Non-natural aldofuranosides as substrates of a β-glucosidase

Article abstract of DOI:10.1016/j.tetasy.2004.11.027

Based on glycosidase inhibitory activities of some known 1,4-iminoalditols, four aldofuranosides, (4-nitro)phenyl β-d-glucofuranoside, -β-d-galactofuranoside, -α-l-idofuranoside and -α-l- altrofuranoside, were identified as possible substrates of glucosid

Full text of DOI:10.1016/j.tetasy.2004.11.027

Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 16 (2005) 159–165
Non-natural aldofuranosides as substrates of a b-glucosidase
Andreas Tauss,a Peter Greimel,a Karen Rupitz,b Andreas J. Steiner,a Arnold E. Stutz,a,*
¨
Stephen G. Withersb,* and Tanja M. Wrodnigga
aGlycogroup, Institut fu¨r Organische Chemie der Technischen Universita¨t Graz, Stremayrgasse 16, A-8010 Graz, Austria
bDepartment of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC, Canada V6T 1Z1
Received 29 October 2004; accepted 16 November 2004
Available online 21 December 2004
Abstract—Based on glycosidase inhibitory activities of some known 1,4-iminoalditols, four aldofuranosides, (4-nitro)phenyl b-D-
glucofuranoside, -b-D-galactofuranoside, -a-L-idofuranoside and -a-L-altrofuranoside, were identified as possible substrates of glu-
cosidases and galactosidases. Three of them were found to be accepted by the b-glucosidase from Agrobacterium sp.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
ceptible to inhibition by five- and seven-membered ring
iminoalditols as well as suitably substituted aminocyclo-
pentanes whose structures are not as obviously suitable
for the purpose. Such compounds include well-known
2,5-dideoxy-2,5-imino-D-mannitol 5, a very powerful
glucosidase inhibitor1 and 1,6-dideoxy-1,6-imino-L-idi-
tol 6, a good inhibitor of a wide range of various
glycosidases.2,3
Iminosugars and related structures, including iminoald-
itols, are powerful reversible inhibitors of glycosidases.
Glycosidases, a class of enzymes well known for their
high specificity for the respective natural substrate, are
inhibited by these close structural relatives of their
substrates/products. For example (Scheme 1), 1-deoxy-
nojirimycin, 1, and castanospermine 2 are powerful
inhibitors of D-glucopyranosidases, 1-deoxymannojiri-
mycin 3 inhibits D-mannosidases and the corresponding
galacto-configured iminoalditol 4 is a strong inhibitor of
galactosidases. Interestingly, over the past two decades,
it has been found that many glycosidases are also sus-
Other examples (Scheme 2), just to mention a few, are
1,4-dideoxy-1,4-imino-L-allitol 7 and its epimer at C-5,
1,4-dideoxy-1,4-imino-D-talitol 8, both of which are
potent inhibitors of human liver lysosomal as well as
Golgi II a-mannosidases4 and 1,4,6-trideoxy-6-fluoro-
1,4-imino-D-mannitol 9, a powerful inhibitor of human
liver mannosidases.5 1,4-Dideoxy-1,4-imino-D-glucitol
10 inhibits6 b-glucosidase from sweet almonds with a
Ki of 125 lM (pH 5.2) being about as potent as 1-deoxy-
nojirimycin 1 with this enzyme at the same pH value.
The corresponding epimer at C-5, 1,4-dideoxy-1,4-imi-
no-L-iditol 11 was found to be a potent inhibitor of
human liver lysosomal a-galactosidase.7 2-Acetamido-
1,2,4-trideoxy-1,4-imino-D-galactitol 12 is a good inhibi-
tor of hexosaminidases.8
H
N
H
N
N
HO
HO
HO
HO
HO
OH
OH
HO
OH
HO
OH
OH
OH
OH
OH
1
2
3
H
N
H
N
H
N
HO
HO
OH
H
HO
H
HO
Based on the traditional and common conclusion that
close relatives of good substrates are frequently good
inhibitors, we envisaged that glycosides, which are struc-
turally related to such Ôunusually shapedÕ inhibitors
might turn out to be suitable substrates for glycosidases.
OH
OH
4
HO
OH
5
6
Scheme 1.
