Controlled Chemoenzymatic Synthesis of Size-Defined Polysaccharides and Macrocyclic

Carbohydrates

Xi Chen, Department of Chemistry, UC Davis1st Annual Workshop of the 10+10 Alliance

Peking University, Peking, ChinaMonday, June 9, 2008

• Chemoenzymatic synthesis of biomedically important

carbohydrates and glycoconjugates

• Functional studies of synthesized compounds (collaboration with

Ajit Varki, UCSD; David Smith/Richard Cummings, Emory U.)

• Crystal structure and mutagenesis studies of

glycosyltransferases and other carbohydrate biosynthetic

enzymes (collaboration with Andy Fisher, UCD)

• Understand enzyme catalytic mechanism

• Generate mutants for efficient chemoenzymatic synthesis

The Chen Glyco Group

Outline• Introduction: carbohydrates and sialic acids• One-pot three-enzyme chemoenzymatic

synthesis of sialosides• Controlled chemoenzymatic synthesis of size-

defined polysaccharides• Synthesis of macrocyclic carbohydrates of

different sizes• Future work and conclusion

GlycosylphosphatidylinositolGlycosylphosphatidylinositol(GPI) Anchor(GPI) Anchor

GlycosphingolipidGlycosphingolipid

Lipid bilayer

N-Glycan

O-Glycan

SialicSialic AcidsAcids

Occurrence of sialic acids on mammalian cell surface

Hemagglutininbinds sialic acid(receptor)

Neuraminidasecleaves sialic acid(enzyme)

RNA

Influenza virus

Influenza virus has two membrane-associated proteins: hemagglutinin and neuraminidase

OO

CO2-

AcHNHO

HOHO OH

Sialic acid(SA)

Host Cell

Life cycle of the influenza virus

Host Cell

Hemagglutininbinds sialic acid to initiate infection

Neuraminidase cleaves sialic acid and liberates new virus

Influenza virus

SA

SA

SASA

SASA

Membrane fusion and release of viral particle

Endocytosis

New viruses assemble at membrane

Replication

Tamiflu (Oseltamivis)Hoffmann-La Roche

OAcHN

NH2.H3PO4

CO2Et

O

OHOH

HOAcHN

HNNH2

+

H2N

CO2-

Relenza (Zanamivir)Glaxo Smithkline

O OR

R4OR5

R9OCOOOR7

R8OR4 = H or acetylR5 = NHAc, OH, NH2, or NH-glycolylR7 = H or acetylR8 = H, acetyl, sulfate, or methylR9 = H, acetyl, lactyl, or phosphate

_

Naturally occurring sialic acid modifications

Complex naturally occurring sialic acid structures

O COO

HONH

HO OHOHOH

HOO

O COO

HOHO

HO OHOHOH

KDN

Three basic forms of naturally occurring sialic acids

Neu5Ac Neu5Gc

__O COO

HONH

HO OHOHOH

O

_

1

6

5 42

3

79 8

Angata T., Varki A., Chem. Rev. 2002, 102, 439-469Schauer, R. Glycoconjugate J. 2000, 17, 485-499

O

HO

OHO

OH

ORO

-O2C

HOAcHN

HO OHHO

O

OH

OHHO

O

OR

O

-O2C

HOAcHN

HO OHHO

O

OH

AcHNHO

O

OR

O

-O2C

HOAcHN

HO OHHO

Siaα2,3GalβOR

Siaα2,6GalβOR Siaα2,6GalNAcαOR

O

HO

OHO

OH

ORO

-O2C

HOAcHN

HO OHO

Siaα2,8Siaα2,3GalβOR

αSia

Common naturally occurring terminal sialosides

Reducing endNon-reducing end

Information on the biological function of sialic acid modification is limited

• Regio- and stereo- selective chemical synthesis of sialosides poses challenge

• Difficult to isolate from natural source in pure forms– Low population– Labile to purification conditions

• How about enzymatic synthesis?

O CO2-

HOAcHN

HO OCMPOHOH

OHO

HNO

CO2-

HO OHOH

HOO

OH(NHAc)ORHO

OH

-O P O O

OHHO

N

NNH2

OO

O-

CMP

OH

O

OH(NHAc)ORHO

O

CMP-Neu5Ac

Neu5Ac-Gal(NAc)-OR

Gal(NAc)-OR

Biosynthesis of sialosides in bacteria

Sialyltransferase

Sialic acid aldolase

CMP-sialic acid synthetase

Neu5Ac synthase

O CO2-

HOAcHN

HO OHOHOH

CTP

PPi

Neu5Ac

OHOHO

NHAc

OH

HO

ManNAcPEP

Pi

Pyruvate

or

Pyruvate

O

HOHN

O

CO2-

HO OHOH

Neu5Ac-Gal(NAc)-OR

OH

O

OH(NHAc)ORHO

O

Modified sialosides

Acetyltransferases, Sulfotransferasesetc.

