Total Synthesis of the Congested, Bisphosphorylated Morganella morganii Zwitterionic Trisaccharide Repeating Unit

Article abstract of DOI:10.1021/jacs.9b06830

Zwitterionic polysaccharides (ZPSs) activate T-cell-dependent immune responses by major histocompatibility complex class II presentation. Herein, we report the first synthesis of a Morganella morganii ZPS repeating unit as an enabling tool in the synthesis of novel ZPS materials. The repeating unit incorporates a 1,2-cis-α-glycosidic bond; the problematic 1,2-trans-galactosidic bond, Gal-β-(1 → 3)-GalNAc; and phosphoglycerol and phosphocholine residues which have not been previously observed together as functional groups on the same oligosaccharide. The successful third-generation approach leverages a first in class glycosylation of a phosphoglycerol-functionalized acceptor. To install the phosphocholine unit, a highly effective phosphocholine donor was synthesized.

Full text of DOI:10.1021/jacs.9b06830

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Article
Total Synthesis of the Congested, Bis-Phosphorylated
Morganella Morganii Zwitterionic Trisaccharide Repeating Unit
D. Jamin Keith, and Steven D. Townsend
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b06830 • Publication Date (Web): 22 Jul 2019
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Total Synthesis of the Congested, Bis-Phosphorylated Morganella Mor-
ganii Zwitterionic Trisaccharide Repeating Unit
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D. Jamin Keith and Steven D. Townsend*
Department of Chemistry, Vanderbilt University, Nashville, TN, 37235
ABSTRACT: Zwitterionic polysaccharides (ZPS) activate T-cell-dependent immune responses by major histocompatibility complex
class II presentation. Herein, we report the first synthesis of a Morganella morganii ZPS repeating unit as an enabling tool in the
synthesis of novel ZPS materials. The repeating unit incorporates a 1,2-cis-a-glycosidic bond; the problematic 1,2-trans-galactosidic
bond, Gal-β(1→3)-GalNAc; and phosphoglycerol and phosphocholine residues which have not been previously observed together as
functional groups on the same oligosaccharide. The successful third generation approach leverages a first in class glycosylation of a
phosphoglycerol functionalized acceptor. To install the phosphocholine unit, a highly effective phosphocholine donor was synthe-
sized.
(a) Carbohydrates bind B cell receptors, initiating a weak T cell-independent response.
INTRODUCTION
carbohydrate
The capsule is a protective structure on the surface of a
number of microbes. Its surface is coated with capsular poly-
saccharides (CPSs), incorporating residues that differ signifi-
cantly in structure from those of the mammalian glycome.1-2
Due to their incongruity, CPSs are ligands for the human im-
mune system.3-4 CPSs are antigens that work by activating B
cells through cross-linking of cell surface receptors (Figure
1A). These B cells are partially activated and differentiate into
plasma B cells that produce low-affinity IgM antibodies. In the
absence of external mediators, only a T-cell independent hu-
moral response is induced.
antigen
T cell -
independent
pathway
IgM
plasma
B cell
B cell
plasma cells produce
short-lived low affinity IgM
(b) Zwitterionic polysaccharides induce a robust T cell-dependent response.
Th receptor
ZPS
CTLs
B cell
Th cell
APC
Unlike other carbohydrate antigens, zwitterionic polysac-
charides (ZPS) possess an alternating charge motif and elicit a
T-cell dependent response from the immune system (Figure
1B).5-6 Mechanistically, an antigen-presenting cell (APC) con-
sumes and lyses the ZPS. Next, antigenic fragments are loaded
onto major histocompatibility class II (MHC-II) receptors. The
APC then presents the resulting antigen/MHC-II complex on
the cell surface. Helper T cells (Th cells) bind to the anti-
gen/MHC-II complex through the T-cell receptor (TCR), initi-
ating cytokine release from the Th cell which activates cytotoxic
T cells (CTLs). B cells are also activated. In a parallel pathway,
activated Th cells bind to antigen bound B cells to produce cy-
tokines that fully activate B cells. This action results in immu-
noglobulin class switching and affinity maturation to produce
high affinity IgG antibodies and memory B cells. Memory B cell
formation is central to a long-lasting immune response.
