Automated Oligosaccharide Synthesis Peter H. Seeberger Peter H. - - PowerPoint PPT Presentation

Automated Oligosaccharide Synthesis

Peter H. Seeberger Peter H. Seeberger

Biopolymers: Overview Biopolymers: Overview

Replication O B X O P O O B O O O

• O

X Translation H C C N H CH C R N H O R O Proteins Nucleic Acids

HO O O O HO NHAc O O HO NHAc OH O HO O HO HO OH OH HO O HO O HO O O

Transcription Oligosaccharides - Glycoconjugates Glycosyltransferases

Genomics Genomics Proteomics Proteomics Glycomics Glycomics

Biopolymer Interactions

Nucleic Acids Proteins

Nucleic Acid - Protein Interactions Protein - Protein Interactions Nucleic Acid - Nucleic Acid Interactions

Carbohydrates

Carbohydrate - Protein Interactions Carbohydrate - Nucleic Acid Interactions Carbohydrate - Carbohydrate Interactions

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 Synthesizer The Automated Oligosaccharide Synthesizer

Blood Group Determinants and Tumor Associated Antigens

O OPiv PivO BnO O O BnO OBn BnO O TCAHN BnO O O O PivO OBn BnO O O BnO OPiv O OBn O O PivOOPiv OBn HO O PivOOPiv OBn O O BnO OBn BnO O TCAHN BnO O O O PivO OBn BnO O O BnO OPiv OBn OH O HO Lex O PivOOPiv OBn O O O OBn BnO O TCAHN BnO O O O PivO OBn BnO O O BnO OPiv OBn PivO O HO O OPiv PivO BnO O O BnO OBn BnO O TCAHN BnO O O O PivOOPiv OBn LeyLex Ley O FmocO BnO OPiv OBn OP O (OBu)2 O FmocO PivO OBn BnO OP O (OBu)2 O OBn LevO FmocO TCAHN OP O (OBu)2 O PivOOPiv OBn O P O (OBu)2 O BnO OBn OFmoc BnO O P (OBu)2 O

• Angew. Chem. Int. Ed. 2004, 43, 602

ancora

O BnO BnO OPiv OBn O O BnO O BnO O O BnO BnO O BnO O BnO BnO OAc BnO

Leishmania Cap - 9 h

O PivOOPiv OBn O O BnO OBn BnO O TCAHN BnO O O O PivO OBn BnO O O BnO OPiv OBn OH O

Lex - 11.5 h

O OPiv PivO BnO O O BnO OBn BnO O TCAHN BnO O O O PivO OBn BnO O O BnO OPiv OBn O PivOOPiv OBn O O

Ley - 14 h Ley-Lex - 23 h

OPiv O O OBn BnO O NHTCA BnO O O O PivO OBn BnO O O BnO OPiv OBn O PivOOPiv OBn O O O BnO OBn BnO O NHTCA BnO O O O PivOOPiv OBn O OPiv PivO BnO O O O BnO NHAc OBn O O BnO NHAc OBn O BnO O OBn O BnO BnO OAc O BnO O BnO BnO OAc BnO O

N-Linked Core Region - 9 h

O O AcO OBn BnO O O BnO OPiv O O O AcO OBn BnO BnO O BnO PivO MeO O

Proteoglycan Linkage Region - 9 h

BnO O BnO PivO BnO O BnO PivO O BnO O O PivO O BnO O BnO BnO OBn O BnO O O PivO BnO O BnO BnO OBn OLev O n = 3

Phytoalexin Elicitor - 16 h

O BnO BnO BnO O O BnO BnO BnO O O O BnO BnO BnO O O BnO BnO BnO O O BnO BnO BnO O O BnO BnO BnO O O BnO BnO BnO O O BnO BnO BnO O O BnO BnO BnO O O BnO BnO BnO OAc