Yu H, Chen X. Org. Biomol. Chem. 2007, 5, 865-872.

Many sialic acid modifications are Post-Glycosylational Modifications (PGMs)

• Chemical synthesis: diverse

• Enzymatic synthesis

– high efficient

– stereo- and regio- specific

• Can be used for direct modifying cell surface, polysaccharides, and glycoconjugates.

Chemoenzymatic synthesis

Outline• Introduction: carbohydrates and sialic acids• One-pot three-enzyme chemoenzymatic

synthesis of sialosides• Controlled chemoenzymatic synthesis of size-

defined polysaccharides• Synthesis of macrocyclic carbohydrates of

different sizes• Conclusion and future work

Chemoenzymaticsynthesis of sialosidesusing bacterial enzymes

Finding right enzymes is the key for successful chemoenzymatic syntheses

• Obtainable: easy expression of active and soluble enzyme in high yield

• Flexible: can tolerate substrate modification

O CO2-

HOR2

R6 OCMPR4R5

OHO

R2

CO2-

R6 R4R5

HOO

OH(NHAc)ORHO

OH

CMPOH

O

OH(NHAc)ORHO

O

CMP-Neu5Ac

Neu5Ac-Gal(NAc)-OR

Gal(NAc)-OR

Sialyltransferase

Neu5Ac lyase

CMP-sialic acid synthetase

O CO2-

HOR2

R6 OHR4R5

CTP

PPi

Neu5Ac

OR4

HO

R2

OH

R6

Man(NAc) derivative

Pyruvate

Chemical modification OHO

HO

NHAc(OH)

OH

HO

Man(NAc)

OR2OHR6

R5

OH

NanA_E. coli_K12_C_His

21 3 4 550 kDa

37 kDa

25 kDa

Lanes: 1, Protein standards; 2, whole cells, before IPTG induction; 3, whole cells, after induction; 4, lysate; 5, Ni-NTA column purified protein.

E. coli sialic acid aldolase (lyase)

Flexible substrate specificity

CO2-

O

CO2-

O

OH

O CO2-

HOAcHN

HO OHOH

OHOHO

NHAcHO

HO Aldolase

H

OH

OAcHN

OHHO

OH

OH

AcHNOH

HOOH

-O2C

OH

O

123

123

78945

6

12

3

78

9

45

6

12

34

56123

4 56

ManNAc Neu5Ac

NeuA_Ecoli_K1_C_His

21 3 4 5

75 kDa

50 kDa

37 kDa

25 kDa

NeuA_Nm_C_His

21 3 4 5

75 kDa50 kDa37 kDa

25 kDa20 kDa

NeuA_SaV_C_His

21 3 4 5

75 kDa50 kDa37 kDa

25 kDa

A. B. C.

CMP-sialic acid synthetases (CSS)

Lanes: 1, Protein standards; 2, Whole cells, before IPTG induction; 3, Whole cells, after induction; 4, Lysate; 5, Ni-NTA column purified protein.

Yu H, et al. Bioorg. Med. Chem. 2004, 12, 6427-6435.Yu H, Ryan W, et al. Biotechnol. Lett. 2006, 28, 107-113.Lewis AL et al. J. Biol. Chem. 2007, 282, 27562-27571.

O CO2-

HOAcHN

HOOHOH

OH

O CO2-

HOAcHN

HO OCMPOHOHCTP PPi

CSS

Neu5Ac CMP-Neu5Ac

Pd2,6ST Photobacterium damsela α2,6-

sialyltransferase

Lanes: 1, Protein standards; 2, whole cells, before IPTG induction; 3, whole cells, after induction; 4, lysate; 5, Ni-NTA column purified protein.

Truncated N-His-tagged Δ15Pd2,6ST

Sun M, Li Y, et al. Biotechnol. Lett. 2008, 30, 671-676.

One-pot three-enzyme chemoenzymaticsynthesis of sialosides

Yu, H., Chokhawala H. A., Huang S., Chen X. Nature Protocols 2006, 1, 2485-2492

Yu H, Huang S, Chokhawala H, et al.Angew. Chem. Int. Ed. 2006, 45, 3938-3944.

