Syntheses of phosphatidyl-β-d-glucoside analogues to probe antigen selectivity of monoclonal antibody 'DIM21'

Article abstract of DOI:10.1016/j.bmc.2008.06.041

Herein, we report the chemical syntheses of a series of phosphatidyl-β-d-glucoside (PtdGlc) analogues, including 6-O-Ac, sn-2-O-Me, phosphorothioate as well as phosphatidylgalactoside and -mannoside derivatives. In the key step, β-glycosyl H-phosphonate w

Full text of DOI:10.1016/j.bmc.2008.06.041

Bioorganic & Medicinal Chemistry 16 (2008) 7210–7217
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Syntheses of phosphatidyl-b-D-glucoside analogues to probe antigen
selectivity of monoclonal antibody ‘DIM21’
Peter Greimel a,b, Milaine Lapeyre a, Yasuko Nagatsuka b, Yoshio Hirabayashi b,c, Yukishige Ito a,
*
a RIKEN, The Institute of Physical and Chemical Research, Advanced Science Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
b RIKEN BSI, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
c CREST, Japan Science and Technology Agency (JST), Kawaguchi, Saitama 332-0012, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Herein, we report the chemical syntheses of a series of phosphatidyl-b-
D
-glucoside (PtdGlc) analogues,
Received 27 May 2008
Revised 19 June 2008
Accepted 20 June 2008
Available online 25 June 2008
including 6-O-Ac, sn-2-O-Me, phosphorothioate as well as phosphatidylgalactoside and -mannoside
derivatives. In the key step, b-glycosyl H-phosphonate was condensed with enantiomerically pure diac-
ylglycerol. Comparison of spectroscopic data with mono-acetylated PtdGlc from natural source confirmed
the presence of an acetyl moiety at position 6. Furthermore, the reactivity of PtdGlc and its analogues
toward monoclonal antibody ‘DIM21’ (MAb DIM21) was evaluated, revealing the crucial structural anti-
gen features for successful MAb DIM21 binding.
Keywords:
Glucose
Phospholipid
Analogues
Antibody
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
an enhanced stability under physiological conditions. Furthermore,
the sn-2-O-methyl PtdGlc (5), the phosphatidylgalactoside (6) and
In 1970, Short and White1 identified phosphatidylglucoside (1,
PtdGlc) in the lipid fraction of Staphylococcus aureus. About 30 years
later, the first isolation2 from mammalian sources was reported by
Nagatsuka et al.,3 suggesting PtdGlc involvement in mammalian cell
differentiation. More recently, Yamazaki et al.,4 successfully
produced a monoclonal antibody (MAb) against PtdGlc, called
‘DIM21’. With the help of MAb DIM21, PtdGlc was visualized and
isolated from rat embryonic brain tissue.5 PtdGlc contained
exclusively 18:0/20:0 fatty acid chains, rarely occurring in known
mammalian lipids. The presence of both stereoisomers, PtdGlc (1)
and its sn-2-epimer 3, in the natural sample was recently
confirmed.6 Interestingly, besides PtdGlc also a 6-O-acetylated
PtdGlc derivative (2) was isolated from rat embryonic brain tissue.5
It was speculated that PtdGlc’s unusual fatty acid chain pattern
might be responsible for its presence within distinct membrane
microdomains on astroglial cells. Moreover, it was suggested that
PtdGlc plays an important role in glial cell development and
differentiation.5 However, the biological roles of PtdGlc and its
derivatives in glial cells remain largely unknown.
the phosphatidylmannoside (7) were also prepared (Scheme 1).
We compared the reactivity of these compounds toward MAb
DIM21 to clarify the key structural requirements for antigen
recognition.
2. Results and discussion
2.1. Syntheses
Our synthetic plan toward PtdGlc (1) and its analogues 2–7, re-
quired the separate preparation of stereochemically well-defined
key intermediates. Subsequent condensation of these key interme-
diates, namely b-glycopyranosyl H-phosphonate and enantiomeri-
cally pure glycerol units, followed by selective removal of the
protecting groups yielded the desired products. PtdGlc (1) and its
stereoisomer 3 were prepared according to literature procedures6
(see Schemes 2 and 3) by condensation of intermediate 21 with
10 and 15, respectively.
O-Methylated glycerol unit 12 was prepared from the enantio-
merically pure PMB protected glycerol 8.7 Silylation of the primary
hydroxyl function was followed by methylation and desilylation,
yielding intermediate 11. DCC-mediated introduction of the stearic
acid residue and carefully controlled removal of the PMB protec-
tion gave the desired glycerol unit 12.
Herein, we report the first syntheses of 6-O-acetylated PtdGlc
(2) confirming the proposed structure of the natural compound.
In addition, we synthesized a series of PtdGlc derivatives, including
the phosphorothioate derivative (4), which was expected to exhibit
6-O-Acetylated glucose (17) and mannose (19) units were pre-
pared via their
* Corresponding author. Tel.: +81 48 467 9430; fax: +81 48 462 4680.
E-mail address: yukito@riken.jp (Y. Ito).
a
-trichloroacetimidates 168 and 18,9 respectively.
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.06.041

P. Greimel et al. / Bioorg. Med. Chem. 16 (2008) 7210–7217
7211
O
O
O
O
CH2 CH3
O
O
CH2 CH3
16
16
OR
O
OH
O
O
O
O P O
S
HO
HO
O
HO
HO
O
CH2 CH3
O
CH2 CH3
18
18
O P O
OH
OH
O
O
O
CH2 CH3
16
O
Na
HNEt3
OH
O
O
4
1 R=H
2 R=Ac
HO
HO
O
CH2 CH3
18
O P O
OH
O
Na
3
O
O
O
CH2 CH3
O
CH2 CH3
16
16
OH
OH
OH
O
O
O P O
O
O
O
HO
HO
OMe
O
CH2 CH3
HO
HO
18
O P O
O
O
O
CH2 CH3
16
OH
HO
OH
OH
O
O
HNEt3
HNEt3
O
O
CH2 CH3
18
7
5
O P O
O
OH
HNEt3
6
Scheme 1. Overview of synthesized phosphatidyl-b-D-glucoside (1) and its analogues (2–7).
Treatment with phosphonic acid triggered trichloroacetimidate
activation and b-phosphonate formation, similarly to previously
reported phosphoric acid diester treatment.10 However, H-phos-
phonates seemed to be more resistant to anomerization compared
to their corresponding phosphates. The galactose H-phosphonate
unit 23 was prepared via a two-step sequence from commercially
available peracetate 22. Firstly, 22 was converted to the corre-
sponding tert-butyl orthoester under Lewis acid-mediated condi-
tions.11 Subsequently, autocatalytic reaction with phosphonic
acid12 gave desired b-phosphonate 23. The glucose H-phosphonate
unit 21 was prepared in an analogous manner.
Linkage of the carbohydrate and glycerol units was furnished in
a unified manner with a ‘one-pot’ procedure through pivalyl chlo-
ride mediated H-phosphonate activation and subsequent iodine-
mediated oxidation.13 As a precursor of the phosphorothioate
derivative 4, compound 30 was synthesized from the coupling
product of 21 and 10 by treatment with Beaucage reagent14 as a
sulfurizing agent. As expected, NMR revealed the presence of both
diastereomers at the newly formed chiral phosphorothioate diester
in a ratio close to 1:1.
attempts to employ a catalytic amount of sodium methoxide were
futile, showing poor selectivity, while guanidine/guanidinium ni-
trate15 exhibited somewhat better selectivity. Most satisfactory
was
a
modified hydrazine-mediated deacetylation protocol16
yielding the desired products within 5 min in 43–78% yield. Perb-
enzylated intermediates 26 and 33 were cleanly deprotected with
H2 and Pd/C,17 in contrast to the report of Ramirez et al.,18 to fur-
nish compounds 27 and 7. In all cases, compounds 25, 27, 29,
and 4–7 were isolated as triethylammonium salts after chromato-
graphic purification with CHCl3–MeOH–Et3N. Since natural PtdGlc
and its derivatives were characterized as sodium salt, synthetic
compounds 25, 27, and 29 were converted to the corresponding so-
dium salts 1, 2 and 3 to allow structure verification. In general,
compounds were poorly soluble in any organic solvents, for exam-
ple, chloroform, methanol, DMSO or ether, as well as in water. NMR
measurements were conducted with dilute solutions predomi-
nantly in DMSO-d6.
