An easy stereospecific synthesis of 1-amino-2,5-anhydro-1-deoxy-D-mannitol and arylamino derivatives

Article abstract of DOI:10.1016/S0008-6215(99)00040-3

1-Amino-2,5-anhydro-1-deoxy-D-mannitol and a series of arylamino derivatives were prepared by nitrous acid deamination of 2-amino-2-deoxy-D-glucose and subsequent reductive amination of the resulting 2,5-anhydro-D-mannose. Some of these compounds showed a

Full text of DOI:10.1016/S0008-6215(99)00040-3

Carbohydrate Research 315 (1999) 339–344
Note
An easy stereospecific synthesis of
1-amino-2,5-anhydro-1-deoxy-
D
-mannitol and arylamino
derivatives
Samantha Claustre a, Fre´de´ric Bringaud b, Laurent Aze´ma a, Rudi Baron a,
a
Jacques Pe´rie´ , Miche`le Willson a,*
a Groupe de Chimie Organique Biologique, UMR CNRS 5623, Uni6ersite´ Paul Sabatier, F-31062 Toulouse, France
b Laboratoire d’Immunologie et Biologie Mole´culaire de Parasites Protozoaires, UPRESA CNRS 5016,
Uni6ersite´ de Bordeaux II, F-33076 Bordeaux, France
Received 5 January 1998; revised 29 January 1999; accepted 29 January 1999
Abstract
1-Amino-2,5-anhydro-1-deoxy-
deamination of 2-amino-2-deoxy-
nose. Some of these compounds showed an enhanced affinity for the hexose transporter of Trypanosoma brucei as
compared to -fructose. © 1999 Elsevier Science Ltd. All rights reserved.
D
-mannitol and a series of arylamino derivatives were prepared by nitrous acid
D
-glucose and subsequent reductive amination of the resulting 2,5-anhydro- -man-
D
D
Keywords: 1-Amino-2,5-anhydro-1-deoxy-
brucei hexoses transporter (THT1)
D-mannitol; 2,5-Anhydro-1-arylamino-1-deoxy-D-mannitol derivatives; Trypanosoma
binding such active compounds to a
tose unit, or to the analogue of b- -fructose,
2,5-anhydro- -mannitol, which also has a sig-
D
-fruc-
1. Introduction
D
D
In the course of a programme devoted to
glycolytic enzymes inhibitors and -glucose
intake in the trypanosome [1], we became
interested in the synthesis of -fructose ana-
logues [2] for the following reasons: (i) the
hexose transporter of Trypanosoma brucei
nificant affinity for the hexose transporter
THT1 [3,4]. Bringaud and Baltz [5,6] have
shown that genes encoding for this transporter
were highly conserved among kinetoplastidae,
a feature which underlines the importance of
this protein as a target for chemotherapy of
trypanomiasis (sleeping sickness). These au-
thors have also shown that these transporters
are different in structure from that of the
erythrocyte GLUT1 as revealed by a distinct
sensitivity towards classical inhibitors; (ii)
three of the enzymes of the glycolytic cascade,
phosphoglucoisomerase, phosphofructokinase
and aldolase, involve a phosphorylated fruc-
tose as substrate or product. In addition, the
D
D
(THT1) recognizes not only
D
-glucose but also
D-fructose, whereas that of human erythrocyte
has only affinity for
D
-glucose [3,4]. Because
of this peculiarity, one can consider the possi-
bility of the specific intake of active com-
pounds into the parasite via the transporter by
* Corresponding author. Fax: +33-5-561-251733.
E-mail address: willson@iris.ups-tlse.fr (M. Willson)
0008-6215/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(99)00040-3

340
S. Claustre et al. / Carbohydrate Research 315 (1999) 339–344
reductive amination with the cyanohydridobo-
rate anion [9].
phosphofructokinase responsible for the syn-
thesis of 2,6-diphospho- -fructose, which is a
powerful effector in the glycolysis pathway,
involves a phosphorylated fructose as sub-
strate. Potential inhibitors for these enzymes
D
2,5-Anhydro-
by nitrous acid deamination of 2-amino-2-de-
oxy- -glucose [10] following a modification of
D
-mannose (1) was prepared
D
the Horton and Philips procedure [8]. We
found it convenient to use sodium nitrite in
the presence of a strong cation-exchange resin
as nitrosating agent, which improved the
workup and the yield.
