Synthesis of novel ammonium and selenonium ions and their evaluation as inhibitors of UDP-galactopyranose mutase

Article abstract of DOI:10.1016/j.carres.2004.07.012

The syntheses of two ammonium salts of 1,4-dideoxy-1,4-imino-d-galactitol containing erythritol sulfate side chains are described. The parent compound is a known inhibitor of the enzyme UDP-galactopyranose mutase (UDP-galactopyranose furanomutase, E.C. 5.

Full text of DOI:10.1016/j.carres.2004.07.012

Carbohydrate
RESEARCH
Carbohydrate Research 339 (2004) 2205–2217
Synthesis of novel ammonium and selenonium ions and
their evaluation as inhibitors of UDP-galactopyranose mutase
Natacha Veerapen,a Yue Yuan,a David A. R. Sandersb and B. Mario Pintoa,
*
aSimon Fraser University, Burnaby, British Columbia, Canada V5A 1S6
bUniversity of Saskatchewan, 110Science Place, Saskatoon, Saskatchewan, Canada S7N 5C9
Received 10 June 2004; accepted 12 July 2004
Available online 13 August 2004
Abstract—The syntheses of two ammonium salts of 1,4-dideoxy-1,4-imino-D-galactitol containing erythritol sulfate side chains are
described. The parent compound is a known inhibitor of the enzyme UDP-galactopyranose mutase (UDP-galactopyranose furano-
mutase, E.C. 5.4.99.9), which is responsible for the conversion of UDP-galactopyranose into UDP-galactofuranose and is presum-
ably protonated in its active form. The side chain was chosen because it is present in a known sulfonium ion a-glucosidase inhibitor,
salacinol. The syntheses of the selenonium analogues derived from 1,4-dideoxy-1,4-seleno-D-galactitol are also described. The syn-
thetic strategy in the syntheses of all four salts involved the nucleophilic attack of a protected derivative of the alditol at the least
hindered carbon of 2,4-O-benzylidene D- or L-erythritol-1,3-cyclic sulfate to give adducts that were subsequently deprotected. The
importance of different protecting groups used in the various syntheses is also highlighted. Enzyme inhibition assays carried out on
these compounds, and the corresponding sulfonium ions synthesized previously, show that they are poor inhibitors of UDP-galacto-
pyranose mutase.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Keywords: Galactofuranose; Galactofuranosyltransferase; UDP-galactopyranose mutase; Inhibitors; Cyclic sulfate; Ammonium ions; Selenonium
ions
1. Introduction
gates,4 and its presence in cell-wall structures appears
to be essential for the survival and infectivity of micro-
organisms. Hence, the enzymes involved in both the for-
mation and incorporation of Galf in bacteria and
parasites have become important drug targets.
UDP-galactofuranose (UDP-Galf) is known to be the
activated precursor for the construction of Galf-contain-
ing oligosaccharides, and it is synthesized from UDP-
galactopyranose (UDP-Galp) in a reaction catalyzed
by UDP-galactopyranose mutase.7,8 Galf is then trans-
ferred onto acceptors to form various glycoconjugates
by galactofuranosyltransferase. UDP-galactopyranose
mutase is essential for the viability of mycobacteria,9
suggesting that the inhibition of either the interconver-
sion of UDP-Galp to UDP-Galf or the transfer of the
Galf residue (galactosylation) is a promising strategy
for the development of new therapeutic agents.
D-Galactofuranose (Galf) residues are found in the cell
walls of many mycobacteria1 and in other microorgan-
isms such as protozoa2 and fungi.3 Galf structures are
components of the oligosaccharide core of both the
glycoinositolphospholipid (GIPL) and lipopeptidophos-
phoglycan (LPPG) of Trypanosoma cruzi as well as the
lipophosphoglycan (LPG) of Leishmania species.4–6
These protozoa are known to cause life-threatening dis-
eases, like Chagas disease and leishmaniasis.4 Mycobac-
teria cause pathogenic diseases like tuberculosis
(Mycobacterium tuberculosis) and leprosy (Mycobacte-
rium leprae).1 Of particular interest is the fact that the
cell walls of most of these mycobacteria consists largely
of internal (1!5)-b-D-Galf or (1!6)-b-D-Galf residues.1
Importantly, Galf is absent in mammalian glycoconju-
The mechanism of UDP-Galp mutase, a flavoprotein
in which the FAD coenzyme is noncovalently bound
to the protein, is still the subject of active investigation.
*
Corresponding author. Tel.: +1-604-291-4327; fax: +1-604-291-5424;
e-mail: bpinto@sfu.ca
0008-6215/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2004.07.012

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N. Veerapen et al. / Carbohydrate Research 339 (2004) 2205–2217
Nonetheless, the direct participation of the flavin coen-
zyme10,11 as well as the formation of the iminium ion
intermediate C11 have been established (Scheme 1).
Whether the reduced flavin transfers a single electron
to the oxacarbenium ion intermediate A (pathway
I)10,12 or acts as a nucleophile (pathway II), as suggested
by Soltero-Higgin et al.,11 to form the coenzyme–sub-
strate adduct B remains uncertain at this time. However,
both the radical mechanism, initially proposed by Full-
erton et al.12 and subsequently supported by Liu and co-
workers10 and that proposed by Soltero-Higgin et al.11
seem to reconcile most of the findings obtained to date.
While the formation of a bicyclic intermediate pro-
posed by Blanchard and co-workers13,14 is now ruled
out, the participation of the 4-OH in the ring formation
is still invoked.10,11 This is contradictory to the results
obtained by Burton et al.15 who found that UDP-[4-F]
Galp, which is expected to inhibit the enzyme mutase if
the 4-OH group were crucial in the ring contraction,
showed no inhibitory activity against the enzyme
galactopyranose mutase in the direction pyranose !
furanose, but it did inhibit the reverse reaction
(furanose ! pyranose).
were potent inactivators of Galp mutase.16 The authors
suggested that the inactivation could result from the for-
mation of a covalent adduct between the enzyme and the
inactivators. It was suggested further that compounds
that lead to oxacarbenium ion-type intermediates or
transition states resembling A and D could function as
inhibitors of the enzyme by forming stable covalent
adducts with the nucleophilic amino acid residues
present in the enzyme active site.16
The second class of enzymes of interest, the galacto-
furanosyltransferases, transfers Galf residues to glycosyl
acceptors to give Galf-containing glycoconjugates and
oligosaccharides. Mechanistically, the reaction catalyzed
by glycosyltransferases is believed to be similar to that
of glycosidases,17 also involving an oxacarbenium-ion-
like transition state.18,19
We report herein the syntheses of 4-imino Galf
ammonium salts 1 and 2 as potential inhibitors of
UDP-Galp mutase and Galf-transferase. This reasoning
is based on the fact that the parent iminogalactitol 7 is a
known inhibitor of Galp mutase20 and is likely to be
protonated in the active site.21 The choice of the erythr-
itol sulfate side chain derives from its presence in the
known glycosidase inhibitors salacinol 822–24 and gha-
vamiol 9.25 The sulfate counterion is also believed to
contribute to the stability of these salts, thereby further
supporting its presence in our designed compounds.
We report also the synthesis of the corresponding
selenonium salts 3 and 4; we have previously reported
the synthesis of the analogous sulfonium ions 5 and
6.26 We propose to test the hypothesis that the perma-
nent positive charge, as well as the shape of the salts
Of interest to us was that the proposed mechanism in-
volves the reversible cleavage of the anomeric C–O bond
with the departure of the nucleotide to form a transient
oxacarbenium ion-like transition state or intermediate,
in accordance with preliminary studies carried out by
Blanchard and co-workers.13,14 The formation of such
a species is further supported by a mechanistic investiga-
tion carried out on fluorinated analogues of UDP-Galf,
which revealed that UDP-[2-F]Galf and UDP-[3-F]Galf
R
OH
O
Me
Me
N
N
O
OH
HO
NH
N
(FADH.)
O
OH
(FADH-)
Me
Me
N
(I)
OH
O
OH
HO
OH
O
OH
OH
O
OH
(II)
HO
HO
N
N
O
R
HO
OUDP
OH
OH
α-UDP-Galp
(FADH-)
O
B
A
HN
UDP-Galp mutase
Me
Me
H
O
Me
OH
Me
HO
HO
O
O
O
OH
N
N
N
O
OH
R
HO
OH
N
N
N
O
R
OUDP
OUDP
OH
OH
OH
OH
O
OH
OH
OH
OH
D
O
HN
OH
HN
(FADH-)
α-UDP-Galf
C
Scheme 1.

