Synthesis and biological evaluation of new mannose 6-phosphate analogues

Article abstract of DOI:10.1016/S0968-0896(02)00264-X

Three new analogues of mannose 6-phosphate (M6P) - a sulphate and two carboxylates - have been synthesized and their affinity toward the M6P/IGFII receptor evaluated by affinity column chromatography. These compounds display strong binding to the receptor

Full text of DOI:10.1016/S0968-0896(02)00264-X

Bioorganic & Medicinal Chemistry 10 (2002) 4051–4056
Synthesis and Biological Evaluation of New Mannose 6-Phosphate
Analogues
Sebastien Vidal,a Marcel Garcia,b Jean-Louis Monteroa and Alain Morerea,*
a
´ ´ ´
Laboratoire de Chimie Biomoleculaire (UMR 5032), Universite Montpellier II, Ecole Nationale Superieure de Chimie de Montpellier,
8 Rue de l’Ecole Normale, F-34296 Montpellier Cedex 05, France
´
INSERM, Unite 540, EMCC, 60 Rue de Navacelles, F-34090 Montpellier, France
b
Received 11 March 2002;accepted 28 June 2002
Abstract—Three new analogues of mannose 6-phosphate (M6P)—a sulphate and two carboxylates—have been synthesized and
their affinity toward the M6P/IGFII receptor evaluated by affinity column chromatography. These compounds display strong
binding to the receptor and therefore are new M6P analogues which may find some dermatological applications, for example
healing of post-surgical scars.
# 2002 Elsevier Science Ltd. All rights reserved.
Results and Discussion
Introduction
Recognition requirements and previous results
Mannose 6-phosphate (M6P) is a recognition marker
involved in the selective targeting of newly synthesized
enzymes to lysosomes.1 While two different mannose
6-phosphate receptors (M6PR) recognize specifically the
M6P residues, only the larger M6P receptor mediates
the endocytosis of extracellular M6P-containing
ligands.2 This receptor is a type I glycoprotein of 275
kDa, which apart from M6P residues, is also known to
bind, through distinct binding sites, retinoic acid3 (RA)
and insulin-like growth factor II4 (IGFII). This receptor
(M6P/IGIIR) is the first example of a protein able to
bind three different classes of ligands, that is a sacchar-
ide (M6P), a peptide (IGFII) and a lipid (RA). More-
over, the M6P/IGFIIR plays a fundamental role in the
control of cell growth in fetal development and carci-
nogenesis.5 In our ongoing research program, we focus
on the ability of the M6P/IGFIIR to recognize and then
internalize M6P analogues. We recently described6 the
synthesis of two mannose 6-phosphonate (M6Pn) ana-
logues (I and II) of M6P (Fig. 1), and evaluated their
affinity towards the M6P/IGFIIR. Isosteric compound I
has been shown to have an affinity to the M6P/IGFIIR
similar to natural M6P, whereas non-isosteric com-
pound II was only weakly recognized. We present
herein the preparation and biological evaluation of
three M6P analogues.
As a continuation of our previous work, we decided to
synthesize other analogues with a few modifications of
the carbohydrate backbone in agreement with some
observations reported in literature.7ꢀ9 Some structural
features were shown to be determinant in the binding of
M6P to the receptor such as : (a) the hydroxyl group at
the 2-position of the pyranose ring must be axial since
glucose 6-phosphate (G6P) is weakly recognized by the
receptor whereas fructose 1-phosphate (F1P) and M6P
bind strongly to the receptor (Fig. 1). The absence of the
hydroxyl group at the same position will also affect the
recognition since 2-deoxy-glucose 6-phosphate (2dG6P)
is as weakly recognized as G6P.7 The substitution at the
anomeric center (b) does not influence the interaction
since F1P is as well recognized as M6P7 (Fig. 1) and (c)
the substitution at the 5-position of the hexopyranoside
ring — anomeric position for the F1P — does not affect
the interaction since F1P is as well recognized as M6P7
(Fig. 2).
