Synthesis of fluorescence-labelled disaccharide substrates of glucosidase II

Article abstract of DOI:10.1016/S0008-6215(03)00255-6

The fluorescence-labelled disaccharides Glcα(1→3)GlcαOR and Glcα(1→3)ManαOR, both substrates for the glycoprotein-processing enzyme glucosidase II, were synthesised via the use of a n-pentenyl-derived linker at the anomeric position. This allowed incorpor

Full text of DOI:10.1016/S0008-6215(03)00255-6

Carbohydrate Research 338 (2003) 1937ꢁ1949
/
www.elsevier.com/locate/carres
Synthesis of fluorescence-labelled disaccharide substrates of
glucosidase II
Ian Cumpstey,a Terry D. Butters,b Richard J. Tennant-Eyles,a Antony J. Fairbanks,a,*
Robert R. France,a Mark R. Wormaldb
a Dyson Perrins Laboratory, Oxford University, South Parks Road, Oxford OX1 3QY, UK
b Department of Biochemistry, Oxford Glycobiology Institute, University of Oxford, South Parks Road, Oxford OX1 3QU, UK
Received 20 February 2003; received in revised form 23 May 2003; accepted 31 May 2003
Abstract
The fluorescence-labelled disaccharides Glca(10
/
3)GlcaOR and Glca(103)ManaOR, both substrates for the glycoprotein-
/
processing enzyme glucosidase II, were synthesised via the use of a n-pentenyl-derived linker at the anomeric position. This allowed
incorporation of a pyrenebutyric acid label, via a sequence of oxidative hydroboration, mesylation, azide displacement, reduction
with concomitant global deprotection, and peptide coupling. Selective activation of a fully armed thioglycoside in the presence of n-
pentenyl glycosides was readily achieved by the use of methyl triflate as promoter.
# 2003 Elsevier Ltd. All rights reserved.
Keywords: Glucosidase II; Glycoprotein processing; n-Pentenyl glycosides; Fluorescence labels
1. Introduction
a-linked glucose residues from the precursor oligosac-
charide Glc2Man9GlcNAc2 (Fig. 1). In particular,
cleavage of the first glucose residue as catalysed by
glucosidase II releases the glycan Glc1Man9GlcNAc2.
The correct folding of the glycoprotein is dependent on
the binding of the lectin chaperones calnexin and
calreticulin to this monoglucosylated moeity.4 The
chaperones do not bind to the non-glucosylated Man9-
GlcNAc2 glycan, so cleavage of the final glucose residue
by glucosidase II stops protein folding. Indeed, if a
protein has not achieved its native conformation at this
point, it is recognized and re-glucosylated by a glucosyl
transferase5 as part of a quality control mechanism to
complete the folding process.6 Conversely, if correct
folding has occurred prior to cleavage of the last glucose
residue by glucosidase II, the protein is not re-glucosy-
lated and it then leaves the ER to continue maturation.
Previous work investigating the kinetics of glucosi-
dase II from rat liver using a-paranitrophenyl glucopyr-
anoside as an artificial substrate, has led to the proposal
that the enzyme contains two active sites.7 Moreover,
these authors have suggested that the Glc2Man9Glc-
NAc2 substrate may bind to these two sites simulta-
neously, resulting in the cleavage of both glucose
linkages before release of the Man9GlcNAc2 glycan.
N-Glycoprotein biosynthesis is initiated by the oligo-
saccharyl transferase (OST)-mediated transfer of a 14-
membered dolichol phosphate bound oligosaccharide
(Glc3Man9GlcNAc2) to particular asparagine residues
(Asn-X-Ser/The).1 Following this transfer sequential
trimming of the oligosaccharide chain occurs that is
initiated by two glycoprotein-processing enzymes, glu-
cosidases I and II, in the endoplasmic reticulum (ER) of
the cell. Subsequently, the action of several other
glycosidases and glycosyl transferases leads to the
eventual production of glycoproteins bearing the famil-
iar variety of N-glycan structures.
Glucosidase I2 is a type II protein found in the ER,
and is responsible for cleavage of the terminal a-(10
/
2)-
linked glucose residue from the Glc3Man9GlcNAc2
glycan. Glucosidase II,3 a heterodimeric enzyme found
in the ER, then sequentially cleaves the remaining two
* Corresponding author. Tel.: ꢀ
/
44-1865-275647; fax: ꢀ44-
/
1865-275674.
E-mail address: antony.fairbanks@chem.ox.ac.uk (A.J.
Fairbanks).
0008-6215/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0008-6215(03)00255-6

1938
I. Cumpstey et al. / Carbohydrate Research 338 (2003) 1937ꢁ1949
/
Fig. 1. Enzymatic cleavages of N-linked glycans by Glucosidase II.
However, the isolation of substantial quantities of the
Glc1Man9GlcNAc2 glycan during treatment of the
Glc2Man9GlcNAc2 substrate with catalytic quantities
of glucosidase II indicated that for the natural substrates
As such the use of an n-pentenyl glycoside to furnish a
sterically undemanding and flexible linker between the
disaccharides and the label seemed ideal.11 Indeed we
recently reported a protocol whereby n-pentenyl glyco-
sides, which themselves are readily available and com-
cleavage of the Glca(10
/
3)Glc linkage was faster than
3)Man linkage.8 This rate
monly used glycosyl donors*thus allowing the
/
cleavage of the Glca(10
/
possibility of extending the synthesis to tri- and higher
oligosaccharides if desired, may be derivatised and
attached to the fluorescent label 2-aminobenzamide (2-
AB) via a reductive amination strategy.12 Herein we
report the synthesis of the two disaccharide substrates
for glucosidase II13 and their fluorescence-labelling via
n-pentenyl-derived linkers.
differential, which may be important to allow the
opportunity for chaperone binding before the removal
of the final glucose unit, could be entirely due to the
different stereochemistry of the respective endo residues
(i.e., gluco vs. manno), or alternatively, could at least in
part be effected by the rest of the oligosaccharide chain.
In order to further investigate the kinetics of glucosidase
II, and whether the rest of the oligosaccharide chain
plays a role in determining this differential rate of
cleavage, the synthesis of two disaccharides representing
the two a-gluco linkages in question, each with a
fluorescence label attached to the terminal anomeric
position via a short a-linker, was undertaken (Fig. 2). It
was also envisaged that these substrates might also be
used for the development of an assay for glucosidase II.
Fluorescence-labelling of enzyme substrates is a valu-
able tool, as it enables the sensitive detection of the small
quantities of material involved in an enzymatic assay.9
When considering the means of attachment of such a
label to the carbohydrate several factors must be
considered; the most important being that the linker
should be long and flexible enough so that the presence
of the label should not affect the kinetics of enzymatic
cleavage. Moreover, direct attachment of a label to the
terminal residue, for example via a reductive amination
strategy10 would necessarily involve destruction of the
terminal pyranose ring, and clearly in this case mean-
ingless kinetic results would ensue.
2. Results and discussion
The labelling strategy adopted involved the synthesis of
the two required disaccharides as their a-n-pentenyl
glycosides, and subsequent installation of the fluores-
cence label by manipulation of the alkene function of
the pentenyl group. Such a strategy is flexible in that
further monosaccharide units may be easily added if
desired, allowing access to higher oligosaccharide sub-
strates of glucosidase II. Synthesis of the Glca(10
/
3)Man disaccharide proceeded as follows. Heating
mannose 1 with camphor sulfonic acid (CSA) in
penten-1-ol produced the a-pentenyl mannoside 2,
which was then treated with benzaldehyde dimethyl
acetal to yield a mixture of monobenzylidene derivative
3 (52% yield) as well as the endo and exo dibenzylidene
derivatives 4a and 4b (46% yield, endo ꢁ
Scheme 1). Selective reductive cleavage of the endo
dibenzylidene derivative 4a with DIBAL at ꢂ40 8C in
/exo ratio 1:1,
/
toluene produced the desired 2-O-benzylated glycosyl
acceptor (5a) (40% yield). Alternatively, benzylation of
the mono-benzylidene derivative 3 under phase-transfer
conditions also gave the required alcohol 5a (45% yield)
along with a minor amount of the undesired 3-O-
benzylated sugar (5b) (20% yield). Therefore, in sum-
mary the most efficient synthesis of 5a could be achieved
by selective mono-benzylidination of 2 to yield 3 and
subsequent selective benzylation. Mindful of the fact
that n-pentenyl glycosides are readily activated under
many of the same sets of conditions as thioglycosides
(for example with sources of Iꢀ) glycosylation of the
Fig. 2. Fluorescence labelled substrates for glucosidase II.