N. Veerapen et al. / Carbohydrate Research 339 (2004) 2205–2217
2207
1–6 could mimic the oxacarbenium-like transition states
of the enzymatic reactions described above, and there-
fore function as inhibitors. We also report their evalua-
tion as inhibitors of UDP-Galp mutase.
Various syntheses of 1,4-dideoxy-1,4-imino-D-galacti-
tol have been previously reported by different research
groups.27–30 However, we used an alternative pathway
to obtain 1,4-dideoxy-1,4-imino-D-galactitol en route
to our target compounds. This method, similar to the
procedure of Lee et al.,27 involved the epimerization at
C-4 of a benzyl-protected glucose derivative 13 as the
key step (Scheme 3). The diol 12, prepared from D-glu-
cose,31 was converted into the dimesylate 13, which was
then reacted with allylamine in an SN2-type reaction to
give the tertiary amine 14 in 83% yield. Unlike Lee
et al.,27 we chose allylamine rather than benzylamine
because it permitted selective deprotection of the nitro-
gen atom, without affecting the benzyl protecting
groups. Subsequent treatment of 14 with WilkinsonÕs
catalyst32 gave the iminogalactitol derivative 15 in 70%
yield.
OH
OH
OH
OH
X
OH
OSO3
X
OH
OSO3
OH
OH
OH
OH
OH
OH
2 X= NH
4 X= Se
6 X= S
1 X= NH
3 X= Se
5 X= S
The coupling reaction of the benzylated derivative 15
with 2,4-O-benzylidene-D-erythritol-1,3-cyclic sulfate
1024 was then investigated. Thus, when a mixture of 15
and the cyclic sulfate 10 in dry acetone was heated at
70ꢁC in the presence of anhydrous potassium carbonate
for 12h, the desired coupled product 16 was obtained in
68% yield (Scheme 4). In an attempt to improve the yield
of the coupling reaction and also to investigate the reac-
tivity of the imino sugar, the deprotected galactitol 7,
obtained by hydrogenolysis of 15, was reacted with the
D-cyclic sulfate 10. Owing to the insolubility of the de-
protected compound 7 in acetone, the reaction was per-
formed in dry methanol. When the mixture was heated
at 60ꢁC for 12h, TLC analysis showed the formation
of two additional polar products as well as the presence
of large amounts of both starting materials. Increasing
OH
OH
H
N
HO
X
HO
OSO3
OH
OH
OH
OH
OH
7
8 X= S
9 X= NH
2. Results and discussion
Retrosynthetic analysis showed that the target com-
pounds could be obtained by the alkylation of the
heteroatom of suitably protected cyclic alditols (Scheme 2).
We chose 2,4-O-benzylidene-D-(10) and L-(11)erythri-
tol-1,3-cyclic sulfates as alkylating agents, given the
success of similar alkylation reactions in our labora-
tory.24,25
OBn
HO
MsCl
6 steps
Ref.30
OH
Pyridine
D-glucose
BnO
OBn
OBn
12
OMs
OBn
OH
N
OMs
OBn
NH2
OH
BnO
OH
X
OSO3
X
OBn
OBn
13
OBn
OBn
OH
OH
OH
+
L
OSO3
OBn
14
OH
OH
OH
OH
H
N
OBn
OH
X= NH, Se
OH
OBn
OBn
HO
HO
O
O
O
Ph
O
O
O
O
O
S
O
S
O
OBn
15
Scheme 2.
Scheme 3.

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N. Veerapen et al. / Carbohydrate Research 339 (2004) 2205–2217
Ph
OH
2'
O
O
3'
4'
1'
3'
OH
4'
2'
1'
+NH
OSO3
-
+NH
OBn
OSO3
-
1
4
OH
H2, Pd/C
AcOH
O
Acetone, K2CO3
O
4
1
3
2
15
O
Ph
+
O
3
2
O
S
OH
OH
OBn
OBn
O
OH
OBn
10
16
1
Ph
O
OH
O
OH
-
+NH
OH
OSO3
-
+NH
OBn
OSO3
O
O
H2, Pd/C
AcOH
O Acetone, K2CO3
Ph
+
15
O
O
S
OH
OH
O
OBn
OBn
OH
OBn
17
2
11
Scheme 4.
R
R
R
R
+NH
OBn
+NH
OBn
N
OBn
N
OBn
ki
kd
kp
OBn
OBn
'
kd
OBn
OBn
OBn
OBn
OBn
OBn
OBn
OBn
OBn
OBn
R = protected cyclic sulfate
kd, kd' = rate of deprotonation of ammonium salt
kp, kp' = rate of protonation of amine
ki = rate of inversion
Scheme 5.
the temperature to 70ꢁC for longer reaction times only
led to the decomposition of both the products and the
cyclic sulfate.33 It would appear that the protection of
the hydroxyl groups as benzyl ethers makes the com-
pound more nucleophilic. Diastereomer 17 was obtained
similarly by the reaction of compound 15 with the L-cy-
clic sulfate 1124 in 74% yield (Scheme 4).
Compounds 16 and 17 were completely characterized
by 2D NMRtechniques. Initially, the NMRstudies for
both 16 and 17 were carried out in CD2Cl2 and CD3OD,
and it was observed that the peaks were broad and over-
lapping in both cases. However, when the NMRstudies
were performed on samples of compounds 16 and 17 in
deuterated methanol, made basic with small amounts of
sodium deuteroxide to give the corresponding amines,
the peaks were more defined. We believe that the broad-
ening of the peaks in the absence of base is due to chem-
ical exchange between the ammonium salts and the
corresponding tertiary amines, a process that is in the
intermediate-exchange regime on the NMRtime scale
(Scheme 5). In the presence of base, only the rapidly
inverting tertiary amine is present, and a conformation-
ally averaged NMRspectrum in the fast-exchange re-
gime is observed.
Deprotection of 16 and 17 was achieved by hydro-
genolysis over Pd/C in 80% acetic acid to yield the dia-
stereomers 1 and 2, respectively. The 1H and 13C
NMRspectra for both diastereomers obtained in
CD3OD were essentially identical, except for small
changes in the chemical shifts. Interestingly, sharp peaks
were obtained for the deprotected compounds 1 and 2
in the absence of base. Apparently, chemical exchange

N. Veerapen et al. / Carbohydrate Research 339 (2004) 2205–2217
2209
between the ammonium salt and the corresponding ter-
tiary amine in compounds 1 and 2 is slower than in com-
pounds 16 and 17 (Scheme 5).
ing to H-10b and H-10a while irradiation of the multiplet
containing H-10a gave rise to an enhancement of the
resonance corresponding to both protons H-1b and
H-4. This study suggests that the stereochemistry at
the nitrogen center is fixed or that chemical exchange
is slow on the NMRtime scale in the deprotected
compounds. The 1D-transient NOE results obtained at
285K did not allow us to determine which pair of
protons is on the same face of the ring. However, a
NOESY experiment carried out at 298K showed corre-
lations between H-4 and H-10b and H-1b and H-10a,
respectively, indicating that the stereochemistry at the
stereogenic nitrogen center in 1 is such that the erythri-
tol side chain is preponderantly trans to the chain at C-5
of the anhydrogalactitol moiety.
The stereochemistry at the stereogenic nitrogen center
in 2 was also established by means of a NOESY exper-
iment. The correlation between H-10 and H-4 confirmed
the trans relationship between the erythritol side chain
and the chain at C-5 of the anhydrogalactitol moiety,
and also confirmed that the compound maintained its
stereochemical integrity.
To verify the hypothesis of chemical exchange, 1D-
transient NOE experiments were performed on 16 and
1 in deuterated methanol at 285K. If chemical exchange
were to occur, exchange peaks would display the same
phase as that of the originally saturated resonance.34
On the other hand, in the absence of any chemical
exchange, only positive NOE enhancements would be
observed.34 For the protected compound 16, when the
multiplets containing H-10a and H-10b were selectively
irradiated in separate experiments, enhancement of
the H-10b and H-10a resonances, respectively, were
observed. The irradiated and observed peaks were of
the same phase, thereby confirming that chemical
exchange, together with NOE transfer, were occurring
on the NMRtime scale ( Fig. 1b and c). It should be
noted that the tentative assignments of the broad
NMRspectrum of 16 (Fig. 1a) are based on the assign-
ments made for the spectrum in basic CD3OD. With
deprotected compound 1, when the multiplets contain-
ing H-1b and H-10a, were selectively irradiated, in
separate experiments, only positive NOE enhancements
of the relevant resonances were observed (Fig. 1e and f).
Irradiation of the multiplet containing H-1b and H-4
gave rise to enhancements in the resonances correspond-
The selenonium analogues 3 and 4 were synthesized
using a similar approach as for their ammonium conge-
ners. However, previous experience with selenonium
salts in our laboratory had shown that the hydrogeno-
lysis of the benzyl ethers in these compounds is difficult
Figure 1. (a) 1D 1H NMRspectrum of 16 in CD3OD, (b) 1D-transient NOE spectrum of 16 in CD3OD upon irradiation of the multiplet containing
H-10b, (c) 1D-transient NOE spectrum of 16 in CD3OD upon irradiation of the multiplet containing H-10a, (d) 1D 1H NMRspectrum of 1 in
CD3OD, (e) 1D-transient NOE spectrum of 1 in CD3OD upon irradiation of the multiplet containing H-1b and H-4 and (f) 1D-transient NOE
spectrum of 1 in CD3OD upon irradiation of the multiplet containing H-10a.

