Controlled Chemoenzymatic Synthesis of Size-Defined Polysaccharides and Macrocyclic Carbohydrates Xi Chen, Department of Chemistry, UC Davis 1 st Annual Workshop of the 10+10 Alliance Peking University, Peking, China Monday, 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
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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
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