novel carbohydrate-based ligands with immunostimulatory properties
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NOVEL CARBOHYDRATE-BASED LIGANDS WITH IMMUNOSTIMULATORY
PROPERTIESCECILIA H MARZABADI, VIKRAM BASAVA AND CONSTANTINE BITSAKTSIS
SETON HALL UNIVERSITYSOUTH ORANGE, NJ USA
2
INTRODUCTION
• THE ZWITTERIONIC POLYSACCHARIDE, PSA1, ISOLATED FROM THE CAPSULE OF BACTEROIDES FRAGILIS, HAS BEEN SHOWN TO STIMULATE BOTH INNATE AND ADAPTIVE IMMUNE RESPONSES IN MAMMALS AND IS BELIEVED TO BE IMPORTANT IN ESTABLISHING A BALANCED IMMUNE SYSTEM WHICH IS MEDIATED BY T-LYMPHOCYTES.
O
O
O
OO
OO
CH3
CH3NH3+
OH
O-
O
OH
NH
OHNHO
OOH
OHOH
OH H
OHAc
Ac
1
3
INTRODUCTION
WORK IN OUR LABORATORY INVOLVES THE SYNTHESIS OF SIMPLER ZWITTERIONIC SACCHARIDE ANALOGUES FOR EVALUATION AS POTENTIAL STIMULANTS OF INNATE AND/OR ADAPTIVE IMMUNITY.
RECENTLY, ONE SUCH ANALOG, 2, HAS BEEN SYNTHESIZED AND HAS BEEN SHOWN TO POSSESS IMMUNOSTIMULATORY PROPERTIES.
O
O
O
O
OO
CH3
CH3
NH3+
OH
O-
O
OH
NHAc
OH
2
4
O
OAc
AcOAcO
O
OH
HOHO
O
OTs
HOHO
O
OTs
AcOAcO
OHOHO
OHO
O
O
O
Br
O
O
N
O
O
O
O
H2N
NaOMe
CH3OH
S ClOO
NO O
ON
NN
LiAlH4
O
CH3COOH
O
NH
Cr O
O
O
O Cr O
O
O
O
N
O
O
IP
NaI; Cs2CO3
NH
O
O
1. NH2NH2. H2O CH3OH
2. 5% HCl
X
LiAlH4 OX
Synthesis of Subunit A: (Proposed Scheme)
SYNTHETIC STUDIES
5
OTsO
HOHO
O
O
OHH
6 eq LiAlH4 / THF
75 oC overnight
82 %
ORTEP representation of the anhydride 3,6-Anhydride molecules showing intermolecular H-bonding
SYNTHETIC STUDIES
6
O
O
OHH
O
O
OHO
I
O
O
OHO
IO
O
O
HO
I+ +
N
O
O
I
CH3OH; CH3CN
O
O
OHH
O
O
OHHO
I
O
O
OHHO
IO
O
OH
HO
I
++
N
O
O
I
CH3CN
OI
O
O
+
OOHO
OO O 48%
O
O
OHHN
O
O
I
CH3CNOI
O
O4Ao sieves
76%
SYNTHETIC STUDIESReactivity of 3,6-Anhydro-D-glucal:
7
O
O
OHH
O
O
OH
S
O
O
OH
SO
HO
HOS
OHO
HO
S
+ + +Ce(NH4)2(NO3)6
SH
CH3CN
O
O
OH
S
O
O
OH
SO
HO
HO
S
+ +
Ce(NH4)2(NO3)6
SH
CH3CN
NaI
Reactivity of 3,6-Anhydro-D-glucal:
SYNTHETIC STUDIES
8
9
SYNTHETIC STUDIES
O
OH
HOHO
NH2.HCl
OHO
OH
HOHO
NH2
OH
O
OH
HOHO
NH
OH
O COOH
O
OAc
AcOAcO
N
OAc
O O
O
OAc
AcOAcO
N
S
O O
NaOCH3
CH3OH
O
O
O
(CH3CO)2O
N
SH
BF3. Et2O
anh CH2Cl2A
Synthesis of Subunit A:
10
SYNTHETIC STUDIESSynthesis of Subunit B:
O
OH
Si
HO
OH
SiCl
N
NH
anhNH
O
OHO
Si
B
11
SYNTHETIC STUDIESSynthesis of Subunit C:
O
OAc OAc
AcO
OAc
OAc
O
OAc OAc
AcO
OAc
S
BF3. Et2O
SH
O
OH OH
HO
OH
SNaOCH3, CH3OH
O
OO
HO
OH
S
H
H
O
ZnCl2
Cl
O
O
OO
O
OH
S
H
O
C6H6; reflux; 4Ao sieves
Sn
OO
OO
HO
O
S
H
O
(major) (minor)
12
SYNTHETIC STUDIESSynthesis of Subunit C (cont):
C
O
OO
O
O
S
H
O
O
O
OO
O
O
S
O
O
O
O
1. 20% CF3COOH; CH2Cl2
O
O
O
2. BF3. Et2O; CH3CN
O
OO
O
OH
S
H
O CH3COCl
N
N
N
13
SYNTHETIC STUDIESCoupling of Subunits A and B:
O
OH
Si
O
OAc
AcOAcO
N
O
O O OSi
O
OAc
AcOAcO
N
O
O O OH
O
OAc
AcOAcO
NO O
S-78oC
SiO
SO O
F F
F
SiO
SO O
F F
F
1.5h
-78oC to 0oC
7h
N
F
O
B A
A+B
N
O
O
I
N
O
O
I
14
O
OAc
AcOAcO
N
O
O O OH
A+B
O
OO
O
O
S
O
O
O
O
O
O
OO
N
OO
OO
O
O
O
O
O
OO
OO
OO
O
anh CH2Cl2
- 40 o
C
N
O
O
I
SiO
SO O
F F
F
C
PS A1 analogue
SYNTHETIC STUDIESCoupling of Subunit A+B with C:
15
BIOLOGICAL EVALUATION
• PROTECTED ANALOG 2 WAS EVALUATED IN ELISA ASSAYS FOR THE PRODUCTION OF THE PROINFLAMMATORY CYTOKINES TNF-A AND IL-6 USING RAW 264.7 AND MOUSE PERITONEAL MACROPHAGES (C57 BL/6, JACKSON)
Innate Immunity:
16
BIOLOGICAL EVALUATION
TNF-A 24hrs
RAWPEC
0
500
1000
1500015LPS***
** ***[T
NF-
] (pg
/ml)
Innate Immunity:
17
BIOLOGICAL EVALUATION
IL-6Innate Immunity:
24hrs
050
100150200250 0
15LPS
N.S.