2.2. Structure verification and characterization
Final deprotection to yield compounds 4–6 required selective
removal of the acetyl groups under conservation of the fatty acid
esters. This apparently challenging strategy was chosen in order
to develop a suitable approach to an initially proposed structure
of PtdGlc, decorated with an arachidonic acid residue.2 In addition,
b-phosphate diesters are poorly tolerant to acidic conditions. Initial
In addition to PtdGlc (1), isolation of a slightly less polar novel
compound from fetal rodent brain was reported.5 MS spectroscopic
data indicated the presence of an additional acetyl group. NMR
data suggested the presence of an acetyl group at position 6 of
the glucose moiety, due to a considerable low-field shift of protons
at position 6 as well as 5 of the glucose moiety in contrast to PtdGlc
R2
O
O
R2
R3
R3
HNEt3
O
O
CH2 CH3
O
CH2 CH3 b,c)
O
16
BnO
BnO
16
g)
O
BnO
BnO
O
O
R1
O
CH2 CH3
OH
OPMB
18
OH
R1
PH
OH
a)
O
NH
CCl3
16, 18
9
10
17, 19
O
OH
OPMB
O
16, 17 R1=OBn; R2=H; R3=OAc
OH
8
d,e,f)
a,c)
18, 19 R1=H; R2=OBn; R3=OBn
O
CH2 CH3
16
OMe
OPMB
OMe
R5
OH
OH
R5
R4
AcO
11
12
OAc
O
HNEt3
OAc
O
h,g)
R4
AcO
O
OAc
O
O
O
OH
OAc
OAc
PH
O
O
CH2 CH3
O
CH2 CH3
b,c)
16
a)
16
20, 22
21, 23
O
OH
OPMB
20, 21 R4=OAc; R5=H
22, 23 R4=H; R5=OAc
O
CH2 CH3
OH
18
OPMB
13
14
15
Scheme 2. Syntheses of glycerol units 10, 12, and 15 and carbohydrate units 17, 19, 21, and 23. Reagents and conditions: (a) DCC, DMAP, stearic acid; (b) DCC, DMAP,
arachidic acid; (c) DDQ; (d) TBDPSCl, imidazole; (e) MeI, NaH; (f) TBAF, AcOH; (g) (i)—H3PO3; (ii)—NEt3, 4 °C; (h) t-BuOH, DMAP, AlCl3.

7212
P. Greimel et al. / Bioorg. Med. Chem. 16 (2008) 7210–7217
O
O
O
O
CH2 CH3
O
O
CH2 CH3
16
a)
a)
16
OR
O
OAc
O
21 + 10
17 + 10
O
O
RO
RO
RO
RO
O
CH2 CH3
O
O
CH2 CH3
18
18
O P O
O P O
OR
OR
O
O
R'
R'
24 R=Ac; R'=HNEt3
25 R=OH; R'=HNEt3
1 R=OH; R'=Na
26 R=Bn; R'=HNEt3
27 R=OH; R'=HNEt3
2 R=OH; R'=Na
b)
c)
d)
c)
O
O
O
O
CH2 CH3
O
O
CH2 CH3
16
16
e)
OR
O
OR
O
O
21 + 10
O
RO
RO
RO
O
CH2 CH3
CH2 CH3
a)
18
18
O P O
O P O
S
b)
21 + 15
RO
OR
OR
30 R=Ac
4 R=OH
O
R'
HNEt3
28 R=Ac; R'=HNEt3
29 R=OH; R'=HNEt3
3 R=OH; R'=Na
b)
c)
O
O
CH2 CH3
16
OR
O
O P O
O
O
RO
OMe
a)
31 R=Ac
5 R=OH
21 + 12
RO
b)
OR
HNEt3
O
O
O
O
CH2 CH3
O
CH2 CH3
OR
O
OR
16
16
OR
O
a)
O
O
O P O
O
OR
23 + 10
O
CH2 CH3
O
CH2 CH3
a) RO
RO
18
18
O P O
RO
19 + 10
OR
O
O
b)
HNEt3
HNEt3
32 R=Ac
6 R=OH
33 R=Bn
7 R=OH
d)
Scheme 3. Linkage and selective deprotection to yield PtdGlc (1) and its derivatives (2–7). Reagents and conditions: (a) (i)—PivCl, pyridine, 0 °C ? rt; (ii)—I2, pyridine/H2O;
(b) N2H4/HOAc = 4:1; (c) DOWEX 50WX8; (d) H2, Pd/C; (e) (i)—PivCl, pyridine, 0 °C ? rt; (ii)—Beaucage reagent.
(1). Comparison of the reported spectral data from natural source
with synthetic 6-O-acetyl PtdGlc derivative (2) revealed good
agreement of NMR- and MS-spectra, confirming the proposed
structure of the natural sample.
Proton NMR spectrum of 25 (see Fig. 1B) acquired in DMSO-d6
provided an interesting insight into the orientation of the hydroxyl
groups of the glucose moiety. Namely, OH-2 exhibited, in contrast
to OH-3, OH-4, and OH-6, an unusual low-field shift to 6.38 ppm.
Saturation of the solvent signal via a selective DANTE pulse se-
quence affected OH-3, OH-4, and OH-6 signals strongly, consistent
with a fast exchange process. On the contrary, the OH-2 signal
showed little influence, indicating a slow exchange process. A pos-
sible explanation for these results would be the presence of an
intramolecular hydrogen bond. This hypothesis is supported by
computer simulations19 of PtdGlc (1), proposing a distance of only
1.7 Å between the proton of OH-2 and one of the non-bridging oxy-
gen of the phosphate moiety (see Fig. 1A). This structural feature
resembles a six-membered ring in slightly distorted chair confor-
mation, likely leading to a considerable reduction of the flexibility
at the phosphate-sugar linkage. Nevertheless, to what extent this
intramolecular hydrogen bond is present in water or in biological
systems is unknown.
Figure 1. (A) Graphical representation of proposed intramolecular H-bond (dotted line) in PtdGlc (1); (B–D) 1H NMR in DMSO-d6 (B) of PtdGlc (25); (C) of phosphorothioate
derivative 4; D) of manno derivative 7 (location of OH-2 indicated by gray area).

P. Greimel et al. / Bioorg. Med. Chem. 16 (2008) 7210–7217
7213
Figure 2. Determination of MAb DIM21 antigen specificity. TLC developed (A) with Orcinol for unspecific visualization; (B) by immunostaining assay to visualize MAb DIM21
immunoreactive spots.
its immunoreactivity with our synthetic PtdGlc derivatives (see
Fig. 2) at concentrations of 1/2–8 nmol by immunostaining assay.4
Immunoreactivity of sn-1,2-di-O-acyl PtdGlc (1) was the strongest,
tightly followed by its diastereomer 3 and phosphatidylgalactoside
(6). In case of phosphatidylmannoside (7), immunoreactivity
was considerably reduced, ensued by 6-O-acetyl PtdGlc (2) and
phosphorothioatidylglucoside (4), exhibiting a further decrease in
reactivity. Interestingly, sn-2-O-methyl PtdGlc (5) exhibited no
detectable reactivity with MAb DIM21.
Figure 3. Visualization of proposed structure–immunoreactivity relationship map
An overview of the immunological assay results was mapped as
shown in Figure 3. The good recognition of phosphatidylgalacto-
side (6) and sn-2,3-di-O-acyl PtdGlc (3) by MAb DIM21 denotes
that its interaction with the antigens is not sensitive to the stereo-
chemistry at position 4 as well as sn-2. As for position 6, modifica-
tion, for example, 2, resulted in a largely abolished recognition by
MAb DIM21. While steric reasons cannot be excluded, partial inter-
ruption of direct antigen-antibody interaction seems more likely to
be responsible. This might also explain the absence of cross reac-
tivity with PI and PGly at low concentrations. Although both posses
very similar features, PGly totally lacks the OH-6 region and PI
might not be able to present a suitable hydroxyl function in the re-
quired area, highlighting the importance of this position for anti-
body recognition.
