Fluorescence labelling of carbohydrates with 2-aminobenzamide (2AB)

Article abstract of DOI:10.1016/S0957-4166(00)00477-8

2-Aminobenzamide (2AB) is a common fluorescence label attached to reducing oligosaccharides by a reductive amination procedure. Chemical investigation of the published literature procedure reveals labelling occurs by the expected mechanism for both protec

Full text of DOI:10.1016/S0957-4166(00)00477-8

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 11 (2000) 4985–4994
Fluorescence labelling of carbohydrates with
2-aminobenzamide (2AB)
Robert R. France,a Ian Cumpstey,a Terry D. Butters,b Antony J. Fairbanksa,* and
Mark R. Wormaldb
aDyson Perrins Laboratory, Oxford University, South Parks Road, Oxford OX1 3QY, UK
bOxford Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road,
Oxford OX1 3QU, UK
Received 23 November 2000; accepted 30 November 2000
Abstract
2-Aminobenzamide (2AB) is a common fluorescence label attached to reducing oligosaccharides by a
reductive amination procedure. Chemical investigation of the published literature procedure reveals
labelling occurs by the expected mechanism for both protected and unprotected glucose derivatives to
yield open-chain carbohydrates rather than result in the formation of any heterocyclic materials. Pentenyl
glucosides may also be readily attached to the 2AB label by a sequence of dihydroxylation, periodate
cleavage and subsequent reductive amination of the resulting aldehyde. 2AB labelling is compatible with
deprotection of both acetate and benzyl protecting groups. © 2001 Elsevier Science Ltd. All rights
reserved.
1. Introduction
Fluorescence labels are extremely useful tools since they allow the monitoring of extremely
low concentrations of particular chemical species involved in a biochemical process of interest.
Fluorescence labelling of carbohydrates is commonly used to visualise oligosaccharides1 after
cleavage from glycoproteins in order either to facilitate purification of the oligosaccharide of
interest,2 or to allow monitoring of the concentration of a particular oligo- or monosaccharide
as the substrate for a particular enzyme.3
We recently became interested in the synthesis of a series of substrates for the glycoprotein
processing enzymes glucosidase I and II and the question arose as to the best method of
attaching fluorescence labels to synthetic substrates for these enzymes. Anthranilamide (2-
aminobenzamide, 2AB) is a frequently used fluorescence label for the visualisation of oligosac-
charide materials.4 We envisaged that attachment of a 2AB label to a carbohydrate by means of
* Corresponding author. Tel: +44-1865-275647; fax: +44-1865-275674; e-mail: antony.fairbanks@chem.ox.ac.uk
0957-4166/00/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(00)00477-8

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an n-pentenyl glycoside5 derived spacer arm would allow a flexible approach since upon
activation n-pentenyl glycosides can act as efficient glycosyl donors. Thus other sugars could be
added before labelling if required by taking advantage of an armed/disarmed approach.6 We
detail herein our investigations into the attachment of a 2-aminobenzamide label to some
glucose derivatives by reductive amination procedures, either directly at the anomeric centre or
via a pentenyl derived spacer arm.
2. Results and discussion
At the outset of our investigations we became aware of the fact that no thorough chemical
investigation concerning the efficiency or mode of linking of the 2AB label to the anomeric
centre of the reducing terminus of an oligosaccharide had been published.7 In fact to our
surprise we were unable to find any reports of reductive amination of aldehydes with 2AB in the
chemical literature. This is doubly surprising considering that 2AB labelling is now considered
a standard practice in many glycobiology laboratories, frequently employing a commercially
available reaction kit. It is well known that tetrahydroquinazolinones can result from reaction
of anthranilamide with aldehydes8 (Fig. 1), or with ketones under more forcing conditions.9 At
the outset it was therefore not completely clear to us that reductive amination ‘as expected’ was
necessarily the sole outcome of previously reported procedures and therefore complete charac-
terisation of the 2AB labelled carbohydrates synthesised was prudent.
Figure 1.
