Chemical Tools for Studying and Perturbing Glycans

Lecture 41

Carolyn R. Bertozzi

UC Berkeley

Monosaccharide"building blocks"

Cytosol

Cell surface glycoconjugates

Substrates

ER/Golgi

Metabolicinterconversions

Carbohydrate-specific receptor

Glycoconjugate assembly

Bertozzi, C. R.; Kiessling, L. L. Science 2001, 291, 2357.

Unnatural

Glycosyltransferase or glycosidase inhibitors

Chemical approaches for perturbing cellular glycans

Lecture Outline

1. Inhibitors of glycosyltransferases and glycosidases- Natural products- Rational design- Library screening

2. Chemical activation of glycosyltransferases

3. Substrate-based methods for perturbing glycosylation- Glycoside primers- Metabolic oligosaccharide engineering

Tunicamycin - An inhibitor of N-linked glycan biosynthesis

• Blocks the transfer of GlcNAc-1-P from UDP-GlcNAc to dolichyl-P (GPT)

• Resistant mutants overproduce GPT

• Km for UDP-GlcNAc is ~3 x 10-6 M, whereas the Ki value for tunicamycin is ~5 x 10-8M

O

O

NHAc

OH

OHHO

O

RCHN

OHO

OH

CH2 O

OHOH

HH

HH

HO

HN

N

O

O

Plant Alkaloids - Natural Inhibitors of Glycosidases

2

Asn

3

4

4

63

6

-mannosidase II-glucosidase II -mannosidase I GlcNAc TI

Asn

3

4

4

63

6

Asn

3

4

4

6

2

3

3

2

2

3

4

4

63

2 2

6

Asn

-glucosidase I

3

3

2

2

3

4

4

63

2 2

6

Asn

±

2

2

3

4

4

63

2 2

6

Asn

Swainsonine

Deoxynojirimycin

Deoxymannojirimycin

Australine

Castanospermine

Kifunensin

Plant Alkaloids

NOH

OHH

OH

Swainsonine-Mannosidase II

N

OHHO

OHH

Castanospermine-Glucosidase I and II

N

OH

H O

HO

CH2OH

Deoxymannojirimycin-Mannosidase I

N

H

OH

OHHO

CH2OH

Australine-Glucosidase I

• Alkaloids contain polyhydroxylated ring systems that mimic the orientation of hydroxyl groups in the natural substrates

• Protonation of the ring nitrogen may mimic the positive charge developed on the ring oxygen during the hydrolytic reaction

HO

Glycosyltransferase inhibitors based on substrates/transition states

O

HO OH

HO O

OHO

OH

ONHAc

NH3C

HOOH

OH

OR

H OH

Fucosyltransferase inhibitor, IC50 = 31 μML. Qiao, B. W. Murray, M. Shimazaki, J . Schultz, C.-H. Wong, J . Am. Chem. Soc. 118, 7653 (1996)

O

OHHO

HOAcHN

O

PO

OHO–

PO–O

O

O

OH OH

N

N

O

NH2

HN

NH

NHR

O

OOH

O

HOHN

ONH

HNO

+H3N

OS

Oligosaccharyl transferase inhibitor, K i = nMP. D. Eason, B. Imperiali, Biochemistry 38, 5430 (1999).

Sialyltransferase inhibitor, K i = 40 nMB. Müller, C. Schaub, R. R. Schmidt,

Angew. Chem. Int. Ed. 37, 2893 (1998)

Problems with most rationally designed inhibitors

• Lack of activity in cells (poor bioavailability)

• Lack of selectivity

Pharmaceutical approach: High-throughput screenfollowed by medicinal chemistry optimization of hits

