Transcript Synthesis and Immunological Application of Glycopeptide

Synthesis and Immunological
Application of HIV and TumorAssociated Carbohydrate Antigens
Michael Giuliano
Gellman Group
December 6th, 2007
Antigens and Vaccines
• immunogen - agent capable of eliciting an immune system response
• antigen - contraction of “antibody generating”
• lipids
OH OH
O
• proteins/peptides
HO
-Tn
NHAc
• carbohydrates
Danishefsky, S.J. et al. Angew. Chem.
Int. Ed. 2000, 39, 836.
Lipid A - E. Coli
Boons, G.-J. et al. J. Am. Chem. Soc. 2007, 129, 5200.
poliovirus
http://www.math.sunysb.edu
2
Immune Response to Foreign Antigens
Antigen
Innate Immune Response
Adaptive Immune Response
• takes place in bloodstream
• Cell - mediated
• Helper T Cells
• Macrophages
• B Cells
• Antibodies (non-specific)
• Cytotoxic T Cells
IgG
IgM
• Generation of specific
antibodies and long-term
immunity
• An effective vaccine illicits both of these responses
3
Types of Vaccines
Type of Vaccine
Type of Antigen
Example
Live Attenuated
whole organism
measles, mumps,
rubella
Inactive - whole
whole organism
poliovirus, hepatitis A,
rabies
Inactive - fractional toxoid protein
structural protein
tetanus, diptheria
hepatitis B, human
papillomavirus, Lyme
pneumococcus,
pure carbohydrate* meningococcus
carbohydrate
conjugate
**Haemophilus
influenzae b
**
** First officially licensed carbohydrate conjugate
vaccines (95-100% clinical efficacy):
• ProHIBIT (1987)
http://phil.cdc.gov
4
Conjugate Vaccines
Carrier
S
c
a
f
f
o
l
d
E
E
E
Adjuvant: an organic or
inorganic immunostimulant
administered with a vaccine
Epitope: The antigen itself
Scaffold: - must not itself be immunogenic
- biomimetic antigen presentation
Carrier: - contains peptide sequence that stimulates cell-mediated immune response
- capable of presenting a high epitope density (i.e. multivalent presentation)
5
Efforts Toward an HIV-1 Vaccine
6
Antibody Evasion by HIV-1
• Dense coating of high-mannose
oligosaccharides excludes the vast majority
of recognizable protein components of gp120
• Facilitates association with
mannose-binding receptors on
antigen-presenting dendritic
cells.
http://www3.niaid.nih.gov
7
The 2G12 Antibody
• Discovered in mid 1990’s - exhibits novel rearrangement of binding domains
Fab
Fc
• Goal: Elicit an immune response with
epitope specificity similar to 2G12
• Typical IgG antigbody;
2 antigen binding sites
}
•IgG 2G12 antibody; 4
antigen binding sites
8
Calarese, D.A. et al. Science. 2003, 300, 2065.
2G12 Recognition of Oligomannose
OH
HO
HO
Early Scaffold Designs:
OH
O
OH
O
HO
HO
= Man9GlcNAc2Asn
O
O
OH
O
HO
O
HO
HO
O
HO O
HO
HO
O
OH
HO
OH
HO
HO
= Man1-2Man
linkage
S
O
N
H
R =
N
S
O
O
O
HO
O
O
OH
OH
O
HO
OH
O
HO
NHAc
O
NH2
NH2
NHAc
O
R
Man9GlcNAc2
HO
HO
O
HO O
HO
HO
O
HO
HO
RR
RR
R
O
R OMe
O
R
R
R
O
R
O
O
O
R
MeO
OEt
Calarese, D.A. et al. Science. 2003, 300, 2065.
Wang, L.-X. et al. Org. Biomol. Chem. 2004, 2, 483.
Wang, L.-X. et al. Chem. Biol. 2004, 11, 127.
OEt
O
MeO
OEt
N
H
O
S S
HN
CO2H
NH
HO2C
O
N
H
9
An Early Semi-Synthetic Epitope Cluster
NHBoc
NHBoc
O
OH
CO2H
O
1) MeOH, NaHCO3, AIBN, hv,
SH
NH2
H
HO
S
H
H
8 steps
OH
O
H
2) NaHCO3, THF,
O
O
Cholic Acid
O
O
N
O
O
S
S
O
N
O
O O
NH
O
NH
O
N
O
N
O
OH
Man9 O
HO
OH
O
AcHN
O
HO
O
SH
O
AcHN
NH
1) 5.25 equiv.
O
O
NH
N
O
O
NH2
NH
O
R
CO2H
R
R
2) MeCN, sodium phosphate buffer; pH 6.6
3) TFA
O
R=
O
S
= Man9GlcNAc2Asn
Wang, L.-X. et al. Org. Biomol. Chem. 2004, 2, 483.
