Synthesis and Inhibition Properties of Conformational Probes for the Mutase-Catalyzed UDP-Galactopyranose/Furanose Interconversion

Article abstract of DOI:10.1002/chem.200305141

UDP-galactose mutase is a flavoenzyme that catalyzes the isomerization of UDP-galactopyranose into UDP-galactofuranose, a key step in the biosynthesis of important bacterial oligosaccharides. Several mechanisms for this unique ring-contraction have been proposed, one of them involving a putative 1,4-anhydrogalactopyranose as an intermediate in the reaction. The purpose of this study was to probe the mutase binding site with conformationally restricted analogues of its substrate. Thus, we describe the straightforward synthesis of two C-glycosidic UDP-galactose derivatives: analogue 1, presenting a galactose moiety locked in a bicyclic ^1,4B boat conformation, and UDP-C-Galf 2, where the galactose residue is locked in the conformation of the mutase substrate. The two molecules were found to be inhibitors of UDP-galactose mutase at levels depending on the redox state of the enzyme. Strong inhibition of the native enzyme, but a low one of the reduced mutase, were observed with UDP-C-Galf 2, whereas 1 displayed intermediate inhibition levels under both native and reducing conditions. These data provide evidence of a significant conformational difference of the mutase binding pocket in the reduced enzyme and in the native one, the enzyme switching from a low Galf-affinity state (reduced enzyme) to a very strong one (native enzyme). It is remarkable that the mutase binds the boat-locked analogue 1 with similar affinities in both its conformational states. These results support a mechanism involving the formation of 1,4-anhydrogalactopyranose as a low-energy intermediate. An alternative explanation would be that the distortion of the galactose moiety during the cycle contraction transiently brings the carbohydrate into a conformation close to a ^1,4B boat.

Full text of DOI:10.1002/chem.200305141

FULL PAPER
Synthesis and Inhibition Properties of Conformational Probes for the Mutase-
Catalyzed UDP-Galactopyranose/Furanose Interconversion
Audrey Caravano,[a] Dominique Mengin-Lecreulx,[b] Jean-Marc Brondello,[c]
Ste¬phane P. Vincent,*[a] and Pierre Sinay*[a]
»
Abstract: UDP-galactose mutase is a
flavoenzyme that catalyzes the isomer-
ization of UDP-galactopyranose into
UDP-galactofuranose, a key step in the
biosynthesis of important bacterial oli-
gosaccharides. Several mechanisms for
this unique ring-contraction have been
proposed, one of them involving a
putative 1,4-anhydrogalactopyranose as
an intermediate in the reaction. The
purpose of this study was to probe the
mutase binding site with conformation-
ally restricted analogues of its substrate.
Thus, we describe the straightforward
synthesis of two C-glycosidic UDP-gal-
actose derivatives: analogue 1, present-
ing a galactose moiety locked in a
bicyclic 1,4B boat conformation, and
UDP-C-Galf 2, where the galactose
difference of the mutase binding pocket
in the reduced enzyme and in the native
one, the enzyme switching from a low
Galf-affinity state (reduced enzyme) to
a very strong one (native enzyme). It is
remarkable that the mutase binds the
boat-locked analogue 1 with similar
affinities in both its conformational
states. These results support a mecha-
nism involving the formation of 1,4-
anhydrogalactopyranose as a low-ener-
gy intermediate. An alternative explan-
ation would be that the distortion of the
galactose moiety during the cycle con-
traction transiently brings the carbohy-
drate into a conformation close to a
1,4B boat.
residue is locked in the conformation
of the mutase substrate. The two mole-
cules were found to be inhibitors of
UDP-galactose mutase at levels depend-
ing on the redox state of the enzyme.
Strong inhibition of the native enzyme,
but a low one of the reduced mutase,
were observed with UDP-C-Galf 2,
whereas 1 displayed intermediate inhib-
ition levels under both native and re-
ducing conditions. These data provide
evidence of a significant conformational
Keywords: C-glycosides
furanose inhibitors
conformations ¥ nucleotides
¥
galacto-
¥
¥ locked
lian cells, thus making the enzymes responsible for their
biosynthesis potential targets for applications in antibiother-
apy.[2, 3] Galactofuranosyl moieties have been found in lip-
opolysaccharides (LPSs) of Gram-negative bacterial outer
membranes, in capsular oligosaccharides of different bacterial
strains, and also in mycobacterial cell walls.[4] Furthermore,
they are key components of the arabinogalactan complex
characteristic of mycobacterial membranes, where they are
present as an oligo-(Galf) made up of over 20 Galf residues
with a typical b-1,5-Galf-b-1,6-Galf repeated unit. The en-
zymes responsible for the galactan biosynthesis are a mutase[5]
catalyzing the isomerization of UDP-galactopyranose into
UDP-galactofuranose and transferases[6] triggering the glyco-
sylation of a growing oligosaccharide acceptor with UDP-Galf
as the donor substrate. Importantly, studies on mutants of
Mycobacterium smegmatis have demonstrated that the inhib-
ition of the arabinogalactan biosynthesis effected through
UDP-Gal mutase inactivation prevents mycobacterial prolif-
eration.[7] Although few Galf-transferases (GalfTs) have been
fully characterized, it has been shown that the same GalfT can
catalyze two subsequent Galf transfers.[8] The most studied
enzyme of the galactan biosynthesis is the UDP-Gal mutase
Introduction
Galactan biosynthesis is emerging as an important research
field at the frontiers of microbiology, enzymology and
bioorganic chemistry.[1] Galactans are oligosaccharides with
galactofuranose (Galf) and galactopyranose (Galp) residues
as main components. Galf moieties are expressed in the
membranes of prokaryotes but are totally absent in mamma-
[a] Dr. S. P. Vincent, Prof. P. Sinay», A. Caravano
¬
Ecole Normale Superieure
Departement de Chimie, UMR-8642 du CNRS
24rue Lhomond, 75231 Paris Cedex 05 (France)
Fax : (‡33)1-44-32-33-97
¬
E-mail: sinay@ens.fr
[b] Dr. D. Mengin-Lecreulx
¬
Enveloppes Bacteriennes et Antibiotiques
IBBMC, UMR-8619 du CNRS
¬
√
Universite de Paris-Sud, Bat. 430
91405 Orsay Cedex (France)
[c] Dr. J.-M. Brondello
¬
¬
Institut de Genetique Humaine
UPR-1142 du CNRS, 141 rue de la cardonille
34396 Montpellier Cedex 05 (France)
5888
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
DOI: 10.1002/chem.200305141
Chem. Eur. J. 2003, 9, 5888 5898

5888 5898
cloned from Escherichia coli,
Klebsiella pneumoniae, and
Mycobacterium
tuberculosis,
the three-dimensional structure
of which is now available.[9] The
mechanism by which this en-
zyme catalyzes this unique iso-
merization, formally a ring-con-
traction, remains very puzzling
to date. First, all characterized
mutases are flavoenzymes, but
the exact role of the FAD
cofactor is far from being well
understood. From a study em-
ploying membrane extracts of
P. charlesii, it was first hypothe-
sized that an oxidation at the
2-position of the galactose moi-
ety would occur during the
Scheme 1. Hypothetical mechanism of the mutase-catalyzed UDP-Galp/UDP-Galf interconversion. a) Mutase-
catalyzed pyranose/furanose interconversion through a putative 1,4-anhydrogalactose intermediate. b) Chemical
models for 1,4-anhydrogalactose opening reactions exclusively giving galactofuranosides.
12]
enzymatic process.[10
However, several UDP-Gal ana-
logues fluorinated at the 2-, 3-, and 4-positions have been
was maintained in its reduced state (thus ruling out the
hypothesis of an oxidation at the 2-position), whereas the
same molecules displayed inhibition properties under non-
reducing conditions.[15] At about the same time, Naismith
et al. concluded from analysis of the three-dimensional
mutase structure that an oxidation/reduction reaction in the
vicinity of the anomeric center is likely to occur.[9] In a more
recent investigation, it was shown that the enzyme can
stabilize the semiquinone form (FADH¥) of the flavin cofactor
and that the fully reduced flavin was FADHÀ.[16] However, no
redox turnover of the flavin cofactor has ever been found. The
specific role of the FAD cofactor thus remains to be defined to
allow both a clear understanding of this unique molecular
rearrangement and the design of mechanism-based inhibitors
of UDP-Gal mutase.
synthesized and tested as inhibitors or substrates of the E. coli
15]
mutase.[13
Interestingly, 2F-Galf-UDP and 3F-Galf-UDP
were found to be substrates of the enzyme when the mutase
Abstract in French: L'UDP-galactose mutase est une flavoen-
¬
zyme catalysant l'isomerisation de l'UDP-galactopyranose en
¬
¬
UDP-galactofuranose (UDP-Galf), une etape cle de la biosyn-
¡
¬
¬
these d'oligosaccharides bacteriens. Plusieurs mecanismes
¬
¬ ¬
¬
reactionnels de cette contraction de cycle ont ete proposes,
l'un d'entre eux faisant intervenir le 1,4-anhydrogalactopyra-
¬
¬
¬
nose comme intermediaire hypothetique. Le but de cette etude
est de sonder le site actif de cette mutase avec des analogues du
Nevertheless, Blanchard et al. have provided interesting
information regarding this enzymatic ring-contraction by
positional isotope exchange (PIX) experiments.[17] Indeed, it
was shown with K. pneumoniae mutase (and later reproduced
with E. coli)[15] that a cleavage of the C1-O(UDP) bond of
UDP-Gal occurs during the enzymatic process. From these
results, it was hypothesized that 1,4-anhydrogalactopyranose
A (Scheme 1) could be an intermediate in the isomerization.
Although this mechanistic suggestion does not take account of
any oxido-reduction process, it may, if confirmed, provide
interesting knowledge about the conformation(s) of the
galactose moiety at the transition(s) state(s) (if a 1,4-
anhydrogalactopyranose is an intermediate in this reaction,
two transition states might be invoked). Thus, to strengthen
this mechanistic hypothesis, a non-enzymatic model of the
mutase-catalyzed isomerization was designed by our group
and showed that 2,3,6-tribenzyl-1,4-anhydrogalactopyranose,
in the presence of a nucleophile and a catalytic amount of
camphorsulfonic acid (CSA), yields an anomeric mixture of
galactosides, exclusively in furanose configurations.[18] Similar
selective ring-opening had previously been described in the
literature and had been explained by stereoelectronic ef-
fects.[19] Given the positions of the lone-pair electrons of the
¬
substrat figes dans des conformations distinctes. Ainsi, nous
¬
¬
avons synthetise deux C-glycosides analogues de l'UDP-
galactose: le nucleotide sucre 1 presentant un groupe galactose
bloque dans une conformation bateau 1,4B, et l'analogue 2 dont
l'unite galactose est bloquee dans une configuration furanose.
Ces deux molecules se sont averees etre des inhibiteurs de
l'UDP-galactose mutase avec des niveaux d'inhibition depen-
dant de l'etat d'oxydo-reduction de l'enzyme. L'UDP-C-Galf 2
inhibe fortement l'enzyme native, mais faiblement l'enzyme
reduite, tandis que l'analogue 1 possede un pouvoir inhibiteur
intermediaire a la fois dans des conditions natives et reduc-
trices. Ces donnees d'inhibition mettent clairement en evidence
des differences significatives de la conformation du site actif de
l'enzyme, celle-ci passant d'un etat de forte affinite pour le Galf
(conditions natives) a un etat de faible affinite (conditions
reductrices). Il est par ailleurs remarquable que ces deux etats
conformationnels possedent des affinites comparables pour le
groupe galactose fige dans une conformation bateau 1,4B. Ces
resultats montrent qu'un mecanisme faisant intervenir le 1,4-
anhydrogalactopyranose en tant qu'intermediaire a basse
energie est possible. Toutefois, ces resultats peuvent egalement
mettre en evidence le fait que l'enzyme peut stabiliser, au cours
de cette isomerisation, une conformation transitoire proche
d'un bateau 1,4B.
¬
¬
¬
¬
¬
¬
¬ ¬
√
¬
¬
¬
¬
¡
¬
¡
¬
¬
¬
¬
¬
¬
¡
¬
¬
¬
¬
¡
¬
¬
¬
¬
¬
¡
¬
¬
¬
¬
¬
À
À
oxygen O-4and O-5 relative to the C1 O4and C1 O5 s
À
bonds, the C1 O5 bond may be considered weakened by
Chem. Eur. J. 2003, 9, 5888 5898
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S. P. Vincent, P. Sinay» et al.
FULL PAPER
stereoelectronic effects, due to its antiperiplanarity with one
of the O-4doublets (Scheme 1).
From the mechanistic investigations (PIX experiments) and
the non-enzymatic model we designed the phosphonate 1 as a
mechanistic probe with which to confirm or rule out the
easily be performed, giving the intermediate exo-glycal 5
(Scheme 2). This reactive enol ether could not be purified by
standard silica gel chromatography. Interestingly, the main
degradation product on silica gel was the cyclization product
6, with a 1,4-anhydrogalactopyranose structure, thus showing
the effective propensity of galactofuranosyl exo-glycals to
cyclize under mild conditions. The intermediate 5 was there-
fore not isolated, but was directly treated, in one-pot fashion,
with an excess of NIS in anhydrous CH2Cl2, to give the
cyclized iodide 7 in 62% from 4. Unfortunately, attempts to
phosphonylate 7 under Arbuzoff ((RO)3P, heat) or Arbuz-
off Becker ((RO)2PONa, heat) conditions proved unsuc-
cessful. This lack of reactivity is probably due to the neopentyl
nature of this primary iodide.