*
To probe this hypothesis, with the aid of Dreiding mod-
els as well as computer assisted molecular modelling
Corresponding authors. Tel.: +43 316 873 8247; fax: +43 316 873
8740 (A.E.S.); e-mail: stuetz@orgc.tu-graz.ac.at
0957-4166/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2004.11.027

160
A. Tauss et al. / Tetrahedron: Asymmetry 16 (2005) 159–165
OH
OH
OH
H
N
H
N
H
N
F
HO
HO
H
H
H
HO
OH
HO
OH
HO
OH
7
8
9
OH
H
OH
OH
H
H
N
H
N
H
HO
HO
HO
N
H
HO
OH
HO
OH
HO
NHAc
10
11
12
Scheme 2.
(Fig. 1, Scheme 3), we identified b-D-gluco-13 and a-L-
idofuranosides 14 as potential substrates for galactosid-
ases and b-D-galacto-15 as well as a-L-altrofuranosides
16 as likely suitable substrates for b-glucosidases.
emulsin (but not E. coli K 12 and bovine liver b-galacto-
sidases) solvolysed phenyl D-gluco- as well as D-
galactofuranosides.
The b-glucosidase from Agrobacterium sp. has substan-
tial b-galactosidase activity and its inhibition by a
variety of iminosugars has been probed. Consequently
The concept was further supported by the work of Yos-
hida and Nobuko9 in 1960s, who had found that almond
Figure 1. Superpositions of (a) pNP b-D-glucofuranoside, (b) pNP a-L-idofuranoside, (c) pNP b-D-galactofuranoside and (d) pNP a-L-
altrofuranoside with pNP b-D-glucopyranoside (green).

A. Tauss et al. / Tetrahedron: Asymmetry 16 (2005) 159–165
161
2.1.3. (4-Nitro)phenyl b- and a-D
-galactofurano-
sides. Known compounds 15 and 29 were prepared
from the corresponding known per-O-benzoylated
galactofuranoses 30 and 31 which were isolated from
the reaction mixture obtained by per-O-benzoylation
of D-galactose following available protocols (Scheme
6).18,19
OH
H
OH
HO
O
O
O
HO
HO
O
O
pNP
pNP
pNP
H
HO
13
OH
HO
OH
14
OH
OH
H
HO
O
O
O
2.1.4. (4-Nitro)phenyl a- and b-L-altrofuranosides. Di-
O-mesylation of 3-O-acetyl-1,2-O-isopropylidene-a-D-
galactofuranose,20 32 (available in three steps from
D-galactose by acid-catalysed reaction with acetone, 3-O-
acetylation of the resulting 1,2:5,6-di-O-isopropylidene-
a-D-galactofuranose21 and standard 5,6-O-deprotection)
led to the corresponding 5,6-di-O-methanesulfonyl
sugar 33. Its reaction with sodium acetate in glacial
acetic acid furnished 3,5,6-tri-O-acetyl-1,2-O-isopropyl-
idene-b-L-altrofuranose 34 which by acetolysis was
converted into an anomeric mixture of per-O-acetyl-
ated altrofuranoses 35 and 36, the latter present only
in very small proportions. Conventional reaction
gave, after chromatography, the corresponding
protected (4-nitro)phenyl a-L-altrofuranoside 37 as a
single product from which pure 16 was obtained by
pNP
H
HO
15
OH
HO
OH
16
Scheme 3.
it was chosen as the initial enzyme to test for the
concept.
Whereas L-ido- as well as L-altrofuranosides have not
been discovered as natural products, as yet, glucofur-
anosides have frequently been isolated from plants, for
example as constituents of glycosylated flavonoids,10
and galactofuranosides have been found in bacterial,
protozoan as well as fungal polysaccharides.11
´
Zemplen saponification followed by chromatography
(Scheme 7).
2.2. Enzymatic studies
2. Results and discussion
2.1. Syntheses
Compounds 13–16 were screened as substrates for Agro-
bacterium sp. b-glucosidase employing (4-nitro)phenyl
b-D-glucopyranoside as the standard (Km = 0.072 mM,
kcat = 141 sꢀ1).
2.1.1. (4-Nitro)phenyl a- and b-D-glucofuranosides. (4-
Nitro)phenyl b- 13 and a-D-glucofuranosides 17 are
known12 and were prepared (Scheme 4) via known
1,2,3,5,6-penta-O-acetyl-a/b-D-glucofuranose13 18, itself
prepared via acetolysis14 of 3,5,6-tri-O-acetyl-1,2-O-iso-
propylidene-a-D-glucofuranose 19.
The best substrate turned out to be the a-L-altrofurano-
side 16 with Km = 1.69 mM and a kcat = 81 sꢀ1, followed
by the b-D-glucofuranoside 13 (Km = 1.33 mM, kcat
=
12 sꢀ1). Significantly less accepted was the a-L-idofur-
anoside 14 (Km = 0.92 mM, kcat = 0.9 sꢀ1). Interestingly,
b-D-galactofuranoside 15 was found not to be a sub-
strate at all.