Wang, L.-X. et al. Org. Biomol. Chem. 2003, 1, 3507.
O
N
H
N
S
O
10
An Early Semi-Synthetic Epitope Cluster
• Competitive Binding (ELISA) Results vs. gp120:
Man9GlcNAc2Asn - IC50= 960 mm
Trivalent cluster - IC50= 21mm
NH2
O
O
O
S
S
S
O
O
O
N
O
N
O
N
O
NH
NH
NH
O
O
O
S
O
S
S
NH
O
O
HO2C
NH
NH
HO2C
HO2C
O
O
HN
O
NH
O
HO
NHAc
NH
O
O
NHAc
NHAc
HO
HO
OHO
HO
O
O
HO
HO
HO
HO
OO
HO
O
O
HOHO
HO O
O
O
HO
O
HO
HO
NHAc
O
NHAc
HO
OH
O
OHO
O
O
HOHO
HO
O
O
OH O
OH
HO
O
O
HO
O
OH O
OH
HO
O
HO
OH
OH
O
O
O
HO HO
HO
HO
OH
O
OH
O
OH
OH
OO
HO
OH
OH
OH
O
O
OH
OH
OO
O
HO
O
OH
OH
OH
OO
OH
OH
O OH
OH
OH
O
OH
O
O
OH
OH
O
HO
HO
O OH
OH
HO
O
O
O
OH
OH
OH
OH
11
OH
O
OHOH
OH
O
O
OH
OH
OHOH
OH
OHOH
O
OH
OH
OHOH
OH
O
OH
O
O
HO
HO
OHOH
O
HO
HO
HO O
HO
Wang, L.-X. et al. Org. Biomol. Chem. 2004, 2, 483.
Calarese, D.A. et al. Science. 2003, 300, 2065.
NHAc
O
OH
O
OH
O
OH
OH
The Importance of Epitope Design
Man9 - dimer
Carbohydrate Inhibition of
2G12 Binding to gp120
Carbohydrate Antigen
IC50 (mM)
Man5GlcNAc
Man6GlcNAc
Man9GlcNAc
Man9 – dimer
Bi - Man9
Tri - Man9
Tetra - Man9
200 (estimated)
70
0.96
0.40
0.13
0.044
0.013
HO
HO
O
Relativ e Binding to
Man9GlcNAc
0.005
0.014
1.0
2.4
7.3
22.0
73.0
O
HN
N
H
CO2H
Bi-Man9
NH
HO2C
O
R
MeO
OEt
Tri-Man9
RR
O
HO
HO
O
HO
HO
O
HO
O
HO
HO
O
HO O
HO
HO
O
OH
OH
HO
HO
HO
HO
HO
O
HO O
HO
HO
O
HO
HO
HO
O
HO
HO
HO
O
OH
O
O
O
O
HO
HO
O
OH
O
HO
OH OH
O
OH
O
HO
Man9GlcNAc
O
OH
OH
O
O
NHAc
HO
HO
HO
O
O
MeO
OEt
OH
O
HO
Tetra-Man9
O
HO
HO
HO
O
OH
R
O
OH
O
HO
O
OH OH
O
OH
O
HO
O
OH
OH
O
HO
OH
Man6GlcNAc
RR
O
O
NHAc
HO
HO
O
HO HO
OH
OR
HO
Man5GlcNAc
O
OH
R
NHAcOEt
HO
HO
O
HO HO
Wang, L.-X. et al. Org. Biomol. Chem. 2004, 2, 483.
Wang, L.-X. et al. Org. Biomol. Chem. 2003, 1, 3507.
Wang, L.-X. et al. Chem Biol. 2004, 11, 127.
