Synthesis of a polyphosphorylated GPI-anchor core structure

Article abstract of DOI:10.1139/v02-160

Using a linear assembly approach a highly differentially protected derivative of the common GPI-anchor core structure (α-D-Man-(1→6)-α-D-Man-(1→2)-α-D-Man-(1→4)- α-D-GlcNH₂-(1→6)-D-myo-inositol) has been synthesized. All mannose donors were pre

Full text of DOI:10.1139/v02-160

1105
Synthesis of a polyphosphorylated GPI-anchor
core structure
Martina Lahmann, Per J. Garegg, Peter Konradsson, and Stefan Oscarson
Abstract: Using a linear assembly approach a highly differentially protected derivative of the common GPI-anchor core
structure (ꢀ-D-Man-(1J6)-ꢀ-D-Man-(1J2)-ꢀ-D-Man-(1J4)-ꢀ-D-GlcNH2-(1J6)-D-myo-inositol) has been synthesized.
All mannose donors were prepared from a common thioglycoside precursor (1), and coupled to GlcN3-myo-inositol ac-
ceptor 5 in a linear five-step glycosylation–deprotection sequence in 49% overall yield, to give the key intermediate 10,
with orthogonal temporary protecting groups at the 6>>, 2>>, 6>, and 2 positions of the trimannoside motif and at the 1
and 2 positions of the inositol part. Consecutive removal of the temporary protecting groups in the trimannoside moiety
followed by phosphorylation, gave a tetraphosphosphate derivative in 60% overall yield. Removal of a camphor acetal
afforded a 1,2-inositol diol, which was converted to a 1,2-cyclic phosphate using commercial methyl dichlorophosphate
(J17, 95%). One-step deprotection using sodium in liquid ammonia afforded the target polyphosphorylated core struc-
ture 18 (60%), which will be tested for metabolic insulin action.
Key words: glycophosphatidylinositols, linear synthesis, glycosylations, inositolphosphoglycans, IPG.
Résumé : Faisant appel à une approche d’assemblage linéaire, on a réalisé la synthèse d’un dérivé, portant des groupes
protecteurs fortement diversifiés, de la structure ancrée fondamentale du GPI, le ꢀ-D-Man-(1J6)-ꢀ-D-Man-(1J2)-ꢀ-D-
Man-(1J4)-GlcNH2-(1J6)-D-myo-inositol. Tous les groupes mannoses donneurs ont été préparés à partir d’un
thioglycoside précurseur commun, 1, et ils ont été couplés à un GlcN3-myo-inositol accepteur, 5, dans une séquence en
cinq étapes de glycosylation-déprotection, avec un rendement global de 49%, pour conduire à l’intermédiaire clé 10
portant des groupes protecteurs orthogonaux temporaires dans les positions 6>>, 2>>, 6> et 2 du motif trimannoside et
dans les positions 1 et 2 de la portion inositol. Les éliminations consécutives des groupes protecteurs temporaires de la
portion mannoside, suivies d’une phosphorylation, on conduit au dérivé tétraphosphate, avec un rendement global de
60%, à partir duquel on a enlevé un acétal du camphre pour obtenir un diol 1,2-inositol qui a été transformé en un
phosphate 1,2-cyclique à l’aide de dichlorophosphate de méthyle commercial (J17, 95%). Une déprotection en une
étape à l’aide de sodium dans l’ammoniac liquide fournit le structure fondamentale phosphorylée recherchée, 18, avec
un rendement de 60% qui a été évaluée pour son action d’insuline métabolique.
Mots clés : glycophosphatidylinositols, synthèse linéaire, glycosylations, inositolphosphoglycanes, IPG.
[Traduit par la Rédaction] Lahmann et al. 1111
Introduction
bone, ꢀ-D-Man-(1J6)-ꢀ-D-Man-(1J2)-ꢀ-D-Man-(1J4)-ꢀ-
D-GlcNH2-(1J6)-D-myo-inositol, but the substitution pattern
varies between species (Fig. 1). Apart from functioning as
protein anchors, a number of functions have been suggested
for the GPI-structures (2, 3), such as they have been impli-
cated as second messengers to insulin (4–6) and poly-
phosphorylated derivatives have been shown to give an
insulin-type effect and have been considered as drug candi-
dates towards diabetes (7).
The novelty and complexity of these structures in combi-
nation with the interest to investigate more in detail their bi-
ological functions have resulted in a number of syntheses of
parts of the backbone, the complete backbone, and also the
backbone with various substituents (8–10). The latter syn-
theses have generally been directed towards a species spe-
cific target molecule not allowing too much variation in the
substitution pattern. Recently, Ley and co-workers (10) pre-
sented a synthesis of the backbone with orthogonal tempo-
rary protecting groups at relevant positions allowing the
continued synthesis of a variant of species specific GPI-
anchor structures. Another feature of their synthesis is that
the orthogonal and one-pot glycosylations are used in the
Ever since the discovery of a new way to anchor proteins
to cell membranes through glycosylphosphatidylinositol
structures (GPI-anchors, see refs. 1–3), these anchor struc-
tures have been of a main research interest, both analytically,
synthetically, and biologically. Isolation and analysis have
shown that these structures are present in almost all species,
and that their structure is not only partly conserved but also
species specific (2, 3). All GPI-anchors found and character-
ized so far contain a common pseudo-pentasaccharide back-
Received 28 August 2001. Published on the NRC Research
Press Web site at http://canjchem.nrc.ca on 29 August 2002.
Dedicated to the memory of Professor Raymond U. Lemieux.
M. Lahmann, P.J. Garegg, and S. Oscarson.1 Department
of Organic Chemistry, Arrhenius Laboratory, Stockholm
University, S-106 91 Stockholm, Sweden.
P. Konradsson. Department of Chemistry/IFM, Linköping
University, S-581 83 Linköping, Sweden.
1Corresponding author (e-mail: s.oscarson@organ.su.se).
Can. J. Chem. 80: 1105–1111 (2002)
DOI: 10.1139/V02-160
© 2002 NRC Canada

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Can. J. Chem. Vol. 80, 2002
Fig. 1. Schematic structure of GPI anchors.
