Synthesis of three C-glycoside analogues of UDP-galactopyranose as conformational probes for the mutase-catalyzed furanose/pyranose interconversion

Article abstract of DOI:10.1002/ejoc.200801249

UDP-galactopyranose mutase (UGM) catalyzes the isomerization of UDP-galactopyranose (UDP-Galp) into UDP-galactofuranose (UDP-Galf), an essential step of the mycobacterial cell wall biosynthesis, Tn order to probe the UGM binding pocket, we synthesized the

Full text of DOI:10.1002/ejoc.200801249

FULL PAPER
DOI: 10.1002/ejoc.200801249
Synthesis of Three C-Glycoside Analogues of UDP-Galactopyranose as
Conformational Probes for the Mutase-Catalyzed Furanose/Pyranose
Interconversion
Audrey Caravano[a][‡] and Stéphane P. Vincent*[a]
Dedicated to Professor Alain Krief
Keywords: Biosynthesis / Phosphorus / Inhibitors / C-Glycosides / Carbohydrates
UDP-galactopyranose mutase (UGM) catalyzes the isomer-
ization of UDP-galactopyranose (UDP-Galp) into UDP-galac-
tofuranose (UDP-Galf), an essential step of the mycobacterial
cell wall biosynthesis. In order to probe the UGM binding
pocket, we synthesized the α- and β-C-glycosidic analogues
of UDP-galacopyranose along with the corresponding UDP-
exo-galactal. Preliminary inhibition evaluation indicated that
UDP-exo-galactal inhibits UGM with a binding affinity sim-
ilar to that of UDP-α-C-galactopyranose.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2009)
(Figure 1). Such an enzymatic isomerization is a key biocat-
alytic process not only because it is essential for the survival
of mycobacteria, but also because its mechanism addresses
questions that are at the heart of enzymology and the chem-
istry of carbohydrates. UDP-galactopyranose mutase
(UGM), the enzyme catalyzing this reaction, is a flavoen-
zyme whose FAD cofactor plays an unprecedented role in
biochemistry.[5,6] Moreover, the binding mode of the galac-
tose residue within the UGM catalytic pocket has raised
many questions and led to different investigations:
potentiometric analysis,[6] STD-NMR experiments corre-
lated with computational studies,[7] mutagenesis,[8] as well
as inhibition studies with analogues of its substrate.[9–13]
In preliminary studies, our approach was to probe the
UGM binding site with conformationally locked molecules
such as 3[11,14] and 4 but also with acyclic iminogalactitol
derivatives.[15] Moreover, we found that UDP-exo-glycals
derived from galactofuranose displayed interesting time-de-
pendent inactivation properties.[12,13] All these molecules
were analogues of the product UDP-Galf 2. Therefore, to
have a global and comparative overview of the binding
mode of the galactose moiety with UGM we need now to
assess the inhibition profiles of C-glycosidic analogues of
UDP-galactopyranose 1 and compare them to boat-locked
galactoside 3 and furanoside 4.
Introduction
Although tuberculosis can be effectively treated by anti-
biotic therapy, it remains a life-threatening infection, not
only in Third World Countries but also in western coun-
tries.[1] Notably, the identification of extreme drug-resistant
tuberculosis strains has recently raised considerable al-
arm.[2] The unusual structure of the cell wall of Mycobacte-
rium tuberculosis, the causative agent of tuberculosis, is now
acknowledged to be a key target for the development of
new therapeutics.[3] This cell wall insures the survival of the
mycobacteria by acting both as a permeability barrier to
the passage of antibiotics and also as a modulator of the
host immune system. Oligo-galactofuranosides (Galf) are
conserved cell wall glycoconjugates of all mycobacteria
whose biosynthesis has attracted much attention for the dis-
covery of novel classes of molecules that would be devel-
oped for the treatment of tuberculosis.[4]
The search for the biosynthetic origin of the Galf resi-
dues has led to the discovery of an unusual enzymatic ring
contraction: the interconversion of UDP-galactopyranose
(UDP-Galp) 1 into UDP-galactofuranose (UDP-Galf) 2,
the universal Galf donor for all oligo-galactofuranosides
[a] University of Namur (FUNDP), Département de Chimie, La-
boratoire de Chimie Bio-Organique,
Here, we report the synthesis of UDP-C-α-Galp 5 and
UDP-C-β-Galp 6, the phosphonate analogues of UDP-gal-
actopyranose, with respectively α and β configurations at
the anomeric position, as well as the synthesis of UDP-
(Z)-exo-galactal 7. Preliminary inhibition profiles are also
described.
rue de Bruxelles 51, 5000 Namur, Belgium
Fax: +32-81724517
E-mail: stephane.vincent@fundp.ac.be
[‡] Current address: ENDOTIS PHARMA, 102 av Gaston Rous-
sel, département de Chimie, bâtiment Pasteur, 93230 Romain-
ville
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Figure 1.C-Glycosidic analogues of UDP-galactose.
densing the lithium anion of dimethyl methylphosphonate
at –78 °C in anhydrous THF to yield intermediate lactol
12a in 63% yield after purification on silica gel (Scheme 2).
We observed that it was important to keep the temperature
at –78 °C during the course of the reaction (30 min) to
minimize the parallel formation of furano-lactol 12b. This
surprising side reaction was in fact a transsilylation of
pyranose 12 through an acyclic intermediate resulting in fu-
ranose formation.
Results and Discussion
Synthesis of UDP-C-β-Galp
In a previous study,[11] we reported a short synthetic se-
quence allowing the preparation of UDP-C-α-Galf 4 (5
steps, 21% global yield) by exploiting the stereoselective hy-
drogenation of protected exo-glycals 8 (Scheme 1). What-
ever the protective group at the 2-position, we always ob-
served high “α” (or 1,2-cis) selectivity.[11,16]
Lactol 12a was then subjected to an elimination reaction
achieved through a procedure described by Lin et al.[18] As
expected, this procedure exclusively gave exo-glycal 14 with
the (Z) configuration in 70% yield. When exo-glycal 14 was
submitted to the hydrogenation conditions we had opti-
mized for the synthesis of α-C-galactofuranosides (Pearl-
man’s catalyst in EtOAc),[11] the reaction was highly stereo-
selective, but this time, in favor of the β-phosphonate (com-
pound 16) with a global yield of 90%. At first sight, this
result surprised us given the α selectivity observed for the
same reaction performed on the corresponding furanose
(Scheme 1).
Scheme 1. Stereoselective preparation of “α”-C-galactofuranosides.
Considering the results obtained in the furanose series,
we reasoned that it should be interesting to develop the
same strategy in the pyranose series to obtain the corre-
sponding α-phosphonate.
The β configuration was established by the coupling con-
Persilylated exo-glycal 14 was first synthesized in two stant between H-1 and H-2 (J = 8.7 Hz), showing a trans
steps from persilylated 1,5-galactonolactone 10[17] by con- relationship between these two protons. In this case, the ste-
Scheme 2. Reagents and conditions: (a) (RЈO)2POCH2Li, THF, –78 °C, 30 min; (b) (CF3CO)2O, Py, THF, 0 °C, 1 h; (c) H2, Pd-
(OH)2/C, EtOAc, room temp., 8 h; (d) UMP-N-methylimidazolium, molecular sieves 4 Å, CH3CN, 0 °C, 5 h then NH4+, AcO– (pH = 7;
250 m).
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Synthesis of Three C-Glycoside Analogues of UDP-Galactopyranose
ric hindrance at the 2-position, which was likely the origin of deprotection is important and cannot be inverted: the
of the α stereospecificity in the furanose series,[11] did not TBS groups prevent saturation of the double bond during
operate. Instead, it might be argued that the β face of pyr- the hydrogenolysis step.
anose 14 is hindered by the two TBS groups at the 4- and
6-positions, thus directing the hydrogen addition from the
α face. As such a remote control is rather unusual for the
addition on exocyclic double bonds, electronic or even
stereoelectronic effects may also be at the origin of this total
“β” stereoselectivity.
Considering this result, we reasoned that it would be ju-
dicious to realize this sequence on a perbenzylated exo-gly-
cal to obtain the C-β-phosphonate in order to shorten the
synthesis and make the complete deprotection of the final
phosphonate sugar easier. We thus synthesized (Z)-exo-gly-
cal 15 in 83% yield in a one-pot sequence from perbenzyl-
Scheme 3. Reagents and conditions: (a) (BnO)2POCH2Li, THF,
–78 °C, 30 min (55%); (b) (CF3CO)2O, Py, THF, 0 °C, 2 h (91%);
(c) H2, Pd/C, Et3N, CH2Cl2, room temp., 1 h 30 min;
ated -galactono-1,5-lactone 11 by following the procedure
described above. The hydrogenation of intermediate 15, cat-
alyzed by Pd(OH)2 yielded β-configured phosphonate 17 as
sole product in quantitative yield. This novel sequence from
11 to 17 was, indeed, much shorter and efficient than the
previous one starting from 10, notably because ring isomer-
ization/expansion could not occur.
A survey of the literature showed us that similar
stereoselectivities had been observed with pyranosides bear-
ing an exocyclic enol ether functionality,[19,20] but the origin
of the selectivity is still obscure. To summarize, hydro-
genation reactions of exo-glycals lead to 1,2-cis adducts
from furanosides and 1,2-trans adducts from pyran-
osides.
Deprotected β-phosphonate 17 was then coupled to acti-
vated UMP in CH3CN by following a procedure described
by Bogachev[21] for the synthesis of nucleosides triphos-
phates and by Kiessling[22] for the challenging synthesis of
UDP-galactofuranose.[23–25] UDP-C-β-Galp 1 was obtained
in 54% yield after size exclusion chromatography and semi-
preparative HPLC purification.