Initial investigations centered on the 2AB labelling of tetrabenzylglucose 1, following the
published protocol of Bigge et al.5 (Scheme 1). Treatment of 1 with sodium cyanoborohydride
in a mixture of dimethyl sulphoxide and acetic acid at 60°C did result in production of the
labelled open-chain sugar 2 as the major reaction product, in an acceptable 70% yield. The
1
open-chain structure of 2 was confirmed by H NMR and mass spectral analysis; in particular
the two H-1 protons could be clearly discerned. It should be noted that a mixture of several
other unidentified minor side products, which were inseparable by flash chromatography, was
also produced during this reaction. This mixture of side products could well contain tetra-
hydroquinazolinone materials, but proper characterisation of the complex mixture proved
difficult. Removal of the benzyl protecting groups from 2 proved more problematic then
expected. Simple catalytic hydrogenation with hydrogen gas and a selection of heterogeneous Pd
catalysts produced no reaction. However refluxing 2 in methanol in the presence of excess
palladium on carbon and ammonium formate for 24 hours finally successfully yielded the
completely deprotected material 3 in 60% yield. 2-Aminobenzamide labelling of glucose 4 itself
was then investigated using the same reaction conditions. Again labelling occurred as expected
and the desired product 3 was obtained, though in a moderate 67% yield; it should be noted that
the isolation and purification of this highly polar compound is not facile. It is therefore clear
that whilst anthranilamide labelling using the published literature procedure does not quantita-

R. R. France et al. / Tetrahedron: Asymmetry 11 (2000) 4985–4994
4987
Scheme 1. (i) NaBH3CN, Me2SO, AcOH, 60°C, 24 h, 70%; (ii) ammonium formate, Pd/C, MeOH, reflux, 24 h, 60%;
(iii) NaBH3CN, Me2SO, AcOH, 60°C, 2 h, 67%
tively result in the formation of a single reaction product, that the major reaction product is
indeed the desired reductively aminated material, rather than a tetrahydroquinazolinone.
Attention then moved towards the use of n-pentenyl glycosides as potential sites for the
attachment of 2AB labels. As previously discussed this approach would be inherently flexible,
since a pentenyl linker arm could also be activated for the addition of other carbohydrate units
if desired. The ultimate goal of this research was the synthesis of a series of fluorescence labelled
oligosaccharides as substrates for a variety of glycosidases. To this end it was also thought that
the pentenyl derived spacer arm would be appropriate mode of attachment for the 2AB label
since the alkyl chain would hopefully be long enough to avoid any appreciable interaction
between the label and the enzyme.
The peracetylated and perbenzylated a-gluco pentenyl glycosides 5a and 5b were synthesised
from glucose following standard literature procedures.10 Conversion of the alkene to an
aldehyde ready for reductive amination was best achieved by the standard two step variation
involving dihydroxylation and periodate cleavage, producing aldehydes 6a and 6b in 87 and 95%
yields, respectively (Scheme 2). Attempted ozonolysis was not clean, and resulted in the
formation of the carboxylic acid as an undesired side product in both cases.
Scheme 2. (i) OsO4, N-methylmorpholine N-oxide, THF; (ii) NaIO4, THF, H2O; (iii) NaBH(OAc)3, DCE, rt, 2AB

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R. R. France et al. / Tetrahedron: Asymmetry 11 (2000) 4985–4994
A screen of conditions for the reductive amination reaction revealed that the best reaction
conditions involved the use of sodium triacetoxyborohydride11 in 1,2-dichloroethane (DCE),
which resulted in the formation of the 2AB labelled a-glucosides 7a and 7b in 80 and 87% yields,
respectively. It should be noted that in the case of the perbenzylated aldehyde 6b the previously
used reaction conditions employing sodium cyanoborohydride resulted in the formation of an
intractable complex mixture of products.
Finally deprotection of both labelled materials was undertaken in an attempt to access the
desired tetrol 8. Deacetylation of 7a occurred rapidly with catalytic methoxide in methanol to
yield 8 in a satisfactory 63% yield (Scheme 3). However, removal of the benzyl protecting groups
of 7b proved problematic. Initial attempts at simple hydrogenation with hydrogen gas and
catalytic Pd black resulted in no reaction. A screen of various catalysts for hydrogenation was
subsequently undertaken. After many unsuccessful attempts it was discovered that once again
complete removal of all benzyl groups could be achieved by prolonged reflux in methanol with
a large excess of ammonium formate and palladium on carbon. Thus finally all benzyl groups
were removed to yield 8 in a satisfactory 76% yield.
