Synthesis of biotinylated bis(D-glucose) derivatives for glucose transporter photoaffinity labelling

Article abstract of DOI:10.1016/S0008-6215(01)00025-8

New diazirine based bis-glucose derivatives for tagging glucose transporters have been synthesised. These included two biotinylated compounds linked either by an aminocaproate or by a cleavable dithiol link. These compounds have been derivatised via a key

Full text of DOI:10.1016/S0008-6215(01)00025-8

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Carbohydrate Research 331 (2001) 119–127
Synthesis of biotinylated bis(
D-glucose) derivatives for
glucose transporter photoaffinity labelling
Makoto Hashimoto,a Yasumaru Hatanaka,b Jing Yang,a Jaswant Dhesi,a
Geoffrey D. Holmana,*
aDepartment of Biology and Biochemistry, Uni6ersity of Bath, Cla6erton Down, Bath BA2 7AY, UK
bResearch Institute for Wakan-Yaka, Toyama Medical and Pharmaceutical Uni6ersity, Sugitani 2630,
Toyama 930-0194, Japan
Received 20 September 2000; accepted 18 January 2001
Abstract
New diazirine based bis-glucose derivatives for tagging glucose transporters have been synthesised. These included
two biotinylated compounds linked either by an aminocaproate or by a cleavable dithiol link. These compounds have
been derivatised via a key skeleton compound that can be easily used for introduction of additional tags. Studies on
the erythrocyte glucose transporter (GLUT1) and the insulin-stimulated adipose cell transporter (GLUT4) have
revealed the biotinylated photoreactive bis-glucose compounds are effective labelling reagents. © 2001 Elsevier
Science Ltd. All rights reserved.
Keywords: Bis-glucose; Biotin; Photolabels; Glucose transporters
1. Introduction
the transporters.3 We have previously found
that a bis-hexose structure has advantages for
photolabelling of the exofacial surface of the
transporters. The bis-structure allows a hydro-
phobic moiety to be inserted in the bridge in a
form that produces a molecule, that overall,
still has a hydrophilic character and is water-
soluble. In addition, the bis-hexoses are bulky
and consequently act as powerful inhibitors of
glucose transport but are not themselves
transported because of steric constraints im-
posed by the bulky substituents. Consequently
as these probes are too hydrophilic to enter
cells through the membrane lipid, they act
specifically at the exofacial binding site. This
bis-structure has previously been reported for
bis-mannose4 linked by a 2-propylamine
bridge and bis-glucose derivatives linked by a
secondary amine bridge.5 The bis-mannose
propyl-2-amine (BMPA) compounds includ-
There are five major glucose transporters in
mammalian tissues (GLUTs 1–5)1 but new
isoforms are being discovered.2 These proteins
catalyse one of the most important cell mem-
brane transport events since glucose plays a
central role in cellular homeostasis and
metabolism.
The GLUT transporters have broad specifi-
cities for hexoses and, in addition to glucose,
other monosaccarides such as galactose and
mannose are transported. Previous studies
have established that bulky groups are toler-
ated around the 4-OH position when the
hexose occupies the exofacial binding sites of
* Corresponding author. Tel.: +44-1225-826874; fax: +
44-1225-826779.
E-mail address: g.d.holman@bath.ac.uk (G.D. Holman).
0008-6215/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(01)00025-8

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M. Hashimoto et al. / Carbohydrate Research 331 (2001) 119–127
ing a biotinylated derivative6 have been partic-
ularly useful in tagging the glucose trans-
porters and therefore we have examined here a
bis-glucose compound with the versatile 2-
propylamine bridge. The choice of photoreac-
tive substituent for studying glucose
transporters was also considered to be impor-
tant. Bis-mannose derivatives in which aryl
azide or benzophenone groups have been in-
troduced have been previously described.7,8
The disadvantages of the aryl azides is that
nucleophilic side reactions reduce the specific-
ity of the labelling reaction while the disad-
vantage of the benzophenone groups is that
long irradiation times are needed to produce a
high yield of cross-linked adduct. By contrast,
diazirine groups have been found to generate
highly reactive carbenes smoothly and specifi-
cally with short irradiation times. Further-
more, we have found that a carbene-
introduced linkage is more stable than those
introduced by the other two species.9 We have
consequently concluded that diazirine is the
most useful photophor10,11 and have synthe-
sised new bis-glucose compounds that contain
this photoreactive group. We have chosen to
introduce biotin groups attached by either
amino-hexanoate or cleavable dithiol links.
The advantage of the latter is that it allows
release, under mild reducing conditions at
moderate temperatures, of the photolabelled
protein from the avidin or streptavidin matrix
used to isolate and purify the protein. We
have found that the release of photolabelled
transporters, tagged with the previously de-
scribed biotin-amino-hexanoate linked bis-
mannose photolabel Bio-LC-ATB-BMPA,
from streptavidin agarose requires that the
adduct be heated at 100 °C for 30 min in the
presence of denaturing SDS.