(d) nBu4NF·3H2O, THF, room temp., 4 h (78%, 2 steps); (e) UMP-
+
N-methylimidazolium, CH3CN, room temp., 12 h then NH4
,
AcO– (pH = 7, 250 m) (67%).
exo-Glycal 19 was then coupled to UMP by using the
Bogachev procedure and target exo-glycal 3 was efficiently
obtained in 67% yield after purification. Compound 7 was
obtained in five steps in 26% global yield from persilylated
-galactono-1,5-lactone 10 (Scheme 3).
In 2004, Schmidt et al. reported the synthesis of novel
UDP-glycal derivatives, such as 3, as transition-state ana-
logue inhibitors of UDP-GlcNAc 2-epimerase.[20] From a
purely synthetic point of view, we managed to improve the
global yield of the synthesis of 3 by appropriate choice of
the protecting groups on the sugar (silyl ethers instead of
acetyl groups) and by proper choice of the coupling pro-
cedure with UMP. On the one hand, the Bogachev pro-
cedure was found to be much more efficient than the stan-
dard morpholidate coupling, because the short reaction
times and the mild conditions avoided side reactions or de-
composition of the sensitive enol ether functionality. On the
other hand, it should be mentioned that the Bogachev pro-
cedure is, technically, much more demanding than the clas-
sical morpholidate coupling, as it requires extremely pure
and anhydrous reagents as well as the preparation of several
intermediate solutions for the activation of UMP.
Synthesis of UDP-exo-Galactal 7
In previous work,[12] we presented the synthesis of UDP-
exo-galactofuranosylglycal with a (Z) configuration that
displayed an interesting time-dependent inactivation of
UDP-galactopyranose mutase. We then prepared their fluo-
rinated analogues,[13] whose UGM inactivation kinetics
Synthesis of UDP-C-α-Galp 5
To achieve the synthesis of α-C-galactosyl derivative 5
ruled out single-electron transfers from the FAD cofactor. (Scheme 4), we used a multistep sequence developed in the
Considering these results, we naturally turned our attention literature by Nicotra et al. for the synthesis of the phos-
to the synthesis of the same molecule in the pyranose series. phonate analogues of -glucose and -mannose 1-phos-
To synthesize UDP-exo-galactal 7, we used the same phates.[26] The key step of this strategy is an α-stereospecific
strategy as that for the preparation of UDP-exo-glycalGalf mercurio-cyclization of a 1,2-dideoxy heptenitol pioneered
previously described in our group.[12] exo-Glycal 18 was by Sinaÿ and collaborators.[27]
synthesized in two steps from lactone 10 (Scheme 3). Then,
The reaction of 2,3,4,6 tetra-O-benzyl--galactose 20
the complete deprotection was achieved first by selective with methylenetriphenylphosphorane gave compound 21 in
debenzylation under standard conditions, directly followed 72% yield according to a known procedure.[28] Enitol 21
by a tetradesilylation with the use of an excess amount of was thus converted into its mercuric derivative with mercu-
tetrabutylammonium fluoride to give phosphonate 19 in ric acetate. As expected, this reaction proceeded stereospe-
78% yield over two steps after ion-exchange chromatog- cifically to afford the 1,2-cis C-glycoside [J(1-2) = 3.7 Hz].
raphy. As in the case of the furanose series,[12] the sequence When this intermediate was treated with diiode, we gener-
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Table 1. Inhibition percentages of UGM from E. coli at [UDP-
Galf] = [inhibitor] = 1 m under nonreducing conditions.
Scheme 4. Reagents and conditions: (a) 20, then (PPh3CH3Br,
BuLi), THF, 0 °C to room temp., 8 h; (b) Hg(OAc)2, THF, room
temp., 20 h then KCl (aq.), room temp., 1 h; (c) I2, DCM, room
temp., 6 h; (d) (EtO)3P, 160 °C, 12 h; (e) TMSI, CCl4, 0 °C, 4 h;
+
(f) UMP-N-methylimidazolium, CH3CN, 0 °C, 1.5 h then NH4
,
AcO– (pH = 7; 250 m).
ated 22 in 90% yield, which was heated at reflux with tri-
ethyl phosphite to afford phosphonate 23 in 93% yield after
chromatography on silica gel. Deprotection of both benzyl
ether and ethyl ester groups could be easily performed with
an excess amount of iodotrimethylsilane at 0 °C in CCl4
to afford expected phosphonate 24 in 84% yield after ion-
exchange chromatography.
The Bogachev coupling of 24 with UMP completed the
synthesis of target molecule 5. As in the case of UDP-C-α-
Galf we previously synthesized,[11] compound 5 was found
to be unstable: its decomposition into cyclic phosphonate
25 during the course of the reaction and its purification
explained the rather poor, but optimized, yield. In pure
water, a decomposition of 10% of nucleotide sugar 5 was
observed when it was kept for 2 h at room temperature. A
reaction time of 1.5 h and a temperature of 0 °C were re-
quired to minimize this side reaction, and desired nucleo-
tide sugar 5 was isolated after size-exclusion chromatog-
raphy and semipreparative HPLC purification in a trieth-
ylammonium acetate buffer.
(native enzyme) conditions. We had applied the same condi-
tions for the measurements of inhibition and/or inactivation
properties of molecules 3 and 4.[11,12] Under reducing con-
ditions, all pyranosides 5, 6, and 7 displayed poor inhibition
properties (Ͻ10%). Therefore, in the whole series depicted
in Scheme 1, constrained analogue 3 remains the best inhib-
itor.[11]
Inhibition percentages under nonreducing conditions are
reported in Table 1, from the best inhibitor (Entry 1) to the
weakest (Entry 5). As expected, β-pyranoside 6 with the
wrong anomeric configuration displayed the lowest inhibi-
tion percentage (8%, Entry 5). Not surprisingly, UDP-C-α-
Galp 5 exhibited lower inhibition percentages (Entry 4)
than UDP-C-α-Galf 4 (Entry 1), as expected from their rel-
ative Km values for UGM and the 92:8 pyranose/furanose
Enzymatic Assay
As a preliminary biological evaluation, we measured in- ratio at the equilibrium (Figure 1, equilibrium between 1
hibition percentages of the interconversion of UDP-Galf and 2).[25]
and UDP-Galp catalyzed by UDP-galactopyranose mutase
Very surprisingly, exo-glycal 7 and α-C-pyranoside 5 ex-
from E. coli. Table 1 compares the inhibition levels of nucle- hibited similar inhibition profiles despite their significant
otide sugars 5, 6, and 7 to those of two C-glycosidic ana- structural differences. Moreover, exo-glycal 7 did not inacti-
[11]
logues of UDP-Galf we previously described:
UDP-C- vate UGM in a time-dependent manner, which is in deep
Galf 4 and constrained analogue 3 locked in a 1,4B boat contrast with the inactivation we observed with all exo-gly-
conformation.
cals in the furanose series.[12,13] This result indicates that
The inhibitory activities of the C-glycosides were evalu- exo-glycal 7 adopts a specific binding mode within the cata-
ated by HPLC following the procedure developed by Liu et lytic cavity that prevents its reaction with the enzyme.
al.:[10] all molecules were tested at a concentration of 1 m Docking and cocrystallization experiments with UGM are
by using UDP-Galf (1 m) as substrate under reducing in progress to explain why this molecule possesses a similar
(upon addition of sodium dithionite) and nonreducing affinity to the C-glycosidic analogue of natural substrate 1.
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Synthesis of Three C-Glycoside Analogues of UDP-Galactopyranose
concentrations were adjusted to allow a conversion of UDP-Galp
between 15 and 25%. Reactions were stopped by freezing the solu-
tion in liquid nitrogen. Residual enzyme activities were then mea-
Conclusions
In conclusion, we described the synthesis of three C-gly-
cosidic analogues of UDP-galactopyranose and compared sured in the presence of inhibitors at three different times, com-
pared with the same experiment conducted without inhibitors, and
averaged.
their inhibition properties, against UGM, with known C-
glycosides in the UDP-galactofuranose series. The binding
affinities of these nucleotide-sugars for UGM significantly
increase in the order: β-galactopyranose Ͻ α-galactopyr-
anose ≈ exo-glycalpyranose Ͻ boat-locked galactose Ͻ α-
galactofuranose. Surprisingly, UDP-exo-galactal derivative
7 was a competitive inhibitor, whereas time-dependent inac-
tivation was expected.
Uridine Diphosphate-C-α-D-Galactopyranose (5): The Bogachev
procedure detailed below for the synthesis of molecule 6 was strictly
followed.
The final crude mixture was purified by size-exclusion chromatog-
raphy (Sephadex G15). Eluent was a 50 m triethylammonium ace-
tate buffer (pH 6.8) to avoid decomposition of compound 5 into
UMP and cyclic phosphonate 25. The appropriate fractions were
pooled and freeze-dried. Compound 5 was further purified by
HPLC by using a C18 column (elution: 1% MeCN in 50 m trieth-
ylammonium acetate buffer pH 6.8 as eluent; flow rate:
1 mLmin–1). Fractions containing 5 were pooled and lyophilized.