I. Cumpstey et al. / Carbohydrate Research 338 (2003) 1937ꢁ
/1949
1939
Scheme 1. Reagents and conditions: (i) penten-1-ol, CSA, 100 8C, 61%; (ii) PhCH(OMe)2, CSA, DMF, 65 8C, 260ꢁ
/280 mbar; 3,
52%; 4a:4b (endo:exo, 1:1), 46%; (iv) DIBAL, toluene, ꢂ
/
40 8C, 40%; (v) BnBr, NaOH, Bu4NHSO4, CH2Cl2, H2O, 41 8C; 5a, 45%;
˚
5b, 20%; (vi) MeOTf, 4 A sieves, Et2O, RT, 58%.
alcohol 5a was then attempted with the fully armed
gluco thioglycoside 6.14 It was found that selective
activation of the thioglycoside could be achieved by
the use of methyl triflate. Thus glycosylation of 5a with
6 was achieved by treatment with methyl triflate, using
diethyl ether as solvent in order to ensure good a-
selectivity, the desired disaccharide 7 was produced in an
acceptable 58% yield, along with a small quantity of the
O-methylated acceptor; no b-disaccharide was isolated.
To the best of our knowledge, this represents only the
second report of the selective activation of a thioglyco-
side in the presence of an n-pentenyl glycoside.15
activator, with ether as solvent, and proceeded with
good stereoselectivity to yield the desired a-disaccharide
12a (80% yield) along with a small quantity of the
undesired b-linked disaccharide (12b, 15% yield).
Previous studies on the attachment of the commonly
used fluorescence label 2-aminobenzamide (2-AB) to an
n-pentenyl glycoside had been achieved via a procedure
of dihydroxylation, periodate cleavage and subsequent
reductive amination.12 Although this procedure had
worked on a monosaccharide, problems had been
encountered therein, particularly with attempted re-
moval of benzyl protecting groups by hydrogenation.
Indeed disappointingly several attempts to use similar
reductive amination methodology with aldehydes de-
rived from the pentenyl disaccharides all met with
failure. Consequently, a different methodology was
sought. Various possibilities were investigated, including
linkage of methylumbellieryl, pyrenebutyryl and dansyl
labels via ether, ester and sulfonamide linkages, respec-
tively. However, as in the 2-AB case, it was found that
the coumarin and pyrene labels were incompatible with
the hydrogenation conditions necessary to remove the
benzyl ether protecting groups. Consequently, it was
seen to be advantageous to attach the fluorescent label
after debenzylation, and so it was necessary to find a
way to attach a label to the deprotected sugar. It was
envisaged that this could be achieved by installation of
A similar reaction sequence was employed for the
synthesis of the required Glca(10
/
3)Glc disaccharide
(Scheme 2). Thus treatment of D-glucose 8 with penten-
1-ol and CSA produced an inseparable mixture of a-
and b-pentenyl glucosides 9a/b (63% yield), which were
treated with benzaldehyde dimethyl acetal to yield the
mono-benzylidene derivatives 10a and 10b (46 and 20%
yields, respectively) which could easily be separated by
column chromatography. Selective benzylation of the
required a-glucoside 10a under phase-transfer condi-
tions yielded the desired 2-O-benzyl alcohol 11a as the
major product, together with some of the undesired 3-O-
benzylated regioisomer 11b. Glycosylation of the re-
quired alcohol 11a using thioglycoside 6 as the glycosyl
donor was again achieved by the use of methyl triflate as
Scheme 2. Reagents and conditions: (i) penten-1-ol, CSA, 140 8C, 63%; (ii) PhCH(OMe)2, CSA, DMF, 65 8C, 260ꢁ280 mbar; 10a,
/
˚
46%; 10b, 20%; (iii) BnBr, NaOH, Bu4NHSO4, CH2Cl2, H2O, 41 8C, 66%, 11a:11b, 1.6:1; (iv) 6, MeOTf, 4 A sieves, Et2O, RT; 12a,
80%; 12b, 15%.

1940
I. Cumpstey et al. / Carbohydrate Research 338 (2003) 1937ꢁ1949
/
Scheme 3. Reagents and conditions: (i) 9-BBN, THF or toluene, 50 8C; (ii) H2O2, NaOH, H2O; 13a, 52%; 13b, 60%; (iii) MsCl,
Et3N, DCM, 0 8C; (iv) NaN3, DMF, RT; 14a, 81%; 14b, 80%; (v) MeOH, AcOH, H2, Pd (10% on carbon), RT; (vi) 16, Et3N, DMF;
15a, 44%; 15b, 79%.
an azide functionality at the terminus of the pentenyl
group which could be reduced to an amine concomitant
with the removal of the benzyl and benzylidene protect-
ing groups; peptide coupling to a fluorescent carboxylic
acid would then yield the desired deprotected labelled
disaccharides.
The rate at which glucosidase II hydrolysed the
Glca1,3Man-pyrene 15a was significantly reduced
(7.6%) as indicated by the appearance of the hydrolysis
product at an earlier retention time (Fig. 3D). The
differential rates of hydrolysis of the two linkages have
To this end, the pentenyl glycosides 7 and 12a were
converted to the alcohols 13a and 13b by regioselective
hydroboration in 52 and 60% yields, respectively.
Surprisingly this reaction required elevated tempera-
tures, and also in the case of the Glca(10
/3)Glc
disaccharide 12a, a change of solvent from THF to
toluene, in order to proceed efficiently. Mesylation of
these primary alcohols, followed by displacement by
azide proceeded smoothly in both cases to yield the
desired azides 14a and 14b (81 and 80% yields,
respectively). Finally, global deprotection with conco-
mitant reduction of the azides was achieved by catalytic
hydrogenation, to give the desired deprotected disac-
charide amines. These were then directly coupled to the
pentafluorophenyl ester of 1-pyrenebutyric acid 16$ to
furnish the desired labelled deprotected disaccharides
15a (44% yield) and 15b (79% yield), respectively
(Scheme 3).
The deprotected pyrene labelled disaccharides were
purified using normal phase HPLC and then assessed as
substrates for glucosidase II (Fig. 3). Incubation for
short times at 37 8C with linkage-specific a-glucosidase
II showed rapid cleavage of the Glca1,3Glc-pyrene 15b
to Glc-pyrene (Fig. 3A). Following a 1 h incubation,
65% of the fluorescent substrate had been hydrolysed to
Glc-pyrene as indicated by the shift in retention time.
Fig. 3. Normal Phase HPLC Analysis of Fluorescent Gluco-
sidase II Substrates. Pyrene disaccharide 15b (Glca1,3Glc, 2
mM) was incubated for 1 h at 37 8C with A, a-glucosidase II;
B, a-glucosidase I; C, buffer alone. Pyrene disaccharide 15a
(Glca1,3Man, 2 mM) was incubated for 1 h at 37 8C with D, a-
glucosidase II; E, a-glucosidase I; F, buffer alone. Hydrolysis
$
Made by treatment of 1-pyrene butyric acid and
pentafluorophenol (4 equiv) with dicyclohexylcarbodiimide
(DCC, 2 equiv) in dichloromethane at room temperature for 1
h 30 min, and subsequent purification by flash column
chromatography.
products were separated at 0.5 mL/min on a 4.6ꢃ250 mm
/
TSK gel amide-80 column at 30 8C using a solvent composed
of 80% acetonitrile, 20% ammonium acetate (100 mM), pH
3.85 and detected at Ex l 345 nm, Ex l 380 nm.