MHC-II
complex
cytokines
T cell -
dependent
pathway
plasma
cell
memory
B cell
+
B cell
MHC-II
IgM
IgG
Th cell
(c) Structures of representative ZPSs.
H3N
CH3
O
OH
O
HO
HO
H3C
O
AcHN
OH
O
COO
OH
O
OH
HO
O
OH
O
O
O
O
O
O
H3N
O
O
O
O
O
HO
Due to their immuno-stimulatory properties, ZPSs have
been studied in a number of immunology settings. For example,
ZPSs function as adjuvants when co-administered with poorly
immunogenic antigens. In studies by the Wack group, ZPSs co-
administered with the tetanus toxoid antigen were observed to
increase antibody titers in vivo.7
P
O
AcHN
O
NHAc
O
P
O
O
H3C
O
O
HO
HO
N
HO
H3C
OH
O
CH3
O
PS-A1 (1)
MM-ZPS (2)
Figure 1. (a)/(b) Immunological response to carbohydrate anti-
gens. (c) Structures of PS-A1 (1) and MM-ZPS (2).
1
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The most common use of ZPSs are in cancer-immunother-
Page 2 of 8
O
CH3
CH3
O
BnO
1
2
3
4
5
6
7
8
P
O
apy as substitutes for carrier proteins in vaccine development.
ZPSs offer several advantages over carrier proteins such key-
hole limpet hemocyanin (KLH).8-13 Carrier protein-antigen con-
jugates are difficult to synthesize and characterize. They also
elicit low levels of IgG antibodies in patients.14-15 It is believed
this profile is due to the strong immune response elicited by car-
rier proteins themselves and their inadvertent suppression of
immune responses to the antigen.16 The most well-characterized
ZPS is PS-A1 (1), a Bacteroides fragilis CPS (Figure 1C).17
Foundational work by the Andreana lab has shown that, when
administered to rodents, PS-A1 conjugates to tumor-associated
carbohydrate antigens (TACAs) elicit high antibody titers.18-21
Moreover, the antibodies are highly selective for their respec-
tive antigen. The Seeberger and Andreana labs have inde-
pendently completed total syntheses of PS-A1 to better charac-
terize its immunostimulatory properties.22-23
6
NiPr2
ii
OH
*
RO
RO
OR
O
O
OH
O
O
OH
HO
HO
OH
HO
P
*
RO
HO
OR
O
iii
OH
OR
O
OH
O
N3
O
O
O
O
H3
N
OCH3
NHAc
i
O
O
R2N
5
CH3
O
OR
H3
H3
C
O
P
7
H3
C
C
O
O
N
O
P
C
N
iv
*
H3
O
X
CH3
O
X
4
PgO
MM-ZPS target repeating unit (3)
Figure 2. First-generation analysis.
9
The synthesis started from Schmidt trichloroacetimidate
8,35 prepared in seven steps from D-galactose (Scheme 1A).
BF3•Et2O mediated glycosylation with known 6-O-TBS galac-
tal acceptor36 9 proceeded selectively at C3 to provide α-10 in
65% yield as the major product. The high a-selectivity can be
attributed to solvent participation in a b-fashion by diisopropyl
ether.37 To avoid temporarily protecting the C3’ position, we
installed the phosphoglycerol unit using phosphoramidite 6.38-40
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Recently, we took note of the Morganii morganella ZPS
(MM-ZPS, 2).24 In vitro binding assays revealed that 2 engages
the MHCII in a manner competitive with PS-A1. Moreover, cel-
lular studies suggest that 2 activates CD4+ T-cells. Antibody
binding assays of hydrolyzed fragments and oligomers of the
repeating unit revealed that the phosphocholine functional
group was the dominant element of the epitope. Isolating MM-
ZPS from the microbial cell surface for further evaluation is
challenged by two problems. First, extraction and purification
are complex and result in the isolation of small, impure batches.
Second, the isolation process can alter the ZPS. Thus, there is
an unmet need to synthesize structurally defined antigens.