This protocol afforded compound 5 as a white solid (31 mg, trieth-
ylammonium salt) in 30% yield. 1H NMR (400 MHz, D2O): δ =
7.96 [d, J(5ЈЈ-6ЈЈ) = 8.0 Hz, 1 H, 6ЈЈ-H], 5.99 [d, J(1Ј-2Ј) = 4.2 Hz,
1 H, 1Ј-H], 5.98 [d, J(5ЈЈ-6ЈЈ) = 8.0 Hz, 1 H, 5ЈЈ-H], 4.47 (m, 1 H,
1-H), 4.40–4.35 (m, 2 H, 2Ј-H, 3Ј-H), 4.29 (m, 2 H, 1-H, 4Ј-H),
4.28–4.18 (m, 2 H, 5Ј-Ha, 5Ј-Hb), 3.99 [dd, J(2-3) = 6.1 Hz, 1 H,
2-H], 3.98 (m, 1 H, 4-H), 3.93 [ddd, J(4-5) = 2.0 Hz, J(5-6a) =
9.2 Hz, J(5-6b) = 4.4 Hz, 1 H, 5-H], 3.79 (m 1 H, 3-H), 3.77 [ABX,
J(5-6a) = 9.2 Hz, J(6a-6b) = 11.6 Hz, 1 H, 6-Ha], 3.68 [ABX, J(5-
6b) = 4.4 Hz, J(6a-6b) = 11.6 Hz, 1 H, 6-Hb], 3.21 (q, J = 7.3 Hz,
CH2, NEt3), 2.25 [ABXXЈ, J(1-1Јa) = 10.8 Hz, J(1Јa-P) = 15.7 Hz,
J(1Јa-1Јb) = 15.7 Hz, 1 H, 1Ј-Ha], 2.13 [ABXXЈ, J(1-1Јb) = 4.0 Hz,
J(1Јb-P) = 20.1 Hz, J(1Јa-1Јb) = 15.7 Hz, 1 H, 1Ј-Hb], 1.29 (t, J =
7.3 Hz, CH3, NEt3) ppm. 31P NMR (101 MHz, D2O): δ = 15.35
[d, J(Pα-Pβ) = 26.3 Hz, Pα] and –11.15 [d, J(Pα-Pβ) = 26.3 Hz, Pβ]
ppm. MS (ESI–): m/z (%) = 563 (100) [M – H]–. HRMS: calcd. for
C16H25N2O16P2 563.0679; found 563.0656.
Experimental Section
Materials and Procedures: All chemicals were purchased from
Sigma, Aldrich, or Fluka and used without further purification.
Tetrahydrofuran, diethyl ether, and toluene were freshly distilled
from sodium benzophenone, dichloromethane over P2O5, and ace-
1
tonitrile over CaH2. H, 13C, and 31P NMR spectra were recorded
with Bruker AC-250 and AMX-400 spectrometers. All compounds
1
were characterized by H, 13C, and 31P NMR spectroscopy as well
1
1
as by H–1H and H–13C correlation experiments. Specific optical
rotations were measured with a Perkin–Elmer 241 Polarimeter in a
1 dm cell. Melting points were determined with a Büchi 535 appa-
ratus. Column chromatography was performed on silica gel Kiesel-
gel Si 60 (40–63 µm). Size-exclusion chromatography was ac-
complished on a Sephadex G15 column (2.5ϫ60 cm) by using a
Pharmacia Biotech Äkta FPLC apparatus, and the fractions con-
taining uridine derivatives were detected by UV. When required,
purifications of nucleotide-sugars were realized by semipreparative
HPLC by using a Waters Delta prep 4000 chromatography system
equipped with a NovaPack C18 (1ϫ10 cm) column (eluent: trieth-
ylammonium acetate 50 m, pH 6.8). For enzymatic assays, a
Waters 600 E analytical apparatus equipped with a Zorbax C18-SB
column (25ϫ0.46 cm, 5 µm) was used (eluent: triethylammonium
acetate 50 m, pH 6.8). UDP-Galf was prepared according to
known procedures.[22,24,25]
Uridine Diphosphate-C-β-D-Galactopyranose (6): A suspension of
UMP (triethylammonium salt, 78.7 mg, 0.185 mmol) in a mixture
of freshly distilled MeCN (950 µL), N,N-dimethylaniline (93 µL,
0.74 mmol), and Et3N (51 µL, 0.37 mmol) was stirred under an ar-
gon atmosphere at 0 °C. Trifluoroacetic anhydride (154 µL,
1.11 mmol) was slowly added dropwise. The reaction was stirred
for a few minutes at room temperature, after which time a red-
brown coloration was observed. Excess trifluoroacetic anhydride
and trifluoroacetic acid were removed from the reaction mixture
under vacuum. In a separate flask, a mixture of N-Me-imidazole
(74 µL, 0.92 mmol) and Et3N (156 µL, 1.11 mmol) in anhydrous
MeCN (150 µL) was cooled to 0 °C, then added to the flask con-
taining the mixed phosphoryl anhydride. The reaction was stirred
for 10 min at 0 °C, after which time a bright yellow solution was
obtained (Solution A). In the meantime, a solution containing 17
(tributylammonium salt, 71.6 mg, 0.153 mmol) and 4 Å molecular
sieves in MeCN (800 µL) was stirred for 30 min at 0 °C (Solution
B). The solution of UMP-N-methylimidazolium (Solution A) was
then added dropwise to the solution containing 17 (Solution B).
The resulting mixture was stirred at 0 °C under an argon atmo-
sphere for 15 h and then quenched with cold aqueous ammonium
formate (3 mL, 250 m, pH 7). After filtration through a Celite
pad, the amines were extracted from the aqueous phase with
CH2Cl2 (3 mL). The organic phase was washed with cold aqueous
ammonium formate (2 mL, 250 m, pH 7), and the combined
aqueous phases were pooled and freeze-dried. The residue was
purified by size-exclusion chromatography (Sephadex G15) eluted
with a 50 m triethylammonium acetate buffer (pH 6.8). The ap-
Atom and Position Numberings: We systematically numbered the
phosphonate methylene group 1Ј and adopted the usual numbering
for carbohydrates from 1 to 6 with 1 for the anomeric position. For
nucleotide-sugars, we used the conventional ribose and pyrimidine
numberings: 1Ј for the anomeric position, 1ЈЈ for the nitrogen atom
linked to the ribose.
Preparation and Purification of UGM: UGM from E. coli was over-
expressed and purified following our procedure.[11]
Enzyme Kinetics and Inhibition Assays: Conditions described by Liu
et al. were followed.[10] All assays were performed by using a potas-
sium phosphate buffer (100 m, pH 6.8) at T = 21 °C. Under re-
ductive conditions, freshly prepared sodium dithionite solutions
were used to allow a final concentration of 20 m. When native
mutase was used (without sodium dithionite), reactions were con-
ducted in the dark under aerobic conditions. All inhibition studies
consisted in measuring the conversion of pure starting UDP-Galf
into UDP-Galp (compared with a commercially available sample)
by analytical HPLC (C18 column, elution by triethylammonium
acetate 50 m, pH 6.8, detection at 262 nm). Substrate and inhibi-
tor concentrations were adjusted at 1 m from titrated mother
solutions. Incubation times were always 12 h at 21 °C. As a control,
the relative concentrations of any species in the reaction mixtures
could be titrated by using UMP as an internal standard. Enzyme
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A. Caravano, S. P. Vincent
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propriate fractions were pooled and freeze-dried. Compound 1 was
further purified by reverse-phase (C18) HPLC with 1% MeCN in
50 m triethylammonium acetate buffer pH 6.8 as eluent and a
flow rate of 1 mLmin–1. This protocol afforded compound 1 as a
white solid (55 mg, triethylammonium salt) in 54% yield. 1H NMR
(400 MHz, D2O): δ = 7.94 [d, J(5ЈЈ-6ЈЈ) = 8.1 Hz, 1 H, 6ЈЈ-H], 6.00
tetra-O-tert-butyldimethylsilyl--galactono-1,5-lactone 10[17]
(1.13 g, 1.78 mmol) in anhydrous THF (6 mL) was added. The tem-
perature was maintained at –78 °C for 30 min after which the mix-
ture was diluted with 1  phosphate buffer at pH 7 (50 mL) and
extracted with CH2Cl2 (2ϫ100 mL). The combined organic phase
was dried with MgSO4, filtered, and concentrated, and the residue