We then turned our attention to the second strategy, based
on the cyclization of a pre-phosphonylated exo-glycal. The
desired enol ethers 10 and 11 were prepared by a straightfor-
involvement of an intermediate possessing a structure close to
a 1,4-anhydrogalactopyranose, or at least to measure the
affinity of this enzyme for a galactose moiety locked in a
1,4B boat conformation. This molecule is made up of a 1,4-
anhydrogalactopyranose substructure, to test the mechanistic
assumption, together with a UDP moiety required for tight
binding to the enzyme. The two substructures are linked
through a methylene group between the anomeric carbon
atom of the constrained sugar and the b-phosphate of UDP, to
provide both chemical and enzymatic stability to the final
nucleotide sugar. Thus, 1 can also be seen as a conformational
probe for this particular glycosyl processing enzyme. For
comparison of the binding affinities, we also report a new
synthesis of UDP-C-Galf 2, the phosphonate analogue of
UDP-Galf, in which the galactose moiety is in the ground
state conformation.
ward two-step procedure from the galactonolactone
4
(Scheme 3). This lactone was condensed with the lithium
anion of dibenzyl (or dimethyl) methylphosphonate to give
the lactol 8 (or 9).[21] The subsequent elimination was achieved
through a slight modification of a procedure recently descri-
bed in the literature.[22, 23] The presence of the unprotected
hydroxy group at the 5-position does not significantly modify
the yields of these two steps. It is worth noting that the lactols
8 and 9 were isolated, characterized, and induced to react in
their furanose forms. Since the 5-OH group is free, an
isomerization to a pyranose form might have been observed.
However, NMR spectral data on lactols 8 and 9, as well as on
exo-glycals 10 and 11, are consistent with furanosides. To
demonstrate the furanosidic structure of the central inter-
mediate 10 unambiguously, we also performed the methyl-
enephosphonylation/elimination sequence on the 1,4-galacto-
nolactone protected at the 5-position as a pivaloyl ester
(compounds 12 to 14, Scheme 3). Removal of the pivaloate
from glycal 14 gave a 5-hydroxylated compound strictly
identical to compound 11. These experiments both confirmed
the structures of compounds 10 and 11 and outlined the
efficiency of this sequence: the exo-glycal 10 possesses a free
5-hydroxy group, ready to cyclize, without protective group
manipulation. The exo-glycals were isolated as single diaster-
eomers with Z configurations, as shown by a NOE between
H-1' (the olefinic proton) and H-2, a result consistent with
Results and Discussion
After retrosynthetic analysis, we reasoned that it should be
advantageous to choose the commercially available 1,4-
galactonolactone 3 as starting material. Two synthetic strat-
egies were then explored, differing in the two key steps that
had to be envisioned: a cyclization yielding the 1,4-anhydro
motif and the installation of the phosphonate group. Indeed,
we first had to define which step, the phosphonylation or the
cyclization, should be performed first. The 2,3,6-tri-O-sily-
lated lactone 4, possessing a free hydroxy group at the
5-position, could easily be obtained on large scale, by a known
procedure (Scheme 2).[20] We then found that, even in the
presence of the 5-hydroxy group, a Tebbe reaction could
24]
literature data.[22 Owing to the electron-withdrawing char-
acter of the phosphoryl moiety, these exo-glycals are stable on
silica gel.
Scheme 2. Synthesis of a non-phosphonylated 1,4-anhydrogalactopyranose substructure. Reagents and conditions: a) acetone, CuSO4; b) TBDMSCl, Im,
DMF; c) AcOH, H2O; d) TBDMSCl, Im, DMF; e) Tebbe reagent, f) silica gel; g) NIS, CH2Cl2. TBDMS ˆ tert-butyldimethylsilyl, Im ˆ imidazole, NIS ˆ N-
iodosuccinimide. TBS ˆ tert-butyldimethylsilyl.
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UDP-Galactopyranose/Furanose
5888 5898
Scheme 3. Synthesis of the 1,4-anhydrogalactopyranose-phosphonate substructure. Reagents and conditions: a) (RO)2POCH2Li, THF; b) Py, (CF3CO)2O;
c) PivCl; d) Dibal-H, CH2Cl2, O8C; e) CSA, CH2Cl2, reflux; f)TBAF, THF; g) H2, Pd/C, MeOH. Piv ˆ pivaloyl, Dibal-H ˆ diisobutylaluminum hydride,
CSA ˆ camphorsulfonic acid.
The phosphonylated glycal 10 readily cyclized in the
presence of CSA in refluxing CH2Cl2 to give the constrained
structure 15 in 88% yield. The 1,4B conformation was con-
90% after 12 h. The only observed side product was the
expected UMP dimer (UppU), in proportions (typically 5%)
highly dependant on the proper drying of the reaction
mixture. We have thus demonstrated the efficiency of this
coupling methodology, allowing the preparation of 1 in good
yield from a hindered neopentyl phosphonate.
firmed by comparison with 1H NMR data of analogous
27]
compounds,[25
with typically small coupling constants
between H-2, H-3, H-4, and H-5 (0.9 Hz < J < 1.2 Hz). The
three tert-butyldimethylsilyl (TBDMS) groups of intermedi-
ate 15 were removed with tetrabutylammonium fluoride
(TBAF) in THF at À58C. Deprotection was completed by
hydrogenolysis under standard conditions, affording 16 in
78% yield for the two deprotection steps.
Anew synthesis of UDP- C-Galf: UDP-C-Galf, the phospho-
nate analogue of UDP-Galf (Scheme 5), was first synthesized
in our laboratory in eight steps by exploiting, as the key
cyclization step, an a-stereospecific iodoetherification.[25]
Here we report a shorter and still stereospecific sequence to
this target molecule. Persilylated (TBS)4-1,4-galactonolactone
17 was subjected to a one-pot sequence affording exo-glycal
18 in 77% yield. Hydrogenation over Pearlman×s catalyst in
AcOEt gave the desired a-phosphonate 19 in 82% yield (no b
product could be isolated or even detected by 31P NMR on the
final crude reaction mixture). The a configuration at the
Coupling to UMP: To circumvent the often problematic
coupling of hindered phosphonates to UMP-morpholidate,
we explored a procedure recently developed by Bogachev for
the large-scale synthesis of nucleoside triphosphates.[28] This
methodology was first exploited by Kiessling et al., for the
synthesis of UDP-Galf, and then by our group for the
synthesis of GDP-hexanolamine.[29, 30] The procedure consists
of the sequential activation of UMP by trifluoroacetic
anhydride (TFAA) followed by the addition of an excess of
N-Me-imidazole, affording a reactive phosphoimidazolium
intermediate in situ. This undergoes a fast nucleophilic
substitution upon phosphonate addition (Scheme 4). This
reaction usually gives good to excellent yields with reaction
times within hours, comparing very favorably with those
required for classical morpholidate couplings. Under opti-
mized conditions, coupling of phosphonate 16 with the
activated UMP afforded the desired UDP-C-1,4-anhydroga-
lactopyranose 1 in 76% yield after size exclusion chromatog-
raphy. From 31P NMR spectra performed on aliquots of crude
reaction mixture, we estimated that the conversion into 1 was
1
1-position could be confirmed by H NMR (NOEs between
H-1 and H-4could be observed in molecules 19 and 20) as well
as by comparison with the previously characterized phospho-
nate 20.[25] The source of the selectivity is probably the steric
hindrance of the TBDMS group at the 2-position. Excess
TMSI in CCl4 quantitatively removed all the protecting
groups. Phosphonate 20 was then coupled to UMP to afford
pure UDP-C-Galf in five steps from commercial 1,4-galacto-
nolactone (Scheme 5). We observed that UDP-C-Galf 2
decomposes through an intramolecular cyclization during
the course of the reaction and the purification, to afford UMP
and the bicyclic phosphonate 21. This side reaction explains
the poorer yield in relation to the same coupling method
affording 1 (Scheme 4), despite almost total consumption of
Scheme 4. Synthesis of nucleotide-sugar 1. DMA ˆ N,N-dimethylaniline.
Chem. Eur. J. 2003, 9, 5888 5898
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S. P. Vincent, P. Sinay» et al.
FULL PAPER
Scheme 5. Synthesis of UDP-C-Galf 2. Reagents and conditions: a) (RO)2POCH2Li, THF, concentrated then THF/Py, (CF3CO)2O; b) H2, Pd(OH)2/C,
AcOEt; c) TMSI, CCl4.
starting phosphonate 20. This tendency was also observed in
the preparation of other cyclic 1,2-syn-hydroxy-pyrophos-
phates such as UDP-Galf[29] and ADP-b-mannoheptopyra-
inhibition properties when the native enzyme was used. The
most significant information provided by this study is in the
relative inhibition levels observed from native to reduced
conditions: strong inhibition by UDP-C-Galf 2 (81 to 91%)
was observed when the native enzyme was used, whereas the
inhibition–with or without preincubation of the inhibitor–
collapsed to 2 14% under reductive conditions. Under the
same assay conditions, the boat-locked molecule 1 displayed a
moderate decrease in the inhibition level on going from the
native enzyme (42 53%) to the reduced one (32 34%),
making 1 a better inhibitor than 2 when the enzyme is
maintained in its optimal catalytic efficiency. These results
corroborate the previously described inhibition levels of
UDP-2F-Galf and UDP-3F-Galf (Table 1), with which strong
inhibition was observed when the native enzyme was used
whereas these fluorinated molecules were either substrates or
poor inhibitors of the reduced enzyme.[15] The inhibitory
activity of the fluorinated analogues of UDP-Galf on the
native enzyme could be explained by the irreversible inacti-
vation of the enzyme, whereas such a phenomenon is not
likely to occur with ether-phosphonates such as 1 and 2. Thus,
our results clearly demonstrate that UDP-galactose mutase
displays two different conformational states, a ™native∫ and a
™reduced∫ state, characterized by high and low affinity,
respectively, for the galactofuranose moiety, and comparable
affinities for the 1,4-anhydrogalactopyranose substructure.
The activity of the native enzyme, without dithionite
addition, is typically very low compared to that of the reduced
one. Photoreduction of the flavin cofactor has been proposed
to explain the residual activity of the enzyme when no
chemical reductant is added.[9] The kinetic and binding
properties of the same enzyme under reducing or native
conditions are very different: the Km values for UDP-Galf
were determined to be 22 mm and 194 mm under reduced and
native conditions, respectively.[15, 33] Similar values could be
reproduced in our hands. A recent report showed that the
flavosemiquinone FADH¥ is stabilized in the presence of
nose[31]
.
Enzymatic assay: Two methods to evaluate UDP-galactose
mutase kinetics have been described: a HPLC method and the
use of radiolabeled UDP-Galf*.[9] The HPLC method was
first described by McNeil et al., and was based on the
separation of UDP-Galp (used as the substrate) and UDP-
Galf (as the product of the enzymatic reaction).[32] Because of
the small amount of furanose formed at the equilibrium (8%),
this method was improved by Liu et al.,[15, 33] by following the
reaction in the reverse direction (Galf to Galp). In this study
we followed Liu×s procedure as strictly as possible to compare
the binding properties of our molecules with those of the
described fluorinated inhibitors.[15] UDP-Gal mutase was
overexpressed and purified by an improved procedure, similar
to that described by Liu et al.,[33] but with the concomitant
expression of chaperones that allowed an increase in the yield
of soluble protein. A very intriguing and important observa-
tion was that fluorinated molecules were found to be
inhibitors of UDP-Gal mutase when the native enzyme was
used, and substrates of the enzyme when reductive conditions
were employed (upon addition of freshly prepared sodium
dithionite).[15] Furthermore, Zhang and Liu showed that their
inhibitors did not display the same inhibition properties if the
inhibitors were preincubated with the enzyme.[15] This binding
and catalytic behavior might suggest significant changes in the
conformation and/or the chemical state of the UDP-galactose
binding pocket, when the enzyme is maintained (or not) in a
reduced form. To provide a complete comparative inhibition
pattern, we measured the residual mutase activities in the
presence of our molecules under both native and reductive
conditions, with and without preincubation. The results are
summarized in Table 1. As in the case of 2-fluoro and 3-fluoro
analogues of UDP-Galf, compounds 1 and 2 displayed better
Table 1. Inhibition percentages of UDP-galactose mutase at [UDP-Galf] ˆ 1 mm and [inhibitor] ˆ 1 mm.
UDP-1,4-anh-Galp (1) [%]
UDP-C-Galf (2) [%]
UDP-2F-Galf[15] [%]
UDP-3F-Galf[15] [%]
[c]
native enzyme, no preincubation[a]
native enzyme, preincubated[b]
reduced enzyme, no preincubation
reduced enzyme, preincubated
4
53
nd3414
32
2
91
81
87 nd
98.6
61
10
2
nd
nd
[a] For conditions, see Experimental Section. [b] The inhibitor was incubated with the enzyme for 1 h at 218C before addition of UDP-Galf. [c] Not
determined.
