A flexible route to mannose 6-phosphonate functionalized derivatives

Article abstract of DOI:10.1080/10426500214297

A new approach for the synthesis of a mannose 6-phosphonate isosteric analog of mannose 6-phosphate is reported. The mannosylphosphonate has been prepared in a multistep synthesis involving an homologation reaction of the methyl α-D-mannopyranoside followed by an Arbuzov reaction between a bromohomomannosyl derivative and the tris(trimethylsilyl)phosphite. This approach, avoiding the deprotection of dialkylphosphonate, allowed us to prepare the mannose 6-phosphonate in good yield. The described method was successfully extended to the preparation of a mannose 6-phosphonate linked to a cholesteryl moiety. This strategy affords a more general route for a wide range of functionalized mannose 6-phosphonate derivatives.

Full text of DOI:10.1080/10426500214297

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A Flexible Route to Mannose 6-Phosphonate
Functionalized Derivatives
Sébastien Vidal a , Jean-Louis Montero a , Alain Leydet a & Alain Morère a
a Laboratoire de Chimie Biomoléculaire , Université Montpellier II, Ecole Nationale
Supérieure de Chimie de Montpellier , Montpellier, France
Published online: 27 Oct 2010.
To cite this article: Sébastien Vidal , Jean-Louis Montero , Alain Leydet & Alain Morère (2002) A Flexible Route to Mannose 6-
Phosphonate Functionalized Derivatives, Phosphorus, Sulfur, and Silicon and the Related Elements, 177:10, 2363-2377, DOI:
10.1080/10426500214297
To link to this article: http://dx.doi.org/10.1080/10426500214297
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Phosphorus, Sulfur and Silicon, 2002, Vol. 177:2363–2377
C
Copyright 2002 Taylor & Francis
1042-6507/02 $12.00 + .00
DOI: 10.1080/10426500290110559
A FLEXIBLE ROUTE TO MANNOSE
6-PHOSPHONATE FUNCTIONALIZED DERIVATIVES
Se´bastien Vidal, Jean-Louis Montero, Alain Leydet,
and Alain More`re
Laboratoire de Chimie Biomole´culaire, Universite´
Montpellier II, Ecole Nationale Supe´rieure de Chimie de
Montpellier, Montpellier, France
(Received March 19, 2002)
A new approach for the synthesis of a mannose 6-phosphonate isos-
teric analog of mannose 6-phosphate is reported. The mannosylphos-
phonate has been prepared in a multistep synthesis involving an
homologation reaction of the methyl -D-mannopyranoside followed
by an Arbuzov reaction between a bromohomomannosyl derivative
and the tris(trimethylsilyl)phosphite. This approach, avoiding the de-
protection of dialkylphosphonate, allowed us to prepare the mannose
6-phosphonate in good yield. The described method was successfully
extended to the preparation of a mannose 6-phosphonate linked to a
cholesteryl moiety. This strategy affords a more general route for a wide
range of functionalized mannose 6-phosphonate derivatives.
Keywords: Glycolipid; lectin; mannose 6-phosphate; mannose
6-phosphate/insulin-like growth factor; mannose 6-phosphonate/
triethyleneglycolcholesteryl; phosphonation; tris(trimethylsilyl)-
phosphite
INTRODUCTION
Mannose 6-Phosphate (M6P) is a recognition marker involved in the
selective targeting of newly synthesized enzymes to lysosomes.1 While
two different receptors recognize specifically the M6P residues, only
the larger M6P receptor mediates the endocytosis of extracellular
This research was supported by the Scientific Council of the Universite´ Montpellier
II and the Fondation pour la Recherche Me´dicale (to S.V.). We also wish to thank Mayoly-
Spindler Laboratories (Chatou—France) for financial support.
Address correspondence to Alain Morere, Laboratoire de Chimie Biomole´culaire
(UMR 5032), Universite´ Montpellier II, Ecole Nationale Supe´rieure de Chimie de
Montpellier, 8 Rue de l’Ecole Normale, F-34296 Montpellier Cedex 05, France. E-mail:
morere@univ-montp2.fr
2363

2364
S. Vidal et al.
M6P-containing ligands.2 This receptor is a type I glycoprotein of
275 kDa, which apart from M6P residues, is also known to bind retinoic
acid3 through distinct binding sites (RA) and insulin-like growth fac-
tor II4 (IGF-II). This receptor is the first example of a protein able to
bind three different classes of ligands, that is, a saccharide (M6P), a
peptide (IGF-II), and a lipid (RA). Moreover, the M6P/IGFIIR plays a
fundamental role in the control of cell growth in fetal development and
carcinogenesis.6 In our ongoing research program, we focus on the abil-
ity of the M6P/IGF-IIR to recognize and then internalize M6P analogs.
We recently described7 the synthesis of two mannose 6-phosphonate
(M6Pn) analogs (I and II) of M6P (Figure 1), and evaluated their affin-
ity towards the M6P/IGFIIR.
Isosteric compound I has been shown to have an affinity to the
M6P/IGFIIR similar to natural M6P, whereas nonisosteric compound
II was only weakly recognized. In our previous syntheses7,8 of the man-
nosyl phosphonate I, the key reaction in the synthetic scheme was the
last step, involving the deprotection of the diethylphosphonate moi-
ety according to Rabinowitz’s procedure.9 However, the conversion of
diethylphosphonates into the corresponding free phosphonic acids us-
ing the method of transesterification by Me3SiBr can be troublesome.
Indeed, degradation problems, partial deprotection, or even complete
failure of the deprotection has been described in several cases, such as
with aminoacids or glycopyranose frameworks.10–12 Recently, these dif-
ficulties were met with the preparation of a glycoconjugate we called
FIGURE 1 Phosphonate analogs of M6P.