2.1.2. (4-Nitro)phenyl a- and b-L-idofuranosides. Con-
ventional 3,5,6-tri-O-acetylation of 1,2-O-isopropylid-
ene-b-L-idofuranose,15 20 (prepared by NaBH4
reduction of 1,2-O-isopropylidene-b-L-idofuranurono-
6,3-lactone,16 21, or from 1,2-O-isopropylidene-3,5,6-
tri-O-methanesulfonyl-a-D-glucofuranose,17 22) to give
triacetate 23 followed by acetolysis gave an anomeric
mixture of 1,2,3,5,6-penta-O-acetyl-L-idofuranoses 24
and 25, which was converted to the corresponding mix-
ture of per-O-acetylated (4-nitro)phenyl furanosides,
from which the pure compounds 26 and 27 were iso-
lated. Individual treatment with NaOMe in methanol
furnished pure samples of free idofuranosides 14 and
28 (Scheme 5).
Previous kinetic studies on Agrobacterium sp. b-glucosi-
dase22 have revealed that the most important interac-
tions between the enzyme and the substrate at the
transition state are those formed with OH-2 and OH-
3, contributing at least 18 and 7 kJmolꢀ1, respectively,
to transition state stabilisation. The modelling depicted
in Figure 1, therefore, sought to optimise these interac-
tions, as well as those with the ring oxygen, which is
known to contribute substantially. Interactions at OH-
4 and OH-6 are relatively less important, contributing
2.5–3 kJmolꢀ1. Interestingly, of the four compounds
OAc
H
OAc
H
OH
AcO
O
O
AcO
a
O
OAc
b, c
HO
+
O
O
pNP
13
H
AcO
O
AcO
OAc
HO
OH
19
18
17
Scheme 4. Reagents and conditions: (a) Ac2O, HOAc, H2SO4; (b) (4-NO2)PhOH, PTSA, PhMe; (c) NaOMe, MeOH.

162
A. Tauss et al. / Tetrahedron: Asymmetry 16 (2005) 159–165
OR
OMs
H
HO
O
H
O
O
RO
O
O
MsO
O
O
H
O
O
RO
O
MsO
O
21
22
20: R =H
23: R =Ac
OAc
H
OAc
H
AcO
O
OAc
OAc
AcO
O
OAc
OAc
a
b
23
+
AcO
24
AcO
25
OAc
OAc
H
AcO
O
OpNP
OAc
AcO
O
OpNP
+
H
AcO
AcO
OAc
27
26
c
c
OH
H
OH
H
HO
O
OpNP
OH
HO
O
OpNP
OH
HO
HO
14
28
Scheme 5. Reagents and conditions: (a) Ac2O, HOAc, H2SO4; (b) (4-NO2)PhOH, PTSA, PhMe; (c) NaOMe, MeOH.
BzO
BzO
BzO
O
OBz BzO
O
OBz
OBz
a
D-galactose
pyranoses
+
H
H
+
BzO
OBz
BzO
30
31
b, c
b, c
HO
HO
HO
O
OpNP HO
OH
O
OpNP
H
H
HO
HO
OH
15
29
Scheme 6. Reagents and conditions: (a) BzCl, pyridine, (b) (4-NO2)PhOH, PTSA, PhMe; (c) NaOMe, MeOH.
modelled in Figure 1, the a-L-altrofuranoside arguably
provided the best overlay of interactions at the 4 and
6-positions, with O-5 of the altro-sugar overlaying nicely
with O-4 of the glucopyranoside and O-6 of each sugar
overlaying well. This is nicely consistent with the fact
that the a-L-altrofuranoside was indeed the best sub-
strate. At the level of modelling conducted it was not
really possible to rank the relative resemblances of the
other sugars, especially when it is considered that the
most important interactions are those formed at the
transition state, in which the sugar likely adopts a half
chair conformation.