Reductive amination of 1 with primary ary-
lamine was carried out at optimum pH 5–6,
resulting in the target arylamino derivatives
4a–f. The stereospecificity of the reaction was
confirmed by 13C NMR spectroscopy of the
crude extract (see Section 2). This method is
are likely to be
view of this, we devised a stereospecific syn-
thesis of 1-amino-2,5-anhydro-1-deoxy-
mannitol and derivatives (Scheme 1). A recent
method described in the literature for the
preparation of 1-amino-2,5-anhydro-1-deoxy-
hexitols requires drastic conditions and leads
to a mixture of compounds [7]. We therefore
considered it preferable to develop an im-
D
-fructofuranose analogues. In
D
-
proved methodology involving 2,5-anhydro-
D-
mannose (1) [8] as starting material and
Scheme 1.

S. Claustre et al. / Carbohydrate Research 315 (1999) 339–344
341
Table 1
Inhibition constants (Ki mM) on THT1 with respect to 2-deoxy-
D
-glucose (2-dGlc)
Compound
D
-Glucose 2-Deoxy-
arabino-
D
-
D
-Fructose 2,5-Anhy-
dro- -man-
D
hexose
nitol
(‘2-deoxy-
D
-
glucose’, 2-dGlc)
Km or Ki
0.76
0.54
4
8.6
(mM)
Compound
1
2
3
4a
4b
4.590.8
4c
4d
4e
4f
Kia (mM)
\100
\100
\100
15.691.5
0.4890.1 1.490.2
190.2 0.290.05
a The Ki values (mM), expressed as means S.D. (n=3 or 4), were determined by inhibition of 2-dGlc uptake using a 30 s time
point assay at 25 °C.
2. Experimental
general and can be applied to different amino
compounds (Scheme 1).
General methods.—Optical rotations were
determined with a Perkin–Elmer 241 polar-
imeter for solutions in water or MeOH. TLC
was conducted on Silica Gel 60 F254 plates (E.
Merck) with appropriate eluents. The prod-
ucts were detected by UV irradiation (u=254
nm) or by spraying the plates with an aqueous
solution of 10% HCl containing 5% ammon-
ium cerium (IV) nitrate or, for amino sugars,
an ethanolic solution of ninhydrin (0.1 g, 100
mL) and heating. Final products were purified
by preparative HPLC on a silica column
Attempts to synthesize 1-amino-2,5-anhy-
dro-1-deoxy- -mannitol (3) from 1 via the
D
Leuckart–Wallach reductive amination with
formamide or ammonium acetate were unsuc-
cessful, probably due to the drastic conditions
required, which involve a treatment at 180–
230 °C in a sealed tube [11]. An alternative
synthesis was therefore developed involving
either hydrogenolysis of the benzylamino–
mannitol derivative 4a or hydrogenolysis of
the intermediate oxime 2 formed from 1 and
hydroxylamine. The latter method turned out
to be simpler since the intermediate did not
need purification.
(Macherey–Nagel Polygoprep C18 12–25 mm
1
60 A). H and 13C NMR spectra in D2O or
˚
CD3OD were recorded at 200 MHz on a
Bruker AC 200 spectrometer. Chemical shifts
(l) were measured in ppm from tetramethylsi-
lane. High-resolution mass spectra were
recorded on an AUTOSPEC 6F [Micromass,
Altricham, UK] instrument using LSIMS (res-
olution 5000–10,000) as ionisation mode and
glycerol, trichloroacetic acid 1% in H2O as
matrix and polyethyleneglycol (PEG) or PEG-
Na as reference. Elementary analyses were
performed on an Eager 200 instrument by the
INP-ENSCT of Toulouse, France.
Affinity measurements on the hexose trans-
porter (Table 1) indicate that: (i) compounds
bearing aliphatic polar substituents at C-1
have no affinity; this result appears particu-
larly surprising for compound 1, largely in the
hydrate form and therefore close structurally
to the reference 2,5-anhydro- -mannitol; (ii)
D
in contrast, those bearing a hydrophobic
phenyl or naphthyl group on nitrogen bind at
the transporter; this affinity is still increased
further—up to 20 times that of the reference
fructose—by substituents on the ring inducing
a dipole, the negative part of which is
external.
2,5-Anhydro-
D
-mannose (1).—A solution
of 2-amino-2-deoxy-
D
-glucose hydrochloride
Other work is in progress for the design of
inhibitors that will allow us to explore the
binding site of this hexose carrier.