Scheme 1. Reagents and conditions: (a) TBDMSCl, pyridine;
(b) BzCl, pyridine; (c) TBDPSCl, pyridine; (d) MBnCl, NaH,
0°C; (e) TBAF, THF; ( f ) AlIBr, NaH, DMF, 0°C.
by DDQ treatment (J 9, 72%) and subsequent glycosylation
with thioglycoside 4 gave the target intermediate 10 in
84% yield and a 49% overall yield over the five steps. The
ꢀ-configuration of all glycosidic linkages was proven by the
1JC,H coupling constants of the anomeric carbons, which all
were above 168 Hz.
In compound 10 all the functionalities are now in place to
allow continued syntheses of many different GPI anchor
structures. To prove the orthogonality of the temporary pro-
tecting groups, the groups were removed one by one in se-
quence to produce a tetraol (Scheme 3). The allyl group was
removed under neutral conditions using an iridium catalyst
followed by hydrolysis of the obtained vinyl ether with NIS–
H2O (15) (J 11, 98%). Removal of the p-methoxybenzyl
protecting group was accomplished by DDQ treatment to
give diol 12 (84%). The benzoate was removed using
Zemplén conditions (J 13, 85%) and finally the silyl group
was cleaved by TBAF treatment to give the tetraol 14 (86%)
in an overall deprotection yield of 60%.
As mentioned, GPI structures have been suggested as in-
sulin second messengers (4, 5). A key structure is believed
to be a 1,2-cyclic phosphate on the insulin moiety obtained
through enzymatic cleavage of the native glycerol fragment
(6). Additional phosphorylation has been shown to enhance
the biological activity (7). Accordingly, the tetraol was
phosphorylated using the amidite approach (16) to give
benzyl protected 6>>>,2>>>,6>>,2>-tetraphosphate derivative 15
in quantitative yield, which was subsequently processed to
give the corresponding 1,2-cyclic phosphate 17 (Scheme 4).
Acidic hydrolysis of the campher acetal (J 16, 46%) fol-
lowed by treatment with commercial methyl dichloro-
phosphate afforded 17 in 95% yield after work-up. Catalytic
hydrogenolysis to remove benzyl protecting group is often
troublesome with phosphate-containing compounds; how-
ever, as found earlier (17, 18), Birch-type reaction condi-
tions smoothly removed all benzyl groups without any
indication (NMR, MALDI-TOF MS) of phosphate migration
or hydrolysis or opening of the cyclic phosphate. Concomi-
tantly, the azido function was reduced, in a one-pot reaction,
to yield target structure 18 in 60% yield.
build-up of the target structures, utilizing the different reac-
tivity of both the uniquely protected donors and the variety
of donors. Although conceptually elegant, such an approach
requires considerable fine-tuning of the reactivity to be suc-
cessful. We herein describe a synthesis, using a linear syn-
thetic approach, of a similar highly and differentially
protected backbone structure that, will allow for the synthe-
ses of most types of species-specific structures from the
same intermediate. Furthermore, the temporary protecting
groups were sequentially removed and the resulting hydroxyl
derivatives were phosphorylated and deprotected to give a
polyphosphorylated structure, which will be investigated for
insulin activity.
Results and discussions
The linear approach can have considerable advantages, es-
pecially since the target structure is a rather small molecule
containing no repeating units (at least no larger than a
monosaccharide). Only monosaccharide donors have to be
prepared and all mannose donors can be prepared from a
common precursor. The protecting-group strategy can be
planned to be optimal (selective and complete) in terms of
protection and deprotection, since almost no considerations
have to be taken to the reactivity or stereochemical influence
of the protecting-group pattern in the various mannose do-
nors. In the coupling reactions, self condensation and
transglycosylation reactions, often found side reactions in
orthogonal glycosylations, are not possible, which results in
excellent glycosylation yields.
As donors we chose thioglycosides (11, 12). All the three
different mannose donors could be obtained in a few high-
yielding steps from the same precursor: ethyl 3,4-di-O-
benzyl-1-thio-ꢀ-D-mannopyranoside (13) (1) (Scheme 1).
Regioselective silylation at the primary position followed by
benzoylation or p-methoxybenzylation gave donors 2 (80%)
and 3 (85%), respectively. Subsequent desilylation and
allylation afforded mannose donor 4 (64% overall yield).
With these donors in hand the pseudo-pentasaccharide could
then be assembled through three efficient glycosylations all
promoted by DMTST in diethyl ether to ensure ꢀ-selectivity
(Scheme 2). Acceptor 5 (14) and donor 2 were coupled to
give pseudo-trisaccharide 6 in 98% yield. Removal of the
TBDMS-protecting group afforded acceptor 7 (85%), which
was condensed with donor 3 to yield the tetrasaccharide 8 in
quantitative yield. Removal of the p-methoxybenzyl group
Conclusion
In summary, a most effective linear synthesis of a highly
differentially protected backbone structure of GPI anchors
has been performed. The temporary protecting groups can be
removed in an orthogonal fashion to allow synthesis of
© 2002 NRC Canada

Lahmann et al.
1107
Scheme 2. Reagents and conditions: (a) DMTST, Et2O, MS (4 Å); (b) TBAF, THF; (c) DDQ, CH2Cl2–H2O.
formed on silica gel (Matrix Silica Si 60A, 35–70m,
Amicon). MALDI-TOF spectra were recorded on a Bruker
Biflex III using PEG 1500 as calibration reference.
Scheme 3. Reagents and conditions: (a) (i) Pd/C, iridium cata-
lyst, H2, (ii) NIS, H2O; (b) DDQ, CH2Cl2–H2O; (c) NaOMe,
MeOH; (d) TBAF, THF.
Ethyl 2-O-benzoyl-3,4-di-O-benzyl-6-O-tert-
butyldimethylsilyl-1-thio-ꢀ-D-mannopyranoside (2)
TBDMSCl (325 mg, 2.16 mmol) and a catalytic amount of
DMAP were added to a solution of ethyl 3,4-di-O-benzyl-1-
thio-ꢀ-D-mannopyranoside (13) (1, 550 mg, 1.36 mmol) in
pyridine (2 mL). The reaction mixture was stirred at room
temperature for about 3 h when benzoyl chloride (1 mL) was
added and stirring was continued for an additional 45 min.
