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Synthesis of Building Blocks for O-Mannosylated Glycans
Catherine Merton and Ke Zhao
Group Pictures
Kate Zhao
Catherine Merton
The Koviach-Côté Lab, Bates College
O-Mannosylated Glycoproteins
• Glycosylation is a post-translational modification that involves the addition of an oligosaccharide to a protein
• Linkage of mannose residue to a serine or threonine residue on a protein through an O-linkage
Praissman, J. L.; Wells, L. Biochemistry, 2014, 53, 3066-3078.
O-mannosylated glycoproteins - History• Originally found in yeast
• First detected in mammalian tissue in 1979
• 50+ proteins have been found to be O-mannosylated • Modification at more than 235 sites • Include proteins involved in cell-cell and cell-matrix adhesion
Praissman, J. L.; Wells, L. Biochemistry, 2014, 53, 3066-3078.
O-Mannosylated Glycoproteins – Prevalence and Function• Comprise approximately 30% of O-linked glycans in the mammalian brain
• Important for neural function and nervous system growth, such as neuron migration, axon path finding, and remyelination
• Disruption of these glycans has links to cancer, congentical muscular dystrophy, multiple sclerosis, mental retardation
• Receptor for leprosy and arenaviruses
Praissman, J. L.; Wells, L. Biochemistry, 2014, 53, 3066-3078.
Project Goals and Purpose
• Develop synthetic pathways, skills and techniques
• For further research • Provide authentic samples for bioanalytical chemists • Collaborate with biological chemist to… • Investigate glycan binding to proteins • Probe antibodies • Investigate peptide conformations
Core m1 Glycans
Ser/Thrα
β1,2
Ser/Thrα
β1,2
β1,4
Ser/Thrα
β1,2
β1,4
α1,3
Ser/Thrα
β1,2
β1,4
α2,3
Ser/Thrα
β1,2
β1,4
α2,3
Ser/Thrα
β1,2
β1,4
β1,3
3S
Core m2 Glycans
Ser/Thrα
β1,2
β1,6
Ser/Thrα
β1,2
β1,4
β1,6
Ser/Thrα
β1,2
β1,4
β1,6
β1,4
α1,3
Ser/Thrα
β1,2
β1,4
β1,6
β1,4
Ser/Thrα
β1,2
β1,4
α2,3
β1,6
Ser/Thrα
β1,2
β1,4
α1,3
β1,6β1
,4
Ser/Thrα
β1,2
β1,4
α1,3
β1,6β1
,4
Ser/Thrα
β1,2
β1,4
α2,3
β1,6β1
,4
Ser/Thrα
β1,2
β1,4
α1,3
β1,6β1
,4
α2,3
Ser/Thrα
β1,2
β1,4
α1,3
β1,6β1
,4
α2,3
Ser/Thrα
β1,2
β1,4
β1,6β1
,4
α2,3
α2,3
Ser/Thrα
β1,2
β1,6β1
,4
β1,3
3SSer/Thr
α
β1,2
β1,4
β1,6β1
,4
β1,3
3SSer/Thr
α
β1,2
β1,4
α1,3
β1,6β1
,4
β1,3
3S
Ser/Thrα
β1,2
β1,4
β1,6β1
,4
α2,3
β1,3
3S
Core m3 Glycans
Kanagawa, M. et al. Cell Rep. 2016, 14, 2209-2223.
Mannose Building Block for m1 glycans
O OHOH
HOHO
HOO OAc
OAc
AcOAcO
OAc
I2, Ac2O 1) HBr/HOAc
2) Et3N, TBAB, MeOH 56%
O OO
AcOAcO
AcO OMe
1) Na°, MeOH
2) NaH, BnBr, TBAI 57%, 2 steps
O OO
BnOBnO
BnO OMeAcOH, H2O, 78% O OH
OAc
BnOBnO
OBn
1.75 g total
10.5 g
Ser/Thrα
β1,2
Ser/Thrα
β1,2
β1,4
Mannose Building Block for m2 glycans
1) NaOMe
2) imid., TBDPSCl 99%
OO
HOHO
TBDPSO
O
OMeNaH, BnBr, TBAI
64%OO
BnOBnO
TBDPSO
O
OMe
AcOH/H2O, 96%OOAc
BnOBnO
AcO
OH
OO
AcOAcO
AcO
O
OMe
1) TBAF
2) Pyr, Ac2O 70%, 2 steps
OO
BnOBnO
AcO
O
OMe
1.82 g
3.2 g
Ser/Thrα
β1,2
β1,6
Ser/Thrα
β1,2
β1,4
β1,6
β1,4
Differentially Protected Galactose Building Block for m1 glycans
Ser/Thrα
β1,2
β1,4
β1,3
3S
OAcO
AcO OAc
O
OAcAcO
AcO OAc
OAc1) HBr/HOAc
2) Et3N, TBAB 43%, 2 steps OO
OMe
Na°, MeOH
quant.
