Workshop on the Future of Glycoscience

Overview Talks on Key Scientific Challenges‐Synthesis, Enzymatic and 

Biological

Robert J. Linhardt, Rensselaer Polytechnic Institute

January 13, 2012

Enzymatic/Chemoenzymatic Synthesis• Glycosidase reversal

– Kinetic vs. thermodynamic control (microscopic reversibility)‐glycosidases run in transglycosylation mode.

– Recombinant enzymes and chemically modified substrates‐Mutant glycosidases run in transglycosylation mode serve as Glycosynthases. 

• Carbohydrate remodeling– Combination of breakdown and synthesis

• Glycosyl transferases– Availability nucleotide donors– Unnatural nucleotide donors

• Scaling synthesis– Microscale– Macroscale

Carbohydrate Remodeling of Glycoproteins

GlcNAcGal

Man

Glyco-engineeredE. coli

a) Expression

b) Glycan trimmingDe-glycosylation

HeterogeneousRecombinant glycoprotein

Homogeneous Glycoproteins

Endo-enzymemutants

Sugar oxazolines

Schwarz, F., Huang, W., Li, C., Schulz, B. L., Lizak, C., Palumbo, A., Numao, S., Neri, D., Aebi, M., Wang, L. X., Nat. Chem. Biol., 6, 264-266 (2010)

Glycosidase Reversal

Kittl, R. Withers, S. G., Carbohydr. Res., 345, 1272–1279 (2010)

Chemoenzymatic synthesis of ultralow molecular weight heparins

12 steps• Et3N/CH3OH/H2O: chemical de-NTFA• N-sulfotransferase: enzymatic N-sufation• C5-epi/2-OST: enzymatic epimerization of GlcA to IdoA/2-O-sulfation• 6-OST: enzymatic 6-O-sulfation• 3-OST: enzymatic 3-O-sulfation

HO3SNH

HO3SNHHO3SNH

HO3SOHO3SNH

HO3SNH

HO3SNH

HO3SNH

HO3SNH

HO3SNH

OSO3H

HO3SO

OHOHOOH

HOOCO

OHOHO

OH

a, b

a. KfiA, UDP-GlcNTFAb. pmHS2, UDP-GlcUA

80%

OHO

OH

HOOC

O

OHOHO

OHR =

OHOHO

OH

HOOCOO

HO

OCF3CONH

OH

R

6

7

a, b, c

c. KfiA, UDP-GlcNAc

OHO

OH

HOOCOO

HO

OCF3CONH

OH

R

OHO

OH

HOOCOO

HO

OCF3CONH

OHOHO

HO

OCH3CONH

OH

d, e, f, g, h

d. Et3N/CH3OH/H2Oe. N-sulfotransferase, PAPSf. C5-epi/2-OST, PAPSg. 6-OST, PAPSh. 3-OST-1, PAPS

OHO

O

OSO3H

R

OHO

OH

COOHOO

OSO3HOHO

HO

OCH3CONH

8

ULMW heparin construct 1

OHO

OH

COOHOO

HO

OCF3CONH

OH

R

OHO

OH

HOOCOO

HO

OCF3CONH

OH

9a, b

HO

OHO

OH

COOHOOHO

O

OH

R

OHO

OH

HOOCOO

HOO

OH

10

HO

d, e

11

a, f

OHO

O

OH

R

OHO

OH

COOHOO

HO

OHO

OHOHO

CF3CONH

OH

OHO

O

OSO3H

R

OHO

OH

COOHOO

OSO3HO

OHOHO

OSO3H

d, e, g, h

ULMW heparin construct 2

80%

70%

80%

80%

80%

90%

Arixtra (if R = -CH3)

OOOH

HO3SO

O

COOH

OOOH

HO3SO

O

COOH

OOOH

HO3SO

O

COOH

AB

C

D E

AB

C

D E

10 steps

Xu, Y., Masuko, S., Takieddin, M., Xu, H., Liu, R., Jing, J., Mousa, S., Linhardt, R. J., Liu, J., Science, 334, 498-501, 2011.

Cofactor Recycling

S. Masada, Y. Kawase, M. Nagatoshi, Y. Oguchi, K. Terasaka, H. Mizukami, FEBSLett., 581,2562–2566 (2007)

M. D. Burkart, M. Izumi, E. Chapman, C.-H. Lin, C.-H. Wong, J. Org. Chem., 65, 5565-5574 (2000)

Natural and Unnatural UDP‐donorsUDP-GlcNAc

• substrate for KfiA• commercially available

UDP-GlcNTFA and others

• substrate for KfiA• unnatural analog of UDP-GlcNAc• also t-Boc, Fmoc, alkynoyl, alkenolyl

O P O P OO O

ONa ONaO

HN

N

O

O

HO OH

OHOHO

NH

OH

F3C

O

M. Weïwer, T. Sherwood, D. E. Green, M. Chen, P. L. DeAngelis, J. Liu, R. J. Linhardt, J. Org. Chem., 73, 7631-7637, 2008.S. Masuko, S. Bera, D. E. Green, M. Weïwer, P. L. DeAngelis, R. J. Linhardt, J. Org. Chem., in press, 2012.

•Not a substrate of native synthases• unnatural analog of UDP-GlcA

O P O P OO O

ONa ONaO

HN

N

O

O

HO OH

OHOHO

HO

COOH

UDP-IdoAUDP-GlcA

• substrate for pmHS2• commercially available

Microscale Enzyme Assisted Synthesis

Microarrays using enzymes

Microfluidics using enzymes

J. G. Martin, M. Gupta, Y. Xu, S. Akella, J. Liu, J. S. Dordick, R. J. Linhardt, J. Am. Chem. Soc., 131, 11041–11048, 2009.