Flexible Route to Mannose 6-Phosphonate
2365
M6Pn/CSA,13 where M6Pn and CSA stand for mannose 6-phosphonate
and cholesteryl-succinyl-anilinyl respectively (Figure 1). Partial depro-
tection of the diethylphosphonate of this molecule and partial cleav-
age of the anomeric bond and/or amide bonds were observed under
Rabinowitz’s procedure. It is worth pointing out that this amphiphilic
steroidal mannose 6-phosphonate incorporated into the lipidic bilayer
of a liposome13 constitute an efficient drug delivery system in selective
targeting of breast cancer cells.
RESULTS AND DISCUSSION
In order to circumvent the Rabinowitz deprotection step in the synthesis
of M6Pn I and its conjugates, we decided to introduce the phosphonate
moiety by an Arbuzov reaction between tris(trimethylsilyl)phosphite
and a halogenohomomannosyl derivative. This attractive approach
could furnish the bis(trimethylsilyl)ester which can be easily hy-
drolyzed with saturated aqueous NaHCO3 solution.14 The key step
of this alternative synthesis involves the preparation of a man-
noheptose derivative by a one carbon chain extension at the C-6
position. Various procedures are described in the literature, and
many reactants can be used such as cyanide anion,15–20 triphenyl-
methoxymethylphosphonium salt,21 or iron-carbonyl-cyclopentadienyl
complexes.22,23 Homologation can be also accomplished via alkyne24–28
or epoxysilane29,30 derivatives. Another method is possible using a cyclic
sulfate intermediate.31
The preparation of the mannose 6-phosphonate was realized
(Scheme 1) via an homologation reaction involving condensation of the
2-lithio-1,3-dithiane32 with the triflate derivative of alcohol 1.33 Com-
pound 2 was then reacted with ceric ammonium nitrate (CAN)34–36
in wet acetonitrile affording a crude aldehyde (1H NMR analysis
gave satisfactory data showing the resonance of the aldehydic pro-
ton at 9.75 ppm) which was reduced by sodium borohydride to fur-
nish the homologated alcohol 3 in a one-pot procedure. Reactions
proceed quickly affording expected compounds in good yields. Conver-
sion of homoalcohol 3 to homoalkylbromide 5 was carried out by re-
action of 3 with methanesulfonylchloride (affording 4 in 95% yield)
followed by bromination with lithium bromide37,38 in butanone, afford-
ing 5 (95%) without further purification. This two-step procedure was
39
preferred to the one-step PPh3/CBr4 method, in order to avoid prob-
lems with the removal of triphenylphosphine oxide byproduct by col-
umn chromatography. The phosphonate moiety was introduced by an
Arbuzov reaction between tris(trimethylsilyl)phosphite and 5, affording

2366
S. Vidal et al.
SCHEME 1 Reagents and conditions: (a) Tf2O, 2,6-di-tert-butyl-4-methyl-
pyridine, CH2Cl2, 40 C, 10 min (85%). (b) 1,3-dithiane, n-BuLi, HMPA, THF,
78 C, 10 min (85%). (c) CAN, wet CH3CN, rt, 15 min. (d) NaBH4, EtOH/H2O,
rt, 5 min (75% for two steps). (e) MsCl, DIEA, CH2Cl2, rt, 15 min (95%). (f)
LiBr, butanone, 85 C, 1 h (95%). (g) P(OSiMe3)3, 160 C, 16 h (85%). (h) H2/Pd-C
10%, EtOH/H2O, rt, 17 h then DOWEX 50WX2 (Na+ form) (90%).
a bis(trimethylsilyl)phosphonate, which was hydrolyzed with saturated
aqueous NaHCO3 solution, giving the salt of the corresponding phos-
phonic acid 6. Benzyl protecting groups were removed by hydrogenoly-
sis over Pd-C (10%) to obtain the title compound I in good overall yield
(29%).
We therefore decided to use the same strategy to prepare the M6Pn-
based glycolipid 13 (Scheme 2) in which the hydrophilic carbohydrate
head is linked to the hydrophobic steroidal tail through a triethyleneg-
lycol (TEG) spacer arm. Compound 3 was deprotected to afford the
mannoheptose derivative 7 which was activated at the 6-position fol-
lowed by peracetylation32,33,40 and subsequent bromination to afford 8
in 55% yield over three steps. The methyl glycoside 8 was then con-
verted to the 1-O-acyl glycoside 9 then activated to provide a gly-
cosyl donor intermediate 10. Koenigs-Knorr glycosylation41,42 of 10,
using known 8-(cholest-5-en-3 -yloxy)-3,6-dioxaoctan-1-ol43 as the gly-
cosyl acceptor, affords the orthoester derivative 11 as the major prod-
uct, which upon subsequent treatment with a Lewis acid, underwent
rearrangement44–49 to furnish the desired O-glycoside 12. These two iso-
mers could easily be differentiated according to their respective NMR
data (Figure 2 and Table I) and also by TLC.