Scheme 3. (i) NaOMe, MeOH, rt, 4 h, 63%; (ii) ammonium formate, Pd/C, MeOH, reflux, 36 h, 76%
In summary in the case of reducing sugars it is clear that labelling at the anomeric centre of
protected carbohydrates with the anomeric hydroxyl free using 2AB and sodium cyanoborohy-
dride mediated reductive amination is reasonably efficient. It is also clear that fluorescence labels
such as 2AB may be attached to pentenyl glycosides by a sequence involving dihydroxylation,
periodate cleavage and reductive amination. In addition pentenyl linked 2AB labels are
compatible with both ester and benzyl ether protecting groups. However the difficulties
encountered during attempted hydrogenation steps should be borne in mind when choosing a
protecting group pattern; in particular the current conditions require large amounts of catalyst
and protracted reaction times. Further investigations into more efficient methods of benzyl
group removal, and into the attachment of various different fluorescence labels to pentenyl
glycosides are currently in progress and will be reported in due course.

R. R. France et al. / Tetrahedron: Asymmetry 11 (2000) 4985–4994
4989
3. Experimental
3.1. General methods
Petrol refers to the fraction of petroleum ether that boils in the range 40–60°C; water was
distilled. All other solvents were used as supplied (analytical grade) without prior purification.
Reactions performed under an atmosphere of hydrogen gas were maintained by an inflated
balloon. All other reagents were used as supplied without prior purification. Thin layer
chromatography (TLC) was performed on glass backed plates coated with 60 F254 silica. Plates
were visualised using a solution of 5% ammonium molybdate in 2 M sulphuric acid, and by
fluorescence where appropriate. Flash column chromatography was performed on Sorbsil C60
40/60 silica. CMAW refers to the eluent system chloroform:methanol:acetic acid:water
(60:30:3:5). Melting points were recorded on a Kofler hot block and are uncorrected. Optical
rotations were recorded on a Perkin–Elmer 241 polarimeter with a path length of 1 dm.
Concentrations are quoted in g/100 ml. Elemental analyses were performed by the microanalysis
services of the Inorganic Chemistry Laboratory (Oxford) and Elemental Microanalysis Ltd
(Devon). Infrared spectra were recorded on a Perkin–Elmer 1750 IR Fourier Transform
spectrophotometer using either thin films on NaCl discs (thin film) or KBr discs (KBr) as stated.
Only the characteristic peaks are quoted. Nuclear magnetic resonance (NMR) spectra were
recorded on a a Bruker DPX 400 (1H: 400 MHz and 13C: 100 MHz), or where stated on Bruker
DRX 500 or AMX 500 (1H: 500 MHz and 13C: 125 MHz), in the deuterated solvent stated. All
chemical shifts (l) are quoted in ppm. Residual signals from the solvents were used as an
internal reference. Low resolution mass spectra (m/z) were recorded on a Micromass Platform
APCI using atmospheric pressure chemical ionisation (APCI), and partial purification by HPLC
with methanol:acetonitrile:water (40:40:20) as eluent. HRMS (electrospray) analyses were per-
formed on a Waters 2790 Micromass LCT electrospray ionisation mass spectrometer or by the
EPSRC Mass Spectrometry Service Centre, Department of Chemistry, University of Wales,
Swansea on a MAT900 XLT electrospray ionisation mass spectrometer.