(4.22 g, 19.6 mmol) in water (100 mL) at
mutarotational equilibrium ($5 h at 25 °C)
was cooled to 0 °C. Sodium nitrite (3.45 g,

342
S. Claustre et al. / Carbohydrate Research 315 (1999) 339–344
1-Amino-2,5-anhydro-1-deoxy-
D
-mannitol
50 mmol) was added in one batch, followed
by cation-exchange resin (Amberlite IR-120,
H+, 100 mL) in portions, the temperature
being maintained at 0–5 °C during the addi-
tion. The mixture was stirred for 4 h at
0 °C, at which point the amino sugar was no
longer detectable. The resin was filtered and
the filtrate was neutralised by addition of an
anion-exchange resin (Dowex 1×8–50,
CO23−). The solution was filtered and the
filtrate was lyophilized to give a hygroscopic,
yellow–white solid (3.06 g, 17 mmol, 87%)
containing 1 and its hydrated form: [h]2D5+
31.8° (c 1,43; MeOH), lit. +33° [10]; Rf 0.43
(4:1 CH2Cl2–MeOH); 1H NMR (D2O): l
8.40 (s, 0.6 H, H-1 aldehyde), 5.05 (d, 1 H,
J1,2 7 Hz, H-1 gem-diol), 4.11 (t, 1 H, J2,3=
J3,4 5 Hz, H-3), 4.01 (t, 1 H, J4,5 5 Hz, H-4),
3.84 (m, 2 H, H-2 and H-5), 3.54–3.74 (2
dd, 2 H, J5,6 6 Hz and J6a,6b 12 Hz, H-6a
and H-6b); 13C NMR (D2O): l 188.2 (C-1
aldehyde), 92.31 (C-1 gem-diol), 86.66 (C-2),
85.92 (C-5), 80.05 (C-3), 79.17 (C-4), 63.70
(C-6); HRMS: Calcd for C6H13O6 [M+
H]+181.0712; Found 181.1394. Anal. Calcd
for C6H11O5 [M+H]+163.0606; Found
162.9981. Anal. Calcd for C6H12O6·1.5H2O:
C, 34.78; H, 7.29. Found: C, 34.56; H, 7.47.
(3)
Procedure A. Oxime 2 (2.63 g, 14.9 mmol)
was dissolved in a solution of formic acid
(4.4% in MeOH); Pd–C 10% (0.75 g) was
added and the reduction was achieved by
hydrogenation (at atmospheric pressure,
room temperature, 10 h). Filtration and
evaporation yielded 3 as a colourless oil
(2.21 g, 13.6 mmol, 91%), purified on cation-
exchange resin (Dowex 1×8, H+) with 0.5
M acetic acid as eluent: [h]2D5+28.7 ° (c 1,
1
MeOH); Rf 0.15 (EtOH+10% AcOH); H
NMR (CD3OD): l 4.06 (t, 1 H, J2,3 =J3,4
5.5 Hz, H-3), 3.96–3.92 (m, 2 H, H-2 and
H-5), 3.90 (dd, 1 H, J4,5 5 Hz, H-4), 3.74 (2
dd, 2 H, J5,6 6 Hz and J6a,6b 12 Hz, H-6a
and H-6b), 3.69 (2 dd, 2 H, J1a,1b 14.5 Hz,
H-1a and H-1b); 13C NMR (CD3OD): l
86.57 (C-5), 81.70 (C-3), 80.49 (C-2), 78.91
(C-4), 63.24 (C-6), 42.76 (C-1); HRMS:
Calcd for C6H14NO4 [M+H]+164.0923;
Found
164.0934.
Anal.
Calcd
for
C6H13NO4·1H2O: C, 39.79; H, 8.35; N, 7.73.
Found: C, 39.93; H, 8.16; N, 7.86.
Procedure B. Pd–C 10% (0.69 g) was
added to a solution of 4a (3.43 g, 13.8
mmol) in MeOH and the suspension was
stirred under H2 at atmospheric pressure,
room temperature for 16 h to yield com-
pound 3 (1.59 g, 9.8 mmol, 71%), identical
to the product obtained in procedure A.
2,5-Anhydro- -mannose oxime (2).—To a
D
solution of 1 (3.06 g, 17 mmol) in MeOH
were successively added hydroxylamine hy-
drochloride (1.76 g, 25.5 mmol) and sodium
acetate (2.79 g, 33.6 mmol). The solution
was stirred for 6 h at room temperature.
The methanol was removed under reduced
pressure and the resulting syrup was dis-
solved in abs EtOH to precipate the salts.