2210
N. Veerapen et al. / Carbohydrate Research 339 (2004) 2205–2217
and low yielding, explaining our choice of p-meth-
oxybenzyl (PMB) ether groups instead. p-Meth-
oxybenzyl (PMB) ethers, while having similar chemical
properties as benzyl ethers, do not require hydrogenoly-
sis. However, they are acid labile and therefore noncom-
pliant with the synthetic route used to make the
protected iminogalactitol derivative 15 (Scheme 3). A
modified synthetic pathway in which the anomeric OH
could be selectively deprotected in the presence of
PMB ethers was therefore desired. Both allyl and pente-
nyl glycosides are suitable options for this modified syn-
thetic route. However, in our hands, allyl deprotection
was low yielding and expensive due to the requirement
of PdCl2 in virtually stoichiometric amounts.
The pentenyl glycoside 18 (Scheme 6), obtained in
75% yield from D-glucose35–37 in a 1:1 a,b ratio, on
the other hand, gave better results. Cleavage of the pen-
tenyl glycoside to the hemiacetal was achieved in high
yields using N-bromosuccinimide (NBS).38 The reaction
was performed in the dark to ensure that no radical
reaction leading to the bromination of the methylene
moiety of the PMB group would occur. The hemiacetal
was then reduced to the diol, which was subsequently
converted to the desired dimesylated compound 21.
Na2Se prepared freshly in situ was used to displace the
primary mesylate. Cyclization then ensued to give the
protected 1,4-dideoxy-1,4-seleno-D-galactitol (22) in
98% yield.
selective as diastereomers were obtained in a 3:2 ratio in
both cases, as judged by the ratio of the benzylidene pro-
ton resonances in the 1H NMRspectra. The NMRspec-
tra of the coupled products proved to be complicated and
difficult to analyze due to a high degree of overlap of the
peaks. In an attempt to improve the stereoselectivity of
the reaction, we carried out the coupling in dry acetone
at 60ꢁC (Scheme 7). Despite the reaction being slower
and lower yielding, we were pleased to observe a net
enhancement in stereoselectivity. Hence, when a mixture
of the selenoether 22 and the D-cyclic sulfate 10 was
heated at 60ꢁC for 48h, the diastereomers were obtained
in a ratio of 10:1. Similar results were observed in the
coupling reaction with the L-cyclic sulfate 11 that
afforded 24 as a mixture of diastereomers in a 8:1 ratio.
The major isomer in both cases was determined, by
means of NOESY experiments, to be that in which the
erythritol side chain is trans to the chain at C-5. These
diastereomers were then treated with trifluoroacetic acid
to give the deprotected compounds in quantitative yields.
Compounds 3 and 4 were fully characterized by 2D
NMRspectroscopy, and the stereochemistry at the ste-
reogenic selenium center in the major diastereomers
was established by means of NOESY experiments.
The iminogalactitol and selenogalactitol derivatives
1–4, as well as their thio congeners 5 and 6, were tested
for their inhibition of UDP-Galp mutase. The HPLC
assay for detecting UDP-Galf and UDP-Galp was ini-
tially performed on enzyme and substrate alone to deter-
mine the optimal time for examining the inhibitors. We
found the addition of ice-cold HCl, followed by immedi-
ate freezing in liquid nitrogen, greatly increased the
reproducibility of our results, presumably by preventing
further enzyme activity while the samples were re-
With the selenoether in hand, the coupling reaction
with the 2,4-O-benzylidene-D- and L-erythritol-1,3-cyclic
sulfates 6 and 7 was first carried out in hexafluoro-2-pro-
panol (HFIP) at 65ꢁC to afford the coupled products as a
mixture of diastereomers in both cases. The coupling
reactions with the D- and L-cyclic sulfates were not very
AcO
AcO
HO
HO
O
OAc
O
OH
NaOMe
MeOH
2 steps
OPent
PMBCl/ NaH
OPent
D-glucose
Ref 35-37
OAc
OH
18
PMBO
PMBO
PMBO
PMBO
OPMB
OH
HO
O
OPMB
O
OPMB
NBS
OH NaBH4
OPent
PMBO
OPMB OPMB
OPMB
OPMB
19
20
OMs
OPMB
Se
OPMB
MsCl
Pyridine
Na2Se
OMs
PMBO
OPMB
OPMB
OPMB OPMB
21
OPMB
22
Scheme 6.

N. Veerapen et al. / Carbohydrate Research 339 (2004) 2205–2217
2211
Ph
O
O
-
O
OSO3
Se
OPMB
O
Ph
22 +
O
Acetone, K2CO3
O
O
S
O
OPMB
OPMB
OPMB
23 trans:cis = 10:1
OH
OH
TFA
OH
OH
-
-
Se+
OH
OSO3
Se+
OH
OSO3
+
OH
OH
OH
OH
OH
OH
Ph
trans-3
cis-3
O
O
-
O
OSO3
Se
OPMB
O
Acetone, K2CO3
O
22 + Ph
O
O
S
O
OPMB
OPMB
OPMB
24 trans:cis = 8:1
OH
OH
TFA
OH
OH
-
Se+
OH
OSO3
-
Se+
OH
OSO3
+
OH
OH
OH
OH
OH
OH
trans- 4
cis-4
Scheme 7.
thawed prior to HPLC analysis. At high UDP-Galp
concentrations, there was a slight evidence of a break-
down product of UDP-Galp (less than 1%), and this
was included in the calculation for UDP-Galp when nec-
essary. A time point of 5min was chosen for the inhibi-
tor studies, as this gave a turnover of 56.8 4.0%,
allowing us to follow any small changes in the rate of
turn-over.
10mM were required to begin to see inhibition (Table 1).
Previous site-directed mutagenesis studies showed that
the presence of a tryptophan (Trp) residue located in
the putative active-site cleft away from the isoallox-
azine ring of the FAD was required for enzyme
activity.39 We believe that this Trp residue forms a
base-stacking interaction with the UDP moiety of the
sugar substrate as has been seen with a number of other
nucleotide–sugar interactions with proteins.19,39–42 UDP
has been demonstrated to be an inhibitor of mutase
Compounds 1–6 proved to be very poor inhibitors
of UDP-galactopyranose mutase. Concentrations of

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N. Veerapen et al. / Carbohydrate Research 339 (2004) 2205–2217
Table 1. Inhibition of UDP-galactopyranose mutase by inhibitors
to UV light and/or sprayed with a solution containing
1% Ce(SO4)2 and 1.5% molybdic acid in 10% aqueous
H2SO4, and heated. Compounds were purified by flash
chromatography on Kieselgel 60 (230–400 mesh). Sol-
vents were distilled before use and were dried as neces-
sary by literature procedures. Solvents were
evaporated under reduced pressure and below 50ꢁC.
High-resolution mass spectra were liquid secondary ion-
ization fast-atom bombardment (LSIMS(FAB)), using
Cs+ ions, run on a Kratos Concept H double-focusing
mass spectrometer at 10000 RP, using meta-NO2-benzyl
alcohol as matrix or, in the case of compounds 1 and 2,
with glycerine as matrix and PEG-sulfate as the mass
reference.
Inhibitor
(10 mM final conc.)
% Turn-overa
% Inhibitionb
1
33.7 4.8
47.3 1.2
57.7 1.6
46.3 4.0
32.0 2.0
56.8 0.6
56.8 4.0
40.7
16.7
2
3
None
18.5
43.7
4
5
6
No inhibitor
None
N/A
a The % turn-over was calculated by integration of the two peaks.
b The % inhibition was calculated from the turn-over of the inhibited
reaction compared to the reactions with no inhibitor. Each result was
measured in triplicate.
(IC50 = 200lM),42 while we have been unable to show
any inhibitory activity of galactose (to 50mM concen-
trations, data not shown).
3.2. Protein over-expression and purification
As all of the molecules 1–6 lack the nucleotide moiety,
a promising strategy to enhance their inhibitory activi-
ties might be to elaborate analogous structures incorpo-
rating nucleotide moieties.
UDP-galactopyranose mutase (UDP-galactopyranose
furanomutase, E.C. 5.4.99.9) from Klebsiella pneumonia
was purified as described previously.39 The protein was
flash-frozen in liquid nitrogen (20lL aliquots) and
stored at 193K. The enzyme does not require glycerol
for storage, provided the aliquots are small and very
rapidly frozen. Protein concentrations were determined
by the method of Bradford.45
3. Experimental section
3.1. General
Optical rotations were measured with a Rudolph
Research Autopol II automatic polarimeter. 1H and
13C NMRspectra were recorded on a Bruker AMX
400 NMRspectrometer at 400.13 and 100.6MHz, for
1H and 13C, respectively. Chemical shifts are given in
ppm downfield from Me4Si for those spectra measured
in CDCl3 or CD3OD and from 2,2-dimethyl-2-silapen-
tane-5-sulfonate (DSS) for those spectra measured in
D2O. Chemical shifts and coupling constants were
obtained from a first-order analysis of the spectra. All
assignments were confirmed with the aid of two-dimen-
sional H/1H COSY, H/13C HMQC, and H NOESY
experiments, using standard Bruker pulse programs.
Processing of the spectra was performed with standard
UXNMR (Bruker) and WINNMR software. Zero filling of
the acquired data (512 t1 values and 2K data points in
t2) led to a final data matrix of 1K · 1K (F1 · F2) data
points. 1D Transient NOE experiments were recorded
at 285K on a Bruker AMX 600 NMRspectrometer.
For each 1D transient NOE spectrum, 1024 scans, pre-
ceded by 16 dummy scans were acquired. 1D transient
NOE experiments were performed with 80-ms Gaus-
sian-shaped pulses (2K data points, truncation level
1%) for selective inversion as described previously.43,44
A corrected mixing time of 100 and 500ms was used
in 1D transient NOE experiments for compound 16
and compound 1, respectively. Analytical thin-layer
chromatography (TLC) was performed on aluminum
plates precoated with Merck silica gel 60F254 as the
adsorbent. The developed plates were air-dried, exposed
3.3. 1,2,3,5,6-Penta-O-benzyl-4-di-O-methanesulfonyl-D-
glucitol (13)
Methanesulfonyl chloride (15mL, 0.19mol) was dis-
solved in pyridine (15mL) and this mixture was added
dropwise to a stirred solution of the diol 12 (2.2g,
41mmol) in dry CH2Cl2 (30mL). TLC analysis after
3h showed the absence of starting material and forma-
tion of a less polar compound. The reaction mixture
was quenched with solid NaHCO3, and the mixture
was stirred for 30min. The mixture was then partitioned
between water (100mL) and CH2Cl2 (50mL). The
CH2Cl2 extract was washed with 5% HCl (2 · 10mL),
water (2 · 10mL), satd aq NaHCO3 solution
(2 · 10mL) and finally with satd aq NaCl solution
(2 · 10mL). The organic extract was then dried
(Na2SO4) and evaporated. The residue was then purified
by flash chromatography using 3:2 hexanes–EtOAc to
1
1
1
give the dimesylated product 13 (2.53g, 89%). [a]D +18
1
(c 1.4, CHCl3); H NMR(CDCl ): d 7.52–7.22 (20H,
3
m, Ar), 5.21 (1H, dd, J4,5 6.8, J4,3 2.9Hz, H-4), 4.79,
4.62 (2H, 2d, JA,B 11.3Hz, CH2Ph), 4.58–4.46 (6H, m,
3 · CH2Ph), 4.31 (1H, dd, J1a,1b 10.9, J1a,2 4.2Hz, H-
1a), 4.25 (1H, dd, J1b,2 4.3, H-1b), 3.96–3.85 (3H, m,
H-5, H-4, H-2), 3.76 (1H, dd, J6a,6b 10.3, J6a,5 5.5Hz,
H-6a), 3.62 (1H, dd, J6b,5 5.6Hz, H-6b), 2.96 (3H, s,
CH3), 2.81 (3H, s, CH3). 13C NMR(CDCl ): d 137.72,
3
137.50, 137.32, 137.22 (4 · Cipso), 128.62–127.80
(20CAr), 81.85 (C-4), 76.60 (2C C-5, C-2), 76.05 (C-3),
74.95, 73.48, 73.03, 72.40 (4 · CH2Ph), 68.72 (C-1),