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UDP-Galactopyranose/Furanose
5888 5898
UDP-galactose and that the fully reduced flavin is the anionic
FADHÀ rather than FADH2, suggesting a crypto-redox
process.[16] Nevertheless, no redox turnover of the flavin
cofactor has ever been detected during the UDP-Galp/UDP-
Galf interconversion, which is also the case for several other
flavoenzymes where the FAD has no apparent redox activ-
ity.[34] The exact role of the FAD cofactor therefore remains
obscure, two different mechanistic assumptions being possi-
ble: 1) the flavin acts as a conformational switch, its bent
conformation, when reduced as FADHÀ, inducing a confor-
mational change in the enzyme catalytic pocket, and 2) single-
electron transfers are part of an activation of the galactose
moiety at the anomeric center, triggering the cycle contrac-
tion.
At first sight, our data suggest that a 1,4B conformation is
not adopted by the galactose moiety at the transition state(s);
otherwise stronger inhibition levels would have been ob-
served in both native and reduced conditions. If a 1,4-
anhydrogalactopyranose A is involved as a low-energy
intermediate (mechanism depicted in Scheme 1), it might
have been expected that 1 and 2 should bind to the enzyme
with similar affinities; that is, with inhibition levels of similar
magnitudes. The fact that a stronger inhibition level was
observed with the boat-locked molecule 1 than with the
corresponding furanose-locked 2 suggests that such a mech-
anism is possible. However, one cannot conclude solely on the
basis of our inhibition study that the postulated mechanism[17]
gives a complete picture of the furanose/pyranose intercon-
version catalyzed by UDP-galactose mutase. An alternative
explanation would be that the distortion of the galactose
moiety during the cycle contraction transiently brings the
carbohydrate into a conformation close to a 1,4B boat to allow
nucleophilic substitutions at the anomeric position from the 4-
and the 5-hydroxy groups. Co-crystallization of the mutase
with molecules 1 and 2 might provide a better understanding
of the conformational changes involved in this enzymatic
reaction.
mechanism involving the formation of 1,4-anhydrogalacto-
pyranose as a low-energy intermediate.
Experimental Section
Materials and procedures: All chemicals were purchased from Sigma,
Aldrich, or Fluka and were used without further purification. Tetrahy-
drofuran, diethyl ether, and toluene were freshly distilled over sodium
benzophenone, dichloromethane over P2O5, and acetonitrile over CaH2.
1H, 13C, and 31P NMR spectra were recorded with Bruker AC 250 and
AMX 400 spectrometers. All compounds were characterized by 1H, 13C,
and 31P NMR, as well as by 1H,1H and 1H,13C correlation experiments.
Specific optical rotations were measured on a Perkin Elmer 241 polar-
imeter in a 1 dm cell. Melting points were determined with a B¸chi 535
apparatus. Column chromatography was performed on silica gel (Kieselgel
Si 60, 40 63 mm). Size exclusion chromatography was carried out on a
Sephadex G15 column (2.5 Â 60 cm) on a Pharmacia Biotech ækta FPLC
apparatus, fractions containing uridine derivatives being detected by UV.
When required, purifications of nucleotide-sugars were achieved by semi-
preparative HPLC, by use of a Waters Delta prep 4000 chromatography
system fitted with a NovaPack C18 (1 Â 10cm) column (eluent triethylam-
monium acetate 50 mm, pH ˆ 6.8). For enzymatic assays, a Waters 600 E
analytical apparatus fitted with a Zorbax C18-SB column (25 Â 0.46 cm,
5 mm) was used (eluent triethylammonium acetate 50 mm, pH ˆ 6.8). UDP-
Galf was prepared by known procedures.[29, 33]
Bacterial strains, plasmid vectors, and growth conditions: The E. coli
strains DH5a (Life Technologies, Inc.) and BL21(DE3)pLysS (Novagen)
were used as hosts for plasmids and for the preparation of the over-
produced Glf enzyme. The pREP4groESL plasmid allowing overproduc-
tion of the bacterial chaperones was obtained from K. Amrein.[35] Unless
otherwise noted, 2YT (Miller) was used as a rich medium and growth was
monitored at 600 nm with a Shimadzu UV-1601 spectrophotometer. For
strains carrying drug resistance genes, antibiotics were used at the following
concentrations (mgmlÀ1): ampicillin (100), kanamycin (35), and chloram-
phenicol (25).
General DNAtechniques : DNA restriction and modification enzymes
were obtained from New England Biolabs and oligonucleotides were
obtained from MWG-Biotech. Small- and large-scale plasmid isolations
from E. coli cells were carried out by the alkaline lysis method, and
standard procedures were used for endonuclease digestions, ligation, and
agarose electrophoresis.[36] E. coli cells were made suitable for trans-
formation with plasmid DNA by treatment with CaCl2.[37]
Construction of plasmids: A plasmid allowing expression of the glf gene
from E. coli in the 6xHis-tagged form under control of the T7 promoter was
constructed as follows. Firstly, a vector pET2160 that derived from pET21d
(Novagen) was constructed by removal (filling-in) of its unique BglII site
and replacement of the NcoI-HindIII polylinker by that from the pQE60
vector (Qiagen), as described previously for the construction of
pTrcHis60.[38] Polymerase chain reaction (PCR) primers were designed to
incorporate a BspLU11I site (in bold) 5' at the initiation codon (under-
lined) of the glf gene (5'-GAGGATTACATGTACGATTATAT-
CATTGTTGGTTC-3'), and a BglII site (in bold) 3' to the gene and
replacing the stop codon (5'-ATAGAAGATCTATCCGTACTCATTA-
TATTTTTCAC-3'). PCR amplification from the E. coli chromosome was
performed in a Thermocycler 60 apparatus (Bio-med) by use of the
polymerase Expand-Fidelity from Roche. The resulting 1.1 Kb product was
purified by use of the Wizard PCR Preps DNA purification kit (Promega),
treated with BspLU11I and BglII, and cloned between the compatible sites
NcoI and BglII of vector pET2160, giving rise to plasmid pMLD200. DNA
sequencing was performed to check that the sequence of the cloned
fragment was correct.
Conclusion
In summary, this study describes the straightforward synthesis
of two C-glycosidic UDP-galactose derivatives: analogue 1,
presenting a galactose moiety locked in a bicyclic 1,4B boat
conformation, and analogue 2, in which the galactose residue
is locked in the conformation of the UDP-Gal mutase
substrate. The two molecules were found to be inhibitors of
the mutase at levels depending on the redox state of the
enzyme. UDP-C-Galf 2 is a strong inhibitor of the native
enzyme but a poor inhibitor of the reduced one, while 1
displayed intermediate inhibition levels under both native and
reducing conditions. These data indicate a significant con-
formational difference in the mutase binding pocket on going
from the reduced enzyme to the native one, the enzyme
switching from a low Galf-affinity (reduced enzyme) to a
strong one (native enzyme). It is remarkable that the mutase
in its two conformational states binds the boat-locked
analogue 1 with related affinities. These results support a
Preparation of crude enzyme: Cells of BL21(DE3)pLysS carrying the two
plasmids pREP4groESL and pMLD200 were grown exponentially at 378C
in 2YT-ampicillin medium (1 L cultures). When the optical density of the
culture reached 1, the temperature of the culture was decreased to 228C
and expression of both the chaperones and the Glf protein was induced by
addition of isopropyl-b-d-thiogalactopyranoside (IPTG) at a final concen-
tration of 1 mm. Growth was continued for 16 h at 228C and cells were
Chem. Eur. J. 2003, 9, 5888 5898
www.chemeurj.org
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
5893

S. P. Vincent, P. Sinay» et al.
FULL PAPER
harvested and washed with 40 mL of cold 20 mm potassium phosphate
buffer, pH 7.4, containing 0.5 mm MgCl2 and 0.1% b-mercaptoethanol
(buffer A). The cell pellet was then resuspended in 20 mL of the same
buffer and disrupted by sonication (sonicator VibraCell, Bioblock, Illkirch,
France) for 10 min in the cold. The resulting suspension was centrifuged at
48C for 30 min at 200000 Â g and the supernatant (20 mL, 350 mg of
proteins) designated as crude enzyme was stored at À208C. SDS-PAGE
analysis of proteins was performed as previously described, by use of 12%
polyacrylamide gels.[39] Protein concentrations were determined by the
method of Bradford,[40] with bovine serum albumin as standard.
raphy (Sephadex G15) eluted with water. The appropriate fractions were
pooled and lyophilized. Compound 1 was further purified by HPLC on a
C18 column with 1% acetonitrile in 50 mm triethylammonium acetate
buffer pH ˆ 6.8 as eluent and a flow rate of 1 mLminÀ1. The retention time
of 1 under these conditions was 12.6 min. This protocol afforded 1 as a
white solid (triethylammonium salt, 61 mg) in 76% yield.
1H NMR (400 MHz, D2O): d ˆ 7.95 (d, J(5'',6'') ˆ 8.0 Hz, 1H; H-6''), 5.97
(d, J(1',2') ˆ 4.1 Hz, 1H; H-1'), 5.95 (d, J(5'',6'') ˆ 8.0 Hz, 1H; H-5''), 4.49
(d, J(3,4) ˆ 1.0 Hz, 1H; H-4), 4.36 (m, 2H; H-2', H-3'), 4.27 (m, 1H; H-4'),
4.22 4.18 (m, 2H; H-5'a, H-5'b), 3.95 (brs, 1H; H-3), 3.83 (dd, 1H; H-5),
3.80 (d, J(2,3) ˆ 0.6 Hz, 1H; H-2), 3.60 (syst. ABX, J(5,6a) ˆ 4.4 Hz,
J(6a,6b) ˆ 12.0 Hz, 1H; H-6a), 3.53 (ABX, J(5,6b) ˆ 5.6 Hz, J(6a,6b) ˆ
12.0 Hz, 1H; H-6b), 3.18 (q, J ˆ 7.3 Hz ; CH2 (Et3N)), 2.51 (ABX, J ˆ
19.1 Hz, J(H,P) ˆ 19.0 Hz, 2H; CH2P), 1.26 (t, J ˆ 7.3 Hz ; CH3
Purification of the 6xHis-tagged Glf enzyme: The one-step purification
procedure was carried out under native conditions, basically by following
the steps in the manufacturer×s (Qiagen, Santa Clarita, Calif.) recommen-
dations: binding of 6xHis-Glf on Ni2‡-nitrilotriacetate-agarose (Ni2‡
-
‡
(Et3N)) ppm; 13C NMR (NH4 salt, 100 MHz, D2O): d ˆˆ 168.48 (C-4''),
NTA), washing with buffer A containing 200 mm KCl and 20 mm imidazole
to remove impurities, and elution of the protein with increasing concen-
trations of imidazole added to buffer A (from 40 to 300 mm; the 6xHis-Glf
protein eluted in 100 mm and 200 mm). The purified protein was then
concentrated to 10 mL on PM10 membranes (Millipore) and dialyzed
overnight against buffer A at 48C. The 6xHis-tagged enzyme prepared in
this manner was more than 95% pure, as estimated by SDS-PAGE.
Glycerol (15%) was added for its storage at À208C.
153.58 (C-2''), 141.76 (C-6''), 106.99 (d, J(1,P) ˆ 4.0 Hz; C-1), 103.00 (C-5''),
89.02 (C-1'), 84.33 (d, J(3,P) ˆ 5.2 Hz; C-3), 83.78 (C-4), 83.32 (d, J(4',P) ˆ
9.0 Hz; C-4'), 78.03 (C-2), 76.87 (C-5), 74.07 (C-3'), 62.58 (C-2'), 65.18 (d,
J(5',P) ˆ 5.5 Hz; C-5'), 62.58 (C-6), 29.59 (d, J(C,P) ˆ 138.5 Hz;
CH2P) ppm; 31P NMR (101 MHz, D2O): d ˆ 8.95 (d, J ˆ 24.7 Hz), À11.43
‡
(d, J ˆ 24.8 Hz) ppm; MS (ESI): m/z (%): 561 (100) [M À H] , 280 (64)
‡
‡
‡
‡
‡
[hB(M À 2H)/2i] , 583 (21) [M À 2H ‡Na] , 599 (21) [M À 2H ‡K] ;
HRMS: found 561.05049; C16H23N2O16P2 calcd (%) 561.05228
Enzyme kinetics and inhibition assays: The conditions described by Liu
et al. were followed.[15] All assays were performed with a potassium
phosphate buffer (100 mm, pH ˆ 6.8) at Tˆ 218C. Under reductive
conditions, freshly prepared sodium dithionite solutions were used to
allow a final concentration of 20 mm. When native mutase was used
(without sodium dithionite), reactions were conducted in the dark under
aerobic conditions. All inhibition studies consisted of measurement of 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 mm, pH ˆ 6.8, detection at 262 nm).
Substrate and inhibitor concentrations were adjusted at 1 mm from titrated
mother solutions. Incubation times were always 1 h at 218C. As a control,
the relative concentrations of any species in the reaction mixtures could be
titrated with UMP as an internal standard. Enzyme concentrations were
adjusted to allow a conversion of UDP-Galp between 5 and 15%.
Reactions were stopped by freezing the solution in liquid nitrogen.
Residual enzyme activities were then measured in the presence of
inhibitors at three different times, compared with the same experiment
conducted without inhibitors, and averaged.