Drastically reduced reactivity of phosphatidylmannoside (7)
may derive from the altered orientation of the sugar residue due
to the absence of the intramolecular hydrogen bond. The phos-
phate moiety itself seems to be essential for antigen recognition,
as indicated by the strong reduction of antibody binding for both
diastereomers of phosphorothioatidylglucoside (4). Predomi-
nantly, one of the diastereomers should be affected, in case primar-
ily steric effects cause decreased recognition. Hence, only a
reduction of about 50% should be observed, due to the presence
of a close to 1:1 mixture of diastereomers. This is not in good
agreement with experimental results, showing a much stronger
reduction of antigen recognition. It seems more likely that recogni-
tion of both diastereomers is strongly affected, for example, due to
a higher charge localization on the sulfur. While the remaining
non-bridging oxygen of the phosphorothioate moiety is involved
in the weakened intramolecular hydrogen bond, the negatively
charged sulfur might not be able to form the necessary electro-
static or hydrogen bond with MAb DIM21 for binding. Conse-
quently, antigen recognition of both diastereomers would be
for MAb DIM21. Gray background pattern indicates essential area for MAb DIM21
binding.
This effect was observed with all synthetic PtdGlc derivatives
bearing a glucose or galactose residue, except the phosphorothio-
ate derivative (4 Rp/Sp, see Fig. 1C), which exhibited a shift value
of only 5.66 ppm for the minor and 5.56 ppm for the major diaste-
reomer. This observation indicates the attenuated capacity of
phosphorothioate to serve as a hydrogen bond acceptor, presum-
ably due to the less electronegative nature of sulfur.20
Not surprisingly, the proton signal of OH-2 from phosphat-
idylmannoside (7, see Fig. 1D) resides at 4.60 ppm, being well
within the range of hydroxyl group protons not involved in intra-
molecular hydrogen bonding. Hence, epimerization at C-2 seems
to disfavor the formation of an intramolecular hydrogen bond,
leading to full flexibility of the phosphate-sugar linkage.
2.3. Structure–activity realtionship (SAR) study
As previously reported,4 MAb DIM21 did not show any cross
reactivity with neutral phospholipids, like phosphatidylcholine
(PC), phosphatidylethanolamine (PE), phosphatidylserine (PS),
sphingomyelin, or glycosylceramide. MAb DIM21 reacted strongly
with natural PtdGlc, a mixture of diastereomers, and exhibited
some cross reactivity with phosphatidylinositol (PI) and phosphat-
idylglycerol (PGly). On the contrary, recent data5 do not exhibit any
cross reactivity of MAb DIM21 with PI or PGly at low nanomolar
concentrations. Probably, the initially reported cross reactivity
might have been caused by the presence of some immunoreactiv-
ities against these lipids in peroxidase-conjugated goat IgG fraction
to mouse immunoglobulins (IgG, IgA, IgM). In order to gain a better
understanding of MAb DIM21 antigen selectivity, we determined

7214
P. Greimel et al. / Bioorg. Med. Chem. 16 (2008) 7210–7217
largely abolished, as observed, indicating the great importance of
the phosphate moiety in antigen recognition.
4.1.1. 6-O-Acetyl-2,3,4-tri-O-benzyl-b-D-glucopyranosyl
hydrogenphosphonate triethylammonium salt (17)
Intriguingly, conversion of ester at position sn-2 to ether, as in
sn-2-O-methyl PtdGlc (5), widely eliminated antibody binding. It
can only be speculated whether this is caused by the absence of vi-
tal hydrophobic interactions between the hydrocarbon tail of the
arachidic acid residue and the antibody or due to the absence of
an important antigen-antibody interaction at the sn-2 carbonyl
group. Presumably, it might be a combination of both effects. Nev-
ertheless, this strongly indicates the importance of the arachidic
acid residue at sn-2 position of PtdGlc for antigen recognition.
Prepared by phosphonylation of compound 16 (85 mg,
0.13 mmol) to give compound 17 as a colorless honey (30 mg,
0.046 mmol, 35% yield). [a]
20 = 27 (c 3.2 in CHCl3); 1H NMR
D
(400 MHz, CDCl3, 25 °C): d = 7.38–7.26 (n.r., 15H; Bn), 7.07 (d, 1J
(H,P) = 641.3 Hz, 1H; HP), 5.17 (dd, J = 7.9 Hz, 3J (H,P) = 9.2 Hz,
1H; H-1), 5.04 (d, J = 11.2 Hz, 1H; Bn), 4.92 (d, J = 10.7 Hz, 1H;
Bn), 4.86 (d, J = 10.7 Hz, 1H; Bn), 4.79 (d, J = 10.5 Hz, 1H; Bn),
4.76 (d, J = 11.0 Hz, 1H; Bn), 4.56 (d, J = 11.0 Hz, 1H; Bn), 4.34 (d,
J = 11.7 Hz, 1H; H-6b), 4.20 (d, J = 12.0 Hz, 1H; H-6a), 3.70 (dd,
J = 8.5 Hz, J = 8.8 Hz, 1H; H-3), 3.59–3.58 (n.r., 2H; H-4, H-5), 3.51
(dd, J = 8.3 Hz, J = 8.5 Hz, 1H; H-2), 2.97 (q, J = 7.3 Hz, 6H; NEt3),
2.01 (s, 3H; Ac), 1.25 (t, J = 7.3 Hz, 9H; NEt3); 13C NMR (100 MHz,
CDCl3, 25 °C): d = 170.69 (1C; Ac), 138.59, 138.41, 137.79 (3C; Bn-
ipso), 128.51, 128.43, 128.27, 128.15, 127.95, 127.85, 127.70,
127.47 (15C; Bn), 97.44 (d, 2J (C,P) = 3.3 Hz, 1C; C-1), 84.72 (1C;
C-3), 82.78 (d, 3J (C,P) = 5.0 Hz, 1C; C-2), 77.17 (1C; C-5), 75.86,
75.15, 74.80 (3C; Bn), 73.17 (1C; C-4), 63.10 (1C; C-6), 45.63 (3C;
NEt3), 21.22 (1C; Ac), 8.98 (3C; NEt3); LRMS (MALDI-TOF, neg)
calcd for C35H48NO9P [MÀNEt3]À: 556.2, found: 556.3
3. Conclusion
MS and NMR spectroscopic data of natural mono-acetylated
PtdGlc derivative5 and synthetic 6-O-acetyl PtdGlc (2) were identi-
cal, thus confirming the proposed presence of the acetyl group at
position 6 of the glucose moiety.
Furthermore, proton NMR data strongly suggested the presence
of an intramolecular hydrogen bond at position OH-2. Computer
simulations proposed one of the non-binding oxygen as a suitable
partner. This prominent feature is conserved in most cases of glu-
cose and galactose derivatives of PtdGlc, leading to a reduced flex-
ibility at the phosphate moiety, likely contributing to its unique
biological role. Interestingly, the presence of a phosphorothioate
moiety (4) significantly weakened the intramolecular hydrogen
bond, while it was totally abolished in case of the mannose deri-
vate (7).
Concerning MAb DIM21, the phosphate moiety and the arachi-
dic acid ester at position sn-2 of PtdGlc and its derivatives serve as
the primary recognition site, additionally supported by the hydro-
xyl function at position 6. Disturbance at one of these three recog-
nition sites will lead to considerable reduction or total abolishment
of antigen recognition. Experimental data indicate that hydroxyl
functions at position 4 as well as ester function at position sn-1/3
are less important for MAb DIM21 binding.