Mannose Oligomer - 23 h

O O OAc BnO BnO O O BnO BnO O O BnO BnO n = 3

Rhamnose Pentamer 10 h

O O OBn OPiv OR BnO O O OBn PivO BnO O BnO OBn BnO BnO

Gb3 - 6.5 h

Automated Synthesis of Complex Structures

Automation of Difficult Glycosylations Automation of Difficult Glycosylations

β-Mannosylation α-Galactosylation α-Sialylation

O BnO O BnO OPiv O OBn O BnO OPiv O OBn BnO O BnO O O OBn BnO O NHTCA O OBn BnO BnO O O OBn PivO OPiv Globo-H Hexasaccharide O BzO OBn O OBz O O TrocHN AcO AcO OAc AcO O O OBn BnO COOMe OAc GM3 Trisaccharide O O BnO BnO BnO O O BnO BnO BnO O O OBn HO O O Ph O OBn O O O OPiv O BnO BnO O O O OBn BnO O O Ph Ph

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

O HO O HO OH O OH O HO OH O OH HO O OH OCeramide O OH HO O NHAc O OH HO HO O O OH HO OH Globo-H O HO O HO OH O OH O HO OH O OH HO O OH OCeramide O OH HO O NHAc O OH HO HO OH Gb5 (SSEA-3) O HO O HO OH O OH O HO OH O OH HO HO OH OCeramide Gb3

Globo-H: Retrosynthesis Globo-H: Retrosynthesis

O OPiv OBn O BnO O O OPiv OBn O BnO O BnO OBn BnO O O NHTCA OBn BnO O O O OBn BnO BnO O PivOOPiv OBn O OPiv OBn O BnO O O OPiv OBn O BnO O BnO OBn BnO HO O OPiv OBn FmocO BnO O OPiv OBn FmocO BnO O BnO OBn BnO FmocO O TCAHN OBn BnO FmocO O OFmoc OBn BnO BnO O PivOOPiv OBn O O P 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 synthesis Gb3 synthesis

O HO TMSOTf DCM

• 15¼

C; Pip., DMF TMSOTf DCM

• 15¼

C; Pip., DMF TMSOTf DCM

• 30¼

C; Pip., DMF; ethylene Grubbs' cat.

O BnO FmocO BnO OPiv O P OBu OBu O O OBn FmocO BnO OPiv O P OBu O OBu O OBn BnO FmocO OBn O CCl3

O BnO O BnO OPiv O OBn O BnO OPiv O OBn BnO HO BnO O Gb3 46% isolated yield

NH

Crude HPLC

Globo-H Synthesis Globo-H Synthesis

Crude HPLC

O BnO O BnO OPiv O OBn O BnO OPiv O OBn BnO O BnO O O O OBn BnO HO TCAHN

• cat. TMSOTf

DCM, -10¼ C 25 min; Pip., DMF

O OBn BnO BnO OFmoc O

• cat. TMSOTf

DCM, -10¼ C 25 min; Grubbs' cat. ethylene

NPh CF3 O PivOOPiv OBn O NPh CF3

O BnO O BnO OPiv O OBn O BnO OPiv O OBn BnO O BnO O O OBn BnO O NHTCA O OBn BnO BnO O O OBn PivO OPiv Globo-H Hexasaccharide

Purified Globo-H Purified Globo-H

O BnO O BnO OPiv O OBn O BnO OPiv O OBn BnO O BnO O O OBn BnO O NHTCA O OBn BnO BnO O O OBn PivO OPiv Globo-H Hexasaccharide 30% overall yield after column chromatography

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

β β-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

O O O Ph BnO OBn O CO2H Tf2O (1 eq.) DTBMP (2 eq.) DCM O O O Ph BnO OBn OTf + O O O HO O O O O O Ph BnO OBn

Elongation of the C3 Position Elongation of the C3 Position

OPiv O BnO BnO AcO O NH CCl3 O OBn BnO OCB O O Ph O OBn O O O OPiv O BnO BnO O O O OBn BnO O O Ph Ph O HO TMSOTf Toluene; NaOMe, MeOH DCM Tf2O, DTBMP; TBAF, THF; O OCB OBn O O O Ph TIPSO Tf2O, DTBMP Ethylene Grubbs' Cat. 60% isolated yield (over 6 steps)

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

Sialic Acid Disaccharide Building Block Sialic Acid Disaccharide Building Block

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

O TrocHN AcO AcO OP(OBn)2 COOMe OAc AcO + TMSOTf (0.15 eq.) O HO OBn BnO O O OBn BnO O TrocHN AcO AcO COOMe OAc AcO O TrocHN AcO AcO OAc AcO O O OBn BnO COOMe OAc O CF3 NPh 86% isolated yield (α πρ οδυχτ) 11:1 (α/β) Ετ ΧΝ, ΜΣ 4 , − 78 … Χ