N. Veerapen et al. / Carbohydrate Research 339 (2004) 2205–2217
2213
68.62 (C-6), 39.98 (OMs), 37.08 (OMs). Anal. Calcd for
C36H42O10S2: C, 61.87; H, 6.06. Found: C, 61.97; H,
6.01.
(C-5), 79.42, 73.29 (2C, 2 · CH2Ph), 72.61 (CH2Ph),
72.06 (C-6), 71.2 (C-4), 70.85 (CH2Ph), 66.46 (C-3),
51.25 (C-1). Anal. Calcd for C37H41NO4: C, 77.98; H,
7.12; N, 2.67. Found: C, 78.11; H, 7.12; N, 2.59.
3.4. N-Allyl-2,3,5,6-tetra-O-benzyl-1,4-dideoxy-1,4-
imino-D-galactitol (14)
3.6. 10-((2,3,5,6-Tetra-O-benzyl-1,4-dideoxy-1,4-imino-D-
galactitol)-4-N-ammonium)-20,40-O-benzylidene-10-de-
oxy-D-erythritol-30-sulfate (16)
The dimesylate 13 (2.53g, 3.6mmol) was dissolved in
allylamine (60mL) and the solution heated to reflux
for 12h. The solvent was then removed under reduced
pressure, and the residue was dissolved in water
(50mL) and extracted with CH2Cl2 (3 · 50mL). The
combined organic extracts were dried (Na2SO4) and
concentrated. Purification of the resulting oil by column
chromatography (2:1 hexanes–EtOAc) gave the desired
tertiary amine 14 as a colorless oil (1.7g, 83%). [a]D
+27 (c 2.9, CHCl3); 1H NMR(CDCl 3): d 7.41–7.22
(20H, m, Ar), 5.96–5.85 (1H, m, NCH2CH@CH2),
5.18 (1H, d, Jtrans = 17.0Hz, NCH2CH@CH2), 5.10
(1H, d, Jcis = 10.1Hz, NCH2CH@CH2), 4.83 (1H, d,
JA,B 11.9Hz, CH2Ph), 4.76 (1H, d, JA,B 11.9Hz,
CH2Ph), 4.45–4.40 (6H, m, 3 · CH2Ph), 4.00 (1H, bs,
H-3), 3.92–3.88 (3H, m, H-2, H-5, H-6a), 3.62 (1H,
dd, J6b,6a 10.8, Jvic 7.6Hz, H-6b), 3.54 (1H, dd, Jgem
13.6, Jvic 4.2Hz, NCH2CH@CH2), 3.21 (1H, d, J1a,1b
10.7Hz, H-1a), 3.05 (1H, dd, NCH2CH@CH2), 2.91
(1H, m, H-4), 2.62 (1H, dd, J1bÀ2 4.4Hz, H-1b). 13C
NMR(CDCl 3): d 139.01, 138.72, 138.31, 138.28 (4 ·
A mixture of the imino sugar 15 (1.1g, 2.1mmol) and
the D-cyclic sulfate 10 (0.7g, 2.6mmol, 1.2equiv) was
dissolved in dry acetone (0.5mL) containing anhyd
K2CO3 (20mg). The mixture was stirred in a sealed tube
in an oil bath (70ꢁC) overnight. The solvent was re-
moved under reduced pressure and the product was
purified by column chromatography (2:1 EtOAc–
MeOH). The coupled product was obtained as a white
1
solid (1.1g, 64%). [a]D +1.2 (c 0.85, CHCl3); H NMR
(CD3OD made basic with NaOD): d 7.40–7.10 (25H,
m, Ar), 5.35 (1H, s, CHPh), 4.54–4.50 (3H, m, 2CH2
Ph, H-40a), 4.36–4.24 (6H, m, 6 CH2Ph), 4.18–4.11
(1H, ddd, J3 ,4 a J3 ,2 9.9, J3 ,4 b Hz, H-30), 3.87 (1H, t,
0
0
0
0
0
0
J2 ,1 a J2 ,1 b 8.7Hz, H-20), 3.85–3.78 (3H, m, H-2, H-3,
H-5), 3.75–3.67 (2H, m, H-40b, H6a), 3.42–3.39 (1H,
m, H-6b), 3.37 (1H, brd, H-10a), 3.24 (1H, d, J1a,1b
10.9Hz, H-1a), 3.13 (1H, dd, J4,5 5.2, J4,3 2.6Hz, H-4),
0
0
0
0
2.82–2.73 (2H, m, H-1b, H-10b). 13C NMR(CD OD):
3
d 142.46, 142.09, 141.88, 141.78, 141.43 (5 · Cipso),
135.61–129.89 (25CAr), 104.67 (CHPh), 86.85 (C-5),
85.29 (C-3), 83.10 (C-20), 82.05 (C-2), 76.48 (CH2Ph),
76.48, 76.01 (2C, CH2Ph), 74.79 (C-6), 74.53 (C-4),
74.39 (C-40), 72.92 (C-30), 72.28 (CH2Ph), 61.59 (C-1),
58.52 (C-10). Anal. Calcd for C45H49O10S: C, 67.91; H,
6.21; N, 1.76. Found: C, 68.04; H, 6.07; N, 2.01.
C
ipso), 135.9 (1C, NCH2CH@CH2), 128.42–127.40
(20CAr), 116.96 (NCH2CH@CH2), 84.18 (C-3), 81.22
(C-5), 79.13 (C-2), 73.18 (CH2Ph), 72.78 (CH2Ph),
72.01 (C-6), 71.15 (C-4), 70.70 (2C, 2 · CH2Ph), 58.78
(NCH2CH@CH2), 57.05 (C-1). Anal. Calcd for
C37H41NO4: C, 78.83; H, 7.33; N, 2.48. Found: C,
78.59; H, 7.45; N, 2.50.
3.7. 10-((2,3,5,6-Tetra-O-benzyl-1,4-dideoxy-1,4-imino-D-
galactitol)-4-N-ammonium)-20,40-O-benzylidene-10-
deoxy-L-erythritol-30-sulfate (17)
3.5. 2,3,5,6-Tetra-O-benzyl-1,4-dideoxy-1,4-imino-D-
galactitol (15)
The tertiary amine 14 (1.7g, 3.3mmol) was dissolved in a
mixture of CH3CN–H2O, 8:1 (100mL). N2 gas was bub-
bled through the solution for 10min, after which Wilkin-
sonÕs catalyst (80mg) was added. The reaction mixture
was heated to reflux for 15h. The solvents were then
removed, and the oil was purified by flash chromato-
graphy (1:1 EtOAc–hexanes) as eluent. The deprotected
amine 15 was obtained as a yellow oil (1.1g, 70%). [a]D
À20 (c 4.3, CHCl3); 1H NMR(CDCl 3): d 7.51–7.25
(20H, m, Ar), 4.78, 4.36 (2H, 2d, JA,B 11.6Hz, CH2Ph),
4.54–4.44 (6H, m, 6 CHPh), 3.97 (1H, m, H-2), 3.88–
3.80 (2H, m, H-3, H-5), 3.76–3.68 (2H, 2 dd, J6a,6b
10.4, J6a,5 6.1, J6b,5 4.6Hz, H-6a, H-6b), 3.13 (1H, d,
J1a,1b 12.5Hz, H-1a), 3.07 (1H, dd, J4,5 5.9, J4,3 3.7Hz,
H-4), 2.97 (1H, dd, J1b,2 5.0Hz, H-1b). 13C NMR
(CDCl3): d 138.58, 138.27, 138.20, 138.05 (4 · Cipso),
128.41–127.52 (20CAr), 85.97 (C-3), 84.51 (C-2), 77.12
A mixture of the imino sugar 15 (0.25g, 0.48mmol) and
the L-cyclic sulfate 11 (0.15g, 0.58mmol, 1.2equiv) was
dissolved in dry acetone (0.5mL) containing anhyd
K2CO3 (10mg) added. The mixture was stirred in a
sealed tube in an oil bath (70ꢁC) overnight. The solvent
was removed under reduced pressure, and the product
was purified by column chromatography (2:1 EtOAc–
MeOH). The coupled product was obtained as a white
1
solid (0.28g, 74%). [a]D +31 (c 1, CHCl3); H NMR
(CD3OD made basic with NaOD): d 7.29–7.12 (25H,
0
m, Ar), 5.44 (1H, s, CHPh), 4.52 (1H, dd, J4 a,4 b 11.1,
0
J4 a,3 5.3Hz, H-40a), 4.49 (2H, d, 2CH2Ph), 4.13 (1H,
0
0
ddd, J3 ,4 b J3 ,2 9.92Hz, H-30), 3.91 (1H, t, J2 ,1 a
0
0
0
0
0
0
J2 ,1 b 8.7Hz, H-20), 3.88–3.85 (2H, m, H-2, H-4),
3.75–3.67 (3H, m, H-40b, H-5, H-6a), 3.42 (1H, dd,
J6a,6b 10.8, J6a,5 6.9Hz, H-6a), 3.28 (1H, brd, H-1a),
3.16 (1H, d, H-10a), 3.01 (1H, dd, J4,5 5.3, J4,3 2.4Hz,
0
0