4.1.2. 2,3,4,6-Tetra-O-benzyl-b-D-mannopyranosyl
hydrogenphosphonate triethylammonium salt (19)
Prepared by phosphonylation of compound 18 (25 mg,
0.036 mmol) to give compound 19 as a colorless honey (13 mg,
0.019 mmol, 52% yield). [a]
20 = 16 (c 1.6 in CHCl3); 1H NMR
D
(400 MHz, CDCl3, 25 °C): d = 7.40–7.18 (n.r., 20H; Bn), 6.92 (d, 1J
(H,P) = 628.8 Hz, 1H; HP), 5.73 (dd, J = 1.4 Hz, 3J (H,P) = 8.7 Hz,
1H; H-1), 4.91 (d, J = 11.0 Hz, 1H; Bn), 4.78 (d, J = 12.5 Hz, 1H;
Bn), 4.74 (d, J = 12.7 Hz, 1H; Bn), 4.63 (d, J = 12.0 Hz, 1H; Bn),
4.58 (s, 2H; Bn), 4.54 (d, J = 11.0 Hz, 1H; Bn), 4.49 (d, J = 12.0 Hz,
1H; Bn), 4.08–3.99 (n.r., 3H; H-3, H-4, H-5), 3.90 (dd, J = 1.5 Hz,
J = 2.7 Hz, 1H; H-2), 3.77 (dd, J = 4.4 Hz, J = 10.7 Hz, 1H; H-6b),
3.71 (dd, J = 1.1 Hz, J = 10.9 Hz, 1H; H-6a), 2.93 (q, J = 7.3 Hz, 6H;
NEt3), 1.23 (t, J = 7.3 Hz, 9H; NEt3); 13C NMR (100 MHz, CDCl3,
25 °C): d = 138.78, 138.65, 138.48 (4C; Bn-ipso), 128.29, 127.96,
127.90, 127.79, 127.56, 127.48 (20C; Bn), 93.35 (d, 2J
(C,P) = 2.5 Hz, 1C; C-1), 79.82 (1C; C-3), 75.49 (d, 3J (C,P) = 5.8 Hz,
1C; C-2), 75.08, 73.54, 72.60, 72.14 (4C; Bn), 75.01 (1C; C-4),
73.10 (1C; C-5), 69.76 (1C; C-6), 45.65 (3C; NEt3), 9.05 (3C;
NEt3); LRMS (MALDI-TOF, neg) calcd for C40H52NO8P [MÀNEt3]À:
604.2, found: 604.3.
4. Experimental
Unless stated otherwise, all chemicals were purchased as re-
agent grade from commercial suppliers and used without further
purification. Dry solvents were purchased from Kanto Chemical
Co., Inc. and used as supplied. Analytical thin layer chromatogra-
phy (TLC) was performed on Merck Silica gel 60 F256 plates, flash
column chromatography on Kanto Chemical Co., Ltd silica gel
60N (40–100 mesh) using indicated solvent systems. Low resolu-
tion mass spectra (LRMS) were recorded on an AXIMA-LNR MALDI
from Shimadzu/KRATOS (MALDI-TOF) or on a Jeol AccuTOF JMS-
T700LCK (ESI-TOF). High resolution mass spectra (HRMS) were re-
corded on the ESI-TOF utilizing TFA as an internal standard. NMR
spectra were obtained either with a JEOL EX-400, ECX-400 or
JNM-ECA-600 spectrometer (1H at 400/600, 13C at 100/150 MHz
and 31P at 160/240 MHz) in the indicated solvents, with chemical
shift referenced to residual non-deuterated solvent. Optical rota-
tions were measured using a JASCO DIP-370 polarimeter.
4.2. General procedure: condensation
A 4% solution of H-phosphonate (1.0–1.25 equiv) in dry THF was
treated with pyridine (50 equiv) and cooled to 0 °C. Quick addition
of a 2% solution of glycerol (1.0–1.25 equiv) in dry THF was fol-
lowed by immediate activation of the condensation with pivalyl
chloride (2.5 equiv) and removal of the ice-bath. The reaction mix-
ture was stirred for 10 min at rt. Subsequently, oxidation was ini-
tiated by addition of iodine solution (2.0 equiv, 0.197 M in
pyridine/water = 95:5) and the reaction mixture was stirred for
30 min at rt. The reaction mixture was quenched by addition of
aq Na2S2O3 solution (1 M). The water layer was washed twice with
CHCl3, and the combined organic layer was washed with NEt3/
H2CO3-buffer (1 M, pH 8.4), dried over Na2SO4, and concentrated
in vacuo. The residue was subjected to flash chromatography on
silica gel (CHCl3/MeOH/NEt3 = 7:1:1).
4.1. General procedure: phosphonylation
A 5% solution of trichloroacetimidate (1.0 equiv) in dry THF was
treated with phosphorous acid solution (7.1 equiv, 0.5 M in dry
THF) and stirred for 5 min at rt. Subsequently, the reaction mixture
was cooled to 0 °C, treated with triethylamine (21 equiv) and kept
without stirring at 0 °C for 1 h. The precipitate was filtered off, and
the filtrate was concentrated in vacuo. The residue was purified by
preparative TLC (toluene/MeOH/NEt3 = 3:1:2.5%).
4.2.1. (2-O-Arachidyl-1-O-stearyl-sn-glycer-3-yl) (6-O-acetyl-
2,3,4-tri-O-benzyl-b-D-glucopyranosyl)phosphate triethylam-
monium salt (26)
Prepared by condensation of compound 17 (15 mg, 23
lmol)
with compound 10 (19 mg, 29 mol) to give compound 26 as a
l

P. Greimel et al. / Bioorg. Med. Chem. 16 (2008) 7210–7217
7215
slightly yellow honey (16 mg, 12
l
mol, 53% yield). 1H NMR
(4C; Ac), 96.41 (d, 2J (C,P) = 4.2 Hz, 1C; C-1), 71.22 (1C; C-3),
71.13 (1C; C-5), 70.57 (d, 3J (C,P) = 7.5 Hz, 1C; sn-2), 69.74 (d, 3J
(C,P) = 7.5 Hz, 1C; C-2), 67.31 (1C; C-4), 63.98 (d, 2J (C,P) = 4.2 Hz,
1C; sn-3), 63.05 (1C; sn-1), 61.35 (1C; C-6), 45.63 (3C; NEt3),
34.61 (1C; Stea-2), 34.42 (1C; Ara-2), 32.25, 30.04, 29.86, 29.69,
29.50, 27.83, 23.04 (30C; Stea-4-17, Ara-4-19), 25.26 (1C; Stea-3),
25.23 (1C; Ara-3), 21.29, 21.03, 20.95 (4C; Ac), 14.49 (2C; Stea-
18, Ara-20), 9.08 (3C; NEt3); LRMS (ESI-TOF, neg) calcd for
(400 MHz, CDCl3, 25 °C): d = 7.37–7.20 (n.r., 15H; Bn), 5.23–5.15
(m, 1H; sn-2), 5.11 (dd, J = 7.8 Hz, 2J (H,P) = 7.8 Hz, 1H; H-1), 5.05
(d, J = 11.2 Hz, 1H; Bn), 4.90 (d, J = 11.0 Hz, 1H; Bn), 4.83 (d,
J = 10.7 Hz, 1H; Bn), 4.75 (d, J = 10.3 Hz, 1H; Bn), 4.73 (d,
J = 10.7 Hz, 1H; Bn), 4.54 (d, J = 11.0 Hz, 1H; Bn), 4.37 (dd,
J = 3.4 Hz, J=11.7 Hz, 1H; H-6b), 4.31 (dd, J = 3.1 Hz, J = 12.1 Hz,
1H; sn-1b), 4.15 (dd, J = 4.0 Hz, J = 11.8 Hz, 1H; H-6a), 4.07 (dd,
J = 7.0 Hz, J = 12.1 Hz, 1H; sn-1a), 3.99–3.93 (m, 2H; sn-3), 3.67
(dd, J = 8.8 Hz, J = 8.8 Hz, 1H; H-3), 3.59–3.56 (m, 1H; H-5), 3.52
(dd, J = 8.3 Hz, J = 10.0 Hz, 1H; H-4), 3.47 (dd, J = 8.3 Hz, J = 8.8 Hz,
1H; H-2), 2.98 (q, J = 7.3 Hz, 6H; NEt3), 2.22 (t, J = 7.7 Hz, 4H;
Stea-2, Ara-2), 1.99 (s, 3H; Ac), 1.56–1.54 (n.r., 4H; Stea-3, Ara-3),
1.26–1.17 (n.r., 69H; Stea-4-17, Ara-4-19, NEt3), 0.85 (t,
J = 6.8 Hz, 6H; Stea-18, Ara-20); 13C NMR (100 MHz, CDCl3,
25 °C): d = 173.35 (1C; Stea-1), 172.93 (1C; Ara-1), 170.67 (1C;
Ac), 138.78, 138.47, 137.86 (3C; Bn-ipso), 128.52, 128.43, 128.24,
128.17, 127.98, 127.86, 127.69, 127.41 (15C; Bn), 98.43 (d, 2J
(C,P) = 4.2 Hz, 1C; C-1), 84.73 (1C; C-3), 82.70 (d, 3J (C,P) = 7.5 Hz,
1C; C-2), 77.44 (1C; C-5), 75.89, 75.14, 74.62 (3C; Bn), 73.25 (1C;
C-4), 70.70 (d, 3J (C,P) = 10.0 Hz, 1C; sn-2), 63.95 (d, 2J
(C,P) = 5.0 Hz, 1C; sn-3), 63.12 (1C; sn-1), 62.89 (1C; C-6), 45.70
(3C; NEt3), 34.61 (1C; Stea-2), 34.42 (1C; Ara-2), 32.26, 30.06,
29.88, 29.70, 29.51, 27.65, 23.05 (30C; Stea-4-17, Ara-4-19),
25.24 (2C; Stea-3, Ara-3), 21.22 (1C; Ac), 14.51 (2C; Stea-18, Ara-
C
61H114NO17P [MÀHNEt3]À: 1061.7, found: 1061.6.
