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

Automated Oligosaccharide Synthesis Peter H. Seeberger Peter H. Seeberger

Biopolymers: Overview Biopolymers: Overview Genomics Glycomics Genomics Proteomics Glycomics Proteomics O O B Glycosyltransferases O X - O P O Transcription O O O O O B HO H O N C C N H CH C HO HO H R R Translation O O X O HO OH OH OH O HO O O O O HO O HO HO HO O Replication NHAc NHAc Proteins Oligosaccharides - Glycoconjugates Nucleic Acids

Biopolymer Interactions Protein - Protein Nucleic Acid - Nucleic Acid Interactions Interactions Nucleic Acid - Protein Interactions Nucleic Proteins Acids Carbohydrate - Nucleic Acid Carbohydrate - Protein Interactions Interactions Carbohydrates 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.

The Automated Oligosaccharide Synthesizer The Automated Oligosaccharide Synthesizer Science 2001, 291 , 1523

Blood Group Determinants and Tumor Associated Antigens OBn BnO OBn OBn BnO OBn BnO BnO OBn OBn BnO BnO HO HO O O O O O O O O O O O O BnO O O O O BnO BnO O BnO O PivO O OPiv PivO OH OPiv TCAHN TCAHN O O OBn OBn O BnO PivOOPiv PivOOPiv Le x PivO Le y OPiv OBn BnO OBn OBn BnO OBn BnO BnO BnO HO O O O O O O O O O O O BnO O BnO O O PivO O PivO OPiv TCAHN TCAHN O OBn O OBn O PivOOPiv BnO PivOOPiv PivO OPiv Le y Le x OBn OBn OBn O O BnO O O O FmocO LevO OP (OBu) 2 OP (OBu) 2 O O P (OBu) 2 BnO FmocO BnO OPiv TCAHN OBn OFmoc BnO O O O OP (OBu) 2 FmocO O P (OBu) 2 O OBn PivO PivOOPiv Angew. Chem. Int. Ed. 2004, 43 , 602

Automated Synthesis of Complex Structures BnO O OAc OLev BnO OBn O OBn BnO O O O BnO BnO O BnO BnO BnO O O BnO BnO BnO BnO PivO O O BnO O OBn O O OBn BnO BnO O O BnO BnO O O BnO OBn O BnO PivO BnO OR BnO OBn O PivO BnO OPiv O BnO O O BnO OAc BnO O O O BnO O BnO O n = 3 O BnO BnO OPiv n = 3 BnO PivO BnO Gb3 - 6.5 h O O O BnO BnO BnO O BnO O BnO BnO PivO OAc O BnO Leishmania Cap - 9 h Phytoalexin Elicitor - 16 h Rhamnose Pentamer BnO 10 h BnO O O BnO OBn OBn BnO OBn BnO BnO OBn BnO OBn BnO OBn BnO BnO O O O O O O O O O BnO O BnO O O O O O O BnO O BnO O PivO O BnO O OPiv O BnO TCAHN PivO O OH OPiv TCAHN OBn BnO O OBn O PivOOPiv BnO Le y - 14 h BnO O Le x - 11.5 h PivOOPiv PivO OPiv O BnO BnO BnO O OBn OBn OBn BnO BnO OBn BnO BnO BnO O BnO O O O O O O O O O O O O BnO O BnO O BnO PivO O OPiv OPiv OAc NHTCA NHTCA BnO O BnO BnO O O OBn O O O OBn BnO BnO PivO PivOOPiv BnO PivOOPiv OPiv BnO O OBn BnO OAc OBn BnO OBn O Le y -Le x - 23 h O O O BnO BnO O O O O O BnO BnO BnO BnO O BnO NHAc NHAc BnO O O O N -Linked Core Region - 9 h BnO OBn OBn BnO BnO MeO BnO O O O O BnO O O O O BnO O BnO BnO ancora AcO AcO OPiv PivO O BnO BnO Proteoglycan Linkage Region - 9 h O Mannose Oligomer - 23 h

Automation of Difficult Glycosylations Automation of Difficult Glycosylations OBn BnO OBn OBn BnO BnO O O O O O BnO BnO O NHTCA O OBn BnO O O O O OBn BnO O BnO OPiv OPiv OPiv PivO Globo-H Hexasaccharide α -Galactosylation OBn O Ph O O O HO BnO O OBn BnO OPiv Ph O BnO β -Mannosylation BnO O O O OBn O Ph O O O O BnO O BnO O BnO BnO O BnO O α -Sialylation AcO OAc COOMe BnO OBn OBn AcO O O O O O TrocHN O BzO AcO OAc OBz GM3 Trisaccharide

Globo-H Series of Tumor-Associated Antigens Globo-H Series of Tumor-Associated Antigens OH HO OH OH HO HO O O O O O HO OH NHAc O O OH HO O O O OH O HO OCeramide HO OH OH OH HO Globo-H OH OH HO HO OH OH HO HO O O O O HO O O HO OH OH OH NHAc O OH O OH HO HO O O O O O O HO OCeramide HO OCeramide HO HO OH OH OH OH Gb5 (SSEA-3) Gb3

Globo-H: Retrosynthesis Globo-H: Retrosynthesis OBn OBn OBn BnO BnO BnO O O O O O BnO BnO NHTCA O OBn OBn O O OBn O O O O BnO PivOOPiv BnO OPiv OPiv Globo-H Hexasaccharide OBn BnO O HO BnO OBn OBn O O O O O BnO BnO OPiv OPiv Gb3 Trisaccharide OBn OBn OBn BnO NPh BnO O O O O FmocO O O P OBu O P OBu O CF 3 BnO FmocO BnO OBu OPiv OBu BnO OFmoc NPh OBn OBn FmocO BnO O O O CF 3 O O O O P OBu O P OBu OBn FmocO BnO OBu OPiv OBu TCAHN PivOOPiv Werz, D. B.; Castagner, B.; Seeberger, P. H. J. Am. Chem. Soc. 2007, 129 , 2770