For work up, the mixture was diluted with toluene, washed
with water and brine, dried (MgSO4), and concentrated. To
remove the main part of pyridine, co-evaporation with toluene
was performed twice before applying the residue to flash
chromatography (light petroleum (bp 40ꢂ65°C) – Et2O, 5:1)
to give 2 (680 mg, 1.09 mmol, 80%). [ꢀ]D +39 (c 1.0,
1
CHCl3). H NMR (CDCl3) ꢁ: 0.06 (s, 6H, Me2Si), 0.92 (s,
9H, t-BuSi), 1.30 (m, 3H, MeCH2S), 2.65 (m, 2H, SCH2),
3.80ꢂ4.10 (m, 5H), 4.54, 4.65, 4.76, 4.92 (4 × d, 4 × 1H,
PhCH2), 5.38 (s, 1H, H-1), 5.68 (br s, 1H, H-2) 7.18–8.12 (m,
13H), 8.12 (m, 2H). 13C NMR (CDCl3) ꢁ: ꢂ 4.9 (Me2Si), 15.1
(MeCH2S), 18.5 (t-BuSi), 25.6 (SCH2), 26.1 (t-BuSi), 62.4,
71.2, 71.8, 73.2, 74.6, 75.3, 78.9 (C2–C6, CH2Ph), 82.5 (C1),
127.8–138.8 (aromatic C), 165.9 (PhCO). Anal. calcd. for
C35H46O6SSi: C 67.49, H 7.44; found: C 67.38, H 7.56.
various substituted anchor structures. The partly deprotected
structures can be polyphosphorylated and deprotected using
Birch-type reaction conditions in high yields, to give syn-
thetic phosphoinositolglycans.
Experimental
All organic solvents were distilled before use, except
Et2O, which was stored over Na. Organic solutions were
dried over MgSO4 before concentration, which was per-
formed under reduced pressure at <40°C (bath temperature).
NMR spectra were recorded at 270 (JEOL), 300, 400, or
600 MHz (Varian) (1H), or at 67.5, 75, or 100 MHz (13C),
unless otherwise stated, in CDCl3. For 1H NMR spectra,
TMS was used as internal standard (ꢁ = 0). 13C NMR spectra
were referred to the chloroform signal (ꢁ = 77.17). 31P NMR
spectra were standardized by using an external H3PO4 sam-
ple (ꢁ = 0). TLC was performed on silica gel 60 F254
(Merck) plates with detection by UV-light and (or) charring
with 8% sulfuric acid. Column chromatography was per-
Ethyl 3,4-di-O-benzyl-6-O-tert-butyldiphenylsilyl-2-O-p-
methoxybenzyl-1-thio-ꢀ-D-mannopyranoside (3)
TBDPSCl (1.3 mL, 4.9 mmol) and a catalytic amount of
DMAP were added to a solution of 1 (1.35 g, 3.34 mmol) in
pyridine (10 mL). The reaction mixture was stirred at room
temperature for about 24 h, then diluted with toluene
and washed with brine and water, dried (MgSO4), and
filtered. The filtrate was concentrated and the pyridine
co-evaporated with toluene. The crude product was purified
by flash-chromatography (toluene–EtOAc, 3:1) to give ethyl
3,4-di-O-benzyl-6-O-tert-butyldiphenylsilyl-1-thio-ꢀ-D-manno-
© 2002 NRC Canada

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Can. J. Chem. Vol. 80, 2002
Scheme 4. Reagents and conditions: (a) (i) DBDIPPA, tetrazole, (ii) MCPBA; (b) TFA; (c) (i) MeOPOCl2, pyridine, (ii) HCl; (d) Na,
NH3 (l).
pyranoside (1.98 g, 3.08 mmol, 92%). [ꢀ]D +70 (c 1.0,
CH2Cl2). 13C NMR (CDCl3) ꢁ: 14.8 (MeCH2S), 19.4 (t-
BuSi), 24.6 (SCH2), 26.7, 26.9 (t-BuSi), 63.2 (C6), 70.1,
72.3, 72.9, 74.7, 75.3, 80.7, (C2–C5, CH2Ph), 82.8 (C1),
127.7–136.0 (aromatic C). A solution of this derivative
(1.47 g, 2.29 mmol) in dry DMF (20 mL) was cooled to
0°C. NaH (190 mg, ~60% dispersed in oil) was added and
the mixture was stirred for 30 min. Freshly prepared p-
methoxybenzyl bromide (1.0 mL), dissolved in DMF
(10 mL), was added dropwise. After 3 h the reaction was
quenched by addition of MeOH (1 mL), diluted with tolu-
ene, washed with water and brine, dried (MgSO4), and con-
centrated. Purification by flash chromatography (toluene–
EtOAc, 18:1) gave 3 (1.60 g, 2.10 mmol, 92%). [ꢀ]D +41
was purified by flash chromatography (toluene J toluene–
EtOAc, 18:1) to yield ethyl 3,4-di-O-benzyl-2-O-p-methoxy-
benzyl-6-O-tert-butyldimethylsilyl-1-thio-ꢀ-D-mannopyranoside
(1.16 g, 1.82 mmol, 94%). [ꢀ]D +25 (c 1.0, CH2Cl2). 13C NMR
(CDCl3) ꢁ: 15.0 (MeCH2S), 18.5 (t-BuSi), 25.0 (SCH2), 26.1
(t-BuSi), 55.4 (MeOBn), 62.9 (C6), 71.6, 72.2, 73.6, 75.3,
76.1, 77.4, 80.5, 81.4 (C1–C5, CH2Ph, MBn), 113.9 (MBn),
127.7–130.4, 138.7, 138.9 (aromatic C), 159.3 (MBn). This
compound (1.10 g, 1.72 mmol) dissolved in a TBAF–THF
solution (3.6 mL, 1 M) was stirred at room temperature
overnight. After concentration, the residue was applied to
flash chromatography (toluene J toluene–EtOAc, 12:1 J
toluene–EtOAc, 3:1) to obtain ethyl 3,4-di-O-benzyl-2-O-p-
methoxybenzyl-1-thio-ꢀ-D-mannopyranoside (780 mg,
1.49 mmol, 87%). 13C NMR (CDCl3) ꢁ: 15.0 (MeCH2S),
25.5 (SCH2), 55.4 (MeOBn), 62.5 (C6), 72.1, 72.2, 72.5,
75.2, 76.2, 80.5, 82.4 (C1–C5, CH2Ph, MBn), 114.0 (MBn),
127.8–129.7, 138.4, 138.4 (aromatic C), 159.2 (MBn). This
compound (780 mg, 1.49 mmol) was dissolved in dry DMF
(5 mL) and cooled to 0°C. NaH (120 mg, -60% dispersed in
oil) and allyl bromide (300 L, 3.55 mmol) were added and
the reaction mixture was allowed to reach room temperature.