Flexible Route to Mannose 6-Phosphonate
2367
SCHEME 2 Preparation of M6Pn/TEGC 13—reagents and conditions:
(a) H2/Pd-C 10%, EtOH/H2O, rt, 16 h, (95%). (b) TsCl, C5H5N, rt, 5 h. (c) Ac2O,
C5H5N, 0 C to rt, 1 h. (d) LiBr, 2-butanone, 85 C, 1 h, (55% over three steps).
(e) Ac2O/AcOH/H2SO4(cat.), rt, 24 h, (85%). (f) HBr/AcOH, CH2Cl2, rt, 16 h.
(g) 8-(cholest-5-en-3 -yloxy)-3,6-dioxaoctan-1-ol, CH2Cl2, AgOTf, sym-collidine,
0 C to rt, 15 min, (45% over both steps). (h) TMSOTf, CH2Cl2, molecular
˚
sieves (3A), 5 C, 5 min, (40%). (i) P(OSiMe3)3, 160 C, 16 h then NaHCO3(aq)
.
(j) NaOMe, MeOH, rt, 16 h, (50% over both steps).
The final step in our synthesis involved an Arbuzov reaction between
the alkyl bromide derivative 12 and tris(trimethylsilyl)phosphite,
followed by deprotection of the acetate under Zemple´n conditions
affording the final M6Pn/TEGC 13 in good yields. Characterization
of this glycolipid was troublesome but evidence of the structure
was provided by NMR spectroscopy (1H and 31P) and also by mass
spectrometric analyses.

2368
S. Vidal et al.
TABLE I Comparison of Compounds 11 and 12 NMR Data
a
b
c
d
e
Compound
J1–2
H-1
C
CH3
CH3
Orthoester 11
Glycoside 12
2.6
1.5
5.44
4.75
124.7
—
—
21.5
1.71
1.91
aCoupling constant, in Hz.
b1H NMR chemical shift, in ppm.
c13C NMR chemical shift for the quaternary carbon (see in Figure 2),
in ppm.
d13C NMR chemical shift for the methyl (see † in Figure 2), in ppm.
e1H NMR chemical shifts for the methyl groups, in ppm.
FIGURE 2 Partial NMR analyses for compounds 11 and 12. a) 13C NMR in
CDCl3 shows the signal corresponding to the quaternary carbon (C ) from the
orthoester derivative 11 while b) does not display this peak for 12. c) 1H NMR
in CDCl3 shows two signals for the acetates and one more singlet corresponding
to the orthoester-methyl (CH3†) group for 11 while d) displays the three signals
corresponding to each acetyl groups for 12.

Flexible Route to Mannose 6-Phosphonate
2369
CONCLUSION
The compound
I was obtained in good overall yield (29%),
demonstrating the usefulness of the Arbuzov phosphonation—using
the tris(trimethylsilyl)phosphite—for the preparation of glycopyra-
nosylphosphonic acids, such as the isosteric analog (I) of mannose
6-phosphate. This strategy was successfully applied to the prepara-
tion of a steroidal derivative M6Pn/TEGC 13. Preparation of lipo-
somes incorporating M6Pn/TEGC 13 and evaluation of their interac-
tions with MCF7 cells now are being investigated in our group with
the aim of developing drug delivery systems. Although the overall
yield is still very low (1.75% over 16 steps starting from methyl -D-
mannopyranoside) for the preparation of M6Pn/TEGC 13, this strategy
provides a more general route to a wide variety of functionalized M6Pn
where the anomeric center will be substituted with any biologically
active molecule during the glycosylation.
EXPERIMENTAL
Analytical TLC were performed using aluminum-coated TLC plates
60-F254 (Merck). Plates were developed with: (1) UV light (254 nm),
(2) immersion in a 10% H2SO4/EtOH solution followed by charring,
(3) immersion in a 5% rhodanine/EtOH solution followed by charring
(for aldehydes) or (4) immersion in a phosphomolybdic solution (for
phosphorus containing compounds). Silica gel column chromatogra-
phy was performed with silica gel 60A (Carlo Erba). Optical rotations
were measured at the sodium D-line with a Perkin-Elmer-241 polarime-
ter. Fast Atom Bombardment (FAB) mass spectra were recorded on a
Jeol JMS-DX300 spectrometer in either positive (FAB+) or negative
(FAB ) modes and using either 3-nitrobenzylic alcohol (NBA) or glyc-
erol/thioglycerol (1:1) mixture (G/T). 1H NMR spectra were recorded on
a Bruker DRX 400 (400 MHz), at 25 C. Chemical shifts ( ) are given
in ppm and referenced using residual solvent signals (7.26 ppm for
CHCl3 and 4.79 ppm for HOD). The following abbreviations were used
to explain the signal multiplicities or characteristics: s (singlet), d (dou-
blet), dd (double doublet), t (triplet), td (triplet doublet), m (multiplet).
13C NMR spectra were recorded on a Bruker DRX 400 (100.6 MHz).
Chemical shifts ( ) are given in ppm relative to TMS as an external
reference. 31P NMR spectra were recorded on a Bruker DPX 200 (81.0
MHz). Chemical shifts ( ) are given in ppm relative to phosphoric acid
(85%) as an external reference.