Tools for studying N-linked glycosylation

OOH

OHO

AcHNNH O

NH

OOOH

OHO

AcHN

OO

HOO

OH

OHO

HOHO

OHO

HOHO

OO

OH

OHO

AcHN

OOHOH

OOH

O

HOOH

HOAcHN

HO

CO2–

OO

OH

OHO

AcHN

OOHOH

OOH

O

HO OHHO

AcHNHO

CO2–

Asn

Conserved core

N-X-S/TConsensus sequence

Affinity reagents ConA, LPHA

Small molecule inhibitors Tunicamycin

Enzymes for cleavage PNGase F

OHO

OAcHN

NH O

CH3(H)O

OOHOH

OHO

OOOH

OO

NHAcO

OHOH

OOH

O

HO OHHO

AcHNHO

CO2–

O

OHOH

OHH3C

O

HO OH

HOAcHN

HO

CO2–

Challenges in studying O-linked glycosylation

NoneConsensus sequence

Small molecule inhibitors None

Enzymes for cleavage Nothing general

Affinity reagents Nothing general

Conserved core

The polypeptide N-acetylgalactosaminyltransferases (ppGalNAcTs) initiate mucin-type O-linked glycosylation

UDP-GalNAc

protein substrate

elaborating glycosyltranferases

Complex mucin-type O-linked glycoproteins

ppGalNAcTs(~24 in human)

Tabak et al.

H2N NH O

HN

CH3 (H)HO

COOH

OHO

AcHNHO

OH

H2N NH O

HN

CH3 (H)O

COOH

OP

OO

OP

O

O OO

N

NH

O

O

HO OH

OHO

AcHNHO

OH

Design and synthesis of a 1338-member uridine-based library

O

OHHO

O

NH

N

O

O SO

O

O

OHHO

HN

NH

N

O

ONH

O

O

OHHO

HN

NH

N

O

O

OOH2N

H2NH2N

HO

R> 400 commercial aldehydes

uridine analogs

O

OHHO

OPO

O

O-P

O

O-

NH

N

O

O

O

O

HO

O

OHHO

NH

N

O

OON

R

uridine

diphosphate mimic

sugarsubstitute

nucleotide sugar substrate

O

OHHO

O

NH

N

O

ONR O

OHHO

HN

NH

N

O

O

OONR S

O

O

O

OHHO

HN

NH

N

O

ONH

O

NR

H

H H

uridine-based library

Winans, K. A.; Bertozzi, C. R. Chem. Biol. 2002, 9, 113-129

Uridine analogs identified as broad spectrum ppGalNAcT inhibitors

O

OH OH

N

NH

O

O

HNO

O

N

OHOH

HO

O

OH OH

N

NH

O

O

ON

HO

HOHO

2-68AKi = 7.50 + 1.02 μM (T-1)

1-68AKi = 7.84 + 0.96 μM (T-1)

mppGalNAcT-1

mppGalNAcT-2

mppGalNAcT-3

mppGalNAcT-4

rppGalNAcT-5

rppGalNAcT-7

rppGalNAcT-10

mppGalNAc-11

1-4GalT-1

1-3GalT

IC50 2-68A (μM)

21

15

40

30

20

22

7

39

> 500

> 500

IC50 1-68A (μM)

24

18

38

20

26

27

6

32

> 500

> 500

KM of donor (μM)

22

25

32

69

51

20

10

20

25

20

Ki of UDP = 380 μM

Hang, H. C., et al. Chem. Biol. 2004, 11, 337-345

1-68A inhibits O-linked but not N-linked glycosylation in Jurkat cells

%M

FI

20

60

100

0

40

80

120

140

160

50 1500 100 200 250

VVA (O-linked)

MAA (2,3NeuAc)

ConA (N-linked)

HPA (O-linked)

[inhibitor] (μM)

Lecture Outline

1. Inhibitors of glycosyltransferases and glycosidases- Natural products- Rational design- Library screening

2. Chemical activation of glycosyltransferases

3. Substrate-based methods for perturbing glycosylation- Glycoside primers- Metabolic oligosaccharide engineering

Glycosyltransferases are residents of the Golgi compartment

cis medial trans TGN

Anatomy of a prototypical glycosyltransferase

Golgi membrane

Golgi lumen

Cytosol

C-terminalcatalytic domain (CAT)

N-terminalcytosolic tail (C)

Stem region (S)