After 2 h the reaction was quenched by addition of MeOH
(1 mL). The mixture was diluted with toluene, washed with
water, dried (MgSO4), and concentrated. Purification by flash
chromatography (toluene–EtOAc, 12:1) gave 4 (792 mg,
1.40 mmol, 94%). [ꢀ]D +57 (c 1.0, CH2Cl2). 1H NMR
(CDCl3) ꢁ: 1.24 (m, 3H, MeCH2S), 2.58 (m, 2H, SCH2),
3.60–3.84 (m, 7H), 3.90–4.18 (m, 4H), 4.48ꢂ4.74 (m, 6H),
4.92 (d, 1H, ArCH2), 5.14 (m, 1H), 5.26 (m, 1H), 5.36 (s,
1H, H-1), 5.94 (m, 1H), 6.80–6.84 (m, 2H), 7.16–7.40 (m,
12H). 13C NMR (CDCl3) ꢁ: 15.2 (MeCH2S), 25.5 (SCH2),
55.4 (MeOBn), 69.4 (C6), 71.6, 71.9, 72.0, 72.5, 75.2, 75.3,
75.8, 80.4, 81.9 (C1–C5, CH2Ph, MBn, allyl), 113.8 (MBn),
116.8 (allyl), 127.86–130.2 (aromatic C), 134.9 (allyl),
138.4, 138.7 (aromatic C), 159.2 (MBn). Anal. calcd. for
C33H40O6S: C 70.18, H 7.14; found: C 69.88, H 7.08.
1
(c 1.0, CHCl3). H NMR (CDCl3) ꢁ: 1.06 (s, 9H, t-BuSi),
1.22 (m, 3H, MeCH2S), 2.56 (m, 2H, SCH2), 3.68ꢂ4.10 (m,
8H), 4.46ꢂ4.76 (m, 5H, ArCH2), 4.92 (d, 1H, ArCH2), 5.38
(s, 1H, H-1), 5.68 (br s, 1H, H-2), 6.74ꢂ7.80 (m, 24H).
13C NMR (CDCl3) ꢁ: 15.0 (MeCH2S), 19.4 (t-BuSi), 25.1
(SCH2), 26.9 (t-BuSi), 55.4 (MeOBn), 63.4 (C6), 71.8, 72.3,
73.5, 75.1, 75.3, 76.5, 80.6, 81.4 (C1–C5, CH2Ph, MBn),
113.9 (MBn), 127.7–138.7 (aromatic C), 159.2 (MBn).
Anal. calcd. for C46H54O6SSi: C 72.40, H 7.13; found:
C 72.27, H 7.28.
Ethyl 6-O-allyl-3,4-di-O-benzyl-2-O-p-methoxybenzyl-1-
thio-ꢀ-D-mannopyranoside (4)
TBDMSCl (920 mg, 6.1 mmol) and a catalytic amount of
DMAP were added to a solution of 1 (1.6 g, 4.0 mmol) in
pyridine (10 mL). The reaction mixture was stirred at room
temperature for about 3 h and then diluted with toluene,
washed with water and dried over MgSO4. After filtration,
the mixture was concentrated and co-evaporated from tolu-
ene. Subsequent purification by flash-chromatography (tolu-
ene–EtOAc, 9:1) of the crude product afforded ethyl 3,4-di-
O-benzyl-6-O-tert-butyldimethylsilyl-1-thio-ꢀ-D-mannopyra-
noside (1.7 g, 3.3 mmol, 83%) as a colourless syrup. A solu-
tion of this derivative (1.00 g, 1.93 mmol) in dry DMF
(20 mL) was cooled to 0°C. NaH (120 mg, -60% dispersed
in oil) was added and the mixture was stirred for 30 min.
Freshly prepared p-methoxybenzyl bromide (750 L) dis-
solved in DMF (10 mL) was added dropwise. After 30 min
the reaction was quenched by addition of MeOH (1 mL), di-
luted with toluene and washed with water and brine. Drying
(MgSO4), filtration, and evaporation gave a residue, which
(2-O-Benzoyl-3,4-di-O-benzyl-6-O-tert-butyldimethylsilyl-
ꢀ-D-mannopyranosyl)-(1J4)-(2-azido-3,6-di-O-benzyl-2-
deoxy-ꢀ-D-glucopyranosyl)-(1J6)-3,4,5-tri-O-benzyl-1,2-
O-(L-1,7,7-trimethyl[2.2.1]bicyclohept-6-yliden)-D-myo-
inositol (6)
A solution of 5 (14) (46 mg, 48 mol) and 2 (30 mg,
48 mol) in dry Et2O (10 mL) was stirred with powdered
© 2002 NRC Canada

Lahmann et al.
1109
molecular sieves (4 Å) under argon for 1 h when DMTST
(41 mg, 158 mol) was added. The reaction, followed by
TLC (toluene–EtOAc, 12:1), was quenched after 3.5 h by
addition of Et3N (100 L), and then the mixture was diluted
with Et2O (20 mL), filtered through Celite, and concen-
trated. The residue was subjected to silica gel column chro-
matography (toluene–EtOAc, 12:1) to obtain 6 (71 mg,
(3,4-Di-O-benzyl-6-O-tert-butyldiphenylsilyl-2-O-p-
methoxybenzyl-ꢀ-D-mannopyranosyl)-(1J2)-(6-O-allyl-
3,4-di-O-benzyl-ꢀ-D-mannopyranosyl)-(1J6)-(2-O-
benzoyl-3,4-di-O-benzyl-ꢀ-D-mannopyranosyl)-(1J4)-(2-
azido-3,6-di-O-benzyl-2-deoxy-ꢀ-D-glucopyranosyl)-
(1J6)-3,4,5-tri-O-benzyl-1,2-O-(L-1,7,7-trimethyl[2.2.1]bi-
cyclohept-6-yliden)-D-myo-inositol (10)
1
47 mol, 98%). [ꢀ]D +42 (c 1.0, CH2Cl2). H NMR (CDCl3)
DDQ (17 mg, 75 mol) was added to a solution of 8
(90 mg, 47 mol) in water saturated CH2Cl2 (5% H2O,
5 mL) and stirred overnight at room temperature. The mix-
ture was diluted with CH2Cl2 (15 mL), washed with water
and Na2S2O3 (10% aq), dried (MgSO4), and concentrated.