T.-J. Park, M.-Y. Lee, J. S. Dordick, R. J. Linhardt, Anal. Biochem., 383, 116–121, 2008.

GlucoseNH4Cl

E. coli K5 Fermentation

Centrifuge Centrifuge

Ion Exchange Column Chitosan

Cells

Waste

Chitosan-Heparosan PEC

Chitosan

NaOH HCl

Chemical N-DeaceytlationChitosan Removal

Dialysis N-Sulfonation

C5 Epimerase2-O-Sulfonation

Reaction

6-O-Sulfonation Reaction

3-O-Sulfonation Reaction

N-Sulfoheparosan

NaH

CO

3

(CH

3 )3 NS

O3

NaC

l + H2 O

Ethano

l

CaC

l 2 Tris

PN

PS P

AP

PN

PS P

AP

PN

PS P

AP

Heparin

DEAE

NaCl

FERMENTATION PURIFICATIONN-DEACETYLATION

N-SULFONATION

O-SULFONATION PURIFICATION

Macroscale Enzyme Assisted Synthesis

100,000‐L fermentationaffording 1 metric ton (10g/L)heparosan, chemically modifiedand enzymatically transfoemed with 4 enzymes (1‐10 kg/each)and  PAPS (1 kg with 5000xrecycling) to afford 2 metric tonsheparin run 50 times/y to meetannual world supply (100 metric tons) at $25,000/kg

U. Bhaskar, E. Sterner, A. M. Hickey, A. Onishi, F. Zhang, J. S. Dordick, R.J. Linhardt, Appl. Microbiol. Biotechnol., in press, 2012.

Metabolic Engineering• Bacterial biosynthesis of complex glycans

– Require extensive pathway engineering and the introduction of compartmental structure

• Yeast biosynthesis of complex glycans– Have a Golgi but are missing portions of human pathways

• Insect cell‐baculovirus expression system– Have a Golgi but are missing portions of human pathways

• Mammalian cell biosynthesis of complex glycans– Difficult to grow at high cell density – Have full biosynthetic pathway but difficult to control/regulate  

• Metabolic Glycoengineering

Bottom‐up Engineering of N‐glycan Biosynthesis in E. coli

Valderrama-Rincon J, Fisher AC, Merritt J, Yao-Yun F, ReadingC, Chhiba KD,Aebi M and DeLisa MP(2011) Nature Chemical Biology (in revision)

Metabolic Flux Analysis

2. Reformulate as 

edge‐node representation.

4. Formulate the constraint‐based mathematical model.

5. Apply an objective function and solve optimization problem.

3. Setup mass balances 

for each node in the network.

1. Annotate all known genes for the genome of interest (E.coli).

Xu P, Ranganathan S, Fowler ZL, Maranas CD, Koffas MA., Metab. Eng., 13, 578-587 (2011)

Biosynthetic Control in the Golgi of Eukaryotes

????

Engineering Eukaryotic CellsYeast                                              Insect cells                                           CHO cells

β‐1,4 GT 

Aumiller JJ, Mabashi-Asazuma H, Hillar A, Shi X, Jarvis DL. Glycobiology2011 in press.

T. U. Gerngross, Nat. Biotechnol., 22, 1409-1414 (2004)

J. Y. Baik, L. Gasimli, B. Yang, P. Datta, F. Zhang, C. A Glass, J. D. Esko, R.J. Linhardt, S. Sharfstein, Metabolic Engineering, 2011, in review

OHO

HO

O

OH

HONHR1

O

CO2-

cellCellcellCell

Hexosamineanalogue

Cell surface display of non-natural glycans

Sialic acid biosynthetic pathway

OR2O

R2OR2O

HN

OR2

R1

O

“R1” groups . . .

. . . .can modulate biological activity

. . . . comprise a tool for glycomics

“Click” reaction

“R2” groups . . .

. . . .increase labeling efficiency

. . . .lead to new biological activities

OHO

HO

O

OH

HONH

O

CO2-

cellCell

Sia5Pent

OHO

HO

O

OH

HONH

O

CO2-

NN+

N- cellCell

Sia5AzOHO

HOHO

HN

OH

R1

O

OO

OO

HN

O

R1

O

O

O

O

OO

O

OO

HN

O

R1

O

O

O

O

O

1 600 2,100

High flux, few “side” effects

“Whole molecule” activity (e.g., NF-κB inhibition)

OHO

OO

HN

O

R1

O

O

O

OO

O

OO

HN

OH

R1

O

O

O

O

Metabolic Glycoengineering

Du J, Meledeo MA, Wang Z, Khanna HS, Paruchuri VD, Yarema KJ. Glycobiology 19, 1382-1401 (2009)

Acknowledgements

• Paul DeAngelis, University of Oklahoma, Oklahoma City• Mathew Delisa, Cornell University, Ithaca• Jonathan Dordick, Rensselaer Polytechnic Institute, Troy• Mattheos Koffas, Rensselaer Polytechnic Institute, Troy• Jian Liu, University of North Carolina, Chapel Hill• Lai‐Xi Wang, University of Maryland, Baltimore• Chi‐Huey Wong, Scripps, La Jolla• Kevin Yarema, Johns Hopkins University, Baltimore