Uridine diphosphate C-a-d-galactofuranose (2): The procedure for the
preparation of 1 was followed. Compound 20 and activated UMP were
allowed to react for 1.5 h at 08C. The eluent for size exclusion chromatog-
raphy was 50 mm triethylammonium acetate buffer (pH ˆ 6.8) to prevent
decomposition of the desired nucleotide-sugar. This protocol afforded 2 as
1
a viscous solid (triethylammonium salt, 35% yield). H, 13C, 31P NMR and
mass spectra were in agreement with those published.[25]
1,4-Anhydro-2,3,6-tri-O-tert-butyldimethylsilyl-1-methyl-a-d-galactopyra-
nose (6): Tebbe×s reagent (0.4m in toluene, 0.55 mL) was added dropwise at
08C to a solution of lactone 4[20] (49 mg, 94 mmol) in an anhydrous mixture
of toluene (1 mL), THF (1 mL), and pyridine (0.4mL). This dark red
solution was stirred for 1 h at 08C and for 30 min at room temperature. The
reaction was stopped by dropwise addition of NaOH (1n, 2.0 mL) at 08C.
The resulting mixture was filtrated through a short pad of celite and
concentrated under vacuum. The resulting enol ether decomposed during
the flash chromatography on silica gel (eluent cyclohexane/AcOEt 9:1) to
give 6 (28 mg, 57%).
1H NMR (400 MHz, CDCl3): d ˆ 4.34 (d, J(3,4) ˆ 1.3 Hz, 1H; H-4), 3.79
(brt, J(2,3) ˆ J(3,4) ˆ 1.2 Hz, 1H; H-3), 3.70 (d, J(2,3) ˆ 1.0 Hz, 1H; H-2),
3.66 (dd, J(5,6a) ˆ 4.4 Hz, J(5,6b) ˆ 9.7 Hz, 1H; H-5), 3.56 (dd, J(5,6a) ˆ
4.4 Hz, J(6a,6b) ˆ 9.7 Hz, 1H; H-6a), 3.38 (t, J(5,6b) ˆ J(6a,6b) ˆ 9.7 Hz,
Atom and position numberings: We have systematically numbered the
phosphonate methylene group as 1' 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: 1' for
the anomeric position, 1'' for the nitrogen atom linked to the ribose.
À
À
1H; H-6b), 1.52 (s, 3H; CH3), 0.95 (s, 9H; Si tBu), 0.94(s, 9H; Si tBu),
À
À
À
0.91 (s, 9H; Si tBu), 0.13 (s, 6H; 2 Â Si Me), 0.12 (s, 6H; 2 Â Si Me), 0.08
Uridine diphosphate-C-a-d-1,4-anhydrogalactopyranose (1): 5'UMP (trie-
thylammonium salt, 59.6 mg, 0.14mmol) was suspended in a mixture of
freshly distilled MeCN (750 mL), N,N-dimethyl-aniline (70 mL, 0.56 mmol),
and Et3N (4 0mL, 0.28 mmol), cooled to 08C and stirred under argon.
Trifluoroacetic anhydride (118 mL, 0.84mmol) was slowly added dropwise
to the flask containing 5'-UMP. The reaction mixture was stirred for a few
minutes at room temperature, after which a red-brown coloration was
obtained. Excess trifluoroacetic anhydride and trifluoroacetic acid were
removed from the reaction mixture under vacuum (by use of an oil pump
and a liquid N2 trap). In a separate flask, a mixture of N-Me-imidazole
(56 mL, 0.70 mmol) in anhydrous MeCN (150 mL) and Et3N (118 mL,
0.84mmol) was prepared, cooled to 0 8C, and then added to the flask
containing the mixed phosphoryl anhydride. The reaction mixture was
stirred for 5 10 min at 08C, after which a bright yellow solution was
obtained. In the meantime, a flask containing 16 (tributylammonium salt,
73 mg, 0.117 mmol) and preactivated 4ä molecular sieves in MeCN
(800 mL) was stirred for 30 min at 08C. The solution of UMP-N-
methylimidazolide was then added dropwise to the solution containing
16. The resulting mixture was stirred at 08C under Ar for 2 h and then for
13 h at room temperature. The reaction was quenched with cold aqueous
ammonium formate (3 mL, 250 mm, 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 mm, pH ˆ 7) and the combined aqueous phases were
pooled and lyophilized. They were purified by size exclusion chromatog-
(s, 6H; 2  Si Me) ppm; 13C NMR (100 MHz, CDCl3): d ˆ 107.13 (C-1),
À
87.38 (C-3), 83.65 (C-4), 80.82 (C-2), 76.53 (C-5), 62.32 (C-6), 25.79, 25.66,
25.55, 18.02, 17.95, 16.11 (CH3), À4.23, À4.49, À4.66, À4.71, À5.45,
‡
À5.47 ppm; MS (DCI-NH3): m/z (%): 536 (100%) [M‡NH4] .
1,4-Anhydro-2,3,6-tri-O-tert-butyldimethylsilyl-1-iodomethyl-a-d-galacto-
pyranose (7): Tebbe×s reagent (0.4m in toluene, 1.4mL) was added
dropwise at 08C to a solution of lactone 4[20] (226 mg, 435 mmol) in an
anhydrous mixture of toluene (4mL), THF (2 mL), and pyridine (0.7 mL).
This dark red solution was stirred for 1 hour at 08C and for 30 min at room
temperature. The reaction was stopped by dropwise addition of NaOH (1n,
2.0 mL) at 08C. The resulting mixture was filtrated through a short pad of
celite and concentrated under vacuum. The crude material was dried twice
by azeotropic evaporation with anhydrous toluene, and then dissolved in
anhydrous CH2Cl2 (5 mL). Molecular sieves (4ä, 300 mg) were added to
this solution, followed by NIS (170 mg, 756 mmol, 1.7 equiv) at 08C. The
resulting mixture was stirred for 45 min at 08C, and then filtrated through
celite, diluted with CH2Cl2 (15 mL), and washed twice with sodium
dithionite (0.5n, 10 mL). The organic phase was dried over MgSO4,
filtered, and concentrated. The residue was purified by silica gel chroma-
tography, eluted with cyclohexane/AcOEt (2:1) to give 7 (174mg, 62%
yield).
1H NMR (400 MHz, CDCl3): d ˆ 4.42 (d, J(3,4) ˆ 1.3 Hz, 1H; H-4), 4.04
(brt, 1H; H-3), 3.87 (d, J(2,3) ˆ 0.9 Hz, 1H; H-2), 3.82 (dd, J(5,6a) ˆ
4.5 Hz, J(5,6b) ˆ 9.9 Hz, 1H; H-5), 3.61 (dd, J(5,6a) ˆ 4.5 Hz, J(6a,6b) ˆ
5894
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2003, 9, 5888 5898

UDP-Galactopyranose/Furanose
5888 5898
À
À
À
1.5 H; Si Me), 0.20 (s, 3H; Si Me), 0.19 (s, 3H; Si Me), 0.15 (s, 4.5H; 2 Â
9.8 Hz, 1H; H-6a), 3.52 (t, J(5,6b) ˆ J(6a,6b) ˆ 9.8 Hz, 1H; H-6b), 3.47
À
À
À
À
Si Me), 0.14(s, 3H; Si Me), 0.12 (s, 1.5H; Si Me), 0.08 (s, 4,5H; Si Me),
À
À
(AB, J(AB) ˆ 11.5 Hz, 2H; CH2 I), 0.94(s, 9H; Si tBu), 0.93 (s, 9H;
0.07 (s, 3H; Si Me) ppm; 13C NMR (100 MHz, CDCl3): d ˆ 104.80 (d,
À
À
À
À
À
Si tBu), 0.91 (s, 9H; Si tBu), 0.14(s, 3H; Si Me), 0.13 (s, 6H; 2 Â Si Me),
J(1,P) ˆ 5.9 Hz; C-1b), 102.48 (d, J(1,P) ˆ 2.8 Hz; C-1a), 83.59 (C-4b), 83.55
(C-4a), 82.36 (d, J(2,P) ˆ 7.8 Hz; C-2b), 80.43 (d, J(2,P) ˆ 5.3 Hz; C-2a),
78.68 (C-3b), 78.53 (C-3a), 70.99 (C-5a), 70.64(C-5 b), 63.75 (C-6b), 63.49 (C-
6a), 52.97 (d, J(C,P) ˆ 6.2 Hz, OCH3b), 52.83 (d, J(C,P) ˆ 6.3 Hz, OCH3a),
52.24(d, J(C,P) ˆ 6.2 Hz, OCH3a), 51.81 (d, J(C,P) ˆ 6.3 Hz, OCH3b), 33.27
(d, J(1',P) ˆ 136.8 Hz; C-1'a), 31.39 (d, J(1',P) ˆ 139.3 Hz; C-1'b), 25.87,
25.83, 25.77, 25.71, 25.66, 18.24, 18.20, 17.92, 17.91, 17.75, À4.30, À4.38,
À4.43, À4.48, À4.57, À4.68, À4.70, À5.39, À5.44, À5.49 ppm; 31P NMR
(101 MHz, CDCl3): d ˆ 30.59 (major diast.), 28.19 (minor diast.) ppm; MS
À
À
À
0.12 (s, 3H; Si Me), 0.09 (s, 3H; Si Me), 0.08 (s, 3H; Si Me) ppm;
13C NMR (100 MHz, CDCl3): D ˆ 105.44 (C-1), 84.89 (C-3), 83.66 (C-4),
81.80 (C-2), 77.20 (C-5), 61.72 (C-6), 25.76, 25.74, 18.05, 17.93, 17.89, 0.52
À
(CH2 I), À4.19, À4.46, À4.71, À4.79, À5.43, À5.44 ppm; MS (DCI-NH3):
‡
m/z (%): 662 (100) [M‡NH4] .
1-(Dibenzyloxyphosphoryl)methyl-2,3,6-tri-O-tert-butyldimethylsilyl-a,b-
d-galactofuranose (8): Butyllithium (3.75 mL, 9.37 mmol, of
a 2.5m
solution in hexane) was added under Ar to a cooled (À708C) solution of
dibenzyl methylphosphonate (2.59 g, 9.37 mmol) in anhydrous THF
(18 mL), followed after 20 min by a solution of 2,3,6-tri-O-tert-butyldime-
thylsilyl-d-galactono-1,4-lactone 4[20] (1.95 g, 3.75 mmol) in anhydrous THF
(4mL). The temperature was maintained at À708C for 10 min, and the
reaction mixture was then allowed to come to À408C over a 1 h period. The
solution was then diluted with phosphate buffer (1m, 70 mL, pH ˆ 7) and
extracted with CH2Cl2 (2 Â 180 mL). The combined organic phases were
dried over MgSO4, filtered, and concentrated. The residue was purified by
silica gel chromatography eluted with cyclohexane/AcOEt (4:1) to give 8
(2.42 g, 81% yield) as a white solid. a/b ratio: 20:80.
‡
‡
(DCI-NH3): m/z (%): 645 (17) [M‡H] , 662 (100) [M‡NH4] ; elemental
analysis calcd (%) for C27H61O9PSi3: C 50.28, H 9.53; found: C 50.27, H 9.61.
(1(1')Z)-1-(Dibenzyloxyphosphoryl)methylidene-2,3,6-tri-O-tert-butyldi-
methylsilyl-d-galactofuranose (10): Pyridine (510 mL, 6.32 mmol) and
trifluoroacetic anhydride (440 mL, 3.16 mmol) were added at 08C to a
solution of 8 (503 mg, 0.63 mmol) in anhydrous THF (20 mL). After 3 h at
08C, the reaction was stopped by addition of saturated aqueous NaHCO3,
and the mixture was extracted with AcOEt (100 mL). The organic phase
was dried over MgSO4, filtered, and concentrated at reduced pressure.
Purification by silica gel chromatography, eluted with cyclohexane/AcOEt
(2:1), afforded 10 in (378 mg, 77% yield) as a colorless syrup.