= Man9GlcNAc2Asn
O
R =
S
O
O
N
H
N
O
S
O
N
H
R OMe
OH OH
O
OH
O
S S
12
Continued Structure-Activity Studies
•
For the 2G12 - gp120 interaction to be exploited there are 2
requirements to be met by synthetic chemists:
1)
Efficient routes to Man9GlcNHAc2 and other oligomannose species
2) Design of a scaffold that presents the given epitope in an orientation
advantageous to binding by 2G12
extremely important for synthetic vaccine design
13
One-Pot Oligomannose Assembly
HO
OBn
BnO
TBDMSO
BnO
HO
OAc
O
OBn
O
OH
NIS, TfOH, CH2Cl2,
4Å MS, 0°C, 24h
O(CH2)2NHCBz
Deprotection:
1) NaOMe, r.t., 2h
2) Pd black, 5% HCO2H/MeOH,
H2, r.t., 24h
SPhCH3
OH
O
HO
HO
HO
OH
O
HO
HO
HO
O
OH
O
O
30%
O
1
NH2
OBn
OH
O
BnO
TBDMSO
BnO
HO
O
BnO
TBDMSO
TBDMSO
OBn
BnO
TBDMSO
1) NIS, TfOH, CH2Cl2 4Å MS, -40°C, 1h
SPhCH3
O
HO
O
SPhCH3
OH
O
HO
HO
HO
BnO
HO
O
O
HO
HO
OBn
NIS, TfOH, CH2Cl2,
O
OH
OH
O
HO O
4Å MS, -10°C, 4h
64%
O(CH2)2NHCBz
4:3, 68% conversion
2) TBAF, THF, r.t., 24h
3) Et3N, Ac2O, DMAP, CH2Cl2, r.t., 2h
Deprotection:
1) 80% HOAc, r.t, 4h
2) NaOMe, r.t., 2h
3) Pd black, 5% HCO2H/MeOH,
H2, r.t., 24h
HO
OBn
OH
O
BnO
TBDMSO
O
OH
OH
O
HO
BnO
O
BnO
TBDMSO
BnO
BnO
TBDMSO
HO
O
O
HO
HO
O
HO
O
HO
HO
SPhCH3
O
O
O
HO O
OH
OH
O
44%
O
3
Wong, C.-H. et al. Angew. Chem. Int. Ed. 2004, 43, 1000.
Wong, C.-H. et al. J. Am. Chem. Soc. 1999, 121, 724.
NH2
2
14
NH2
One-Pot Oligomannose Assembly
HO
HO
O
HO
HO
HO
HO
OBn
OH
O
BnO
TBDMSO
BnO
HOHO
O
BnO
TBDMSO
HO
OBn
BnO
TBDMSO
OH
O
1) NIS, TfOH, CH2Cl2 4Å MS, -40°C, 1h
SPhCH3
OBn
O
O
OH
O
HO O
HOHO
BnO
HO
O
O O
HO
O
SPhCH3
O
HO O HO
HO
60%
O
4
NIS, TfOH, CH2Cl2,
4Å MS, -20°C, 2h
NH2
O(CH2)2NHCBz
4:3, 68% conversion
2) TBAF, THF, r.t., 24h
3) Et3N, Ac2O, DMAP, CH2Cl2, r.t., 2h
OBn
OH
O
BnO
TBDMSO
BnO
O
BnO
TBDMSO
BnO
BnO
TBDMSO
OH
Deprotection:
1) NaOMe, r.t., 2h
2) Pd black, 5% HCO2H/MeOH,
H2, r.t., 24h
HO
O
O
HOHO
O
OH
O
HO
HO
HO
HO O
HO
HOHO
O
HO
HO
O
HO
O
O O HO
HO
HO
SPhCH3
HOHO
O
O
O O
HO O
OH
O
41%
O
5
Wong, C.-H. et al. Angew. Chem. Int. Ed. 2004, 43, 1000.
Wong, C.-H. et al. J. Am. Chem. Soc. 1999, 121, 724.
15
NH2
Glycosyl Donor Activation
oxocarbenium glycosyl donor
PGO
O
PGO
PGO
PGO
PGO
glycosyl acceptor
PGO
OH
O
PGO
OH
O
PGO
PGO
-H+
OH
O
PGO
SPhCH3
O
PGO
PGO
SPhCH3
PGO
O
PGO
PGO
PGO
I
O
OPG
O
PGO
OPG
O
PGO
PGO
N
PGO
PGO
PGO
O
S
OTf
PhCH3
H
S
Succinimide
I
PhCH3
I
O
N
H
O
16
Fraser-Reid, B. Tetrahedron Lett. 1990, 31, 4313.
Evaluation
Carbohydrate Inhibition of
2G12 Binding to gp120
Carbohydrate Antigen
Man9GlcNAc
1
2
3
4
5
% Inhibition at 0.5mM
Antigen
42
8
16
58
38
48
= mannose
1
2
3
Wong, C.-H. et al. Angew. Chem. Int. Ed. 2004, 43, 1000.
Wong, C.-H. et al. J. Am. Chem. Soc. 1999, 121, 724.
Wilson, I. A.; Wong, C.-H. et al. Proc. Natl. Acad. Sci. USA. 2005, 102, 13372.