I. Cumpstey et al. / Carbohydrate Research 338 (2003) 1937ꢁ
/1949
1941
been observed using glucosylated oligosaccharides8,16
,
using 5% w/v ammonium molybdate in 2 M sulphuric
acid. Flash column chromatography was carried out
using Sorbsil C60 40/60 silica. CMAW (CHCl3ꢁ
MeOHꢁAcOHꢁwater) used as an eluant was prepared
in the following ratio (CHCl3ꢁMeOHꢁAcOHꢁH2O,
and is in support of the data presented here. Impor-
tantly, no hydrolysis of the fluorescent disaccharides
was seen using purified a-glucosidase I or buffer alone
(Fig. 3B, C, E, F).
/
/
/
/
/
/
In summary, two fluorescence-labelled disaccharide
substrates for glucosidase II have been successfully
synthesized by means of selective activation of a
thioglycoside donor in the presence of a pentenyl
glycoside; in both cases installation of a fluorescence
label was achieved by derivatisation of the anomeric
pentenyl chain and peptide coupling with an activated
ester of pyrene butyric acid. Both deprotected disac-
charides have been demonstrated to be substrates for
glucosidase II, and differential rates of cleavage ob-
served; the GlcGlc disaccharide 15b being cleaved more
rapidly than the GlcMan disaccharide 15a. Detailed
investigation of the relative rates of cleavage of each by
glucosidase II is currently underway, and the results will
be reported in due course.
60:30:3:5). Solvents and reagents were dried and purified
before use according to standard procedures under an
atmosphere of argon; MeOH was distilled from sodium
hydride, dichloromethane (DCM) and toluene were
distilled from CaH2, Py was distilled from CaH2 and
stored over potassium hydroxide, and Et2O and THF
were distilled from a solution of sodium benzophenone
ketyl immediately before use.
3.2. Pent-4?-enyl a-D-mannopyranoside (2)
Camphor sulfonic acid (110 mg, 0.47 mmol) was added
to a stirred suspension of -mannose 9 (8.5 g, 47.2
mmol) in pent-4-en-1-ol (40 g) and the reaction mixture
stirred at 100 8C for 20 h. After this time TLC (EtOAcꢁ
D
/
MeOH, 4:1) indicated the formation of a major product
(Rf 0.4). Pentenol (30 g) was recovered by vacuum
distillation using a dry ice condenser, and the remaining
brown residue was partitioned between water (200 mL)
and CH2Cl2 (200 mL). The aqueous layer was concen-
trated in vacuo and the resulting foam purified by flash
3. Experimental
3.1. General methods
Melting points were recorded on a Kofler hot block and
are uncorrected. Proton nuclear magnetic resonance
(dH) spectra were recorded on a Bruker DPX 400 (400
MHz) or on a Varian Gemini 200 (200 MHz) spectro-
meter. Carbon nuclear magnetic resonance (dC) spectra
were recorded on a Bruker AC 200 (50.3 MHz), or on a
Bruker DPX 400 (100.6 MHz) spectrometer. Multi-
plicities were assigned using DEPT sequence. All
chemical shifts are quoted on the d-scale in parts per
million (ppm). All NMR experiments were performed at
a probe temperature of 30 8C. Infrared spectra were
chromatography (EtOAc then EtOAcꢁ
give a-pentenyl glycoside 2 (7.64 g, 61%) as a pale yellow
oil; [a]2D2
54.4 (c, 1.0 in
CHCl3)]17; dH (400 MHz, CDCl3) 1.66 (2 H, m,
OCH2CH2), 2.08 (2 H, m, OCH2CH2CH2), 3.41 (1 H,
dt, Jgem 9.6 Hz, J 6.4 Hz, OCHH?CH2), 3.51 (1 H, br,
H-5), 3.65 (1 H, dt, J 6.7 Hz, OCHH?CH2), 3.76 (1 H,
/
MeOH, 9:1) to
ꢀ
/
64.2 (c, 0.5 in CHCl3) [Lit. ꢀ
/
br, H-6) 3.84 (1 H, br, H-3) 3.89ꢁ
4, H-6?), 4.81 (1 H, s, H-1), 4.96ꢁ
CH2), 5.80 (1 H, ddt, JE 17.0 Hz, JZ 10.2 Hz, J 6.6 Hz,
CH ÄCH2).
/3.97 (3 H, m, H-2, H-
/
5.05 (2 H, m, CHÄ
/
/
recorded on a PerkinꢁElmer 150 Fourier Transform
/
spectrophotometer. Low resolution mass spectra were
recorded a VG Micromass Platform using either atmo-
spheric pressure chemical ionisation (APCI), or negative
ion electrospray (ESꢂ) or positive ion electrospray
(ESꢀ). High resolution mass spectra (electrospray)
were performed 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 spectro-
3.3. Pent-4?-enyl 4,6-O-benzylidene-a-
mannopyranoside (3), pent-4?-enyl endo-2,3:4,6-di-O-
benzylidene-a- -mannopyranoside (4a) and pent-4?-enyl
exo-2,3:4,6-di-O-benzylidene-a- -mannopyranoside (4b)
D-
D
D
Tetrol 2 (5.4 g, 20.5 mmol), benzaldehyde dimethyl
acetal (7.1 mL, 47.0 mmol) and CSA (95 mg, 0.41 mmol)
were dissolved in DMF (30 mL) and the mixture was put
onto a rotary evaporator at 65 8C and 260ꢁ
/280 mbar.
After 5 h 30 min, TLC (petrolꢁEtOAc, 3:1) indicated
/
meter. Optical rotations were measured on a Perkinꢁ
/
the formation of a major product (Rf 0.9) and small
amounts of starting material (baseline) and a minor
product (Rf 0.1). The mixture was concentrated in
vacuo, and the residue dissolved in CH2Cl2 (400 mL).
Elmer 241 polarimeter with a path length of 1 dm.
Concentrations are given in g/100 mL. Microanalyses
were performed by the microanalytical services of the
Inorganic Chemistry Laboratory, Oxford. Thin layer
chromatography (TLC) was carried out on Merck
This solution was washed with water (2ꢃ150 mL) and
/
brine (100 mL), dried (MgSO4), filtered and concen-
trated in vacuo. The residue was purified by flash
Kieselgel 0.22ꢁ
/
0.25 mm thickness glass-backed sheets,
pre-coated with 60F254 silica. Plates were developed
chromatography (petrolꢁEtOAc, 8:1) to afford a 1:1
/

1942
I. Cumpstey et al. / Carbohydrate Research 338 (2003) 1937ꢁ1949
/
mixture of endo and exo dibenzylidene derivatives 4a/4b
(4.0 g, 46%) as a waxy solid. Recrystallisation from
EtOH gave the exo dibenzylidene 4b as a white,
reaction allowed to warm to room temperature (rt).