[d, J(1Ј-2Ј) = 4.1 Hz, 1 H, 1Ј-H], 5.99 [d, J(5ЈЈ-6ЈЈ) = 8.1 Hz, 1 H, was purified by chromatography on silica gel (cyclohexane/EtOAc,
5ЈЈ-H], 4.40–4.37 (m, 2 H, 2Ј-H, 3Ј-H), 4.29 (m, 1 H, 4Ј-H), 4.26 9:1Ǟ8.5:1.5) to furnish pyranose 12a (855 mg, 63% yield) and fu-
[ABXXЈ, J(4Ј-5Јa) = 2.6 Hz, J(5Јa-5Јb) = 11.7 Hz, J(5Јa-P) = ranose 12b (230 mg, 17% yield) as colorless oils. [α]2D2 = +4.3 (c =
4.6 Hz, 1 H, 5Ј-Ha], 4.19 [ABXXЈ, J(4Ј-5Јb) = 2.8 Hz, J(5Јa-5Јb) =
11.7 Hz, J(5Јb-P) = 5.7 Hz, 1 H, 5Ј-Hb], 3.93 [d, J(3-4) = 3.4 Hz,
1 H, 4-H], 3.82 [ABX, J(5-6a) = 8.9 Hz, J(6a-6b) = 12.7 Hz, 1 H,
6a-H], 3.67–3.63 (m, 2 H, 5-H et 6-Hb), 3.63 [dd, J(2-3) = 9.5 Hz,
0.8, CHCl3). 1H NMR (400 MHz, CDCl3): δ = 5.34 (br., 1 H, OH-
1), 4.21 (s, 1 H, 4-H), 4.14 [dd, J(2-3) = 9.4 Hz, J(3-4) = 1.8 Hz, 1
H, 3-H], 3.96 [d, J(2-3) = 9.4 Hz, 1 H, 2-H], 3.87 [dd, J(5-6b) =
5.5 Hz, 1 H, 5-H], 3.79 [d, J(H-P) = 11.1 Hz, 3 H, O CH3], 3.72
J(3-4) = 3.4 Hz, 1 H, 3-H], 3.57 [qd, J(1-2) = J(1-P) = J(1-1Јb) = [d, J(H-P) = 10.9 Hz, 3 H, O CH3], 3.65 [t, J(5-6a) = J(6a-6b) =
9.5 Hz, J(1-1Јa) = 2.5 Hz, 1 H, 1-H], 3.45 [t, J(1-2) = J(2-3) =
9.5 Hz, 1 H, 2-H], 3.21 (q, J = 7.3 Hz, CH2, Et3N), 2.34 [ABXXЈ,
J(1-1Јa) = 2.5 Hz, J(1Јa-1Јb) = 15.4 Hz, J(1Јa-P) = 19.5 Hz, 1 H,
CH2P], 2.06 [ABXXЈ, J(1-1Јb) = 9.5 Hz, J(1Јa-1Јb) = 15.4 Hz,
9.5 Hz, 1 H, 6-Ha], 3.54 [ABX, J(5-6b) = 5.5 Hz, J(6a-6b) =
9.5 Hz, 1 H, 6-Hb], 2.57 [ABX, J(1Јa-1Јb) = 14.9 Hz, J(1Јa-P) =
18.0 Hz, 1 H, 1Ј-Ha], 1.95 [ABX, J(1Јa-1Јb) = 14.9 Hz, J(1Јb-P) =
18.6 Hz, 1 H, 1Ј-Hb], 0.98 (s, 18 H, 2 Si-tBu), 0.95 (s, 9 H, Si-tBu),
J(1Јb-P) = 22.0 Hz, 1 H, CH2P], 1.23 (t, J = 7.3 Hz, CH3, Et3N) 0.90 (s, 9 H, Si-tBu), 0.20 (s, 3 H, Si-Me), 0.19 (s, 3 H, Si-Me), 0.17
ppm. 13C NMR (100 MHz, D2O): δ = 166.57 (C-4ЈЈ), 152.22 (C-
(s, 6 H, 2 Si-Me), 0.12 (s, 3 H, Si-Me), 0.09 (s, 3 H, Si-Me), 0.07
2ЈЈ), 141.99 (C-6ЈЈ), 103.06 (C-5ЈЈ), 88.89 (C-1Ј), 83.58 [d, J(4Ј-P) (s, 3 H, Si-Me), 0.06 (s, 3 H, Si-Me) ppm. 13C NMR (100 MHz,
= 9.1 Hz, C-4Ј], 79.12 (C-5), 76.47 [d, J(1-P) = 5.4 Hz, C-1], 74.21 CDCl3): δ = 98.00 [d, J(1-P) = 8.7 Hz, C-1], 73.90 [d, J(2-P) =
(C-3), 74.15 (C-3Ј), 72.10 [d, J(2-P) = 13.9 Hz, C-2], 70.00 (C-2Ј), 13.4 Hz, C-2], 72.61 (C-5), 72.43 (C-4), 72.16 [d, J(3-P) = 4.4 Hz,
69.84 (C-4), 65.09 [d, J(5Ј-P) = 3.4 Hz, C-5Ј], 62.14 (C-6), 47.08 C-3], 60.39 (C-6), 53.45 [d, J(C-P) = 5.4 Hz, OMe], 51.57 [d, J(C-
(CH2, Et3N), 31.29 [d, J(CH2-P) = 140.0 Hz, CH2P], 8.62 (CH3,
Et3N) ppm. 31P NMR (101 MHz, D2O): δ = 15.08 [d, J(Pα-Pβ) = C(CH3)3], 26.33 [Si-C(CH3)3], 26.07 [Si-C(CH3)3], 25.75 [Si-C-
25.3 Hz, Pα], –11.35 [d, J(Pα-Pβ) = 25.3 Hz, Pβ] ppm. MS (ESI–): (CH3)3], 19.36 [Si-C(CH3)3], 18.65 [Si-C(CH3)3], 18.25 [Si-C-
m/z (%) = 563 (100) [M – H]–. HRMS: calcd. for C16H25N2O16P2 (CH3)3], 18.00 [Si-C(CH3)3], –2.13 (Si-Me), –3.22 (Si-Me), –3.69
P) = 6.1 Hz, OMe], 34.17 [d, J(1Ј-P) = 133.9 Hz, C-1Ј], 26.99 [Si-
563.0679; found 563.0689.
(Si-Me), –4.55 (Si-Me), –4.38 (Si-Me), –4.69 (Si-Me), –5.33 (Si-
Me), –5.37 (Si-Me) ppm. 31P NMR (101 MHz, CDCl3): δ =
32.09 ppm. MS (DCI-NH3): m/z (%) = 759 (45) [M + H]+, 776
(30) [M + NH4]+, 741 (100) [M – H2O + H]+. HRMS: calcd. for
C33H79O9NSi4P 776.4570; found 776.4560.
UDP-[1(1Ј)Z]-exo-Glycal-
D
-Galactopyranose (7): The procedure for
the preparation of 5 and 6 was followed. The reaction time between
19 (30 mg, 0.043 mmol) and activated UMP (0.056 mmol) was 15 h
at room temperature. The final exo-glycal was first purified by size-
exclusion chromatography [Sephadex G15, eluent: 50 m trieth-
ylammonium acetate buffer (pH 6.8)] and then by reverse phase
C18 HPLC (eluent: 1% MeCN in 50 m triethylammonium acetate
buffer pH 6.8; flow rate: 1 mLmin–1). This protocol afforded nucle-
otide-sugar 7 as a viscous solid (19 mg, triethylammonium salt) in
67% yield. 1H, 13C, 31P NMR, and mass spectra were in agreement
with those published.[20] 1H NMR (400 MHz, D2O): δ = 7.96 [d,
J(5ЈЈ-6ЈЈ) = 8.1 Hz, 1 H, 6ЈЈ-H], 5.97 [d, J(1Ј-2Ј) = 4.0 Hz, 1 H, 1Ј-
H], 5.96 [d, J(5ЈЈ-6ЈЈ) = 8.1 Hz, 1 H, 5ЈЈ-H], 5.52 [d, J(1exo-P) =
10.5 Hz, J(1exo-2) = 1.6 Hz, 1 H, 1-Hexo], 4.36 (m, 2 H, 2Ј-H, 3Ј-
H), 4.27–4.18 (m, 4 H, 4Ј-H, 5Ј-Ha, 5Ј-Hb, 2-H), 4.09 [d, J(3-4) =
3.1 Hz, 1 H, 4-H], 3.92 (m, 2 H, 5-H, 6-Ha), 3.76 [t, J(5-6b) = J(6a-
6b) = 7.8 Hz, 1 H, 6-Hb], 3.71 [dd, J(2-3) = 10.1 Hz, J(3-4) =
3.1 Hz, 1 H, 3-H], 3.19 (q, J = 7.3 Hz, CH2, Et3N), 1.27 (t, J =
7.3 Hz, CH3, Et3N) ppm. 13C NMR (100 MHz, D2O): δ = 166.62
(C-4ЈЈ), 165.63 [d, J(1-P) = 1.7 Hz, C-1], 152.20 (C-2ЈЈ), 142.05 (C-
6ЈЈ), 103.01 (C-5ЈЈ), 100.32 [d, J(1exo-P) = 186.3 Hz, C-1exo], 88.74
(C-1Ј), 83.66 [d, J(4Ј-P) = 9.0 Hz, C-4Ј], 80.55 (C-5), 74.15 (C-3Ј),
73.35 (C-3), 69.98 (C-2Ј), 69.37 (C-4), 68.80 [d, J(2-P) = 12.3 Hz,
C-2], 65.01 [d, J(5Ј-P) = 5.3 Hz, C-5Ј], 61.63 (C-6), 47.03 (CH2,
Et3N), 8.59 (CH3, Et3N) ppm. 31P NMR (101 MHz, D2O): δ =
3.04 [d, J(Pα-Pβ) = 24.3 Hz, Pα], –11.56 [d, J(Pα-Pβ) = 24.3 Hz,
Pβ] ppm. MS (FAB+): m/z (%) = 585 (50) [M + Na]+, 601 (100)
[1(1Ј)Z]-2,3,4,6-Tetra-O-tert-butyldimethylsilyl-1-deoxy-1-(dimeth-
oxyphosphoryl)methylidene-D-galactopyranose (14): To a solution of
compound 12a (784 mg, 1.03 mmol) in anhydrous THF (10.5 mL)
at 0 °C was successively added pyridine (836 µL, 10.3 mmol) and
trifluoroacetic anhydride (719 µL, 5.17 mmol). The resulting solu-
tion was stirred at 0 °C for 1 h. A saturated aqueous solution of
NaHCO3 was then added and extracted with EtOAc (100 mL). The
organic layer was dried with MgSO4, filtered, and concentrated un-
der reduced pressure. Purification by silica-gel chromatography (cy-
clohexane/EtOAc, 8:2) afforded exo-glycal 14 (536 mg, 70% yield)
as a colorless syrup. [α]2D2 = +54.4 (c = 1.0, CHCl3). 1H NMR
(400 MHz, CDCl3): δ = 5.22 [dd, J(1Ј-2) = 0.8 Hz, J(1Ј-P) =
13.5 Hz, 1 H, 1Ј-H], 4.39 [d, J(2-3) = 8.2 Hz, 1 H, 2-H], 4.33 [t,
J(3-4) = J(4-5) = 2.0 Hz, 1 H, 4-H], 3.89 [t, J(5-6a) = J(6a-6b) =
6.5 Hz, 1 H, 6-Ha], 3.85 [ddd, J(4-5) = 2.0 Hz, J(5-6a) = 6.5 Hz,
J(5-6b) = 8.1 Hz, 1 H, 5-H], 3.79 [AX, J(5-6b) = 8.1 Hz, 1 H, 6-
Hb], 3.75 [d, J(H-P) = 11.3 Hz, 3 H, OMe], 3.74 [d, J(H-P) =
11.3 Hz, 3 H, OMe], 3.71 [dd, J(2-3) = 8.2 Hz, J(3-4) = 2.0 Hz, 1
H, 3-H], 0.96 (s, 9 H, Si-tBu), 0.95 (s, 9 H, Si-tBu), 0.94 (s, 9 H,
Si-tBu), 0.91 (s, 9 H, Si-tBu), 0.19 (s, 3 H, Si-Me), 0.15 (2 s, 6 H,
2 Si-Me), 0.14 (2 s, 6 H, 2 Si-Me), 0.11 (s, 3 H, Si-Me), 0.09 (s, 3
H, Si-Me), 0.08 (s, 3 H, Si-Me) ppm. 13C NMR (100 MHz, CDCl3):
δ = 170.44 [d, J(1-P) = 1.3 Hz, C-1], 94.50 [d, J(1Ј-P) = 191.3 Hz,
C-1Ј], 82.12 (C-5), 75.43 (C-3), 71.39 [d, J(2-P) = 13.4 Hz, C-2],
69.75 (C-4), 60.72 (C-6), 52.41 [d, J(C-P) = 5.4 Hz, OMe], 51.93
[M +K]+, 607 (50) [M
C16H24O16N2P2Na 585.0499; found 585.0495.