2370
S. Vidal et al.
Methyl 2,3,4-Tri-O-benzyl-6-deoxy-6-(1,3-dithianyl)-
- D-mannopyranoside 2
To a solution of 133 (1 g, 2.16 mmol) and 2,6-di-tert-butyl-4-
methylpyridine (555 mg, 2.7 mmol) dissolved in CH2Cl2 (5 mL) was
added dropwise triflic anhydride (633 L, 2.37 mmol), at 40 C. After
stirring for 30 min, the reaction was neutralized with NaHCO3 (50 mL,
1 g/100 mL H2O) and the aqueous layer was extracted with CH2Cl2
(2 50 mL). Organic layers were combined, dried (Na2SO4), filtered,
and evaporated under reduced pressure. The resulting residue was pu-
rified by silica gel column chromatography (hexane then hexane/ether
4:1) affording a pale pink oil (1.09 g, 85%). To a solution of 1,3-dithiane
(706 mg, 5.87 mmol) dissolved in THF (2 mL) was added dropwise
HMPA (300 l, 1.67 mmol). The mixture was cooled to 78 C before
addition of n-BuLi (5 mL, 5.87 mmol, 1.6 M in hexane). After 5 min, a
solution of the triflate derivative previously prepared (1 g, 1.67 mmol)
in THF (5 mL) was added dropwise at 78 C. After 10 min, the re-
action mixture was quenched with NH4Cl (50 mL, 1 g/100 mL H2O)
and the aqueous layer was extracted with CH2Cl2 (2 50 mL). Organic
layers were combined, dried (Na2SO4), filtered, and evaporated under
reduced pressure. The resulting residue was purified by silica gel col-
umn chromatography (hexane/ether 95:5 then 3:1) affording 2 as pale
20
yellow oil (807 mg, 85%). R f = 0.67 (hexane/ether 2:3). [ ]D = +20.8
(c 0.72/CHCl3). 1H NMR (CDCl3): = 1.80 2.20 (m, 3H, H-6 CH2CH2S),
2.30 2.50 (m, 1H, H-60), 2.70 3.00 (m, 4H, CH2S), 3.41 (s, 3H, OCH3),
3.73 (t, 1H, J = 9.3 Hz, H-4), 3.84 (dd, 1H, J = 1.9 and 2.8 Hz, H-2),
3.90 4.05 (m, 2H, H-3 H-5), 4.32 (dd, 1H, J = 3.7 and 11.0 Hz, H-
7), 4.65 (s, 2H, CH2Ph), 4.69 (d, 1H, J = 12.0 Hz, CH2Ph), 4.72 (d, 1H,
H-1), 4.76 (d, 1H, J = 12.5 Hz, CH2Ph), 4.83 (d, 1H, CH2Ph), 5.01 (d, 1H,
CH2Ph), 7.20 7.50 (m, 15H, 15H-ar). 13C NMR (CDCl3): = 24.0, 24.3,
29.0, 36.5, 42.1, 54.0, 67.0, 70.0–73.0 (3C), 73.7, 77.7, 79.2, 98.1, 125.0–
127.0 (15C), 137.0–137.5 (3C). MS (FAB+, NBA): m/z: 589 [M + Na]+,
567 [M + H]+, 535 [M OCH3]+, 475 [M PhCH2]+, 91 [PhCH2]+.
Methyl 2,3,4-Tri-O-benzyl-6-deoxy- -D - manno-
heptopyranoside 3
To a solution of 2 (440 mg, 0.78 mmol) dissolved in CH3CN/H2O (15:1,
16 mL) was added ceric ammonium nitrate (1.285 g, 2.34 mmol). After
stirring at rt for 15 min, the reaction mixture was evaporated under re-
duced pressure before diluting with EtOAc (100 mL). The organic layer
was washed with H2O (2 75 mL) and the organic layer extracted with
EtOAc (2 100 mL). Organic layers were combined, dried (Na2SO4),

