOHO H3N

OH

OO

OHOH

OH

OHOHO

OHO

OHOHO

OOH

OHOHO

OO

OHOHO

OHOH

POO

O

PO O

NH2

OH

O

X

OOHO H3N

OH

OO

OHOH

OH

OHOHO

OHO

OHOHO

OOH

OHOHO

OHOP

OO

O

PO O

NH2

OH

O

X

OOHO H3N

OH

OO

OHOH

OH

OHOHO

OHO

OHOHO

OOH

OHOHO

OHOH

PO O

OH

O

X

OOHO H3N

OH

OO

OHOH

OH

OHOHO

OHO

OHOHO

OHOH

PO O

OH

O

X

OOHO H3N

OH

OO

OHOH

OH

OHOHO

OHOH

PO O

OH

O

X

OHOHO H3N

OH

OO

OHOH

OHPO O

OH

O

X

Chem. Eur. J. 2005, 11, 2493.

sera

Cy3

Incubation

High Throughput Detection of Anti-GPI Antibodies on MicroarraysHigh Throughput Detection of Anti-GPI Antibodies on Microarrays

Kamena, J.F.; Tamborrini, M.; Liu, X.; Pluschke, G.; Seeberger, P.H. submitted

Read Out Quantification

Mean Anti-GPI IgG Titres in Human SeraMean Anti-GPI IgG Titres in Human Sera

Malaria endemic area of Burkina Faso Malaria non-exposed Europeans

Kamena, J.F.; Tamborrini, M.; Liu, X.; Pluschke, G.; Seeberger, P.H. submitted

GPI Microarray Results - SummaryGPI Microarray Results - Summary

• Fine specificities and titers differ between exposed and naive populations

• Children of mothers with specific antibodies have no antibodies

• Disease specific antibodies decline in migrants to about 40% in three years

Specific GPI Antibodies Protect Adults in Endemic Areas from Severe Disease

Induction of GPI-specific Antibodies Should Protect Naive Individuals and Small Children from Severe Disease

Development of an Anti-Toxin Malaria VaccineDevelopment of an Anti-Toxin Malaria Vaccine

1) Vaccination experiments in mice using additional synthetic antigens

• Scale-up and process development for synthetic antigen by

100 g - 5 kg?????????Current Ancora

1 - 100 g87.5272.70Initial Ancora

10 -100 mg79.5260.26Seeberger Lab

ScaleYield/Step Linear StepsTotal YieldSynthesis

Ancora

3) Conjugation and formulation agreement with major vaccine manufacturer

4) Toxicology and preclinical studies

5) Selection of sites for active and passive immunization trials

How do Merozoites Enter Red Blood Cells?

How Does P. falciparum Initiate the Inflammatory Response?

ETHS. BufaliX. LiuC. NotiL. HosseinA. AdibekianL. KroeckK. GeyerR. CastelliP. BindschädlerT. HorlacherM. OberliP. SeifD. EspositoY. GuoP. Stallforth

Dr. F. KamenaDr. N. AzzouzDr. F. CarellDr. B. CastagnerDr. S. HanashimaDr. R. WadaDr. H. WippoDr. BoonyarattanakalinDr. T. GustafssonDr. R. GilmourDr. K. Raghavendra

SNFKGFETHEU (ERA-NET, Marie-Curie)A. von Humboldt FoundationDFG (Emmy Noether Program)NIH (HL-64799, HL-62598)HFSPEMBORoche FoundationFondation Bay

ancora

CollaborationsDr. BeckerProf. SchofieldProf. JensenProf. TextorProf. PluschkeProf. ChatterjeeProf. SchachnerProf. Hengartner

The Burnham InstituteDr. P. WangDr. F. WallnerDr. S. Takashima

ETH Zurich GroupETH Zurich Group