Gb3 synthesis Gb3 synthesis OBn BnO OBn O FmocO BnO O CCl 3 FmocO OBn O O BnO FmocO OBu OBn O P BnO O P OBu BnO NH OBu OPiv OPiv O O OBu O HO TMSOTf TMSOTf TMSOTf DCM DCM DCM BnO O OBn O -15¼ C; -15¼ C; -30¼ C; BnO Pip., DMF; Pip., DMF Pip., DMF O HO O O BnO O ethylene BnO Grubbs' cat. OPiv OPiv Gb3 46% isolated yield Crude HPLC

Globo-H Synthesis Globo-H Synthesis NPh OBn BnO NPh O CF 3 O OBn O BnO OBn OBn BnO O CF 3 BnO O PivOOPiv OFmoc O O cat. TMSOTf cat. TMSOTf O HO DCM, -10¼ C DCM, -10¼ C BnO TCAHN O OBn 25 min; 25 min; BnO Pip., DMF Grubbs' cat. O O O ethylene BnO O BnO OPiv OPiv OBn BnO OBn OBn BnO BnO O O O O O BnO BnO NHTCA O O OBn BnO O O O OBn O BnO O BnO OPiv OPiv OPiv PivO Globo-H Hexasaccharide Crude HPLC

Purified Globo-H Purified Globo-H OBn BnO OBn OBn BnO BnO O O O O O BnO BnO O NHTCA O OBn BnO O O O OBn O BnO O BnO OPiv OPiv OPiv PivO 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 -Mannosylation β -Mannosylation OBn Ph O O Tf 2 O (1 eq.) OBn O Ph O O DTBMP (2 eq.) BnO O + BnO DCM O O OTf O CO 2 H O HO OBn O Ph O O O O BnO -No pre-activation necessary -Compatible with linker olefin -No acceptor by-product formation Kim, K. S.; Kim, J. H., Lee, Y. J.; Lee Y. J.; Park, J. J. Am. Chem. Soc. 2001, 123 , 8477-8481

Elongation of the C3 Position Elongation of the C3 Position OPiv BnO O AcO OBn BnO OBn Ph O Ph O O O O O O CCl 3 OBn OPiv O BnO Ph O BnO TIPSO O O OBn O OCB O O NH OCB Ph O TMSOTf Ethylene O O BnO O Grubbs' Cat. Toluene; Tf 2 O, DTBMP; HO Tf 2 O, DTBMP BnO O NaOMe, MeOH TBAF, THF; DCM 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 AcO AcO OAc OAc OBn OP(OBn) 2 COOMe BnO OBn BnO + TMSOTf (0.15 eq.) AcO AcO O O O COOMe TrocHN TrocHN O Ετ ΧΝ, ΜΣ 4 , − 78 … Χ HO O AcO AcO 11:1 (α/β) 86% isolated yield ( α πρ οδυχτ) 1. ΠηΙ(ΟΑχ) 2 , ΒΦ 3 〈ΟΕτ 2 , ∆ΧΜ; Αχ 2 Ο, πψ ρ ιδινε 2. Ν 2 Η 4 〈ΑχΟΗ, ∆ΜΦ 3. ΧΦ 3 Χ(ΝΠη)Χλ , ΧσΧΟ 3 , ∆ΧΜ AcO OAc COOMe BnO OBn NPh AcO O O TrocHN O OAc O CF 3 AcO 76% οϖ ερ 4 στ επσ Hanashima, S.; Castagner, B.; Esposito, D., Nokami, T.; Seeberger, P. H. Org. Lett. 2007, 9 , 1777.

Solution Phase Sialyl Lewis X Synthesis Solution Phase Sialyl Lewis X Synthesis AcO OAc COOMe OBn BnO NPh O AcO O (1.5 eq.) AcO OAc TrocHN O COOMe OAc O CF 3 AcO TMSOTf BnO + OBn BnO OBn OBn AcO O BnO CH 2 Cl 2 O TrocHN O BnO OBn O O O OBn O BnO -15 ¼ C, 93% O O O O NHAlloc LevO O AcO BzO HO O O O OAc NHTCA LevO 6 OBz OBz O NHAlloc BzO NHTCA OBz 6 OBz OC(NPh)CF 3 O OBn OAc AcO OAc OAc COOMe Yb(OTf) 3 N 2 H 4 á H 2 O BnO OBn MS 4 , CH 2 Cl 2 BnO AcO OBn AcOH, pyridine OBn O BnO O TrocHN dioxane, Et 2 O O O allyl alcohol O O O O O O NHR 95% AcO O 88% BzO OAc NHTCA OBz 6 OBz O OBn OAc OAc HO OH COOH HO OH HO OH HO OH O HO O AcHN O O O O O O O O NH 2 HO O HO OH NHAc OH 6 OH O OH OH OH Sialyl Lewis X

Time Allocation During Oligosaccharide Synthesis Time Allocation During Oligosaccharide Synthesis Building Oligosaccharide Depro- Solution Solution Block Assembly tection Phase Phase Synthesis weeks - months weeks - months days Building Automated Solid Phase Automated Solid Phase Depro- Block (currently) (currently) tection Synthesis weeks - months 1 day days Building Automated Solid Phase Automated Solid Phase Block (soon) (soon) Synthesis 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 Synthesize antigen Identify unique antigen Conjugate and formulate M. tuberculosis Avian flu Bacterial antigens Preclinical Challenge Test Immunogenicity Development animal model Leishmania, HIV Malaria Cancer antigens ancora B. anthracis Clinical Development

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