While it is general practice in the field to synthesize repeating
units that are ready for chemical polymerization, our strategy
differs in that we plan to use the repeating unit as ink to “print”
unnatural ZPS nanomaterials.25 Indeed, three-dimensional (3D)
printing is a frontier area of nanoscience due to its ability to
produce 3D objects in advanced applications in areas ranging
from tissue engineering to biomaterials. We hypothesize that
nanoprinting can be used to synthesize ZPS materials, in the ab-
sence of traditional conjugation handles, through deposition of
the ZPS onto polyelectrolyte (PE) complexes with nanometer
precision in all dimensions. Described herein is the first step in
that process, the total synthesis of an MM-ZPS trisaccharide.
Scheme 1. (a) First-generation approach, (b) Alternative donor
leading to undesired orthoester product.
(a) First-generation approach
BnO
BnO
OBn
O
OBn
O
BnO
BnO
OTBS
O
HO
HO
a. BF3•Et2O, iPr2O
OTBS
O
O
CCl3
+
HO
N3
N3
65% α-only
NH
O
[gram-scale]
8
9
10
7 steps
from D-Gal
4 steps
from D-Gal
b. 6, Tetrazole
then
mCPBA
87%
[gram-scale]
O
O
CH3
CH3
CH3
O
CH3
O
O
OBn
O
BnO
BnO
O
O
OBn
O
BnO
BnO
O
P
P
OBn
OTBS
O
c. DMDO
OBn
OTBS
O
O
O
N3
N3
O
O
11
O
12
O
CH3
CH3
Ph
O
O
Ph
O
O
OBn
O
O
OBn
OTBS
BnO
BnO
P
O
O
O
d. ZnCl2
HO
O
O
O
AcHN
N3
OiPr
O
O
13
OH
AcHN
RESULTS AND DISCUSSION
OiPr
14
At the planning stage, we were drawn to repeating unit 3
which features a tetra-substituted galactose residue housing
three of the repeating unit’s four charges (Figure 2). This mol-
ecule also contains two sites of phosphorylation, including a
phosphocholine residue which is rarely observed on carbohy-
drates.26 Each residue is galactose derived and one of the glyco-
sidic bonds is a Gal-β-(1→3)-GalNAc linkage. Regardless of
conditions employed, installing this 1,2-trans-galactosyl bond
is regularly accompanied by orthoester formation (when partic-
ipating groups are located at C2).27-32 Based on these consider-
ations, our first-generation approach focused on stepwise func-
tionalization of a central epoxygalactal residue 5. After forming
the 1,2-cis-a-glycosidic bond (GalN3-α-(1→3)-Gal) (step i), we
planned to install the phosphoglycerol arm using phospho-
ramidite 6 (step ii). Installation of the 1,2-trans-b-glycosidic
bond and second oxygen-phosphorus bond would hinge on ring
opening of the epoxide (steps iii and iv, respectively).33-34
(b) Orthoester formation
O
CH3
CH3
O
i) DMDO
O
OBn
O
OBn
BnO
BnO
e.
11
ii) EtSH, TFAA
f. 11, NIS/TMSOTf
P
O
OTBS
O
iii) Ac2O
O
57% over 3 steps
N3
SEt
O
[gram-scale]
OAc
15
O
CH3
CH3
O
O
O
OBn
O
O
O
BnO
BnO
CH3
not observed
desired
OBn
P
O
CH3
Ph
OTBS
O
O
O
g. TfOH or TMSOTf
OBn
O
BnO
N3
OBn
P
O
O
O
OTBS
O
O
O
Ph
observed
undesired
BnO
O
O
N3
H3C
O
O
O
O
O
OH
AcHN
OiPr
16
14
O
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Reagents and conditions: (a) BF3·Et2O, 4:1 iPr2O/CH2Cl2, -40
of the galactosamine acceptor as an azide.27, 50 We incorporated
this maneuver into a second-generation approach targeting 3
(Figure 3). The plan called for Gal-β-(1→3)-GalNAc glyco-
sidic bond formation early in the synthesis (step i). Following
manipulation of the azide (step ii) we would install the a-gly-
cosidic bond (step iii). The final steps would involve installation
of the phosphoglycerol (step iv) and phosphocholine (step v)
arms. Installing the phosphoglycerol unit in the latter-stages of
the synthesis would minimize the number of subsequent steps
requiring handling and characterization of phosphorus epimers.