4.2.4. (2-O-Arachidyl-1-O-stearyl-sn-glycer-3-yl) (2,3,4,6-tetra-
O-benzyl-b-D-mannopyranosyl)phosphate triethylammonium
salt (33)
Prepared by condensation of compound 19 (13 mg, 19
with compound 10 (15 mg, 23 mol) to give compound 33 as a
slightly yellow honey (11 mg, 8.1
mol, 43% yield). 1H NMR
lmol)
l
l
(400 MHz, CDCl3, 25 °C): d = 7.38–7.16 (n.r., 20H; Bn), 5.69 (d, 3J
(H,P) = 7.6 Hz, 1H; H-1), 5.20–5.18 (n.r., 1H; sn-2), 4.89 (d,
J = 11.0 Hz, 1H; Bn), 4.74, 4.58 (s, 4H; Bn), 4.61 (d, J = 12.0 Hz,
1H; Bn), 4.52 (d, J = 11.5 Hz, 1H; Bn), 4.47 (d, J = 12.0 Hz, 1H; Bn),
4.34 (dd, J = 2.8 Hz, J = 12.1 Hz, 1H; sn-1b), 4.10 (dd, J = 7.0 Hz,
J = 12.1 Hz, 1H; sn-1a), 4.04–3.92 (n.r., 6H; H-2, H-3, H-4, H-5, sn-
3), 3.72–3.67 (m, 2H; H-6), 2.89 (q, J = 7.2 Hz, 6H; NEt3), 2.24 (t,
J = 7.3 Hz, 1H; Stea-2), 2.23 (t, J = 7.6 Hz, 1H; Ara-2), 1.54 (n.r.,
2H; Stea-3, Ara-3), 1.24–1.17 (n.r., 73H; Stea-4-17, Ara-4-19,
NEt3), 0.87 (t, J = 6.7 Hz, 6H; Stea-18, Ara-20); 13C NMR
(100 MHz, CDCl3, 25 °C): d = 173.34 (1C; Stea-1), 172.94 (1C; Ara-
1), 138.75, 138.66, 138.53, 138.32 (4C; Bn-ipso), 128.34, 128.28,
128.03, 127.86, 127.82, 127.61, 127.47 (20C; Bn), 94.31 (d, 2J
(C,P) = 3.3 Hz, 1C; C-1), 79.69 (1C; C-3), 75.46 (d, 3J (C,P) = 7.5 Hz,
1C; C-2), 75.10, 73.55, 72.62, 72.10 (4C; Bn), 74.94 (1C; C-4),
72.96 (1C; C-5), 70.63 (d, 3J (C,P) = 7.5 Hz, 1C; sn-2), 69.83 (1C; C-
6), 63.96 (d, 2J (C,P) = 3.3 Hz, 1C; sn-3), 63.03 (1C; sn-1), 45.52
(3C; NEt3), 34.62 (1C; Stea-2), 34.43 (1C; Ara-2), 32.26, 30.06,
29.88, 29.71, 29.50, 28.13, 23.06 (30C; Stea-4-17, Ara-4-19),
25.23 (2C; Stea-3, Ara-3), 14.51 (2C; Stea-18, Ara-20), 9.53 (3C;
NEt3); LRMS (ESI-TOF, neg) calcd for C81H130NO13P [MÀHNEt3]À:
1253.8, found: 1253.8.
20), 8.95 (3C; NEt3); LRMS (ESI-TOF, neg) calcd for C76H126NO14
P
[MÀHNEt3]À: 1205.8, found: 1205.8.
4.2.2. (2-O-Methyl-1-O-stearyl-sn-glycer-3-yl) (2,3,4,6-tetra-O-
acetyl-b-
Prepared by condensation of compound 21 (49 mg, 96
with compound 12 (29 mg, 77 mol) to give compound 31 as a
slightly yellow amorphous solid (46 mg, 52
mol, 68% yield). 1H
D-glucopyranosyl)phosphate triethylammonium salt (31)
l
mol)
l
l
NMR (400 MHz, CDCl3, 25 °C): d = 5.26 (dd, J = 8.1 Hz, 3J
(H,P) = 8.1 Hz, 1H; H-1), 5.19 (dd, J = 9.5 Hz, J = 9.5 Hz, 1H; H-4),
5.07 (dd, J = 9.8 Hz, J = 9.8 Hz, 1H; H-3), 5.00 (dd, J = 8.1 Hz,
J = 9.5 Hz, 1H; H-2), 4.27 (dd, J = 3.3 Hz, J = 11.8 Hz, 1H; sn-1b),
4.21 (dd, J = 4.2 Hz, J = 12.5 Hz, 1H; H-6b), 4.14 (dd, J = 2.2 Hz,
J = 12.5 Hz, 1H; H-6a), 4.05 (dd, J = 6.3 Hz, J = 11.7 Hz, 1H; sn-1a),
3.95 (ddd, J = 4.9 Hz, 3J (H,P) = 6.3 Hz, J = 11.0 Hz, 1H; sn-3b), 3.86
(ddd, J = 4.5 Hz, 3J (H,P) = 6.3 Hz, J = 12.2 Hz, 1H; sn-3a), 3.80
(ddd, J = 2.3 Hz, J = 4.0 Hz, J = 9.9 Hz, 1H; H-5), 3.60–3.57 (m, 1H;
sn-2), 3.40 (s, 3H; OMe), 2.98 (q, J = 7.2 Hz, 6H; NEt3), 2.29 (t,
J = 7.6 Hz, 2H; Stea-2), 2.04, 2.03, 2.00, 1.97 (s, 12H; Ac), 1.60–
1.54 (m, 2H; Stea-3), 1.27 (t, J = 7.2 Hz, 9H; NEt3), 1.23 (n.r., 28H;
Stea-4-17), 0.85 (t, J = 6.7 Hz, 3H; Stea-18); LRMS (ESI-TOF, neg)
calcd for C42H78NO16P [MÀHNEt3]À: 781.4, found: 781.3.