– H +
2Na]+. HRMS: calcd. for
2,3,4,6-Tetra-O-tert-butyldimethylsilyl-1-(dimethoxyphosphoryl)- [d, J(C-P) = 5.7 Hz, OMe], 26.28 [Si-C(CH3)3], 26.07 [Si-C(CH3)3],
methyl-α-
D
-galactopyranose (12a): To a cooled (–78 °C) solution of
26.02 [Si-C(CH3)3], 25.76 [Si-C(CH3)3], 18.64 [Si-C(CH3)3], 18.47
[Si-C(CH3)3], 18.17 [Si-C(CH3)3], 18.06 [Si-C(CH3)3], –3.93 (Si-
Me), –4.20 (Si-Me), –4.36 (Si-Me), –4.47 (Si-Me), –4.77 (Si-Me),
–4.92 (Si-Me), –5.35 (Si-Me), –5.42 (Si-Me) ppm. 31P NMR
freshly distilled dimethyl methylphosphonate (291 µL, 2.67 mmol)
in anhydrous THF (10 mL) was added butyllithium (1.0 mL,
2.49 mmol, 2.5  in hexane). After 20 min, a solution of 2,3,4,6-
1776
www.eurjoc.org
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2009, 1771–1780

Synthesis of Three C-Glycoside Analogues of UDP-Galactopyranose
(101 MHz, CDCl3): δ = 21.11 ppm. MS (DCI-NH3): m/z (%) = 741
(100) [M + H]+. HRMS: calcd. for C33H74O8Si4P 741.4198; found
741.4196.
[dd, J(5-6a) = 8.7 Hz, J(5-6b) = 5.4 Hz, 1 H, 5-H], 2.39 [ABXXЈ,
J(1-1Јa) = 1.6 Hz, J(1Јa-1Јb) = 15.4 Hz, J(1Јa-P) = 20.5 Hz, 1 H,
1Ј-Ha], 1.82 [ABXXЈ, J(1-1Јb) = 11.1 Hz, J(1Јa-1Јb) = 15.4 Hz,
J(1Јb-P) = 29.7 Hz, 1 H, 1Ј-Hb], 0.96 (s, 9 H, Si-tBu), 0.94 (s, 9 H,
Si-tBu), 0.93 (s, 9 H, Si-tBu), 0.89 (s, 9 H, Si-tBu), 0.18 (s, 3 H, Si-
Me), 0.16 (s, 3 H, Si-Me), 0.15 (s, 3 H, Si-Me), 0.14 (s, 3 H, Si-
Me), 0.11 (s, 3 H, Si-Me), 0.08 (s, 3 H, Si-Me), 0.05 (s, 3 H, Si-
Me), 0.04 (s, 3 H, Si-Me) ppm. 13C NMR (100 MHz, CDCl3): δ =
78.95 (C-5), 77.58 [d, J(3-P) = 3.1 Hz, C-3], 76.55 [d, J(1-P) =
6.3 Hz, C-1], 72.30 [d, J(2-P) = 15.8 Hz, C-2], 71.89 (C-4), 60.61
(C-6), 52.70 [d, J(C-P) = 6.0 Hz, OMe], 51.80 [d, J(C-P) = 6.2 Hz,
OMe], 28.85 [d, J(1Ј-P) = 141.1 Hz, C-1Ј], 26.92 (Si-tBu), 26.30 (Si-
tBu), 26.02 (Si-tBu), 25.75 (Si-tBu), 19.30 (Si-tBu), 18.60 (Si-tBu),
18.12 (Si-tBu), 18.01 (Si-tBu), –2.26 (Si-Me), –3.31 (Si-Me), –3.77
(Si-Me), –4.13 (2 Si-Me), –4.20 (Si-Me), –4.63 (Si-Me), –5.40 (Si-
Me) ppm. 31P NMR (101 MHz, CDCl3): δ = 33.00 ppm. MS (DCI-
NH3): m/z (%) = 743 (100) [M + H]+, 760 (20) [M + NH4]+.
HRMS: calcd. for C33H76O8Si4P 743.4355; found 743.4357.
[1(1Ј)Z]-2,3,4,6-Tetra-O-benzyl-1-deoxy-1-(dibenzyloxyphosphoryl)-
methylidene-D-galactopyranose (15): To a cooled (–78 °C) solution
of freshly distilled dibenzyl methylphosphonate (481 mg,
1.74 mmol) in anhydrous THF (8 mL) was added butyllithium
(704 µL, 1.74 mmol, 2.5  in hexanes). After 20 min, a solution of
2,3,4,6-tetra-O-benzyl--galactono-1,4-lactone 11 (469 mg,
0.87 mmol) in anhydrous THF (1.2 mL) was added. The tempera-
ture was maintained at –78 °C for 10 min after which the mixture
was allowed to reach –40 °C over a period of 1 h. The solution was
then diluted with 1  phosphate buffer at pH 7 (15 mL) and ex-
tracted with CH2Cl2 (2ϫ40 mL). The combined organic phase was
dried with MgSO4, filtered, and concentrated. To a solution of this
crude residue containing intermediate 13 in anhydrous THF
(10 mL) was added pyridine (703 µL, 8.7 mmol) and trifluoroacetic
anhydride (604 µL, 4.35 mmol) at 0 °C. The resulting solution was
allowed to reach room temperature over a period of 1 h. Solvents
were then evaporated under vacuum, and the crude was diluted
with CH2Cl2 (20 mL) and washed with a saturated aqueous solu-
tion of NaHCO3. The organic layer was dried with MgSO4, fil-
tered, and concentrated under reduced pressure. Purification by sil-
ica-gel chromatography (cyclohexane/EtOAc, 1:1) afforded exo-gly-
C-(1-Deoxy-β-D-galactopyranosyl)methyl Phosphonic Acid (17):
Compound 15 (240 mg, 0.30 mmol) was dissolved in a mixture of
EtOAc/MeOH (1:1.8 mL) and vigorously stirred at room tempera-
ture under a H2 atmosphere (1.5 bar) with palladium hydroxide
(240 mg, 20% Pd on carbon, wet) and Bu3N (108 µL, 0.60 mmol).