After 2 h, TLC (EtOAcꢁpetrol, 1:2) indicated formation
of two products (Rf 0.45 and 0.6) and remaining starting
material (Rf 0.8). The reaction was cooled to ꢂ40 8C
and further diisobutyl aluminium hydride (0.52 mL of
1.5 M solution in toluene, 0.78 mmol) was added. After
a further 2 h, TLC indicated the formation of a major
product (Rf 0.6). The reaction was quenched with
MeOH (2 mL) and the mixture partitioned between
CH2Cl2 (50 mL) and water (50 mL). The organic layer
was washed with brine (50 mL), dried (MgSO4), filtered
and concentrated in vacuo. The residue was purified by
/
crystalline solid, mp 131ꢁ
CHCl3); nmax (KBr disk) 1641 (w, CÄ
MHz, CDCl3) 1.67ꢁ1.74 (2 H, m, OCH2CH2), 2.11ꢁ
/
136 8C; [a]2D2
ꢀ
/
11.5 (c, 1.0 in
/
/
C) cmꢂ1; dH (400
/
/
2.17 (2 H, m, OCH2CH2CH2), 3.45 (1 H, dt, Jgem 9.7
Hz, J 6.4 Hz, OCHH?CH2), 3.75 (1 H, dt, J 6.6 Hz,
OCHH?CH2), 3.81ꢁ3.93 (3 H, m, H-4, H-5, H-6), 4.16
/
(1 H, d, J2,3 5.4 Hz, H-2), 4.35 (1 H, dd, J5,6? 3.0 Hz, J6,6?
8.3 Hz, H-6?), 4.66 (1 H, dd, J3,4 7.6 Hz, H-3), 5.00 (1 H,
dd, Jgem 1.7 Hz, JZ 10.2 Hz, CHÄ
JE 17.1 Hz, CHÄCHEHZ, 5.11 (1 H, s, H-1), 5.65 (1 H, s,
PhCH), 5.81 (1 H, ddt, J 6.7 Hz, CH ÄCH2), 6.30 (1 H,
s, PhCH), 7.34ꢁ7.55 (10 H, m, ArÃH); dC (CDCl3) 28.4,
30.2 (2ꢃ t, OCH2CH2CH2), 60.4, 75.5, 75.6, 77.6 (4ꢃ
d, C-2, C-3, C-4, C-5), 67.4, 68.9 (22ꢃ t, C-6,
OCH2CH2CH2), 97.5, (d, C-1), 102.1, 103.1 (2ꢃ d,
2ꢃ PhCH), 115.4 (t, CHÄCH2), 126.3, 126.5, 128.5,
128.7, 129.4 (5ꢃ d, 10ꢃ ArÃCH), 137.4, 138.9 (2ꢃ s,
2ꢃ ArÃC), 138.0 (d, CHÄ
CH2); m/z (APCIꢀ) 425
(Mꢀ OC5H9, 6%). (Found: C,
Hꢀ, 100), 339 (Mꢂ
/CHEHZ) 5.04 (1 H, dd,
/
flash chromatography (petrolꢁEtOAc, 6:1) to afford the
/
/
desired 2-O-benzylated compound 5a (120 mg, 40%
(43% over recovered starting material)) as a colourless
oil, which crystallised from petrol to give a white,
/
/
/
/
/
crystalline solid, mp 68ꢁ
0.5 in CHCl3); nmax (KBr disk) 3392 (b, OH), 1641 (w,
CÄ 1.71 (2 H, m,
C) cmꢂ1; dH (400 MHz, CDCl3) 1.64ꢁ
OCH2CH2CH2), 2.10ꢁ2.16 (2 H, m, OCH2CH2CH2),
/
71 8C (petrol); [a]D22
ꢀ16.0 (c,
/
/
/
/
/
/
/
/
/
/
/
/
/
/
2.39 (1 H, d, J 8.0 Hz, OH-3), 3.39 (1 H, dt, Jgem 9.6 Hz,
J 6.4 Hz, OCHH?CH2), 3.70 (1 H, dt, J 6.6 Hz,
/
/
70.43; H, 6.78. C25H28O6 requires C, 70.73; H, 6.65%).
The mother liquor was a concentrated to give colourless
oil which had a 1H NMR spectrum consistent with a 6:1
OCHH?CH2), 3.79ꢁ3.86 (3 H, m, H-2, H-5, H-6), 3.92
/
(1 H, m, H-4), 4.12 (1 H, m, H-3), 4.26 (1 H, m, H-6?),
4.71, 4.76 (2 H, ABq, JAB 11.7 Hz, PhCH2), 4.83 (1 H, d,
J1,2 1.1 Hz, H-1), 5.00 (1 H, dd, Jgem 1.7 Hz, JZ 11.1 Hz,
endo ꢁexo mixture of dibenzylidenes 4a and 4b. Partial
/
NMR data for 4a follows: dH (200 MHz, CDCl3) 1.73 (2
H, m, OCH2CH2CH2), 2.15 (2 H, m, OCH2CH2CH2),
CHÄ
5.59 (1 H, s, PhCH), 5.81 (1 H, ddt, CH Ä
7.55 (10 H, m, ArÃH); dC (CDCl3) 28.4, 30.2 (2ꢃ
OCH2CH2CH2), 63.5, 68.8, 78.7, 79.6 (4ꢃ d, C-2, C-3,
/
CHEHZ 5.04 (1 H, dd, JE 17.1 Hz, CHÄ
CH2), 7.29ꢁ
t,
/
CHEHZ,
/
/
3.48 (1 H, dt, OCHH?CH2), 4.28ꢁ
H, at), 4.98ꢁ5.11 (2 H, m, CHÄCH2), 5.18 (1 H, s, H-1),
5.54 (1 H, s, PhCH), 5.83 (1 H, ddt, CH ÄCH2), 5.98 (1
H, s, PhCH), 7.37ꢁ H). Further
/
4.33 (2 H, m), 4.50 (1
/
/
/
/
/
/
C-4, C-5), 67.1, 68.8, 73.8 (3ꢃ
OCH2CH2CH2), 98.5, 102.2 (2ꢃ
115.3 (t, CHÄ
(5ꢃ d, ArÃCH), 137.0 (2ꢃ
CH2); m/z (APCIꢀ) 875 (2Mꢀ
9), 427 (Mꢀ H, 100), 341 (Mꢂ
Hꢀ, 9), 425 (Mꢂ
/
t, C-6, PhCH2,
d, C-1, PhCH),
/
7.58 (10 H, m, ArÃ
/
/
elution of the flash column afforded the monobenzyli-
dene derivative 3 (3.8 g, 52%) as a gelatinous solid; [a]D22
/
CH2), 126.5, 128.2, 128.5, 128.8, 129.3
s, ArÃC), 138.1 (d, CHÄ
Naꢀ, 5), 449 (MꢀNaꢀ,
OC5H9,
/
/
/
/
/
ꢀ
/
44.2 (c, 1.25 in CHCl3) [Lit. ꢀ
CHCl3)]18; dH (200 MHz, CDCl3) 1.65ꢁ
OCH2CH2CH2), 2.09ꢁ2.21 (2 H, m, OCH2CH2CH2),
2.60 (2 H, br s, 2ꢃ OH), 3.45 (1 H, dt, Jgem 9.6 Hz, J
6.3 Hz, OCHH?CH2), 3.73 (1 H, dt, J 6.5 Hz,
OCHH?CH2), 3.81ꢁ4.26 (5 H, m, H-2, H-3, H-4, H-5
and H-6), 4.29 (1 H, d, J 5.6 Hz, H-6?), 4.88 (1 H, s, H-
1), 4.99ꢁ5.11 (2 H, m, CHÄCH2) 5.60 (1 H, s, PhCH),
5.83 (1 H, ddt, JE 16.8 Hz, JZ 10.1 Hz, J 6.4 Hz, CH Ä
CH2), 7.34ꢁ7.55 (5 H, m, ArÃH).