3.2. 2-(N-(1-Deoxy-2,3,4,6-tetra-O-benzyl- -glucitol-1-yl)amino)benzamide 2
D
Tetrabenzylglucose 1 (500 mg, 0.92 mmol) and anthranilamide (190 mg, 1.39 mmol) were
dissolved in DMSO (3.5 ml) and AcOH (1.5 ml). After 30 min sodium cyanoborohydride (260
mg, 4.33 mmol) was added and the reaction mixture was heated to 60°C. The mixture was
stirred at 60°C for 24 h, at which point TLC (ethyl acetate:petrol, 3:2) showed the formation of
a major product (Rf 0.4) and the absence of starting material (Rf 0.6). The reaction mixture was
cooled and partitioned between diethyl ether (100 ml) and 1.0 M HCl (100 ml). The organic
layer was washed with brine (50 ml), dried (MgSO4), filtered and concentrated in vacuo. The
residue was purified by flash column chromatography (ethyl acetate:petrol:triethylamine, 1:2:0.5)
to afford 2 (430 mg, 70%) as a colourless oil; [h]2D1 +0.4 (c, 1.0 in CHCl3); wmax (thin film) 3344
(NH), 1654 (CꢀO), 1616, 1579 (CꢀO, NH) cm−1; lH (400 MHz, CDCl3) 3.21 (1H, br, s, OH),
3.27 (1H, dd, J1,1% 13.5 Hz, J1,2 6.7 Hz, H-1), 3.45 (1H, dd, J1%,2 4.3 Hz, H-1%), 3.64 (2H, d, J5,6
4.5 Hz, 2×H-6), 3.79 (1H, dd, J3,4 4.1 Hz, J4,5 6.8 Hz, H-4), 3.90 (1H, dd, J2,3 5.4 Hz, H-3),
3.98–4.02 (1H, m, H-2), 4.03–4.07 (1H, m, H-5), 4.49, 4.53 (2H, ABq, JAB 11.9 Hz, PhCH2),
4.57, 4.61 (2H, ABq, JAB 11.5 Hz, PhCH2), 4.62, 4.71 (2H, ABq, JAB 11.3 Hz, PhCH2), 4.69 (2H,
s, PhCH2), 5.77 (2H, br, s, NH2), 6.57–6.61 (2H, m, 2×ArH), 7.19–7.36 (22H, m, 22×ArH); lC

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R. R. France et al. / Tetrahedron: Asymmetry 11 (2000) 4985–4994
(CDCl3) 43.9 (t, C-1), 71.3 (d, C-5), 71.7 (t, C-6), 73.7, 73.9, 75.0 (3×t, 4×PhCH2), 78.0 (d, C-2),
78.1 (d, C-4), 79.4 (d, C-3), 112.5, 115.1, 133.9 (3×d, 3×ArC), 114.0, 150.5 (2×s, 2×ArC), 128.2,
128.3, 128.4, 128.7, 128.9, 128.9, 129.0 (7×d, 21×ArC), 138.4, 138.5 (2×s, 5×ArC), 172.8 (s,
CONH2); m/z (APCI+) 717 (MK+H2O, 14%); HRMS calcd for C41H45O6N2 (MH+): 661.3277;
found: 661.3280.
3.3. 2-(N-(1-Deoxy-
D
-glucitol-1-yl)amino)benzamide 3
3.3.1. Method 1
A mixture of compound 2 (100 mg, 0.15 mmol), palladium on carbon (322 mg, 3.0 mmol),
and ammonium formate (334 mg, 5.3 mmol) were stirred together in methanol (5 ml) and the
reaction mixture heated under reflux. After 24 h, TLC (CMAW) showed the formation of a
major product (Rf 0.5). The reaction mixture was cooled, filtered, and the residue purified by
flash column chromatography (CMAW) to afford 3 (27 mg, 60%) as a crystalline white solid,
mp 171–174°C (EtOH); [h]2D4 −0.3 (c, 0.5 in H2O); vmax (KBr) 3400, 3252 (br, OH, NH), 1650
(CꢀO); lH (500 MHz, CD3OD/D2O, 1:1) 3.24 (1H, dd, J1,1% 13.1 Hz, J1,2 8.0 Hz, H-1), 3.45 (1H,
dd, J1%,2 4.1 Hz, H-1%), 3.66 (1H, dd, J5,6 5.6 Hz, J6,6% 11.6 Hz, H-6), 3.68 (1H, dd, J3,4 2.1, J4,5
8.8 Hz, H-4), 3.76–3.79 (1H, m, H-5), 3.82 (1H, dd, J5,6% 3.1 Hz, H-6%), 3.87 (1H, dd, J2,3 5.4 Hz,
H-3), 4.02 (1H, dat, H-2), 6.71 (1H, at, J 7.5 Hz, ArH), 6.86 (1H, d, J 8.1 Hz, ArH), 7.38–7.41
(1H, m, ArH), 7.56 (1H, dd, J 7.9 Hz, J 1.5 Hz, ArH); lC (CD3OD/D2O, 1:1) 45.6, 48.6 (2×t,
C-1, C-6), 71.0, 71.6, 71.7, 72.1 (4×d, C-2, C-3, C-4, C-5), 112.6, 116.1, 129.3, 133.9 (4×d,
4×ArC), 149 (s, ArC), 174.3 (s, CONH2); m/z (APCI+) 323 (MNa+, 9), 301 (MH+, 63), 120
(100%); HRMS calcd for C13H21O6N2 (MH+): 301.1400; found: 301.1399.