None of the furanosides was accepted by the b-galacto-
sidase from Xanthomonas manihotis, a Family 35
enzyme, which is quite specific for b-D-galactopyrano-
sides. This is consistent with the fact that this enzyme
is more specific with respect to the interactions formed

A. Tauss et al. / Tetrahedron: Asymmetry 16 (2005) 159–165
163
OH
H
OMs
OAc
HO
O
O
MsO
a
O
O
AcO
b
O
O
c
H
H
AcO
O
AcO
O
AcO
O
32
33
34
OAc
H
OAc
H
OR
H
AcO
O
OAc
OAc
AcO
O
OAc
OAc
RO
O
OpNP
OR
d, e
+
AcO
35
AcO
36
RO
37
16
R = Ac
R = H
Scheme 7. (a) MesCl, pyridine, CH2Cl2; (b) NaOAc, HOAc, DMF; (c) Ac2O, HOAc, H2SO4; (d) (4-NO2)PhOH, PTSA, PhMe; (e) NaOMe, MeOH.
at O-4 and O-6 in the pyranoside substrates. Earlier
studies had shown that interactions at the 6-position
contribute at least 20 kJmolꢀ1, with similar values being
likely for the 4-position, based upon rates with the
gluco-substrate.23 As expected, considering the ÔwrongÕ
anomeric configurations, compounds 13–16 were not
hydrolysed by the a-galactosidase from green coffee
beans (Family 27).
station by Silicon Graphics using the BGFS-minimiser
and the Tripos Force Field.
4.1. (4-Nitro)phenyl a- and b-D-glucofuranosides
Compounds 17 and 13, as well as (4-nitro)phenyl a-
and b-D-galactofuranosides 15 and 29 were prepared
by available methods as mentioned in Results and
discussion.
3. Conclusion
4.2. (4-Nitro)phenyl 2,3,5,6-tetra-O-acetyl-a-L-idofur-
anoside 26 and (4-nitro)phenyl 2,3,5,6-tetra-O-acetyl-b-L-
idofuranoside 27
In conclusion, some of the furanosides tested, especially
the altrofuranoside with the Agrobacterium sp. b-gluco-
sidase, were remarkably good substrates. However, this
does not appear to be a completely general phenome-
non, with success possibly being related more to the pro-
miscuity of the enzyme tested.
To a solution of 1,2,3,5,6-penta-O-acetyl-a/b-L-idofura-
nose 24 and 2525 (1.56 g, 4.0 mmol) in toluene (40 mL),
(4-nitro)phenol (2.60 g, 18.7 mmol) and (4-toluene)sul-
fonic acid (75 mg) were added and the mixture was kept
under reflux in a Dean–Stark apparatus for 3 h. After
cooling to ambient temperature, the solution was
washed with 5% aqueous bicarbonate, dried (Na2SO4),
the solvent was removed under reduced pressure and
the oily residue was chromatographed on silica gel
(cyclohexane/ethyl acetate 4:1, v/v) to give, as the non-
polar component, syrupy b-anomer 27 (263 mg, 14%).
4. Experimental
Optical rotations were measured on a JASCO Digital
Polarimeter or with a Perkin–Elmer 341 with a path
length of 10 cm. NMR spectra were recorded at 200 as
well as 500 MHz (1H), and at 50 and 125 MHz (13C).
CDCl3 was employed for protected compounds and
D2O as well as MeOH-d4 for free sugars. Chemical shifts
are listed in delta employing residual, not deuterated,
solvent as the internal standard. The signals of aromatic
substituents as well as protecting groups were found in
the expected regions and are not listed explicitly. Struc-
tures of crucial intermediates were unambiguously
assigned by 1D-TOCSY and HSQC experiments. TLC
was performed on precoated aluminium sheets (E.
Merck 5554). Compounds were detected by staining
with concd H2SO4 containing 5% vanillin.
Found: C, 51.22; H, 4.99; C20H23NO12 requires: C,
20
D
51.18; H, 4.94; ½aꢁ ¼ þ67:9 (c 4.6, CH2Cl2); dH
(CDCl3) 6.05 (d, 1H, J1,2 4.6 Hz, H-1), 5.68 (dd, 1H,
J2,3 8.3 Hz, J3,4 8 Hz, H-3), 5.16 (ddd, 1H, J4,5 2.0 Hz,
0
J5,6 5.1 Hz, J5,6 7.5 Hz, H-5), 5.14 (dd, 1H, H-2), 4.64
0
(dd, 1H, H-4), 4.24 (dd, 1H, J6,6 11.5 Hz, H-6), 4.15
(dd, 1H, H-60); dC (CDCl3) 96.5 (C-1), 75.1, 74.9, 73.3,
68.0 (C-2, C-3, C-4, C-5), 62.4 (C-6).
Next eluted was syrupy a-anomer 26, (480 mg, 25.6%),
Found: C, 51.11; H, 5.00; C20H23NO12 requires: C,
20
D
(CDCl3) 5.65 (s, 1H, H-1), 5.48 (n.r., 1H, H-3), 5.24
51.18; H, 4.94; ½aꢁ ¼ ꢀ153:1 (c 4.6, CH2Cl2); dH
For column chromatography Silica Gel 60 (E. Merck)
was used.