4.2.5. (R/S)-(2-O-Arachidyl-1-O-stearyl-sn-glycer-3-yl) (2,3,4,6-
tetra-O-acetyl-b-D-glucopyranosyl)phosphorothioate triethylam-
monium salt (30 RP/SP)
A solution of compound 21 (49 mg, 96 lmol) in dry THF (2 mL)
was treated with pyridine (310
Quick addition of a solution of compound 10 (50 mg, 77
dry THF (3 mL) was followed by immediate activation of the linkage
with pivalyl chloride (24 L, 0.19 mmol) and removal of the ice-
l
L, 3.9 mmol) and cooled to 0 °C.
lmol) in
l
bath. Thereactionmixturewasstirredfor 10 minat rt. Subsequently,
phosphorothioate formation was initiated by addition of a Beaucage
reagent14 solution (10 mL, 0.76 mmol, 76 mM in dry THF), and the
reaction mixture was stirred for 7.5 min at rt. The reaction mixture
was quenched by addition of NEt3/H2CO3-buffer (1 M, pH 8.4), and
extracted twice with CHCl3. The combined organic layer was dried
over Na2SO4 and concentrated in vacuo. The residue was subjected
to flash chromatography on silica gel (CHCl3/MeOH/
NEt3 = 25:1:0.5%) to give an inseparable mixture of compound 30
RP and compound 30 SP as a slightly yellow amorphous solid
4.2.3. (2-O-Arachidyl-1-O-stearyl-sn-glycer-3-yl) (2,3,4,6-tetra-O-
acetyl-b-D-galactopyranosyl) phosphate triethylammonium salt
(32)
Prepared by condensation of compound 23 (71 mg, 14
with compound 10 (60 mg, 92 mol) to give compound 32 as a
slightly yellow amorphous solid (75 mg, 64
mol, 70% yield). 1H
lmol)
l
l
NMR (400 MHz, CDCl3, 25 °C): d = 5.37 (dd, J = 2.0 Hz, 1H; H-4),
5.25 (d, J = 7.8 Hz, 3J (H,P) = 7.8 Hz, 1H; H-1), 5.20 (n.r., 1H; sn-2),
5.19 (dd, J = 7.9 Hz, J = 10.1 Hz, 1H; H-2), 5.01 (dd, J = 3.5 Hz,
J = 10.1 Hz, 1H; H-3), 4.35 (dd, J = 3.2 Hz, J = 12.0 Hz, 1H; sn-1b),
4.14 (dd, J = 7.1 Hz, J = 11.0 Hz, 1H; H-6b), 4.11 (dd, J = 7.1 Hz,
J = 12.0 Hz, 1H; sn-1a), 4.06 (dd, J = 6.1 Hz, J = 11.0 Hz, 1H; H-6a),
4.01–3.93 (m, 3H; H-5, sn-3), 2.97 (q, J = 7.2 Hz, 6H; NEt3), 2.26
(t, J = 7.4 Hz, 2H; Stea-2), 2.25 (t, J = 7.6 Hz, 2H; Ara-2), 2.11, 2.03,
2.00, 1.94 (s, 12H; Ac), 1.56–1.55 (n.r., 4H; Stea-3, Ara-3), 1.26–
1.16 (n.r., 69H; Stea-4-17, Ara-4-19, NEt3), 0.85 (t, J = 6.8 Hz, 6H;
Stea-18, Ara-20); 13C NMR (100 MHz, CDCl3, 25 °C): d = 173.39
(1C; Stea-1), 172.95 (1C; Ara-1), 170.30, 170.20, 169.95, 169.76
(72 mg, 61 lmol, 80% yield). [a]
20 = 9.5 (c 8.3 in CHCl3); 1H NMR
D
(400 MHz, CDCl3, 25 °C): d = 5.42 (dd, J = 8.1 Hz, 3J (H,P) = 11.0 Hz,
1H; H-1 (R/S)), 5.36 (dd, J = 8.2 Hz, 3J (H,P) = 11.4 Hz, 1H; H-1 (R/
S)), 5.24–5.19 (m, 4H; H-4 (R/S), sn-2 (R/S)), 5.08 (dd, J = 9.5 Hz,
J = 9.8 Hz, 2H; H-3 (R/S)), 5.03–4.99 (m, 2H; H-2 (R/S)), 4.33 (dd,
J = 3.2 Hz, J = 12.0 Hz, 1H; sn-1b (R/S)), 4.32 (dd, J = 2.9 Hz,
J = 12.2 Hz, 1H; sn-1b (R/S)), 4.22 (dd, J = 4.0 Hz, J = 12.6 Hz, 2H; H-
6b (R/S)), 4.17–4.07 (m, 8H; H-6a (R/S), sn-1a (R/S), sn-3 (R/S)), 3.78
(m, 2H; H-5 (R/S)), 2.90 (q, 12H; NEt3), 2.27–2.22 (m, 8H; Stea-2 (R/

7216
P. Greimel et al. / Bioorg. Med. Chem. 16 (2008) 7210–7217
S), Ara-2(R/S)), 2.05, 2.03, 2.02, 1.99, 1.99, 1.96, 1.96(s, 24H;Ac(R/S)),
1.56 (n.r., 8H; Stea-3 (R/S), Ara-3 (R/S)), 1.24–1.20(n.r., 138H; Stea-4-
17 (R/S), Ara-4-19 (R/S), NEt3), 0.85 (t, J = 6.8 Hz, 12H; Stea-18 (R/S),
Ara-20 (R/S)); 13C NMR (100 MHz, CDCl3, 25 °C): d = 173.37, 173.35
(2C; Stea-1 (R/S)), 172.95, 172.91 (2C; Ara-1 (R/S)), 170.53, 170.03,
169.96, 169.71, 169.56, 169.50, 169.44 (8C; Ac (R/S)), 96.47 (d, 2J
(C,P) = 3.3 Hz, 2C; C-1 (R/S)), 73.21 (2C; C-4 (R/S)), 72.32, 72.19 (4C;
C-5 (R/S)), 71.97 (d, 3J (C,P) = 8.3 Hz, 2C; C-2 (R/S)), 71.75 (d, 3J
(C,P) = 9.2 Hz, 2C; C-2 (R/S)), 70.46 (d, 3J (C,P) = 8.3 Hz, 1C; sn-2 (R/
S)), 70.30 (d, 3J (C,P) = 10.0 Hz, 1C; sn-2 (R/S)), 68.64, 68.55 (2C; C-3
(R/S)), 64.57 (d, 2J (C,P) = 5.7 Hz, 1C; sn-3 (R/S)), 64.52 (d, 2J
(C,P) = 5.0 Hz, 1C; sn-3 (R/S)), 63.08, 62.89 (2C; sn-1 (R/S)), 62.18,
61.97 (2C; H-6 (R/S)), 46.09 (6C; NEt3), 34.62, 34.60 (2C; Stea-2 (R/
S)), 34.43, 34.39 (2C; Ara-2 (R/S)), 32.24, 30.03, 29.86, 29.68, 29.49,
23.03 (56C; Stea-4-17, Ara-4-19 (R/S)), 25.24 (2C; Stea-2 (R/S)),
25.22 (2C; Ara-2 (R/S)), 21.31, 21.11, 21.08, 20.96 (8C; Ac (R/S)),
14.48 (4C; Stea-18, Ara-20 (R/S)), 9.79 (6C; NEt3); LRMS (ESI-TOF,
neg) calcd for C61H114NO16PS [MÀHNEt3]À: 1077.6, found: 1077.6.