/
53.5 (c, 1.1 in
/
/
/1.79 (2 H, m,
/
/
/
/
8%). (Found: C, 70.26; H, 7.31. C25H30O6 requires C,
70.40; H, 7.09); together with undesired 3-O-benzylated
/
material 5b (15 mg, 5%) as a colourless oil; [a]D22
ꢀ
/
45.9
(c, 1.25 in CHCl3); nmax (film) 3468 (OH), 1641 (CÄC)
cmꢂ1; dH (400 MHz, CDCl3) 1.66ꢁ
1.73 (2 H, m,
OCH2CH2CH2), 2.10ꢁ2.16 (2 H, m, OCH2CH2CH2),
/
/
/
/
/
/
/
2.66 (1 H, s, OH-2), 3.44 (1 H, dt, Jgem 9.7 Hz, J 6.4 Hz,
OCHH?CH2), 3.71 (1 H, dt, J 6.6 Hz, OCHH?CH2),
/
/
3.84ꢁ3.90 (2 H, m, H-5, H-6), 3.94 (1 H, dd, J2,3 3.4 Hz,
/
3.4. Pent-4?-enyl 2-O-benzyl-4,6-O-benzylidene-a-
mannopyranoside (5a) and pent-4?-enyl 3-O-benzyl-4,6-
O-benzylidene-a- -mannopyranoside (5b)
D
-
J3,4 9.6 Hz, H-3), 4.07 (1 H, m, H-2), 4.11 (1 H, m, H-4),
4.28 (1 H, m, H-6?), 4.74, 4.88 (2 H, ABq, JAB 11.8 Hz,
PhCH2), 4.87 (1 H, s, H-1), 4.99, (1 H, dd, JZ 10.2 Hz,
D
Jgem 1.5 Hz, CHÄ
CHÄCHEHZ, 5.63 (1 H, s, PhCH), 5.81 (1 H, ddt, J 6.8
Hz, CH2CH ÄCH2), 7.29ꢁ7.52 (10 H, m, 10ꢃArÃH);
dC (CDCl3) 28.4, 30.2 (2ꢃ t, OCH2CH2CH2), 63.3,
70.0, 75.9, 79.0 (4ꢃ d, C-2, C-3, C-4, C-5), 67.1, 68.9,
73.2 (3ꢃ t, C-6, PhCH2, OCH2CH2CH2), 100.1, 101.7
(2ꢃ d, C-1, PhCH), 115.3 (t, CHÄCH2), 126.3, 128.1,
128.5, 128.7, 129.1 (5ꢃ d, ArÃCH), 137.8, 138.2 (2ꢃ s,
/
CHEHZ 5.04 (1 H, dd, JE 17.1 Hz,
3.4.1. Method 1 selective reduction. The Â
/
6:1 endoꢁ
/
exo
/
mixture of dibenzylidene derivatives 4a/4b (as detailed
above, 300 mg, 0.71 mmol) was dissolved in freshly
distilled toluene (3 mL) in a flame-dried flask. The
solution was stirred under Ar and cooled to ꢂ
Diisobutyl aluminium hydride (0.52 mL of 1.5 M
/
/
/
/
/
/
/
40 8C.
/
/
/
solution in toluene, 0.78 mmol) was added and the
/
/
/

I. Cumpstey et al. / Carbohydrate Research 338 (2003) 1937ꢁ
/1949
1943
ArÃ
/
C), 138.1 (d, CHÄ
/
CH2); m/z (APCIꢂ) 425 (Mꢂ
Bn, 77), 317 (44), 121 (100); (Found: C,
69.90; H, 7.32; C25H30O6 requires C, 70.40; H, 7.09).
/
H,
16.9 Hz, JZ 10.2 Hz, J 6.6 Hz, CH Ä
H, m, ArÃ
OCH2CH2CH2), 64.0, 70.8, 72.9, 77.4, 77.5, 78.9, 79.8,
81.4 (8ꢃ d, C-2a, C-3a, C-4a, C-5a, C-2b C-3b, C-4b C-
5b), 67.1, 68.4, 69.0, 70.7, 73.5, 73.9, 75.0, 75.6 (8ꢃ t, C-
6a, C-6b, 5ꢃ PhCH2, OCH2CH2), 97.1, 99.7, 102.5
(3ꢃ d, C-1a, C-1b, PhCH), 115.3 (t, CHÄCH2), 126.6,
127.4, 127.7, 128.0, 128.2, 128.4, 128.6, 128.7, 129.5 (9ꢃ
d, 30ꢃ ArÃCH), 137.7, 138.4, 138.5, 138.7 (4ꢃ s, 6ꢃ
ArÃC), 138.2 (d, CHÄ
CH2); m/z (APCIꢀ) 966 (Mꢀ
/
CH2), 6.97ꢁ
/
7.52 (30
6), 335 (Mꢂ
/
/
H); dC (CDCl3) 28.4, 30.2 (2ꢃ t,
/
/
3.4.2. Method 2 selective benzylation. Monobenzylidene
derivative 3 (200 mg, 0.60 mmol) was dissolved in
CH2Cl2 (10 mL) and tetrabutyl ammonium hydrogen-
sulfate (41 mg, 0.12 mmol) and benzyl bromide (0.11
mL, 0.96 mmol) were added. An aq soln of sodium
hydroxide (50 mg in 1 mL) was added and the mixture
/
/
/
/
/
/
/
/
/
/
/
was refluxed at 41 8C. After 24 h, TLC (EtOAcꢁ
/petrol,
NHꢀ4 ; 68); 949 (Mꢀ
/
Hꢀ, 9%), (APCIꢂ) 983 (MꢀClꢂ,
/
1:2) indicated formation of two products (Rf 0.45 and
0.6) and starting material (Rf 0.1). The reaction mixture
was cooled and partitioned between CH2Cl2 (20 mL)
and water (20 mL). The organic layer was washed with
98%). (HRMS Calcd. For C59H68NO11 (MNHꢀ4 )
966.479238. Found 966.478812).
3.6. Pent-4?-enyl 4,6-O-benzylidene-a-D-glucopyranoside
water (2ꢃ
centrated in vacuo. The residue was purified by flash
chromatography (30ꢁ40 petrolꢁEtOAc, 8:1) to afford
/
20 mL), dried (Na2SO4), filtered and con-
(10a) and pent-4?-enyl 4,6-O-benzylidene-b-D-
glucopyranoside (10b)
/
/
the desired 2-O-benzylated material 5a (115 mg, 45%) as
a white crystalline solid together with the undesired 3-O-
benzylated material 5b (52 mg, 20%) as a colourless oil,
identical to the materials previously described.
Camphor sulfonic acid (200 mg, 0.86 mmol) was added
to a stirred suspension of -glucose 8 (10.0 g, 55.5
mmol) in 4-penten-1-ol (40 mL) and the mixture was
D
refluxed at 140 8C for 48 h. After this time, TLC
(EtOAcꢁ
product (Rf 0.4) and the absence of starting material (Rf
0). Pentenol (Â30 g) was recovered by vacuum distilla-
tion using a dry ice condenser. Triethylamine (10 drops)
was added to the remaining brown residue, and the
mixture concentrated in vacuo. The resulting brown oil
was purified by flash chromatography (EtOAc then
/
MeOH, 4:1) indicated the formation of a major
3.5. Pent-4?-enyl 2,3,4,6-tetra-O-benzyl-a-
glucopyranosyl-(103)-2-O-benzyl-4,6-O-benzylidene-a-
-mannopyranoside (7)
D-
/
/
D
Pentenyl glycoside 3 (300 mg, 0.70 mmol) and thioglyco-
side 614 (456 mg, 0.72 mmol) were dissolved in freshly
distilled ether (15 mL) in a flame-dried flask. The
˚
mixture was stirred under Ar with activated 4 A
molecular seives for 30 min. Methyl triflate (0.4 mL,
3.52 mmol) was added and the mixture stirred at rt.