• 1. ΠηΙ(ΟΑχ)2, ΒΦ

3〈ΟΕτ 2,

∆ΧΜ; Αχ2Ο, πψ ρ ιδινε

• 2. Ν2Η4〈ΑχΟΗ, ∆ΜΦ
• 3. ΧΦ

3Χ(ΝΠη)Χλ

, ΧσΧΟ3, ∆ΧΜ 76% οϖ ερ 4 στ επσ

Solution Phase Sialyl Lewis X Synthesis Solution Phase Sialyl Lewis X Synthesis

O TrocHN AcO AcO OAc AcO O O OBn BnO COOMe OAc O BzO OBz OBn O NHAlloc O OBz O OBn BnO O LevO NHTCA O BnO O O BzO OBz OBn O NHAlloc O OBz O OBn BnO O LevO NHTCA O BnO HO N2H4á H2O AcOH, pyridine allyl alcohol 95% O AcHN HO HO OH HO O O OH HO COOH OH O HO OH OH O NH2 O OH O OH HO O O NHAc O HO O O OBn OC(NPh)CF3 OAc OAc O OH OH OH O OBn OAc OAc O TrocHN AcO AcO OAc AcO O O OBn BnO COOMe OAc O BzO OBz OBn O NHR O OBz O OBn BnO O O NHTCA O BnO O Yb(OTf)3 MS 4 , CH2Cl2 dioxane, Et2O CH2Cl2

• 15 ¼

C, 93% 88% O TrocHN AcO AcO OAc AcO O O OBn BnO COOMe OAc O CF3 NPh + TMSOTf (1.5 eq.) 6 6 6 6 Sialyl Lewis X

Time Allocation During Oligosaccharide Synthesis Time Allocation During Oligosaccharide Synthesis

Building Block Synthesis Oligosaccharide Assembly Depro- tection Solution Solution Phase Phase Building Block Synthesis Depro- tection Automated Solid Phase Automated 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 Antigens Synthetic Carbohydrate Antigens Development of Vaccine Candidates Against Development of Vaccine Candidates Against Parasites, Bacteria and Cancer Parasites, Bacteria and Cancer

Carbohydrate Vaccine Development Path Carbohydrate Vaccine Development Path

Conjugate and formulate

• M. tuberculosis

Identify unique antigen Clinical Development Synthesize antigen Avian flu Bacterial antigens Test Immunogenicity Cancer antigens Preclinical Development

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Malaria Challenge animal model Leishmania, HIV

• B. anthracis

An Anti-Toxin Malaria Vaccine An Anti-Toxin Malaria Vaccine

QuickTime and a ª Photo - 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

O O HO

+H3N

OH O O O OH OH OH O HO HO OH O O HO HO O OH O HO HO O O O HO HO HO HO P

• O

O P O- O(CH2)2NH3

+

O O O HO

+H3N

OH O O O OH OH OH O HO HO OH O O HO HO O OH O HO HO O O O HO HO HO HO 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

1896 1896 Golgi Postulates Malaria Toxin Golgi Postulates Malaria Toxin 2002 2002 Toxin Identified Toxin Identified

An Anti-Toxin Malaria Vaccine An Anti-Toxin Malaria Vaccine

Glycosyl Phosphatidyl Inositol (GPI): Structure

>GPI-proteins >Free GPIs GPI-proteins GPI anchor

O O HO H2N OH OH O O OH OH OH O HO HO OH O O HO HO O OH O HO HO OH O P OH O O P O OH O H N OR OR' Protein Glycosylation EtN-Phosphorylation Acylation O NHR 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.