After 24 h, the catalyst was removed by filtration through a pad of
Celite, and the filtrate was concentrated. Compound 17 (189 mg,
cal 15 (582 mg, 83% yield) as a colorless oil.[α]2D2 = +66.8 (c = 1.0,
1
CHCl3). H NMR (400 MHz, CDCl3): δ = 7.41–7.30 (m, 30 H, H tributylammonium salt) was obtained quantitatively as a white hy-
1
arom.), 5.51 [dd, J(1Ј-2) = 1.5 Hz, J(1Ј-P) = 13.7 Hz, 1 H, 1Ј-H],
5.07 [AX, J(H-P) = 8.2 Hz, 4 H, 2CH2Ph phosphonate], 5.01 [AB,
J(H-H) = 11.2 Hz, 1 H, CH2Ph], 4.84 [AB, J(H-H) = 11.2 Hz, 1
H, CH2Ph], 4.80 [AB, J(H-H) = 11.9 Hz, 1 H, CH2Ph], 4.76 [AB,
J(H-H) = 11.9 Hz, 1 H, CH2Ph], 4.75 [AB, J(H-H) = 11.2 Hz, 1
groscopic solid. [α]2D2 = +7.8 (c = 0.53, H2O, 0.85 equiv. Bu3N). H
NMR (400 MHz, D2O): δ = 3.92 [d, J(3-4) = 3.4 Hz, 1 H, 4-H],
3.74 [ABX, J(5-6a) = 3.6 Hz, J(6a-6b) = 10.2 Hz, 1 H, 6-Ha], 3.67–
3.62 (m, 2 H, 5-H, 6-Hb), 3.60 [dd, J(2-3) = 9.4 Hz, J(3-4) = 3.4 Hz,
1 H, 3-H], 3.50 [qd, J(1-2) = J(1-P) = J(1-1Јb) = 9.4 Hz, J(1-1Јa)
H, CH2Ph], 4.65 [AB, J(H-H) = 11.2 Hz, 1 H, CH2Ph], 4.51 [ddd, = 2.8 Hz, 1 H, 1-H], 3.40 [t, J(1-2) = J(2-3) = 9.4 Hz, 1 H, 2-H],
J(1Ј-2) = 1.5 Hz, J(2-3) = 9.7 Hz, J(2-P) = 3.5 Hz, 1 H, 2-H], 4.47 3.11 [m, CH2 (d), Bu3N], 2.17 [ABXXЈ, J(1-1Јa) = 2.8 Hz, J(1Јa-
[AB, J(H-H) = 11.7 Hz, 1 H, CH2Ph], 4.42 [AB, J(H-H) = 11.7 Hz,
1 H, CH2Ph], 4.14 [d, J(4-5) = 0.8 Hz, 1 H, 4-H], 3.88 [td, J(4-5)
1Јa) = 15.4 Hz, J(1Јa-P) = 19.0 Hz, 1 H, 1Ј-Ha], 1.82 [ABXXЈ, J(1-
1Јb) = 9.4 Hz, J(1Јa-1Јa) = 15.4 Hz, J(1Јb-P) = 21.9 Hz, 1 H, 1Ј-
= 1.2 Hz, J(5-6a,b) = 6.5 Hz, 1 H, 5-H], 3.75 [t, J(5-6a) = J(6a-6b) Hb], 1.65 [m, CH2 (c), Bu3N], 1.35 [sex, J(H-H) = 7.3 Hz, CH2 (b),
= 8.9 Hz, 1 H, 6-Ha], 3.72 [dd, J(2-3) = 9.7 Hz, J(3-4) = 2.7 Hz, 1 Bu3N], 0.91 [t, J(H-H) = 7.3 Hz, CH3 (a), Bu3N] ppm. 13C NMR
H, 3-H], 3.63 [ABX, J(5-6b) = 5.3 Hz, J(6a-6b) = 8.9 Hz, 1 H, 6-
Hb] ppm. 13C NMR (100 MHz, CDCl3): δ = 168.32 [d, J(1-P) =
(100 MHz, D2O): δ = 78.92 (C-5), 76.75 (C-1), 74.10 (C-3), 72.20
[d, J(2-P) = 10.9 Hz, C-2], 69.63 (C-4), 61.96 (C-6), 52.97 [CH2
1.4 Hz, C-1], 138.21, 137.85, 137.57, 137.44 (4 Cq arom.), 136.67 (d)], 31.68 [d, J(1Ј-P) = 131.4 Hz, C-1Ј], 25.51 [CH2 (c)], 19.61 [CH2
[d, J(C-P) = 7.2 Hz, Cq arom.], 136.64 [d, J(C-P) = 7.2 Hz, Cq
arom.], 128.37–127.44 (30 CH arom.), 94.42 [d, J(1Ј-P) = 191.7 Hz,
(b)], 13.13 [CH3(a)] ppm. 31P NMR (101 MHz, D2O): δ =
21.77 ppm. MS (FAB–): m/z (%) = 257 (100) [M – H]–. HRMS:
C-1Ј], 81.61 (C-3), 78.30 (C-5), 76.44 [d, J(2-P) = 12.8 Hz, C-2], calcd. for C7H14O8P 257.0426; found 257.0433.
74.78 (CH2Ph), 74.50 (CH2Ph), 73.72 (C-4), 73.38 (CH2Ph), 72.54
[1(1Ј)Z]-1-Deoxy-1-(dibenzyloxyphosphoryl)methylidene-2,3,4,6-
tetra-O-tert-butyldimethylsilyl-D-galactopyranose (18): To a cooled
(CH2Ph), 67.64 (C-6), 67.04 [d, J(C-P) = 5.6 Hz, CH2Ph], 66.70 [d,
J(C-P) = 5.7 Hz, CH2Ph] ppm. 31P NMR (101 MHz, CDCl3): δ =
18.88 ppm. MS (DCI-NH3): m/z (%) = 797 (70) [M + H]+, 814
(100) [M + NH4]+. HRMS: calcd. for C49H50O8P 797.3243; found
797.3239.
(–78 °C) solution of freshly distilled dibenzyl methylphosphonate
(1.74 g, 63.10 mmol) in anhydrous THF (250 mL) was added butyl-
lithium (25.3 mL, 63.10 mmol, 2.5  in hexanes). After 20 min, was
added a solution of 2,3,4,6-tetra-O-tert-butyldimethylsilyl--galac-
tono-1,5-lactone 10[17] (2 g, 3.15 mmol) in anhydrous THF
Dimethyl C-(2,3,4,6-tetra-O-tert-butyldimethylsilyl-β-D-galacto-
pyranosyl)methanephosphonate (16): Compound 14 (200 mg, (12 mL). After 30 min at –78 °C the mixture was diluted with 1 
0.27 mmol) was dissolved in ethyl acetate (20 mL) and vigorously
stirred at room temperature under a H2 atmosphere (1.5 bar) in the
presence of Pd(OH)2 (92 mg, 20% Pd on carbon, wet). After 8 h,
phosphate buffer at pH 7 (100 mL) and extracted with CH2Cl2
(2ϫ200 mL). The combined organic phase was dried with MgSO4,
filtered, and concentrated, and the residue was purified by
the catalyst was removed by filtration through a pad of Celite, chromatography on silica gel (cyclohexane/EtOAc, 9:1) to yield the
and the filtrate was concentrated. The residue was purified by
lactol intermediate (1.58 g, 55% yield) as a colorless oil. To a solu-
tion of this lactol (702 mg, 0.77 mmol) in anhydrous THF (7.7 mL)
at 0 °C was successively added pyridine (624 µL, 7.72 mmol) and
chromatography on silica gel (cyclohexane/EtOAc, 7:3Ǟ5:5) to give
compound 16 (172 mg, 90% yield) as a white solid. [α]2D4 = –11.0
1
(c = 0.8, CHCl3). M.p. 64–65 °C. H NMR (400 MHz, CDCl3): δ trifluoroacetic anhydride (537 µL, 3.86 mmol). The resulting solu-
= 4.16 (s, 1 H, 4-H), 3.79 [t, J(1-2) = J(2-3) = 8.7 Hz, 1 H, 2-H], tion was stirred at 0 °C for 2 h, stopped by adding an aqueous
3.74 [d, J(H-P) = 10.9 Hz, 3 H, OMe], 3.72 [d, J(H-P) = 11.0 Hz,
saturated solution of NaHCO3 and extracted with EtOAc
3 H, OMe], 3.66 [t, J(5-6a) = J(6a-6b) = 8.7 Hz, 1 H, 6-Ha], 3.62 (100 mL). The organic layer was dried with MgSO4, filtered, and
[d, J(2-3) = 8.7 Hz, 1 H, 3-H], 3.62–3.55 (m, 2 H, 1-H, 6-Hb), 3.45 concentrated under reduced pressure. Purification by silica-gel
Eur. J. Org. Chem. 2009, 1771–1780
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
1777

A. Caravano, S. P. Vincent
FULL PAPER
chromatography (cyclohexane/EtOAc, 8.5:1.5) afforded 18 (626 mg,
drous THF (20 mL) at –30 °C under an argon atmosphere was
added very slowly n-butyllithium (1.5 mL, 3.71 mmol, 2.5  in hex-
91% yield) as a colorless oil. [α]2D2 = +63.8 (c = 1.0, CHCl3). 1H
NMR (400 MHz, CDCl3): δ = 7.37–7.30 (m, 10 H, H arom.), 5.40 ane), and the solution was stirred for 30 min at 0 °C. In a separate
[dd, J(1Ј-2) = 1.4 Hz, J(1Ј-P) = 13.7 Hz, 1 H, 1Ј-H], 5.10 [ABX, flask, a solution of methylenetriphenylphosphosphorane in dry
J(H-P) = 8.2 Hz, J(H-H) = 12.2 Hz, 1 H, CH2Ph], 5.08 [AX, J(H- THF (40 mL) prepared at 0 °C from methylenetriphenylphospho-
P) = 8.1 Hz, 2 H, CH2Ph], 5.05 [ABX, J(H-P) = 7.9 Hz, J(H-H) =
12.2 Hz, 1 H, CH2Ph], 4.49 [ddd, J(1Ј-2) = 1.4 Hz, J(2-3) = 9.1 Hz,
nium bromide (2.65 g, 7.42 mmol) and a solution of n-butyllithium
(2.8 mL, 7.0 mmol, 2.5  in hexane) was added at 0 °C. The mix-
J(2-P) = 2.7 Hz, 1 H, 2-H], 4.28 [d, J(3-4) = 1.8 Hz, 1 H, 4-H], ture was stirred 8 h at room temperature and then cooled to 0 °C.