OHO
HO OH
OO
OMe
OHO
OO
OO
OMe
Ph
PhCH(OMe)2, p-TsOH
21%
LevOH, EDC, DMAP,
quant
OLevO
OO
OO
OMe
Ph
Et3SiH, TFA, 41%
OLevO
HO OBn
OO
OMe
1. NaH, BnBr
HOAc/H2O
OLevO
BnO OBn
AcOOH
8 g
Differentially Protected Galactose For m1 Glycan
O
OAcAcO
OAc OAc
OAcPrSH, BF3OEt2 O
OAcAcO
OAc OAc
SPrNaOMe O
OHHO
OH OH
SPr
O
OHHO SPr
LevOH, DMAP, EDCI Pyr, Ac2OO
O
Ph
O
OHLevO SPr
OO
Ph
O
OAcLevO SPr
OO
Ph
PhCH(OMe)2
p-TsOH
Jaipuri, Firoz A.; Pohl, N. L. Org. Biomolec. Chem. 2008, 6, 2686-2691.
Ser/Thrα
β1,2
β1,4
β1,3
3S
O
OAcAcO
AcO OAc
OO
OAcAcO
AcO OAc
OHO
OAcAcO
OAc OAc
OAcDBU, Cl3CCNN2H4
56% 33%CCl3
NH
Terminal Galactose For m1 and m2 Glycans
Ser/Thrα
β1,2
β1,4 Ser/Thr
α
β1,2
β1,4
β1,6
β1,4Ser/Thr
α
β1,2
β1,4
β1,6
GlcNc, terminal and differentially protected For m1 and m2 Glycans
O OH
NH3ClHO
HO
OH
MeO
O
H
O OAc
NH3ClAcO
AcO
OAcEt3N, Cl3C
O
ClO OAc
NHTCAAcO
AcO
OAc
O SPr
NHTCAAcO
AcO
OAcPrSH, BF3•OEt2
69%1) Na°, MeOH
2) PhCH(OMe)2, p-TsOH51%, 2 steps
O SPr
NHTCAHOO
OPh
LevOH, EDCI, DMAPO SPr
NHTCAAcOO
OPh
Et3SiH, TFA
91%
O SPr
NHTCAAcO
HO
OBn
Pyr, Ac2O, 95%
O SPr
NHTCAAcO
LevO
OBn
1)
2) Pyr, Ac2O, 96% 3) HCl, 52%
85%
85%
Ser/Thrα
β1,2
Ser/Thrα
β1,2
β1,6
Ser/Thrα
β1,2
β1,4
Ser/Thrα
β1,2
β1,4
β1,6
β1,4
Ser/Thrα
β1,2
β1,4
β1,6
Xylose For m3 Glycan
O
OHHO
HO OHPyr, Ac2O
quant.
O
OAcAcO
AcO OAc PrSH, BF3•OEt2 O
OAcAcO
AcO SPr
O
OAcAcO
AcO SPrNa°, MeOH
41%, 2 steps1:1 mix of anomers
O
OHHO
HO SPrPivCl, DMAP O
OHPivOHO SPr
Watanabe, H.; Nakada, M. J. Am. Chem. Soc. 2008, 130, 1150.
Attempted Glycosylation
O
OAcAcO
AcO OAc
O CCl3
NH
O SPr
NHTCAAcOHO
OBn TMSOTfDCM
0°CO
OAcAcO
AcO OAc
SPr
Ser/Thrα
β1,2
β1,4
Ser/Thrα
β1,2
β1,4
β1,6
β1,4
Ser/Thrα
β1,2
β1,4
β1,6
Acknowledgment
Research reported in this publication was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM0103423.