The crude product was purified by flash chromatography
(toluene–EtOAc, 6:1) to give 60 mg (34 mol, 72%) of (6-
O-allyl-3,4-di-O-benzyl-ꢀ-D-mannopyranosyl)-(1J6)-(2-O-
benzoyl-3,4-di-O-benzyl-ꢀ-D-mannopyranosyl)-(1J4)-(2-
azido-3,6-di-O-benzyl-2-deoxy-ꢀ-D-glucopyranosyl)-(1J6)-
3,4,5-tri-O-benzyl-1,2-O-(L-1,7,7-trimethyl[2.2.1]bicyclohept-
6-yliden)-D-myo-inositol (9). [ꢀ]D +51 (c 1.0, CH2Cl2).
13C NMR (CDCl3) ꢁ: 10.0, 20.6, 20.9, 27.2, 30.0, 45.0, 45.3,
48.2, 51.8 (Cph), 63.4ꢂ80.8 (Cino1–Cino6, C2–C6, C2>–C6>,
C2>>–C6>>, CH2Bn, allyl), 95.6 (C1), 98.4 (C1>>), 99.1 (C1>),
116.9 (allyl), 118.2 (Cph-acetal), 125.3–138.7 (aromatic C,
allyl), 165.3 (PhCO). A solution of 9 (300 mg, 168 mol)
and 3 (130 mg, 170 mol) in dry Et2O (80 mL) was stirred
with powdered molecular sieves (4 Å) under argon for 1 h.
Then DMTST (150 mg, 0.58 mmol) was added. The reaction,
monitored by TLC (light petroleum (bp 40–60°C) – Et2O,
1:1), was quenched after 45 min by addition of Et3N
(500 L) and stirred for another 20 min. The mixture was di-
luted with Et2O (20 mL), filtered through Celite, and con-
centrated. The residue was subjected to silica gel column
chromatography (toluene–EtOAc, 12:1) to obtain 350 mg
ꢁ: 0.02 (s, 6H, Me2Si), 0.74ꢂ2.10 (m, 27H, t-BuSi, Cph),
3.24ꢂ4.98 (m, 29H), 5.50 (s, 1H), 5.61 (d, J = 1 Hz, 1H)
5.66 (s, 1H), 7.00–7.66 (m, 38H), 8.06 (d, 2H). 13C NMR
(CDCl3) ꢁ: ꢂ 4.9 (Me2Si), 10.1, 20.7, 20.9 (Cph), 26.2 (t-
BuSi), 27.2, 30.1, 44.9, 45.3, 48.2, 51.8 (Cph), 61.8, 63.4,
68.6–76.7, 78.3–80.8 (Cino1–Cino6, C2–C6, C2>–C6>,
CH2Bn), 95.7 (C1) 99.3 (C1>), 118.1 (Cph-acetal), 127.5–
139.1 (aromatic C), 165.4 (PhCO). Anal. calcd. for
C90H105N3O16Si: C 71.45, H 7.00; found: C 71.28, H 6.84.
(6-O-Allyl-3,4-di-O-benzyl-2-O-p-methoxybenzyl-ꢀ-D-
mannopyranosyl)-(1J6)-(2-O-benzoyl-3,4-di-O-benzyl-ꢀ-
D-mannopyranosyl)-(1J4)-(2-azido-3,6-di-O-benzyl-2-
deoxy-ꢀ-D-glucopyranosyl)-(1J6)-3,4,5-tri-O-benzyl-1,2-
O-(L-1,7,7-trimethyl[2.2.1]bicyclo-hept-6-yliden)-D-myo-
inositol (8)
TBAF (55 L, 1 M solution in THF) was added to a solu-
tion of 6 (71 mg, 47 mol) in freshly distilled THF (5mL).
After 6 h additional TBAF (55 L) was added and after 24 h
30 L more. Although the reaction was not complete (as
monitored by TLC (toluene–EtOAc, 12:1) after 48 h the
mixture was diluted with toluene, washed with water, dried
(MgSO4), and concentrated. The residue was applied onto a
silica gel column and eluted (toluene–EtOAc, 15:1) to give
2-O-benzoyl-3,4-di-O-benzyl-ꢀ-D-mannopyranosyl)-(1J4)-
(2-azido-3,6-di-O-benzyl-2-deoxy-ꢀ-D-glucopyranosyl)-(1J6)-
3,4,5-tri-O-benzyl-1,2-O-(L-1,7,7-trimethyl[2.2.1]bicyclohept-
6-yliden)-D-myo-inositol (7, 56 mg, 40 mol, 85%). [ꢀ]D
+35 (c 1.0, CH2Cl2). 13C NMR (CDCl3) ꢁ: 10.2, 20.7, 20.9
(Cph), 27.2, 30.1, 45.0, 45.3, 48.2, 51.8 (Cph), 62.0, 63.6,
68.1–76.9, 78.3–80.7 (Cino1–Cino6, C2–C6, C2>–C6>,
CH2Bn), 95.6 (C1) 99.1 (C1>), 118.1 (Cph-acetal), 127.5–
138.4 (aromatic C), 165.2 (PhCO). A solution of 4 (28 mg,
50 mol) and 7 (75 mg, 54 mol) in dry Et2O (10 mL) was
stirred with powdered molecular sieves (4 Å) under argon
for 1 h, when DMTST (40 mg, 155 mol) was added. The
reaction, monitored by TLC (light petroleum (bp 40–60°C) –
Et2O, 1:1), was quenched after 1.5 h by addition of Et3N
(100 L) and stirred for a further 20 min. The mixture was
diluted with Et2O (20 mL), filtered through Celite, and con-
centrated. The residue was subjected to silica gel column
chromatography (toluene–EtOAc, 12:1) to obtain 8 (95 mg,
50 mol, quant.). [ꢀ]D +41 (c 1.0, CH2Cl2). 1H NMR
(CDCl3) ꢁ: 0.78–1.96 (m, 16H, Cph), 3.30–5.20 (m, 52H),
5.50 (s, 1H), 5.61 (d, J = 1 Hz, 1H) 5.66 (s, 1H), 5.81 (m,
1H), 6.78 (d, 2H), 7.00–7.45 (m, 48H), 8.00 (d, 2H).