In our consideration of the route to produce
new bis-glucose photolabels we thought it use-
ful to prepare a key intermediate that could
subsequently be used to introduce new tags as
required. In this paper we describe the synthe-
sis of novel amino BOC protected diazirine-
based photoreactive bis-glucose derivative as
this key intermediate. Removal of a BOC
protecting group leaves an amine group that
can then be easily coupled to any probe moi-
ety containing a carboxylate group via amide
bond formation. To determine the utility of
the approach we studied the interaction of the
biotinylated bis-glucose photoaffinity probes
with the human erythrocyte transporter,
GLUT1 and the with the adipose cell trans-
porter, GLUT4.
2. Results and discussion
Synthesis of bis-glucose photoreacti6e
probes.—The route for synthesis of the bis-
glucose compounds is shown in Fig. 1. 2,3,5,6-
Di-O-isopropylidene-
D
-glucose
dimethyl
acetal (1)12 was converted to the 4-O-linked
dimer 2 with 1,3-dichloro-2-propanone O-
benzyloxime. This crosslinking reagent has
been previously used to synthesise bis- -man-
D
nose-2-propylamine.13 The reaction proceeds
rapidly to a monochloro intermediate (which
could be isolated and characterised) and then
more slowly to the bis- compound. These
conversions can be followed by thin layer
chromatography (TLC). Following purifica-
tion, 2 was hydrogenated using Raney nickel
as catalyst to give the amine 3 in good yield.
The protecting acetal groups were then re-
moved by acid hydrolysis to generate the 1,3-
bis(glucos-4-yloxy) 2-propylamine compound
(4).
Fig. 1. (i) (a) NaH, DMF, room temperature; (b) 1,3-
dichloro-2-propane benzyloxime, room temperature, 74%. (ii)
H2, Raney nickel, ethanol, H2O, NH4OH, room temperature,
94%. (iii) 1 M HCl, 100 °C, quant.

M. Hashimoto et al. / Carbohydrate Research 331 (2001) 119–127
121
Fig. 2. (i) BBr3, CH2Cl2, room temperature, 64%. (ii) 2-[2-(2-tert-Butoxycalbonylaminoethoxy)ethoxy]ethyl bromide, K2CO3,
n-Bu4NI, DMF, 60 °C, 91%. (iii) n-Bu4NMnO4, pyridine, room temperature, 90%. (iv) N-Hydroxysuccinimide, dicyclohexylcar-
bodiimide, CH2Cl2, CH3CN, room temperature, 99%.
Fig. 3. (i) 0.1 M NaHCO3, DMF, room temperature, 88%. (ii) 50% Trifluoroacetic acid, CH2Cl2, 0 °C, 86%. (iii) N-Biotinylamino
caproic acid N-hydroxysuccinimide ester, 0.1 M NaHCO3–DMF, room temperature, 37%. (iv) ([2-(Biotinyl)ethylamido]-3,3%-
dithiodipropionic acid N-hydroxysuccinimide ester, 0.1 M NaHCO3–DMF, room temperature, 40%.
sis of the key skeleton compound 11, we
required the novel compound 9, the produc-
tion of which, itself required modifications of
previously described syntheses (Fig. 2). We
were able to achieve ether cleavage of 515 with
BBr3 without protection of the aldehyde
group of this diazirine compound. Phenol
alkylation with the BOC protected ethylene
glycol spacer14 then afforded 7 in good yield.
The aldehyde group of 7 was then oxidised to
the carboxylic acid in 8 which was then con-
verted to its N-hydroxy-succinimide ester
derivative 9 (Fig. 2).
We have previously reported the synthesis
of a diazirinyl aryl carboxylate with a
polyether spacer linked to biotin14 that can be
used to biotin tag proteins directly. However,
we considered it more useful in the present
study to synthesise from 4, a photoreactive
skeleton compound first and then subse-
quently couple this to biotin via ether
aminocaproate or dithiol linkers (Figs. 2 and
3). Many more applications of the intermedi-
ate 11 are then possible as it can be further
substituted with any additional tags as re-
quired for additional applications. For synthe-

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M. Hashimoto et al. / Carbohydrate Research 331 (2001) 119–127
The condensation of compound 4 and 9 in
smoothly with a moderate yield of the desired
product.
nonaqueous conditions (triethylamine or mor-
pholine in DMF) was slow and only produced
low yields of 10 (less than 20%), possibly due
to low solubility of the bis-glucose-2-propy-
lamine compound in DMF We therefore used
a water–DMF mixture for coupling. The bis-
glucose-2-propylamine compound and the di-
azirine were dissolved in 0.1 M NaHCO3 and
DMF, respectively. The reaction proceeded
The BOC protected skeleton was stable on
storage and could be conveniently deprotected
by TFA treatment for immediate coupling to
an appropriate biotin-NHS ester as required.