Another important structural feature was shown to play
an important role in the recognition phenomenon,
namely the distance between the negative charge and the
pyranose ring. We demonstrated6 that this distance
must be in the same range as that in the M6P, that is
four atoms from the 5-position (Fig. 2) since the isos-
teric M6Pn analogue is very well recognized by the
M6P/IGFIIR whereas the non isosteric M6Pn analogue
*Corresponding author. Tel.: +33-4-6714-4345;fax: +33-4-6714-
4344;e-mail: morere@univ-montp2.fr
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00264-X

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S. Vidal et al. / Bioorg. Med. Chem. 10 (2002) 4051–4056
Figure 1. Relation of the distance between the negative charge and the pyranose ring.
derivative 2. This compound could be prepared easily
by a two steps synthesis (Scheme 1) starting from
known methyl 2,3,4-tri-O-benzyl-a-d-mannopyrano-
side11 by treatment with sulfur trioxide/pyridine com-
plex12 affording
1
in 90% yield. Subsequent
hydrogenolysis13 of the benzyl protecting groups furn-
ished 2 in 85% yield. It is worth mentioning that pur-
ification of this compound could be achieved by
quenching the reaction with Na2CO3 (1 M) then wash-
ing off the crude solid using methanol in which Na2CO3
is insoluble. This purification method will allow for the
preparation in bulk quantities.
We also decided to prepare two carboxylic acids in
order to further investigate the structure–activity rela-
tionships of the M6P/IGFIIR. These derivatives can be
obtained by using a Swern oxidation14 at the 6-position
of mannose affording an aldehyde function which was
then reacted under Wittig–Horner conditions15 to fur-
nish the corresponding conjugated esters. Another
approach is the condensation of a malonate diester with
a 6-deoxy-6-halogeno carbohydrate derivative under
basic conditions.16 Although both of these methods
have been extensively described in literature for the
preparation of various octoglycosuronic acids,17 none
of these focused on the synthesis of the deprotected
acids and their subsequent biological evaluation. We
therefore chose to use the Wittig–Horner method and
selected two different protecting groups — namely ben-
zyl and trimethylsilyl ethers — in order to obtain both
the aliphatic carboxylic acid derivative 5 and the con-
jugated carboxylic acid derivative 8 respectively, as
outlined in Scheme 2.
Figure 2. Three main structural requirements for reaching high affinity
toward the M6P/IGFIIR. Influence of the substitution at the (a) 2-
position, (b) anomeric center and (c) 5-position.
was very weakly recognized. We recently prepared two
b-hydroxyphosphonate analogues of M6P and demon-
strated that they were not recognized by the M6P/
IGFIIR.10 While the replacement of the phosphomo-
noester linkage by a methylene group does not affect the
recognition phenomenon,6 further substitution at the
6-position lessens the interaction of the analogues
obtained with the receptor, probably due to steric hin-
drance.10
Concerning the negative charge, it has been shown8 that
a singly charged phosphodiester analogue of M6P (with
one methyl ester on the phosphate moiety) displayed
very similar affinity toward the receptor than M6P. This
result demonstrates that only a single negative charge is
necessary for the binding to the M6P/IGFIIR.
The Swern oxidation14 of methyl 2,3,4-tri-O-benzyl-a-d-
mannopyranoside11 afforded the corresponding alde-
hyde (not isolated), which was then reacted with trie-
thylphosphonoacetate sodium salt affording the
conjugated ester 3 in 80% yield over both steps. The
hydrogenolysis of benzyl protecting groups afforded 4
in 98% yield and subsequent saponification of the ester
moiety furnished 5 in 95% yield. In the same fashion,
methyl 2,3,4-tri-O-trimethylsilyl-a-d-mannopyranoside18
was oxidized14 to the corresponding aldehyde and reac-
ted under the same Wittig–Horner conditions to obtain
6 in 85% over both steps. Mild acid hydrolysis of the
trimethylsilyl protecting groups afforded 7 in 95% yield
and saponification of the ester moiety furnished 8 in
95% yield.
Preparation of the three new M6P analogues
According to these observations, we decided to synthe-
size three different M6P analogues which will display
only one negative charge and respect the geometrical/
functional requirements discussed above. The first of
these compounds will replace the M6P phosphorus
atom for a sulfur atom to obtain the sulphate monoester

S. Vidal et al. / Bioorg. Med. Chem. 10 (2002) 4051–4056
4053
Scheme 1. Reagents and conditions: (i) SO3/C5H5N, C5H5N, 4 h;(ii) H 2, Pd(OH)2/C (20%), EtOH/H2O (1:1), 24 h.
slightly lower affinity. The difference in affinities
between the carboxylic derivatives 5 (5 mM) and 8 (2.5
mM) might be related to a better defined geometry of
the double bond present in 8, in comparison with the
single bond present in 5.