1H NMR (400 MHz, CDCl3): d ˆ 7.40 7.33 (m, 12.5H; H arom.), 5.14 5.05
(m, 4H; CH2Phb), 5.00 4.93 (m, 1H; CH2Pha), 4.84 (brs, 1H; OH-1b), 4.57
(brs, 0.25H; OH-1a), 4.42 (d, J(2,3) ˆ 2.4Hz, 0.25H; H-2 a), 4.25 (dd,
0.25H; H-3a), 4.24 (d, J(3,4) ˆ 4.0 Hz, J(4,5) ˆ 1.8 Hz, 1H; H-4b), 4.15 (t,
J(3,4) ˆ 4.0 Hz, J(2,3) ˆ 3.8 Hz, 1H; H-3b), 4.10 (d, J(2,3) ˆ 3.8 Hz, 1H;
H-2b), 4.07 (t, J(3,4) ˆ J(4,5) ˆ 2.6 Hz, 0.25H; H-4a), 3.71 3.53 (m, 3.75H;
H-5a,b, H-6aa,b, H-6ba,b), 3.28 (d, J(5,OH) ˆ 7.3 Hz, 1H; OH-5b), 3.08 (d,
J(5,OH) ˆ 5.5 Hz, 0.25H; OH-5a), 2.70 (ABX, J(1'a,1'b) ˆ 15.3 Hz,
J(1'a,P) ˆ 18.4Hz, 0.25H; H-1 'aa), 2.55 (ABX, J(1'a,1'b) ˆ 15.3 Hz,
J(1'b,P) ˆ 19.0 Hz, 0.25H; H-1'ba), 2.52 (ABX, J(1'a,1'b) ˆ 15.4Hz,
J(1'a,P) ˆ 19.2 Hz, 1H; H-1'ab), 2.29 (ABX, J(1'a,1'b) ˆ 15.4Hz,
[a]2D3 ‡55.4(c ˆ 1.2, CHCl3); 1H NMR (400 MHz, CDCl3): d ˆ 7.38 7.30
(m, 10H; H arom.), 5.09 5.01 (m, 4H; CH2Ph), 4.75 (dd, J(1',2) ˆ 1.5 Hz,
J(1',P) ˆ 12.4Hz, 1H; H-1 '), 4.53 (ddd, J(1',2) ˆ 1.5 Hz, J(2,P) ˆ 4.1 Hz,
J(2,3) ˆ 6.6 Hz, 1H; H-2), 4.44 (d, J(3,4) ˆ 6.3 Hz, 1H; H-4), 4.35 (dd,
J(2,3) ˆ 6.6 Hz, J(3,4) ˆ 6.3 Hz, 1H; H-3), 3.73 3.64(m, 3H; H-5, H-6a,
À
À
À
H-6b), 0.95 (s, 9H; Si tBu), 0.93 (s, 9H; Si tBu), 0.91 (s, 9H; Si tBu), 0.17
À
À
À
(s, 3H; Si Me), 0.14(2 Â s, 6 H; 2 Â Si Me), 0.12 (s, 3H; Si Me), 0.06 (s,
6H; 2  Si Me) ppm; 13C NMR (100 MHz, CDCl3): d ˆ 174.29 (d, J(1,P) ˆ
À
2.0 Hz; C-1), 136.50 (d, J(C,P) ˆ 7.2 Hz ; Cq arom.), 136.40 (d, J(C,P) ˆ
7.2 Hz ; Cq arom.), 128.45 127.75 (10 Â CH arom.), 84.35 (C-4), 83.81 (d,
J(1',P) ˆ 193.5 Hz; C-1'), 78.38 (d, J(2,P) ˆ 12.5 Hz; C-2), 75.67 (C-3), 70.36
(C-5), 67.20 (d, J(C,P) ˆ 5.2 Hz; CH2Ph), 66.90 (d, J(C,P) ˆ 5.2 Hz;
CH2Ph), 62.82 (C-6), 25.81, 25.78, 25.70, 18.12, 17.83, 17.81, À4.01, À4.1,
À4.28, À4.56, À5.44, À5.53 ppm; 31P NMR (101 MHz, CDCl3): d ˆ
À
J(1'b,P) ˆ 17.9 Hz, 1H; H-1'bb), 0.97 (s, 2.25H; Si tBu), 0.93 (s, 9H;
À
À
À
Si tBu), 0.92 (2 Â s, 11.25H; 2 Â Si tBu), 0.90 (s, 9H; Si tBu), 0.89 (s,
À
À
À
2.25H; Si tBu), 0.23 (2 Â s, 1.5 H; 2 Â Si Me), 0.17 (s, 3H; Si Me), 0.16 (s,
À
À
À
3H; Si Me), 0.15 (2 Â s, 7.5 H; 4Si Me), 0.12 (s, 0.75H; Si Me), 0.06 (2 Â s,
6H; 2 Â Si Me), 0.04(s, 0.75H; Si Me) ppm; 13C NMR (100 MHz,
CDCl3): d ˆ 136.61 (d, J(C,P) ˆ 6.5 Hz; Cq arom.a), 136.26 (d, J(C,P) ˆ
6.4Hz; C q arom.b), 136.20 (d, J(C,P) ˆ 6.5 Hz; Cq arom.a), 136.01 (d,
J(C,P) ˆ 6.5 Hz; Cq arom.b), 128.81 127.60 (CH arom.), 104.60 (d, J(1,P) ˆ
6.0 Hz; C-1b), 102.72 (d, J(1,P) ˆ 6.0 Hz; C-1a), 83.65 (C-4a), 82.79 (C-4b),
82.45 (d, J(2,P) ˆ 7.7 Hz; C-2b), 80.15 (d, J(2,P) ˆ 4.2 Hz; C-2a), 78.50 (C-
3a), 78.27 (C-3b), 71.09 (C-5a), 70.45 (C-5b), 67.84(d, J(C,P) ˆ 5.9 Hz;
CH2Pha), 67.64(d, J(C,P) ˆ 6.0 Hz; CH2Phb), 67.26 (d, J(C,P) ˆ 6.3 Hz;
CH2Phb), 66.50 (d, J(C,P) ˆ 6.3 Hz; CH2Pha), 63.58 (C-6b, C-6a), 34.20 (d,
J(1',P) ˆ 137 Hz; C-1'a), 32.90 (d, J(1',P) ˆ 138.5 Hz; C-1'b), 26.26, 26.07,
25.87, 25.80, 25.78, 25.72, 18.21, 17.89, 17.77, À4.30, À4.42, À4.44, À4.47,
À4.57, À4.64, À5.38, À5.41, À5.44 ppm; 31P NMR (101 MHz, CDCl3): d ˆ
29.36 (major diast.), 26.58 (minor diast.) ppm; MS (DCI-NH3): m/z (%):
‡
À
À
20.69 ppm; MS (DCI-NH3): m/z: 779 [M‡H] ; elemental analysis calcd
(%) for C39H67O8PSi3: C 60.12, H 8.67; found: C 60.03, H 8.76.
(1(1')Z)-2,3,6-Tri-O-tert-butyldimethylsilyl-1-(dimethoxyphosphoryl)me-
thylidene-d-galactofuranose (11): Triethylamine (320 mL, 2.28 mmol) and
trifluoroacetic anhydride (475 mL, 3.42 mmol) were added at 08C to a
solution of 9 (489 mg, 0.76 mmol) in anhydrous pyridine (10 mL). The
resulting solution was allowed to reach room temperature for a period of
2 h. Solvents were evaporated, and the crude mixture was diluted with
CH2Cl2 (20 mL) and washed with saturated aqueous NaHCO3. The organic
phase was dried over MgSO4, filtered, and concentrated at reduced
pressure. The purification by silica gel chromatography, eluted with
cyclohexane/AcOEt (1:1), afforded 11 (300 mg, 63%) as a white solid.
M.p. 95 968C; [a]2D4 ‡50.3 (c ˆ 1.0, CHCl3); 1H NMR (400 MHz, CDCl3):
d ˆ 4.68 (dd, J(1',2) ˆ 1.5 Hz, J(1',P) ˆ 12.0 Hz, 1H; H-1'), 4.55 (ddd,
J(1',2) ˆ 1.5 Hz, J(2,P) ˆ 4.0 Hz, J(2,3) ˆ 6.3 Hz, 1H; H-2), 4.45 (d,
J(3,4) ˆ 5.9 Hz, 1H; H-4), 4.35 (t, J(2,3) ˆ J(3,4) ˆ 6.3 Hz, 1H; H-3), 3.76
(d, J(H,P) ˆ 11.6 Hz, 3H; OCH3), 3.74(d, J(H,P) ˆ 11.3 Hz, 3H; OCH3),
‡
‡
‡
797 (25) [M‡H] , 814(37)
[
M‡NH4] , 779 (100) [M‡H À H2O] ;
elemental analysis calcd (%) for C39H69O9PSi3: C 58.76, H 8.72; found: C
58.61, H 8.84.
2,3,6-Tri-O-tert-butyldimethylsilyl-1-(dimethoxyphosphoryl)methyl-a,b-d-
galactofuranose (9): The procedure used for the preparation of 8 was
followed. The residue was purified by silica gel chromatography eluted with
cyclohexane/AcOEt (2:1) to give 9 (65% yield) as a colorless syrup. a/b
ratio: 35:65.
À
3.81 3.69 (m, 3H; H-5, H-6a, H-6b), 0.98 (s, 9H; Si tBu), 0.93 (s, 9H;
À
À
À
À
Si tBu), 0.92 (s, 9H; Si tBu), 0.18 (s, 3H; Si Me), 0.17 (s, 3H; Si Me), 0.14
(s, 3H; Si Me), 0.09 (s, 9H; 3Si Me) ppm; 13C NMR (100 MHz, CDCl3):
d ˆ 174.31 (d, J(1,P) ˆ 2.0 Hz; C-1), 84.71 (C-4), 82.82 (d, J(1',P) ˆ
193.3 Hz; C-1'), 78.46 (d, J(2,P) ˆ 12.5 Hz; C-2), 75.80 (C-3), 70.43 (C-5),
62.91 (C-6), 52.44 (d, J(C,P) ˆ 5.6 Hz, OCH3), 51.99 (d, J(C,P) ˆ 5.5 Hz,
OCH3), 25.82, 25.77, 25.69, 18.16, 17.86, 17.82, À4.05, À4.19, À4.29, À4.58,
À5.44, À5.57 ppm; 31P NMR (101 MHz, CDCl3): d ˆ 22.60 ppm; MS (DCI-
À
À
1H NMR (400 MHz, CDCl3): d ˆ 4.32 (d, J(2,3) ˆ 2.5 Hz, 0.5H; H-2a), 4.25
(dd, J(3,4) ˆ 3.8 Hz, J(4,5) ˆ 1.9 Hz, 1H; H-4b), 4.23 (t, J(2,3) ˆ J(3,4) ˆ
2.5 Hz, 0.5H; H-3a), 4.16 (tdd, J(2,3) ˆ 2.7 Hz, J(3,4) ˆ 3.8 Hz, J(3,P) ˆ
0.8 Hz, 1H; H-3b), 4.07 (d, J(2,3) ˆ 2.7 Hz, 1H; H-2b), 4.05 (m, 0.5H;
H-4a), 3.82 (d, J(H,P) ˆ 11.2 Hz, 3H; OCH3b), 3.81 (d, J(H,P) ˆ 11.1 Hz,
1.5H; OCH3a), 3.79 (d, J(H,P) ˆ 10.9 Hz, 3H; OCH3b), 3.74(d, J(H,P) ˆ
10.9 Hz, 1.5H; OCH3a), 3.74 3.58 (m, 4.5H; H-5 a,b, H-6aa/b, H-6ba/b), 2.56
(ABX, J(1'a,1'b) ˆ 15.3 Hz, J(1'a,P) ˆ 18.5 Hz, 0.5H; H-1'aa), 2.46 (ABX,
J(1'a,1'b) ˆ 15.3 Hz, J(1'b,P) ˆ 19.1 Hz, 0.5H; H-1'ba), 2.45 (ABX,
J(1'a,1'b) ˆ 15.4Hz, J(1'a,P) ˆ 19.1 Hz, 1H; H-1'ab), 2.25 (ABX,
‡
‡
NH3): m/z (%): 627 (100) [M‡H] , 644 (70) [M‡NH4] ; elemental analysis
calcd (%) for C27H59O8PSi3: C 51.72, H 9.48; found: C 51.57, H 9.64.
2,3,6-Tri-O-tert-butyldimethylsilyl-5-O-pivaloyl-d-galactono-1,4-lactone
(12): 2,3,6-Tri-O-tert-butyldimethylsilyl-d-galactono-1,4-lactone 4[20] (1 g,
1.92 mmol) was dissolved in anhydrous pyridine (15 mL) under Ar
atmosphere. Excess pivaloyl chloride (0.9 mL, 7.29 mmol) and a catalytic
amount of 4-dimethylaminopyridine (DMAP) (94 mg, 0.77 mmol) in
anhydrous pyridine (1.5 mL) were slowly added to this solution, and the
resulting mixture was stirred for an additional 12 h at room temperature.
À
J(1'a,1'b) ˆ 15.4Hz, J(1'b,P) ˆ 18.1 Hz, 1H; H-1'bb), 0.96 (s, 4.5H; Si t-
À
À
À
Bu), 0.95 (s, 9H; Si tBu), 0.93 (s, 4.5H; Si tBu), 0.92 (s, 9H; Si tBu), 0.91
À
À
À
(s, 9H; Si tBu), 0.90 (s, 4.5H; Si tBu), 0.23 (s, 3H; 2 Â Si Me), 0.22 (s,
Chem. Eur. J. 2003, 9, 5888 5898 www.chemeurj.org
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
5895

S. P. Vincent, P. Sinay» et al.
FULL PAPER
The solution was washed with saturated aqueous NaHCO3 and brine, and
dried over MgSO4. After solvent evaporation, the residue was purified by
silica gel chromatography eluted with cyclohexane/AcOEt (96:4) to give 12
(1.12 g, 96.5% yield) as a white solid.