One-pot three-enzyme chemoenzymaticsynthesis of α2,6-linked sialosides

O

OH

HOOH

OOO

HOOH

OH

O N3

OCO2

-

HOAcHN

O OHOH

O

OH

O

OH

HOOH

OOO

HOOH

OH

O N3

OCO2

-

HOHO

AcO OHOH

O

OH

HOOH

OOO

HOOH

OH

O N3

OCO2

-

HOAcO

AcO OHOH

OOH

HOOH

OOO

HOOH

OH

O N3

OCO2

-

HONH

AcO OHOH

HOO

O

OH

HOOH

OOO

HOOH

OH

O N3

OCO2

-

HOHO

HO OH

O

OH

HOOH

OOO

HOOH

OH

O N3

OCO2

-

HONH

HO OHOH

CbzHNO

O

OH

HOOH

OOMe

OCO2

-

HOHO

N3 OHOH

O

OH

HOOH

OOMe

OCO2

-

HON3

HO OHOH

O

OH

HOOH

O

OMe

OCO2

-

HOAcHN

N3 OHOH

OOH

HOOH

O

OMeO

CO2-

HONH

HO OHOH

N3O

OOH

HOOH

OOMe

OCO2

-

HONH

HO OHOH

OO

O

OH

HONHAc

O

O N3

OCO2

-

HOAcHN

HO OHOH

O

OH

HOAcHN

O

O N3

OCO2

-

HOAcHN

HO OHOH

OHO

OOH

O

OOHO

OH

OH

O N3

OCO2

-

HOAcHN

HO OHOH

O

-O2C

HOAcHN

HO OHOH

O

OH

HOOH

OOO

HONHAc

OH

OH

OCO2

-

HOAcHN

HO OHOH

OOH

HOOH

OOO

HOOH

OH

O N3

OCO2

-

HOAcO

HO OHHO

OOH

HOOH

OOO

HOOH

OH

O N3

OCO2

-

HONH

HO OHHO

HOO

O

OH

HOOH

OOO

HOOH

OH

O N3

OCO2

-

HOHO

HO OHHO

O

OH

HOOH

OOO

HOOH

OH

O N3

OCO2

-

HOAcHN

HO OHOH

O

OH

HOOH

OOO

HOOH

OH

O N3

OCO2

-

HOMeO

HO OHOH

O

OH

HOOH

OOO

HOOH

OH

O N3

OCO2

-

HONH

HO OHHO

AcOO

O

OH

HOOH

OOO

HOOH

OH

O N3

OCO2

-

HONH

HO OHOH

MeOO

O

OH

HOOH

OOO

HOOH

OH

O N3

OCO2

-

HOAcHN

AcO OHHO

Yields: 61-99%

Scale > 20 mg

OHOHO

OH

OH

HO

OHOHO

NHAc

OH

HO

OHOHO

HN

OH

HO OHO

OHOHO

OAc

OH

HO

OHOHO

OMe

OH

HO

OHOHO

HN

OH

HO OAcO

OHOHO

HN

OH

HO OMeO

OHOHO

NHAc

OH

AcO

Yu H, Huang S, Chokhawala H, et al.Angew. Chem. Int. Ed. 2006, 45, 3938-3944.

Pasteurella multocida sialyltransferase-1 Pm0188Ph (PmST1)

Truncated C-His-tagged Δ24PmST1

21 3 4 5

50 kDa37 kDa

75 kDa

25 kDa

Lanes: 1, Protein standards; 2, whole cells, before IPTG induction; 3, whole cells, after induction; 4, lysate, after induction; 5, Ni-NTA column purified protein.

Yu H, Chokhawala H, et al. J. Am. Chem. Soc. 2005, 127, 17618-17619.

OOH

OHO

HOOH

OOHHO

OHO

O

-O2C

HOHO

HO HO OH

OOH

OHO

HO

OOHHO

OHO

O N3O

-O2C

HOHO

HO HO OH

OOH

OHO

HO

OOHHO

OHO O

OO

O O

-O2C

NHHO

HO HO OH

O

OOH

OHO

HO

OOHHO

OHO O N3

O

-O2C

NHHO

HO HO OH

HOO

OOH

OHO

HO

OOHHO

OHO O

OO

O

-O2C

NHHO

HOHO OH

N3O

OOH

OHO

HOOMe

OOHHO

OHO

O

-O2C

NHHO

HO HO OH

N3O

O NO2O

OHHO

OOH

O

-O2C

HOHO

HO HO OH

OOH

OHO

HO

OOHHO

OHO OMe

O

-O2C

AcHNHO

N3HO OH

OOH

OHO

HO

OOHHO

OHO OMe

O

-O2C

HOHO

N3OHOH

OOH

OHO

HO

OOHHO

OHO O N3

O

-O2C

NHHO

HO HO OH

MeOO

OOH

OHO

HO

OOHHO

OHO

N3O

-O2C

NHHO

HO HO OH

HOO

65%

80%

90%

82%

80%

75%

75%

91%

88%

90%

81%

Yu H, Chokhawala H, et al.J. Am. Chem. Soc. 2005,127, 17618-17619.