After filtration, complete removal of solvent
gave 2 (2.63 g, 14.9 mmol, 87%): [h]2D5+9.9°
(c 1, MeOH); Rf 0.56 (4:1 CH2Cl2–MeOH);
1H NMR (CD3OD): l 8.49 (s, 1 H, N–OH),
7.41 (d, 1 H, J1,2 7.3 Hz, H-1), 4.38 (dd, 1
N-Aryl-1-amino-2,5-anhydro-1-deoxy-1-
mannitols (4a–f)
D
-
General procedure. NaBH3CN (0.7 equiv)
was added to a solution of 1 (1 equiv) and
the primary arylamine (1 equiv) in absolute
MeOH at pH 5.8. The solution was stirred
at room temperature while the pH was
maintained at 5.8 by dropwise addition of
0.5 M HCl in MeOH until the starting ma-
terial was no longer detectable. The mixture
was concentrated to a small volume by rota-
tory evaporation under reduced pressure and
resulting 4 was purified by preparative
HPLC using various proportion of water–
MeCN as eluent.
H, J2,3 4.8 Hz, H-2), 4.11 (dd, 1 H, J3,4
7
Hz, H-3), 4.03 (t, 1 H, H-4), 3.90 (m, 1 H,
H-5), 3.69 (2 dd, 2 H, J5,6 6 Hz and J6a,6b 12
Hz, H-6a and H-6b); 13C NMR (CD3OD): l
150.51 (C-1), 85.65 (C-5), 82.04 (C-2), 81.14
(C-3), 78.67 (C-4), 63.26 (C-6); HRMS:
Calcd for C6H12NO5 [M+H]+178.0715;
Found 178.0712.
2,5 - Anhydro - 1 - benzylamino - 1 - deoxy - -
D
mannitol (4a).—From benzylamine (2 g; 18.7
mmol); HPLC purification (95:5 water–
MeCN) gave a colourless oil (3.49 g; 13.8

S. Claustre et al. / Carbohydrate Research 315 (1999) 339–344
343
1
mmol; 74%): [h]2D5+75.3 ° (c 1.1, MeOH); H
NMR (CD3OD): l 7.45 (m, 5 H, H-2% to
H-6%), 4.02 (t, 1 H, J2,3=J3,4 4.5 Hz, H-3),
3.92 (m, 2 H, H-2 and H-5), 3.85 (dd, 1 H, J4,5
5 Hz, H-4), 3.59–3.70 (4 dd, 4 H, J6a,6b 12 Hz
and J1a,1b 14.5 Hz, H-6a, H-6b, H-1a and
H-1b), 3.20 (d, 2 H, J7a,7b 12 Hz, H-7%a and
H-7%b); 13C NMR (CD3OD): l 133.38 (C-1%),
131 and 131.09 (C-3% and C-5%), 130.24 (C-2%
and C-6%), 130.03 (C-4%), 86.39 (C-5), 81.07
(C-2), 80.86 (C-3), 78.74 (C-4), 63.22 (C-6),
50.43 (C-7), 44.40 (C-1); HRMS: Calcd for
C13H19NO4Na [M+Na]+ 276.1212; Found
276.1220. Anal. Calcd for C13H19NO4·0.5H2O:
C, 59.56; H, 7.70; N, 5.34. Found: C, 59.71;
H, 7.42; N, 5.22.
274.0846; Found 274.0848. Anal. Calcd for
C12H16ClNO4·1.5H2O: C, 48.02; H, 6.38; N,
4.67. Found: C, 47.94; H, 6.51; N, 4.58.
2,5-Anhydro-1-deoxy-1-(m-nitrophenylam-
ino)- -mannitol (4d).—From 3-nitroaniline
D
(0.48 g; 3.5 mmol); HPLC purification (4:1
water–MeCN) gave an orange oil (0.87 g;
3.08 mmol; 88%): [h]2D5 +76° (c 1.1, MeOH);
1H NMR (CD3OD): l 7.42 (m, 2 H, H-2% and
H-6%), 7.27 (t, 1 H, J4%,5%=J5%,6% 8.3 Hz, H-5%),
7.03 (dd, 1 H, J2%,4%=J4%,6% 2 Hz, H-4%), 3.98 (m,
3 H, H-2, H-3 and H-4), 3.86 (m, 1 H, H-5),
3.61–3.70 (2 dd, 2 H, J5,6 6 Hz and J6a,6b 12
Hz, H-6a and H-6b), 3.30–3.35 (2 dd, 2 H,
J1a,1b 14.5 Hz and J1,2 6 Hz, H-1a and H-1b);
13C NMR (CD3OD): l 151.19 (C-1%), 151 (C-
3%), 130.84 (C-5%), 119.90 (C-6%), 112.10 (C-4%),
107.27 (C-2%), 85.25 (C-5), 83.12 (C-2), 80.35
(C-3), 78.86 (C-4), 63.29 (C-6), 46.75 (C-1);
HRMS: Calcd for C12H17N2O6 [M+H]+
285.1087; Found 285.1087. Anal. Calcd for
C12H16N2O6·1H2O: C, 47.71; H, 6.18; N, 9.27.