(d, J(1,P) ˆ 2.5 Hz; C-1), 85.80 (C-4), 81.67 (d, J(1',P) ˆ 194.3 Hz; C-1'),
79.97 (d, J(2,P) ˆ 13.3 Hz; C-2), 76.31 (C-3), 71.74(C-5), 60.47 (C-6), 52.13
(d, J(C,P) ˆ 5.1 Hz, OCH3), 51.99 (d, J(C,P) ˆ 5.3 Hz, OCH3), 29.69, 27.10,
25.78, 25.73, 25.65, 18.15, 17.80, À4.21, À4.27, À4.31, À4.41, À5.47,
À5.59 ppm; 31P NMR (101 MHz, CDCl3): d ˆ 21.16 ppm; MS (DCI-
m.p. 35 368C; [a]2D4 ‡2.5 (c ˆ 1.01, CHCl3); 1H NMR (400 MHz, CDCl3):
d ˆ 5.01 (ddd, J(4,5) ˆ 1.1 Hz, J(5,6a) ˆ 7.1 Hz, J(5,6b) ˆ 7.3 Hz, 1H; H-5),
4.48 (dd, J(4,5) ˆ 1.1 Hz, J(3,4) ˆ 7.0 Hz, 1H; H-4), 4.43 (d, J(2,3) ˆ 7.0 Hz,
1H; H-2), 4.13 (t, J(2,3) ˆ J(3,4) ˆ 7.0 Hz, 1H; H-3), 3.74 3.72 (m, 2H;
‡
NH3): m/z (%): 711 (100) [M‡H] ; elemental analysis calcd (%) for
C32H67O9PSi3: C 54.05, H 9.50; found: C 53.81, H 9.80.
1,4-Anhydro-1-(dibenzyloxyphosphoryl)methyl-2,3,6-tri-O-tert-butyldime-
thylsilyl-a-d-galactopyranose (15): Camphorsulfonic acid (272 mg,
1.17 mmol) was added to a solution of 10 (537 mg, 0.69 mmol) and
molecular sieves (4ä, 900 mg) in dry dichloromethane (21 mL). The
reaction mixture was heated at reflux under Ar for 17 hours, and then
filtered through a pad of celite. The filtrate was washed with saturated
aqueous NaHCO3, dried over MgSO4, filtered, and concentrated. The
residue was chromatographed on silica gel with cyclohexane/AcOEt (4:1)
as eluent. The product 15 was obtained as a colorless oil (596 mg, 74%
yield), along with 15' (65 mg, 14% yield), the product of monodesilylation
at the 6-position. Compounds 15 and 15' could be pooled for the removal of
the TBDMS groups.
À
À
H-6a, H-6b), 1.23 (s, 9H; Piv), 0.95 (s, 9H; Si tBu), 0.93 (s, 9H; Si tBu),
À
À
À
0.91 (s, 9H; Si tBu), 0.24(s, 3H; Si Me), 0.18 (s, 3H; Si Me), 0.13 (s, 3H;
À
À
À
Si Me), 0.12 (s, 3H; Si Me), 0.10 (s, 3H; Si Me), 0.09 (s, 3H;
Si Me) ppm; 13C NMR (100 MHz, CDCl3): d ˆ 177.49 (C O , Piv),
172.88 (C-1), 78.78 (C-4), 76.53 (C-2), 75.42 (C-3), 70.03 (C-5), 59.59 (C-
6), 39.0, 27.09, 25.71, 25.64, 18.13, 18.06, 17.76, À4.19, À4.29, À4.77, À5.49,
À
ˆ
‡
À5.57 ppm; MS (DCI-NH3): m/z (%): 622 (100) [M‡NH4] ; elemental
analysis calcd (%) for C29H60O7Si3: C 57.57, H 10.00; found: C 57.70, H
10.00.
2,3,6-Tri-O-tert-butyldimethylsilyl-1-(dimethoxyphosphoryl)methyl-5-O-
pivaloyl-a,b-d-galactofuranose (13): The procedure used for the prepara-
tion of 8 was applied. The residue was purified by silica gel chromatography
eluted with cyclohexane/AcOEt (2:1) to give 13 (511 mg, 65% yield) as a
colorless oil. a/b ratio: 45:55.
[a]2D0 ‡26.6 (c ˆ 1.2, CHCl3); 1H NMR (400 MHz, CDCl3): d ˆ 7.38 7.30
(m, 10H; H arom.), 5.16 5.03 (2ABX, 4H; CH2Ph), 4.46 (d, J(3,4) ˆ
1.5 Hz, 1H; H-4), 4.12 (t, J(2,3) ˆ J(3,4) ˆ 1.3 Hz, 1H; H-3), 3.69 (brs,
1H; H-2), 3.66 (dd, J(5,6a) ˆ 4.5 Hz, J(5,6b) ˆ 9.9 Hz, 1H; H-5), 3.53
(ABX, J(5,6a) ˆ 4.5 Hz, J(6a,6b) ˆ 9.8 Hz, 1H; H-6a), 3.45 (ABX,
J(5,6b) ˆ J(6a,6b) ˆ 9.8 Hz, 1H; H-6b), 2.50 (ABX, J(1'a,1'b) ˆ 15.7 Hz,
J(1'a,P) ˆ 19.5 Hz, 1H; H-1'a), 2.43 (ABX, J(1'a,1'b) ˆ 15.7 Hz,
1H NMR (400 MHz, CDCl3): d ˆ 5.31 (dt, J(4,5) ˆ 9.1 Hz, J(5,6a,b) ˆ
3.9 Hz, 0.8H; H-5a), 5.08 (q, J(4,5) ˆ 4.7 Hz, J(5,6a,b) ˆ 5.0 Hz, 1H;
H-5b), 4.39 (s, 0.8H; H-2a), 4.31 (t, J(3,4) ˆ J(4,5) ˆ 4.7 Hz, 1H; H-4b), 4.08
(d, J(3,4) ˆ 0.9 Hz, 0.8H; H-3a), 4.07 (d, J(2,3) ˆ 2.9 Hz, 1H; H-2b), 4.02 (d,
J(4,5) ˆ 9.1 Hz, 0.8H; H-4a), 3.96 (ddd, J(2,3) ˆ 2.9 Hz, J(3,4) ˆ 4.6 Hz,
J(3,P) ˆ 0.7 Hz, 1H; H-3b), 3.83 (d, J(H,P) ˆ 11.1 Hz, 3H; OCH3b), 3.79 (d,
J(H,P) ˆ 11.3 Hz, 3H; OCH3b), 3.76 (d, J(H,P) ˆ 11.2 Hz, 2.4H; OCH3a),
À
À
J(1'b,P) ˆ 19.4Hz, 1H; H-1 'b), 0.93 (s, 9H; Si tBu), 0.92 (s, 9H; Si tBu),
À
À
À
0.90 (s, 9H; Si tBu), 0.13 (s, 6H; 2 Â Si Me), 0.11 (s, 3H; Si Me), 0.05 (s,
9H; 3Si Me) ppm; 13C NMR (100 MHz, CDCl3): d ˆ 136.54(d, J(C,P) ˆ
À
6.5 Hz; Cq arom.), 136.38 (d, J(C,P) ˆ 6.5 Hz; Cq arom.), 128.46 127.88
(10  CH arom.), 105.32 (d, J(1,P) ˆ 4.3 Hz; C-1), 86.72 (d, J(3,P) ˆ 6.4Hz;
C-3), 84.09 (C-4), 79.99 (C-2), 76.49 (C-5), 67.58 (d, J(C,P) ˆ 5.9 Hz;
CH2Ph), 67.05 (d, J(C,P) ˆ 6.1 Hz; CH2Ph), 61.94(C-6), 27.57 (d, J(1',P) ˆ
141.7 Hz; C-1'), 25.76, 25.75, 25.72, 18.03, 17.90, 17.86, À4.21, À4.52, À4.66,
À4.77, À5.46 ppm; 31P NMR (101 MHz, CDCl3): d ˆ 24.66 ppm; MS (DCI-
3.72 (d, J(H,P) ˆ 10.9 Hz, 2.4H; OCH3a), 3.81 3.72 (m, 5.4H; H-5a/b
,
H-6aa/b, H-6ba/b), 2.51 (AX, J(1',P) ˆ 18.3 Hz, 1.6H; H-1'aa, H-1'ba), 2.30
(ABX, J(1'a,1'b) ˆ 15.4Hz, J(1'a,P) ˆ 18.1 Hz, 1H; H-1'ab), 2.24(ABX,
J(1'a,1'b) ˆ 15.4Hz, J(1'b,P) ˆ 18.1 Hz, 1H; H-1'bb), 1.25 (2  s, 16.2H;
À
À
À
Piva/b), 0.98 (s, 7.2H; Si tBu), 0.95 (s, 7.2H; Si tBu), 0.93 (s, 7.2H; Si tBu),
À
À
À
0.92 (s, 9H; Si tBu), 0.91 (2 Â s, 18H; 2 Â Si tBu), 0.19 (s, 2.4H; Si Me),
‡
NH3): m/z (%): 779 (100) [M‡H] ; elemental analysis calcd (%) for
À
À
À
0.17 (s, 2.4H; Si Me), 0.16 (s, 3H; Si Me), 0.14(2 Â s, 8.4H; 3Si Me), 0.13
C39H67O8PSi3: C 60.12, H 8.67; found: C 60.01, H 8.76.
À
À
(2 Â s, 5.4H; 2 Â Si Me), 0.09 (2 Â s, 5.4H; 2 Â Si Me), 0.08 (s, 5.4H; 2 Â
13
Si Me); C NMR (100 MHz, CDCl3): d ˆ 178.47 (C O , Pivb), 177.64
Analytical data for molecule 15': [a]2D0 ‡84.6 (c ˆ 1.9, CHCl3); 1H NMR
(400 MHz, CDCl3): d ˆ 7.39 7.35 (m, 10H; H arom.), 5.19 5.02 (2  ABX,
4H; CH2Ph), 4.57 (d, J(3,4) ˆ 1.4Hz, 1H; H-4), 3.94 (ABX, J(5,6a) ˆ
2.0 Hz, J(6a,6b) ˆ 12.9 Hz, 1H; H-6a), 3.79 (brs, 1H; H-3), 3.78 (brt,
J(5,6a,b) ˆ 2.0 Hz, 1H; H-5), 3.68 (brs, 1H; H-2), 3.64(ABX, J(5,6b) ˆ
2.0 Hz, J(6a,6b) ˆ 12.9 Hz, 1H; H-6b), 2.61 (ABX, J(1'a,1'b) ˆ 15.7 Hz,
J(1'a,P) ˆ 19.4Hz, 1H; H-1 'a), 2.45 (ABX, J(1'a,1'b) ˆ 15.7 Hz,
À
ˆ
ˆ
(C O, Piva), 104.82 (d, J(1,P) ˆ 6.2 Hz; C-1b), 104.12 (C-1a), 84.24 (d,
J(2,P) ˆ 8.7 Hz; C-2b), 83.04(C-4 a), 81.69 (C-4b), 79.32 (d, J(2,P) ˆ 2.9 Hz;
C-2a), 78.71 (C-3a), 78.67 (C-3b), 72.69 (C-5a), 71.99 (C-5b), 62.40 (C-6a),
61.66 (C-6b), 53.11 (d, J(C,P) ˆ 5.9 Hz, OCH3b), 52.89 (d, J(C,P) ˆ 6.3 Hz,
OCH3b), 51.71 (d, J(C,P) ˆ 6.0 Hz, OCH3a), 51.65 (d, J(C,P) ˆ 6.0 Hz,
OCH3a), 38.91, 35.21 (d, J(1',P) ˆ 136.4Hz; C-1 'a), 31.80 (d, J(1',P) ˆ
137.2 Hz; C-1'b), 27.29, 27.25, 25.85, 25.83, 25.76, 25.74, 25.63, 18.26, 18.20,
17.98, 17.96, 17.83, 17.81, À4.26, À4.37, À4.40, À4.49, À4.72, À4.74, À4.78,
À4.88, À5.39, À5.41, À5.47 ppm; 31P NMR (101 MHz, CDCl3): d ˆ 31.07
(major diast.), 28.18 (minor diast.) ppm; MS (DCI-NH3): m/z (%): 746
À
À
J(1'b,P) ˆ 18.1 Hz, 1H; H-1'b), 0.95 (s, 9H; Si tBu), 0.93 (s, 9H; Si tBu),
À
À
À
0.15 (s, 3H; Si Me), 0.14(s, 3H; Si Me), 0.12 (s, 3H; Si Me), 0.09 (s, 3H;
Si Me) ppm; 13C NMR (100 MHz, CDCl3): d ˆ 136.19 (d, J(C,P) ˆ 6.7 Hz;
À
Cq arom.), 136.17 (d, J(C,P) ˆ 6.6 Hz; Cq arom.), 128.49 128.04 (10  CH
arom.), 104.97 (d, J(1,P) ˆ 9.7 Hz; C-1), 87.70 (d, J(3,P) ˆ 12.6 Hz; C-3),
84.33 (C-4), 80.41 (d, J(2,P) ˆ 3.2 Hz; C-2), 77.37 (C-5), 67.63 (d, J(C,P) ˆ
6.1 Hz; CH2Ph), 67.39 (d, J(C,P) ˆ 6.0 Hz; CH2Ph), 63.73 (C-6), 27.78 (d,
J(1',P) ˆ 142.3 Hz; C-1'), 25.66, 17.86, 17.83, À4.17, À4.48, À4.73,
À4.76 ppm; 31P NMR (101 MHz, CDCl3): d ˆ 27.26 ppm; MS (DCI-NH3):
‡
‡
(100) [M‡NH4] , 728 (45) [M‡NH4 À H2O] ; elemental analysis calcd (%)
for C32H69O10PSi3: C 52.71, H 9.54; found: C 52.53, H 9.71.