Conclusions
In conclusion, three new M6P analogues have been
synthesized in excellent yields and these compounds are
now being prepared on the 100 g scale for in vivo bio-
logical evaluation. The synthesis of both carboxylic
acids (5 and 8) is now being adapted for the preparation
of new amphiphilic M6P steroidal analogues in order to
generate new drug delivery systems as powerful as the
M6Pn cholesteryl conjugate previously described in a
recent paper.20 Another exciting potential application of
these compounds is in dermatological treatments, since
M6P and M6Pn were shown21 to reduce the size of
scars. The carboxylic acid analogues 5 and 8 are expec-
ted to display the same property and the preparation
described herein presents a very efficient synthesis of
these compounds for large-scale applications in various
dermatological treatments (post-surgery scars, wrinkles,
etc.).
Experimental
General aspects
All solvents were dried prior to use according to stan-
dard methods.22 Analytical TLC were performed using
aluminum-coated TLC plates 60-F254 (Merck). Plates
were developed with (1) UV light (254 nm), and (2)
immersion in a 10% H2SO4/EtOH solution followed by
charring or (3) immersion in a 5% rhodanine/EtOH
solution followed by charring (for aldehydes). Silica gel
column chromatography was performed with silica gel
60A (Carlo Erba). Optical rotations were measured at
the sodium d-line with a Perkin–Elmer-241 polarimeter.
IR Spectra were obtained on Perkin–Elmer FT-1600
spectrometer in solution. Fast Atom Bombardment
(FAB) mass spectra were recorded on a Jeol JMS-
DX300 spectrometer in either positive (>0) or negative
(<0) modes and using either 3-nitrobenzylic alcohol
Scheme 2. Reagents and conditions: (i) (COCl)2, DMSO, i-Pr2NEt,
ꢀ60 ꢁC, THF;(ii) (EtO) 2(O)PCH2CO2Et, NaH, THF;(iii) H 2, Pd/C
(10%), EtOH/H2O (1:1), 4 h;(iv) 2 M NaOH, 1 h;(v) HCl (1 M in
THF), 5 min.
Biological evaluation of the three new M6P analogues
With these three new M6P analogues in our hands, we
needed to determine their relative affinities toward the
M6P/IGFIIR. These biological assays were based on
the method we described before.10 Pentamannose
6-phosphate Sepharose columns19 were loaded with the
receptor then eluted with analogues 2, 5 and 8. The
results obtained are presented in Figure 3.
1
(NBA) or glycerol/thioglycerol (1:1) mixture (G/T). H
We observed that both the sulphate monoester 2 and
the conjugated carboxylic acid 8 displayed similar affi-
nities (2.5 mM) to the M6P (Table 1) while 5 displayed a
NMR spectra were recorded on a Bruker DRX 400
(400 MHz), at 25 ꢁC. Chemical shifts (d) are given in
ppm and referenced using residual solvent signals (7.24

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S. Vidal et al. / Bioorg. Med. Chem. 10 (2002) 4051–4056
Figure 3. Affinities of M6P analogues toward M6P/IGFIIR. M6P/IGFIIR from calf serum was retained on phosphomannan Sepharose columns
and eluted with the indicated concentrations of mannose 6-phosphate (M6P test) and compounds 2, 5 and 8. The eluted protein was analyzed in a
10% SDS polyacrylamide gel followed by silver-staining. Mw, molecular weight markers;boxes indicate the eluted M6P/IGFIIR.
Table 1. Relative affinities observed for compounds 2, 5 and 8
mannan Sepharose.19 The M6P/IGFIIR was purified
from fetal calf serum on a phosphomannan–Sepharose
affinity column, according to the method previously
described.23 SDS polyacrylamide gel electrophoresis was
performed according to Laemmli24 and proteins visua-
lized using a silver-staining kit (Biorad).