13C NMR (CDCl3) ꢁ: 10.0 (Cph), 20.6, 20.9, 27.2, 30.0,
45.0, 45.4, 48.2, 51.8 (Cph), 55.4 (MeOBn), 63.6ꢂ80.8
(Cino1ꢂCino6, C2ꢂC6, C2>ꢂC6>, C2>>ꢂC6>>, CH2Bn, MBn,
allyl), 95.6 (C1), 98.4 (C1>>), 99.1 (C1>), 113.7 (MBn),
116.7 (allyl), 118.2 (Cph-acetal), 125.3–138.7 (aromatic C,
allyl), 159.0 (MBn), 165.4 (PhCO). Anal. calcd. for
1
(141 mol, 84%) of 10. [ꢀ]D +21 (c 1.0, CH2Cl2). H NMR
(CDCl3) ꢁ: 0.78–1.96 (m, 25H, t-BuSi, Cph), 3.30–5.20 (m,
61H), 5.28 (d, J = 2 Hz, 1H), 5.50 (s, J = 2 Hz, 1H), 5.58 (d,
J = 4 Hz, 1H), 5.63 (m, 1H), 5.71 (m, 1H), 6.78 (d, 2H),
7.00–8.00 (m, 72H). 13C NMR (CDCl3) ꢁ: 10.0 (Cph), 19.6
(t-BuSi), 20.6, 20.9, 21.7 (Cph), 27.2 (t-BuSi, Cph), 30.0,
45.0, 45.3, 48.2, 51.8 (Cph), 55.3 (MeOBn), 63.4–80.8
(Cino1–Cino6, C2–C6, C2>–C6>, C2>>–C6>>, C2>>>-C6>>>,
CH2Bn, MBn, allyl), 95.5 (C1), 98.7 (C1>>), 98.8 (C1>>>),
99.2 (C1>), 113.6 (MBn), 116.3 (allyl), 118.2 (Cph-acetal),
125.4–139.0 (aromatic C, allyl), 158.9 (MBn), 165.3
(PhCO). Anal. calcd. for C151H165N3O27Si: C 73.07, H 6.70;
found: C 72.93, H 6.75.
(3,4-Di-O-benzyl-6-O-tert-butyldiphenylsilyl-ꢀ-D-manno-
pyranosyl)-(1J2)-(3,4-di-O-benzyl-ꢀ-D-mannopyranosyl)-
(1J6)-(2-O-benzoyl-3,4-di-O-benzyl-ꢀ-D-mannopyranosyl)-
(1J4)-(2-azido-3,6-di-O-benzyl-2-deoxy-ꢀ-D-glucopyra-
nosyl)-(1J6)-3,4,5-tri-O-benzyl-1,2-O-(L-1,7,7-tri-
methyl[2.2.1]bicyclohept-6-yliden)-D-myo-inositol (12)
Compound 10 (700 mg, 282 mol) was dissolved in
freshly distilled THF (10 mL) and stirred 10 min with a cat-
alytic amount of Pd/C. The filtered solution was degassed
and set under argon. After addition of 1,5-cycloocta-
dienebis(methyldiphenylphosphine)iridium(I)·PF6 (27 mg,
32 mol), the solution was saturated with hydrogen until the
intensive red colour of the mixture had disappeared (approx.
after 20 min). The solution was stirred for 1 h under argon at
C
115H125N3O22: C 72.65, H 6.63; found: C 72.38, H 6.60.
© 2002 NRC Canada

1110
Can. J. Chem. Vol. 80, 2002
room temperature, then NIS (420 mg, 1.87 mmol) and H2O
(1.75 mL) were added. After 2.5 h the reaction mixture was
diluted with EtOAc, washed consecutively with 10% aque-
ous Na2S2O3, NaHCO3, dried (MgSO4), and concentrated.
The crude product was purified by flash chromatography
(toluene–EtOAc, 12:1) to give (3,4-di-O-benzyl-6-O-tert-
butyldiphenylsilyl-2-O-p-methoxybenzyl-ꢀ-D-mannopyrano-
syl)-(1J2)-(3,4-di-O-benzyl-ꢀ-D-mannopyranosyl)-(1J6)-
(2-O-benzoyl-3,4-di-O-benzyl-ꢀ-D-mannopyranosyl)-(1J4)-
(2-azido-3,6-di-O-benzyl-2-deoxy-ꢀ-D-glucopyranosyl)-(1J6)-
3,4,5-tri-O-benzyl-1,2-O-(L-1,7,7-trimethyl[2.2.1]bicyclohept-
6-yliden)-D-myo-inositol (11, 674 mg, 276 mol, 98%). [ꢀ]D
+30 (c 1.0, CH2Cl2). 13C NMR (CDCl3) ꢁ: 10.0 (Cph), 19.6
(t-BuSi), 20.6, 20.9 (Cph), 27.2 (t-BuSi), 30.0, 45.0, 45.3,
48.2, 51.8 (Cph), 55.4 (MeOBn), 61.9–80.8 (Cino1–Cino6,
C2–C6, C2>–C6>, C2>>–C6>>, C2>>>–C6>>> CH2Bn, MBn), 95.5
(C1), 98.7 (C1>>), 98.8 (C1>>>), 99.1 (C1>), 113.6 (MBn),
118.2 (Cph-acetal), 125.4–138.7 (aromatic C), 158.9 (MBn),
165.3 (PhCO). DDQ (80 mg, 350 mol) dissolved in CH2Cl2
(2 mL) was added to an ice cooled solution of 11 (650 mg,
266 mol) in water saturated CH2Cl2 (5% H2O, 25 mL) and
stirred for 4 h, an additional 30 mg of DDQ in portions of
10 mg were added until the conversion was complete. The
mixture was diluted with CH2Cl2 (100 mL); washed with
water, Na2S2O3 (10% aq), and NaHCO3; dried (MgSO4);
and concentrated. The crude product was purified by flash
chromatography (toluene–EtOAc, 12:1) to give 12 (520 mg,
224 mol, 84%). [ꢀ]D +28 (c 1.0, CH2Cl2). 1H NMR
(CDCl3) ꢁ: 0.80–1.96 (m, 25H, t-BuSi, Cph), 3.30ꢂ4.90 (m,
52H), 5.19 (s, 1H), 5.52 (s, 1H), 5.59 (d, J = 3 Hz, 1H), 5.63
(m, 1H), 7.00–8.00 (m, 70H). 13C NMR (CDCl3) ꢁ: 10.0
(Cph), 19.5 (t-BuSi), 20.6, 20.9 (Cph), 27.1 (t-BuSi), 30.1,
45.0, 45.3, 48.2, 51.8 (Cph), 61.9–80.8 (Cino1–Cino6, C2–C6,
C2>–C6>, C2>>–C6>>, C2>>>–C6>>>, CH2Bn), 95.5 (C1), 98.7
(C1>>), 99.1 (C1>), 100.9 (C1>>>), 118.2 (Cph-acetal),
125.4ꢂ138.7 (aromatic C), 165.3 (PhCO).
