Inhibition of the D-fructose transporter protein GLUT5 by fused-ring glyco-1,3-oxazolidin-2-thiones and -oxazolidin-2-ones

Article abstract of DOI:10.1016/S0008-6215(03)00007-7

The glucose transporter 5 (GLUT5) - a specific D-fructose transporter - belongs to a family of facilitating sugar transporters recently enlarged by the human genome sequencing. Prompted by the need to develop specific photolabels of these isoforms, we have studied the interaction of conformationally locked D-fructose and L-sorbose derived 1,3-oxazolidin-2-thiones and 1,3-oxazolidin-2-ones to provide a rational basis for an interaction model. The inhibition properties of the D-fructose transporter GLUT5 by glyco-1,3-oxazolidin-2-thiones and glyco-1,3-oxazolidin-2-ones is now reported. In vitro, the fused-rings systems tested showed an efficient inhibition of GLUT5, thus bringing new insights on the interaction of D-fructose with GLUT5.

Full text of DOI:10.1016/S0008-6215(03)00007-7

Carbohydrate Research 338 (2003) 711–719
www.elsevier.com/locate/carres
Inhibition of the
D-fructose transporter protein GLUT5 by
fused-ring glyco-1,3-oxazolidin-2-thiones and -oxazolidin-2-ones
Jolanta Girniene,a Arnaud Tatiboue¨t,a,* Algirdas Sackus,c Jing Yang,b
Geoffrey D. Holman,b Patrick Rollina
aICOA, UMR 6005, Uni6ersite´ d’Orle´ans, BP 6759, F-45067 Orle´ans, France
bDepartment of Biology and Biochemistry, Uni6ersity of Bath, Bath BA2 7AY, UK
cDepartment of Organic Chemistry, Kaunas Uni6ersity of Technology, LT-3028 Kaunas, Lithuania
Received 24 September 2002; received in revised form 20 December 2002; accepted 28 December 2002
Abstract
The glucose transporter 5 (GLUT5)—a specific
recently enlarged by the human genome sequencing. Prompted by the need to develop specific photolabels of these isoforms, we
have studied the interaction of conformationally locked -fructose and -sorbose derived 1,3-oxazolidin-2-thiones and 1,3-oxazo-
lidin-2-ones to provide a rational basis for an interaction model. The inhibition properties of the -fructose transporter GLUT5
by glyco-1,3-oxazolidin-2-thiones and glyco-1,3-oxazolidin-2-ones is now reported. In vitro, the fused-rings systems tested showed
D-fructose transporter—belongs to a family of facilitating sugar transporters
D
L
D
an efficient inhibition of GLUT5, thus bringing new insights on the interaction of
Science Ltd. All rights reserved.
D-fructose with GLUT5. © 2003 Elsevier
Keywords: 1,3-Oxazolidine-2-one; 1,3-Oxazolidine-2-thione;
D-Fructose; L-Sorbose; GLUT5
1. Introduction
members of the third class C sub-group of transporters
which also includes the uncharacterised GLUT12.
However, a member of the class C sub-group is specific
for myo-inositol.5
An improved characterisation of the GLUTs will
require a more complete analysis of interaction with
Recent human genome sequencing has led to realize
that the family of facilitating sugar transporters
(GLUTs: glucose transporters) in mammals is more
extensive than the group of five members (GLUTs 1–5)
described a decade ago. Now the GLUTs are known to
be a more complex family of 13 isomeric proteins that
is divisible into three sub-groups based on sequence
similarities.1 The class A group consists of the glucose
transporters (GLUTs 1–4) which preferentially trans-
D
-glucose and
cificities and functions of these isoforms. A range of
-glucose analogues has been used to define the ana-
logue specificities of the class A sub-group6–9 but the
analysis of the class B ( -fructose specific) and class C
-glucose and -fructose specific) sub-groups requires
D-fructose analogues that define the spe-
D
D
port
several similar isoforms. GLUT5 is known to be spe-
cific for -fructose and it has therefore been assumed
D-glucose. The class B group includes GLUT5 and
(D
D
the development of a new range of analogues and
potentially isoform-specific inhibitors. The sugar trans-
porters of trypanosomes and plasmodium also facilitate
D
(based on sequence similarity) that GLUT7 and
GLUT9 have similar specificity. GLUT8 and GLUT10
uptake of both D-glucose and D
-fructose.10,11 Design of
transport both
D-glucose and D
-fructose2–4 and are
anti-parasitic reagents based on selective inhibition of
these transporters, excluding those of the mammalian
hosts,12,13 will require detailed comparisons of the bind-
ing site specificity.
* Corresponding author. Tel.: +33-2-38494854; fax: +33-
For initial development and analysis of
D-fructose
2-38417281
analogues, we have utilised a system in which GLUT5
E-mail address: arnaud.tatibouet@univ-orleans.fr (A. Tati-
boue¨t).
is expressed in Chinese Hamster Ovary (CHO) cells.14,15
0008-6215/03/$ - see front matter © 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0008-6215(03)00007-7

712
J. Girniene et al. / Carbohydrate Research 338 (2003) 711–719
gesting that this molecule adopts a position in the
binding site in which C-6 is trailing (top middle panel,
We have previously found that a major difficulty in
analysis of -fructose interactions with its transporters
is its ability to exist in a,b-pyranose or a,b-furanose
forms (Fig. 1). By using both -fructofuranoside and
-fructopyranoside analogues as inhibitors of -fruc-
D
Fig. 1). However, other
are also well recognized, such as 1,2-O-isopropylidene-
b- -fructopyranose, may adopt a conformation in
D-fructose derivatives which
D
D
D
D
which C-1 and C-2 are trailing (top left panels, Fig. 1).
Conformationally restricting the number of ring con-
formations was considered to be important in the devel-
opment of new analogues with large substituted groups.
In addition, we wished to explore the possibility that
tose transport in CHO-GLUT5 cells, we have been able
to establish that both ring forms are tolerated.
However, L-sorbose, which is predominantly present
in solution in the pyranose form, is not well recognized.