EtOAcꢁMeOH, 9:1) to give an a/b mixture of pentenyl
/
glycosides 9a/b (9.2 g, 63%) as a brown oil, which was
used directly without further characterisation. The
mixture of glycosides 9a/b (9.2 g, 34.8 mmol), benzalde-
hyde dimethyl acetal (6.00 mL, 40.0 mmol) and CSA
(160 mg, 0.70 mmol) were dissolved in DMF (30 mL)
and the mixture was put onto the rotary evaporator at
After 19 h, TLC (petrolꢁEtOAc, 4:1) indicated the
/
formation of a major product (Rf 0.45) and very little
remaining starting materials (Rf 0.25 and 0.5). The
reaction was quenched with Et3N (1 mL) and stirred for
a further 30 min. The mixture was filtered through
Celite† and concentrated in vacuo. The residue was
65 8C and 260ꢁ280 mbar. After 7 h, TLC (EtOAc)
/
indicated the formation of two products (Rf 0.5 and 0.6)
and small amounts of starting material (Rf 0.1). Et3N
(10 drops) was added, and the mixture concentrated in
vacuo. The residue was dissolved in CH2Cl2 (200 mL)
purified by flash chromatography (petrolꢁ
to afford the desired a-disaccharide 7 (385 mg, 58%) as a
colourless oil; [a]D22
76.9 (c, 0.32 in CHCl3); nmax (film)
1640 (w, CÄ 1.68
C) cmꢂ1; dH (400 MHz, CDCl3) 1.61ꢁ
(2 H, m, OCH2CH2CH2), 2.07ꢁ2.12 (2 H, m,
/
EtOAc, 7:1)
ꢀ
/
and washed with water (2ꢃ
dried (MgSO4), filtered and concentrated in vacuo. The
residue was purified by flash chromatography (petrolꢁ
/100 mL) and brine (50 mL),
/
/
/
/
OCH2CH2CH2), 3.37 (1 H, dt, Jgem 9.6 Hz, J 6.5 Hz,
OCHH?CH2), 3.52 (1 H, dd, J1,2 3.3 Hz, J2,3 9.6 Hz, H-
EtOAc, 1:1) to afford the desired a-pentenyl glycoside
10a as a white solid (5.3 g, 46% (62% over recovered
starting material)) which was recrystallised from ether/
2b) 3.58ꢁ
/
3.70 (4 H, m, OCHH?CH2, H-4b, H-6b, H-6?b);
3.78ꢁ3.81 (2 H, m, H-2a, H-5b) 3.84ꢁ
/
/3.89 (2 H, m, H-5a,
petrol; mp 87 8C [Lit. 90 8C]19; [a]D22
CHCl3) [Lit. ꢀ
CDCl3) 1.74ꢁ1.81 (2 H, m, OCH2CH2CH2), 2.14ꢁ
ꢀ
/
105.3 (c, 1.0 in
96.5 (c, 1.13 in CHCl3)];19 dH (400 MHz,
2.20
H-6a) 3.99 (1 H, at, J 9.3 Hz, H-3b), 4.20 (1 H, m, H-6a?);
4.31, 4.55 (2 H, ABq, JAB 12.3 Hz, PhCH2), 4.35 (1 H,
at, J 8.2 H-4a), 4.37 (1 H, m, H-3a), 4.42, 4.84 (2 H,
ABq, JAB 10.9 Hz, PhCH2), 4.43, 4.60 (2 H, ABq, JAB
12.1 Hz, PhCH2), 4.73, 4.96 (2 H, ABq, JAB 10.9 Hz,
PhCH2), 4.77, 4.92 (2 H, ABq, JAB 11.9 Hz, PhCH2),
/
/
/
(3 H, m, OCH2CH2CH2, OH-2), 2.62 (1 H, d, J 2.0 Hz,
OH-3), 3.49 (1 H, dt, OCHH?CH2), 3.52 (1 H, at, J 9.3
Hz, H-4), 3.64 (1 H, m, H-2), 3.76 (1 H, at, J 10.2 Hz,
H-6), 3.77ꢁ
at, J 9.2 Hz, H-3), 4.29 (1 H, dd, J5,6? 4.6 Hz, J6,6? 9.9
Hz, H-6?), 4.89 (1 H, d, J1,2 4.0 Hz, H-1), 5.00ꢁ5.09 (2
/
3.87 (2 H, m, H-5, OCHH?CH2), 3.95 (1 H,
4.80 (1 H, s, H-1a), 4.98ꢁ
(1 H, s, PhCH), 5.54 (1 H, d, H-1b), 5.78 (1 H, ddt, JE
/
5.04 (2 H, m, CHÄ
/
CH2), 5.47
/

1944
I. Cumpstey et al. / Carbohydrate Research 338 (2003) 1937ꢁ1949
/
H, m, CHÄ
16.9 Hz, JZ 10.2 Hz, J 6.6 Hz, CH Ä
H, m, ArÃH); and the undesired b-pentenyl glycoside
10b as a white solid (2.3 g, 20% (27% over recovered
starting material)) which was recrystallised from etherꢁ
EtOAc; mp 148 8C [Lit. 144ꢁ 51.9 (c,
145 8C];19 [a]2D2
1.0 in CHCl3) [Lit. ꢂ
43.8 (c, 1.16 in CHCl3)];19 dH (400
MHz, CDCl3) 1.72ꢁ1.80 (2 H, m, OCH2CH2CH2),
2.14ꢁ2.19 (2 H, m, OCH2CH2CH2), 2.54 (1 H, d, J
2.4 Hz, OH-2), 2.72 (1 H, d, J 2.2 Hz, OH-3), 3.44ꢁ3.62
/
CH2), 5.55 (1 H, s, PhCH), 5.83 (1 H, ddt, JE
OCH2CH2CH2), 97.6, 102.0 (2ꢃ
115.3 (t, CHÄCH2), 126.6, 127.9, 128.3, 128.5, 128.8,
129.2, 129.4 (7ꢃ d, ArÃCH), 137.4, 138.2 (2ꢃ s, ArÃ
C), 138.2 (d, CHÄ H,
CH2); m/z (APCIꢂ) 851 (2Mꢂ
42), 425 (Mꢂ Bn, 32), 207 (62), 113
/
d, C-1, PhCH),
/
CH2), 7.36ꢁ7.52 (5
/
/
/
/
/
/
/
/
/
/
/
H, 59), 335 (Mꢂ
/
/
ꢂ
/
(100%); (HRMS Calcd. For C25H31O6 (MHꢀ)
427.212064. Found 427.211859); together with pure 3-
O-benzylated material 11b as a white, crystalline solid,
/
/
/
mp 107ꢁ
CHCl3); dH (400 MHz, CDCl3) 1.73ꢁ
OCH2CH2CH2), 2.13ꢁ2.19 (2 H, m, OCH2CH2CH2),
/
108 8C (ether/petrol); [a]D22
ꢀ
/
59.4 (c, 0.88 in
/
/1.80 (2 H, m,
(4 H, m, H-5, H-2, H-4, OCHH?CH2), 3.80 (1 H, at, J
10.2 Hz, H-6), 3.84 (1 H, m, H-3), 3.94 (1 H, dt, Jgem 9.5
Hz, J 6.6 Hz, OCHH?CH2), 4.36 (1 H, dd, J5,6? 4.9 Hz,
/
2.24 (1 H, d, J 8.4 Hz, OH-2), 3.50 (1 H, dt, Jgem 9.7 Hz,
J 6.5 Hz, OCHH?CH2), 3.65 (1 H, at, J 9.2 Hz, H-4),
J
6,6? 10.5 Hz, H-6?), 4.40 (1 H, d, J1,2 7.8 Hz, H-1), 4.99ꢁ
5.08 (2 H, m, CHÄCH2), 5.55 (1 H, s, PhCH), 5.83 (1 H,
ddt, JE 16.9 Hz, JZ 10.2 Hz, J 6.7 Hz, CH ÄCH2), 7.37ꢁ
7.51 (5 H, m, ArÃH).