TransmembraneDomain (T)

CTS = Encodes Golgi localizationCAT = Encodes catalytic activity

Exploiting the requirement of Golgi localization for small molecule switching of enzyme activity

Small moleculebinding proteins

Golgilocalized

SecretedGolgi

localized

Golgilocalized

Kohler, J. J.; Bertozzi, C. R. Chem. Biol. 2003, 10, 1303-1311

Rapamycin mediates association of FKBP and FRB

Rapamycin

J. Liang, J. Choi, & J. Clardy. Acta Cryst. (1999) D55, 745-752

O

OHOO

N

O

O

OCH3

OH

HO

O

OCH3

O

O

FucT7

Leukocyte

L-Selectin

Endothelial cell

Fucosyltransferase 7 is involved in selectinligand biosynthesis

{

O

CO2–OH

OH

AcHNHO

O

HO OH

O OOH

OOSO3

–

ONHAc

OH3C

HOOH

OH

O

O

HO

AcHN

O

HO OH

O OOH

O

HO

OCO2–OH OH

AcHNHO

O

HO OH

OOH

OH3C

HOOH

OH

HO O O

OSO3–

OAcHN

FucT7 constructs for CHO cell transfection

Tail TM Stem Catalytic domain

CTS (1-51)CAT (39-342)

N C

Tail TM Stem

Catalytic domain

N CFRB or FKBPn = 1-3

FKBP or FRB

n = 1-3CN

Rap

O

CO2–OH

OH

AcHNHO

O

HO OH

O OOH

OOH

ONHAc

OH3C

HOOH

OH

ORHOO

CO2–OH

OH

AcHNHO

O

HO OH

O OOH

OOH

HONHAc

ORHO

FucT7GDP-Fuc

FucT7 activity in CHO cells can be monitored bythe expression of sialyl Lewis x

Sialyl Lewis x(detected with mAb HECA-452)

sLex

No Rapamycin: FucT7 is “off”

+ Rapamycin: FucT7 is “on”

sLex

Lecture Outline

1. Inhibitors of glycosyltransferases and glycosidases- Natural products- Rational design- Library screening

2. Chemical activation of glycosyltransferases

3. Substrate-based methods for perturbing glycosylation- Glycoside primers- Metabolic oligosaccharide engineering

Monosaccharide"building blocks"

Cytosol

Cell surface glycoconjugates

Substrates

ER/Golgi

Metabolicinterconversions

Carbohydrate-specific receptor

Glycoconjugate assembly

Bertozzi, C. R.; Kiessling, L. L. Science 2001, 291, 2357.

Unnatural

Chemical approaches for perturbing cellular glycans

Glycoside Primers - Substrate Mimicry

• Prepare compounds that resemble biosynthetic intermediates

• Conjugate to a hydrophobic aglycone to enhance uptake and activity

• Alkylation or acylation also improve bioavailability

HOO

H

H

HO

H

H

OHH

O

Sarkar et al. (1995) Proc. Natl. Acad. Sci. USA 92: 3323

-Napthyl xyloside

Cell

O

2S

6S

NS NS

6S

Xyloside primers block proteoglycan glycosylation

2S

6S

NS NS

6S

Proteoglycan

O

2 S

6S

NS NS

6SO

2S

6S

NSNS

6S

O

O

Xylose HydrophobicAglycone

Xyloside

Fritz & Esko (2001) Methods Mol. Biol. 171:309

Types of primers

Glycoside Pathway affected

Xyl- -O R GlycosaminoglycansGlycolipids

Gal- -O R Glycosaminoglycans

GalNAc- -O R - O linked chains found on glycoproteins and mucins

GlcNAc- -O R Polylactosaminoglycans

Peracetylated Gal4GlcNAc - -O R LewisX

Peracetylated GlcNAc3Gal - -O R LewisX antigens

Gal GlcNAc

O

OAcAcO

OAc OAcO

NHAcAcO

OAc

O O

Naphthalenemethanol

Metabolic oligosaccharide engineering

X

X

X

X Y

Y

Unnatural substrate

Bioorthogonal functional

group

Exogenousreagent

Labeling enables:• detection• enrichment

Dube, D. H.; Bertozzi, C. R. Curr. Opin. Chem. Biol. 2003, 7, 616-625

The Staudinger ligation is highly selective and “bioorthogonal”