2214
N. Veerapen et al. / Carbohydrate Research 339 (2004) 2205–2217
H-4), 2.93 (1H, dd, J1 b,1 a 14.0, J1 b,2 8.5Hz, H-10b),
2.76 (1H, dd, J1b,1a 11.1Hz, H-1b). 13C NMR(CD 3OD):
d 140.89, 140.53, 140.27, 140.20, 139.96 (50Cipso),
130.03–128.50 (25CAr), 103.35 (CHPh), 85.85 (C-3),
83.55 (C-20), 81.03 (C-5), 80.89 (C-2), 74.99 (CH2Ph),
74.69 (CH2Ph), 73.29 (CH2Ph), 73.17 (CH2Ph), 72.99
(C-6), 72.89 (C-4), 71.33 (C-40), 70.98 (C-30), 60.17 (C-
1), 59.13 (C-10). Anal. Calcd for C45H49NO10S: C,
67.91; H, 6.21; N, 1.76. Found: C, 67.67; H, 6.14; N,
1.82.
82.24 (C-3), 79.39 (C-4), 79.34 (C-2), 75.12 (C-5),
71.14 (C-20), 67.43 (C-6), 64.49 (C-40), 64.18 (C-10),
62.84 (C-1). HRMS Calcd for C10H22NO10S [M+1]:
m/z 348.0964. Found: m/z 348.0959.
0
0
0
0
3.10. Pent-4-enyl 2,3,5,6-tetra-O-p-methoxybenzyl-a,b-D-
glucofuranoside (19)
To a solution of pent-4-enyl 2,3,5,6-tetra-O-acetyl-a,b-
D-glucofuranoside 18 (3.9g, 9.4mmol) in MeOH
(50mL) was added 1M solution of sodium methoxide
in MeOH (0.02mol, 20mL). The mixture was stirred
overnight at room temperature and then neutralized
with Rexyn 101 [H+]. The Rexyn was filtered off and
the filtrate was concentrated to a straw-colored syrup.
To a stirring solution of this syrup in dry DMF
(50mL) at 0ꢁC, NaH (3g, 47mmol, 5equiv) was added
in small portions. p-Methoxybenzylchloride (7.3g,
47mmol, 5equiv) was then added and the mixture stir-
red at room temperature for 4h. The reaction mixture
was quenched with MeOH and the solution diluted with
water. This mixture was extracted with CH2Cl2
(3 · 30mL). The organic extracts were dried (Na2SO4)
and evaporated to an oil that was purified by column
chromatography (2:1 hexanes–EtOAc) to give 19 as a
mixture of anomers in a 1:1 ratio. (5.7g, 84%). [a]D
À18.5 (c 2.0, CHCl3); 1H NMRof one anomer (CDCl 3):
3.8. 10-(1,4-Dideoxy-D-1,4-imino-galactitol)-4-N-ammo-
nium)-10-deoxy-D-erythritol-30-sulfate (1)
Compound 16 (900mg, 1.1mmol) was dissolved in
AcOH–H2O (5mL) and stirred with Pd–C (100mg)
under H2 (100psi). After 48h, the reaction mixture
was filtered through a pad of Celite, which was subse-
quently washed with MeOH. The combined filtrates
were concentrated and the residue was purified by
column chromatography (1:1 EtOAc–MeOH) to give
the product as an off-white solid (240mg, 62%). [a]D
1
+5.9 (c 0.15, MeOH); H NMR(CD OD): d 4.29 (1H,
3
ddd, J3 ,2 5.9, J3 ,4 a J3 ,4 b 4.1Hz, H-30), 4.16 (1H,
0
0
0
0
0
0
ddd, J2 ,1 a 8.1Hz, J2 ,1 b 7.9, H-20), 4.10 (1H, dd, H-2),
0
0
0
0
4.01 (1H, bs, H-3), 3.91 (2H, dd, ddd, H-40a, H-5),
3.81 (1H, dd, J4 b,4 a 12.1Hz, H-40b), 3.74 (1H, dd,
J6a,6b 11.60, J6a,5 4.3Hz, H-6a), 3.64 (1H, dd, J6b,5
4.3Hz, H-6b), 3.61 (1H, dd, H-10a), 3.54 (1H, d, J1a,1b
11.8Hz, H-1a), 3.25 (1H, dd, J1b,2 3.9Hz, H-1b), 3.19
0
0
d
7.33–6.72 (16H, m, Ar), 5.88–5.74 (1H, m,
OCH2CH2CH2CH@CH2), 5.05–4.89 (m, 3H, H-1,
OCH2CH2CH2CH@CH2), 4.65 (1H, d, JA,B 10.9Hz,
CHPh), 4.54–4.36 (8H, m, 7 CHPh, H-3), 4.25 (1H,
dd, J4,5 8.9, J4,3 4.6Hz, H-4), 4.02 (1H, m, H-5), 3.93
(1H, bd, H-2), 3.80 (3H, s, OCH3), 3.79 (3H, s,
OCH3), 3.78 (3H, s, OCH3), 3.77 (3H, s, OCH3), 3.70–
3.58 (m, 3H, H-6a, H-6b, OCHCH2CH2CH@CH2),
3.42–3.34 (1H, m, OCHCH2CH2CH@CH2), 2.14–2.02
(2H, OCH2CH2CH2CH@CH2), 1.74–1.61 (2H, m,
0
0
(1H, d, J4,5 7.0Hz, H-4), 2.98 (1H, dd, J1 a,1 b 13.1Hz,
13
H-10b). C NMR(CD OD): d 84.08 (C-30), 81.65 (C-
3
3), 80.30 (C-4), 79.55 (C-2), 74.99 (C-5), 72.06 (C-20),
67.38 (C-6), 64.78 (C-10), 64.61 (C-1), 64.41 (C-40).
HRMS Calcd for C10H22NO10S [M+1]: m/z 348.0964.
Found: m/z 348.0961.
3.9. 10-(1,4-Dideoxy-D-1,4-imino-galactitol)-4-N-ammo-
nium)-10-deoxy-L-erythritol-30-sulfate (2)
OCH2CH2CH2CH@CH2). 13C NMR (CDCl3):
d
159.55, 159.32, 159.21, 159.15 (4 · Cpara), 138.53 (OCH-
CH2CH2CH@CH2), 131.39, 131.06, 130.42, 129.92
(4 · Cipso), 129.54, 129.51, 129.46, 129.40 (4 · Cortho),
114.95 (OCHCH2CH2CH@CH2), 114.06–113.78 (4 ·
Compound 17 (190mg, 0.24mmol) was dissolved in 4:1
AcOH–H2O (5mL) and stirred with Pd–C (40mg) under
H2 (100psi). After 48h, the reaction mixture was filtered
through a pad of Celite, which was subsequently washed
with MeOH. The combined filtrates were concentrated
and the residue was purified by column chromatography
(1:1 EtOAc–MeOH) to give the product as an off-white
solid (72mg, 86%). [a]D +24.9 (c 0.75, MeOH); 1H NMR
C
meta), 107.80 (C-1), 85.52 (C-2), 80.63 (C-3), 80.11
(C-4), 76.41, 73.19, 72.29, 71.66 (4 · CH2PH), 71.51
(C-5), 70.86 (C-6), 68.05 (OCHCH2CH2CH@CH2),
55.57–55.37 (4 · OCH3), 30.50 (OCHCH2CH2CH@
CH2), 29.05 (OCHCH2CH2CH@CH2). Anal. Calcd for
C38H46O10: C, 68.86; H, 7.00. Found: C, 68.95; H, 7.14.
0
0
0
0
0
0
(CD3OD): d 4.26 (1H, ddd, J3 ,2 6.3, J3 ,4 b J3 ,4 a 4.3Hz,
H-30), 4.11–4.06 (2H, m, H-20, H-2), 4.01 (1H, bs, H-3),
3.91 (1H, ddd, H-5), 3.88 (1H, dd, H-40a)3.81 (1H, dd,
3.11. 2,3,5,6-Tetra-O-(p-methoxybenzyl)-D-glucitol (20)
J4 b,4 a 12.1Hz, H-40b), 3.73 (1H, dd, J6a,6b 11.5, J6a,5
To a cold solution of 19 (5.7g, 7.8mmol) in 40% aq
CH3CN (80mL) was added NBS (3.48g, 19.5mmol,
2.5equiv). The reaction mixture was stirred at 0ꢁC for
1h in the dark and then quenched with a 10% Na2S2O3
solution. The mixture was concentrated to 20mL and
0
0
4.7Hz, H-6a), 3.63 (1H, dd, J6b,5 5.5Hz, H-6b), 3.40
0
0
(1H, d, J1a,1b 11.0Hz, H-1a), 3.26 (1H, d, J1 a,1 b
10.1Hz, H-10a), 3.16 (1H, dd, H-10b), 3.14–3.09 (2H,
m, H-1b, H-4). 13C NMR(CD 3OD): d 84.08 (C-30),