(br s, 1H, H-2), 5.24 (ddd, 1H, J4,5 5.3 Hz, J5,6 4.3 Hz,
0
J6,6 11.8 Hz, H-6), 3.95 (dd, 1H, H-60); dC (CDCl3)
103.2 (C-1), 80.3, 79.7, 74.4, 68.7 (C-2, C-3, C-4, C-5),
62.6 (C-6).
J5,6 6.8 Hz, H-5), 4.68 (dd, 1H, H-4), 4.21 (dd, 1H,
0
Molecular modelling was performed as previously de-
scribed24 employing Sybyl versions on an Octane work-

164
A. Tauss et al. / Tetrahedron: Asymmetry 16 (2005) 159–165
4.3. (4-Nitro)phenyl a-L-idofuranoside 14
upy material can immediately be used in the next step.
An analytical sample was obtained by chromatography.
Found: C, 37.28; H, 5.35; C13H22O11S2 requires:
To a 5% methanolic solution of 26 (500 mg, 1.07 mmol),
a 1 M NaOMe solution (three drops) was added at 0 ꢁC.
After completion of the deprotection (TLC, ethyl
acetate), the solution was neutralised with ion exchange
resin Amberlite IR 120 [H+], filtered and concentrated
under reduced pressure. The residue was dissolved in
ethyl acetate and filtered over a short plug of silica
gel to give pure 14 (290 mg, 90%). Found: C, 47.89;
20
C, 37.32; H, 5.30; ½aꢁ ¼ ꢀ22:8 (c 3.8, CHCl3); dH
D
(CDCl3) 5.99 (d, 1H, J1,2 3.7 Hz, H-1), 5.12 (d, 1H,
J2,3 1.5 Hz, H-3), 5.05 (ddd, 1H, J4,5 8.8 Hz, J5,6
0
3.7 Hz, J5,6 5.5 Hz, H-5), 4.62 (d, 1H, H-2), 4.60 (dd,
0
1H, J6,6 12.1 Hz, H-6), 4.24 (dd, 1H, H-4), 3.42 (dd,
1H, H-60); dC (CDCl3) 106.2 (C-1), 84.3, 83.9, 78.9,
76.6 (C-2, C-3, C-4, C-5), 67.9 (C-6).
H, 5.05; C12H15NO8 requires: C, 47.84; H, 5.02;
20
½aꢁ ¼ ꢀ17:1 (c 2.1, MeOH); dH (MeOH-d4) 5.65 (s,
4.7. 3,5,6-Tri-O-acetyl-1,2-O-isopropylidene-b-L-altro-
furanose 34
D
1H, H-1), 4.39 (m, 1H, H-4), 4.37 (br s, 1H, H-2), 4.22
(d, 1H, J3,4 4.4 Hz, H-3), 3.96 (ddd, 1H, J5,6 4.8 Hz,
0
0
J5,6 5.9 Hz, H-5), 3.67 (dd, 1H, J6,6 11.2 Hz, H-6),
3.58 (dd, 1H, H-60); dC (MeOH-d4) 107.6 (C-1), 85.1,
82.7, 77.4, 72.7 (C-2, C-3, C-4, C-5), 64.3 (C-6).
To a 5% solution of 33 (5.10 g, 12.2 mmol) in DMF,
NaOAc (25 g) and Ac2O (75 mL) were added and the
mixture was stirred at 130 ꢁC until TLC showed
completed conversion of the starting material. The mix-
ture was allowed to reach ambient temp, solids were
removed by filtration and the filtrate was concen-
trated under reduced pressure. CH2Cl2 was added to
the residue and the organic layer was washed consecu-
tively with 5% aqueous HCl and satd aqueous bicarbon-
ate, dried (Na2SO4) and filtered. Solvents were
removed under reduced pressure and the residue was
purified on silica gel to give 34 (3.80 g, 90%). Found:
4.4. (4-Nitro)phenyl b-L-idofuranoside 28
To
a
2% methanolic solution of 27 (200 mg,
0.426 mmol), a 1 M NaOMe solution (three drops)
was added at 0 ꢁC. After completion of the deprotection
(TLC, ethyl acetate), the solution was neutralised with
ion exchange resin Amberlite IR 120 [H+], filtered and
concentrated under reduced pressure. The residue was
dissolved in ethyl acetate and filtered over a short plug
of silica gel to give pure 28 (52 mg, 40%). Found: C,
C, 51.95; H, 6.47; C15H22O9 requires: C, 52.02; H,
20
D
1H, J1,2 3.7 Hz, H-1), 5.32 (ddd, 1H, J4,5 7.3 Hz, J5,6
6.40; ½aꢁ ¼ þ54:3 (c 1.2, CHCl3); dH (CDCl3) 5.92 (d,
47.78; H, 5.00; C12H15NO8 requires: C, 47.84; H, 5.02;
20
D
0
2.6 Hz, J5,6 4.8 Hz, H-5), 5.12 (s, 1H, H-3), 4.56 (d,
½aꢁ ¼ þ49:7 (c 1.8, MeOH); dH (MeOH-d4) 5.79 (d,
0
1H, J1,2 4.2 Hz, H-1), 4.44 (dd, 1H, J2,3 6.3 Hz, J3,4
6.6 Hz, H-3), 4.36 (dd, 1H, H-2), 4.29 (dd, 1H, J4,5
1H, H-2), 4.17 (dd, 1H, J6,6 12.1 Hz, H-6), 4.09
(dd, 1H, H-60), 4.08 (dd, 1H, H-4); dC (CDCl3) 106.4
(C-1), 84.5, 83.4, 77.3, 69.9 (C-2, C-3, C-4, C-5), 62.5
(C-6).