4
5
17
Assembly of Branched Oligosaccharides
OHOTBDMS
O
BnOO
SPhCH3
AcO
BnO
O
BnO O
AcO
BnO
O
OAc
OBn
BnO
O
1) NBS, H2O, Acetone
2) Cl3CCN, DBU, CH2Cl2
OBn
OTBDMS
O
AcO
BnO
O
BnO O
3) TBDMSOTf, Et2O, -60°C,
AcO
BnO
OBn
O
OAc
OTBDMS
OBn
O
BnO
HO
SPhCH3
SPhCH3
O(CH2)5NHCbz
AcO
BnO
O
BnO O
AcO
BnO
O
BnO O
AcO
1) NIS, TfOH, CH2Cl2,
BnO
O
OAc 4Å MS -20°C, 2h
BnO
OH
HO
O
HO
HO
O
HO O
HO
HO
O
OH
OH
HO
HO
2) AcOH, THF, TBAF, 1h
3) 25% w/v NaOMe, MeOH, 48h
4) MeOH, HCO2H, Pd-black, H2, 24h
OH
O
O
HO
OH O
HO
HO
O
OH
O
O
6
O(CH2)5NHCbz
O
OH
HO
HO
HO
1) NBS, H2O, Acetone
2) Cl3CCN, DBU, CH2Cl2
3) TBDMSOTf, Et2O, -40°C,
BnO
AcO
SPhCH3
AcO
BnO
O
BnO O
AcO
BnO
O
OAc
OBn
1) NBS, H2O, Acetone
2) Cl3CCN, DBU, CH2Cl2
OBn
OAc
O
OBn
O
BnO
AcO
O
3) TBDMSOTf, Et2O, -60°C,
OH
BnO
BnO
BnO
O
O
OBn
OBn
O
OBn
O
SPhCH3
BnO
O
BnO
BnO
O
OBn
OBn
OBn
O
OH
O
OHOTBDMS
HO
O
BnOO
HO
OH
O
O(CH2)5NHCbz
O
AcO
HO
BnO
O
BnO O
HO
AcO
O
BnO
OH
O
BnO O
O
AcO
HO
O
BnO
O
OAc
HO
O
BnO
HO
O
HO OH
4) 25% w/v NaOMe, MeOH, 48h
HO
O
5) MeOH, HCO2H, Pd-black, H2, 24h
HO
HO
SPhCH3
OH O
HO
HO
OH O
HO
HO
Wilson, I. A.; Wong, C.-H. et al. Proc. Natl. Acad. Sci. USA. 2005, 102, 13372.
Wong, C.-H. et al. J. Am. Chem. Soc. 1999, 121, 724.
HO
OH
HO
HO
O
O
OH
O
7
O(CH2)5NHCbz
O
18
Assembly of Branched Oligosaccharides
1) NBS, H2O, Acetone
2) Cl3CCN, DBU, CH2Cl2
3) TBDMSOTf, Et2O, -40°C,
BnO
AcO
SPhCH3
AcO
BnO
O
BnO O
AcO
BnO
O
OAc
OBn
1) NBS, H2O, Acetone
2) Cl3CCN, DBU, CH2Cl2
3) TBDMSOTf, Et2O, -60°C,
OH
OBn
O
BnO
HO
SPhCH3
OBn
OAc
O
BnO
O
BnO
AcO
O
O
BnO
O
AcO
BnO
O
BnO O
AcO
BnO
O
OAc
OBn
OHOTBDMS
O
BnOO
OBn
O
AcO
BnO
BnO OO
AcO
BnO
O
BnO O
AcO
BnO
O
OAc
BnO
SPhCH3
O(CH2)5NHCbz
4) 25% w/v NaOMe, MeOH, 48h
5) MeOH, HCO2H, Pd-black, H2, 24h
HO
HO
OH OH
O
OH
O
HO
HO
O
O
HO
O
HO
HO
O
HO O
HO
HO
O
OH
OH
HO
HO
O
HO
HO
HO
HO
HO
HO
HO
OH
O
O
O(CH2)5NHCbz
O
HO
OH O
Wilson, I. A.; Wong, C.-H. et al. Proc. Natl. Acad. Sci. USA. 2005, 102, 13372.
Wong, C.-H. et al. J. Am. Chem. Soc. 1999, 121, 724.
OH
O
O
8
O
O
19
Glycosyl Donor Activation
oxocarbenium glycosyl donor
PGO
PGO
OH
O
PGO
OH
O
O
PGO
PGO
PGO
PGO
PGO
PGO
-H+
OH
O
PGO
glycosyl acceptor
SPhCH3
O
PGO
PGO
SPhCH3
PGO
OPG
O
OPG
PGO
PGO
O
PGO
PGO
O
PGO
PGO
O
PGO
PGO
CCl3
O
NH
Ph
PGO
CCl3
NH
Ph(H3C)2Si
CH3
CH3
Si
OTf
PGO
I
O
OPG
O
PGO
O
PGO
PGO
N
OPG
PGO
PGO
O
S
OTf
PhCH3
H
S
Succinimide
I
PhCH3
I
O
N
H
O
20
Frasier-Reid, B. Tetrahedron Lett. 1990, 31, 4313.