N.S.N.S.
N.S.
[IL-6
] (pg
/ml)
48hrs
RAWPEC
0100200300400500
015LPS**
****[IL
-6] (
pg/m
l)
18
BIOLOGICAL EVALUATION
• THE MOLECULE SYNTHESIZED WAS ALSO TESTED IN SPLEEN CELLS FROM THE SAME MICE LOOKING FOR T AND B CELL PROLIFERATION, LEVELS OF IFN-G IN THE SUPERNATANTS (INDICATIVE OF A T CELL RESPONSE), AND POLYCLONAL IGM AND IGG LEVELS IN THE SUPERNATANTS L (INDICATIVE OF A B CELL RESPONSE) WERE MEASURED.
• NO T CELL OR B CELL RESPONSES WERE OBSERVED.
Adaptive Immunity:
19
BIOLOGICAL EVALUATION
TO ELUCIDATE THE BACTERIAL TOLL LIKE RECEPTOR RESPONSIBLE FOR CYTOKINE PRODUCTION, THE PERITONEAL MACROPHAGES FROM TLR2 AND TLR4 KNOCKOUT MICE (FEMALE, 4-6 WEEK OLD C57BL6) WERE DOSED WITH COMPOUND 2 AND, TNF-A AND IL6 LEVELS WERE MEASURED OVERTIME IN THE SUPERNATENTS.
Innate Immunity: TLR2 vs TLR4 Ligand
20
BIOLOGICAL EVALUATIONTLR KO MICE: TNF- Production2 hrs
WT
TLR-2ko
TLR-4ko
0
100
200
300
400 015LPS
*****
[TN
F-
] (pg
/ml)
24 hrs
WT
TLR-2ko
TLR-4ko
0
200
400
600 015LPS
****
[TN
F-
] (pg
/ml)
21
BIOLOGICAL EVALUATIONTLR KO MICE: IL-6 Production24hrs
WT
TLR-2ko
TLR-4ko
0
1000
2000
3000 015LPS
****
[IL-6
] (pg
/ml)
48hrs
WT
TLR-2ko
TLR-4ko
0
1000
2000
3000 015LPS
****
[IL-6
] (pg
/ml)
22
THE EXTENT OF BINDING OF ANALOGUE 2 TO THE TLR-4 RECEPTORS WAS ILLUSTRATED BY TREATING THE PERITONEAL EXUDATE CELLS (PEC) WITH DIFFERENT CONCENTRATIONS OF THE COMPOUND. THE CELLS WERE PRE-INCUBATED WITH THE COMPOUND, AFTER WHICH WERE WASHED. TO THE WASHED CELLS WAS ADDED THE ANTIBODY FOLLOWED BY THE PHYTOERYTHRIN DYE AND, THE NUMBER OF THE COLOURED CELLS WAS OBSERVED BY FLOW CYTOMETRY.
TLR-4 EXPRESSION: Flow CytometryBIOLOGICAL EVALUATION
23
BIOLOGICAL EVALUATIONTLR-4 EXPRESSION: Flow Cytometry
TLR-4
24
PECs
contro
lg/m
l
1g/m
l
5g/m
l
10
0
20
40
60
80N.S.***
% T
LR-4
exp
ress
ion
BIOLOGICAL EVALUATIONTLR-4 EXPRESSION: Flow Cytometry
25
BIOLOGICAL EVALUATIONTLR-2 EXPRESSION: Flow Cytometry
26
BIOLOGICAL EVALUATION
• A REDUCTION IN THE PRODUCTION OF BOTH TNF-A AND IL-6 WAS OBSERVED IN ONLY THE TLR4 KNOCKOUT MICE SUGGESTING THAT SIGNALING OF 2 OCCURS THROUGH TLR4. FURTHERMORE, THE MAGNITUDE OF TLR4 SIGNALING WITH 2 WAS GREATER THAN THE KNOWN TLR4 AGONIST, E. COLI LPS (LIPOPOLYSACCHARIDE)
SUMMARY: TLR KO MICE
27
CONCLUSIONS
• THE DESIRED ZWITTERIONIC POLYSACCHARIDE, AN ANALOGUE OF PSA1, WAS SUCCESSFULLY SYNTHESIZED WITH BOTH AMINO AND CARBOXYLATE MOIETIES
• THE BIOLOGICAL EVALUATION CLEARLY DENOTES THAT THE ANALOGUE 2 SHOWS IMMUNOSTIMULATORY PROPERTIES
• ANALOGUE 2 WAS FOUND TO STIMULATE THE INNATE IMMUNE SYSTEM, AND NOT THE ADAPTIVE IMMUNE SYSTEM
• AN UNEXPECTED AND AN EFFICIENT SYNTHESIS OF 3, 6-ANHYDRO-D-GLUCAL PAVES A ROUTE TO THE SYNTHESIS OF INTERESTING NEW CARBOHYDRATE MOIETIES
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