3.81 (m, 1 H, 6-Ha), 3.72 (m, 2 H, 5-H, 6-Hb), 3.69 [dd, J(2-3) = A saturated aqueous solution of NH4Cl (50 mL) was added, and
9.1 Hz, J(3-4) = 1.8 Hz, 1 H, 3-H], 0.97 (s, 9 H, Si-tBu), 0.96 (2 s, the mixture was warmed to room temperature and diluted with
18 H, 2 Si-tBu), 0.89 (s, 9 H, Si-tBu), 0.21 (s, 3 H, Si-Me), 0.17 (s,
3 H, Si-Me), 0.16 (s, 6 H, 2 Si-Me), 0.15 (s, 3 H, Si-Me), 0.09 (s, 3
H, Si-Me), 0.05 (s, 3 H, Si-Me), 0.02 (s, 3 H, Si-Me) ppm. 13C
NMR (100 MHz, CDCl3): δ = 171.32 (C-1), 136.88 [d, J(C-P) =
6.6 Hz, Cq arom.], 136.85 [d, J(C-P) = 7.1 Hz, Cq arom.], 128.33–
127.45 (10 CH arom.), 94.63 [d, J(1Ј-P) = 192.2 Hz, C-1Ј], 81.23
CH2Cl2. The organic layer was separated and washed with water
(5 mL), dried with MgSO4, filtered, and concentrated. The residue
was purified by silica-gel chromatography (cyclohexane/EtOAc,
9:1Ǟ8:2) to yield 21 (1.4 g, 72% yield) as a colorless oil. [α]2D2
=
1
–7.6 (c = 1.01, CHCl3); ref.[30] [α]1D8 = –10.8 (c = 1.2, CHCl3). H
NMR (400 MHz, CDCl3): δ = 7.44–7.26 (m, 20 H, H arom.), 5.98
(C-5), 75.55 (C-3), 70.63 [d, J(2-P) = 13.0 Hz, C-2], 70.49 (C-4), [ddd, J(2-3) = 7.9 Hz, J(2-1trans) = 17.7 Hz, J(2-1cis) = 10.3 Hz, 1
67.01 [d, J(C-P) = 5.2 Hz, CH2Ph], 66.52 [d, J(C-P) = 5.3 Hz,
CH2Ph], 60.03 (C-6), 26.41 [Si-C(CH3)3], 26.16 [Si-C(CH3)3], 26.02
H, 2-H], 5.44 [dd, J(1trans-1cis) = 1.2 Hz, J(2-1trans) = 17.7 Hz, 1
H, 1-Htrans], 5.40 [dd, J(1trans-1cis) = 1.2 Hz, J(2-1cis) = 17.7 Hz,
[Si-C(CH3)3], 25.72 [Si-C(CH3)3], 18.78 [Si-C(CH3)3], 18.51 [Si- 1 H, 1-Hcis], 4.86 (s, 2 H, CH2Ph), 4.75 [AB, J(H-H) = 11.8 Hz, 1
C(CH3)3], 18.18 [Si-C(CH3)3], 17.96 [Si-C(CH3)3], –3.74 (Si-Me),
–3.93 (Si-Me), –4.22 (Si-Me), –4.47 (Si-Me), –4.80 (Si-Me), –5.02
(Si-Me), –5.41 (Si-Me), –5.42 (Si-Me) ppm. 31P NMR (101 MHz,
H, CH2Ph], 4.58 [AB, J(H-H) = 12.0 Hz, 1 H, CH2Ph], 4.52 [AB,
J(H-H) = 11.5 Hz, 1 H, CH2Ph], 4.50 [AB, J(H-H) = 12.0 Hz, 1
H, CH2Ph], 4.46 [AB, J(H-H) = 11.5 Hz, 1 H, CH2Ph], 4.45 [AB,
CDCl3): δ = 19.59 ppm. MS (DCI-NH3): m/z (%) = 893 (100) [M J(H-H) = 11.8 Hz, 1 H, CH2Ph], 4.23 (m, 1 H, 6-H), 4.18 [dd, J(2-
+ H]+. HRMS: calcd. for C45H82O8Si4P 893.4824; found 893.4820.
3) = 7.9 Hz, J(3-4) = 3.9 Hz, 1 H, 3-H], 3.93–3.89 (m, 2 H, 4-H, 5-
H), 3.63 [ABX, J(6-7a) = 6.2 Hz, J(7a-7b) = 9.4 Hz, 1 H, 7-Ha],
3.58 [ABX, J(6-7a) = 6.5 Hz, J(7a-7b) = 9.4 Hz, 1 H, 7-Hb], 3.14
[d, J(6-OH) = 5.0 Hz, 1 H, OH-6] ppm. 13C NMR (100 MHz,
CDCl3): δ = 138.17, 138.11, 138.07, 137.94 (4 Cq arom.), 135.66
(C-2), 128.25–127.49 (20 CH arom.), 119.10 (C-1), 82.00 (C-4),
80.65 (C-3), 76.49 (C-5), 75.14 (CH2Ph), 73.05 (CH2Ph), 73.01
(CH2Ph), 71.11 (C-7), 70.21 (CH2Ph), 69.58 (C-6) ppm. MS (DCI-
NH3): m/z (%) = 556 (100) [M + NH4]+.
{[1(1Ј)Z]-1-Deoxy-1-methylidene- -galactopyranosyl}phosphonic
D
Acid (19): A solution of phosphonate 18 (578 mg, 0.65 mmol) dis-
solved in anhydrous CH2Cl2 (30 mL) with Et3N (182 µL,
1.30 mmol) and 10% activated Pd/C as catalyst (65 mg) was vigor-
ously stirred for 1.5 h at room temperature under a H2 atmosphere
(1 bar). The suspension was then filtered through a pad of Celite,
and the filtrate was concentrated to give a viscous oil. Tetrabu-
tylammonium fluoride (850 mg, 2.73 mmol) was added at 0 °C to
a solution of this compound dissolved in distilled THF (30 mL).
After 4 h at room temperature, the reaction mixture was concen-
trated under reduced pressure, dissolved in a minimum amount of
water, filtered through paper, and the aqueous layer was freeze-
dried. The residue was dissolved in a minimum amount of water
and applied to Dowex 50WX8–200 (Na+ form) column (eluent:
H2O). The appropriate fractions (TLC conditions EtOH/NH4OH/
H2O, 5:3:1) were pooled and freeze-dried. The compound was then
purified by size-exclusion chromatography (Sephadex G15, eluent:
H2O) and then applied to a Dowex 50WX8–200 (Bu3NH+ form)
column. The desired fractions were combined and freeze-dried to
give compound 19 (317 mg, 78% yield, bistributylammonium salt)
as a white hygroscopic solid. [α]2D1 = +36.4 (c = 0.52, H2O, 2 equiv.
C-(2,3,4,6-Tetra-O-benzyl-α-D-galactopyranosyl)iodomethane (22):
To a solution of compound 21 (1.14 g, 2.12 mmol) in dry THF
(24 mL) under an argon atmosphere was added mercuric diacetate
(1.35 g, 4.24 mmol). The reaction mixture was stirred at room tem-
perature for 20 h. Et3N was then added (3 drops) followed by KCl
(316 mg, 4.24 mmol) solubilized in a minimum amount of water.
After 1 h at room temperature, the reaction mixture was diluted
with diethyl ether (100 mL), and the aqueous layer was extracted
with diethyl ether (3ϫ20 mL). The combined organic layer was
dried with MgSO4 and the solvents evaporated. The crude mixture
was purified by flash chromatography on silica gel (cyclohexane/
EtOAc, 8:2) to yield intermediate mercurio adduct (1.52 g, 96%
yield) as a colorless oil. To a stirred solution of this mercurio deriv-
ative (1.35 g, 3.0 mmol) in anhydrous CH2Cl2 (10 mL) was added
I2 in dry CH2Cl2 (15 mL, C = 0.5 , 7.6 mmol) dropwise under
an argon atmosphere. After 6 h at room temperature, the reaction
mixture was quenched with an aqueous solution of Na2S2O3, with
saturated NaCl, and then with water. The combined organic layer
was dried with MgSO4, filtered, and concentrated. The residue was
purified by flash chromatography on silica gel (cyclohexane/EtOAc,
1
Bu3N). H NMR (400 MHz, D2O): δ = 5.41 [dd, J(1Ј-2) = 1.8 Hz,
J(1Ј-P) = 10.8 Hz, 1 H, 1Ј-H], 4.21 [ddd, J(1Ј-P) = 1.8 Hz, J(2-3)
= 10.1 Hz, J(2-P) = 3.3 Hz, 1 H, 2-H], 4.08 [d, J(3-4) = 3.2 Hz, 1
H, 4-H], 3.91 [ABX, J(5-6a) = 7.8 Hz, J(6a-6b) = 11.1 Hz, 1 H, 6a-
H], 3.85 (dd, 1 H, 5-H), 3.75 [ABX, J(5-6b) = 2.9 Hz, J(6a-6b) =
11.1 Hz, 1 H, 6-Hb], 3.68 [dd, J(2-3) = 10.1 Hz, J(3-4) = 3.2 Hz, 1
H, 3-H], 3.12 [m, CH2(d), Bu3N], 1.66 [m, CH2(c), Bu3N], 1.36 [sex,
J(H-H) = 7.4 Hz, CH2(b), Bu3N], 0.92 [t, J(H-H) = 7.4 Hz, CH3(a),
Bu3N] ppm. 13C NMR (100 MHz, D2O): δ = 164.44 (C-1), 101.54
[d, J(1Ј-P) = 176.6 Hz, C-1Ј], 80.66 (C-5), 73.55 (C-3), 69.33 (C-4),
68.77 [d, J(2-P) = 11.3 Hz, C-2], 61.63 (C-6), 53.01 [CH2(d), Bu3N],
25.55 [CH2(c), Bu3N], 19.63 [CH2(b), Bu3N], 13.12 [CH3(a), Bu3N]
ppm. 31P NMR (101 MHz, D2O): δ = 10.91 ppm. MS (FAB–): m/z
(%) = 255 (100) [M – H]–. HRMS: calcd. for C7H12O8P 255.0270;
found 255.0265.