1H; sn-1b), 3.96 (dd, J = 6.2 Hz, J = 11.7 Hz, 1H; sn-1a), 3.74 (ddd,
J = 5.5 Hz, 3J (H,P) = 5.5 Hz, J = 11.0 Hz, 1H; sn-3b), 3.69 (ddd,
J = 5.2 Hz, 3J (H,P) = 6.2 Hz, J = 11.2 Hz, 1H; sn-3a), 3.64 (d,
J = 11.7 Hz, 1H; H-6b), 3.52–3.49 (m, 1H; sn-2), 3.42 (dd,
J = 6.2 Hz, J = 11.7 Hz, 1H; H-6a), 3.30 (s, 3H; OMe), 3.12 (dd,
J = 8.2 Hz, J = 8.9 Hz, 1H; H-3), 3.09 (ddd, J = 2.3 Hz, J = 5.5 Hz,
J = 9.4 Hz, 1H; H-5), 3.04 (dd, J = 8.9 Hz, J = 9.6 Hz, 1H; H-4), 3.00
(dd, J = 7.6 Hz, J = 8.9 Hz, 1H; H-2), 2.46 (q, J = 7.1 Hz, 6H; NEt3),
2.29 (t, J = 7.2 Hz, 2H; Stea-2), 1.53–1.49 (m, 2H; Stea-3), 1.23
(n.r., 28H; Stea-4-17), 0.94 (t, J = 7.2 Hz, 9H; NEt3), 0.85 (t,
J = 7.2 Hz, 3H; Stea-18); 31P NMR (240 MHz, DMSO-d6, 25 °C):
d = À1.28 (1P; P); HRMS (ESI-TOF, neg) calcd for C348H70NO12
P
[MÀHNEt3]À: 613.3353, found: 613.3344.
4.3.3. (2-O-Arachidyl-1-O-stearyl-sn-glycer-3-yl) b-D-galacto-
pyranosyl phosphate triethylammonium salt (6)
Prepared by deacetylation of compound 32 (26 mg, 22
give compound 6 as a white amorphous solid (10 mg, 10
l
mol) to
l
mol, 43%
yield). 1H NMR (600 MHz, DMSO-d6, 25 °C): d = 6.23 (d, J = 1.5 Hz,
1H; OH-2), 5.08–5.05 (m, 1H; sn-2), 4.61 (d, J = 5.5 Hz, 1H; OH-3),
4.55 (dd, J = 6.6 Hz, 3J (H,P) = 7.6 Hz, 1H; H-1), 4.54 (dd, J = 5.5 Hz,
J = 6.6 Hz, 1H; OH-6), 4.29 (dd, J = 3.0 Hz, J = 12.1 Hz, 1H; sn-1b),
4.24 (d, J = 4.0 Hz, 1H; OH-4), 4.07 (dd, J = 7.3 Hz, J = 11.8 Hz, 1H;
sn-1a), 3.79 (ddd, J = 5.3 Hz, 3J (H,P) = 6.0 Hz, J = 11.3 Hz, 1H; sn-
3b), 3.73 (ddd, J = 5.3 Hz, 3J (H,P) = 6.0 Hz, J = 11.3 Hz, 1H; sn-3a),
3.59 (dd, J = 3.5 Hz, J = 4.0 Hz, 1H; H-4), 3.51 (ddd, J = 6.0 Hz,
J = 6.6 Hz, J = 10.9 Hz, 1H; H-6b), 3.44 (ddd, J = 5.3 Hz, J = 5.8 Hz,
J = 10.9 Hz, 1H; H-6a), 3.33–3.27 (n.r., 8H; H-2, H-5, NEt3), 3.24
(ddd, J = 3.5 Hz, J = 5.8 Hz, J = 9.3 Hz, 1H; H-3), 2.28 (t, J = 7.1 Hz,
2H; Stea-2), 2.25 (t, J = 6.8 Hz, 2H; Ara-2), 1.52–1.47 (n.r., 4 H;
Stea-3, Ara-4), 1.23 (s, 69H; Stea-4-17, Ara-4-19, NEt3), 0.85 (t,
J = 7.1 Hz, 6H; Stea-18, Ara-19); 31P NMR (240 MHz, DMSO-d6,
25 °C): d = À0.88 (1P; P); HRMS (ESI-TOF, neg) calcd for
4.3. General procedure: deacetylation
A 2% solution of acetylatedphosphate diester (1.0 equiv) in a mix-
ture of CHCl3/MeOH = 1:2.5 was treated with fresh N2H4/HOAc solu-
tion (equal volume as CHCl3; prepared by slow (Caution!—
exothermic reaction) treatment of hydrazine monohydrate (2.04 g,
40.0 mmol) with glacial acetic acid (0.627 g, 10.4 mmol)) and stirred
for 5 min at rt. Subsequently, the reaction mixture was quenched
with NEt3/H2CO3-buffer (1 M, pH 8.4). The resulting mixture was
washed twice with CHCl3/MeOH = 2:1, the combined organic layer
was dried over Na2SO4 and concentrated in vacuo. The residue was
purified by preparative TLC (CHCl3/MeOH/NEt3 = 5:1:1%) and subse-
quent gel filtration on LH-20 (CHCl3/MeOH/NEt3 = 1:1:0.5%).
4.3.1. (R/S)-(2-O-Arachidyl-1-O-stearyl-sn-glycer-3-yl) b-
D
-
C
53H106NO13P [MÀHNEt3]À: 893.6119, found: 893.6107.
glucopyranosyl phosphorothioate triethylammonium salt (4 RP/
SP)
4.4. General procedure: hydrogenation
Prepared by deacetylation of compound 30 RP/SP (55 mg,
47
l
mol) to give an inseparable mixture of 4 RP and compound 4 SP
A 0.5% solution of benzylated phosphate diester (1.0 equiv) in
ethanol was hydrogenated in the presence of palladium on acti-
vated charcoal (1.7 equiv; 10% as Pd) under an atmosphere of H2
at ambient pressure. The reaction mixture was stirred for 6 h,
and subsequently filtered over Celite. The filtrate was concentrated
in vacuo and the residue was purified by gel filtration on LH-20
(CHCl3/MeOH/NEt3 = 1:1:0.1%).
as a white amorphous solid (27 mg, 27
l
mol, 57% yield). 1H NMR
(600 MHz, DMSO-d6, 25 °C): d = 5.66 (d, J = 2.2 Hz, 1H; OH-2 (R/S)),
5.56 (d, J = 2.2 Hz, 1H; OH-2 (R/S)), 5.11–5.10 (m, 1H; sn-2 (R/S)),
5.08–5.07 (m, 1H; sn-2 (R/S)), 4.87 (d, J = 4.9 Hz, 1H; OH-3 (R/S)),
4.86 (d, J = 4.9 Hz, 1H; OH-3 (R/S)), 4.84 (d, J = 4.4 Hz, 1H; OH-4 (R/
S)), 4.83 (d, J = 4.9 Hz, 1H; OH-4 (R/S)), 4.82 (dd, J = 7.1 Hz, 3J
(H,P) = 8.8 Hz, 1H; H-1 (R/S)), 4.77 (dd, J = 7.7 Hz, 3J (H,P) = 8.8 Hz,
1H; H-1 (R/S)), 4.48 (t, J = 6.0 Hz, 1H; OH-6 (R/S)), 4.46 (t, J = 6.3 Hz,
1H; OH-6 (R/S)), 4.29 (dd, J = 2.7 Hz, J = 12.1 Hz, 1H; sn-1b (R/S)),
4.27 (dd, J = 3.0 Hz, J = 11.8 Hz, 1H; sn-1b (R/S)), 4.09 (dd, J = 7.4 Hz,
J = 11.8 Hz, 1H; sn-1a (R/S)), 4.07 (dd, J = 7.7 Hz, J = 12.1 Hz, 1H; sn-
1a (R/S)), 3.93–3.91 (m, 2 H; sn-3b (R/S)), 3.87–3.86 (m, 1H; sn-3a
(R/S)), 3.80–3.79 (m, 1H; sn-3a (R/S)), 3.66–3.59 (m, 2H; H-6b (R/
S)), 3.46–3.40 (m, 2H; H-6a (R/S)), 3.32 (n.r., 12H; NEt3), 3.17–3.11
(n.r., 2H; H-3 (R/S)), 3.09–3.05 (n.r., 4H; H-5 (R/S), H-4 (R/S)), 3.03–
2.99 (n.r., 2H; H-2 (R/S)), 2.28 (t, 4H; Stea-2 (R/S)), 2.25 (t, 4H; Ara-
2 (R/S)), 1.50–1.49 (n.r., 8H; Stea-3 (R/S), Ara-3 (R/S)), 1.23 (n.r.,