Flexible Route to Mannose 6-Phosphonate
2371
filtered, and evaporated under reduced pressure. The crude oil was
dissolved in absolute ethanol (10 mL) before addition of a solution of
NaBH4 (75 mg, 1.95 mmol) in EtOH/H2O (1:1, 20 mL). The reaction
mixture was stirred at rt for 5 min before being neutralized with HCl
(1 M) and then diluted with H2O (100 mL). The aqueous layer was ex-
tracted with CH2Cl2 (3 100 mL). Organic layers were combined, dried
(Na2SO4), filtered, and evaporated under reduced pressure. The result-
ing residue was purified by silica gel column chromatography (hexane/
ether 7:3 then 1:1) affording 3 as pale yellow oil (278 mg, 75%). R f = 0.44
20
1
(hexane/ether 1:4). [ ]D = + 21.5 (c 0.65/CHCl3). H NMR (CDCl3):
= 1.80 2.30 (m, 2H, H-6 H-60), 3.35 (s, 3H, OCH3), 3.75 3.95 (m,
6H, H-2 H-3 H-4 H-5 H-7 H-70), 4.65 (s, 2H, CH2Ph), 4.66 (d, 1H, J =
10.9 Hz, CH2Ph), 4.70 (d, 1H, J = 1.7 Hz, H-1), 4.73 (d, 1H, J = 12.4
Hz, CH2Ph), 4.82 (d, 1H, CH2Ph), 5.05 (d, 1H, CH2Ph), 7.30 7.50 (m,
15H, 15H-ar). 13C NMR (CDCl3): = 34.2, 55.2, 61.6, 71.9, 72.5, 73.4,
74.9, 75.7, 78.6, 80.6, 99.5, 128.0 129.0 (15C), 138.6–138.9 (3C). MS
(FAB+, NBA): m/z: 501 [M + Na]+, 479 [M + H]+, 447 [M OCH3]+, 91
[PhCH2]+.
Methyl 2,3,4-Tri-O-benzyl-6-deoxy-7-O-methanesulfonyl-
-D-manno-heptopyranoside 4
To a solution of 3 (250 mg, 0.52 mmol) dissolved in CH2Cl2 (5 mL) was
added DIEA (116 L, 0.68 mmol) and then methanesulfonyl chloride
(50 L, 0.63 mmol). After stirring for 15 min at rt, the reaction mixture
was diluted with CH2Cl2 (150 mL) and the organic layer was washed
with NaHCO3 (10% aqueous, 2 100 mL), dried (Na2SO4), filtered, and
evaporated under reduced pressure affording 4 as a white oil (275 mg,
1
95%). R f = 0.55 (hexane/ether 1:4). H NMR (CDCl3): = 1.80 2.05
(m, 1H, H-6), 2.25 2.50 (m, 1H, H-60), 2.95 (s, 3H, CH3SO2), 3.34 (s,
3H, OCH3), 3.60 4.00 (m, 4H, H-2 H-3 H-4 H-5), 4.35 4.50 (m, 2H,
H-7 H-70), 4.65 (s, 2H, CH2Ph), 4.66 (d, 1H, J = 11.0 Hz, CH2Ph), 4.71
(d, 1H, J = 1.8 Hz, H-1), 4.73 (d, 1H, J = 12.3 Hz, CH2Ph), 4.81 (d, 1H,
CH2Ph), 5.01 (d, 1H, CH2Ph), 7.30 7.50 (m, 15H, 15H-ar). 13C NMR
(CDCl3): = 31.7, 37.8, 55.3, 67.0, 67.8, 72.5, 73.4, 75.0, 75.7, 78.7, 80.7,
99.5, 128.0–129.0 (15C), 138.0–139.0 (3C). MS (FAB+, NBA): m/z: 579
[M + Na]+, 557 [M + H]+, 525 [M OCH3]+, 91 [PhCH2]+.
Methyl 2,3,4-Tri-O-benzyl-7-bromo-6,7-dideoxy-
-D-manno-heptopyranoside 5
A solution of 4 (200 mg, 0.36 mmol) and LiBr (625 mg, 7.2 mmol) in
2-butanone (10 mL) was stirred at 85 C for 1 h. The reaction mixture