N. Veerapen et al. / Carbohydrate Research 339 (2004) 2205–2217
2215
the residue extracted with EtOAc (3 · 30mL). The
organic extracts were combined, dried (Na2SO4) and
concentrated. The residue was purified by column chro-
matography (1:1 hexanes–EtOAc) to give the hemiacetal
as a colorless syrup (4.13g, 80%). NaBH4 (0.75g,
25mmol, 4equiv) was added in small amounts to a cold
solution of the hemiacetal in MeOH (50mL). The reac-
tion mixture was then stirred at room temperature for
2h, after which the solvent was evaporated. The residue
was dissolved in water and extracted with EtOAc
(3 · 30mL). The organic extracts were dried (Na2SO4)
and concentrated. The crude diol was purified by col-
umn chromatography (1:1 hexanes–EtOAc) to afford
5.6Hz, H-6b), 2.97 (3H, s, CH3), 2.83 (3H, s, CH3). 13C
NMR(CDCl 3): d 159.69, 159.66, 159.56, 159.52
(4 · Cpara), 130.60–129.51 (4 · Cipso, 4 · Cortho), 114.2–
114.0 (4 · Cmeta), 82.56 (C-4), 76.87 (C-5), 76.47 (C-2),
75.64 (C-3), 74.66, 73.32, 72.79, 72.24 (4 · CH2Ph),
69.13 (C-1), 68.67 (C-6), 55.49 (4 · OCH3), 39.23
(OMs), 37.27 (OMs). Anal. Calcd for C40H50O14S2: C,
58.66; H, 6.15. Found: C, 58.68; H, 6.04.
3.13. 1,4-Anhydro-2,3,5,6-tetra-O-(p-methoxybenzyl)-4-
seleno-D-galactitol (22)
Selenium metal (0.32g, 4.1mmol) was added to liquid
NH3 (60mL) in a À50ꢁC bath, and small pieces of so-
dium (0.20g) were added until a blue color appeared.
NH3 was removed by warming on a water bath, and
DMF was added, and removed under high vacuum to
remove the rest of NH3. A solution of the mesylated
compound 21 (2.2g, 2.7mmol) in DMF (20mL) was
added and the mixture was stirred under Ar in a 50ꢁC
bath for 3h. The reaction mixture was taken up in water
(50mL) and extracted with CH2Cl2 (3 · 25mL). The
combined extracts were washed with water (50mL)
and brine (50mL), then dried (MgSO4) and concen-
trated. The crude product was purified by flash chroma-
tography (3:1 hexanes–EtOAc) to give a light-yellow oil
(1.5g, 80%). [a]D À37.7 (c 6.0, CHCl3); 1H NMR
(CDCl3): d 7.26–6.81 (16H, m, Ar), 4.61, 4.26 (2H, 2d,
JA,B 11.1Hz, CH2Ph), 4.54, 4.47 (2H, 2d, JA,B 11.4Hz,
CH2Ph), 4.43–4.39 (4H, m, 4 · CHPh), 4.09 (1H, ddd,
H-2), 3.94 (1H, dd, J2,3 J3,4 6.7Hz, H-3), 3.82–3.80
(1H, m, H-5), 3.80 (3H s, OCH3), 3.79 (3H, s, OCH3),
3.78 (3H, s, OCH3), 3.75 (3H,s, OCH3), 3.63 (1H, dd,
J4,5 6.3Hz, H-4), 3.51–3.44 (2H, m, H-6a, H-6b), 2.80
(1H, dd, J1a,1b 9.8, J1a,2 5.6Hz, H-1a), 2.96 (1H, dd,
J1b,2 7.6Hz H-1b). 13C NMR(CDCl 3): d 159.20,
159.15, 155.28, 159.20 (4 · Cpara), 132.74–127.99
(4 · Cipso, 4 · Cortho), 113.74–111.06 (4 · Cmeta), 85.69
(C-2), 84.15 (C-3), 78.24 (C-5), 72.99–71.38
(4 · CH2Ph), 70.52 (C-6), 56.23, 55.24 (4 · OCH3),
44.25 (C-4), 21.77 (C-1). Anal. Calcd for C38H44O8Se:
C, 64.49; H, 6.27. Found: C, 64.49; H, 6.19.
20 as a colorless syrup (3.2g, 78%). [a]D À20.5 (c 8.0,
1
CHCl3); H NMR(CDCl ): d 7.28–7.01 (16H, m, Ar),
3
4.64 (1H, d, JA,B 11.3Hz, CHPh), 4.55–4.44 (5H, m, 5
CHPh), 4.30–4.24 (2H, m, CHPh), 3.90–3.85 (2H, m,
H-3, H-4), 3.83 (1H, dd, J1a,1b 10.2, J1a,2 2.9Hz,
H-1a), 3.78 (3H, s, OCH3), 3.77 (3H, s, OCH3), 3.75
(6H, s, 2 · OCH3), 3.78–3.70 (m, 2H, H-6a, H-6b),
3.67 (1H, dd, J1b,2 4.9Hz, H-1b), 3.63–3.57 (2H, m,
H-5, H-2), 3.20–2.81 (2H, bs, 2 · OH). 13C NMR
(CDCl3): d 159.55, 159.49, 159.40, 159.36 (4 · Cpara),
130.83, 130.53, 130.41, 130.35 (4 · Cipso), 129.95,
129.79, 129.65, 129.58 (4 · Cortho), 114.04–113.93
(4 · Cmeta), 77.98, 77.86 (C-5, C-2), 75.36 (C-3), 73.37,
73.29, 71.94, 71.63 (4 · CH2Ph), 70.30 (C-1), 69.44 (C-
4), 60.88 (C-6), 55.46 (4 · OCH3). Anal. Calcd for
C38H46O10: C, 68.86; H, 7.00. Found: C, 68.95; H, 7.14.
3.12. 1,4-Di-O-methanesulfonyl-2,3,5,6-tetra-O-(p-meth-
oxybenzyl)-D-glucitol (21)
Methanesulfonyl chloride (15mL, 0.19mol) was dis-
solved in pyridine (15mL), and this mixture was added
dropwise to a stirred solution of the diol 20 (2.2g,
3.3mmol) in dry CH2Cl2 (30mL). TLC analysis after
3h showed the absence of starting material and formation
of a less polar compound. The reaction mixture was
quenched with solid NaHCO3, and the mixture was stir-
red for 30min. The mixture was then partitioned between
water (100mL) and CH2Cl2 (50mL). The CH2Cl2 extract
was washed with water (2 · 10mL), satd aq NaHCO3
solution (2 · 10mL) and finally with satd aq NaCl solu-
tion (2 · 10mL). The organic extract was then dried
(Na2SO4) and evaporated. The residue was then purified
by flash chromatography using 3:2 hexanes–EtOAc to
give the dimesylated product 21 (2.2g, 82%). [a]D +17.5
(c 4.0, CHCl3); 1H NMR(CDCl 3): d 7.28–6.78 (16H, m,
Ar), 5.14 (1H, dd, J4,5 7.2, J4,3 2.8Hz, H-4), 4.71, 4.38
(2H, 2d, JA,B 11.1Hz, CH2Ph), 4.54–4.40 (6H, m,
3 · CH2Ph), 4.24 (1H, dd, J1a,1b 10.8, J1a,2 5.9Hz,
H-1a), 4.15 (1H, dd, J1b,2 4.1, H-1b), 3.85–3.81 (2H, m,
H-5, H-3), 3.80 (3H, s, OCH3), 3.79 (3H, s, OCH3), 3.78
(3H, s, OCH3), 3.73 (4H, m, OCH3, H-2), 3.68 (1H, dd,
J6a,6b 10.1, J6a,5 5.9Hz, H-6a), 3.56 (1H, dd, J6b,5
3.14. 10-((2,3,5,6-Tetra-O-(p-methoxybenzyl)-1,4-dide-
oxy-1,4-seleno-D-galactitol)-4-Se-selenonium)-20,40-O-
benzylidene-10-deoxy-D-erythritol-30-sulfate (23)
The seleno sugar 22 (110mg, 0.15mmol) and the D-cy-
clic sulfate 10 (48mg, 0.17mmol, 1.2equiv) was dis-
solved in dry acetone (1mL) containing anhydrous
K2CO3 (10mg). The mixture was stirred in a sealed tube
in an oil bath (60ꢁC) for 48h. The solvent was removed
under reduced pressure, and the product was purified by
column chromatography (2:1 EtOAc–MeOH). The
coupled product was obtained as
a white solid
(100mg, 68%). [a]D +1.9 (c 0.53, CHCl3); 1H NMR