Compd
Minimum concentration needed to
observe the eluted M6P/IGFIIR (mM)
M6P
M6Pn I
2.5
1a
2
5
8
2.5
5
2.5
Methyl 6-O-Sulfonato-2,3,4-tri-O-benzyl-ꢀ-D-mannopyr-
anoside (1). To a solution of SO3/C5H5N complex (335
mg, 2.09 mmol) in C5H5N (5 mL), a solution of methyl
2,3,4-tri-O-benzyl-a-d-mannopyranoside11 (540 mg,
1.16 mmol) in C5H5N (5 mL) was added. Reaction was
stirred for a period of 4 h after which it was neutralized
with Na2CO3(aq) (1 M, 10 mL) and concentrated under
reduced pressure. The salts were washed with anhy-
drous MeOH (50 mL) and filtered. The filtrate was
concentrated under reduced pressure and the residue
purified by silica gel column chromatography (EtOAc/
MeOH 9:1) affording 1 (590 mg, 90%) as a white foam.
Rf 0.70 (EtOAc/MeOH 3:1). [a]D +58.3 (c=0.9/
CHCl3). 1H NMR (CDCl3), d: 3.21 (s, 3H, OCH3);
3.62–3.75 (m, 1H, H-5);3.72–3.78 (m, 1H, H-2);3.82
(dd, 1H, J=2.1, 9.3 Hz, H-3);4.11 (t, 1H, H-4);4.38–
4.47 (m, 2H, H-6 and H-60);4.52 (s, 2H, C H2Ph);4.62–
5.00 (m, 5H, CH2Ph and H-1);7.10–7.40 (m, 15H, H-
ar). 13C NMR (CDCl3), d: 55.5 (OCH3);66.7–77.7 (C-6
and CH2Ph);70.5–80.3 (C-2, C-3, C-4 and C-5);99.7
aResult previously described.6
ppm for CHCl3 and 4.79 ppm for HOD). The following
abbreviations were used to explain the signal multi-
plicities or characteristics: s (singlet), d (doublet), dd
(double doublet), ddd (double double doublet), t (tri-
plet), td (triplet doublet), q (quartet), m (multiplet). 13C
NMR spectra were recorded on a Bruker DRX 400
(100.6 MHz). Chemical shifts (d) are given in ppm rela-
tive to TMS as an external reference. 29Si NMR spectra
were recorded on a Bruker DPX 200 (39.8 MHz). Che-
mical shifts (d) 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 (d) are
given in ppm relative to phosphoric acid (85%) as an
external reference. The pentamannose 6-phosphate was
functionalized with b-(p-aminophenyl)ethylamine, then
reduced with sodium tetrahydroborate, and finally cou-
pled on CNBr activated Sepharose leading to phospho-
IV
(C-1);127.9–129.1 ( CH–Ar);137.8–138.7 (C ). MS
ar

S. Vidal et al. / Bioorg. Med. Chem. 10 (2002) 4051–4056
4055
(FAB>0, NBA), m/z: 589 [M+Na]+, 567 [M+H]+,
487 [MꢀSO3+H]+, 455 [MꢀSO3ꢀOCH3]+, 91
[CH2Ph]+. MS (FAB<0, NBA): m/z: 543 [MꢀNa]ꢀ.
Ethyl (methyl 6,7-dideoxy-ꢀ-D-manno-octopyranoside)
uronate (4). A suspension of 3 (80 mg) and Pd/C (10%,
100 mg) in EtOH/H2O (1:1, 20 mL) was vigorously
stirred under an H2 atmosphere for 4 h. The reaction
was filtered through a Celite pad and concentrated
under reduced pressure affording pure 4 (39 mg, 98%)
as a yellow oil. Rf 0.55 (EtOAc/MeOH 9:1). [a]D +63.6
Methyl 6-O-sulfonato-ꢀ-D-mannopyranoside (2). A sus-
pension of 1 (530 mg) and Pd(OH)2/C (20%, 350 mg) in
EtOH/H2O (1:1, 50 mL) was vigorously stirred under an
H2 atmosphere for 24 h. The reaction was filtered
through a Celite pad and concentrated under reduced
pressure affording pure 2 (240 mg, 85%) as a white
powder. Mp 156–159 ꢁC. Rf 0.50 (CH2Cl2/MeOH 2:1).
1H NMR (D2O), d: 3.29 (s, 3H, OCH3);3.56 (t, 1H,
J=9.4 Hz, H-4);3.65 (dd, 1H, J=2.9 Hz, H-3);3.71
(ddd, 1H, J=2.2, 5.3 Hz, H-5);3.81 (dd, 1H, J=1.7 Hz,
H-2);4.10 (dd, 1H, J=11.2 Hz, H-6);4.23 (dd, 1H, H-
60);4.64 (d, 1H, H-1). 13C NMR (D2O), d: 55.2 (OCH3);
66.7 (C-4);67.6 (C-6);70.2 (C-2);70.7,70.8 (C-3 and C-
5);101.3 (C-1). MS (FAB <0, G/T), m/z: 295 [MꢀH]ꢀ,
273 [MꢀNa]ꢀ. C7H13NaO9S (296.02): calcd C 28.38, H
4.42;found C 27.85, H 4.58.