The biotinylated compounds studied here in-
cluded compounds with aminocaproate and
dithiol spacers, compounds 12 and 13, respec-
tively (Fig. 3).
Photoaffinity labelling of human GLUT1.—
Erythrocytes provide a rich source of GLUT1,
which is present as 3–5% of the membrane
protein. GLUT1 was purified as previously
described.16 Purified GLUT1 was then mixed
with stoichiometric amount of compound 12
or 13, and the samples were then irradiated
and the protein was resolved by SDS-PAGE
and transferred to nitrocellulose as described
in Section 3. Extravidin peroxidase conjuga-
tion revealed strong biotinylated bands at ap-
proximately 50 kDa for both compounds 12
and 13 and these signals were competitively
reduced in the incubations with
D
-glucose
Fig. 4. Chemiluminescence detection of photoaffinity labelled
human GLUT1. Compounds 12 and 13 were used to photola-
bel GLUT1 purified from human erythrocytes. Glucose (0.17
M) was added as a competitor for the binding site during the
photolabelling reaction (lanes 2 and 4). The susceptibility of
the dithiol bridge in GLUT1 photolabelled with compound 13
to reducing agent was examined by adding 0.5 M dithiothrei-
tol at room temperature for 15 min after the photolabelling
reaction. Photolabelled samples were resolved by SDS-PAGE
on 10% gels run under reducing conditions (compound 12)
and non-reducing conditions (compound 13) and then trans-
ferred to nitrocellulose for analysis of the biotin signal by
using avidin-HRP and ECL detection.
(Fig. 4). Under the labelling conditions em-
ployed, the signal was therefore specific for
the binding site of GLUT1. One of our aims
was to test the feasibility of using compound
13 in experiments in which the biotin moiety is
removed by a reducing agent that breaks the
dithiol spacer arm. We therefore incubated the
GLUT1 samples (phototagged with 13) either
in the absence or presence of dithiothreitol
and resolved the labelled proteins under non-
reducing conditions (Fig. 4). The biotin group
was found to be cleaved under quite mild
conditions and therefore, this compound is
likely to be useful in future studies in which
release of biotin-tagged protein or binding-site
peptides under mild conditions is required.
Photolabelling of insulin-stimulated GLUT4
in adipocytes.—To determine the affinity of
the interaction between the ligands 12 and 13
and GLUT4, each of the compounds was
Fig. 5. Determination of half maximal inhibition constants
tested as a competitive inhibitor of 2-deoxy- -
D
for bis
-glucose in insulin-stimulated rat adipose cells was deter-
D
-glucose ligands. The uptake of tracer 2-deoxy-[14C]-
glucose uptake into insulin-stimulated rat
adipocytes (Fig. 5). Ki values or half-maximal
inhibition constants for 12 and 13 were 195
and 142 mM, respectively. The Ki value for 12
was similar to, but slightly better than that of
the equivalent bis-mannose compound Bio-
D
mined at the indicated concentrations of compounds 12 (ꢀ)
and 13 (ꢁ). The rate constants (mean and S.E.M. are shown)
for uptake in the presence (V) and absence (V0) of inhibitor
(I) which were then used to calculate the Ki according to the
equation V0/V=1+I/Ki. Results shown are from three ex-
periments.

M. Hashimoto et al. / Carbohydrate Research 331 (2001) 119–127
123
3. Experimental
General methods.—Silica Gel for column
chromatography was Kieselgel 60 (E. Merck,
230–400 mesh). Structural characterisations
of synthetic products were performed with a
JEOL JNM GX-400 spectrometer (1H NMR)
and a Micromass Autospec (MS). Elemental
analysis was carried out by the microanalysis
service (Bath University). The purity of those
compounds that were not available in suffi-
cient quantity for elemental analysis were de-
termined by a combination of diazirine UV
detection, biotin detection and hexose detec-
tion techniques applied to the compounds on
TLC plates. Furthermore, no extraneous lines
were detected in the NMR spectra and UV
spectra and extinction coefficients at 350 nM
were consistent with those previously estab-
lished compounds containing the diazirine
moiety. (Extravidin-peroxidase conjugate was
obtained from Sigma. Chemiluminescence de-
tection reagent and Hyper-film ECL were ob-
tained from Amersham. Nitrocellulose
membrane (BioTrace NT) was obtained from
GelmanScience.)