Found: C, 47.63; H, 6.18; N, 9.32.
2,5-Anhydro-1-deoxy-1-phenylamino- -man-
D
nitol (4b).—From aniline (0.51 g; 5.5 mmol);
HPLC purification (9:1 water–MeCN) gave a
hygroscopic, pale-yellow solid (1.02 g; 4.3
1
mmol; 78%): [h]2D5+35.6° (c 1.15, H2O); H
NMR (CD3OD): l 7.10 and 6.66 (2 m, 2 H
and 3 H, H-2% to H-6%), 4.01 (m, 3 H, H-3, H-4
and H-2), 3.86 (m, 1 H, H-5), 3.75–3.59 (2 dd,
2 H, J5,6 6 Hz, and J6a,6b 12 Hz, H-6a and
H-6b), 3.40–3.19 (2 dd, 2 H, J1a,1b 14,5 Hz
and J1,2 6 Hz, H-1a and H-1b); 13C NMR
(CD3OD): l 150.08 (C-1%), 130.19 (C-2% and
C-6%), 118.55 (C-3% and C-5%), 114.40 (C-4%),
85.11 (C-5), 83.18 (C-2), 80.61 (C-3), 78.99
(C-4), 63.39 (C-6), 47.41 (C-1); HRMS: Calcd
for C12H18NO4 [M+H]+ 240.1236; Found
240.1234. Anal. Calcd for C12H17NO4·1H2O:
C, 56.05; H, 7.45; N, 5.45. Found: C, 55.93;
H, 7.6; N, 5.34.
2,5-Anhydro-1-deoxy-1-(1-naphthylamino)-
D-mannitol (4e).—From naphthylamine (0.43
g; 3 mmol); HPLC purification (MeCN) gave
a colourless oil (0.35 g; 1.2 mmol; 40%):
[h]2D5+15.3° (c 1.2, MeOH); 1H NMR
(CD3OD): l 7.77 and 7.63 (2 m, 2 H, H-4% and
H-5%), 7.30–7.09 (m, 4 H, H-2%, H-3%, H-6% and
H-7%), 8.47 (dd, 1 H, J7%,8% 7.7 Hz and J6%,8%
2
Hz, H-8%), 4.20 (t, 1 H, H-3), 4.11 (t, 1 H, J4,5
6.5 Hz, H-4), 3.95 (m, 1 H, H-5), 3.73–3.68
(m, 3 H, J5,6 6.5 Hz, J6a,6b 12 Hz, H-2, H-6a
and H-6b), 3.35–3.30 (2 dd, 2 H, J1a,1b 14 Hz
and J1,2 6 Hz, H-1a and H-1b); 13C NMR
(CD3OD): l 143.54 (C-1%), 134.27 (C-9%),
128.46 (C-3%), 126.66 (C-5%), 125.79 (C-6%),
124.73 (C-8%), 123.69 (C-10%), 120.50 (C-7%),
117.59 (C-4%), 104.67 (C-2%), 83.72 (C-5), 82.16
(C-2), 79.83 (C-3), 77.86 (C-4), 62.23 (C-6),
46.37 (C-1); HRMS: Calcd for C16H20NO4
[M+H]+ 290.1392; Found 290.1369. Anal.