9:1) to yield 22 (1.16 g, 90% yield) as a pale yellow oil. [α]2D2
=
+16.1 (c = 0.5, CHCl3). 1H NMR (400 MHz, CDCl3): δ = 7.42–
7.32 (m, 20 H, H arom.), 4.75 [AB, J(H-H) = 12.0 Hz, 1 H,
CH2Ph], 4.74 [AB, J(H-H) = 11.8 Hz, 1 H, CH2Ph], 4.68 [AB, J(H-
H) = 10.0 Hz, 1 H, CH2Ph], 4.65 [AB, J(H-H) = 10.2 Hz, 1 H,
CH2Ph], 4.64 [AB, J(H-H) = 12.7 Hz, 1 H, CH2Ph], 4.63 [AB, J(H-
H) = 12.7 Hz, 1 H, CH2Ph], 4.61 [AB, J(H-H) = 11.8 Hz, 1 H,
CH2Ph], 4.59 [AB, J(H-H) = 12.0 Hz, 1 H, CH2Ph], 4.17 [ddd, J(1-
2) = 3.6 Hz, J(1-1Јa) = 6.3 Hz, J(1-1Јb) = 8.6 Hz, 1 H, 1-H], 4.11
3,4,5,7-Tetra-O-benzyl-D-galactohept-1-enitol (21): To a solution of
2,3,4,6-tetra-O-benzyl--galactose 20[29] (2 g, 3.71 mmol) in anhy- (m, 1 H, 5-H), 4.08 [d, J(3-4) = 2.7 Hz, 1 H, 4-H], 4.01 [dd, J(1-2)
1778
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© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2009, 1771–1780

Synthesis of Three C-Glycoside Analogues of UDP-Galactopyranose
= 3.6 Hz, J(2-3) = 6.6 Hz, 1 H, 2-H], 3.97 [ABX, J(5-6a) = 7.2 Hz,
J(6a-6b) = 10.7 Hz, 1 H, 6-Ha], 3.79 [ABX, J(5-6b) = 4.4 Hz, J(6a-
6b) = 10.7 Hz, 1 H, 6-Hb], 3.76 [dd, J(2-3) = 6.6 Hz, J(3-4) =
1.36 [sex, J(H-H) = 7.4 Hz, CH2(b), Bu3N], 0.92 [t, J(H-H) =
7.4 Hz, CH3(a), Bu3N] ppm. 13C NMR (100 MHz, D2O): δ = 72.20
(C-5), 72.11 [d, J(1-P) = 2.4 Hz, C-1], 69.98 (C-3), 69.16 (C-4),
2.7 Hz, 1 H, 3-H], 3.46 [ABX, J(1-1Јa) = 6.3 Hz, J(1Јa-1Јb) = 68.57 [d, J(2-P) = 10.9 Hz, C-2], 61.16 (C-6), 53.01 [CH2(d)], 24.72
10.4 Hz, 1 H, 1Ј-Ha, CH2-I], 3.36 [ABX, J(1-1Јb) = 8.6 Hz, J(1Јa- [d, J(1Ј-P) = 140.0 Hz, C-1Ј], 25.55 [CH2(c)], 19.63 [CH2(b)], 13.12
1Јb) = 10.4 Hz, 1 H, 1Ј-Hb, CH2-I] ppm. 13C NMR (100 MHz,
[CH3(a)] ppm. 31P NMR (101 MHz, D2O): δ = 21.99 ppm. MS
CDCl3): δ = 138.28, 138.24, 138.20, 137.80 (4 Cq arom.), 128.41– (FAB–): m/z (%) = 257 (100) [M – H]–. HRMS: calcd. for
127.46 (20 CH arom.), 75.79 (C-3), 75.71 (C-2), 73.65 (C-4), 73.45 C7H14O8P 257.0426; found 257.0423.
(CH2Ph), 73.23 (CH2Ph), 73.08 (C-5), 72.91 (CH2Ph), 72.88
C-α-D
-Galactopyranose-1,2-cyclophosphonate (25): 1H NMR
(CH2Ph), 71.59 (C-1), 66.78 (C-6), 2.73 (C-1Ј, CH2-I) ppm. MS
(DCI-NH3): m/z (%) = 665 (50) [M + H]+, 682 (100) [M + NH4]+.
HRMS: calcd. for C35H38O5I 665.1754; found 665.1759. C35H37IO5
(664.59): calcd. C 63.26 H 5.61; found C 63.66 H 5.75.
(400 MHz, D2O): δ = 4.83 [tdd, J(1-P) = 3.3 Hz, J(1-1Јa) = 11.1 Hz,
J(1-2) = J(1-1Јb) = 7.8 Hz, 1 H, 1-H], 4.34 [dd, J(1-2) = 7.8 Hz,
J(2-3) = 8.9 Hz, 1 H, 2-H], 3.96 [dd, J(3-4) = 3.1 Hz, J(4-5) =
1.3 Hz, 1 H, 4-H], 3.90 (m, 1 H, 5-H), 3.87 [dd, J(2-3) = 8.9 Hz,
J(3-4) = 3.1 Hz, 1 H, 3-H], 3.76 [ABX, J(5-6a) = 7.3 Hz, J(6a-6b)
= 11.7 Hz, 1 H, 6-Ha], 3.71 [ABX, J(5-6b) = 4.8 Hz, J(6a-6b) =
11.7 Hz, 1 H, 6-Hb], 2.07 [ABXXЈ, J(1-1Јa) = 11.1 Hz, J(1Јa-P) =
17.8 Hz, J(1Јa-1Јb) = 14.1 Hz, 1 H, 1Ј-Ha], 1.96 [ABXXЈ, J(1-1Јb)
= 7.8 Hz, J(1Јb-P) = 11.2 Hz, J(1Јa-1Јb) = 14.1 Hz, 1 H, 1Ј-Hb]
ppm. 13C NMR (100 MHz, D2O): δ = 76.68 [d, J(2-P) = 2.0 Hz,
C-2], 72.90 [d, J(1-P) = 13.6 Hz, C-1], 72.75 (C-5), 71.08 [d, J(3-P)
= 1.4 Hz, C-3], 68.92 (C-4), 61.32 (C-6), 22.00 [d, J(1Ј-P) =
115.5 Hz, C-1Ј] ppm. 31P NMR (101 MHz, D2O): δ = 36.44 ppm.
MS (FAB–): m/z (%) = 239 (100) [M – H]–. HRMS: calcd. for
C7H12O7P 239.0321; found 239.0331.
Diethyl C-(2,3,4,6-tetra-O-benzyl-α-D-galactopyranosyl)methane-
phosphonate (23): A solution of iodide 22 (1 g, 1.5 mmol) in triethyl
phosphite (10.3 mL, 60.2 mmol) was heated at reflux (160 °C) for
12 h under an argon atmosphere. The solvent was then removed
under reduced pressure, and the residue was purified by flash
chromatography on silica gel (cyclohexane/EtOAc, 5:5) to afford
compound 23 (0.94 g, 93% yield) as a colorless oil. [α]2D2 = +32.5
1
(c = 1.0, CHCl3). H NMR (400 MHz, CDCl3): δ = 7.38–7.32 (m,
20 H, H arom.), 4.78–4.52 (m, 8 H, 4CH2Ph), 4.52 (m, 1 H, 1-H),
4.14 [qd, J(H-H) = 7.0 Hz, J(H-P) = 2.7 Hz, 2 H, OCH2CH3], 4.12
[qd, J(H-H) = 7.0 Hz, J(H-P) = 2.7 Hz, 2 H, OCH2CH3], 4.12 (m,
1 H, 5-H), 4.09 [dd, J(3-4) = 2.7 Hz, J(4-5) = 3.2 Hz, 1 H, 4-H],
3.96 [dd, J(1-2) = 4.0 Hz, J(2-3) = 7.1 Hz, 1 H, 2-H], 3.91 [ABX,
J(5-6a) = 6.6 Hz, J(6a-6b) = 10.1 Hz, 1 H, 6-Ha], 3.79 [ABX, J(5-
6b) = 5.2 Hz, J(6a-6b) = 10.1 Hz, 1 H, 6-Hb], 3.71 [dd, J(2-3) =
7.1 Hz, J(3-4) = 2.7 Hz, 1 H, 3-H], 2.22 [AXXЈ, J(1-1Јa) = 3.1 Hz,
J(1Јa-P) = 18.8 Hz, 1 H, 1Ј-Ha], 2.20 [AX, J(1Јb-P) = 18.2 Hz, 1
H, 1Ј-Hb], 1.34 [t, J(H-H) = 7.0 Hz, 3 H, OCH2CH3], 1.30 [t, J(H-
H) = 7.0 Hz, 3 H, OCH2CH3] ppm. 13C NMR (100 MHz, CDCl3):
δ = 138.32, 138.24, 138.17, 137.96 (4 Cq arom.), 128.25–127.36 (20
CH arom.), 76.18 (C-2), 76.07 (C-3), 73.57 (C-4, C-5), 73.18
(CH2Ph), 73.11 (CH2Ph), 73.01 (CH2Ph), 72.64 (CH2Ph), 67.04 (C-
5), 68.94 (C-6), 61.58 [d, J(C-P) = 6.4 Hz, OCH2CH3], 61.48 [d,
J(C-P) = 6.3 Hz, OCH2CH3], 24.72 [d, J(1Ј-P) = 140.0 Hz, C-1Ј],
16.32 (OCH2CH3), 16.26 (OCH2CH3) ppm. 31P NMR (101 MHz,
CDCl3): δ = 29.50 ppm. MS (DCI-NH3): m/z (%) = 675 (100) [M
+ H]+, 692 (20) [M + NH4]+. HRMS: calcd. for C39H48O8P
675.3087; found 675.3092.
Acknowledgments
This work was supported by the F.N.R.S. (FRFC grant no.
2.4.625.08.F), the University of Namur, and the Centre National
de la Recherche Scientifique (CNRS).
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1
0.7 equiv. Bu3N). H NMR (400 MHz, D2O): δ = 4.39 [tdd, J(1-2)
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Received: December 16, 2008
Published Online: February 18, 2009
1780
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© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2009, 1771–1780