Fig. 6. Quantification of GLUT4 labelling. Rat adipose cells
were UV irradiated in the presence of compound 12 at the
indicated concentrations. Cell membrane protein was then
isolated and solubilised. Streptavidin beads was then used to
precipitate the biotin-tagged GLUT4, which was resolved by
SDS-PAGE and detected using an anti-GLUT4 antibody,
peroxidase coupled secondary antibody and ECL (A). The
ECL signal was quantified by comparison with a GLUT4
standard and the extent to which the labelling became satu-
rated with increasing ligand concentrations was determined
(B).
1,3-Bis(2,3, 5, 6-di-O-isopropylidene- -glucose
D
dimethylacetal-4-yloxy)-2-propane-O-benzyl-
oxime (2).—2,3,5,6-Di-O-isopropylidene glu-
copyranosyl dimethyl acetal (1)12 (2.36 g, 7.7
mmol) was dissolved in DMF (15 mL).
Sodium hydride (60%, 0.34 g, 8.5 mmol) was
added to this solution at rt. After bubbling
had ceased, 1,3-dichloro-2-propanone O-
benzyloxime13 (0.89 g, 3.9 mmol) was added.
The reaction mixture was stirred at rt for 2 h.
Additional NaH (0.09 g, 2.2 mmol) was then
added. The reaction mixture was stirred for a
further 1 h, quenched by AcOH (0.3 mL) and
partitioned between CH2Cl2 and water. The
aqueous phase was further extracted into
CH2Cl2 twice. The combined organic layer
was washed with 1 M HCl, saturated
NaHCO3 and satd NaCl, and then dried over
Na2SO4. After filtration, the product was con-
centrated. The residue was purified with silica
column chromatography (4:1 n-hexane–
EtOAc) to afford a colourless oil (2.21 g,
LC-ATB-BMPA (Ki of 273 mM Ref. 6). We
have also determined the most appropriate
concentration of 12 to use in GLUT4-tagging
experiments (Fig. 6). To do this we have pho-
tolabelled, washed and solubilised the adipose
cell protein as described in Section 3 and then
precipitated the biotin-tagged protein with
streptavidin-agarose. A wash step then al-
lowed removal of the non-biotinylated
GLUT4. The precipitated material was next
resolved by SDS-PAGE and an antibody to
the C-terminal section of GLUT4 was used to
detect the tagged protein (Fig. 6(A)). Only a
single labelled band was obtained and the
amount of GLUT4 was quantified by refer-
ence with a standard containing a known
amount of this protein.17 This quantification
(Fig. 6(B)) revealed that maximal tagging with
biotin occurred when 200–400 mM ligand was
used, a value that is consistent with the
affinity constant revealed in the transport inhi-
bition experiments.
1
74%). H NMR (CDCl3): l 7.28–7.38 (5 H,
m, Ph), 5.07 (2 H, s, NOCH2Ph), 4.58 (4 H, s,
OCH2CꢀNO), 4.35 (2 H, d, J=6.0 Hz, H-1),

124
M. Hashimoto et al. / Carbohydrate Research 331 (2001) 119–127
2 - Hydroxy - 4 - [3 - (trifluoromethyl) - 3H-
diazirin-3-yl] benzaldehyde (6).—2-Methoxy-
4.30 (2 H, ddd, J=5.0, 6.0, 6.0 Hz, H-5), 4.17
(2 H, dd, J=6.0, 7.6 Hz, H-2), 4.00–3.95 (6
H, m, H-3 and 6), 3.62 (2 H, dd, J=2.6, 5.0
Hz, H-4), 3.40 (12 H, s, OCH3), 1.37 (24 H, s,
C(CH3)2); FABMS: m/z 772 ([M+H]+); HR-
FABMS: Calcd for C38H62NO15 ([M+H]+)
772.4119; Found 772.4110. Anal. Calcd for
C38H61NO15 C, 59.13; H, 7.97; N, 1.81.
Found: C, 59.2; H, 7.82; N, 1.88.
4-[3-(trifluoromethyl)-3H-diazirin-3-yl]
ben-
zaldehyde (5)15 (3.396 g, 13.9 mmol) was
dissolved in CH2Cl2 at −20 °C. BBr3 (1.4 mL,
14.8 mmol) was added in portions. The reac-
tion mixture was stirred at 0 °C for 1 h and
water was added to quench the reaction. The
organic layer was washed with saturated
NaCl, dried over MgSO4, filtered and then
concentrated. The residue was purified with
silica column chromatography (5:1 n-hexane–
CH2Cl2) to afford a pale yellow oil (2.048 g,
1,3-Bis(2,3,5, 6-di-O-isopropylidene-
D-glu-
cose dimethylacetal-4-yloxy)-2-propylamine
(3).—Compound 2 (2.02 g, 2.6 mmol) and
Raney nickel (59%, 8 mL) were suspended in
16:8:1 water–EtOH–NH3 (20 mL). The reac-
tion mixture was vigorously stirred at rt under
a hydrogen atmosphere for 19 h. The mixture
was filtered through Celite and was then con-
centrated. The residue was partitioned be-
tween EtOAc and water. The organic phase
was washed with saturated NaCl, dried over
Na2SO4, filtered and then concentrated to af-
1
64%). H NMR (CDCl3): l 9.90 (1 H, s,
CHO), 7.59 (1 H, d, J=7.9 Hz, Ph), 6.07 (1
H, d, J=7.9 Hz, Ph), 6.75 (1 H, s, Ph). EIMS:
m/z 230 ([M]+). HRMS Calcd for
C9H5F3N2O2 ([M]+): 230.0303; Found:
230.0291.