(c 1.1/CHCl3). IR (CH2Cl2): n: 1728 cmꢀ1, 3407 cmꢀ1
.
1H NMR (CDCl3), d: 1.26 (t, 3H, J=7.1 Hz,
OCH2CH3);1.65–1.95 (m, 1H, H-6);2.10–2.30 (m, 1H,
H-60);2.30–2.65 (m, 2H, H-7 and H-7 0);3.30–3.45 (m, 1H,
H-5);3.33 (s, 3H, OC H3);3.51 (t, 1H, J=8.9 Hz, H-4);
3.71 (dd, 1H, J=3.2 Hz, H-3);3.89 (dd, 1H, J=1.3 Hz, H-
2);4.14 (q, 2H, OC H2CH3);4.64 (d, 1H, H-1). 13C NMR
(CDCl3), d: 13.1 (OCH2CH3);25.5 (C-6);28.7 (C-7);53.9
(OCH3);59.6 (O CH2CH3);69.4–70.2 (C-2 C-3 C-4 and C-
5);99.9 (C-1);173.5 ( CO2Et). MS (FAB>0, NBA), m/z:
287 [M+Na]+, 265 [M+H]+, 233 [MꢀOCH3]+.
Sodium (methyl 6,7-dideoxy-ꢀ-D-manno-octopyranosi-
de)uronate (5). Compound 4 (30 mg) was dissolved in
2 M NaOH (3 mL) and the mixture was stirred for 1 h
and neutralized with ion exchange resin (Dowex
50WX2, H+ form, 7 g) until the pH was 4–5. The resin
was washed wih H2O (100 mL) and filtered off. The fil-
trate was freeze dried from H2O affording 5 (28 mg,
95%) as a light brown foam. Rf 0.36 (i-PrOH/NH4OH/
H20 8:1:1). [a]D +43.3 (c0.3/H2O). 1H NMR (DMSO-d6),
d: 1.40–1.60 (m, 1H, H-6);1.85–2.45 (m, 3H, H-6 0 H-7 and
H-70);3.15–3.45 (m, 2H, H-3H-4 and H-5);3.21 (s, 3H,
OCH3);3.30–3.45 (m, 1H, H-3);3.58 (dd, 1H, J=1.5, 3.2
Hz, H-2);4.46 (d, 1H, H-1). 13C NMR (DMSO-d6), d:
27.7 (C-6);31.1 (C-7);54.8 (O CH3);71.0–71.9 (C-2 C-3 C-
4 and C-5);101.8 (C-1);175.6 ( CO2Na). MS (FAB>0,
NBA), m/z: 259 [M+H]+, 237 [M-Na]+. MS (FAB<0,
NBA), m/z: 235 [MꢀNa]ꢀ. C9H15NaO7 (258.07): calcd
C 41.87, H 5.86;found C 41.44, H 5.99.
Ethyl [methyl (E)-2,3,4-tri-O-benzyl-6,7-dideoxy-ꢀ-D-
manno-oct-6-enopyranoside]uronate (3). To a cooled
(ꢀ60 ꢁC) solution of oxalyl chloride (106 mL, 1.23
mmol) in THF (2 mL), DMSO (190 mL, 2.69 mmol) was
added dropwise. After 10 min, a THF (4 mL) solution
of methyl 2,3,4-tri-O-benzyl-a-d-mannopyranoside11
(520 mg, 1.12 mmol) was added dropwise. After an
additional 20 min, i-Pr2NEt (960 mL, 5.6 mmol) was
added dropwise. Reaction was allowed to reach rt, stir-
red for 2 h and then concentrated under reduced pres-
sure. The residue was diluted with CH2Cl2 (130 mL) and
the organic layer washed with brine (100 mL), dried
(Na2SO4), filtered and concentrated under reduced
pressure. The crude aldehyde was used for the next step
without further purification.