(1(1')Z)-2,3,6-Tri-O-tert-butyldimethylsilyl-1-(dimethoxyphosphoryl)me-
thylidene-5-O-pivaloyl-d-galactofuranose (14): Trifluoroacetic anhydride
(266 mL, 1.91 mmol) was added to a solution of 13 (278 mg, 0.38 mmol) in
anhydrous pyridine (4mL). The resulting solution was stirred at 0 8C
overnight. Solvents were evaporated and the crude mixture was diluted
with CH2Cl2 (10 mL) and washed with saturated aqueous NaHCO3. The
organic phase was dried over MgSO4, filtered, and concentrated at reduced
pressure. Purification by silica gel chromatography, eluted with cyclo-
hexane/AcOEt (2:1), afforded 14 (108 mg, 40%) as a colorless oil.
‡
m/z (%): 665 (100) [M‡H] ; elemental analysis calcd (%) for C33H53O8P-
si2: C 59.61, H 8.03; found: C 59.41, H 8.23.
(1,4-Anhydro-a-d-galactopyranosyl)methyl phosphonic acid (16): A sol-
ution of 15 (186 mg, 0.24mmol) in THF (10 mL) was cooled to À208C.
Tetrabutylammonium fluoride (226 mg, 0.72 mmol) was added, and the
solution was stirred for 16 h at À58C. The solvent was then removed from
the reaction mixture under reduced pressure. The residue was first purified
by flash chromatography on silica gel with AcOEt/EtOH (9:1) as eluent
and secondly repurified by flash chromatography with acetone/CH2Cl2
(8:2) to afford 16a (86 mg, 83%) as a colorless oil.
[a]2D4 ‡34.8 (c ˆ 1.0, CHCl3); 1H NMR (400 MHz, CDCl3): d ˆ 5.10 (ddd,
J(4,5) ˆ 3.5 Hz, J(5,6a) ˆ 7.3 Hz, J(5,6b) ˆ 4.9 Hz, 1H; H-5), 4.59 (dd,
J(3,4) ˆ 4.8 Hz, J(4,5) ˆ 3.5 Hz, 1H; H-4), 4.58 (d, J(1',P) ˆ 10.2 Hz, 1H;
H-1'), 4.55 (dt, J(1',2) ˆ J(2,P) ˆ 1.2 Hz, J(2,3) ˆ 4.8 Hz, 1H; H-2), 4.07 (t,
J(2,3) ˆ J(3,4) ˆ 4.8 Hz, 1H; H-3), 3.82 (ABX, J(5,6a) ˆ 7.3 Hz, J(6a,6b) ˆ
9.9 Hz, 1H; H-6a), 3.76 (ABX, J(5,6b) ˆ 4.9 Hz, J(6a,6b) ˆ 9.9 Hz, 1H;
H-6b), 3.74(d, J(H,P) ˆ 11.4Hz, 3H; OCH 3), 3.73 (d, J(H,P) ˆ 11.4Hz,
1
[a]2D4 ‡33.7 (c ˆ 1.0, CHCl3); H NMR (400 MHz, CD3OD): d ˆ 7.56 7.52
(m, 10H; H arom.), 5.29 5.18 (2  ABX, 4H; CH2Ph), 4.64 (d, J(3,4) ˆ
1.5 Hz, 1H; H-4), 4.00 (t, J(2,3) ˆ J(3,4) ˆ 1.3 Hz, 1H; H-3), 3.97 (dd,
J(5,6a) ˆ 6.0 Hz, J(5,6b) ˆ 4.9 Hz, 1H; H-5), 3.88 (d, 1H; H-2), 3.72 (ABX,
J(5,6a) ˆ 6.0 Hz, J(6a,6b) ˆ 11.6 Hz, 1H; H-6a), 3.67 (ABX, J(5,6b) ˆ
4.9 Hz, J(6a,6b) ˆ 11.6 Hz, 1H; H-6b), 2.84(ABX, J(1'a,1'b) ˆ 15.9 Hz,
J(1'a,P) ˆ 19.2 Hz, 1H; H-1'a), 2.80 (ABX, J(1'a,1'b) ˆ 15.9 Hz,
À
À
3H; OCH3), 1.23 (s, 9H; Piv), 0.95 (s, 9H; Si tBu), 0.93 (s, 9H; Si tBu),
À
À
À
0.91 (s, 9H; Si tBu), 0.24(s, 3H; Si Me), 0.18 (s, 3H; Si Me), 0.13 (s, 3H;
À
À
À
Si Me), 0.12 (s, 3H; Si Me), 0.10 (s, 3H; Si Me), 0.09 (s, 3H;
Si Me) ppm; C NMR (100 MHz, CDCl3): d ˆ 177.67 (C O, Piv), 172.50
13
À
ˆ
5896
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2003, 9, 5888 5898

UDP-Galactopyranose/Furanose
5888 5898
J(1'b,P) ˆ 19.0 Hz, 1H; H-1'b) ppm; 13C NMR (100 MHz, CD3OD): d ˆ
137.90 (d, J(C,P) ˆ 6.0 Hz; Cq arom.), 137.87 (d, J(C,P) ˆ 6.3 Hz; Cq arom.),
129.91 129.45 (10 CH arom.), 107.08 (d, J(1,P) ˆ 5.9 Hz; C-1), 86.98 (d,
J(3,P) ˆ 8.1 Hz; C-3), 85.84(C-4), 79.86 (d, J(2,P) ˆ 1.6 Hz; C-2), 78.86 (C-
5), 69.36 (d, J(C,P) ˆ 6.5 Hz; CH2Ph), 69.28 (d, J(C,P) ˆ 6.5 Hz; CH2Ph),
63.79 (C-6), 28.36 (d, J(1',P) ˆ 141.8 Hz; C-1') ppm; 31P NMR (101 MHz,
m.p 60 618C; [a]1D9 ‡9.4(c ˆ 1.1, CHCl3); 1H NMR (400 MHz, CDCl3):
d ˆ 4.51 (ddd, J(1',2) ˆ 1.3 Hz, J(2,3) ˆ 3.8 Hz, J(2,P) ˆ 4.6 Hz, 1H; H-2),
4.48 (dd, J(1',2) ˆ 1.3 Hz, J(1',P) ˆ 10.3 Hz, 1H; H-1'), 4.39 (t, J(3,4) ˆ
J(4,5) ˆ 3.8 Hz, 1H; H-4), 4.33 (t, J(2,3) ˆ J(3,4) ˆ 3.8 Hz, 1H; H-3), 3.89
(ddd, J(4,5) ˆ 3.8 Hz, J(5,6a) ˆ 7.4Hz, J(5,6b) ˆ 5.0 Hz, 1H; H-5), 3.74
(ABX, J(5,6a) ˆ 7.4Hz, J(6a,6b) ˆ 10.0 Hz, 1H; H-6a), 3.70 (d, J(H,P) ˆ
11.6 Hz, 3H; OMe), 3.69 (d, J(H,P) ˆ 11.4Hz, 3H; OMe), 3.65 (ABX,
‡
CDCl3): d ˆ 26.94ppm; MS (DCI-NH 3): m/z (%): 437 (100) [M‡H] , 4 54
‡
À
J(5,6b) ˆ 5.0 Hz, J(6a,6b) ˆ 10.0 Hz, 1H; H-6b), 0.92 (s, 9H; Si tBu), 0.90
(20) [M‡NH4] ; elemental analysis calcd (%) for C21H25O8P: C 57.80, H
À
À
À
5.77; found: C 57.74, H 5.94.
(s, 9H; Si tBu), 0.89 (s, 9H; Si tBu), 0.88 (s, 9H; Si tBu), 0.15 (s, 3H;
À
À
À
À
Si Me), 0.14(s, 6H; 2 Â Si Me), 0.11 (s, 3H; Si Me), 0.10 (s, 3H; Si Me),
Compound 16a (55 mg, 0.13 mmol) in a mixture of MeOH (5 mL) and
diisopropylamine (18 mL, 0.13 mmol) was stirred overnight at room
temperature under H2 atmosphere (1 bar) with 10% activated Pd/C
(16 mg) as catalyst. After being filtered through a celite pad, the solution
was concentrated to dryness to yield 16 as its monodiisopropylammonium
salt (43 mg, 94% yield).
À
À
À
0.09 (s, 3H; Si Me), 0.08 (s, 3H; Si Me), 0.07 (s, 3H; Si Me) ppm;
13C NMR (100 MHz, CDCl3): d ˆ 173.12 (d, J(1,P) ˆ 2.8 Hz; C-1), 89.26 (C-
4), 80.99 (d, J(2,P) ˆ 13.7 Hz; C-2), 80.39 (d, J(1',P) ˆ 194.9 Hz; C-1'), 76.47
(C-3), 72.55 (C-5), 63.99 (C-6), 52.00 (d, J(C,P) ˆ 5.4Hz, OMe), 51.86 (d,
J(C,P) ˆ 5.6 Hz, OMe), 25.88, 25.77, 25.65, 25.63, 18.25, 18.09, 17.79, 17.71,
À3.72, À3.88, À4.32, À4.37, À5.00, À5.46, À5.53 ppm; 31P NMR
(101 MHz, CDCl3): d ˆ 22.01 ppm; MS (DCI-NH3): m/z (%): 741 (100)
[a]2D0 ‡28.0 (c ˆ 1.0, H2O); 1H NMR (400 MHz, CD3OD): d ˆ 4.44 (brs,
1H; H-4), 4.07 (brs, 1H; H-3), 3.92 (t, J(5,6a) ˆ J(5,6b) ˆ 6.1 Hz, 1H;
H-5), 3.87 (d, J(2,3) ˆ 0.7 Hz, 1H; H-2), 3.66 3.64(d, J(5,6a,b) ˆ 6.1 Hz,
2H; H-6a, H-6b), 3.63 (sept, J ˆ 6.5 Hz, 2H; (CH3)2CH), 2.51 (ABX,
J(1'a,1'b) ˆ 14.7 Hz, J(1'a,P) ˆ 18.1 Hz, 1H; H-1'a), 2.45 (ABX,
J(1'a,1'b) ˆ 14.7 Hz, J(1'b,P) ˆ 18.7 Hz, 1H; H-1'b), 1.51 (d, J ˆ 6.5 Hz,
12H; (CH3)2CH) ppm; 13C NMR (100 MHz, CD3OD): d ˆ 108.77 (d,
J(1,P) ˆ 1.4 Hz; C-1), 86.69 (C-3), 84.59 (C-4), 80.31 (C-2), 78.60 (C-5),
64.17 (C-6), 48.66, 48.59, 33.07 (d, J(1',P) ˆ 129.4Hz; C-1 '), 19.66,
19.65 ppm; 31P NMR (101 MHz, D2O): d ˆ 15.80 ppm; MS (negative
‡
[M‡H] ; elemental analysis calcd (%) for C33H73O8PSi4: C 53.47, H 9.93;
found: C 53.51, H 9.93.
Dimethyl (1-deoxy-2,3,5,6-tetra-O-tert-butyldimethylsilyl-a-d-galactofura-
nosyl)methyl phosphonate (19): Compound 18 (1 g, 1.35 mmol) was
dissolved in ethyl acetate (105 mL) and was vigorously stirred at room
temperature under H2 atmosphere (1.5 bar) with palladium hydroxide
(470 mg, 20% Pd on carbon, wet) as catalyst. After 24 h, the catalyst was
removed by filtration through
a pad of celite and the filtrate was
concentrated. The residue was purified by chromatography on silica gel
with cyclohexane/AcOEt (4:1) as eluent. Product 19 was obtained as a
colorless oil (826 mg, 82% yield).
‡
ESI): m/z (%): 255 (100) [M À H] ; HRMS found 255.02751; C7H12O8P
calcd 255.02698.
[a]1D9 À10.6 (c ˆ 1.1, CHCl3); 1H NMR (400 MHz, CDCl3): d ˆ 4.32 (tdd,
J(1,2) ˆ 2.8 Hz, J(1,1'a,b) ˆ 6.3 Hz, J(1,P) ˆ 9.7 Hz, 1H; H-1), 4.05 (d,
J(3,4) ˆ 1.1 Hz, 1H; H-3), 3.88 (d, J(1,2) ˆ 2.8 Hz, 1H; H-2), 3.79 (m, 1H;
H-5), 3.78 (d, J(H,P) ˆ 10.9 Hz, 3H; OMe), 3.77 (d, J(H,P) ˆ 10.8 Hz, 3H;
OMe), 3.72 (ABX, J(5,6a) ˆ 3.7 Hz, J(6a,6b) ˆ 10.5 Hz, 1H; H-6a), 3.69
(dd, J(3,4) ˆ 1.1 Hz, J(4,5) ˆ 6.9 Hz, 1H; H-4), 3.59 (ABX, J(5,6b) ˆ
5.4Hz, J(6a,6b) ˆ 10.5 Hz, 1H; H-6b), 2.17 (AXX', J(1,1'a,b) ˆ 6.3 Hz,
2,3,5,6-Tetra-O-tert-butyldimethylsilyl-d-galactono-1,4-lactone (17): d-
Galactono-1,4-lactone (2.69 g, 15.1 mmol) was dissolved in DMF (40 mL)
and treated with imidazole (7.3 g, 107 mmol) and tert-butyldimethylsilyl
chloride (13.4g, 89.2 mmol) at 70 8C for 5 h under argon. The solution was
then allowed to warm to room temperature and stirred overnight. The
reaction mixture was concentrated to dryness under vacuum, diluted with
CH2Cl2 (800 mL), washed with water (2 Â 100 mL), and dried over MgSO4.