O BnO BnO OBn O O BnO BnO O BnO O BnO BnO O TIPSO O BnO BnO OBn BnO R O BnO BnO OBn AcO O NH Cl3C O BnO BnO OAc BnO O NH Cl3C O BnO BnO OAc TIPSO O NH Cl3C O BnO BnO OBn BnO O NH Cl3C O HO

• 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 yield 9 hours 0.1 equiv TMSOTf

• 1. NBS, CH3CN/H2O (67%)
• 2. DBU, Cl3CCN (75%)

CH2Cl2, 4 MS O HO BnO N3 OBn O O O OBn OBn OBn R = NH CCl3 R = 89% O BnO BnO OBn O O BnO BnO O BnO O BnO BnO O TIPSO O BnO BnO OBn BnO O O BnO N3 OBn O O O OBn OBn OBn

Synthesis of a Malaria Vaccine Candidate Synthesis of a Malaria Vaccine Candidate

Nature, 2002, 418, 785

O O HO H3N OH O OO OH OH OH O HO HO OH O O HO HO O OH O HO HO O O O HO HO OH OH P O O O P O O KLH-glycan N H NH S N O O KLH O O HO H3N OH O OO OH OH OH O HO HO OH O O HO HO O OH O HO HO O O O HO HO OH OH P O O O P O O NH3 S NH 1. pH 8.0

• 2. maleimide-activated KLH

H3N O O SH maleimide-activated KLH H3N O O S N O O KLH KLH-cysteine O BnO BnO OBn O O BnO BnO O BnO O BnO BnO O TIPSO O BnO BnO OBn BnO O O BnO N3 OBn OR ROO OBn OBn OBn NPO NHCbz O CN C(CH3)2 OH CSA, HOCH2CH2OH (81%) O BnO BnO OBn O O BnO BnO O BnO O BnO BnO O TIPSO O BnO BnO OBn BnO O O BnO N3 OBn OR ROO OBn OBn OBn Cl2P(O)OMe, Pyr 88% O O BnO N3 OBn O OO OBn OBn OBn O BnO BnO OBn O O BnO BnO O BnO O BnO BnO O OR O BnO BnO OBn BnO P O 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 NHCbz O CN O

Survival and parasitaemia Cerebral Histology Systemic Pathology

Vaccines vs Vaccines vs Controls Controls

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

QuickTime and a ª Photo - JPEG decompressor are needed to see this picture.

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

O O HO H3N OH OO OH OH OH O HO HO OH O O HO HO O OH O HO HO O O O HO HO OH OH P O O O P O O NH2 OH O X O O HO H3N OH OO OH OH OH O HO HO OH O O HO HO O OH O HO HO OH O P O O O P O O NH2 OH O X O O HO H3N OH OO OH OH OH O HO HO OH O O HO HO O OH O HO HO OH OH P O O OH O X O O HO H3N OH OO OH OH OH O HO HO OH O O HO HO OH OH P O O OH O X O O HO H3N OH OO OH OH OH O HO HO OH OH P O O OH O X O HO HO H3N OH OO OH OH OH P O O OH O X

• Chem. Eur. J. 2005, 11, 2493.

sera

Cy3

Incubation

High Throughput Detection of Anti-GPI Antibodies on Microarrays High 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 Sera Mean 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 - Summary GPI 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 Vaccine Development 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 g 87.5 27 2.70 Initial Ancora 10 -100 mg 79.5 26 0.26 Seeberger Lab Scale Yield/Step Linear Steps Total Yield Synthesis

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?

ETH

• S. Bufali
• X. Liu
• C. Noti
• L. Hossein
• A. Adibekian
• L. Kroeck
• K. Geyer
• R. Castelli
• P. Bindschädler
• T. Horlacher
• M. Oberli
• P. Seif
• D. Esposito
• Y. Guo
• P. Stallforth
• Dr. F. Kamena
• Dr. N. Azzouz
• Dr. F. Carell
• Dr. B. Castagner
• Dr. S. Hanashima
• Dr. R. Wada
• Dr. H. Wippo
• Dr. Boonyarattanakalin
• Dr. T. Gustafsson
• Dr. R. Gilmour
• Dr. K. Raghavendra

SNF KGF ETH EU (ERA-NET, Marie-Curie)

• A. von Humboldt Foundation

DFG (Emmy Noether Program) NIH (HL-64799, HL-62598) HFSP EMBO Roche Foundation Fondation Bay

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Collaborations

• Dr. Becker
• Prof. Schofield
• Prof. Jensen
• Prof. Textor
• Prof. Pluschke
• Prof. Chatterjee
• Prof. Schachner
• Prof. Hengartner

The Burnham Institute

• Dr. P. Wang
• Dr. F. Wallner
• Dr. S. Takashima

ETH Zurich Group ETH Zurich Group