2 - [2 - [2 - [2 - (2 - tert - Butoxycarbonylamino-
ethoxy)ethoxy]ethoxy] - 4 - [3 - (trifluoromethyl)-
3H-diazirin-3-yl] benzaldehyde (7).—Com-
pound 6 (2.001 g, 8.69 mmol), 2-[2-(2-tert-
butoxycalbonylaminoethoxy)ethoxy]ethyl bro-
mide,14 K2CO3 (1.999 g, 14.4 mmol) and
nBu4NI (0.403 g, 1.08 mmol) were suspended
in DMF (109 mL). The suspension was heated
at 60 °C for 12 h, concentrated and parti-
tioned between EtOAc and water. The organic
layer was washed with 1 M HCl, satd
NaHCO3 and satd NaCl, and then dried over
MgSO4. After filtration, the product was con-
centrated and subjected to silica column chro-
matography (1:1 n-hexane–EtOAc) to afford
1
ford a colourless oil (1.63 g, 94%). H NMR
(CDCl3): l 4.36 (2 H, d, J=6.2 Hz, H-1), 4.23
(2 H, ddd, J=5.0, 6.0, 6.0 Hz, H-5), 4.14 (2
H, dd, J=6.2, 7.4 Hz, H-2), 4.03–3.98 (6 H,
m, H-3 and 6), 3.67 (1 H, t, J=5.9 Hz,
CHNH2), 3.61 (2 H, dd, J=2.4, 5.0 Hz, H-4),
3.48 (4 H, d, J=5.9 Hz, OCH2), 3.40 (12 H,
s), 1.37 (24 H, s), 4.36 (4 H, d, J=6.3 Hz),
4.3–3.5 (14 H, m), 4.00 (4 H, d, J=6.3 Hz),
3.45 (12 H, s, OCH3), 3.20 (1 H, m), 1.76 (1
H, br), 1.42, 1.40, 1.34 (total 24 H, each s,
C(CH3)2). FABMS: m/z 668 ([M+H]+); HR-
FABMS: Calcd for C31H58N014 ([M+H]+)
668.3857; Found 668.3848. Anal. Calcd For
C31H57NO14·0.5 H2O: C, 55.02; H, 8.64; N,
2.07. Found: C, 55.2; H, 8.52; N, 2.09.
1
a pale yellow oil (3.658 g, 91%). H NMR
(CDCl3): l 10.48 (1 H, s, CHO), 7.85 (1 H, d,
J=8.2 Hz, Ph), 6.86 (1 H, d, J=8.2 Hz, Ph),
6.74 (1 H, s, Ph), 4.27 (2 H, t, J=4.7 Hz),
3.92 (2 H, t, J=4.7 Hz), 3.71 (2 H, m), 3.64 (2
H, m), 3.54 (2 H, m), 3.32 (2 H, m), 1.43 (9 H,
s, C(CH3)3). FABMS m/z 462 ([M+H]+).
HRFABMS Calcd for C20H27F3N3O6 ([M+
H]+): 462.1582; Found: 462.1844.
1,3-Bis( -glucopyranos-4-yloxy)-2-propyl-
D
amine (4).—Compound 3 (0.56 g, 0.83 mmol)
was suspended in 1 M HCl (2 mL) and heated
at 100 °C for 3 h. The reaction mixture was
neutralised with Amberlite IRA-93 (OH form)
and the resin was removed by filtration. The
filtrate was concentrated to afford light brown
2 - [2 - [2 - [2 - (2 - tert - Butoxycarbonylamino-
ethoxy)ethoxy]ethoxy] - 4 - [3 - (trifluoromethyl)-
3H-diazirin-3yl] benzoic acid (8).—Compound
1
oil (0.39 g, quant.). H NMR (CD3OD): l
7
(2.135 g, 4.63 mmol) and tetra-
5.05 (1 H, d, J=3.6 Hz, 1-a, 4.40 (1 H, d,
J=7.6 Hz, 1-b), 3.4–4.0 (17 H, m). FABMS
m/z 416 ([M+H]+). HRFABMS Calcd for
C15H30NO12 ([M+H]+): 416.1768. Found:
416.1745.
butylammonium permanganate (2.485 g, 6.87
mmol) were dissolved in Py (45 mL) at rt and
stirred for 2 h. The reaction mixture was
diluted with toluene and washed with 1 M
Na2SO4,
1
M
HCl and satd NaCl.