Evaluation
Carbohydrate Inhibition of
2G12 Binding to gp120
Carbohydrate Antigen
% Inhibition at 0.5mM
Antigen
42
49
68
63
Man9GlcNAc
6
7
8
= mannose
6
7
Wilson, I. A.; Wong, C.-H. et al. Proc. Natl. Acad. Sci. USA. 2005, 102, 13372.
Wong, C.-H. et al. J. Am. Chem. Soc. 1999, 121, 724.
8
21
Insights from Binding Studies
• Tightest binding oligosaccharide was Man9GlcNAc2
• Scaffold-conjugated or surface-immobilized epitopes
bound 2G12 tighter than soluble ones.
OH
HO
OH
HO
OH
HO
HO
O
HO
OH
OH
O
O
HO
HO
HO
O
OH
OH
HO
HO
OH
OH
O
O
HO
HO
OH
OH
O
HO
O
OH
O
O
O
O
O
HO
O
• Co-crystal structures of branched oligosaccharides and
2G12 confirmed high specificity for Man1-2Man linkages.
O
OH
O
O
HO
OH
Man9GlcNAc2
O
OH
AcHN
O
HO
OH
a
AcHN
O
NH2
b
a) Man1-2Man arm in
major binding site
a
b) Branches in secondary
binding sites
Wilson, I. A.; Wong, C.-H. et al. Proc. Natl. Acad. Sci. USA. 2005, 102, 13372.
Calarese, D.A. et al. Science. 2003, 300, 2065.
22
Fully Synthetic Man9GlcNHAc2
OH
HO
HO
BnO
BnO
OH
O
OH
O
HO
HO
O
O
OH
HO
HO
HO
HO
HO
O
O
O
BnO
OH
OH
O
O
O
OH
HO
HO
BnO
BnO
O
O
HO
BnO
O
OH
O
HO
HO
HO
O
O
HO
O
HO
OBn
O
BnO
BnO
O
HO
HO
OBn
OAc
O
O
HO
BnO
BnO
OH
O
HO
NHAc
O
OBn
NH2
Ph
O
OAc
EtS
O
BnO
HO
BnO
BnO
BnO
BnO
BnO
O
O
HO
OBn
OBn
O
O
BnO
PhSO2HN
OBn
O
O
BnO
PhSO2HN OTBS
Chitobiose Trisaccharide
BnO
BnO
O
HO O
SPh
O
NHAc
OBn
O
O
O
O
O
AcO O
BnO
Danishefsky, S.J. et al. Angew. Chem. Int. Ed. 2004, 43, 2562.
Danishefsky, S.J. et al. Tetrahedron Lett.. 2003, 44, 1791.
23
Synthesis of the Chitobiose Trisaccharide
OBn
OBn
HO
BnO
1) Ac2O, DMAP
O
OBn
I
AcO
BnO
2) I(coll)2ClO4,
PhSO2NH2, 4Åms
O
EtSH/LHMDS, DMF AcO
O
BnO
88%
62%
NHSO2Ph
SEt
NHSO2Ph
OBn
HO
BnO
1) MeOTf, DTBP,
4Åms, CH2Cl2
O
OBn
AcO
BnO
OBn
O
O
O
BnO
70%
O
O
PMBO
OBn
HO
BnO
OBn
O
O
O
BnO
OTBS
3) TBSOTf, 2,6-lutidene, CH2Cl2 PhSO2HN
PhSO2HN
53-60%
4) NaOMe/MeOH
PhSO2HN
Ph
1) I(coll)2ClO4, PhSO2NH2
2) Et3N, H2O/THF
OBn
O
S
O
Ph
Ph
1) Tf2O, DTBMP, CH2Cl2, -78°C
2) CAN, MeCN/H2O
OBn
OBn
O
O
O
HO
O
BnO
63%
Danishefsky, S.J. et al. Tetrahedron Lett.. 2003, 44, 1791.
Seeberger, P.H.; Danishefsky, S.J. et al. Chem. Eur. J. 1997, 3, 1617.