One-pot three-enzyme chemoenzymaticsynthesis of α2,3-linked sialosides

OOH

OHO

HOOH

OOHHO

OHO

O

-O2C

AcHNHO

HO HO OH

OOH

OHO

HON3

OOHHO

OHO

O

-O2C

AcHNHO

HO HO OH

OOH

OHO

HO

OOHHO

OHO

O N3O

-O2C

AcHNHO

HO HO OH

OOH

OHO

HO

OOHHO

OHO O

OO

O

-O2C

AcHNHO

HO HO OH

OOH

OHO

HOOMe

OOHHO

OHO

O

-O2C

AcHNHO

HO HO OH

OMeO

OHHO

OHO

O

-O2C

AcHNHO

HO HO OH

N3OOHHO

OHO

O

-O2C

AcHNHO

HO HO OH

OO

OHHO

OHO

N3O

-O2C

AcHNHO

HO HO OH

OHOOHHO

OHO

O

-O2C

AcHNHO

HO HO OH

OOH

OHO

NHAcOH

OOHHO

OHO

O

-O2C

AcHNHO

HO HO OH

OMe

OOHHO

OHO

O

-O2C

AcHNHO

HO HO OH

92%

84%

91%

75%

79%

84%

82%

79%

76%

95%

62%

Yields 62-95%

Scale > 20 mg

What have we learned?

• Bacterial sialoside biosynthetic enzymes have flexible substrate specificity

• Chemoenzymatic method is a powerful approach to obtaining complex carbohydrates

• Can the chemoenzymatic method can be further applied for the novel synthesis?

Outline• Introduction: carbohydrates and sialic acids• One-pot three-enzyme chemoenzymatic

synthesis of sialosides• Controlled chemoenzymatic synthesis of size-

defined polysaccharides• Synthesis of macrocyclic carbohydrates of

different sizes• Conclusion and future work

OOHO2C

HOHO

OHO OH

O

OOH

OOHHO

HOO

HO

OOOH

HOHO

OH O

O CO2HHOO

HOOH

Some carbohydrate structures containing non-terminal KDN

A polysaccharide from Sinorhizobiumfredii SVQ293, a thiamine auxotrophicmutant of S. fredii HH103.

An oligosaccharide, a cell wall component of Streptomyces sp. MB-8 (Gram-positive bacterium)

A cell wall component of Streptomycessp. VKM Ac-2090 isolated from scab lesions of potato.

SourceRepeating Units

Proposed routes for the synthesis of polysaccharides containing internal sialic acids by sialyltransferase-catalyzed block transfer of

oligosaccharide repeating units

OO

OHOH

HOHO

HO

COONa

OOHHO

HOHO

CTP PPi

CMP-Sialic acid Synthetase

CMPα2,3/α2,6SiaT

OO

OHOH

HOHO

OCMPCOONa

OOHHO

HOHO

OO

HOHO

OH

OH

OOHHO

HOHO

COONa

Oaldolase

Galβ1-9KDNα2-3/α2-6Galβ1-9KDNα2,3/α2,6SiaTn

Galβ1-9KDNα2-3/α2-6

Galβ1-9KDN

Yu H, Chen X. Org. Lett. 2006, 8, 2393-2396

CO2-

O

CO2-

O

OH

O CO2-

HOAcHN

HO OHOH

OHOHO

NHAcHO

HO Aldolase

H

OH

OAcHN

OHHO

OH

OH

AcHNOH

HOOH

-O2C

OH

O

123

123

78945

6

12

3

78

9

45

6

12

34

56123

4 56

ManNAc Neu5Ac

Aldolase-Catalyzed Synthesis of Disaccharides Containing Sialic Acid at Reducing End

Hai

Aldolase-catalyzed synthesis of sialic acid-containing disaccharidesA general approach

Huang S, Yu H, Chen X. Angew. Chem. Int. Ed. Engl. 2007, 5, 2249-2253.