M. Hashimoto et al. / Carbohydrate Research 331 (2001) 119–127
125
Hz, Ph), 6.89 (1 H, s, Ph), 5.23 (1 H, d, J=3.6
Hz, 1-a), 4.44 (1 H, d, J=7.6 Hz, 1-b), 4.40–
3.20 (29 H, m), 1.37 (9 H, s, C(CH3)3).
FABMS: m/z 875 ([M+H]+). HRFABMS
The organic layer was dried over MgSO4,
filtered and concentrated to afford a yellow oil
1
(1.988 g, 90%). H NMR (CDCl3): l 8.16 (1
H, d, J=8.2 Hz, Ph), 6.94 (1 H, d, J=8.2
Hz, Ph), 6.78 (1 H, s, Ph), 4.37 (2 H, m), 3.92
(2 H, m), 3.72 (2 H, m), 3.65 (2 H, m), 3.55 (2
H, m), 3.32 (2 H, m), 1.42 (9 H, s, s, C(CH3)3).
FABMS: m/z 478 ([M+H)+). HRFABMS
Calcd
for
C35H54F3N4O18
([M+H]+):
875.3385; Found 875.3370.
N-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]-
4 - [3 - (trifluoromethyl) - 3H - diazirin - 3 - yl]-
Calcd
for
C20H27F3N3O7
([M+H]+):
benzoyl]1,3 - bis( - glucopyranos - 4 - yloxy) - 2-
D
478.1801; Found: 478.1799.
propylamine (11).—Trifluoroacetic acid (1
mL) was added to compound 10 (0.181 g, 0.21
mmol) in CH2Cl2 (1 mL) at 0 °C and the
mixture was stirred for 0.5 h. The reaction
mixture was concentrated and then redis-
solved in water (2 mL). The solution was
passed through a 3 mL column of Amberlite
IRA-93 (OH form) and the product was
washed through with distilled water. The elu-
ate was concentrated to afford colourless oil
(0.138 g, 86%). FABMS m/z 775 ([M+H]+).
N - [2 - [2 - [2 - (2 - (N - biotinylcaproylamino)-
ethoxy)ethoxy]ethoxy] - 4 - [3 - (trifluoromethyl)-
3H - diazirin - 3 - yl]benzoyl]1,2 - bis(gluco-
pyranosyl-4-yloxy)-2-propylamine (12).—N-
biotinyl caproic acid succinimide ester (0.301
g, 0.66 mmol) was dissolved in DMF (2 mL).
To this solution compound 11 (0.464 g, 0.60
mmol) in 0.1 M NaHCO3 (2 mL) was added
at rt and the reaction mixture was stirred for
12 h. The reaction mixture was then concen-
trated. The residue was dissolved in water and
applied to a column of Amberlite IRN-150L
(2 mL, neutral form) and the product was
washed through the column with distilled wa-
ter. The eluate was then concentrated to af-
ford pale-yellow oil. The eluate was
concentrated and the product was purified
with silica column chromatography (13:5:1
CHCl3–CH3OH–water) to afford a colourless
oil (0.271 g, yield 37%). 1H NMR (CD3OD): l
7.95 (1 H, d, J=8.2 Hz, Ph), 7.02 (1 H, d,
J=8.2 Hz, Ph), 6.85 (1 H, s, Ph), 5.10 (1 H, d,
J=3.6 Hz, 1-a), 4.58 (1 H, d, J=7.6 Hz,
1-b), 4.40–2.80 (37 H, m), 1.80–1.20 (10 H,
m). FABMS: m/z 1114 ([M+H]+). HR-
2 - [2 - [2 - [2 - (2 - tert - Butox6carbonylamino-
ethoxy)ethoxy]ethoxy] - 4 - [3 - (trifluoromethyl)-
3H-diazirin-3-yl] benzoic acid N-hydroxysuc-
cinimide ester (9).—N-hydroxysuccinimide
(0.490 g, 4.26 mmol) and compound 8 (1.837
g, 3.85 mmol) were dissolved in 5:1 CH2Cl2–
CH3CN (12 mL) at rt. Dicyclohexylcarbodi-
imide (0.877 g, 4.25 mmol) in CH2Cl2 was
added. The reaction mixture was stirred at rt
for 3 h, filtered, and then concentrated. The
residue was partitioned between EtOAc and
water. The organic layer was washed with
saturated NaCl, dried over MgSO4, filtered
and concentrated to afford a yellow oil (2.186
1
g, 99%). H NMR (CDCl3): l 8.03 (1 H, d,
J=8.2 Hz, Ph), 6.87 (1 H, d, J=8.2 Hz, Ph),
6.76 (1 H, s, Ph), 4.24 (2 H, m), 3.91 (2 H, m),
3.72 (2 H, m), 3.61 (2 H, m), 3.53 (2 H, m),
3.31 (2 H, m), 2.90 (s, 4 H, CH2CH2), 1.43 (9
H, s, C(CH3)3). FABMS: m/z 575 ([M+H]+);
HRFABMS Calcd for C24H30F3N4O9 ([M+
H]+): 575.1965; Found: 575.1973. Anal. Calcd
For C24H29F3N4O9·0.5 H2O: C, 49.40; H, 5.18;
N, 9.60. Found: C, 49.55; H, 5.35; N, 9.35.
