1. Reyan Coskun1, Regis Saliba1, Nicola L.B. Pohl1
1Indiana University, Department of Chemistry, Bloomington, IN 47405, USA
Toward Efficient Chemoenzymatic Syntheses of Sialyl-α-2,3-Lactose/Lactosamine
Assisted by a Fluorous-Tag Purification
Retrosynthesis
Fluorous Tag Synthesis
O
OHHO
O
OH
O O
OH
HO
NHAc
O
HOOC
HO
AcHN
HO
HO HO
OLinker
Enzyme
O
COOH
OH
Enzymatic
O
COOH
OH
OH+CTP
OCMP + O O
O OLinker
NHAc
OH
HO
HO
OH
OH
Galactoycation/
Deprotection
OOHO
OLinker
NHAc
OAc
AcO AcO
AcO
OAc
OH
OAc
OLG
+
NHAc
AcHN
HO
HO OH
HO
HO OH
Lac-R SLac-R
Neu5Ac
CTP
PmST
NmCSS
(NH4)2CO3 100 mM
pH = 8.5
37oC
Entry R Changed Conditions Conversion (by
mass)?
1 Buffering (pH 8.5) 0%
2 Triton-X
DMSO
0%
4 No Linker 100%
O
O
C8F17
O
O
C8F17
O Ph
OH I C8F17+
AIBN
55%
C8F17 OH
I EtO2
LiAlH4
C8F17 OH
C8F17 OMs O OHC8F17
MsCl
NEt3
CH2Cl2
KOH
Bu4NBr
DMF
73% 98%
72%
GluNAc Synthesis
O
PivO
NHAc
O
O
C8F17
HO
OPiv
O
OAc
AcO
AcO
AcO
O CCl3
NH
BF3.OEt2
DCM
+
28%
O
OAcAcO
OAc
O O
OPiv
PivO
NHAc
O
C8F17
AcO
Na
MeOH
O
OHOH
OH
O O
OPiv
PivO
NHAc
O
C8F17
HO
53%
O
OH
HO
HO
ClH.H2N
OH NaOH
O
OH
HO
HO
N
OH
OMe
O
OMe
in water
O
OAc
AcO
AcO
N
OAc
Ac2O
OMe
O
HCl
O
OAc
AcO
AcO
NH2HCl
OAc
AcCl
NET3
O
OAc
AcO
AcO
NHAc
OAc
Linker
Yb(OTF)3
DCM, ∆
O
OAc
AcO
AcO
NHAc
OLinkerDCM
Na
MeOH
O
OH
HO
HO
NHAc
OLinker
O
OH
HO
AcO
NHAc
OLinker
85%
pyridine
81%
83%
83% 30%
2 steps
O
OPiv
HO
PivO
NHAc
OLinker
1)(Bu)2Si(OTF)3, DMF, 32%
2) Ac2O, pyridine, 29%
3) NEt3.3HF, THF, 18%
(Bu)2Si(OTF)3
DCM
32%
pyridine
O
OAc
HO
AcO
NHAc
OLinker
Ph3Bi(OTF)2
DCM
+ O
OAc
OAc
AcO
AcO SET
O O
O OLinker
NHAc
OH
AcOAcO
HO
O OO OLinker
NHAc
OH
AcOHO
HO
TRIS.HCl
pH 8.8
37°C
OH
OH
OH
OH
O
OHHO
O
OH
O O
OH
HO
NHAc
O
HOOC
HO
AcHN
HO
HO HO
OLinker
Na
MeOH
NMCSS
PmST
MgCl2
α-2,3 SLacNAc Synthesis
LacNAc Synthesis Optimization
O
OHHO
O
OH
O O
OH
HO
NHAc
OHO
HOOC
HO
AcHN
HO
HO HO
O
HO
OH
O O
OH
HO
NHAc
OH
OO
HOOC
HO
AcHN
HO
HO HO
HO
α-2,3-linkage
α-2,6-linkage
Background
ConclusionReferences
α-2,3 and α-2,6-linkage SLacNAc in H2
HA Human Receptors
Future Directions
(1)
(2)
α-2,3 (1) and α-2,6 (2) linkage SLacNAc
Influenza A virus subtype H3N2 has been the cause of a type 2
pandemic (Hong Kong flu, 1968-1969), and as a seasonal
influenza, kills over 36,000 people in the U.S. every year (Lin, Y.
P., et al, 2012). After passing from pigs to birds, the transfer of the
virus from avian to human represents a critical infection period.
H3N2 is passed on from birds to humans when influenza
hemagglutinin’s binding preference mutates from sialyl-α-2,3-
lactosamine (SLacNAc)-receptors found in avian intestinal tracks
to sialyl-α-2,6-lactosamine-receptors found in human airway
epithelia. Presently, it has been observed that the virus mutates
based on the linkage present. Thus, very little is known about how
this change happens. Synthesizing the α-2,3-SLacNAc found in
birds could help elucidate the particularities of the virus’s starting
mutation, and on the particularities of this specific linkage to
preventing the influenza from transmission across species.
(1) Chen, G.-S. and N. L. Pohl (2008). "Synthesis of Fluorous Tags for Incorporation of Reducing Sugars into a Quantitative Microarray Platform." Organic
Letters 10(5): 785-788.
(2) Myszka, H., et al. (2003). "Synthesis and induction of apoptosis in B cell chronic leukemia by diosgenyl 2-amino-2-deoxy-beta-D-glucopyranoside
hydrochloride and its derivatives." Carbohydrate Research 338(2): 133-141.
(3) Liu, J., et al. (2009). "Structures of receptor complexes formed by hemagglutinins from the Asian Influenza pandemic of 1957." Proceedings of the National
Academy of Sciences 106(40): 17175-17180.
(4) Lin, Y. P., et al. (2012). "Evolution of the receptor binding properties of the influenza A(H3N2) hemagglutinin." Proceedings of the National Academy of
Sciences 109(52): 21474-21479.
(5) Stencel-Baerenwald, J. E., et al. (2014). "The sweet spot: defining virus-sialic acid interactions." Nat Rev Micro 12(11): 739-749.
α-2,3 and α-2,6-linked SLacNAc interacts with avian H2 hemagglutinin receptors (trans
conformation). (A) A/dk/Ontario/77 H2 HA, in complex with α-2,3-linked SLacNAc. (B)
A/Singapore/1/57 H2 HA, in complex with α-2,6-linked SLacNAc.
• Sialylation is chemically challenging. Enzymatically, the
sialylation is a more preferable reaction.
• But through enzymatic glycosylation, there are many other
compounds that are difficult to purify.
• The fluorous linker, using FSPE techniques and affinity
chromatography, can be purified easier because only
compounds with the fluorous tag (just the target sugar)
passes through.
Proc Natl Acad Sci U S A. 2009 Oct 6; 106(40): 17175–17180.
The initial SLacNac synthesis saw little conversion by mass from LacNAc to SLacNAc
maybe due to the fluorous tag incompatibility and insolubility. After trying different R groups
and conditions, future directions involve further spacer additions and smaller fluorous tags.
Current results showed that the CMP-Neu5Ac synthetase’s (CSS) sialylation of LacNAc +
the C8F17 linker with Neu5Ac did not proceeded. The main observed problem was the
potential LacNAc + C8F17 linker’s solubility and pH incompatibly with enzymatic
glycosylation. Overall, future trials involve optimization for these conditions and running the
reactions on an automated platform.
(1)
(2)