After solvent evaporation, the residue was purified by flash chromatog-
raphy with cyclohexane/AcOEt (25:1 to 23:1) as eluent to give 17 (9.39 g,
98% yield) as a white solid.
À
J(1',P) ˆ 18.0 Hz, 2H; H-1'a, H-1'b), 0.95 (s, 9H; Si tBu), 0.92 (s, 18H; 2 Â
À
À
À
À
Si tBu), 0.89 (s, 9H; Si tBu), 0.15 (s, 3H; Si Me), 0.14(s, 3H; Si Me), 0.12
À
À
À
(s, 3H; Si Me), 0.11 (s, 6H; 2 Â Si Me), 0.10 (s, 3H; Si Me), 0.08 (s, 6H;
2  Si Me) ppm; 13C NMR (100 MHz, CDCl3): D ˆ 88.55 (C-4), 79.41 (d,
À
M.p. 49 508C; [a]2D3 À11.4(c ˆ 1.0, CHCl3); 1H NMR (400 MHz, CDCl3):
d ˆ 4.45 (t, J(2,3) ˆ J(3,4) ˆ 6.1 Hz, 1H; H-3), 4.38 (d, J(2,3) ˆ 6.1 Hz, 1H;
H-2), 4.35 (dd, J(3,4) ˆ 6.1 Hz, J(4,5) ˆ 1.7 Hz, 1H; H-4), 3.85 (ddd,
J(4,5) ˆ 1.7 Hz, J(5,6a) ˆ 7.7 Hz, J(5,6b) ˆ 5.8 Hz, 1H; H-5), 3.70 (ABX,
J(5,6a) ˆ 7.7 Hz, J(6a,6b) ˆ 9.7 Hz, 1H; H-6a), 3.66 (ABX, J(5,6b) ˆ
J(2,P) ˆ 8.0 Hz; C-2), 78.80 (C-3), 75.26 (C-1), 74.50 (C-5), 63.43 (C-6),
52.38 (d, J(C,P) ˆ 6.5 Hz, OMe), 52.22 (d, J(C,P) ˆ 6.4Hz, OMe), 26.10,
26.02, 25.90, 25.62, 25.25 (d, J(1',P) ˆ 141.4 Hz; C-1'), 18.52, 18.30, 18.08,
17.73, À4.15, À4.25, À4.31, À4.36, À4.49, À4.90, À5.17, À5.28 ppm; 31P
NMR (101 MHz, CDCl3): d ˆ 32.52 ppm; MS (DCI-NH3): m/z (%): 743
‡
‡
À
5.8 Hz, J(6a,6b) ˆ 9.7 Hz, 1H; H-6b), 0.95 (s, 9H; Si tBu), 0.92 (s, 9H;
(40) [M‡H] , 760 (100) [M‡NH4] ; elemental analysis calcd (%) for
À
À
À
À
Si tBu), 0.91 (s, 9H; Si tBu), 0.90 (s, 9H; Si tBu), 0.23 (s, 3H; Si Me),
C33H75O8PSi4: C 53.33, H 10.17; found: C 53.29 10.30.
À
À
À
0.18 (s, 3H; Si Me), 0.16 (s, 3H; Si Me), 0.15 (s, 3H; Si Me), 0.14(s, 3H;
(1-Deoxy-a-d-galactofuranosyl)methyl phosphonic acid (20): Trimethylsil-
yl iodide (0.9 mL, 6.14mmol) was added at 0 8C to a solution of 19 (304mg,
0.41 mmol) in anhydrous carbon tetrachloride (24 mL). The solution was
stirred for 4h at 0 8C and was then allowed to come to room temperature
for a period of 1 h. The solvent was evaporated and the residue was dried
overnight in vacuo. The residue was dissolved in water and extracted three
times with ether, and the combined aqueous phases were pooled and
lyophilized. The resulting product was dissolved in a minimum amount of
Si Me), 0.11 (s, 3H; Si Me), 0.09 (s, 6H; 2 Â Si Me) ppm; 13C NMR
(100 MHz, CDCl3) ˆ 173.51 (C-1), 81.53 (C-4), 76.91 (C-2), 75.59 (C-3),
71.52 (C-5), 63.04(C-6), 25.83, 25.76, 25.74, 25.62, 18.18, 18.15, 18.08, 17.77,
À3.42, À4.13, À4.16, À4.32, À4.71, À4.85, À5.45, À5.49 ppm; MS (DCI-
À
À
À
‡
NH3): m/z (%): 652 (100) [M‡NH4] ; elemental analysis calcd (%) for
C30H66O6Si4: C 56.73, H 10.47; found: C 56.87, H 10.58.
(1(1')Z)-2,3,5,6-Tetra-O-tert-butyldimethylsilyl-1-(dimethoxyphosphoryl)-
methylidene-d-galactofuranose (18): Butyllithium (2.5m solution in hex-
ane, 7.9 mL, 19.8 mmol) was added under argon to a cooled (À708C)
solution of freshly distilled dimethyl methylphosphonate (2.24mL,
20.7 mmol) in anhydrous THF (41 mL), followed after 20 min by a solution
of 17 (5.24g, 8.26 mmol) in anhydrous THF (8 mL). The temperature was
maintained at À708C for 10 min, and the mixture was then allowed to come
to À408C over a 1 h period. The solution was then diluted with phosphate
buffer ((1m, pH ˆ 7, 190 mL) and extracted with CH2Cl2 (2  400 mL). The
combined organic phases were dried over MgSO4, filtered, concentrated,
and dried overnight in vacuo. The crude product was then dissolved at 08C
in anhydrous THF (58 mL), and pyridine (6.7 mL, 82.6 mmol) and
trifluoroacetic anhydride (5.8 mL, 41.7 mmol) were added. The resulting
solution was stirred at 08C for 3 h, quenched by addition of saturated
aqueous NaHCO3, and extracted with AcOEt (400 mL). The organic phase
was dried over MgSO4, filtered, and concentrated at reduced pressure.
Purification by silica gel chromatography, eluted with cyclohexane/AcOEt
(3:1), afforded 18 (4.7g, 77%) as a white solid.
‡
water and applied to a Dowex 50WX8 200 column (Bu3NH form). The
appropriate fractions (detected by TLC EtOH/NH4OH/H2O 5:3:1) were
pooled and lyophilized to give 20 as a white hygroscopic solid (bistribu-
tylammonium salt, 223 mg, 87% yield). Analysis corresponded to those
previously published.[25]
Acknowledgement
This work was supported by the Centre National de la Recherche
¡
¬ ¬ ¬ ¡
Scientifique (CNRS) as well as by the Ministere Delegue a la Recherche
et aux Nouvelles Technologies (Ph.D. grant to A.C.).
[1] J. B. Houseknecht, T. Lowary, Curr. Opin. Chem. Biol. 2001, 5, 677
82.
Chem. Eur. J. 2003, 9, 5888 5898
www.chemeurj.org
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
5897

S. P. Vincent, P. Sinay» et al.
FULL PAPER
[2] L. L. Pedersen, S. J. Turco, Cell. Mol. Life Sci. 2003, 60, 259 66.
[3] L. Kremer, G. S. Besra, Expert. Op. Inv. Drug 2002, 11, 1033 49.
[4] R. Kˆplin, J. R. Brisson, C. Whitield, J. Biol. Chem. 1997, 272, 4121
28.
[21] A. Dondoni, A. Marra, C. Pasti, Tetrahedron: Asymm. 2000, 11, 305
17.
[22] W.-B. Yang, C.-F. Chang, S.-H. Wang, C.-F. Teo, C.-H. Lin, Tetrahe-
dron Lett. 2001, 42, 4657 60.
[5] P. M. Nassau, S. L. Martin, R. E. Brown, A. Weston, D. Monsey, M. R.
McNeil, K. Duncan, J. Bacteriol. 1996, 178, 1047 52.
[6] L. Kremer, L. G. Dover, C. Morehouse, P. Hitchin, M. Everett, H. R.
Morris, A. Dell, P. J. Brennan, M. R. McNeil, C. Flaherty, K. Duncan,
G. S. Besra, J. Biol. Chem. 2001, 276, 26430 40.
[7] F. Pan, M. Jackson, Y. Ma, M. R. McNeil, J. Bacteriol. 2001, 183, 3991
98.
[8] A. K. Pathak, V. Pathak, W. J. Suling, S. S. Gurcha, C. B. Morehouse,
G. S. Besra, J. A. Maddry, R. C. Reynolds, Bioorg. Med. Chem. 2002,
10, 923 28.
[23] W.-B. Yang, C.-Y. Wu, C.-C. Chang, S.-H. Wang, C.-F. Teo, C.-H. Lin,
Tetrahedron Lett. 2001, 42, 6907 10.
[24] W.-B. Yang, Y.-Y. Yang, Y.-F. Gu, S.-H. Wang, C.-F. Chang, C.-H. Lin,
J. Org. Chem. 2002, 67, 3773 82.
[25] J. Kovensky, M. McNeil, P. Sinay», J. Org. Chem. 1999, 64, 6202 05.
[26] C. Schaller, P. Vogel, Helv. Chim. Acta 2000, 83, 193 232.
[27] K. Kraehenbuehl, S. Picasso, P. Vogel, Helv. Chim. Acta 1998, 81,
1439 79.
[28] V. S. Bogachev, Russ. J. Bioorg. Chem. 1996, 22, 599 604.
[29] A. L. Marlow, L. L. Kiessling, Org. Lett. 2001, 3, 2517 19.
[30] S. P. Vincent, L. N. Gastinel, Carbohydr. Res. 2002, 337, 1039 42.
[31] A. Zamyatina, S. Gronow, C. Oertelt, M. Puchberger, H. Brade, P.
Kosma, Angew. Chem. 2000, 112, 4322 25; Angew. Chem. Int. Ed.
2000, 39, 4150 53.
[9] D. A. R. Sanders, A. G. Staines, S. A. McMahon, M. R. McNeil, C.
Whitfield, J. H. Naismith, Nature Struct. Biol. 2001, 8, 858 63.
[10] W. S. Fobes, J. E. Gander, Biochem. Biophys. Res. Commun. 1972, 49,
76 83.
[11] M. Johnson, J. E. Gander, Biochim. Biophys. Acta 1978, 523, 9 18.
[12] G. Stevenson, B. Neal, D. Liu, M. Hobbs, N. H. Packer, M. Batley,
J. W. Redmond, L. Lindquist, P. Reeves, J. Bacteriol. 1994, 176, 4144
56.
[13] J. N. Barlow, J. S. Blanchard, Carbohydr. Res. 2000, 328, 473 80.
[14] A. Burton, P. Wyatt, G.-J. Boons, J. Chem. Soc. Perkin Trans. 1 1997,
2375 83.
[15] Q. Zhang, H.-w. Liu, J. Am. Chem. Soc. 2001, 123, 6756 66.
[16] S. W. B. Fullerton, S. Daff, D. A. R. Sanders, W. J. Ingledew, C.
Whitfield, S. K. Chapman, J. H. Naismith, Biochemistry 2003, 42,
2104 09.
[32] R. Lee, D. Monsey, A. Weston, K. Duncan, C. Rithner, M. McNeil,
Anal. Biochem. 1996, 242, 1 7.
[33] Q. Zhang, H.-w. Liu, J. Am. Chem. Soc. 2000, 122, 9065 70.
[34] S. Bornemann, Nat. Prod. Rep. 2002, 19, 761 72.
[35] K. E. Amrein, B. Takacs, M. Stieger, J. Molnos, N. A. Flint, P. Burn,
Proc. Natl. Acad. Sci. USA 1995, 92, 1048 52.
[36] J. Sambrook, E. F. Fritsch, T. Maniatis, Molecular cloning: a laboratory
manual, 2nd ed., Cold Spring Harbor, NY, 1989.
[37] M. Dagert, S. D. Ehrlich, Gene 1979, 6, 23 28.
[38] F. Pompeo, J. v. Heijenoort, D. Mengin-Lecreulx, J. Bacteriol. 1998,
180, 4799 803.
[17] J. N. Barlow, M. E. Girvin, J. S. Blanchard, J. Am. Chem. Soc. 1999,
121, 6968 69.
[39] U. K. Laemmli, M. Favre, J. Mol. Biol. 1973, 80, 575 99.
[40] M. M. Bradford, Anal. Biochem. 1976, 72, 248 54.
[18] J. Kovensky, P. Sinay», Eur. J. Org. Chem. 2000, 3523 5.
¬
¡
[19] V. Jaouen, A. Jegou, A. Veyrieres, Synlett 1996, 1218 20.
[20] T. Kr¸lle, C. d. l. Fuente, L. Pickering, R. T. Aplin, K. E. Tsitsanou,
S. E. Zographos, N. G. Oikonomakos, R. J. Nash, R. C. Griffiths,
G. W. J. Fleet, Tetrahedron: Asymm. 1997, 8, 3807 20.
Received: May 15, 2003 [F5141]
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