We now consider that these interactions may occur
L-sorbose analogues (in contrast to L-sorbose itself)
because
D-fructofuranose is a relatively symmetrical
might be good inhibitors if they could be locked into a
furanose ring form.
molecule and can be recognized by the binding site on
either face: the anomeric center leading (top middle
panel, Fig. 1) or the anomeric center trailing (top right
panel, Fig. 1).
ranose) may offer similar hydrogen bonding groups to
the transporter (left panels).
D-Fructopyranose (but not L-sorbopy-
2. Results and discussion
We address these issues here by studying the properties
of a new series of conformationally locked carbohy-
drates in which a fused furanose ring is established by
formation of 1,3-oxazolidin-2-thione (OZT) or 1,3-oxa-
zolidin-2-one (OZO) derivatives. The simplest OZT al-
dopentose fused ring systems 1–4 (Scheme 1) are
usually prepared in a single step, using potassium thio-
cyanate under acidic conditions with fair to good yields
and high specificity for the furanose ring.16 Attempts to
attain the same selectivity and yields from the keto-
hexoses were not as successful and, due to purification
problems between possible OZT isomers, a three steps
sequence was required to obtain pure OZT derivatives 5
and 617 (Schemes 2 and 3). After usual processing,
yielding mixtures of OZT isomers, silylation of the
crude product afforded separable mixtures on column
chromatography. Silylated products 7 and 8 were iso-
lated in moderate yields, respectively 20 and 54% in a
two step-process. Acidic deprotections lead to pure
OZT derivatives 5 and 6 in, respectively 90 and 70%
yields. A more efficient and less expensive approach
Of a large range of O-allyl substituted
D-fructoses,
only 6-O-allyl- -fructofuranose is well recognized, sug-
D
Fig. 1.
forms and proposed orientation in relation to the GLUT5
binding site (shaded) residues. The furanose form of -fruc-
D-Fructose and L-sorbose in pyranose and furanose
D
tose is more symmetrical than the pyranose form and it is
proposed that the furanose-form can occupy positions in the
binding site (shaded) either with the anomeric center leading
(top-middle panel) or trailing (top-right panel). In order to
tolerate bulky substitutions at the anomeric center, the latter
binding-site-occupancy state may occur. The pyranose form
D-fructose may be restricted to occupying the site with the
anomeric center trailing (top-left panel). By contrast,
sorbopyranose is not well tolerated but we explore in this
study whether its furanose-derivatives may be good inhibitors
(bottom panels).
was developed to synthesize -sorbose OZT 6 using a
L
4,6-O-isopropylidene protection leading to 9 (with a
similar yield as for 7), followed by a smooth deprotec-
tion in AcOH–water to afford 6 in 87% yield.
In order to get the 1-O substituted derivatives 12–15
(Schemes 2 and 3), a four-step sequence was used. The
of
L
-
first two steps involved acetonation of both
D-fructose
and -sorbose followed by either standard O-benzyla-
L
tion of the primary alcohol group to obtain 10a and
11a, or O-allylation to 10b and 11b in reasonable
yields.
Hydrolysis of the isopropylidene groups under acidic
conditions (trifluoroacetic acid) then application of the
efficient OZT preparation procedure led to the target
D-fructose 12 and 13 (Scheme 2) and L-sorbose deriva-
tives 14 and 15 (Scheme 3). In all cases only one
furanose anomeric form of OZT was detected and
isolated.
Scheme 1. Synthesis of oxazolidinethiones derived from pen-
toses.

J. Girniene et al. / Carbohydrate Research 338 (2003) 711–719
713
Scheme 2. Oxazolidinethiones derived from
D-fructose.
Scheme 3. Synthesis of oxazolidinethiones derived from L-sorbose.
zylated OZO 22 and 23, which were submitted to
hydrogenolysis under standard conditions leading to
OZO derivatives 24 and 25.18
Considering the results obtained by analysing the
ability of the 14 OZT and OZO compounds to inhibit
We initially planned access to OZO 22–25 (Scheme
4) by reacting 1-O-alkyl ketoses with potassium cya-
nate, which would produce the desired products.18
Quite unexpectedly, the yields observed were rather low
and not reproducible. Therefore, having in hand the
readily obtained OZT compounds, we decided to con-
vert them into the corresponding OZO derivatives. We
found that a direct conversion of OZT derivatives 12,
14 to OZO derivatives 22, 23 by oxidation was not
possible, so that a multi-step process was needed
(Scheme 4). Keeping in mind the oxidation route, silyl
ether protection of the hydroxyls in OZT 12, 14 to
afford 16, 17, then BOC protection of the thionocarba-
mate function to 18, 19 was carried out. At this stage,
oxidation was undertaken to produce in reasonable
yields OZO 20, 21. The final acidic hydrolysis of all
protecting groups afforded good yields of the 1-O-ben-
D-fructose transport (Table 1), several important con-
clusions have been reached regarding the specificity of
GLUT5.
The half-maximal inhibition (Ki) values of the OZT
derived pentoses 1–4 are \90 mM. Values in this
range indicate that no significant inhibition of
D-fruc-
tose transport has occurred. However, when the Ki
values of the pentoses are compared with the Ki value
for
the C-1 hydroxymethyl group of
(as this carbon site is absent in the pentose derivatives).
This is particularly the case for the -arabinose deriva-
D-fructose-OZT (5; Ki=25 mM), the importance of
D-fructose is evident
D

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J. Girniene et al. / Carbohydrate Research 338 (2003) 711–719
Scheme 4. Synthesis of
D-fructose and L-sorbose derived oxazolidinones.
(Fig. 1) seems fairly consistent. Further structural stud-
ies on fructopyranose locked conformations and func-
tional modifications of the best inhibitor 23 are under
investigations with a view to develop more efficient
inhibitors and biochemical tools.
tive as the configuration of the C-1 to C-4 hydroxyls is
the same as in -fructose. Moreover, comparisons be-
tween -fructose-OZT compounds (5, 12, 13) and the
equivalent -sorbose-OZT derivatives (6, 14, 15) in
which the C-5 hydroxymethyl group has the opposite
configuration, indicate a better inhibition constant for
the latter derivatives. In addition, these results indicate
D
D
L
that if
furanose ring form, then these are well recognized (in
contrast to -sorbose itself, which is predominately in
L-sorbose analogues can be forced to adopt a
Table 1
Interaction of the OZT and OZO derived sugars with the
GLUT5 transporter
L
the pyranose ring form).