1
2
3
4
5
6
7
8
°C→ 25 °C, 1 h, 4 Å MS, 65% α-only; (b) 6 (1.5 equiv), 1H-
tetrazole (1.5 equiv), CH3CN, 0 °C → 25 °C, 0.5 h then mCPBA
(1.5 equiv), CH2Cl2, -78 °C → 0 °C, 2 h, 87%; (c) DMDO (1.2
equiv), CH2Cl2, -78 °C → 0 °C, 1 h, then 13 (1.2 equiv), ZnCl2
(2.5 equiv), -78 °C → 25 °C, >3 days, <5%; (d) DMDO (1.2
equiv), CH2Cl2, 0 °C, 20 min, then EtSH (28 equiv), TFAA (0.1
equiv), CH2Cl2, -78 °C → 0 °C, 1 h then Ac2O (10 equiv), Et3N
(10 equiv), DMAP (0.1 equiv) CH2Cl2, 0 °C, 10 h, 57% over 3
steps; (e) 13 (2.5 equiv), NIS (1.2 equiv) TMSOTf (0.15 equiv),
CH2Cl2, -78 °C → 25 °C, 2.5 h, <5%; (f) TMSOTf (0.15 equiv)
or TfOH (0.15), CH2Cl2, -78 °C→ 25 °C, 2.5 h.
9
O
10
11
12
13
14
15
16
17
18
19
20
21
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23
24
25
26
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CH3
O
BnO
CH3
P
O
BnO
BnO
OBn
O
NiPr2
6
iv
Subsequent oxidation of the intermediate phosphorus (III)
species with mCPBA provided phosphate ester 11 in 87% yield
as an inseparable mixture of phosphorus epimers. Next, epoxi-
dation of the glycal with DMDO gave intermediate epoxide
12.34 Unfortunately, ring opening the epoxide was intractable.
To circumvent this issue, glycal 11 was converted to thioglyco-
side donor 15 using a sequence of epoxidation, thiylation, and
acylation of the resultant C2’ alcohol (Scheme 1B).41 Unfortu-
nately, glycosylation of 15 with acceptor 13 provided orthoester
16 instead of glycoside 14. Based on literature precedence, we
hypothesized the orthoacetate could be rearranged to the desired
glycoside under Kochetkov conditions.42-43 However, exposing
16 to acidic media resulted in hydrolysis and decomposition
over prolonged reaction times. Derivatives containing electron
deficient acyl protecting groups at C2 were also examined as
donors (see SI.) as electron withdrawing groups have been
shown to slow competitive orthoester formation.37 Orthoester
formation dominated regardless of the protecting group at C2.
O
CCl3
i
N3
HO
OBn
OBn
OBn
O
NH
8
O
MM-ZPS target
repeating unit (3)
OCH3
O
HO
iii
CH3
N
OH
NHAc
H3C
17
O
v
H3C
P
O
ii
X
X
PgO
4
Figure 3. Second-generation analysis.
Donor 18 and acceptor 19 were synthesized from D-galac-
tose in six51 and twelve steps respectively (see SI). Glycosyla-
tion between donor 18 and 19 was followed by saponification
to provide b-linked disaccharide 20 in 91% yield over two steps
(Scheme 2). Subsequent acetonide protection of the C3’-C4’ al-
cohols and PMB protection of the C2’ alcohol (selected to allow
for C4’ protecting group orthogonality in subsequent steps)
gave fully protected disaccharide 21. Exchange of the C2 amine
protecting group at this stage proved critical as the C2 and C2’’
amines are differentially functionalized in the final product.
Thus, we planned to use a GalN3 based glycosyl donor to estab-
lish the trisaccharide core. LiAlH4 reduction of the C2 azide and
protection of the resulting amine as its diacetyl imide afforded
22 in quantitative yield.
As we terminated the first-generation approach, it was
clear the Gal-β-(1→3)-GalNAc bond was more difficult to in-
stall than we anticipated. In fact, with few exceptions, a large
number of routine glycosylation procedures favor orthoester
formation over glycosidic bond formation.27-29, 31-32, 44-49 One ob-
scure tactic used to generate this bond is to mask the C2-amine
Scheme 2. Second-generation approach.