3.3.2. Method 2
Glucose 4 (205 mg, 1.14 mmol) and anthranilamide (230 mg, 1.69 mmol) were dissolved in
DMSO (3.5 ml) and AcOH (1.5 ml). The reaction mixture was heated to 60°C and sodium
cyanoborohydride (320 mg, 5.01 mmol) was added. The mixture was stirred at 60°C for 6 h, at
which point TLC (CMAW) showed the formation of a major product (Rf 0.5) and the absence
of starting material (Rf 0.2). The reaction mixture was cooled and the solvents removed in
vacuo. The residue was dissolved in 1.0 M HCl (20 ml) and stirred for 1 h. The solvent was then
removed in vacuo and the residue purified by flash column chromatography (CMAW) to afford
3 (227 mg, 67%) as a colourless oil, identical to the material described above.
3.4. 4-Oxobutyl 2,3,4,6-tetra-O-acetyl-h- -glucopyranoside 6a
D
Peracetylated pentenyl glycoside 5a10 (1.5 g, 3.61 mmol) and N-methylmorpholine N-oxide
(60% solution in water, 1.1 ml, 5.61 mmol) were dissolved in THF (6 ml). Osmium tetroxide (11
mg, 0.043 mmol) was added and the reaction mixture was stirred for 17 h, at which point TLC
(ethyl acetate) indicated the formation of a major product (Rf 0.2) together with some remaining
starting material (Rf 0.7). The reaction mixture was quenched by the addition of sodium
thiosulphate (4 ml of a saturated aqueous solution), and then stirred for a further 7 h. The
mixture was diluted with ethyl acetate (100 ml) and washed with water (100 ml). The combined
aqueous extracts were re-extracted with ethyl acetate (100 ml), and the combined organic layers
washed with brine (100 ml), dried (MgSO4), filtered and concentrated in vacuo. The residue was
dissolved in a mixture of THF (8 ml) and water (1 ml), and sodium periodate (1.9 g, 9.02 mmol)

R. R. France et al. / Tetrahedron: Asymmetry 11 (2000) 4985–4994
4991
was then added. After 16 h, TLC (ethyl acetate:petrol:triethylamine, 2:3:0.25) showed the
formation of a major product (Rf 0.3). The reaction mixture was diluted with ethyl acetate (100
ml), washed with water (100 ml) and brine (100 ml), dried (MgSO4), filtered and concentrated
in vacuo. The residue was purified by flash column chromatography (ethyl acet-
ate:petrol:triethylamine, 2:3:0.25) to give aldehyde 6a (1.3 g, 87%) as a colourless oil; [h]2D1 +102
(c, 1.1 in CHCl3); wmax (thin film) 1750 (OCOCH3) cm−1; lH (400 MHz, CDCl3) 1.93–2.17 (2H,
m, OCH2CH2
3.41–3.49 (1H, m, OCHa
10.3 Hz, J5,6 4.2 Hz, J5,6% 2.3 Hz, H-5), 4.08 (1H, dd, J6,6% 12.4 Hz, H-6), 4.25 (1H, dd, H-6%), 4.85
(1H, dd, J1,2 4.1 Hz, J2,3 10.3 Hz, H-2), 5.02–5.09 (1H, m, H-4), 5.05 (1H, d, H-1), 5.46 (1H, at,
6
CH2), 2.01, 2.03, 2.07, 2.09 (12H, 4×s, 4×CH3), 2.51–2.58 (2H, m, OCH2CH2CH2
6 ),
6
HbCH2CH2), 3.72–3.82 (1H, m, OCHaHbCH2CH2), 4.00 (1H, ddd, J4,5
6
J 9.8 Hz, H-3), 9.80 (1H, t, J 1.4 Hz, CH2CH
6 O); lC (CDCl3) 20.5, 20.6, 20.7 (3×q, 4×CH3), 24.4,
30.3, 60.4, 67.4 (4×t, OCH2CH2CH2, C-6), 67.4, 68.4, 70.1, 70.8 (4×d, C-2, C-3, C-4, C-5), 95.7
6
6
6
(d, C-1), 169.6, 170.3, 170.7, 177.5 (4×s, 4×C6 O2CH3), 201.5 (s, CH2C6
HO); m/z (APCI+) 457
(MK+, 6), 441 (MNa+, 12), 169 (100%); anal. found: C, 49.64; H, 6.14; C18H26O11·H2O requires:
C, 49.54; H, 6.47%; HRMS calcd for C18H30O11N (MNH+4): 436.1819; found: 436.1817.