/
3.70ꢁ3.89 (5 H, m, OCHH?CH2, H-2, H-3, H-5, H-6),
4.29 (1 H, dd, J5,6? 4.6 Hz, J6,6? 10.0 Hz, H-6?), 4.83, 4.97
(2 H, ABq, JAB 11.6 Hz, PhCH2), 4.90 (1 H, d, J1,2 3.9
/
/
/
/
/
Hz, H-1), 4.99ꢁ
PhCH), 5.81 (1 H, ddt, JE 17.0 Hz, JZ 10.2 Hz, J 6.6 Hz,
CH ÄCH2), 7.28ꢁ7.52 (10 H, m, 10ꢃ ArÃH); dC
(CDCl3) 28.5, 30.3 (2ꢃ t, OCH2CH2CH2), 62.7, 72.6,
79.2, 82.0 (4ꢃ d, C-2, C-3, C-4, C-5), 67.9, 69.0, 74.9
(3ꢃ t, C-6, PhCH2, OCH2CH2), 99.0, 101.3 (2ꢃ d, C-
1, PhCH), 115.4 (t, CHÄCH2), 126.2, 127.9, 128.2,
128.5, 128.6, 129.0, 129.2 (7ꢃ d, ArÃCH), 137.6, 138.8
(2ꢃ s, ArÃC), 138.1 (d, CHÄ
CH2); m/z (APCIꢂ) 425
(MꢂH, 7), 317 (32), 113 (100%). (Found: C, 70.25; H,
6.99; C25H30O6 requires C, 70.40; H, 7.09%).
/
5.08 (2 H, m, CHÄ/CH2), 5.58 (1 H, s,
3.7. Pent-4?-enyl 2-O-benzyl-4,6-benzylidene-a-
glucopyranoside (11a) and pent-4?-enyl 3-O-benzyl-4,6-
benzylidene-a- -glucopyranoside (11b)
D-
/
/
/
/
/
D
/
/
/
The a-diol 10a (1.55 g, 4.6 mmol) was dissolved in
CH2Cl2 (50 mL), and tetrabutylammonium hydrogen-
sulfate (310 mg, 0.92 mmol) and benzyl bromide (0.88
mL, 7.4 mmol) were added. An aq soln of sodium
hydroxide (250 mg in 5 mL) was added and the mixture
/
/
/
/
/
/
/
was refluxed at 41 8C. After 46 h, TLC (EtOAcꢁpetrol,
/
1:2) indicated the formation of a major product (Rf 0.4)
and remaining starting material (Rf 0.1). The reaction
mixture was cooled, and partitioned between CH2Cl2
(50 mL) and water (50 mL). The organic layer was
washed with water (50 mL) and brine (20 mL), dried
(Na2SO4), filtered and concentrated in vacuo. The
3.8. Pent-4?-enyl 2,3,4,6-tetra-O-benzyl-a-
3)-2-O-benzyl-4,6-O-benzylidene-a-
-glucopyranoside (12a) and pent-4?-enyl 2,3,4,6-tetra-O-
D-
glucopyranosyl-(10
/
D
benzyl-b-
benzylidene-a-
D-glucopyranosyl-(10
/
3)-2-O-benzyl-4,6-O-
D
-glucopyranoside (12b)
residue was purified by flash chromatography (petrolꢁ
EtOAc, 4:1) to afford a mixture of the desired 2-O-
benzylated material 11a and undesired 3-O-benzylated
/
The alcohol 11a (50 mg, 0.117 mmol) and thioglycoside
614 (74 mg, 0.117 mmol) were dissolved in freshly
distilled ether (3 mL) in a flame-dried flask. The mixture
˚
was stirred under Ar with activated 4 A molecular seives
for 30 min. Methyl triflate (0.066 mL, 0.585 mmol) was
added and the mixture stirred at rt. After 17 h 30 min,
material 11b (1.30 g, 66%) (11aꢁ
1H NMR spectroscopy). Fractional crystallisation from
etherꢁpetrol gave the pure desired 2-O-benzylated
material 11a as a white, crystalline solid, mp 47ꢁ49 8C
(ether/petrol); [a]D22
65.2 (c, 0.25 in CHCl3); nmax (film)
3400 (br, OH), 1641 (w, CÄ
C) cmꢂ1; dH (400 MHz,
CDCl3) 1.72ꢁ1.79 (2 H, m, OCH2CH2CH2), 2.15ꢁ2.21
(2 H, m, OCH2CH2CH2), 2.53 (1 H, d, J 2.0 Hz, OH-3),
3.37 (1 H, dt, Jgem 9.7 Hz, OCHH?CH2), 3.47 (1 H, dd,
J1,2 3.6 Hz, J2,3 9.3 Hz, H-2), 3.52 (1 H, at, J 9.4 Hz, H-
4), 3.68 (1 H, dt, J 6.5 Hz, OCHH?CH2), 3.71 (1 H, at, J
10.2 Hz, H-6), 3.85 (1 H, adt, J5,6? 4.8 Hz, J 10.0 Hz, H-
5), 4.18 (1 H, dt, H-3), 4.25 (1 H, dd, J6,6? 10.1 Hz, H-6?),
/
11b, 1.6:1 as shown by
/
/
TLC (petrolꢁEtOAc, 4:1) indicated the formation of a
/
ꢀ
/
major product (Rf 0.2) and a minor product (Rf 0.3) and
no remaining starting materials (Rf 0.15, 0.5). The
reaction was quenched with Et3N (0.16 mL) and stirred
for a further 30 min. The mixture was filtered through
Celite† and concentrated in vacuo. The residue was
/
/
/
purified by flash chromatography (petrolꢁ
to afford the desired a-disaccharide 12a (89 mg, 80%) as
a colourless oil; [a]D22
98.2 (c, 0.11 in CHCl3); nmax
(film) 1640 (CÄ
C) cmꢂ1; dH (400 MHz, CDCl3) 1.74ꢁ
1.81 (2 H, m, OCH2CH2CH2), 2.17ꢁ2.23 (2 H, m,
OCH2CH2CH2), 3.38ꢁ3.47 (3 H, m, OCHH?CH2, H-6b,
H-6b?); 3.49 (1 H, dd, J1,2 3.6 Hz, J2,3 9.7 Hz, H-2b) 3.61ꢁ
/EtOAc, 6:1)
ꢀ
/
/
/
4.69, 4.77 (2 H, ABq, PhCH2), 4.75 (1 H, d, H-1), 4.99ꢁ
5.08 (2 H, m, CHÄCH2), 5.53 (1 H, s, PhCH), 5.83 (1 H,
ddt, JE 16.9 Hz, JZ 10.2 Hz, J 6.6 Hz, CH ÄCH2), 7.30ꢁ
7.53 (10 H, m, ArÃH); dC (CDCl3) 28.5, 30.2 (2ꢃ t,
OCH2CH2CH2), 62.2, 70.1, 79.9, 81.4 (4ꢃ d, C-2, C-3,
C-4, C-5), 67.7, 69.0, 73.1 (3ꢃ t, C-6, PhCH2,
/
/
/
/
/
/
/
/
/
3.76 (4 H, m, OCHH?CH2, H-4b, H-2a, H-6a), 3.80 (1 H,
at, J 9.4 Hz, H-4a) 3.91 (1 H, m, J5,6 10.0 Hz, J5,6? 4.8