matic C). Compound 13 (350 mg, 157 mol) was dissolved
in THF (10 mL) and TBAF (200 L, 1 M in THF) was
added. The mixture was stirred at room temperature over-
night, then concentrated, and purified by flash chromatogra-
phy (toluene–EtOAc, 3:1J2:1) to give 267 mg (135 mol,
1
86%) of 14. [ꢀ]D +50 (c 1.0, CH2Cl2). H NMR (CDCl3) ꢁ:
0.80–2.04 (m, 16H, Cph), 3.34ꢂ4.90 (m, 52H), 5.03 (s, 1H),
5.06 (s, 1H), 5.28 (s, 1H), 5.58 (d, J = 2 Hz, 1H), 7.00–7.40
(m, 55H). 13C NMR (CDCl3) ꢁ: 10.1, 20.7, 20.9, 27.3, 30.1,
45.0, 45.4, 48.2, 51.8 (Cph), 62.0–81.0 (Cino1–Cino6, C2–C6,
C2>–C6>, C2>>–C6>>, C2>>>–C6>>>, CH2Bn), 95.5 (C1), 99.5
(C1>), 100.8, 101.2 (C1>>, C1>>> ), 118.2 (Cph-acetal), 127.5–
128.6 and 137.5–138.6 (aromatic C). Anal. calcd. for
C
117H131N3O25: C 71.00, H 6.67; found: C 70.85, H 6.65.
(2,6-Di-[ammoniumphosphate]-ꢀ-D-mannopyranosyl)-
(1J2)-(6-[ammoniumphosphate]-ꢀ-D-mannopyranosyl)-
(1J6)-(2-[ammoniumphosphate]-ꢀ-D-mannopyranosyl)-
(1J4)-(2-amino-2-deoxy-ꢀ-D-glucopyranosyl)-(1J6)-D-
myo-inositol 1,2-cyclic phosphate ammonium salt (18)
1H-Tetrazole (115 mg, 1.64 mmol) was added to a solu-
tion of 14 (230 mg, 116 mol) and dibenzyl diisopropyl-
phosphoramidite (225 L, 700 mol) in CH2Cl2 (3.5 mL).
After 20 min the reaction mixture was cooled to 0°C and
70% m-chloroperbenzoic acid (230 mg) was added. The
mixture was stirred an additional 15 min and then diluted
with CH2Cl2 and washed with Na2S2O3 (10% aq), NaHCO3
and, water. After drying (MgSO4), filtration, and concentra-
tion, the crude product was subjected to flash chromatogra-
phy (toluene J toluene–EtOAc, 20 J 25%) to yield (3,4-di-
O-benzyl-2,6-bis-O-dibenzyloxyphosphoryl-ꢀ-D-mannopyrano-
syl)-(1J2)-(3,4-di-O-benzyl-6-O-dibenzyloxyphosphoryl-ꢀ-
D-mannopyranosyl)-(1J6)-(3,4-di-O-benzyl-2-O-dibenzyl-
oxyphosphoryl-ꢀ-D-mannopyranosyl)-(1J4)-(2-azido-3,6-di-
O-benzyl-2-deoxy-ꢀ-D-glucopyranosyl)-(1J6)-3,4,5-tri-
O-benzyl-1,2-O-(L-1,7,7-trimethyl[2.2.1]bicyclohept-6-yliden)-
D-myo-inositol (15, 364 mg, 116 mol, quant.). [ꢀ]D +23
(c 1.0, CH2Cl2). 13C NMR (CDCl3) ꢁ: 10.2, 20.8, 21.0, 27.4,
30.2, 45.2, 45.5, 48.3, 51.9 (Cph), 63.3–80.8 (Cino1–Cino6,
C2–C6, C2>–C6>, C2>>–C6>>, C2>>>–C6>>>, CH2Bn), 95.5 (C1),
98.9 (C1>), 99.4, 99.9 (C1>>, C1>>>), 118.3 (Cph-acetal),
127.2–138.6 (aromatic C). 31P NMR (CDCl3) ꢁ: 4 signals
between –0.5 and +0.5. TFA (1 mL) was added at 0°C to a
solution of 15 (300 mg, 100 mol) in CH2Cl2 (10 mL). The
mixture was stirred for 18 h (room temperature), quenched
by the addition of Et3N (1 mL), and concentrated. The
residue was purified by flash chromatography (toluene–EtOAc,
2:1) to give (3,4-di-O-benzyl-2,6-bis-O-dibenzyloxyphos-
phoryl-ꢀ-D-mannopyranosyl)-(1J2)-(3,4-di-O-benzyl-6-O-
dibenzyloxyphosphoryl-ꢀ-D-mannopyranosyl)-(1J6)-(3,4-
di-O-benzyl-2-O-dibenzyloxyphosphoryl-ꢀ-D-mannopyrano-
syl)-(1J4)-(2-azido-3,6-di-O-benzyl-2-deoxy-ꢀ-D-glucopyrano-
syl)-(1J6)-3,4,5-tri-O-benzyl-D-myo-inositol (16, 132 mg,
46 mol, 46%). [ꢀ]D +32 (c 1.0, CH2Cl2). 13C NMR (CDCl3) ꢁ:
53.6, 64.2, 66.3–81.7 (Cino1–Cino6, C2–C6, C2>–C6>, C2>>–
C6>>, C2>>>–C6>>>, CH2Bn), 98.2, 98.7 (C1, C1>>), 98.9, 99.0,
99.7, 99.7 (C1>, C1>>>), 127.2–128.7, 135.6–137.8, 137.9–138.5
(aromatic C). 31P NMR (CDCl3) ꢁ: 4 signals between –0.5
and +0.5. Methyl dichlorophosphate (50 L, 50 mmol) was
dissolved in dry pyridine (1 mL) at 15°C (water bath). The
mixture was stirred for 20 min, after which a solution of 16
(3,4-Di-O-benzyl-ꢀ-D-mannopyranosyl)-(1J2)-(3,4-di-O-
benzyl-ꢀ-D-mannopyranosyl)-(1J6)-(3,4-di-O-benzyl-ꢀ-D-