H3C
OBn
O
OBn
O
OBn
O
OBn
O
AcO
AcO
BnO
HO
HO
HO
BnO
O
OBn
O
OBn
O
O
BnO
O
H3C
c. 2,2-DMP (93%)
a. TMSOTf (91%)
b. NaOMe (>95%)
[gram-scale]
OCH3
OCH3
O
OCH3
d. PMB-Cl (94%)
AcO
N3
N3
OH
OPMB
N3
O
NH
CCl3
[gram-scale]
18
19
20
21
6 steps
from D-Gal
12 steps
from D-Gal
e. 1) LAH
2) Ac2O
3) AcCl
>95%, 3 steps
[gram-scale]
OBn
O
BnO
BnO
H3C
H3C
OBn
O
OBn
O
HO
HO
BnO
O
OBn
O
OBn
O
O
BnO
OBn
O
OH OBn
O
BnO
O
f. CuCl2•H2O
g. 8, TMSOTf
N3
OCH3
OCH3
OCH3
O
O
O
40% α-only
[gram-scale]
58%; 27% RSM
[gram-scale]
OPMB
NAc2
OPMB
NAc2
NAc2
OPMB
23
22
24
h. i) 6, tetrazole
ii) mCPBA
85%
[gram-scale]
O
O
CH3
CH3
CH3
CH3
O
O
O
O
O
O
BnO
BnO
BnO
OBn
OBn
O
i. oxidation
P
P
OBn
OBn
O
OBn
OBn
O
O
BnO
O
BnO
O
OBn
O
OBn
O
BnO
O
O
OCH3
OCH3
N3
N3
O
O
NAc2
NAc2
OPMB
OH
25
26
3
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Reagents and conditions: (a) TMSOTf (0.15 equiv), CH2Cl2, 0 °C → 25 °C, 1 h, β-only, 91%; (b) NaOCH3, CH3OH, 25 °C, 2 h,
>95%; (c) 2,2-DMP, p-TsOH, CH2Cl2, 25 °C, 0.5 h, 93%; (d) PMBCl (1.2 equiv), NaH (2.0 equiv), DMF, 0 °C → 25 °C, 1 h, 94%;
(e) LiAlH4, THF, 0 °C → 25 °C, 1 h then Ac2O, pyr., DMAP, 0 °C → 25 °C, 2 h then AcCl, DIPEA, 2:3 CH2Cl2/CH3CN, µwave 85
°C, 3 h, >95% over three steps; (f) CuCl2·H2O, CH3CN, 0 °C → 25 °C, 3 h, 58% with 27% RSM; (g) 8 (2.5 equiv), TMSOTf (0.15
equiv), CH2Cl2, -78 °C→ -40 °C, 2.5 h, a-only, 40%; (h) 6 (1.5 equiv), 1H-tetrazole (1.5 equiv), CH3CN, 0 °C → 25 °C, 0.5 h then
mCPBA (1.5 equiv), CH2Cl2, -78 °C → 0 °C, 2 h, 85%.
reaction, use of a phosphoglycerol containing acceptor (step i).
We opted to use thioglycoside donors to form each glycosidic
bond due to their stability and scalability. The trisaccharide
Next, deprotection of the C3’-C4’ acetonide with
CuCl2·H2O gave C3’-C4’ diol acceptor 23 in modest yield
9
backbone would be constructed from its non-reducing end to its
along with recovered starting material which could be recycled.
10
11
12
13
14
15
16
17
18
19
20
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22
23
24
25
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reducing end to minimize waste of the more valuable acceptor
Conventional methods of acetonide removal (80% AcOH, p-
TsOH/MeOH, aq. TFA, etc.) proved unsuccessful and resulted
in hydrolysis of the glycosidic bond. Leveraging the enhanced
(steps ii & iii). The final challenge would be installation of the
phosphocholine unit (step iv), which has thus far proved elu-
sive. Thus, we anticipated developing a new reagent to install
nucleophilicity of the equatorial C3’-alcohol over the axial C4’-
this motif.
alcohol, we examined glycosylation with imidate donor 8.