To a suspension of NaH (95%, 57 mg, 2.24 mmol) in
THF (2 mL) was added triethylphosphonoacetate (540
mL, 2.69 mmol). Reaction was stirred for 10 min, then a
solution of the crude aldehyde in THF (5 mL) was
added dropwise. The mixture was stirred for 30 min
before concentrating under reduced pressure. The resi-
due was diluted with CH2Cl2 (130 mL) and the organic
layer washed with brine (100 mL), dried (Na2SO4),
filtered and concentrated under reduced pressure. The
residue was purified by silica gel column chroma-
tography (hexane/ether 3:2!1:1) to afford 3 (477 mg,
80%) as a yellow oil. Rf 0.62 (hexane/ether 1:1). [a]D
Ethyl [methyl (E)-2,3,4-tri-O-trimethylsilyl-6,7-dideoxy-ꢀ-
D-manno-oct-6-enopyranoside]uronate (6). To a cooled
(ꢀ60 ꢁC) solution of oxalyl chloride (340 mL, 4.02
mmol) in THF (2 mL), DMSO (620 mL, 8.78 mmol) was
added dropwise, after 10 min, a THF (5 mL) solution of
methyl
2,3,4-tri-O-trimethylsilyl-a-d-mannopyrano-
side18 (1.5 g, 3.66 mmol) was added dropwise. After an
additional 20 min, i-Pr2NEt (3.13 mL, 18.3 mmol) was
added dropwise. The reaction mixture was kept at
ꢀ60 ꢁC for 10 min then allowed to reach rt, stirred for 3 h
and diluted with CH2Cl2 (180 mL). The organic layer was
washed with brine (75 mL), dried (Na2SO4), filtered and
concentrated under reduced pressure. The crude aldehyde
was used for next step without further purification. To a
suspension of NaH (95%, 185 mg, 7.32 mmol) in THF (2
mL) was added triethyl phosphonoacetate (1.76 mL, 8.78
mmol). After 10 min, a THF (10 mL) solution of the crude
aldehyde was added dropwise. Reaction was stirred for 10
min, solvent was evaporated and the residue diluted
with CH2Cl2 (180 mL). The organic layer was washed
with brine (75 mL), dried (Na2SO4), filtered and con-
centrated under reduced pressure. The residue was pur-
ified by silica gel column chromatography (light
petroleum/ether 1:1) affording 6 (1.48 g, 85%) as a color-
less oil. Rf 0.41 (light petroleum/ether 4:1). [a]D +57.5 (c
+77.9 (c 0.95/CHCl3). IR (NaCl film), n: 1661 cmꢀ1
,
1712 cmꢀ1. H NMR (CDCl3), d: 1.33 (t, 3H, J=7.0
Hz, OCH2CH3);3.35 (s, 3H, OC H3);3.82 (t, 1H, J=9.3
Hz, H-4);3.85–3.90 (m, 1H, H-2);3.99 (dd, 1H, J=2.9
Hz, H-3);4.23–4.34 (m, 1H, H-5);4.28 (q, 2H,
1
OCH2CH3);4.60–5.00 (m, 7H, CH Ph and H-1);6.31
2
(dd, 1H, J=1.1, 15.7 Hz, H-7);7.22 (dd, 1H, J=4.7 Hz,
H-6);7.23–7.50 (m, 15H, H-ar). 13C NMR (CDCl3), d:
13.2 (OCH2CH3);53.8 (O CH3);59.3 (O CH2CH3);69.4–
79.0 (C-2, C-3, C-4 and C-5);71.3, 71.8, 74.4 ( CH2Ph);
98.2 (C-1);121.1 (C-7);126.5–127.7 ( CH-Ar);136.9–
137.4 (CIaVr );143.1 (C-6);165.3 ( CO2Et). MS (FAB>0,
NBA), m/z: 531 [M-H]+, 501 [MꢀOCH3]+, 91
[CH2Ph]+.