The benzyl and allyl derivatives of both
and L-sorbose have higher affinity than the C-1-unsub-
D-fructose
Compound
Derivative type
Ki (mM)
stituted compounds. Consideration of the ways in
which the binding site could tolerate the bulky benzyl
and allyl groups, supports our hypothesis. Bulky part
of the furanose derivatives may occupy a position out
of the binding site. Thus, the C-6 hydroxymethyl group
D
D
-fructose
-arabinose-OZT
15.592.9
104.5921.3
122.8927.6
106.2927.1
109.9924.3
32.693.6
20.893.8
24.993.2
12.491.5
14.592.1
17.496.7
8.591.7
1
2
3
L
-arabinose-OZT
D
D
D
D
D
-xylose-OZT
-ribose-OZT
-fructose-Bn-OZT
-fructose-allyl-OZT
-fructose-OZT
4
12
13
5
14
15
6
22
24
23
25
of
L-sorbose may occupy the position normally occu-
pied by C-1 of b-
D-fructofuranose (Fig. 1, top-middle
panel). In that way the C-1 bulky groups may occupy
an opening in the binding-site cleft in which steric
constraints are less critical (Fig. 1, right panels).
L
L
L
-sorbose-Bn-OZT
-sorbose-allyl-OZT
-sorbose-OZT
Finally, replacing sulfur by oxygen from an OZT to
an OZO derivative, leads to more potent inhibitors by
factors of 2–4 especially for derivative 23 (Ki=3.1
mM) which is the best inhibitor of this study. Sulfur
thus appears to hamper the association by possibly
lowering some hydrogen interaction with the protein.15
In summary from these studies, it appears that
locked conformations of ketofuranoses provide better
D
D
-fructose-Bn-OZO
-fructose-OZO
11.993.4
3.190.5
7.291.0
L
L
-sorbose-Bn-OZO
-sorbose-OZO
Uptake of 1 mM [14C]-
GLUT5 was measured at a range of
tions (usually serial increases in concentration up to 40 mM).
The Ki values were calculated by least squares fitting to the
D
-fructose into CHO cells expressing
D-fructose concentra-
interacting molecules (by factor 6) with
D-fructose
equation Vo/V=1+I/Ki (where Vo and V are the
D-fructose
transporter GLUT5 than those previously reported.14
In view of our previous studies and the present one, the
uptake rate constants in the absence and presence of in-
hibitor, respectively). The tabulated values are the mean and
S.E.M. of 3–5 independent experiments.7
proposed interaction model of
D-fructose with GLUT5

J. Girniene et al. / Carbohydrate Research 338 (2003) 711–719
715
3. Experimental
3.1. General methods
0.06 (4s, 18 H, CH3Si); 13C NMR: l 188.8 (CS), 100.9
(C-2), 93.1 (C-3), 88.6 (C-5), 76.1 (C-4), 63.8 (C-1), 62.2
(C-6), 26.0 (CH3), 25.9 (CH3), 25.7 (CH3), 18.4 (CMe3),
18.0 (CMe3), −4.3 and −4.7 (CH3Si); ISMS (+): m/z
564.5 [M+H]+, 586.5 [M+Na]+. Anal. Calcd for
C25H53NO5SSi3: C, 53.24; H, 9.47; N, 2.48. Found: C,
53.02; H, 9.52; N, 2.55.
Melting points were determined on a Bu¨chi 510 appara-
tus and are uncorrected. 1H and 13C NMR spectra were
recorded at 250 and 62.5 MHz, respectively (Bruker
Avance DPX-250 instrument) in CDCl3 (unless other-
wise stated) with Me4Si as internal standard; for other
solvents, the residual peak was used as internal stan-
dard. Whenever appropriate, signal assignments were
deduced by DEPT, COSY and HETCOR NMR exper-
iments. Optical rotations were measured at 20 °C on a
Perkin–Elmer 141 polarimeter using a sodium lamp.
Low resolution mass spectra (MS) were recorded by the
ICOA Analytical Service on a Perkin–Elmer SCIEX
API 300 (ISMS). Elemental analysis was performed by
the micro-analysis service at the University of Bath and
by the analytical service of the CNRS, Vernaison.
Analytical TLC was carried out on precoated Silica Gel
60F-254 plates (E. Merck) and spots were visualised by
UV light (254 nm) and developed by charring after a
5% H2SO4 ethanolic solution spray. Column chro-
matography was performed on Silica Gel SI 60 (43–60
mm) (E. Merck) either with petroleum ether–EtOAc
mixtures (19:1 eluent A, 1–1 B, 2–8 C) or with eluent
D: 4:1 EtOAc–MeOH.
3.4. 1,4,6-Tri-O-tert-butyldimethylsilyl-2-N,3-O-thiocar-
bonyl-a-L-sorbofuranosylamine (8)
White powder (3.0 g, 54%); mp: 145–148 °C; [h]D20
1
−16° (c 1.1, MeOH); H NMR: l 7.28 (s broad, 1 H,
NH), 4.75 (s, 1 H, H-3), 4.38 (d, 1 H, J4,5 2.5 Hz, H-4),
4.03 (ddd, 1 H, J5,6a 6.0, J5,6b 6.6 Hz, H-5), 3.84 (d, 1 H,
J1a,1b 11.0 Hz, H-1a), 3.80 (dd, 1 H, J6a,6b 10.4 Hz,
H-6a), 3.73 (dd, 1 H, H-6b), 3.71 (d, 1 H, H-1b), 0.89
and 0.88 (s, 27 H, CH3), 0.13, 0.11, 0.07, 0.06, and 0.05
(5s, 18 H, CH3Si); 13C NMR: l 189.2 (CS), 100.2 (C-2),
91.3 (C-3), 82.3 (C-5), 73.9 (C-4), 63.7 (C-1), 60.0 (C-6),
26.0 (CH3), 25.9 (CH3), 25.7 (CH3), 18.5 (CMe3), 18.3
(CMe3), −4.7, −5.1 and −5.4 (CH3Si); ISMS (+):
m/z 564.5 [M+H]+, 586.5 [M+Na]+. Anal. Calcd for
C25H53NO5SSi3: C, 53.24; H, 9.47; N, 2.48. Found: C,
52.82; H, 9.49; N, 2.42.