While optimized conditions gave a complex mixture of every
OH
HO
HO
possible glycosylation product, the desired α-anomer 24 was the
major product in 40% yield. Efforts to improve this reaction by
esterifying the C4’ position proved futile as the modification
curtailed the nucleophilicity of the C3’ alcohol. Having estab-
lished the trisaccharide core, we next focused on phosphoryla-
tion. The C4’ phosphoglycerol residue was installed by cou-
pling 24 with phosphoramidite 6 and subsequently oxidizing at
phosphorus using mCPBA. 25 was isolated in 85% yield. At
this stage, completion of the MM-ZPS 2 required installation of
the C2’ phosphocholine residue and global deprotection. Unfor-
tunately, oxidative removal of the C2’ PMB ether was unsuc-
cessful. Both traditional (DDQ,52 CAN) and non-traditional
(HCl/HFIP,53 TfOH,54-55 CBr4-TPP,56-57 Silver(I),58 thermoly-
sis,59 MgBr2,59 and homogeneous electron transfer60) conditions
proved ineffective. Reassessing the route, we hypothesized the
central galactose residue was too hindered to allow for installa-
tion of all substituents. Accordingly, we identified a new target
repeating unit where the phosphoglycerol residue was shifted to
the reducing end (Figure 4). This modification transfers a sub-
stituent away from the central residue and eliminates synthesis
of a repeating unit with an unnatural O-alkyl capping unit at the
reducing end.
O
OH
OH OH
HO
O
OH
O
O
O
H3N
O
O
O
P
OH
NHAc
O
P
O
H3C
O
O
N
MM-ZPS target
repeating unit (2)
H3C
CH3
O
Ph
O
Ph
O
Ph
O
O
O
O
OBn
O
STol
O
O
PMBO
STol
HO
O
O
STol
TBSO
P
AcO
N3
BnO
BzO
30
TCAHN
31
29
iii
OBn
ii
32
i
iv
O
O(CH2)2CN
Me3N
P
BPh4
NiPr2
33
Figure 5. Third-generation analysis.
The third-generation approach commenced with an NIS-
TMSOTf promoted coupling of thioglycoside 31 to phospho-
glycerol acceptor 32 (see SI). After glycosylation, NaOCH3 me-
b
diated removal of the C3 acetate gave -34 in 93% yield over
two steps (Scheme 3). Presumably, neighboring group partici-
pation from the C2 trichloroacetamide assisted in exclusive for-
mation of the β-anomer. Subsequent NIS-TMSOTf promoted
glycosylation with thioglycoside 30 occurred in 91% yield to
give 35.
O
OH
2nd Generation
Target 27
O
O
O
OH
HO
HO
P
O
OH
HO
O
O
OH
O
O
O
H3N
O
NHAc
O
P
OH
H3C
O
O
N
O
H3C
O
O
OH
HO
CH3
O
P
O
OH
O
OH
HO
HO
O
O
H3N
O
O
O
3rd Generation
Target 28
NHAc
O
P
OH
H3C
O
O
N
H3C
O
O
O
O
OH
CH3
O
HO
P
O
OH
HO
O
H3N
O
O
O
O
P
H3C
O
N
H3C
CH3
O
Figure 4. Target repeating unit frameshift.
At the planning stage (Figure 5), it was clear that the syn-
thesis of this new target (2) would maintain its novelty as the
strategy would now feature an unprecedented glycosylation
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Journal of the American Chemical Society
Scheme 4. (a) Phosphocholine coupling and (b) completion of
Scheme 3. Third-generation approach.
Ph
1
2
3
4
5
6
7
8
the total synthesis.