2372
S. Vidal et al.
was cooled to rt then diluted with CH2Cl2 (130 mL) and the organic layer
was washed with H2O (2 100 mL), dried (Na2SO4), filtered, and evap-
orated under reduced pressure affording 5 as a pale yellow oil (185 mg,
20
95%). R f = 0.68 (hexane/ether 2:3). [ ]D = +28.6 (c 0.21/CHCl3). 1H
NMR (CDCl3): = 2.00–2.25 (m, 1H, H-6), 2.35–2.50 (m, 1H, H-60),
3.41 (s, 3H, OCH3), 3.86 (dd, 1H, J = 1.8 and 3.1 Hz, H-2), 3.96 (dd,
1H, J = 8.9 Hz, H-3), 3.55–3.90 (m, 4H, H-4 H-5 H-7 H-70), 4.67 (s,
2H, CH2Ph), 4.69 (d, 1H, J = 11.0 Hz, CH2Ph), 4.74 (d, 1H, H-1), 4.77
(d, 1H, J = 12.4 Hz, CH2Ph), 4.85 (d, 1H, CH2Ph), 5.02 (d, 1H, CH2Ph),
7.30–7.50 (m, 15H, 15H-ar). 13C NMR (CDCl3): = 28.7, 34.0, 53.9,
67.9, 71.1, 71.9, 73.5, 74.1, 77.2, 79.3, 98.0, 126.5–128.0 (15C), 137.0–
137.5 (3C). MS (FAB +, NBA): m/z: 563 [M + Na]+, 509 [M OCH3]+,
449 [M PhCH2]+, 417 [M PhCH2 OCH3]+, 91 [PhCH2]+.
Diammonium Methyl 2,3,4-Tri-O-benzyl-6-deoxy-
6-dihydroxyphosphonylmethylene- -D-
mannopyranoside 6
A solution of 5 (130 mg, 0.24 mmol) dissolved in tris(trimethylsilyl)
phosphite (50 eq, 11.96 mmol) was stirred at 160 C for 16 h. The re-
action mixture was then diluted with CH2Cl2 (150 mL) and poured
into satd. NaHCO3 (100 mL) at 0 C. The organic layer was dried
(Na2SO4), filtered, and evaporated under reduced pressure. The re-
sulting residue was purified by silica gel column chromatography
(i-PrOH/NH4OH/H2O 6:3:1) affording 6 as white foam after freeze dry-
ing from H2O (118 mg, 85%). R f = 0.50 (i-PrOH/NH4OH/H2O 6:3:1).
1H NMR (CDCl3): = 1.50–2.30 (m, 4H, H-6 H-60 H-7 H-70), 3.17 (s,
3H, OCH3), 3.40–3.85 (m, 4H, H-2 H-3 H-4 H-5), 4.40–4.90 (m, 7H, H-1
3CH2Ph), 6.70–7.00 (m, 8H, 2NH4), 7.10–7.40 (m, 15H, 15H-ar). 13C
NMR (CDCl3): = 23.3 (d, J = 138.6 Hz), 24.3 (d, J = 2.3 Hz), 53.7,
70.6–79.3 (3C), 70.7 (d, J = 15.5 Hz), 70.8, 71.0, 74.1, 97.7, 126.4–127.9
(15C), 137.1–137.7 (3C). 31P NMR (CDCl3): = 27.2 (s, P(O)(ONH4)2).
MS (FAB +, NBA): m/z: 565 [M 2NH4+Na]+, 543 [M 2NH4+H]+, 511
[M 2NH4 OCH3]+, 91 [PhCH2]+.
Disodium Methyl 6-Deoxy-6-dihydroxyphosphonyl-
methylene- -D-mannopyranoside I
A suspension of 6 (100 mg, 0.24 mmol) and Pd-C 10% (250 mg) in
EtOH/H2O (1:1, 15 mL) was stirred at rt under H2 atmosphere for
16 h. The reaction mixture was filtered through celite and the filtrate
evaporated under reduced pressure. The residue was dissolved in H2O
(30 mL) and ion exchange resin (Dowex 50WX2, Na+ form, 10 g) was

Flexible Route to Mannose 6-Phosphonate
2373
added and the solution was kept at rt for 24 h before filtering-off the
resin and freeze drying from H2O to afford I as a white foam (48 mg,
90%). R f = 0.16 (i-PrOH/NH4OH/H2O 6:3:1). 1H NMR (D2O): = 1.20–
2.10 (m, 4H, H-6 H-60 H-7 H-70), 3.26 (s, 3H, OCH3), 3.30–3.70 (m, 3H,
H-3 H-4 H-5), 3.79 (dd, 1H, J = 1.4 and 3.2 Hz, H-2), 4.59 (d, 1H, H-1).
13C NMR (D2O): = 24.2 (d, J = 134.4 Hz), 25.4 (d, J = 2.2 Hz), 55.0,
70.2–70.8 (3C), 72.7 (d, J = 17.1 Hz), 101.1 (C-1). 31P NMR (D2O):
=
26.7 (s, P(O)(ONa)2). MS (FAB+, NBA): m/z: 273 [M 2Na + 3H]+. MS
(FAB , NBA): m/z: 271 [M 2Na H] .
Methyl 6-Deoxy- -D-manno-heptopyranoside 7
A suspension of 3 (1.84 g, 3.84 mmol) and Pd-C 10% (250 mg) in
EtOH/H2O (1:1, 20 mL) was stirred at rt under H2 atmosphere for 16 h.
The reaction mixture was filtered through celite and the filtrate evap-
orated under reduced pressure affording 7 as a white foam (760 mg,
95%). R f = 0.50 (CH2Cl2/MeOH 4:1). 1H NMR (D2O): = 1.50–1.70 (m,
1H, H-6), 1.90–2.10 (m, 1H, H-60), 3.27 (s, 3H, OCH3), 3.38 (t, 1H, J =
9.5 Hz, H-4), 3.45–3.75 (m, 4H, H-3 H-5 H-7 H-70), 3.81 (dd, 1H, J = 1.6
and 3.4 Hz, H-2), 4.58 (d, 1H, H-1).
Methyl 2,3,4-Tri-O-acetyl-7-bromo-6,7-dideoxy-
-D-manno-heptopyranoside 8
To a solution of 7 (760 mg, 3.65 mmol) in C5H5N (20 mL) was added a so-
lution of TsCl (1.05 g, 5.48 mmol) in C5H5N (10 mL). After stirring for 5 h
at rt, acetic anhydride (5 mL) was added at 0 C. The reaction was stirred
at rt for 1 h then poured into satd. NaHCO3 (150 mL). The aqueous layer
was extracted with CH2Cl2 (3 150 mL). Organic layers were combined,
dried (Na2SO4), filtered, and evaporated under reduced pressure and
C5H5N was coevaporated with toluene (3 100 mL). The residue was
dissolved in 2-butanone (50 mL) and LiBr (6.35 g, 73.07 mmol) was
added. The reaction was stirred at 85 C for 1 h then cooled to rt and di-
luted with H2O (100 mL). The aqueous layer was extracted with CH2Cl2
(3 100 mL). Organic layers were combined, dried (Na2SO4), filtered,
and evaporated under reduced pressure. The resulting residue was pu-
rified by silica gel column chromatography (light petroleum/ether 9:1
then 2:3) to afford 8 (800 mg, 55%). R f = 0.57 (light petroleum/ether
1
2:3). H NMR (CDCl3): = 1.80–2.05 (m, 2H, H-6 H-60), 1.87 (s, 3H,
CH3CO), 1.95 (s, 3H, CH3CO), 2.02 (s, 3H, CH3CO), 3.34 (s, 3H, OCH3),
3.40–3.50 (m, 2H, H-7 H-70), 3.88 (td, 1H, J = 3.3 and 9.8 Hz, H-5), 4.58
(d, 1H, J = 1.7 Hz, H-1), 4.99 (t, 1H, H-4), 5.11 (dd, J = 3.4 Hz ,H-2),
5.18 (dd, 1H, H-3).