3.5. 4,6-O-Isopropylidene-2-N,3-O-thiocarbonyl-a-L-sor-
bofuranosylamine (9)
3.2. General protocol for the synthesis of silylated
OZT derivatives 7 and 8 from
D-fructose and
L-sorbose
The crude mixture obtained from L-sorbose (1.66 g, 8
mmol) under the general protocol for OZT formation
(7 and 8) was diluted in dry acetone (50 mL), cooled in
an ice bath and concd H2SO4 (95%, 0.3 mL) was added.
After 24 h at rt, the mixture was made neutral with
Et3N, the solvent removed and the residue purified on
silica gel (eluent B), yielding 9 as a pale yellow foam
The ketose (1 equiv) was suspended in water (0.5 M)
then KSCN (2.05 equiv) and 12 N HCl 37% (2.2 equiv)
were added. The pink solution obtained was warmed
up to 50 °C for 5 days. Water was removed by co-evap-
oration with toluene and the crude mixture suspended
in acetone. Salts were removed and the solvent evapo-
rated. The dry crude mixture was silylated with TBDM-
SCl (33 mmol) and imidazole (33 mmol) in DMF (50
mL) at room temperature (rt) for 48 h. Extraction with
EtOAc was conducted twice, the organic layers were
collected and washed with water ×5 then brine, and
dried with MgSO4. The resulting mixture was concen-
trated under diminished pressure then purified by
column chromatography using eluent A, yielding 7 and
8, respectively.
1
(1.3 g, 54%) was obtained; [h]2D0 −73° (c 2, MeOH); H
NMR: l 7.94 (s broad, 1 H, NH), 4.95 (s, 1 H, H-3),
4.55 (d, 1 H, J4,5 2.1 Hz, H-4), 4.20–3.98 (m, 4 H, H-6,
H-1a, H-5), 3.88 (dd, 1 H, J1a,1b 11.6 Hz, H-1b), 2.97 (s
broad, 1 H, OH), 1.47 (s, 3 H, CH3), 1.39 (s, 3 H, CH3);
13C NMR: l 189.1 (CS), 100.4 (CMe2), 98.4 (C-2), 90.4
(C-3), 72.4 (C-4), 72.1 (C-5), 62.9 (C-1), 59.5 (C-6);
ISMS (+): m/z 262 [M+H]+. Anal. Calcd for
C10H15NO5S: C, 45.97; H, 5.79; N, 5.36. Found: C,
46.23; H, 6.10; N, 5.41.
3.3. 1,4,6-Tri-O-tert-butyldimethylsilyl-2-N,3-O-thiocar-
bonyl-b-D-fructofuranosylamine (7)
3.6. General protocol for the synthesis of OZT deriva-
tives 5 and 6
White powder (1.3 g, 20%); [h]2D0 −19° (c 0.5, MeOH);
1H NMR: l 7.18 (s broad, 1 H, NH), 4.83 (s, 1 H, H-3),
4.51 (s, 1 H, H-4), 4.13–4.04 (m, 1 H, H-5), 3.82 (d, 1
H, J1a,1b 10.9 Hz, H-1a), 3.73 (d, 1 H, H-1b), 3.64 (dd,
1 H, J6a,5 5.3, J6a,6b 10.7 Hz, H-6a), 3.64 (dd, 1 H, J6b,5
8.9, H-6b), 0.88 (s, 27 H, CH3), 0.13, 0.11, 0.07 and
Deprotection of silyl groups in 7 and 8 was effected at
0.3 M in 9:1 TFA–water for 24 h. Acetal deprotection
of 9 was effected in 4:1 AcOH–water. Solvent were
removed by co-evaporation with MeOH or toluene and
the crude product purified by flash chromatography
with EtOAc, yielding 5 and 6, respectively.

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J. Girniene et al. / Carbohydrate Research 338 (2003) 711–719
1
3.7. 2-N,3-O-Thiocarbonyl-b-
D-fructofuranosylamine (5)
MeOH); H NMR (CD3OD): l 5.91 (ddt, 1 H, Jb,a 5.7,
Jb,gZ 11.3, Jb,gE 17.3 Hz, H-ballyl), 5.36–5.14 (m, 2 H,
H-gallyl), 4.94 (d, 1 H, J3,4 1.3 Hz, H-3), 4.35 (dd, 1 H,
J4,5 2.5 Hz, H-4), 4.12–4.04 (m, 3 H, H-5, Haallyl), 3.70
(d, 1 H, J1a,1b 10.4 Hz, H-1a), 3.62 (d, 1 H, H-1b), 3.52
(d, 2 H, J5,6 6.6 Hz, H-6); 13C NMR (67.5 MHz,
Me2SO): l 190.4 (CS), 135.4 (Cb), 117.8 (Cg), 101.2
(C-2), 93.8 (C-3), 88.7 (C-5), 76.7 (C-4), 73.4 (Ca), 70.5
(C-1), 62.5 (C-6); ISMS (+): m/z 262 [M+H]+, 284
[M+Na]+. Anal. Calcd for C10H15NO5S: C, 45.97; H,
5.79. Found: C, 46.10; H, 5.94.
(0.17 g, 90%); [h]2D0 −10° (c 1, MeOH); 1H NMR
(CD3OD): l 4.98 (d, 1 H, J3,4 1.3 Hz, H-3), 4.36 (dd, 1
H, J4,5 2.5 Hz, H-4), 4.10 (dt, 1 H, J5,6 6.5 Hz, H-5),
3.78 (d, 1 H, J1a,1b 11.9 Hz, H-1a), 3.69 (d, 1 H, H-1b),
3.53 (d, 2 H, H-6); 13C NMR (CD3OD): l 190.5 (CS),
102.5 (C-2), 93.5 (C-3), 88.8 (C-5), 76.8 (C-4), 62.9
(C-1), 62.6 (C-6); ISMS (+): m/z 222 [M+H]+. Anal.