4056
S. Vidal et al. / Bioorg. Med. Chem. 10 (2002) 4051–4056
1.07/CHCl3). IR (CHCl3), n: 1660 cmꢀ1, 1728 cmꢀ1. 1H
NMR (CDCl3), d: 0.10, 0.12, 0.15 (3s, 27H, Si(CH3)3);
1.25 (t, 3H, J=7.1 Hz, OCH2CH3);3.32 (s, 3H, OC H3);
3.65–3.85 (m, 3H, H-2H-3 and H-4);4.09 (ddd, 1H,
J=1.7, 4.8, 8.5 Hz, H-5);4.21 (q, 2H, OC H2CH3);4.53
(d, 1H, J=1.4 Hz, H-1);6.16 (dd, 1H, J=15.7 Hz, H-7);
7.07 (dd, 1H, H-6). 13C NMR (CDCl3), d: 0.4–1.4
(Si(CH3)3);14.6 (OCH 2CH3);55.1 (O CH3);60.6
(OCH2CH3);71.8–73.8 (C-2, C-3, C-4 and C-5);102.4
References and Notes
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4. (a) Schmidt, B.;Kiecke-Siemsen, C.;Waheed, A.;Braulke,
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(C-1);122.0 (C-7);145.7 (C-6);166.7 (
CO2Et). 29Si
NMR (CDCl3), d: 17.6, 18.3, 19.9 (3s, Si(CH3)3). MS
(FAB>0, NBA), m/z: 551 [M+SiMe3]+, 501
[M+Na]+, 478 [M]+, 447 [MꢀOCH3]+, 73 [SiMe3]+.
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Ethyl [methyl (E)-6,7-dideoxy-ꢀ-D-manno-oct-6-enopyr-
anoside]uronate (7). Compound 6 (400 mg) was dissolved
in a solution of HCl (1 M in THF, 20 mL) and the mixture
was stirred for 5 min. The reaction was quenched with
saturated NaHCO3(aq) until the pH was 8–9. The aqueous
layer was extracted with CH2Cl2 (3ꢂ70 mL). The organic
layers were combined, dried (Na2SO4), filtered and con-
centrated under reduced pressure. Compound 7 (230 mg,
95%) was obtained pure as a colorless oil. Rf 0.65 (EtOAc/
MeOH 9:1). [a]D +65.0 (c 0.6/CH2Cl2). IR (CH2Cl2), n:
1716 cmꢀ1, 3410 cmꢀ1. 1H NMR (CDCl3), d: 1.25 (m, 3H,
OCH2CH3);3.34 (s, 3H, OC H3);3.58 (t, 1H, J=9.4 Hz, H-
4);3.78 (dd, 1H, H-3);3.92 (dd, 1H, J=1.4, 3.2 Hz, H-2);
4.05–4.25 (m, 3H, H-5 and OCH2CH3);4.45 (m, 1H, O H);
4.73 (d, 1H, H-1);4.83 (m, 1H, O H);4.91 (m, 1H, O H);
6.19 (dd, 1H, J=1.7, 15.8 Hz, H-7);7.11 (dd, 1H, J=4.5
Hz, H-6). 13C NMR (CDCl3), d: 13.1 (OCH2CH3);54.0
(OCH3);59.7 (O CH2CH3);69.0–70.2 (C-2, C-3, C-4 and C-
5);100.2 (C-1);120.9 (C-7);143.5 (C-6);166.2 ( CO2Et). MS
(FAB>0, NBA), m/z: 285 [M+Na]+, 263 [M+H]+, 231
[MꢀOCH3]+.
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Sodium [methyl (E)-6,7-dideoxy-ꢀ-D-manno-oct-6-eno-
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1
H2O 8:1:1). [a]D +52.0 (c1.0/H2O). H NMR (D2O), d:
3.28 (s, 3H, OCH3);3.47 (t, 1H, J=9.6 Hz, H-4);3.67
(dd, 1H, J=3.4 Hz, H-3);3.84 (dd, 1H, J=1.7 Hz, H-
2);3.95–4.10 (m, 1H, H-5);4.66 (d, 1H, H-1);6.04 (dd,
1H, J=0.9, 15.7 Hz, H-7);6.54 (dd, 1H, J=6.7 Hz, H-6).
13C NMR (D2O), d: 54.0 (OCH3);68.8–70.8 (C-2, C-3, C-
4 and C-5);100.2 (C-1);128.0 (C-7);138.7 (C-6);172.6
(CO2Na). MS (FAB>0, NBA), m/z: 257 [M+H]+. MS
(FAB<0, NBA), m/z: 233 [MꢀNa]ꢀ. C9H13NaO7
(256.06): calcd C 42.19, H 5.11;found C 41.75, H 5.29.
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Purification of
We are grateful to the Fondation pour la Recherche
Medicale (to S.V.) and to the Conseil Scientifique de
l’Universite Montpellier II for financial support. We
also wish to thank Mayoly-Spindler Laboratories
(Chatou, France) for financial support.