PhSO2HN
OBn
O
BnO
O
PhSO2HN
OTBS
24
Synthesis of Man9GlcNHAc2
BnO OAc
O
BnO
BnO
EtS
BnO
BnO
BnO
BnO
O
BnO O
BnO
BnO
O
BnO O
BnO
BnO
AcOO
BnO
Ph
OBn
OBn
O
O
O
HO
O
BnO
PhSO2HN
BnO
BnO
O
O
BnO
PhSO2HN
OTBS
BnO
BnO
O
PhSO2HN
O
OBn
O
O
BnO
PhSO2HN
O
O
PhSO2HN
51% over 3 steps
O
BnO
O
PhSO2HN
OTBS
BnO
BnO
O
BnO O
BnO
BnO
AcO O
BnO
SPh
(BrC6H4)3NSbCl6, CH3CN, 10h
Sinay radical activation
OH
HO
HO
OH
O
OH
O
HO
HO
O
HO
HO
O
HO O
HO
HO
O
OH
OH
4) Ac2O, sat. aq. NaHCO3, 40°C, 48h
HO
5) sat. aq. NH HCO (Kochetkov amination) HO
4
OTBS
O
1) NaOMe/MeOH, 12h
2) TBAF, HOAc, THF, 0°C, 1h
3) Na/NH3, -78°C, 2h
OBn
O
O
BnO
OH
O
HO
O
OBn
O
BnO
O
BnO
BnO
BnO
BnO
OBn
O
O
BnO
O
OBn
O
BnO
O
BnO
BnO
O
BnO O
BnO
BnO
O
OAc
OBn BnO
BnO
BnO
BnO
BnO
O
BnO O
BnO
BnO
AcO O
BnO
O
OBn
OBn
O
O
O
3
HO
O
O
OH
OH
O
HO
O
HO
OH
O
HO
NHAc
O
NH2
NHAc
82% prior to amination
HO
HO
O
HO O
HO
HO
HO O
HO
25
Danishefsky, S.J. et al. Angew. Chem. Int. Ed. 2004, 43, 2562.
O OBn
O
BnO
BnO
O
BnO O
BnO
BnO
O
OAc
OBn
OH
2) BH3, Bu2OTf, THF, 0°C, 7h
OBn
OAc
O
OBn
O
BnO
O
1) MeOTf, DTBP, CH2Cl2,
-40°C - r.t.12h
OBn
OBn
O
O
Sinay Radical Activation/Glycosylation
OH
O
OBn
O
BnO
O
O
BnO
O
BnO
OBn
O
OBn
O
O
O
(BrC6H4)3NSbCl6, CH3CN, 10h
OBn
O
O
BnO
S
O
O
Ph
+
PhS
Br
SPh
SbCl6
N
Br
Br
O
BnO
O
OBn
O
O
BnO
O
+
BnO
O
PhS
SPh
S
Ph
OH
Sinay, P. et al. Carbohydr. Res. 1992, 236, 73.
Sinay, P. et al. Synlett. 1990, 572.
Danishefsky, S.J. et al. Angew. Chem. Int. Ed. 2004, 43, 2562.
OBn
O
BnO
O
O
26
Conjugate Vaccine Design Considerations
Carrier
S
c
a
f
f
o
l
d
E
E
E
•Three independently conducted binding studies and two crystallographic studies all
arrived at the same conclusions:
• Man9GlcNHAc2, in particular, the highly branched
nonamannose portion, bound the tightest to 2G12 and
likely in a multivalent fashion.
• Carrier and scaffold must be designed such that:
• antigens are presented similarly to their arrangement on the surface
of HIV-1
• each has the appropriate immunogenicity
27
Vaccine Candidates - Wang
• Keyhole Limpet Hemocyanin (KLH) and tetanus toxoid used as carrier/cell-mediated
immunostimulant.
• Very long, hydrophobic linker regions.
= Man9GlcNAc2Asn
O
R =
S
O
O
N
H
N
S
O
RR
RR
O
O
O
H
N
N
R
H
L E T I G I FK SNAK I YQGC
SH
Tetanus Toxoid (830-844) - Th epitope
O
SH
HS
SH
1) 50 mM phosphate buffer (pH 7.2),
CH3CN, 5 mM EDTA
SH
2) 0.1 M Cysteine, phoshpate buffer
KLH
HS
50 mM phosphate buffer (pH 6.6), CH3CN
O
N
H
O
R
O
O
R
S
R
O
N
O
CGQ Y I K A N S K F I G I T E L
• Rabbits were innoculated with either
peptide or KLH conjugates with adjuvant
SH
• Sera taken after four doses showed
high IgG titer…
RR
O
O
R
KLH
…mostly specific for the linkers!
R
O
N
O
n = 300-400
Wang, L.-X. et al. Bioconjugate Chem.. 2006, 17, 493.