3.5. 4-Oxobutyl 2,3,4,6-tetra-O-benzyl-h- -glucopyranoside 6b
D
Perbenzylated pentenyl glycoside 5b10 (454 mg, 0.75 mmol) and N-methylmorpholine N-oxide
(60% solution in water, 0.21 ml, 1.11 mmol) were dissolved in THF (15 ml). Osmium tetroxide
(8.1 mg, 0.032 mmol) was added and the reaction mixture was stirred for 18 h, at which point
TLC (ethyl acetate) indicated a major product (Rf 0.4) together with some starting material (Rf
0.75). The reaction mixture was quenched by the addition of sodium thiosulphate (8 ml of a
saturated aqueous solution), and then stirred at room temperature for a further 6 h. The
reaction mixture was diluted with ethyl acetate (100 ml) and washed with water (2×50 ml). The
combined aqueous extracts were re-extracted with ethyl acetate (100 ml), and the combined
organic layers were washed with brine (100 ml), dried (MgSO4), filtered and concentrated in
vacuo. The residue was dissolved in a mixture of THF (8 ml) and water (1 ml), and sodium
periodate (317 mg, 1.48 mmol) was added. After 15 h, TLC (ethyl acetate) showed the formation
of a major product (Rf 0.75), and the absence of any diol (Rf 0.4). The reaction mixture was
diluted with ethyl acetate (200 ml), washed with water (2×100 ml) and brine (50 ml), dried
(MgSO4), filtered and concentrated in vacuo. The residue was purified by flash column
chromatography (ethyl acetate:petrol, 1:3) to give aldehyde 6b (427 g, 95%) as a colourless oil;
[h]2D1 +36 (c, 1.0 in CHCl3); wmax (thin film) 1723 (CꢀO) cm−1; lH (400 MHz, CDCl3) 1.91–2.06
(2H, m, OCH2CH2
OCHaHbCH2CH2), 3.56 (1H, dd, J1,2 3.6 Hz, J2,3 9.7 Hz, H-2), 3.61–3.76 (5H, m, H-4, H-5, H-6,
H-6%, OCHaHbCH2CH2), 3.96 (1H, at, J 9.3 Hz, H-3), 4.47, 4.84 (2H, ABq, JAB 10.7 Hz,
6 CH2), 2.54–2.59 (2H, m, OCH2CH2CH6 2), 3.43 (1H, dt, Jgem 9.9 Hz, J 6.2 Hz,
6
6
PhCH2), 4.47, 4.61 (2H, ABq, JAB 12.3 Hz, PhCH2), 4.63, 4.79 (2H, ABq, JAB 12.1 Hz, PhCH2),
4.73 (1H, d, H-1), 4.83, 4.99 (2H, ABq, JAB 10.8 Hz, PhCH2), 7.12–7.38 (20H, m, ArH), 9.77
(1H, t, J 1.3 Hz, HCꢀO); lC (CDCl3) 22.1, 40.7, 66.9, 68.4, 73.3, 73.4, 75.1, 75.7 (8×t, 4×PhCH2,
OC
127.7, 127.9, 127.9, 128.0, 128.0, 128.3, 128.4, 128.4 (10×d, 20×ArC), 137.8, 138.1, 138.2, 138.8
(4×s, 4×ArC), 202.0 (d, CH2C
HO); m/z (APCI+) 633 (MNa+, 74), 113 (100%); HRMS calcd for
C38H46O7N (MNH+4): 628.3274; found: 628.3279.