Entry Starting disaccharide Product Yield (%)

A Galα1,2Man Galα1,5KDN 0B Galβ1,2Man Galβ1,5KDN 0C Galα1,4Man Galα1,7KDN ~5D Galβ1,4Man Galβ1,7KDN 38E Glcβ1,4Man Glcβ1,7KDN 35F Galα1,5Man Galα1,5KDN 62G Galβ1,5Man Galβ1,8KDN 85H Galα1,6Man Galα1,9KDN 81I Manα1,6Man Manα1,9KDN 78J Glcα1,6Man Glcα1,9KDN 65K Glcβ1,6Man Glcβ1,9KDN 83L Galα1,2ManNGc Galα1,5Neu5Gc 36M Galβ1,2ManNGc Galβ1,5Neu5Gc 34

Proposed routes for the synthesis of polysaccharides containing internal sialic acids by sialyltransferase-catalyzed block transfer of

oligosaccharide repeating units

OO

OHOH

HOHO

OH

COONa

OOHHO

HOHO

CTP PPi

CMP-Sialic acid Synthetase

CMPα2,3/α2,6SiaT

OO

OHOH

HOHO

OCMPCOONa

OOHHO

HOHO

OO

HOHO

OH

OH

OOHHO

HOHO

COONa

Oaldolase

Galβ1-9KDNα2-3/α2-6Galβ1-9KDNα2,3/α2,6SiaTn

Galβ1-9KDNα2-3/α2-6

Galβ1-9KDN

Chemical synthesis

x?

Chemoenzymatic synthesis of CMP-activated disaccharide analog

OHO

HOHO

HNN3

O

OH

CTP, Mg2+O

HO OHOH

HN

O

OCMP

HO

CO2-

N3

O

CO2-

O

Aldolase, NmCSS

CuI, DIPEAH2O:CH3CN (1:1)83% overall yield

OOAc

OAcAcO

AcO

OO

OAc

OAcAcO

AcO

N NN

OHO OH

OH

NH

O

OCMP

HO

CO2-

CMP-(AcGal-Sia)

Muthana S, Yu H, Huang S, Chen X. J. Am. Chem. Soc. 2007, 129, 11918-11919.

CMP

OHO OH

OH

NHO

-O2C

HO

OO

OAc

OAcAcO

AcO

N NN O

O

O

OHHO

OH

OO

OAc

OAcAcO

AcO

N NN

OHO OH

OH

NHO

OCMP

HOCO2

-

OO

HO

OHHO

OH

OHO OH

OH

NHO

-O2C

HO

OO

OH

OHHO

HO

N NN O O

O

OHHO

OH

Pd2,6ST

Repeat

Deacetylation

CMP-(AcGal-Sia)

n = 0, 1, 2, 3 and 4

(HGal-Sia)-GalβOR

(AcGal-Sia)GalβOR

OHO OH

OH

NHO

-O2C

HO

OO

OH

OHHO

HO

N NN O

O

O

OHHO

OH

OHO OH

OH

NHO

-O2C

HON NN O

O

O

OHHO

OH

n(HGal-Sia)(Gal-Sia)n-GalβOR

Sialyltransferase-Catalyzed Block Transfer of Disaccharide Analogs

Muthana S, Yu H, Huang S, Chen X.J. Am. Chem. Soc.2007, 129, 11918-11919.

Outline• Introduction: carbohydrates and sialic acids• One-pot three-enzyme chemoenzymatic

synthesis of sialosides• Controlled chemoenzymatic synthesis of size-

defined polysaccharides• Synthesis of macrocyclic carbohydrates of

different sizes• Conclusion and future work

Outline• Introduction: carbohydrates and sialic acids• One-pot three-enzyme chemoenzymatic

synthesis of sialosides• Controlled chemoenzymatic synthesis of size-

defined polysaccharides• Synthesis of macrocyclic carbohydrates of

different sizes• Conclusion and future work

Conclusion

• Bacterial sialoside biosynthetic enzymes have flexible substrate specificity and are important tools in synthesizing complex carbohydrates

• Novel chemoenzymatic method has been developed for the controlled synthesis of polysaccharides and macrocyclic carbohydrates of different sizes

Future work

• Synthesize naturally occurring polysaccharides as vaccine candidates using chemically synthesized CMP-activated disaccharides

• Synthesize macrocyclic carbohydrates of larger size

• Introducing functional groups as chemical handle into macrocyclic structures for further conjugation

• Function studies of macrocyclic carbohydrate

Saddam MuthanaDr. Hai Yu Harshal ChokhawalaShengshu HuangDr. Hongzhi Cao

AcknowledgementsFinancial Supports:NSF CAREER 0548235Mizutani foundation for glycoscienceThe Arnold and Mabel Beckman FoundationNIH – R01GM076360