28
Vaccine Candidates - Danishefsky
• Scaffold Assembly and Conjugation:
OH
HO
OH
HO
OH
HO
HO
O
HO
OH
OH
O
O
HO
HO
O
OH
OH
HO
HO
HO
OH
OH
HO
HO
OH
OH
O
O
O
HO
O
O
O
O
O
O
O
OH
HO
O
O
O
HO
OH
D
OH
A
P
O
F
A
O
P
OH
OH
C
D
Y
Y
AcHN
P
SStBu
O
OH
=
O
HO
HN
O
O
AcHN
O
A
O
NH
1) MESNa, DIEA, DMF, H2O (85%)
2) HEPES buffer
F
A
O
D
P
C
D
Y
P
A
Y
P
SStBu
OH
AcHN
O
OH
O
NH2
HO
O
NH
D
OH
P
AcHN
OH
AcHN
O
AcHN
HATU,DIEA,DMSO
O
HO
O
HO
OH
P
A
O
HO
O
HO
D
N
O
OMPC
n = ~ 103
OMPC = meningococcus outer membrane protein
O
NH2
R
O
N
H
O
N
O
H
N
R
N
H
R
O
H
N
O
R
N
N
H
R
O
O
O
O
R
H
N
N
R
O
Danishefsky, S.J. et al. J. Am. Chem. Soc. 2007, 129, 11042.
Dumy, P. et al. Org. Lett. 2003, 5, 243.
Bogusky, M. J,. et al. Biochemistry. 2003, 42, 3214.
O
N
H
R
H
N
R
O
N
O
N
H
H
N
R
O
29
Vaccine Candidates - Danishefsky
• Key features and evaluation:
• Conjugation to a proven, superior immunostimulatory
carrier
• Short, biomimetically structured linker
O
HO
AcHN
O
HO
O
HO
OH
OH
AcHN
O
AcHN
HN
O
O
D
O
O
NH
C
D
Y
OH
D
F
A
• In vivo immunological
evaluations are currently
underway.
NH
A
P
P
O
• OMPC conjugate showed
stronger binding to 2G12 than
any construct evaluated
previously
P
A
Y
P
O
S
N
O
OMPC
n = ~ 103
Danishefsky, S.J. et al. J. Am. Chem. Soc. 2007, 129, 11042.
Wilson, C.B. Eur. J. Immunol. 2001, 31, 2373.
30
Developing an Effective HIV-1 Vaccine
RR
O
O
R
KLH
R
O
N
O
n = 300-400
• There is now proof of principle that a synthetic carbohydrate-based vaccine is feasible.
• Lessons learned about carrier, scaffold, and linker strucutres:
• dense epitope presentation
• minimal exposure of linker and/or scaffold
• choice of carrier/method of delivery
Is there a logical next step?
31
Multi-Component Anti-Tumor
Vaccines:
A Future Direction for HIV?
32
Vaccines as Cancer Therapy
• Correlation between tumor metastasis and re-occurrence and glycoprotein overexpression
• Tumor cell surfaces are heavily coated with specific antigens and yet remain
immunogenically silent
Goal: Break the immune system’s tolerance for tumor
carbohydrate antigens.
• Because tumor cell membranes
often exhibit a diversity of
antigens, a conjugate vaccine
mirroring this may have the best
chance for success.
Carrier
S
c
a
f
f
o
l
d
E1
E2
E3
33
Danishefsky, S.J. et al. Angew. Chem. Int. Ed. 2000, 39, 836.
Tumor-Associated Carbohydrate Antigens
-Tn
ovarian, breast, colon, epithelial
Lew isy
colon, liver, prostate, ovarian
Mbr1/Globo-H
breast, colon, prostate, lung, ovarian,
small cell lung
OH
HO
OH
O
HO
Tissue
OH
CH3
O
OH OH
Antigen
O
OH OH
O
O
O
OH
H3C
HO
OH
OH OH
O
O(CH2)3
O
O
OH
O
OH
-Tn
HO
Lewisy
OH OH
O
HO
O
O
O
NHAc
O
AcHN
OH OH
OH OH
O(CH2)3
O
HO
O OH
H3C
MBr1/Globo-H
O
O
OH
HO
OH
HO
O
OH HO
OH
O
O(CH2)3
OH
Danishefsky, S.J. et al. Angew. Chem. Int. Ed. 2000, 39, 836.
34
Glycoamino Acid Synthesis
Antigen
1) O3, CH2Cl2, MeOH, pyr.
O
Antigen
O
2) Me2S
3) TMG, THF,
CO2TSE
NHBoc
O
(MeO)2 P
NHBoc
CO2CH2CH2TMS
5 mol% (S, S)-Et-DuPOHS-Rh,
Antigen
THF, 50 psi H2
O
NHBoc
P
(S-S)-Et-DuPHOS =
CO2TSE
Yield: 93-99%
P
d.r. > 20:1
35
Danishefsky, S.J. et al. J. Am. Chem. Soc. 2001, 123, 1890.