6 H2C6 H2C6 H2, C-6), 70.3, 77.6, 80.0, 82.0 (4×d, C-2, C-3, C-4, C-5), 97.1 (d, C-1), 127.6, 127.7,
6

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R. R. France et al. / Tetrahedron: Asymmetry 11 (2000) 4985–4994
3.6. 2-(N-(4-(2,3,4,6-Tetra-O-acetyl-h- -glucopyranosyloxy)butyl)amino)benzamide 7a
D
Aldehyde 6a (120 mg, 0.29 mmol) and anthranilamide (39 mg, 0.29 mmol) were dissolved in
DCE (5 ml) and sodium triacetoxyborohydride (85 mg, 0.40 mmol) was added. The mixture was
stirred at room temperature for 4 h, at which point TLC (ethyl acetate:petrol, 3:2) showed the
formation of a major product (Rf 0.2) and the absence of any starting material (Rf 0.4). The
reaction mixture was partitioned between ether (100 ml) and water (100 ml) and the aqueous
layer was re-extracted with ether (100 ml). The combined organic layers were washed with brine
(50 ml), dried (MgSO4), filtered and concentrated in vacuo. The residue was purified by flash
column chromatography (ethyl acetate:petrol:triethylamine, 3:2:0.25) to afford 7a (125 mg, 80%)
as a colourless oil; [h]2D1 +85 (c, 1.1 in CHCl3); wmax (thin film) 3475 (NH), 3359 (NH), 1749
(OCOCH3), 1657 (CONH2) cm−1; lH (400 MHz, CDCl3) 1.68–1.79 (4H, m, OCH2CH2
2.01, 2.03, 2.04, 2.09 (12H, 4×s, 4×CH3), 3.22–3.23 (2H, m, OCH2CH2CH2CH2), 3.47–3.50 (1H,
m, OCHaHbCH2CH2CH2), 3.73–3.77 (1H, m, OCHaHbCH2CH2CH2), 4.01 (1H, ddd, J4,5 10.2
6 CH6 2CH2),
6
6
6
Hz, J5,6 2.3 Hz, J5,6% 4.4 Hz, H-5), 4.08 (1H, dd, J6,6% 12.3 Hz, H-6), 4.24 (1H, dd, H-6%), 4.87 (1H,
dd, J1,2 3.7 Hz, J2,3 10.2 Hz, H-2), 5.06 (1H, at, J 9.7 Hz, H-4), 5.07 (1H, d, H-1), 5.48 (1H, at,
H-3), 6.57–7.41 (4H, m, 4×ArH); lC (CDCl3) 20.7 (q, 4×CH3), 25.7, 27.0, 42.6, 61.9, 68.5 (5×t,
OC
114.5, 128.3, 133.6 (4×d, 4×ArC), 112.9, 150.1 (2×s, 2×ArC), 169.7, 170.2, 170.3, 170.7, 172.1
(5×s, 4×C
OCH3, CONH2); m/z (APCI+) 595 (MK+H2O, 21), 561 (MNa+, 45), 539 (MH+, 100%);
HRMS calcd for C25H35O11N2 (MH+): 539.2241; found: 539.2241.
6 H2C6 H2C6 H2C6 H2, C-6), 67.1, 68.5, 70.2, 70.8 (4×d, C-2, C-3, C-4, C-5), 95.8 (d, C-1), 111.9,
6
3.7. 2-(N-(4-(2,3,4,6-Tetra-O-benzyl-h- -glucopyranosyloxy)butyl)amino)benzamide 7b
D
Aldehyde 6b (200 mg, 0.33 mmol) and anthranilamide (45 mg, 0.33 mmol) were dissolved in
DCE (5 ml). Sodium triacetoxyborohydride (97 mg, 0.46 mmol) was added and the mixture
stirred for 6 h, at which point TLC (ethyl acetate:petrol:triethylamine, 3:2:0.25) showed the
formation of a major product (Rf 0.3) and the absence of any starting material (Rf 0.5). The
reaction mixture was stirred with sodium hydrogen carbonate (5 ml of a saturated aqueous
solution) for 15 min and then partitioned between ether (100 ml) and water (100 ml). The
aqueous layer was re-extracted with ether (100 ml) and the combined organic extracts were
washed with brine (50 ml), dried (MgSO4), filtered and concentrated in vacuo. The residue was
purified by flash column chromatography (ethyl acetate:petrol:triethylamine, 3:2:0.25) to afford
7b (209 mg, 87%) as a colourless oil; [h]2D1 +36 (c, 1.0 in CHCl3); wmax (thin film) 3341 (NH), 1652
(CꢀO) cm−1; lH (400 MHz, CDCl3) 1.70–1.80 (4H, m, OCH2CH2
OCH2CH2CH2CH2), 3.44–3.48 (1H, m, OCHaHbCH2CH2CH2), 3.57 (1H, dd, J1,2 3.6 Hz, J2,3 9.8
Hz, H-2), 3.60–3.74 (4H, m, OCHaHbCH2CH2CH2, H-4, H-6, H-6%), 3.76–3.79 (1H, m, H-5),
6 CH6 2CH2), 3.19–3.21 (2H, m,
6
6
6
3.99 (1H, at, J 9.2 Hz, H-3), 4.47, 4.62 (2H, ABq, JAB 12.3 Hz, PhCH2), 4.48, 4.84 (2H, ABq,
JAB 10.8 Hz, PhCH2), 4.65, 4.79 (2H, ABq, JAB 12.3 Hz, PhCH2), 4.77 (1H, d, H-1), 4.82, 5.00
(2H, ABq, JAB 10.8 Hz, PhCH2), 5.73 (2H, br, s, NH2), 6.57 (1H, at, J 7.6 Hz, ArH), 6.72 (1H,
d, J 8.6 Hz, ArH), 7.14–7.38 (22H, m, ArH); lC (CDCl3) 25.9, 27.0, 42.8, 67.8, 68.5, 73.2, 73.5,
75.1, 75.7 (9×t, C-6, 4×PhCH2, OC6 H2C6 H2C6 H2C6 H2), 70.2, 77.7, 80.1, 82.0 (4×d, C-2, C-3, C-4,
C-5), 97.0 (d, C-1), 127.5, 127.7, 127.8, 127.9, 128.0, 128.3, 128.3, 128.4, 133.5 (9×d, 24×ArC),
137.9, 138.2, 138.3, 138.9 (4×s, 6×ArC), 172.1 (s, CONH2); m/z (APCI+) 754 (MNa+, 88), 732
(MH+, 100%); anal. found: C, 74.01; H, 6.95; N, 3.96; C45H50O7N2 requires: C, 73.95; H, 6.90;
N, 3.83%.