O
O
a) Phosphocholine coupling
OBn
OBn
a. NIS, TMSOTf (93%)
O
O
HO
O
STol
O
P
AcO
P
O
O
b. NaOMe (>95%)
O
40
OBn
Cl
NHTCA
O
O
P
O
RO
N(CH3)3
RO
O
[gram-scale]
RO
O
P
32
31
-or-
(iPr)2N
O
OH
H3C
H3C
O
O
39
42
O
N
O
P
43
CH3
O
41
Ph
O
O
O
b) Synthesis of choline donor
OBn
c. 30, NIS, TMSOTf
O
OBn
O
O
91% β-only
9
HO
P
O
O(CH2)2CN
a. HO(CH2)2N(CH3)3·BPh4
Me3N
OBn
P
NHTCA
[gram-scale]
(iPr2N)2PO(CH2)2CN
O
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1H-tetrazole
MeCN
0 °C, 3 h
71%
BPh4
NiPr2
34
33
44
Ph
O
Ph
O
O
O
OBn
c) Completion of the total synthesis
O
O
OBn
O
e. 29, NIS, TMSOTf
O
O
RO
O
P
OBn
71% 2.4:1α/β
[gram-scale]
NHTCA
OAc
Ph
O
O
Ph
O
Ph
35, R = TBS
36, R = H
d. HF-pyridine (69%)
O
O
[gram-scale]
O
O
PMBO
OBn
a. i) 33, 1H-tetrazole
ii) TBHP
O
OBn
38
O
O
O
N3
O
O
P
Ph
O
iii) DBU
75% over 3 steps
OBn
NH
O
P
O
H3C
H3C
O
O
O
O
N
Ph
Ph
CCl3
CH3
O
45
O
O
O
O
OH
HO
O
PMBO
OBn
O
OBn
O
O
N3
O
OH
O
O
O
OH OH
HO
HO
OH
O
P
b. Pd/C, H2
O
O
OBn
NHTCA
OR
H3N
O
O
O
O
O
89%
P
OH
NHAc
O
P
O
37, R = Ac
38, R = H
H3C
H3C
O
O
f. NaOMe (>95%)
[gram-scale]
N
MM-ZPS (2)
155 mg
CH3
O
Reagents and conditions: (a) NIS (2.0 equiv), TMSOTf (0.15
equiv), CH2Cl2, 4 Å MS, 0 °C→ 25 °C, 1.0 h, β-only, 93%; (b)
NaOCH3, CH3OH, 25 °C, 2 h, >95%; (c) 30 (1.5 equiv), NIS
(1.5 equiv), TMSOTf (0.10 equiv), CH2Cl2, 4 Å MS, -78 °C,
1.0 h, β-only, 91%; (d) HF-pyridine (10.0 equiv), pyridine, 25
°C, 3 h, 69%; (e) 29 (1.4 equiv), NIS (2.0 equiv), TMSOTf (0.15
equiv), 2.4:1 CH2Cl2/iPr2O, 4 Å MS, -60 °C→ 0 °C, 5.0 h, 2.4:1
α/β, 71%; (f) NaOCH3, CH3OH, 25 °C, 2 h, >95%.
Reagents and conditions: (a) 33 (1.2 equiv), 1H-tetrazole (1.2
equiv), CH3CN, 0.5 h, 25 °C then TBHP (1.2 equiv), CH3CN, 0
°C, 1 h then DBU (5.0 equiv), CH2Cl2, 25 °C, 18 h, 78% over
three steps; (b) Pd/C (15% w/w), H2 (balloon), CH3OH, 25 °C,
88 h, 89%.
To introduce this residue we synthesized a new phospho-
choline donor, choline-2-cyanoethyl N,N-diisopropylphospo-
ramidate tetraphenylborate 44, which functioned very well
when using tetrazole as an activator. After formation of the ox-
ygen-phosphorus bond (33 + 38), subsequent oxidation to the
phosphate was achieved using tert-butyl hydroperoxide
(TBHP). Finally, exposure to DBU led to removal of the cyano-
ethyl group, via retro-aldol reaction, to provide the desired
phosphodiester 45 in 75% yield over the three-steps.
Again, the high β-selectivity can be attributed to neighbor-
ing group participation, this time from the C2’-acetate. Next,
we moved forward with liberating the C3’ alcohol, which would
serve as the acceptor in the final glycosylation event. To our
surprise, we encountered difficulties while removing the silyl
ether. The most productive conditions, HF•pyridine buffered by
additional pyridine, provided acceptor 36 in 69% yield. A third
NIS-TMSOTf promoted glycosylation with thioglycoside 29
(see SI) gave trisaccharide 37 in 71% yield as a 2.4 to 1 mixture
of α and β anomers. A number of α-selective additives (DMF,
thioethers, and ethers) were evaluated in the reaction. Ulti-
mately, 25 equivalents of diisopropyl ether was the most α-se-
lective additive. Finally, NaOCH3 mediated removal of the C2’
acetate provided trisaccharide 38 in 95% yield.