PhP

Ph

OCH3

O

N3

Phosphine(nucleophile)

Azide(electrophile)

PhP

Ph

O

NHO

Ligation product

OO

HO

AcHN

HO

OH

OO

HO

AcHN

HO

OH

OO

HO

AcHN

HO

OH

OO

HO

AcHN

HO

OH

p53 Tau

RNApol II Myc

Many nuclear and cytosolic proteins are transiently modified with -O-GlcNAc

• Complete repertoire of O-GlcNAcylated proteins?• Sites of O-GlcNAcylation?

OOAc

AcO

NHAcO

OAc

ON3

OOHO

NHHO

OH

ON3

OOHO

NHHO

OH

ON3

OOHO

NHHO

OH

ON3

OOHO

NHHO

OH

ON3

Cell

GlcNAcylated proteins

System-wide analysis of -GlcNAcylated proteins

GlcNAcGlcNAc

GlcNAc GlcNAc

FLAG

FLAG

FLAG

FLAG

H3CO

Ph2P DYKDDDDK

O

FLAG

Vocadlo, D. J., et al. PNAS 2003, 100, 9116-9121

GlcNAz

Unnatural ManNAc analogs are metabolized to unnatural sialic acids in cells

OHO

HO

HNHO

OH

O

CO2–

HO OHOH

HN

HO

O

O

O

Unnatural analogof ManNAc

Cell

Cell

Unnatural sialic acid

OHO

HO

HNHO

OH

O

n

W. Reutter et al.

OHO

HO

HNHO

OH

O

O

Mahal, L. K.; Yarema, K. J.; Bertozzi, C. R. Science 1997, 276, 1125-1128Luchansky, S. J.; Goon, S.; Bertozzi, C. R. ChemBioChem 2004, 5, 371-374

O

CO2–

HO OHOH

HN

HO

OH

ON3

O

CO2–

HO OHHO

AcHNHO O

CO2–

HO OHO

AcHNHO O

CO2–

HO OHO

AcHNHO

O

Polysialic acid

n

(n = 10-200)

neural cell adhesion molecule(NCAM)

OHO

HO

HN

HO OH

O

ManNBut

O

CO2–

HO OHHO

BuHNHO

O

Unnatural sialic acids can serve as PSA chain terminators

Mahal, L. K., et al. Science 2001, 294, 380-382

The Staudinger ligation can target sialylatedcells with chemical probles

OHO

HO

HNHO

OH

O

CO2–

HO OHOH

HN

HO

ON3

O

N3

OManNAz

Cell

Cell

SiaNAz

NH

O

O

OCH3

PPh2Probe

O

CO2–

HO OHOH

HN

HO

O

O

CellNH

O

ONH

PPh2

O

Probe

Saxon, E.; Bertozzi, C. R. Science 2000, 287, 2007-2010

Imaging changes in glycan expression within living animals

FLAG

O

CO2–

HO OHOH

HN

HO

O

N3

O

Cell

NH

O

O

OCH3

PPh2Probe

O

CO2–

HO OHOH

HN

HO

O

O

CellNH

O

ONH

PPh2

O

Probe

• Glycosylation changes associated with disease

• Glycosylation changes during development

OAcO

AcO

AcO HN

OAc

N3

O

Monosaccharide"building blocks"

Cytosol

Cell surface glycoconjugates

Substrates

ER/Golgi

Metabolicinterconversions

Carbohydrate-specific receptor

Glycoconjugate assembly

Bertozzi, C. R.; Kiessling, L. L. Science 2001, 291, 2357.

Chemical approaches for perturbing cellular glycans

Glycan arrays can be used to probe lectin/enzyme specificity

“GlycoChip” from Glycominds