R. R. France et al. / Tetrahedron: Asymmetry 11 (2000) 4985–4994
4993
3.8. 2-(N-(4-(h-
3.8.1. Method 1
D
-Glucopyranosyloxy)butyl)amino)benzamide 8
Compound 7a (60 mg, 0.11 mmol) was dissolved in methanol (3 ml), a solution of sodium (20
mg, 0.87 mmol) in methanol (2 ml) was added and the mixture stirred. After 4 h, TLC (CMAW)
indicated the formation of a major product (Rf 0.4) and the absence of starting material (Rf 1.0).
The mixture was concentrated in vacuo and the residue purified by flash column chromatogra-
phy (CMAW) to afford 8 (26 mg, 63%) as a colourless oil; [h]2D6 +57 (c, 0.7 in MeOH); wmax (thin
film) 3420 (br, OH), 1660 (CꢀO) cm−1; lH (400 MHz, D2O) 1.56–1.66 (4H, m,
OCH2CH2
3.39–3.44 (1H, m, OCH
(1H, m, H-5), 3.55 (1H, at, H-3), 3.58–3.64 (1H, m, OCHaHb
Hz, J6,6% 12.4 Hz, H-6), 3.68 (1H, dd, J5,6% 2.4 Hz, H-6%), 4.76 (1H, d, H-1), 6.89 (1H, at, J 7.6
Hz, ArH), 6.94 (1H, d, J 8.2 Hz, ArH), 7.39 (1H, at, J 7.4 Hz, ArH), 7.52 (1H, d, J 7.8 Hz);
6
CH2
6
CH2), 3.07–3.20 (2H, m, OCH2CH2CH2CH2
6
), 3.26 (1H, at, J 9.6 Hz, H-4),
6
aHbCH2CH2CH2), 3.40 (1H, dd, J1,2 3.8 Hz, J2,3 9.9 Hz, H-2), 3.48–3.53
6
CH2CH2CH2), 3.60 (1H, dd, J5,6 5.3
lC (D2O) 21.3, 24.8 (2×t, OCH2C6 H2C6 H2CH2), 45.6 (t, OCH2CH2CH2C6 H2), 60.8 (t,
OC6 H2CH2CH2CH2), 69.9 (d, C-4), 71.6 (d, C-2), 72.1 (d, C-5), 73.4 (d, C-3), 98.4 (d, C-1), 116.5,
120.4, 129.5, 134.1 (4×d, ArC), 145.6 (s, ArC), 173.7 (s, CONH2); m/z (APCI+) 393 (MNa+, 54),
371 (MH+, 100%); HRMS calcd for C17H27O7N2 (MH+): 371.1818; found: 371.1811.
3.8.2. Method 2
Compound 7b (250 mg, 0.34 mmol), 10% palladium on carbon (300 mg, 0.28 mmol), and
ammonium formate (324 mg, 5.1 mmol) were stirred together in methanol (5 ml) and the
reaction mixture heated to 65°C. After 24 h, TLC (CMAW) showed the formation a major
product (Rf 0.4). The reaction mixture was cooled, filtered, concentrated in vacuo and the
residue purified by flash column chromatography (CMAW) to afford 3 (96 mg, 76%) as a
colourless oil identical to the material described above.
Acknowledgements
We gratefully acknowledge the use of the EPSRC Mass Spectrometry Service (Swansea, UK)
and the Chemical Database Service (CDS) at Daresbury, UK.
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