2374
S. Vidal et al.
1,2,3,4-Tetra- O-acetyl-7-bromo-6,7-dideoxy-D-manno-
heptopyranoside 9
A solution of 8 (100 mg) in AcOH/Ac2O (4:1, 10 mL) and two drops
of conc. H2SO4 was stirred at rt for 24 h. The reaction mixture was
neutralized with satd. NaHCO3 (100 mL) and the aqueous layer was
extracted with EtOAc (3 100 mL). Organic layers were combined,
dried (Na2SO4), filtered, and evaporated under reduced pressure. The
resulting residue was purified by silica gel column chromatography
(light petroleum/ether 95:5 then 3:7) affording 9 as a pale yellow oil
(91 mg, 85%) and a portion of 8 (15 mg) was recovered. R f = 0.61 (light
petroleum/ether 3:7). 1H NMR (CDCl3): = 1.80–2.00 (m, 2H, H-6 H-60),
2.00 (s, 3H, CH3CO), 2.07 (s, 3H, CH3CO), 2.15 (s, 3H, CH3CO), 3.40–
3.70 (m, 2H, H-7 H-70), 4.08 (td, 1H, J = 3.0 and 9.9 Hz, H-5), 5.16
(t, 1H, H-4), 5.26 (dd, 1H, J = 1.8 and 3.5 Hz, H-2), 5.34 (dd, 1H, H-3),
5.99 (d, 1H, H-1). 13C NMR (CDCl3): = 20.9, 21.1, 21.2, 29.2, 33.9,
68.4–69.6 (4C), 90.6, 170.1, 170.2, 170.3, 170.4. MS (FAB+, NBA): m/z:
447 [M + Na]+, 365 [M OAc]+, 345 [M Br]+, 245 [M 2OAc 2H]+, 203
[M 2OAc CH3CO H]+, 43 [CH3CO]+.
2,3,4-Tri-O-acetyl-7-bromo-6,7-dideoxy-1,2- O-
[8-(cholest-5-en-3 -yloxy)-3,6-dioxaoctan-1-
yloxyethylidene]- -D-manno-heptopyranose 11
A solution of 9 (220 mg, 0.52 mmol) and HBr (5.7 M in AcOH, 2.28 mL,
13 mmol) in CH2Cl2 (15 mL) was stirred at rt for 16 h. The reaction
mixture was poured into satd. NaHCO3 (50 mL) and the aqueous layer
was extracted with CH2Cl2 (3 70 mL). Organic layers were combined,
dried (Na2SO4), filtered, and evaporated under reduced pressure af-
fording 10 used for next step as the crude product. To a solution of
8-(cholest-5-en-3 -yloxy)-3,6-dioxaoctan-1-ol43 (287 mg, 0.57 mmol)
and AgOTf (146 mg, 0.57 mmol) dissolved in CH2Cl2 (5 mL) was added
sym-collidine (76 L, 0.57 mmol) and a solution of 10 in CH2Cl2 (15 mL).
After stirring at rt for 15 min, the reaction mixture was filtered through
celite and the filtrate evaporated under reduced pressure. The result-
ing residue was purified by silica gel column chromatography (light
petroleum/ether 4:1 then ether) affording 11 as a white gum (206 mg,
45%). R f = 0.41 (light petroleum/ether 1:4). 1H NMR (CDCl3): = 0.67
(s, 3H, H-18Chol), 0.86 (d, 6H, J = 6.6 Hz, H-26Chol), 0.91 (d, 3H, J =
6.4 Hz, H-21Chol), 0.99 (s, 3H, H-19Chol), 0.80–2.50 (m, 30H, 28H-Chol
H-6 H-60), 1.71 (s, 3H, CH3†), 2.07 (s, 3H, CH3CO), 2.11 (s, 3H, CH3CO),
3.10–3.30 (m, 1H, H-3Chol), 3.45–3.70 (m, 15H, OCH2CH2O H-5 H-7
H-70), 4.59 (dd, 1H, J = 2.6 and 3.5 Hz, H-2), 5.00–5.20 (m, 2H, H-3