Scaffold Assembly
“Multigen”
36
Danishefsky, S.J. et al. J. Am. Chem. Soc. 2001, 123, 1890.
Evaluation of Multigen-KLH Conjugates
• Five groups of mice were innoculated (three times), respectively, with multigen
conjugate, each individual antigen conjugate, and a mixture of the individual
conjugates of all three antigens (all with QS21 plant extract adjuvant).
• Increased titers of both IgM (nonspecific) and IgG (specific)
antibodies were observed for the construct itself and for each of the
three antigens individually. . .
OH OH
O
HO
H3C
O
O
OH OH
OH OH
O
O
O
O
NHAc
OH
O OH
OH OH
HO
OH
HO
OH OH
H3C
HO
OH
O
O
OH
O
O
O
H
N
AcHN
OH OH
O
O
AcHN
O
OH
OH
CH3
HO
OH
O
HO
HO
O
O
OH HO
O
O
OH
O
OH
O
NHAc
O
O
O
N
H
H
N
O
H
N
S
O
N
KLH
O
O
OH
OH
n = 300-400
A specific immune response for three
distinct carbohydrate antigens.
Danishefsky, S.J. et al. Proc. Natl. Acad. Sci. USA. 2002, 99, 13699.
Danishefsky, S.J. et al. J. Am. Chem. Soc. 2001, 123, 1890.
37
Assembly of a Three Component Vaccine
OH OH
O
Th Cell epitope
AcHN
H2N CKLFAVWKITYKDTG
PGS
B Cell epitope
HO
SPh
O
H2N CTSAPHD T RPAP
O
NH2
HS
Native Chemical Ligation
OH OH
O
TLR-2 epitope
HO
AcHN
O
H2N CKLFAVWKITYKDTGCTSAPHD T RPAP
O
NH2
O
Native Chemical Ligation
HS
O
OH OH
SKKKKG
S
O
O
HN
O
O
14
14
O
14
HO
O
O
O
O
O
SKKKKGCKLFAVWKITYKDTGCTSAPHD T RPAP
S
O
AcHN
HN
NH2
O
14
14
14
Boons, G.-J. et al. Nature Chemical Biology. 2007, 3, 663.
Boons, G.-J. et al. Angew. Chem. Int. Ed. 2005, 44, 5985.
SPh
38
Evaluation of a Vesicle-based Vaccine
OH OH
O
HO
O
O
O
O
HN
NH2
O
14
14
O
SKKKKGCKLFAVWKITYKDTGCTSAPHD T RPAP
S
O
AcHN
Hydration of glycolipopeptide with:
14
egg phosphatidyl choline
egg phosphatidyl glycerol
cholesterol
HEPES buffer
• Seven groups of five mice were immunized four times with
the vesicle construct shown at right or combinations of its
individual components
IgG response to the vesicle construct was in vast excess compared
to that of IgM:
• memory-induced antibody production
• robust T cell response
• cytokine and complement activation via TLR-2 (adjuvant effects)
Boons, G.-J. et al. Nature Chemical Biology. 2007, 3, 663.
Boons, G.-J. et al. Angew. Chem. Int. Ed. 2005, 44, 5985.
39
Conclusions
• At the very least, proof of principle has been achieved in
the development of a gp120 oriented HIV-1 vaccine.
• Structural mimicry of the epitope itself and its
environment are key.
• Well-established single-antigen anti-cancer vaccines have
been advanced to a new realm.
• Multi-component vaccines are employing both
multiple antigens and multiple kinds of
immunogenic epitopes.
40
Future Directions
• The synergistic combinations of antigenic epitopes and
immunogens in a structurally biomimetic fashion is likely
applicable to HIV-1 vaccine development and may provide
chemists and biologists with new tools to study and fight
other infectious diseases.
• The design of new methods of antigen/immunogen display
and delivery may allow for the medical exploitation of new
protein-carbohydrate, protein-protein, and cell-cell
interactions.
41
Acknowledgements
Professor Samuel Gellman
Josh Price
Dr. W. Seth Horne
Soo Hyuk Choi
Li Guo
Melissa Boersma
Dr. Pil Seok Chae
Johnathan Zhang
Kim Peterson
Will Pomerantz
Jay Steinkruger
Erik Hadley
Emily Blamer
Holly Haase
Dr. Brendan Mowery
Matt Windsor
Dr. Felix Friere-Iribarne
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Brooke Richardson Dr. Myung-Ryul Lee
Emily English
Becca Splain
Lisa Johnson
Aaron Almeida
Lexie Dillon
Dmitri Svetlov
Ryan Drake
Melissa Hardy
42
Questions?
43