N - [2 - [2 - [2 - (2 - tert - Butoxycarbonylamino-
ethoxy)ethoxy] ethoxy]-4-[3-(trifluoromethyl)-
3H - diazirin - 3 - yl]benzoyl] - 1,3 - bis(
pyranose-4-yloxy)-2-propylamine
D
- gluco-
(10).—
Compound 9 (0.162 g, 0.28 mmol) was dis-
solved in DMF (2.5 mL) and compound 4
(0.162 g, 0.17 mmol) was added in 3 mL of 0.1
M NaHCO3. The reaction mixture was stirred
at rt for 12 h. The product was then applied to
a column of Amberlite IRN-150L (2 mL, neu-
tral form) and was washed through the
column with water. The eluate was concen-
trated and the product was purified with silica
column chromatography (13:5:1 CHCl3–
CH3OH–water) to afford a colourless oil
FABMS
m/z
([M+H]+)
1114.4478;
Calcd
for
C46H71F3N7O19S:
1114.4530.
Found
N - [2 - [2 - [2 - (2 - (2 - Biotinyl)ethylamido]-
3,3% - dithiodipropionylamino)ethoxy)ethoxy]-
ethoxy] - 4 - [3 - (trifluoromethyl) - 3H - diazirin-
1
(0.131 g, 88%). H NMR (D2O): l 7.88 (1
H, d, J=8.2 Hz, Ph), 7.05 (1 H, d, J=8.2

126
M. Hashimoto et al. / Carbohydrate Research 331 (2001) 119–127
T-TBS for 40 mm. After washing 6 times with
3-yl]benzoyl]1,3-bis( -glucopyranos-4-yloxy)-
D
T-TBS for 5 min, the membrane was im-
mersed in chemiluminescence reagents for 1
min and exposed to film for 0.5 min.
2 - propylamine (13).—[2 - (Biotinyl)ethyl-
amido]-3,3%-dithiodipropionic acid N-hydroxy-
succinimide ester (0.105 g, 0.18 mmol) was
dissolved in DMF (1 mL). To this solution,
compound 11 (0.163 g, 0.21 mmol) in 0.1 M
NaHCO3 (0.5 mL) was added at rt and the
reaction mixture was stirred for 12 h. The
reaction mixture was then concentrated. The
residue was dissolved in water and applied to
a column of Amberlite IRN-150L (2 mL, neu-
tral form) and the product was washed
through the column with distilled water. The
eluate was then concentrated to afford pale
yellow oil. The eluate was concentrated and
the product was purified with silica column
chromatography (50:25:1 CHCl3 –CH3OH–
water) to afford a colourless oil (0.089 g, yield
Photoaffinity labelling of GLUT4.—Rat
adipocytes isolation, Ki measurements, and
UV irradiation for labelling experiments were
carried out as previously described. After la-
belling, cell samples were homogenised in
HES buffer (255 mM sucrose, 1 mM EDTA,
20 mM HEPES, 1 mg/mL antipain, aprotinin,
pepstatin, leupeptin and 100 mM 4-(2-
aminoethyl) benzenesulfonyl fluoride hydro-
chloride (AEBSF), pH 7.2). Homogenates
were washed once with HES buffer and sub-
jected to centrifugation (554,000g for 30 min
at 4 °C) to obtain a total membrane fraction.
This pellet was solubilised in PBS pH 7.2, with
2% of Thesit (C12E9) and protease inhibitors
(antipain, aprotinin, pepstatin, and leupeptin
each at a concentration of 1 mg/mL and 100
mM AEBSF). The samples were solubilised for
50 min at 4 °C with rotation and were then
subjected to centrifugation (20,000g for 20
min at 4 °C). Biotinylated proteins in the su-
pernatants were either immuno-precipitated
using a GLUT4 antiserum as previously
described6 or were precipitated with strep-
tavidin beads (Pierce, Rockford, IL). Follow-
ing GLUT4 immuno-precipitation complexes
were released into electophoresis sample
buffer (62.5 mM Tris, pH 6.8, 2% SDS, 10%
glycerol) at rt. The steptavidin precipitates
were washed four times with PBS buffer con
taining 1% Thesit with protease inhibitors,
four times with PBS containing 0.1% Thesit
plus protease inhibitors and once in PBS.