Flexible Route to Mannose 6-Phosphonate
2375
H-4), 5.30–5.38 (m, 1H, H-6Chol), 5.44 (d, 1H, H-1). 13C NMR (CDCl3):
= 12.2, 15.6–41.7 (C-Chol CH3† C-6), 21.1, 21.2, 29.1, 50.6, 56.5, 57.2,
62.4, 66.2, 67.7, 69.0, 70.3, 70.9, 71.0, 71.1, 71.3, 76.8, 79.9, 97.9, 124.7
(C ), 141.4, 170.2, 170.7.
8-(Cholest-5-en-3 -yloxy)-3,6-dioxaoctan-1-yl-2,3,4-tri-
O-acetyl-7-bromo-6,7-dideoxy- -D-manno-
heptopyranoside 12
˚
To a suspension of 11 (160 mg, 0.18 mmol) and molecular sieves (3A) in
CH2Cl2 (5 mL) was added dropwise TMSOTf (3.3 L, 18 mol) between
5 C and 0 C (ice-salt bath). The suspension was stirred for 5 min be-
fore neutralizing with Et3N (50 L) and diluting with H2O (50 mL).
The aqueous layer was extracted with CH2Cl2 (3 50 mL). Organic
layers were combined, dried (Na2SO4), filtered, and evaporated under
reduced pressure. The resulting residue was purified by silica gel col-
umn chromatography (light petroleum/ether 9:1 then ether) affording
12 as a white gum (64 mg, 40%). R f = 0.58 (light petroleum/ether 1:4).
1H NMR (CDCl3): = 0.60 (s, 3H, H-18Chol), 0.79 (d, 3H, J = 6.6 Hz,
H-26Chol), 0.80 (d, 3H, H-26Chol), 0.84 (d, 3H, J = 6.5 Hz, H-21Chol), 0.92
(s, 3H, H-19Chol), 0.70–2.35 (m, 28H, 28H-Chol), 1.85–1.95 (m, 1H, H-6),
1.91 (s, 3H, CH3CO), 1.99 (s, 3H, CH3CO), 2.07 (s, 3H, CH3CO), 2.00–
2.10 (m, 1H, H-60), 3.05–3.15 (m, 1H, H-3Chol), 3.40–3.50 (m, 1H, H-7),
3.50–3.70 (m, 13H, OCH2CH2O H-70), 3.97 (td, 1H, J = 2.5 and 9.9 Hz,
H-5), 4.75 (d, 1H, J = 1.5 Hz, H-1), 5.04 (t, 1H, H-4), 5.21 (dd, 1H, J =
3.5 Hz, H-2), 5.25–5.30 (m, 1H, H-6Chol), 5.27 (dd, 1H, H-3). 13C NMR
(CDCl3): = 12.3, 19.1–42.7 (C-Chol), 21.1, 21.2, 21.5, 29.2, 34.9, 50.6,
56.6, 57.2, 62.6, 67.7, 67.9, 69.5, 69.8, 70.1, 70.5, 71.0, 71.2, 71.3, 79.9,
97.8, 121.9, 141.4, 170.2, 170.3, 170.5.
Disodium 8-(Cholest-5-en-3 -yloxy)-3,6-dioxaoctan-1-yl-
6-deoxy-6-dihydroxyphosphonylmethylene-
-D-mannopyranoside M6Pn/TEGC 13
A solution of 12 (67 mg, 76 mol) dissolved in tris(trimethylsilyl)
phosphite (5 mL) was stirred at 160 C for 16 h. The reaction mix-
ture was then diluted with CH2Cl2 (15 mL) and poured into satd.
NaHCO3 (25 mL) at 0 C. The aqueous layer was extracted with CH2Cl2
(3 50 mL). Organic layers were combined, dried (Na2SO4), filtered,
and evaporated under reduced pressure. The crude mixture (show-
ing a single signal by 31P NMR spectroscopy at 27 ppm) was dis-
solved in MeOH (5 mL) and NaOMe (35 mg, 610 mol) was added.

2376
S. Vidal et al.
After 24 h, the reaction was neutralized with 1 M HCl and evapo-
rated under reduced pressure. The resulting residue was purified by
silica gel column chromatography (i-PrOH/NH4OH/H2O 6:3:1), then
treated with ion exchange resin (Dowex 50WX2, Na+ form, 5 g) af-
fording M6Pn/TEGC 13 (31 mg, 50%). R f = 0.52 (i-PrOH/NH4OH/H2O
6:3:1). 1H NMR (D2O): = 0.70–2.30 (m, 47H, H-Chol H-6 H-60 H-7 H-
70), 3.20–4.00 (m, 17H, OCH2CH2O H-2 H-3 H-4 H-5 H-3Chol), 5.30–5.40
(m, 1H, H-6Chol). 31P NMR (D2O): = 23.0 (s, P(O)(ONa)2). -MS (FAB+,
G/T): m/z: 803 [M + H]+, 781 [M Na + 2H]+. MS (FAB , G/T): m/z: 757
[M 2Na + H] .
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