2216
N. Veerapen et al. / Carbohydrate Research 339 (2004) 2205–2217
(CD2Cl2) data for the major diastereomer trans-23: d
7.25–6.80 (21H, m, Ar), 5.49 (1H, s, CHPh), 4.50–4.41
(3H, m, H-30, H-2, H-4), 4.40–4.31 (8H, m, 7 CHPh,
H-3), 4.25 (1H, d, JA,B 11.5Hz, CHPh), 4.22 (1H, dd,
vent was then evaporated, and the residue was purified
by column chromatography (1:1 EtOAc–MeOH) to give
the product as a white solid (37mg, 90%). [a]D +11.9 (c
0.25, MeOH); H NMR(CD OD) for major trans iso-
1
3
J1 a,1 b 12.4, J1 a,2 3.1Hz, H-10a), 4.15 (1H, dd, J4 a,4 b
mer: d 4.60 (1H, ddd, J2,3 3.9Hz, H-2), 4.41 (1H, dd,
J3,4 3.9Hz, H-3), 4.32–4.24 (2H, m, H-20, H-30), 4.11–
0
0
0
0
0
0
9.4, J4 a,3 1.3Hz, H-40a), 4.07 (1H, ddd, J2 ,3 9.3,
0
0
0
0
J2 ,1 a J2 ,1 b 3.2Hz, H-20), 3.88 (1H, dd, H-10b), 3.88–
3.76 (13H, m, H-1a, 4 · OCH3), 3.75–3.70 (2H, m, H-
5, H-40b), 3.49 (1H, dd, H-1b), 3.45 (1H, dd, J6a,6b
10.8, J6a,5 3.5Hz, H-6a), 3.41 (1H, dd, J6b,5 4.4Hz, H-
6b). 13C NMR(CD 2Cl2): d 160.0, 159.98, 159.94,
159.75 (4 · Cpara), 137.09 (1 · Cipso), 130.39–126.77 (21
CAr), 114.16 (2C), 114.13, 114.04 (4 · Cmeta), 101.95
(CHPh), 83.44 (C-3), 83.07 (C-2), 76.95 (C-20), 73.76
(C-5), 73.49, 72.16, 72.04, 71.82 (4 · CH2Ph), 70.27 (C-
4), 69.48 (C-6), 69.42 (C-40), 67.62 (C-30), 45.13 (C-10),
44.55 (C-1). Anal. Calcd for C45H56O14SSe: C, 60.05;
H, 5.76. Found: C, 60.03; H, 5.61.
4.04 (2H, m, H-4, H-5), 3.98 (1H, dd, J1 a,1 b 12.1,
0
0
0
0
0
0
0
J1 a,2 3.5Hz, H-10a), 3.92 (1H, dd, J4 a,4 b 12.2, J4 a,3
0
3.8Hz, H-40a), 3.86 (1H, dd, J1 b,2 6.7Hz, H-10b), 3.81
(1H, dd, J4 b,3 4.1Hz, H-40b), 3.76 (1H, d, J6a,6b 11.7,
J6a,5 3.5Hz, H-6a), 3.72–3.67 (2H, m, H-1a, H-6b),
3.57 (1H, dd, J1b,2 4.2Hz, H-1b). 13C NMR(CD OD):
d 80.25 (C-30), 77.60 (C-3), 70.88 (C-2), 68.96 (C-4),
66.58 (C-5), 60.57 (C-20), 64.77 (C-6), 60.59 (C-40),
48.66 (C-10), 44.92 (C-1). HRMS: Calcd for
C10H21O10SSe [M+1]: m/z 413.0021. Found: m/z
413.0022.
0
0
0
0
0
0
0
0
3
3.17. 10-(1,4-Dideoxy-D-1,4-seleno-galactitol)-4-Se-
selenonium)-10-deoxy-L-erythritol-30-sulfate (4)
3.15. 10-((2,3,5,6-Tetra-O-(p-methoxybenzyl)-1,4-dide-
oxy-1,4-seleno-D-galactitol)-4-Se-selenonium)-20,40-O-
benzylidene-10-deoxy-L-erythritol-30-sulfate (24)
Compound 24 (150mg, 0.15mmol) was dissolved in
TFA (5mL) and stirred for 1h at room temperature.
The solvent was then evaporated, and the residue was
purified by column chromatography (1:1 EtOAc–
MeOH) to give the product as a white solid (58mg,
1
92%). [a]D +5.7 (c 0.15, MeOH); H NMR(CD OD)
data for major trans isomer: d 4.64 (1H, ddd, J2,3
4.1Hz, H-2), 4.44 (1H, dd, J3,4 3.8Hz, H-3), 4.30 (1H,
ddd, J2 ,1 a J2 ,3 6.1, J2 ,1 b 2.4Hz, H-20), 4.26
(1H, ddd, H-30), 4.18 (1H, dd, J4,5 5.2Hz, H-4), 4.01
The seleno sugar 22 (150mg, 0.21mmol) and the L-cyclic
sulfate 11 (70mg, 0.25mmol, 1.2equiv) was dissolved in
dry acetone (1mL) containing anhydrous K2CO3
(10mg). The mixture was stirred in a sealed tube in an
oil bath (60ꢁC) for 48h. The solvent was removed under
reduced pressure, and the product was purified by col-
umn chromatography (2:1 EtOAc–MeOH). The coupled
3
0
0
0
0
0
0
product was obtained as a white solid (128mg, 62%).
1
[a]D +2.9 (c 0.35, CHCl3); H NMR(CD Cl2) data for
the major diastereomer trans-24: d 7.42–6.80 (21H, m,
0 0
(1H, ddd, J5,6a J5,6a 12.1Hz, H-5), 3.94 (1H, dd, J4 a,4 b
12.1, J4 a,3 3.8Hz, H-40a), 3.91–3.88 (2H, m, H-10a,
2
0 0
Ar), 5.35 (1H, s, CHPh), 4.56 (1H, dd, J4 a,4 b 11.0,
0
H-10b), 3.83 (1H, dd, J4 b,3 3.8Hz, H-40b), 3.79–3.67
(3H, m, H-1a, H-6a, H-6b), 3.57 (1H, dd, J1b,1a 12.2,
0
0
0
0
J4 a,3 5.4Hz, H-40a), 4.47, 4.28 (2H, d, JA,B 11.3Hz,
CH2Ph), 4.41–4.38 (1H, m, H-2), 4.37–4.29 (9H, m,
0
J1b,2 4.2Hz, H-1b). 13C NMR(CD OD): d 81.92 (C-
3
6CHPh, H-3, H-4, H-30), 4.10 (1H, ddd, J2 ,3 9.8,
30), 81.67 (C-3), 79.43 (C-2), 72.62 (C-4), 70.38 (C-5),
67.64 (C-20), 65.98 (C-6), 61.83 (C-40), 48.63 (C-10),
45.89 (C-1). Anal. Calcd for C10H20O10SSe: C, 29.20;
H, 4.90. Found: C, 29.00; H, 4.63.
0
0
J2 ,1 a 5.5, J2 ,1 b 4.6Hz, H-20), 3.99 (1H, dd, J1 a,1 b
12.2, H-10a), 3.87 (1H, dd, H-10b), 3.79, 3.78, 3.77,
3.76 (12H, 4s, 4 · OCH3), 3.72–3.66 (2H, m, H-5, H-
0
0
0
0
0
0
40b), 3.62 (1H, dd, J1 a,1 b 12.2, J1 a,2 2.8Hz, H-1a),
3.47–3.40 (3H, m, H-1b, H-6a, H-6b). 13C NMR
(CD2Cl2): d 160.0, 159.98, 159.94, 159.75 (4 · Cpara),
137.09 (1 · Cipso), 130.13–126.39 (21 CAr), 114.16 (2C),
114.13, 114.04 (4 · Cmeta), 101.56 (CHPh), 83.56 (C-2),
82.65 (C-3), 76.76 (C-20), 73.68 (C-5), 73.44, 72.19,
71.94, 71.86 (4 · CH2Ph), 70.28 (C-4), 69.70 (C-40),
69.45(C-6), 68.37 (C-30), 47.94(C-10), 44.66 (C-1). Anal.
Calcd for C45H56O14SSe: C, 60.05; H, 5.76. Found: C,
60.00; H, 5.49.
0
0
0
0
3.18. Enzyme assays
Enzyme assays were carried out following a procedure
similar to that described by Zhang and Liu.46 Mutase
(20lg/mL) in 100mM MOPS, pH8.0 was pre-incubated
with 20mM freshly prepared Na2S2O on ice for 1min.
Inhibitors were then added and incubated for an addi-
tional minute. UDP-Galf (63lM) was then added and
the reaction was allowed to proceed at room tempera-
ture. The reactions were stopped at different times by
addition of ice-cold HCl and immediate freezing in liq-
uid nitrogen. Samples were analyzed by HPLC (Agilent
3.16. 10-(1,4-Dideoxy-D-1,4-seleno-galactitol)-4-Se-
selenonium)-10-deoxy-D-erythritol-30-sulfate (3)
1100 series) using
a 5-lm Luna C18(2) column
Compound 23 (100mg, 0.1mmol) was dissolved in TFA
(5mL) and stirred for 1h at room temperature. The sol-
(4.6 · 250mm, Phenomenex). The column was run iso-
cratically with 1.5% acetonitrile in 50mM Et3NHOAc