Calcd for C7H11NO5S: C, 38.00; H, 5.01; N, 6.33.
Found: C, 38.05; H, 5.05; N, 6.18.
3.12. 1-O-Benzyl-2-N,3-O-thiocarbonyl-a-
nosylamine (14)
L-sorbofura-
3.8. 2-N,3-O-Thiocarbonyl-a-L-sorbofuranosylamine (6)
(0.19 g, 70%) from 8 and (0.20 g, 87%) from 9; [h]D20
−22° (c 1.1, MeOH); H NMR (CD3OD): l 4.91 (s, 1
1
Yellow foam (1.8 g, 74%); [h]2D0 −30° (c 2, MeOH); H
NMR (Me2SO–D2O): l 10.85 (s broad, 1 H, NH),
7.48–7.21 (m, 5 H, PhꢀH), 4.81 (s, 1 H, H-3), 4.60 (d,
1 H, J 12.2 Hz, CH2Ph), 4.55 (d, 1 H, CH2Ph), 4.18 (d,
1 H, J4,5 2.5 Hz, H-4), 3.76 (ddd, 1 H, J5,6a 4.7, J5,6b 6.2
Hz, H-5), 3.69 (d, 1 H, J1a,1b 10.4 Hz, H-1a), 3.66 (dd,
1 H, J6a,6b 11.6 Hz, H-6a), 3.56 (d, 1 H, H-1b), 3.53 (dd,
1 H, H-6b); 13C NMR (Me2SO): l 188.0 (CS), 137.7
(C), 128.4 (CH), 127.7 (CH), 127.6 (CH), 99.0 (C-2),
90.2 (C-3), 81.5 (C-5), 72.72 (CH2), 72.6 (C-4), 69.1
(C-1), 58.4 (C-6); ISMS (+): m/z 312.5 [M+H]+,
334.5 [M+Na]+. Anal. Calcd for C14H17NO5S: C,
54.01; H, 5.50. Found: C, 54.03; H, 5.63.
1
H, H-3), 4.31 (d, 1 H, J4,5 2.8 Hz, H-4), 3.99–3.94 (m,
1 H, H-5), 3.90–3.78 (m, 3 H, H-6, H-1a), 3.70 (d, 1 H,
J1a,1b 11.9 Hz, H-1b); 13C NMR (CD3OD): l 190.9
(CS), 101.7 (C-2), 92.0 (C-3), 82.6 (C-5), 74.8 (C-4),
62.7 (C-1), 60.4 (C-6); ISMS (+): m/z 222 [M+H]+.
Anal. Calcd for C7H11NO5: C, 38.00; H, 5.01; N, 6.33.
Found: C, 38.29; H, 5.21; N, 6.23.
3.9. General protocol for the synthesis of 1,3-oxazo-
lidine-2-thiones 12–15 from 1-O-alkylated ketoses
The 1-O-alkylated ketose (1 equiv) was suspended in
water (0.08 M), then KSCN (2.05 equiv) and 12 N HCl
(2.4 equiv) were added. The pink solution obtained was
maintained at 50 °C during 48 h. Water was removed
by co-evaporation with toluene and the crude mixture
purified by column chromatography (eluent C), yielding
12–15.
3.13. 1-O-Allyl-2-N,3-O-thiocarbonyl-a-L-sorbofura-
nosylamine (15)
1
Yellow foam (3.9 g, 75%); [h]2D0 −29° (c 1, MeOH); H
NMR (CD3OD): l 5.91 (ddt, 1 H, Jb,a 5.7, Jb,gZ 11.0,
Jb,gE 17.3 Hz, H-ballyl), 5.36–5.15 (m, 2 H, H-gallyl), 4.88
(s, 1 H, H-3), 4.31 (d, 1 H, J4,5 2.8 Hz, H-4), 4.12–4.05
(m, 2 H, Haallyl), 3.95 (ddd, 1 H, J5,6a 4.7, J5,6b 6.6 Hz,
H-5), 3.85 (dd, 2 H, J6a,6b 11.9 Hz, H-6), 3.76 (dd, 2 H,
H-6), 3.73 (d, 1 H, J1a,1b 10.7 Hz, H-1a), 3.64 (d, 1 H,
H-1b); 13C NMR (Me2SO): l 190.8 (CS), 135.4 (Cb),
117.8 (Cg), 100.6 (C-2), 92.4 (C-3), 82.7 (C-5), 74.8
(C-4), 73.5 (Ca), 70.4 (C-1), 60.4 (C-6); ISMS (+): m/z
262 [M+H]+. Anal. Calcd for C10H15NO5S: C, 45.97;
H, 5.79. Found: C, 46.02; H, 5.89.
3.10. 1-O-Benzyl-2-N,3-O-thiocarbonyl-b-D-fructofura-
nosylamine (12)
Yellow foam (0.62 g, 100%); [h]2D0 −24° (c 2, MeOH);
1H NMR (Me2SO–D2O): l 7.48–7.23 (m, 5 H, PhꢀH),
4.87 (d, 1 H, J3,4 0.9 Hz, H-3), 4.60 (d, 1 H, J 12.2 Hz,
CH2Ph), 4.55 (d, 1 H, CH2Ph), 4.25 (s, 1 H, H-4), 3.93
(ddd, 1 H, J5,4 2.5 Hz, H-5), 3.66 (d, 1 H, J1a,1b 10.4 Hz,
H-1a), 3.56 (d, 1 H, H-1b), 3.37 (dd, 1 H, J5,6a 6.3, J6a,6b
11.3 Hz, H-6a), 3.30 (dd, 1 H, H-6b); 13C NMR
(Me2SO): l 187.7 (CS), 137.7 (C), 128.4 (CH), 127.8
(CH), 127.6 (CH), 99.5 (C-2), 91.8 (C-3), 87.1 (C-5),
74.9 (C-4), 72.6 (CH2), 69.3 (C-1), 60.7 (C-6); ISMS
(+): m/z 312.5 [M+H]+, 334.5 [M+Na]+. Anal.