0
3.0 Hz, H-4), 3.93 (ddd, 1H, J5,6 6.4 Hz, J5,6 6.2 Hz,
H-5), 3.62 (m, 2H, H-6, H-60); dC (MeOH-d4) 101.0
(C-1), 80.1, 78.7, 76.5, 71.5 (C-2, C-3, C-4, C-5), 63.8
(C-6).
4.8. 1,2,3,5,6-Penta-O-acetyl-a-L-altrofuranose 35
4.5. 3-O-Acetyl-1,2-O-isopropylidene-a-D-galactofura-
nose 32
A mixture of 34 (2.36 g, 6.81 mmol), glacial acetic acid
(3.6 mL), Ac2O (0.5 mL), and H2SO4 (0.21 mL) was
kept at ambient temp until TLC indicated completed
conversion of the starting material. The mixture was
poured on ice and extracted with dichloromethane.
The organic layer was consecutively washed with satd
aqueous bicarbonate and brine and dried (Na2SO4).
By chromatography, pure 35 (2.30 g, 86%) was obtained
A solution of 3-O-acetyl-1,2:5,6-di-O-isopropylidene-a-
D-galactofuranose20 (6.71 g, 22.2 mmol) in 50% aqueous
AcOH was stirred at ambient temp for 10 h. The sol-
vents were removed under reduced pressure and the res-
idue was brought to pH 7 with 3% aqueous bicarbonate.
The aqueous phase was extracted with ethyl acetate to
as a syrup. Found: C, 49.15; H, 5.75; C16H22O11
20
D
give oily 32 (5.20 g, 89.3%). Found: C, 50.31; H, 7.01;
requires: C, 49.23; H, 5.68; ½aꢁ ¼ ꢀ53:5 (c 1.9,CHCl3);
20
D
C11H18O7 requires: C, 50.38; H, 6.92; ½aꢁ ¼ þ42:6 (c
dH (CDCl3) 6.63 (s, 1H, H-1), 5.21 (ddd, 1H, J4,5
0
6.5 Hz, J5,6 3.3 Hz, J5,6 6.1 Hz, H-5), 5.15 (d, J3,4
1.5, CHCl3); dH (CDCl3) 5.93 (d, 1H, J1,2 4.0 Hz, H-
1), 5.03 (d, 1H, J3,4 1.5 Hz, H-3), 4.63 (d, 1H, H-2),
4.32 (m, 1H, H-4), 3.84 (m, 1H, H-5), 3.81–3.60 (m,
2H, H-6, H-60), 3.15 (br s, 1H, OH), 2.85 (br s, 1H,
OH); dC (CDCl3) 105.7 (C-1), 86.6, 84.8, 78.0, 70.8 (C-
2, C-3, C-4, C-5), 63.5 (C-6).
0
6.3 Hz, H-3), 5.06 (s, 1H, H-2), 4.39 (dd, 1H, J6,6
12.5 Hz, H-6), 4.26 (dd, 1H, H-4), 4.05 (dd, 1H, H-60);
dC (CDCl3) 99.5 (C-1), 83.4, 80.5, 76.3, 70.1 (C-2, C-3,
C-4, C-5), 62.4 (C-6).