138H; Stea-4-17 (R/S), Ara-4-19 (R/S), NEt3), 0.85 (t, J = 6.9 Hz,
12H; Stea-18 (R/S), Ara-20 (R/S)); 31P NMR (240 MHz, DMSO-d6,
25°C): d = À5.51, À4.42 (2P; P (R/S)); HRMS (ESI-TOF, neg) calcd for
4.4.1. (2-O-Arachidyl-1-O-stearyl-sn-glycer-3-yl) (6-O-acetyl-b-
D
-glucopyranosyl) phosphate triethylammonium salt (27)
Prepared by hydrogenation of compound 26 (15 mg, 11 mol) to
give compound 27 as a white solid (11 mg, 11
mol, quant. yield). 1H
l
l
NMR (400 MHz, DMSO-d6, 25 °C): d = 6.31 (d, J = 2.7 Hz, 1H; OH-2),
5.10 (d, J = 5.4 Hz, 1H; OH-4), 5.07 (n.r., 1H; sn-2), 4.94 (d,
J = 4.5 Hz, 1H; OH-3), 4.62 (dd, J = 7.6 Hz, 3J (H,P) = 7.6 Hz, 1H; H-
1), 4.29 (dd, J = 1.8 Hz, J = 11.7 Hz, 1H; sn-1b), 4.25 (d, J = 12.6 Hz,
1H; H-6b), 4.06 (dd, J = 7.0 Hz, J = 11.2 Hz, 1H; sn-1a), 3.97 (dd,
J = 6.7 Hz, J = 11.7 Hz, 1H; H-6a), 3.79 (ddd, J = 5.6 Hz, 3J
(H,P) = 5.8 Hz, J = 11.5 Hz, 1H; sn-3b), 3.71 (ddd, J = 5.8 Hz, 3J
(H,P) = 5.8 Hz, J = 11.5 Hz, 1H; sn-3a), 3.44 (ddd, J = 1.8 Hz,
J = 6.7 Hz, J = 8.1 Hz, 1H; H-5), 3.33 (n.r., 6H; NEt3), 3.14 (ddd,
J = 5.1 Hz, J = 8.8 Hz, J = 10.3 Hz, 1H; H-3), 3.05 (ddd, J = 5.8 Hz,
J = 8.8 Hz, J = 9.9 Hz, 1H; H-4), 3.01 (ddd, J = 1.6 Hz, J = 7.6 Hz,
J = 8.9 Hz, 1H; H-2), 2.26 (t, J = 6.7 Hz, 2H; Stea-2), 2.25 (t,
J = 7.2 Hz, 2H; Ara-2), 2.00 (s, 3H; Ac), 1.50–1.49 (n.r., 4H; Stea-3,
Ara-3), 1.23 (n.r., 60H; Stea-4-17, Ara-4-19), 1.05 (t, J = 7.0 Hz, 9H;
NEt3), 0.85 (t, J = 6.5 Hz, 6H; Stea-18, Ara-20); 31P NMR (160 MHz,
DMSO-d6, 25 °C): d = À1.63 (1P; P); HRMS (ESI-TOF, neg) calcd for
C
53H106NO12PS [MÀHNEt3]À: 909.5891, found: 909.5886.
4.3.2. (2-O-Methyl-1-O-stearyl-sn-glycer-3-yl) b-D-glucopyranosyl-
phosphate triethylammonium salt (5)
Prepared by deacetylation of compound 31 (11 mg, 11
lmol) to
give compound 5 as a white amorphous solid (6.9 mg, 8.7
lmol,
78% yield). 1H NMR (600 MHz, DMSO-d6, 25 °C): d = 4.62 (dd, 3J
(H,P) = 6.9 Hz, J = 7.6 Hz, 1H; H-1), 4.16 (dd, J = 2.7 Hz, J = 11.7 Hz,
C
55H108NO14P [MÀHNEt3]À: 935.6229, found: 935.6239.

P. Greimel et al. / Bioorg. Med. Chem. 16 (2008) 7210–7217
7217
4.4.2. (2-O-Arachidyl-1-O-stearyl-sn-glycer-3-yl) b-
mannopyranosyl phosphate triethylammonium salt (7)
Prepared by hydrogenation of compound 33 (11 mg, 8.1
give compound 7 as a white amorphous solid (7.8 mg, 7.8
D
-
many) and developed simultaneously with CHCl3/MeOH/HOAc/
water = 65:35:4:4. Subsequently, one TLC plate was sprayed with
Orcinol reagent for visualization of sugar residue. The second TLC
plate was soaked in 1% BSA in PBS for 1 h to block non-specific
antibody binding, then reacted with DIM21 antibody, prepared
l
mol) to
l
mol, 97%
yield). 1H NMR (600 MHz, DMSO-d6, 25 °C): d = 5.15 (dd, J = 1.8 Hz, 3J
(H,P) = 8.3 Hz, 1H; H-1), 5.06–5.02 (m, 1H; sn-2), 4.60 (d, J = 4.0 Hz,
1H; OH-2), 4.59 (d, J = 5.0 Hz, 1H; OH-4), 4.39 (d, J = 6.6 Hz, 1H;
OH-3), 4.34 (dd, J = 5.0 Hz, J = 5.0 Hz, 1H; OH-6), 4.30 (dd,
J = 2.5 Hz, J = 12.1 Hz, 1H; sn-1 b), 4.08 (dd, J = 5.5 Hz, J = 11.1 Hz,
1H; sn-1a), 3.74 (ddd, J = 5.0 Hz, 3J (H,P) = 5.8 Hz, J = 11.1 Hz, 1H;
sn-3b), 3.68 (ddd, J = 5.0 Hz, 3J (H,P) = 6.0 Hz, J = 10.8 Hz, 1H; sn-
3a), 3.59 (ddd, J = 2.1 Hz, J = 5.4 Hz, J = 11.5 Hz, 1 H; H-6b), 3.57–
3.56 (m, 1H; H-2), 3.49–3.35 (n.r., 4H; H-3, H-4, H-5, H-6a), 3.33
(n.r., 6H; NEt3), 2.27 (t, J = 7.3 Hz, 2H; Stea-2), 2.25 (t, J = 7.6 Hz,
2H; Ara-2), 1.52–1.46 (n.r., 4H; Stea-3, Ara-3), 1.23 (n.r., 69H; Stea-
4-17, Ara-4-19, NEt3), 0.85 (t, J = 7.1 Hz, 6H; Stea-18, Ara-20); 31P
NMR (240 MHz, DMSO-d6, 25 °C): d = À2.52 (1P; P); HRMS (ESI-
TOF, neg) calcd for C53H106NO13P [MÀHNEt3]À: 893.6119, found:
893.6118.
according to literature procedure,5 (40
lg/mL, diluted with ‘Can
Get Signal’ solution 1 (Toyobo)) overnight. After three repeated
washings with PBS for 5 min, the TLC plate was incubated with
HRP-conjugated anti-mouse Igs (antibody to IgA, IgG, and IgM)
(Cappel, 1:500 with ‘Can Get Signal’ solution 2 (Toyobo)) for at
least 3 h. The immunoreactive spots were visualized by 4-chloro-
1-naphthol substrate.
Acknowledgments
This work was partially supported by the Presidential Research
Fund of RIKEN. The authors thank Ms. Akemi Takahashi for techni-
cal assistance and Dr. Hiroyuki Koshino for acquiring high field
NMR.
4.5. General procedure: anion exchange
Supplementary data
A 0.2% solution of phosphore diester triethylammonium salt (1.0
equiv) in a mixture of CHCl3/MeOH = 1:1 was passed over an ion ex-
change resin column (Dowex 50WX8 sodium form, 100–200 mesh).
Subsequently, the column was extensively rinsed with CHCl3/
MeOH = 1:1 mixture, and the combined eluate was concentrated
in vacuo.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2008.06.041.
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Samples were spotted with indicated concentration on two
independent TLC plates (Polygram Sil G, Macherey-Nagel, Ger-