Calcd for C14H17NO5S: C, 54.01; H, 5.50. Found: C,
53.62; H, 5.52.
3.14. General protocol for the preparation of silylated
16 and 17
The OZT (1 equiv), imidazole (5 equiv) and TBDMSCl
(2.5 equiv) were dissolved in DMF (10 mL) and the
mixture stirred overnight at rt. After completion, water
was added and the resulting solution was extracted
twice with CH2Cl2. Collected organic layers were
washed abundantly with water then once with brine
and subsequently dried with MgSO4 and evaporated. A
short column chromatography (eluent A) afforded the
protected OZT derivatives 16 and 17.
3.11. 1-O-Allyl-2-N,3-O-thiocarbonyl-b-D-fructofura-
nosylamine (13)
Pale yellow foam (2.5 g, 100%); [h]2D0 −39° (c 1,

J. Girniene et al. / Carbohydrate Research 338 (2003) 711–719
717
3.15. 1-O-Benzyl-4,6-di-O-tert-butyldimethylsilyl-2-N,3-
O-thiocarbonyl-b- -fructofuranosylamine (16)
4.05 (m, 1 H, H-5), 3.62 (d, 1 H, H-1b), 3.61 (dd, 1 H,
J5,6a 4.4, J6a,6b 10.7 Hz, H-6a), 3.42 (dd, 1 H, J5,6b 8.8
Hz, H-6b), 1.52 (s, 9 H, CH3), 0.87 (s, 9 H, CH3), 0.85
D
1
Yellow oil (1.8 g, 100%); [h]2D0 −55° (c 2, MeOH); H
NMR: l 8.55 (s broad, 1 H, NH), 7.28–7.12 (m, 5 H,
PhꢀH), 4.74 (s, 1 H, H-3), 4.52 (d, 1 H, J 12.2 Hz,
CH2Ph), 4.47–4.38 (m, 2 H, H-4, CH2Ph), 4.05–3.94
(m, 1 H, H-5), 3.59 (d, 1 H, J1a,1b 10.4 Hz, H-1a), 3.56
(dd, 1 H, J5,6a 5.3, J6a,6b 10.4 Hz, H-6a), 3.49 (d, 1 H,
H-1b), 3.37 (ft, 1 H, H-6b), 0.79 (s, 9 H, CH3), 0.76 (s,
9 H, CH3), 0.01, 0.00, −0.01 and −0.03 (4s, 12 H,
CH3Si); 13C NMR: l 188.4 (CS), 137.0 (C), 128.5 (CH),
128.0 (CH), 127.8 (CH), 100.0 (C-2), 92.9 (C-3), 88.4
(C-5), 75.9 (C-4), 73.6 (CH2), 69.5 (C-1), 61.9 (C-6),
25.9 (CH3), 25.6 (CH3), 18.2 (CMe3), 17.8 (CMe3),
−5.0, −5.3, −5.4 (CH3Si); ISMS (+): m/z 540.5
[M+H]+, 562 [M+Na]+. Anal. Calcd for
C26H45NO5SSi2: C, 57.84; H, 8.40; N, 2.59. Found: C,
57.61; H, 8.59; N, 2.49.
(s, 9 H, CH3), 0.09, 0.08 and 0.04 (3s, 12 H, CH3Si); 13
C
NMR: l 184.0 (CS), 148.9 (CO), 137.1 (C), 128.6 (CH),
128.1 (CH), 127.8 (CH), 101.9 (C-2), 90.5 (C-3), 89.1
(C-5), 85.1 (CMe3), 76.0 (C-4), 73.5 (CH2), 68.9 (C-1),
62.0 (C-6), 27.9 (CH3), 25.9 (CH3), 25.7 (CH3), 18.2
(CMe3), 17.9 (CMe3), −4.9, −5.2, −5.3 (CH3Si);
ISMS (+): m/z 662.5 [M+Na]+. Anal. Calcd for
C31H53NO7SSi2: C, 58.18; H, 8.35; N, 2.19. Found: C,
58.02; H, 8.38; N, 2.09.
3.19. 1-O-Benzyl-2-N-tert-butoxycarbonyl-4,6-di-O-
tert-butyldimethylsilyl-2-N,3-O-thiocarbonyl-a-
L-sorbo-
furanosylamine (19)
1
Yellow oil (0.42 g, 95%); [h]2D0 −36° (c 2, MeOH); H
NMR: l 7.39–7.23 (m, 5 H, PhꢀH), 4.68 (s, 1 H, H-3),
4.59 (d, 1 H, J 12.2 Hz, CH2Ph), 4.46 (d, 1 H, CH2Ph),
4.37 (d, 1 H, J4,5 2.5 Hz, H-4), 4.19 (d, 1 H, J1a,1b 10.0
Hz, H-1a), 4.00 (td, 1 H, J5,6 6.3 Hz, H-5), 3.76 (d, 2 H,
H-6), 3.51 (d, 1 H, H-1b), 1.52 (s, 9 H, CH3), 0.87 (s, 9
H, CH3), 0.83 (s, 9 H, CH3), 0.08, 0.06, 0.04 and 0.03
(4s, 12 H, CH3Si); 13C NMR: l 184.4 (CS), 149.1 (CO),
137.2 (C), 128.7 (CH), 128.2 (CH), 128.0 (CH), 101.2
(C-2), 89.0 (C-3), 85.2 (CMe3), 82.8 (C-5), 74.3 (C-4),
73.6 (CH2), 68.9 (C-1), 59.8 (C-6), 28.0 (CH3), 26.0
(CH3), 25.7 (CH3), 18.4 (CMe3), 18.0 (CMe3), −4.8,
−5.2, −5.3 (CH3Si); ISMS (+): m/z 640.5 [M+H]+.