mannopyranosyl)-(1J4)-(2-azido-3,6-di-O-benzyl-2-
deoxy-ꢀ-D-glucopyranosyl)-(1J6)-3,4,5-tri-O-benzyl-1,2-
O-(L-1,7,7-trimethyl[2.2.1]bicyclohept-6-yliden)-D-myo-
inositol (14)
Compound 12 (490 mg, 211 mol) was deacetylated un-
der Zemplén conditions. To speed up the reaction, the mix-
ture was warmed to 45°C. After 3 days the reaction mixture
was neutralized with H+ ion exchange resin, filtered, and
concentrated. The residue was purified by flash-
chromatography (toluene–EtOAc, 6:1J3:1) to obtain (3,4-
di-O-benzyl-6-O-tert-butyldiphenylsilyl-ꢀ-D-mannopyrano-
syl)-(1J2)-(3,4-di-O-benzyl-ꢀ-D-mannopyranosyl)-(1J6)-
(3,4-di-O-benzyl-ꢀ-D-mannopyranosyl)-(1J4)-(2-azido-3,6-
di-O-benzyl-2-deoxy-ꢀ-D-glucopyranosyl)-(1J6)-3,4,5-
tri-O-benzyl-1,2-O-(L-1,7,7-trimethyl[2.2.1]bicyclohept-6-yliden)--
D-myo-inositol (13, 400 mg, 180 mol, 85%). [ꢀ]D +40
(c 1.0, CH2Cl2). 13C NMR (CDCl3) ꢁ: 10.0 (Cph), 19.6
(t-BuSi), 20.6, 20.9 (Cph), 27.1 (t-BuSi), 30.1, 45.0, 45.3,
48.2, 51.8 (Cph), 62.0–80.6 (Cino1–Cino6, C2–C6, C2>–C6>,
C2>>–C6>>, C2>>>–C6>>>, CH2Bn), 95.4 (JC1,H1 = 173 Hz, C1),
99.1 (JC1,H1 = 168 Hz, C1>), 100.2, 101.4 (JC1,H1 = 176,
169 Hz, C1>>, C1>>>), 118.2 (Cph-acetal), 127.2–138.5 (aro-
© 2002 NRC Canada

Lahmann et al.
1111
(48 mg, 16.6 mol) in dry pyridine (500 L) was added. Af-
ter 2 h the starting material was consumed (TLC: CHCl3–
MeOH, 9:1) and the reaction was quenched by addition of
NaHCO3. The mixture was transferred into a 100-mL round
bottom flask (using approx. 10 mL pyridine) and concen-
trated. The residue was dissolved and partitioned between
CHCl3 (60 mL) and water (10 mL). HCl (1 M, aq.) was
added until the solution was pH 1 (4 mL). The aqueous
phase was extracted with CHCl3, and the combined organic
phases were dried (MgSO4) and concentrated. After co-
evaporation two times from EtOH, the crude product (3,4-
di-O-benzyl-2,6-bis-O-dibenzyloxyphosphoryl-ꢀ-D-manno-
pyranosyl)-(1J2)-(3,4-di-O-benzyl-6-O-dibenzyloxy-phosphoryl-
ꢀ-D-mannopyranosyl)-(1J6)-(3,4-di-O-benzyl-2-O-dibenzyl-
oxyphosphoryl-ꢀ-D-mannopyranosyl)-(1J4)-(2-azido-3,6-
di-O-benzyl-2-deoxy-ꢀ-D-glucopyranosyl)-(1J6)-3,4,5-tri-
O-benzyl-D-myo-inositol 1,2-cyclic phosphate (17, 48 mg,
95%) was obtained, which was immediately used for the
next reaction step. Raw material 17 dissolved in dry THF
(2 mL) was dropped into liquid ammonia (20 mL). A cata-
lytic amount of sodium was added to the white emulsion,
which turned rapidly into a deep blue solution. After 2 min,
NH4Cl (s) was carefully added until the solution turned
white again. The liquid ammonia was allowed to evaporate.
The dry residue was dissolved in water (20 mL), and washed
once with Et2O (10 mL). After concentration, the crude
product was purified by size exclusion chromatography
(G15, eluent water containing 1% n-BuOH) to give, after
freeze-drying, 18 (12 mg, 10 mol, 60%). 1H NMR (D2O) ꢁ:
3.38–4.60 (m, 31H), 5.14 (s, 1H), 5.25 (s, 1H), 5.49 (1H).
13C NMR (D2O) ꢁ: 55.1, 60.9, 64.8, 64.9, 67.2, 67.3, 70.3–
73.3, 74.8, 75.0, 76.6, 77.8, 79.5, 80.2, 82.2 (Cino1–Cino6,
C2–C6, C2>–C6>, C2>>–C6>>, C2>>>–C6>>>), 96.8 (C1), 99.4
(C1>>), 100.5, 101.5 (C1>, C1>>>). 31P NMR (D2O) ꢁ: 0.3, 0.9
(PO(OH)2), 15.8 (cyclic phosphate). MALDI-TOF MS: 1209.2
[M + H]+, 1227.2 [M + H + NH4]+, 1231.2 [M + Na]+.
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Acknowledgments
Financial support from EU (Contract Number ERB
FMRX CT96 0025) and from the Swedish Natural Science
Research Council are gratefully acknowledged.
© 2002 NRC Canada