The Chen Glyco Group
Transcript of The Chen Glyco Group
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