4.9. (4-Nitro)phenyl 2,3,5,6-tetra-O-acetyl-a-L-altrofur-
anoside 37
4.6. 3-O-Acetyl-1,2-O-isopropylidene-5,6-di-O-metha-
nesulfonyl-a-D-galactofuranose 33
To a solution of 35 (2.0 g, 5.1 mmol) in toluene (70 mL),
(4-nitro)phenol (3.2 g, 23 mmol) and (4-toluene)sulfonic
acid (20 mg) were added and the mixture was kept under
reflux in a Dean–Stark apparatus for 3 h. After cooling
to ambient temperature, the solution was washed with
5% aqueous bicarbonate, dried (Na2SO4), the solvent
To a solution of 32 (4.2 g, 16.0 mmol) in pyridine
(30 mL), methanesulfonyl chloride (4.2 g, 2.3 equiv)
was added at 0 ꢁC and the mixture was kept at ambient
temp for 10 h. MeOH was added and the solution was
concentrated under reduced pressure. The resulting syr-

A. Tauss et al. / Tetrahedron: Asymmetry 16 (2005) 159–165
165
dideoxy-2,5-imino-d-mannitol (DMDP); Springer: Vienna,
New York, 2002; pp 393–426.
2. Moris-Varas, F.; Qian, X.-H.; Wong, C.-H. J. Am. Chem.
Soc. 1996, 118, 7647–7652.
3. Le Merrer, Y.; Poitout, L.; Depezay, J.-C.; Dosbaa, I.;
Geoffroy, S.; Foglietti, M.-J. Bioorg. Med. Chem. 1997, 5,
519–533.
4. Fleet, G. W. J.; Son, J. C.; Green, D. S. C.; Cenci di Bello,
I.; Winchester, B. Tetrahedron 1988, 44, 2649–2655; Al
Daher, S.; Fleet, G. W. J.; Namgoong, S. K.; Winchester,
B. Biochem. J. 1989, 258, 613–615.
was removed under reduced pressure and the oily resi-
due was chromatographed on silica gel (cyclohexane/
ethyl acetate 4:1, v/v) to give syrupy 37 (1.25 g, 52%).
Found: C, 51.22; H, 4.98; C20H23NO12 requires: C,
20
D
5.76 (s, 1H, H-1), 5.32 (ddd, 1H, J5,6 2.6 Hz, J5,6 4.8 Hz,
51.18; H, 4.94; ½aꢁ ¼ ꢀ65:3 (c 1.8, CHCl3); dH (CDCl3)
0
H-5), 5.33–5.28 (m, 3H, H-2, H-3, H-4), 4.17 (dd, 1H,
0
J6,6 12.1 Hz, H-6), 4.09 (dd, 1H, H-60); dC (CDCl3)
104.0 (C-1), 82.0, 81.2, 76.3, 70.1 (C-2, C-3, C-4, C-5),
62.5 (C-6).
5. Winchester, B.; Al Daher, S.; Carpenter, N. C.; Cenci di
Bello, I.; Choi, S. S.; Fairbanks, A.; Fleet, G. W. J.
Biochem. J. 1993, 290, 743–749.
4.10. (4-Nitro)phenyl a-L-altrofuranoside 16
6. Kuszmann, J.; Kiss, J. Carbohydr. Res. 1986, 153, 45–
53.
7. Lundt, I.; Madsen, R.; Al Daher, S.; Winchester, B.
Tetrahedron 1994, 50, 7513–7520.
8. Furneaux, R. H.; Lynch, G. P.; Way, G.; Winchester, B.
Tetrahedron Lett. 1993, 34, 3477–3480; Liessem, B.;
Giannis, A.; Sandhoff, K.; Nieger, M. Carbohydr. Res.
1993, 250, 19–30.
9. Yoshida, K.; Nobuko, I. Chem. Pharm. Bull. 1971, 19, 6–
10, and references cited therein.
10. For example: DÕagostino, M.; Senatore, F.; De Feo, V.;
De Simone, F. Fitoterapia 1995, 66, 283–284; Kang, S. S.;
Woo, W. S. Arch. Pharm. Res. 1982, 5, 13–15; Roshchin,
Y. V.; Shinkarenko, A. L.; Oganesian, E. T. Khimiya
Prirodnykh Soedinenii 1970, 6, 472.
11. For example: Brennan, P.; Nikaido, H. Ann. Rev.
Biochem. 1995, 64, 29–48; de Lederkremer, R. M.; Colli,
W. Glycobiology 1995, 5, 547–552; Unkefer, C. J.; Gander,
J. J. Biol. Chem. 1990, 265, 685–691.
To a 1% solution of 37 (320 mg, 0.68 mmol) in dry
MeOH, 1 M NaOMe (three drops) was added at 0 ꢁC.