N. Veerapen et al. / Carbohydrate Research 339 (2004) 2205–2217
2217
17. Unlingil, U. M.; Zhou, S.; Yuwaraj, S.; Sarkar, M.;
Schachter, H.; Rini, J. M. EMBO J. 2000, 19, 5269–
5280.
18. Saxena, I. M.; Brown, R. M., Jr.; Fevre, M.; Geremia,
R. A., B.; Henrissat, B. J. Bacteriol. 1995, 177, 1419–1424.
19. Charnock, S. J.; Davies, J. Biochemistry 1999, 38,
6380–6385.
buffer, pH6.8 at a flow rate of 1mL/min. The two sugars
separated with base line resolution, and the extent of
conversion was determined by integration of the peaks
and Eq. 1:
ðarea of UDP À Galp peakÞ=½ðarea of UDP
20. Lee, R. E.; Smith, M. D.; Nash, R. J.; Griffiths, R. C.;
McNeil, M.; Grewal, R. K.; Yan, W.; Besra, G. S.;
Brennan, P. J.; Fleet, W. J. Tetrahedron Lett. 1997, 38,
6733–6736.
À Galp peakÞ þ ðarea of UDP À Galf peakÞꢀ ð1Þ
21. Iminosugars as Glycosidase Inhibitors: Norjirimycin and
Beyond; Stutz, A. E., Ed.; Wiley-VCH: Weinheim: New
Acknowledgements
¨
York, 1999.
We are grateful to Dr. A. Ghavami for synthesizing the
L-cyclic sulfate 11, Dr. B. D. Johnston for helpful dis-
cussions, and to the Natural Sciences and Engineering
Research Council of Canada for financial support. We
also acknowledge Dr. Jim Naismith for his gift of the
K. pneumonia mutase construct and for performing
some preliminary enzyme inhibition experiments. The
UDP-Galf used in these experiments was a generous gift
from Dr. Laura Kiessling.
22. Yoshikawa, M.; Murakami, T.; Shimada, H.; Matsuda,
H.; Yamahara, J.; Tanabe, G.; Muraoka, O. Tetrahedron
Lett. 1997, 38, 8367–8370.
23. Yoshikawa, M.; Murakami, T.; Yashiro, K.; Matsuda, H.
Chem. Pharm. Bull. 1998, 46, 1339–1340.
24. Ghavami, A.; Johnston, B. D.; Pinto, B. M. J. Org. Chem.
2001, 66, 2312–2317.
25. Ghavami, A.; Johnston, B. D.; Jensen, M. T.; Svensson,
B.; Pinto, B. M. J. Am. Chem. Soc. 2001, 123, 6268–6271.
26. Ghavami, A.; Chen, J. J.; Pinto, B. M. Carbohydr Res.
2004, 339, 401–407.
27. Lee, R. E.; Smith, M. D.; Pickering, L.; Fleet, W. J.
Tetrahedron Lett. 1999, 40, 8689–8692.
28. Bernotas, R. C. Tetrahedron Lett. 1990, 31, 469–472.
29. Lombardo, M.; Fabbroni, S.; Trombini, C. J. Org. Chem.
2001, 66, 1264–1268.
30. Pham-Huu, D. P.; Gizaw, Y.; Miller, J. N.; Petrus, L.
Tetrahedron Lett. 2002, 43, 383–385.
31. Ito, H.; Matsumoto, M.; Yoshizawa, T.; Takao, K.;
Kobayashi, S. Tetrahedron Lett. 1997, 38, 9009–9012.
32. Laguzza, B. C.; Ganem, B. Tetrahedron Lett. 1981, 22,
1483–1486.
References
1. Brennan, P. J.; Nikaido, H. Annu. Rev. Biochem. 1995, 64,
29–63.
2. de Lederkremer, R. M.; Casal, O. L.; Alves, J. M.; Colli,
W. FEBS Lett. 1980, 116, 25–29.
3. Notermans, S.; Veeneeman, G. H.; van Zuylen, C. W. E.
M.; Hoogerhout, P.; van Boom, J. H. Mol. Immunol. 1988,
25, 975–979.
33. Yuasa, H.; Takada, J.; Hashimoto, H. Tetrahedron Lett.
2000, 41, 6615–6618.
34. Pinto, B. M.; Johnston, B. D.; Nagelkerke, R.; Juaristi, E.;
4. de Lederkremer, R. M.; Colli, W. Glycobiology 1995, 5,
547–552.
5. de Lederkremer, R. M.; Bertello, L. E. Curr. Pharm.
Design. 2001, 7, 1165–1179.
´
Gonzalez, E. A. Can. J. Chem. 1989, 12, 2067–2070.
35. Furneaux, R. H.; Rendle, M. P.; Sims, I. M. J. Chem.
Soc., Perkin Trans. 1 2000, 2011–2014.
6. De Arruda, M. V.; Colli, W.; Zingales, B. Eur. J. Biochem.
1989, 182, 413–421.
36. Gurjar, M. K.; Reddy, L. K.; Hotha, S. J. Org. Chem.
2001, 66, 4657–4660.
37. Velty, R.; Benvegnu, T.; Gelin, M.; Privat, E.; Plusquellec,
D. Carbohydr Res. 1997, 299, 7–14.
38. Mootoo, D. R.; Date, V.; Fraser-Reid, B. J. Am. Chem.
Soc. 1988, 110, 2662.
7. Stevenson, G.; Neal, B.; Liu, D.; Hobbs, M.; Packer, N.
H.; Batley, M.; Redmond, J. W.; Lindquist, L.; Reeves, P.
J. Bacteriol. 1994, 176, 4144–4156.
8. Nassau, P. M.; Martin, S. L.; Brown, R. E.; Weston, A.;
Monsey, D.; McNeil, M. R.; Duncan, K. J. Bacteriol.
1996, 178, 1047–1052.
39. Sanders, D. A. R.; Staines, A. G.; McMahon, S. A.;
McNeil, M. R.; Whitfield, C.; Naismith, J. H. Nat. Struct.
Biol. 2001, 8, 858–863.
40. Busch, C.; Hofmann, F.; Gerhard, R.; Aktories, K. J.
Biol. Chem. 2000, 275, 13228–13234.
41. Gastinel, L. N.; Cambillau, C.; Bourne, Y. EMBO J. 1999,
18, 3546–3557.
42. Scherman, M. S.; Winans, K. A.; Stern, R. J.; Jones, V.;
Bertozzi, C. R.; McNeil, M. R. Antimicrob. Agents and
Chemo. 2003, 47, 378–383.
43. Weimar, T.; Meyer, B.; Peters, T. J. Biomol. NMR. 1993,
3, 399–414.
44. Bauer, C.; Freeman, R.; Frenkiel, T.; Keeler, J.; Shaka, A.
J. J. Magn. Reson. 1984, 58, 442–457.
45. Bradford, M. M. Anal. Biochem. 1976, 72, 248–254.
46. Zhang, Q.; Liu, H. J. Am. Chem. Soc. 2000, 122,
9065–9070.
9. Pan, F.; Jackson, M.; Ma, Y.; McNeil, M. J. Bacteriol.
2001, 183, 3991–3998.
10. Huang, Z.; Zhang, Q.; Liu, H. Bioorg. Chem. 2003, 31,
494–502.
11. Soltero-Higgin, M.; Carlson, E. E.; Gruber, T. D.;
Kiessling, L. L. Nat. Struct. Biol. 2004, 11, 539–543.
12. Fullerton, S. W. B.; Daff, S.; Sanders, D. A. R.; Ingledew,
W. J.; Whitfield, C.; Chapman, S. K.; Naismith, J. H.
Biochemistry 2003, 42, 2104–2109.
13. Barlow, J. N.; Blanchard, J. S. Carbohydr. Res. 2000, 328,
473–480.
14. Barlow, J. N.; Girvin, M. E.; Blanchard, J. S. J. Am.
Chem. Soc. 1999, 121, 6968–6969.
15. Burton, A.; Wyatt, P.; Boons, G. J. J. Chem. Soc., Perkin
Trans. 1 1997, 2375–2382.
16. Zhang, Q.; Liu, H. J. Am. Chem. Soc. 2001, 123,
6756–6766.