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.