Electrophoresis sample buffer was added to
each pellet. The sample was then heated to
95 °C for 30 min. The samples were subjected
to centrifugation (2,300g for 1 min) and the
supernatants were removed. The pellets were
washed with additional electrophoresis sample
buffer, heated to 95 °C for 30 min and resub-
jected to centrifugation. Mercaptoethanol was
added (10% final concentration) to the above
samples in the electrophoresis sample buffer
and these were then subjected to SDS-PAGE
(10% gel). Proteins were transferred to nitro-
cellulose membranes. Membranes were
blocked with 5% non-fat milk in TBS-0.1%
1
40%). H NMR (CD3OD): l 7.42 (1 H, d,
J=8.2 Hz, Ph), 7.10 (1 H, d, J=8.2 Hz, Ph),
6.90 (1 H, s, Ph), 5.23 (1 H, d, J=3.6 Hz,
1-a), 4.60 (1 H, d, J=7.6 Hz, 1-b), 4.50–4.20
(4 H, m), 4.00–3.20 (28 H, m), 3.0–2.50 (14
H, m), 2.25 (2 H, m), 1.80–1.40 (6 H, m).
FABMS: m/z 1235 ([M+H]+). HRFABMS
m/z ([M+H]+) Calcd for C48H74F3N8O20S3:
1235.4117; Found 1235.4134.
Photoaffinity labelling of GLUT1.—Human
GLUT1 was prepared as described by Bald-
win and Lienhard.16 GLUT1 (10 mg) and com-
pound 12 or 13 (0.25 nmol) were suspended in
0.1 M NaCl, 1 mM EDTA, 50 mM sodium
phosphate buffer pH 7.4 (23 mL) at rt for 10
mm. Where indicated in the figure legends,
D-glucose was included for competitive inhibi-
tion. The mixture was irradiated three times
for 30 s with 30 s intervals between successive
irradiations at 0 °C in a Rayonet photochemi-
cal reactor using 300 nm lamps. The solution
was then centrifuged at 400,000g at 4 °C for
30 mm. The pellet was resuspended in (20 mL)
of 1 mM EDTA, 50 mM Tris pH 7.4. A
sample (0.5 mg) was then subjected to elec-
trophoresis on a 10% acrylamide gel and
transferred to nitrocellulose. The nitrocellu-
lose was blocked for 1 h in 10% skimmed milk
suspended with 0.1% Tween 20, Tris-buffered
saline pH 7.4 (T-TBS), washed twice with
T-TBS for 5 min and immersed in Extravidin
peroxidase (a 1:4000 dilution of the manufac-
turers solution) with 1% skimmed milk in

M. Hashimoto et al. / Carbohydrate Research 331 (2001) 119–127
127
4. Holman, G. D.; Midgley, P. J. W. Carbohydr. Res. 1985,
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Tween (TBS-T) and washed six times with
TBS-T. Membranes were then incubated with
affinity purified anti-GLUT4 C-terminal
antibody18 in TBS-T containing 1% BSA (2 h
at rt), followed by washing (6 times in TBST)
and detection using secondary antibody linked
to horseradish peroxidase. GLUT4 protein
was visualised with enhanced chemilumines-
cence (ECL). Pmoles of labelled GLUT4 were
determined by comparison with a GLUT4
standard protein as described.17
5. Abbadi, M.; Holman, G. D.; Morin, C.; Rees, W. D.;
Yang, J. Tetrahedron Lett. 1999, 40, 5861–5864.
6. Koumanov, F.; Yang, J.; Jones, A. E.; Hatanaka, Y.;
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Biochim. Biophys. Acta 1986, 855, 115–126.
8. Holman, G. D.; Karim, A. R.; Karim, B. Biochim. Bio-
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9. Yoshida, E.; Nakayama, H.; Hatanaka, Y.; Kanaoka, Y.
Chem. Pharm. Bull. 1990, 38, 982–987.
10. Clark, A. E.; Holman, G. D. Biochem. J. 1990, 269,
615–622.
11. Gillingham, A. K.; Koumanov, F.; Hashimoto, M.; Hol-
man, G. D. In Membrane Transport. A Practical Ap-
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London, 2000; pp. 193–207.
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Acknowledgements
This work was supported by the Medical
Research Council (UK), the Wellcome Trust
and Diabetes (UK).
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174, 39–50.
17. Ryder, J. W.; Yang, J.; Galuska, D.; Rinco´n, J.; Bjo¨rn-
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