Anal. Calcd for C31H53NO7SSi2: C, 58.18; H, 8.35; N,
2.19. Found: C, 58.04; H, 8.46; N, 2.14.
3.16. 1-O-Benzyl-4,6-di-O-tert-butyldimethylsilyl-2-N,3-
O-thiocarbonyl-a-L-sorbofuranosylamine (17)
1
Yellow oil (3.1 g, 85%); [h]2D0 −21° (c 2, MeOH); H
NMR: l 7.51 (s broad, 1 H, NH), 7.39–7.24 (m, 5 H,
PhꢀH), 4.76 (s, 1 H, H-3), 4.62 (d, 1 H, J 12.2 Hz,
CH2Ph), 4.52 (d, 1 H, CH2Ph), 4.38 (d, 1 H, J4,5 2.5 Hz,
H-4), 4.02 (ddd, 1 H, J5,6a 5.9, J5,6b 6.6, J6a,6b 10.0 Hz,
H-5), 3.80 (dd, 1 H, H-6a), 3.74 (dd, 1 H, H-6b), 3.69
(d, 1 H, J1a,1b 10.4 Hz, H-1a), 3.58 (d, 1 H, H-1b), 0.87
(s, 9 H, CH3), 0.84 (s, 9 H, CH3), 0.09, 0.05 and 0.04
(3s, 12 H, CH3Si); 13C NMR: l 188.9 (CS), 137.0 (C),
128.6 (CH), 128.2 (CH), 128.0 (CH), 99.1 (C-2), 91.4
(C-3), 82.0 (C-5), 73.9 (CH2), 73.8 (C-4), 69.6 (C-1),
59.7 (C-6), 26.0 (CH3), 25.7 (CH3), 18.4 (CMe3), 18.0
(CMe3), −4.8, −5.2, −5.3 (CH3Si); ISMS (+): m/z
540.5 [M+H]+. Anal. Calcd for C26H45NO5SSi2: C,
57.84; H, 8.40; N, 2.59. Found: C, 57.59; H, 8.46; N,
2.40.
3.20. General protocol for the preparation of the car-
bonyl derivatives 20 and 21
The N-Boc protected OZT (1 equiv) was dissolved in
CH2Cl2 (1.2 M) and cooled to 0 °C; m-chloroperben-
zoic acid (70–75%) (3 equiv) and NaHCO3 (3.1 equiv)
were added and the suspension was stirred for 3 h at
0 °C. After dilution with water and DCM extraction
(2×), the collected organic phases were washed with
NaHCO3, water and brine, then dried over MgSO4.
After solvent evaporation, the crude mixture obtained
was purified by chromatography (eluent A).
3.17. General protocol for preparation of the tert-bu-
toxycarbonyl derivatives 18 and 19
The above OZT (1 equiv) was reacted with Boc2O (1.5
equiv) in dry Py (0.3 M) for 24 h at rt. After concentra-
tion under diminished pressure, the crude mixture was
purified by flash column chromatography (eluent A).
3.21. 1-O-Benzyl-2-N-tert-butoxycarbonyl-4,6-di-O-
tert-butyldimethylsilyl-2-N,3-O-carbonyl-b-D-fructofura-
nosylamine (20)
3.18. 1-O-Benzyl-2-N-tert-butoxycarbonyl-4,6-di-O-
tert-butyldimethylsilyl-2-N,3-O-thiocarbonyl-b-
furanosylamine (18)
D
-fructo-
Oil (0.27 g, 70%); [h]2D0 −28° (c 1.7, MeOH); 1H NMR:
l 7.38–7.25 (m, 5 H, PhꢀH), 4.59 (d, 1 H, J3,4 1.6 Hz,
H-3), 4.58 (d, 1 H, J 12.2 Hz, CH2Ph), 4.52 (d, 1 H,
CH2Ph), 4.44 (dd, 1 H, J4,5 2.8 Hz, H-4), 4.20 (d, 1 H,
J1a,1b 9.7 Hz, H-1a), 4.06–3.98 (m, 1 H, H-5), 3.64 (dd,
1 H, J6a,6b 10.8, J5,6a 4.4 Hz, H-6a), 3.60 (d, 1 H, H-1b),
1
Yellow oil (0.68 g, 100%); [h]2D0 −15° (c 2, MeOH); H
NMR: l 7.38–7.24 (m, 5 H, PhꢀH), 4.76 (s, 1 H, H-3),
4.59 (d, 1 H, J 12.2 Hz, CH2Ph), 4.54–4.46 (m, 2 H,
H-4, CH2Ph), 4.18 (d, 1 H, J1a,1b 9.7 Hz, H-1a), 4.14–

718
J. Girniene et al. / Carbohydrate Research 338 (2003) 711–719
3.52 (dd, 1 H, J5,6b 7.2 Hz, H-6b), 1.50 (s, 9 H, CH3),
0.87 (s, 9 H, CH3), 0.86 (s, 9 H, CH3), 0.10, 0.08 and
0.04 (3s, 12 H, CH3Si); 13C NMR: l 151.1 (CO), 148.6
(CO), 137.4 (C), 128.6 (CH), 128.1 (CH), 127.8 (CH),
97.6 (C-2), 88.2 (C-5), 85.9 (C-3), 84.3 (CMe3), 76.4
(C-4), 73.5 (CH2), 69.3 (C-1), 62.1 (C-6), 28.1 (CH3),
26.0 (CH3), 25.8 (CH3), 18.0 (CMe3), −4.8, −4.9,
−5.2, −5.3 (CH3Si); ISMS (+): m/z 646 [M+Na]+,
624 [M+H]+. Anal. Calcd for C31H53NO8Si2: C, 59.68;
H, 8.56; N, 2.24. Found: C, 59.33; H, 8.58; N, 2.23.