With the complete core structure in hand, the final maneu-
ver in the synthesis was a remarkable, exhaustive catalytic hy-
drogenation of 45. This reaction served to remove three benzyl-
idene acetals, one PMB ether, and three benzyl ethers. The re-
ductive environment also converted the C2 trichloroacetamide
to the acetamide and the C2’’ azide to an amine. Starting from
330 mg (0.2 mmol) of 45, purification using size exclusion
chromatography (P2-Biogel) gave 155 mg of MM-ZPS 2 in
89% isolated yield. Charge deconvoluted ESI FT ICR (electron
spray ionization Fourier transformation ion cyclotron reso-
nance) mass spectrometry revealed molecular mass spectral
peaks in agreement with the desired calculated mass (calc’d
With the trisaccharide core in hand, we arrived at the most
critical step of the synthesis, installation of the phosphocholine
residue (Scheme 4). The initial approach to phosphorylation fo-
cused on using the well-established cyclic phospholanes 40 and
41. In theory, following phosphorylation, the 5-membered ring
of 42 can be opened by trimethylamine to provide the phospho-
choline unit. Unfortunately, this line of inquiry was met by two
challenges. First, the C2’ alcohol of 38 proved to be both too
sterically hindered and poorly nucleophilic to couple with either
phospholane. Secondly, cyclic phospholanes are vulnerable to
heat, moisture, and silica gel chromatography.
1
[M+H] for C28H55N3O23P2 = 864.2780, found 864.2772). H
NMR coupling constants revealed the presence of one a glyco-
sidic bond at C1’’ (J = 5.33, 3.6 Hz) and two b glycosidic bonds
at C1’(J = 4.70, 7.9 Hz) and C1 (J = 4.46, 7.6 Hz). 2-D NMR
experiments, including 1H-31P HSQC, enabled full assignment
of the ZPS’s glycosidic bonds and phosphorus functionality.
Additionally, the NMR data from the synthesized repeating unit
5
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Journal of the American Chemical Society
Page 6 of 8
2 is in complete agreement with the data reported for the natural
MM-ZPS polymer (Table S1).
ASSOCIATED CONTENT
1
2
3
4
5
6
7
8
9
SUPPORTING INFORMATION
The supporting information is available free of charge on the ACS
Publications website at DOI:
CONCLUSION
Experimental procedures, characterization data, and NMR spectra
(PDF)
In summary, we have completed the first total synthesis of
the repeating unit of MM-ZPS 2. Key steps include (1) early
stage phosphoglycerol glycosylation occurring in high yield
and excellent b-selectivity, (2) formation of the challenging
Gal-β-(1→3)-GalNAc bond without generating any undesired
orthoacetate byproduct, and (3) installation of the phosphocho-
line group using a cholinoxy-cyanoethoxy-diisopropylamino-
phosphine reagent. As studies with the naturally occurring MM-
ZPS demonstrated that single repeating units do not elicit an
immune response, current efforts are focused on using the syn-
thetic repeating unit to synthesize non-natural ZPS materials.
Studies regarding the synthesis and immunological properties
of these materials are currently underway.
AUTHOR INFORMATION
10
11
12
13
14
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16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Corresponding Author
* steven.d.townsend@vanderbilt.edu
ACKNOWLEDGMENTS
This work was supported, in part, by the National Institutes of
Health under Grant No. 1R35GM133602. The Mass Spectrometry
Research Center at Vanderbilt University is acknowledged for ac-
quisition of high-resolution mass spectral data. Dr. Markus Voehler
and Dr. Donald Stec are acknowledged for assistance with NMR
experiments.
12.
Wang, Z. G.; Williams, L. J.; Zhang, X. F.; Zatorski, A.;
Kudryashov, V.; Ragupathi, G.; Spassova, M.; Bornmann, W.; Slovin,
S. F.; Scher, H. I.; Livingston, P. O.; Lloyd, K. O.; Danishefsky, S. J.,
Polyclonal antibodies from patients immunized with a globo H-keyhole
limpet hemocyanin vaccine: isolation, quantification, and
characterization of immune responses by using totally synthetic
immobilized tumor antigens. Proc Natl Acad Sci U S A 2000, 97 (6),
2719-24.
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1,2-trans-β-galactoside
OH
HO
HO
O
OH
O
OH
HO
O
OH
O
O
O
H3N
N
O
O
O
P
O
1,2-cis-α-galactoside
NHAc
O
P
O
H3C
H3C
O
O
phosphoglycerol
residue
CH3
O
9
phosphocholine
residue
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
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35
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39
40
41
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