Calcd for C16H19NO4: C, 66.47; H, 6.62; N,
4.84. Found: C, 66.15; H, 6.77; N, 4.79.
2,5 - Anhydro - 1 - (m - chlorophenylamino) - 1-
deoxy- -mannitol (4c).—From 3-chloroani-
D
line (0.64 g; 5 mmol); HPLC purification (7:3
water–MeCN) gave a pale-brown oil (0.98 g;
3.6 mmol; 72%): [h]2D5 +48.6° (c 1.24, MeOH);
1H NMR (CD3OD): l 7.03 (t, 1 H, J4%,5%=J5%,6%
8 Hz, H-5%), 6.64 (t, 1 H, J2%,4%=J2%,6% 2.5 Hz,
H-2%), 6.57 (2dd, 2 H, H-4% and H-6%), 4.01–
3.97 (m, 3 H, H-3, H-4 and H-2), 3.79 (m, 1
H, H-5), 3.67 (2 dd, 2 H, J5,6 6 Hz, and J6a,6b
12 Hz, H-6a and H-6b), 3.42 (2 dd, 2 H, J1a,1b
14.5 Hz and J1,2 6 Hz, H-1a and H-1b); 13C
NMR (CD3OD): l 151.63 (C-1%), 135.92 (C-
3%), 131.24 (C-5%), 117.54 (C-4%), 113.37 (C-2%),
112.3 (C-6%), 85.19 (C-5), 83.15 (C-2), 80.48
(C-3), 78.96 (C-4), 63.33 (C-6), 46.83 (C-1);
HRMS: Calcd for C12H17ClNO4 [M+H]+
2,5-Anhydro-1-deoxy-1-(4-nitro-1-naphthyl-
amino)- -mannitol (4f).—From 4-nitro-1-
D
naphthylamine (0.6 g; 3.2 mmol); HPLC
purification (9:1 water–MeCN) gave an or-
ange solid (0.5 g; 1.5 mmol; 47%): [h]2D5+2° (c
1
1.1, MeOH); H NMR (CD3OD): l 8.88 (dd,
1 H, J7%,8% 8 Hz and J6%,8% 2 Hz, H-2%), 8.39 (d, 1

344
S. Claustre et al. / Carbohydrate Research 315 (1999) 339–344
H, J2%,3% 8.3 Hz, H-8%), 8.12 (d, 1 H, J5%,6% 7 Hz,
H-5%), 7.63 and 7.46 (2 t, 2 H, H-6% and H-7%),
6.59 (d, 1 H, H-3%), 4.62 (1 H, NH), 4.21 (dd,
1 H, H-3), 4.09–3.96 (m, 3 H, H-2, H-5 and
H-4), 3.77–3.58 (4 dd, 4 H, J5,6 6.5 Hz, J6a,6b
13 Hz, J1a,1b 14.5 Hz and J1,2 6 Hz, H-1a,
H-1b, H-6a and H-6b); 13C NMR (CD3OD): l
152.66 (C-1%), 134.72 (C-4%), 131.27 (C-6%),
130.80 (C-7%), 129.93 (C-10%), 126.60 (C-3%),
125.31 (C-8%), 123.84 (C-9%), 122.86 (C-5%),
102.24 (C-2%), 86.13 (C-5), 83.32 (C-2), 80.79
(C-3), 79.03 (C-4), 63.23 (C-6), 44.12 (C-1);
HRMS: Calcd for C16H19N2O6 [M+H]+
335.1243; Found 335.1231. Anal. Calcd for
C16H18N2O6: C, 57.52; H, 5.43; N, 8.38.
Found: C, 57.71; H, 5.31; N, 8.50.
inhibiting analogues were determined from the
relationship V0/V=1+([I]/Ki) given by
Eisenthal et al. [14], where V0 and V are the
uninhibited and inhibited initial rates respec-
tively and [I] is the concentration of inhibitor.
Acknowledgements
Financial support for this project was pro-
vided by the European, Science, Research and
Development Commission (IC 18 CT 960079)
and the GDR CNRS-DRET no. 1077.
References
Determination of inhibition constants (Ki) of
the analogues with the Trypanosoma brucei
glucose transporter.—Long slender blood-
stream forms of Trypanosoma brucei strain
427 were grown in rats and isolated by DEAE
ion-exchange chromatography, as previously
described by Lanham et al. [12]. The cells were
washed three times in a phosphate-buffered
saline solution (PBS: 0.15 M NaCl, 5 mM
potassium phosphate, pH 7.4) at 4 °C and
resuspended in PBS at a concentration of 109
cells ml−1. After incubation for 2 min at
25 °C, 100 mL of cell suspension were incu-
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bated during 30 s with 2-deoxy-
cose as substrate (100 mM, 0.1 mCi per point)
and various 2,5-anhydro- -mannitol ana-
D
-[1-3H]-glu-
D
logues as inhibitors (0.01–10 mM). The up-
take was stopped by centrifugation, spinning
the cells through an oil cushion of 1-bromodo-
decane, as described by Wille et al. [13]. The
amount of radioactivity contained in the cell
pellet was determined by liquid scintillation
counting. Inhibition constant (Ki) values of
.