3.25. 1-O-Benzyl-2-N,3-O-carbonyl-a-L-sorbofuranosy-
lamine (23)
Oil (0.075 g, 50%): [h]2D0 −16.5° (c 1, MeOH); 1H
NMR (CD3OD): l 7.42–7.24 (m, 5 H, PhꢀH), 4.69 (s,
1 H, H-3), 4.61 (s, 2 H, CH2ꢀPh), 4.23 (d, 1 H, J4,5 2.8
Hz, H-4), 4.05 (ddd, 1 H, J5,6a 4.7, J5,6b 6.6 Hz, H-5),
3.85 (dd, 1 H, J6a,6b 11.6 Hz, H-6a), 3.76 (dd, 1 H,
H-6b), 3.74 (d, 1 H, J1a,1b 10.0 Hz, H-1a), 3.62 (d, 1 H,
H-1b); 13C NMR (CD3OD): l 159.9 (CO), 139.0 (C),
129.4 (CH), 128.9 (CH), 128.8 (CH), 97.2 (C-2), 87.8
(C-3), 82.0 (C-5), 75.0 (C-4), 74.6 (CH2), 71.2 (C-1),
60.6 (C-6); ISMS (+): m/z 318 [M+Na]+, 296 [M+
H]+. Anal. Calcd for C14H17NO6: C, 56.95; H, 5.80; N,
4.74. Found: C, 56.55; H, 5.85; N, 4.69.
3.22. 1-O-Benzyl-2-N-tert-butoxycarbonyl-4,6-di-O-
tert-butyldimethylsilyl-2-N,3-O-carbonyl-a-L-sorbofura-
nosylamine (21)
1
Oil (0.21 g, 90%); [h]2D0 −36° (c 2, MeOH); H NMR:
l 7.40–7.25 (m, 5 H, PhꢀH), 4.59 (d, 1 H, J 12.2 Hz,
CH2Ph), 4.51 (s, 1 H, H-3), 4.48 (d, 1 H, CH2Ph), 4.28
(d, 1 H, J4,5 2.5 Hz, H-4), 4.25 (d, 1 H, J1a,1b 9.7 Hz,
H-1a), 4.2 (td, 1 H, J5,6 6.0 Hz, H-5), 3.78 (d, 2 H,
H-6), 3.47 (d, 1 H, H-1b), 1.52 (s, 9 H, CH3), 0.87 (s, 9
H, CH3), 0.85 (s, 9 H, CH3), 0.09, 0.08, 0.05 and 0.04
(4s, 12 H, CH3Si); 13C NMR: l 151.7 (CO), 148.7 (CO),
137.3 (C), 128.6 (CH), 128.1 (CH), 127.9 (CH), 97.3
(C-2), 84.2 (CMe3), 84.1 (C-5), 82.3 (C-3), 76.3 (C-4),
73.5 (CH2), 69.0 (C-1), 59.9 (C-6), 28.0 (CH3), 26.0
(CH3), 25.7 (CH3), 18.4 (CMe3), 18.4 (CMe3), −4.8,
−5.1, −5.2, −5.3 (CH3Si); ISMS (+): m/z 646.5
[M+Na]+. Anal. Calcd for C31H53NO8Si2: C, 59.68;
H, 8.56; N, 2.24. Found: C, 59.31; H, 8.52; N, 2.22.
3.26. General method for the preparation of 24 and 25
A suspension of Pd/C 10% (0.3 g) in a MeOH (2 mL)
solution of the 1-O-benzyl-OZO (0.43 g, 1.46 mmol)
was stirred under H2 overnight. After filtration over
celite and chromatography (eluent D) the fully depro-
tected OZO was isolated.
3.27. 2-N,3-O-Carbonyl-b-D
-fructofuranosylamine (24)18
Colorless oil (0.291 g, 100%); [h]2D0 −21° (c 1, MeOH);
Lit.18: [h]2D0 −26 (c 0.9, EtOH).
3.28. 2-N,3-O-Carbonyl-a-L-sorbofuranosylamine (25)
Colorless oil (0.293 g, 100%): [h]2D0 −8° (c 1, MeOH);
1H NMR (CD3OD): l 4.69 (s, 1 H, H-3), 4.24 (d, 1 H,
J4,5 2.8 Hz, H-4), 4.04 (ddd, 1 H, J5,6a 6.6, J5,6b 4.7 Hz,
H-5), 3.86 (dd, 1 H, J6a,6b 11.6 Hz, H-6a), 3.75 (dd, 1 H,
H-6b), 3.74 (d, 1 H, J1a,1b 11.6 Hz, H-1a), 3.49 (d, 1 H,
H-1b); 13C NMR (CD3OD): l 160.1 (CO), 98.3 (C-2),
87.5 (C-3), 82.1 (C-5), 75.1 (C-4), 63.4 (C-1), 60.6 (C-6);
ISMS (+): m/z 228 [M+Na]+, 206 [M+H]+. Anal.
Calcd for C7H11NO6: C, 40.98; H, 5.40; N, 6.83.
Found: C, 41.27; H, 5.57; N, 6.77.
3.23. General protocol for the preparation OZO 22
and 23
The N-Boc OZO 20 and 21 (0.47 g, 0.75 mmol) was
suspended in water (0.3 mL), then TFA (2.7 mL) was
added. The reaction was stirred at rt until completion;
after co-evaporation with toluene, the crude mixture
was purified by column chromatography (eluent C).
3.24. 1-O-Benzyl-2-N,3-O-carbonyl-b-
D-fructofuranosy-
lamine (22)
1
Oil (0.145 g, 65%); [h]2D0 −17° (c 1, MeOH); H NMR
(Me2SO): l 8.77 (s, 1 H, NH), 7.35–7.30 (m, 5 H), 5.61
(d, 1 H, J4,OH 4.2 Hz, OH), 4.90 (t, 1 H, J5,OH 5.5 Hz,
OH), 4.56 (s, 3 H, H-3, CH2ꢀPh), 4.12 (s broad, 1 H,
H-4), 3.86–3.80 (m, H-5), 3.60 (d, 1 H, J1a,1b 10.2 Hz,
H-1a), 3.52 (d, 1 H, H-1b), 3.40–3.33 (m, 2 H, H-6);
13C NMR (CD3OD): l 156.4 (CO), 137.9 (C), 128.3
(CH), 127.6 (CH), 127.5 (CH), 95.7 (C-2), 86.6 (C-3),
86.1 (C-5), 75.4 (C-4), 72.5 (CH2), 70.1 (C-1), 61.2
(C-6); ISMS (+): m/z 318 [M+Na]+, 296 [M+H]+.
Anal. Calcd for C14H17NO6: C, 56.95; H, 5.80; N, 4.74.
Found: C, 56.51; H, 5.84; N, 4.71.
Acknowledgements
We are grateful to the CNRS for financial support
and the MRC (UK) and Diabetes UK for grant
support.
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