After completion of the deprotection (TLC, ethyl ace-
tate), the solution was neutralised with ion exchange re-
sin Amberlite IR 120 [H+], filtered and concentrated
under reduced pressure. The residue was dissolved in
ethyl acetate and filtered over a short plug of silica gel
to give pure 16 (180 mg, 87.6%). Found: C, 47.78; H,
5.05; C12H15NO8 requires: C, 47.84; H, 5.02;
20
D
1H, H-1), 5.32 (ddd, 1H, J5,6 4.0 Hz, J5,6 6.2 Hz, H-
½aꢁ ¼ ꢀ132:1 (c 1.4, MeOH); dH (MeOH-d4) 5.69 (s,
0
5), 4.90–4.80 (m, 3H, H-2, H-3, H-4), 3.65 (dd, 1H,
0
J6,6 11.4 Hz, H-6), 3.59 (dd, 1H, H-60); dC (MeOH-d4)
106.5 (C-1), 86.3, 82.2, 76.8, 71.9 (C-2, C-3, C-4, C-5),
63.0 (C-6).
4.11. Enzyme kinetics
12. Furneaux, R. H.; Martin, B.; Rendle, P. M.; Taylor, C. M.
Carbohydr. Res. 2002, 337, 1999–2004.
13. Furneaux, R. H.; Rendle, P. M.; Sims, I. M. J. Chem.
Soc., Perkin Trans. 1 2000, 2011–2014, and references
cited therein.
14. Ferrier, R. J.; Haines, S. R. J. Chem. Soc., Perkin Trans. 1
1984, 1675–1681.
15. Meyer, A. J.; Reichstein, T. Helv. Chim. Acta 1946, 29,
152–159.
All kinetic studies were performed essentially as de-
scribed previously.22 Kinetic studies were performed at
37 ꢁC in 45 mM sodium phosphate buffer, pH 7.0 con-
taining 0.1% bovine serum albumin. Enzyme concentra-
tions ranging from 0.00019 to 0.35 mgmLꢀ1 were used,
depending on the substrate hydrolysis rates studied.
Substrate concentrations ranging from approximately
0.5 · Km to 5 · Km were employed wherever possible.
Reactions were followed in a UV–vis spectrophotometer
by measuring the change in absorbance of light at
400 nm. Data were analysed by direct fit of the rates ob-
served to the Michaelis Menten equation using the pro-
gramme GraFit.
16. Albert, R.; Dax, K.; Link, R. W.; Stutz, A. E. Carbohydr.
Res. 1983, 118, c5–c6.
¨
17. Helferich, B.; Gnuchtel, A. Chem. Ber. 1938, 71, 712–
¨
718.
18. DÕAccorso, N. B.; Thiel, I. M. E. Carbohydr. Res. 1984,
129, 43–53.
19. Varela, O.; Marino, C.; de Lederkremer, R. M. Carbo-
hydr. Res. 1986, 155, 247–251.
20. Legler, G.; Pohl, S. Carbohydr. Res. 1986, 155, 119–129.
21. Rauter, A. P.; Ramoa-Riberio, F.; Fernandes, A. C.;
Figueiredo, J. A. Tetrahedron 1995, 51, 6529–6532, and
references cited therein.
22. Namchuk, M. N.; Withers, S. G. Biochemistry 1995, 34,
16194–16202.
23. Blanchard, J. MSc thesis. University of British Columbia,
2000.
24. Andersen, S. M.; Ekhart, C.; Lundt, I.; Stutz, A. E.
¨
Acknowledgements
Financial support by the Austrian Fonds zur Fo¨rderung
der Wissenschaflichen Forschung (FWF), Vienna, Pro-
ject P 15726-N03, as well as from the Protein Engineer-
ing Network of Centres of Excellence of Canada
(PENCE) is gratefully acknowledged.
Carbohydr. Res. 2000, 326, 22–33; Andersen, S. M.; Ebner,
M.; Ekhart, C. W.; Gradnig, G.; Legler, G.; Lundt, I.;
References
Stutz, A. E.; Withers, S. G.; Wrodnigg, T. Carbohydr. Res.
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1997, 301, 155–166.
25. Hung, S.-C.; Wang, C.-C.; Chang, S.-W.; Chen, C.-S.
Tetrahedron Lett. 2001, 42, 1321–1324.
1. Wrodnigg, T. M. In Timely Research Perspectives in Carbo-
hydrate Chemistry; Schmid, W., Stutz, A. E., Eds.; From
¨
Liana to a Glycotechnology Tool: 25 years of 2,5-imino-2,5-