/
/
Hz, H-5a), 3.97 (1 H, at, J 9.4 Hz, H-3b), 4.20ꢁ4.26 (2
/

I. Cumpstey et al. / Carbohydrate Research 338 (2003) 1937ꢁ
/1949
1945
H, m, H-5b, H-6?a); 4.29, 4.33, 4.38 (3 H, 3ꢃ
PhCHH?), 4.41 (1 H, at, J 9.3 Hz, H-3a), 4.55ꢁ
H, m, 4ꢃ PhCHH?), 4.79, 4.81 (2 H, 2ꢃ d, 2ꢃ
PhCHH?), 4.87 (1 H, d, J1,2 3.7 Hz, H-1a), 4.99ꢁ5.10
(2 H, m, CHÄCH2), 5.48 (1 H, s, PhCH), 5.59 (1 H, d,
H-1b), 5.85 (1 H, ddt, JE 16.9 Hz, JZ 10.2 Hz, J 6.6 Hz,
CH ÄCH2), 6.91ꢁ7.42 (30 H, m, ArÃH); dC (CDCl3)
28.5, 30.2 (2ꢃ t, OCH2CH2CH2), 62.0, 69.6, 72.8, 75.6,
77.6, 78.8, 81.8, 83.0 (8ꢃ d, C-2a, C-3a, C-4a, C-5a, C-
2b, C-3b, C-4b, C-5b), 67.7, 68.0, 69.2, 71.0, 73.0, 73.3,
74.8, 75.6 (8ꢃ t, C-6a, C-6b, 5ꢃ PhCH2, OCH2CH2),
96.3, 97.5, 102.5 (3ꢃ d, C-1a, C-1b, PhCH), 115.2 (t,
CHÄCH2), 126.4, 126.6, 127.4, 127.6, 127.8, 128.0,
128.3, 128.5, 128.6, 129.6 (10ꢃ d, ArÃCH), 137.3,
137.9, 138.0, 139.1, 139.2 (5ꢃ s, 6ꢃ ArÃC), 138.3 (d,
Naꢀ, 16), 966 (Mꢀ
/
d, 3ꢃ
4.64 (4
/
mL) was added. The reaction mixture was heated to
50 8C and stirred under Ar. After 18 h, the reaction
mixture was cooled to rt and ice-cold water (1 mL) was
added. Then aq H2O2 (35% v/v, 0.5 mL) and aq NaOH
(2 M, 0.75 mL) were added dropwise. The reaction
mixture was stirred for a further 12 h after which time
/
/
/
/
/
/
/
/
/
TLC (cyclohexaneꢁEtOAc, 1:1) indicated no further
/
/
consumption of starting material (Rf 0.7) and the
formation of a major product (Rf 0.3). The mixture
was dissolved in ether (30 mL) and washed with water
(30 mL). The aqueous layer was re-extracted with ether
(30 mL) and the combined organic extracts dried
(MgSO4), filtered and concentrated in vacuo. The
residue was purified by flash column chromatography
/
/
/
/
/
/
/
/
/
/
(cyclohexaneꢁ
52, 57% over recovered starting material) as a clear oil;
[a]2D5
67.9 (c, 0.68 in CHCl3); nmax (thin film) 3409 (br,
OH/NH) cmꢂ1; dH (400 MHz, CDCl3) 1.37ꢁ
1.45 (2 H,
m, OCH2CH2CH2), 1.53ꢁ1.60 (4 H, m,
OCH2CH2CH2CH2), 3.38 (1 H, m, OCHH?CH2), 3.53
(1 H, dd, J1,2 3.6 Hz, J2,3 9.7 Hz, H-2b), 3.57ꢁ3.71 (6 H,
m, H-6b, H-6?b; H-4b, OCHH?CH2, CH2CH2O), 3.82ꢁ
3.84 (2 H, m, H-5b, H-2a), 3.86ꢁ3.88 (2 H, m, H-5a, H-
/
EtOAc, 3:1) to afford alcohol 13a (19 mg,
CHÄ
/
CH2); m/z (APCIꢀ) 971 (Mꢀ
/
NHꢀ4 ; 18); 307 (31), 121 (100%). (HRMS Calcd. For
C59H68NO11 (MNHꢀ4 ) 966.479238. Found 966.481418);
together with undesired b-disaccharide 12b (29 mg, 15%)
ꢀ
/
/
/
as a colourless oil; [a]D22
ꢀ
/
29.5 (c, 1.95 in CHCl3); nmax
C) cmꢂ1; dH (400 MHz, CDCl3)
1.79 (2 H, m, OCH2CH2CH2), 2.15ꢁ2.21 (2 H, m,
(film) 1640 (w, CÄ
/
/
1.72ꢁ
/
/
/
OCH2CH2CH2), 3.24 (1 H, adt, J4,5 9.2 Hz, J 3.0 Hz, H-
5b), 3.36 (1 H, dt, Jgem 9.8 Hz, J 6.6 Hz, OCHH?CH2),
/
6a), 4.00 (1 H, at, J 9.3 Hz, H-3b), 4.22 (1 H, d, J5,6? 5.3
Hz, H-6?a); 4.31, 4.57 (2 H, ABq, JAB 12.0 Hz, PhCH2),
3.50 (1 H, at, J 3.82 Hz, H-2b), 3.55ꢁ3.66 (6 H, m,
/
OCHH?CH2, H-2a, H-3b, H-4b, H-6b, H-6b?); 3.68 (1 H,
at, J 9.4 Hz, H-4a), 3.69 (1 H, at, J 10.2 Hz, H-6a), 3.86
(1 H, adt, J5,6? 4.7 Hz, J 9.9 Hz, H-5a), 4.21 (1 H, dd,
4.36ꢁ4.41 (2 H, m, H-3a, H-4a), 4.43, 4.60 (2 H, ABq,
/
JAB 12.2 Hz, PhCH2), 4.43, 4.86 (2 H, ABq, JAB 11.0
Hz, PhCH2), 4.75, 4.98 (2 H, ABq, JAB 10.4 Hz,
PhCH2), 4.80, 4.94 (2 H, ABq, JAB 12.3 Hz, PhCH2),
4.82 (1 H, d, J1,2 1.4 Hz, H-1a), 5.49 (1 H, s, PhCH),
J
6,6? 10.1 Hz, H-6?); 4.38 (1 H, at, J 9.1 Hz, H-3a), 4.47,
a
4.70 (2 H, ABq, JAB 11.7 Hz, PhCH2), 4.49 (2 H, s,
PhCH2), 4.53, 4.77 (2 H, ABq, JAB 10.7 Hz, PhCH2),
4.64 (1 H, d, J 3.8 Hz, H-1a), 4.75, 5.08 (2 H, ABq, JAB
10.9 Hz, PhCH2), 4.79, 4.93 (2 H, ABq, JAB 10.9 Hz,
5.55 (1 H, d, H-1b), 7.00 (2 H, ad, J 6.7 Hz, ArÃ
7.14ꢁ7.39 (26 H, m, ArÃH), 7.47 (2 H, ad, J 6.7 Hz,
ArÃH); dC (100.6 MHz, CDCl3) 22.9 (t,
OCH2CH2CH2), 29.0, 32.4 (2ꢃ t,
OCH2CH2CH2CH2), 62.6 (t, C-6b), 64.0 (d, C-5a),
67.7, 68.5, 68.9 (3ꢃ t, C-6a, 2ꢃ CH2CH2O), 70.6,
73.3, 73.8, 74.9, 75.5 (5ꢃ t, 5ꢃ PhCH2), 70.8 (d, C-5b),
73.3 (d, C-3a), 77.3, 77.5 (2ꢃ d, C-4b, C-2a), 79.1 (d, C-
/
H),
/
/
/
PhCH2), 4.89 (1 H, d, J1,2 7.7 Hz, H-1b), 4.99ꢁ
m, CHÄCH2), 5.48 (1 H, s, PhCH), 5.83 (1 H, ddt, JE
17.0 Hz, JZ 10.2 Hz, J 6.6 Hz, CH ÄCH2), 7.13ꢁ7.43 (30
H, m, ArÃH); dC (100 MHz, CDCl3) 28.5, 30.2 (2ꢃ t,
OCH2CH2CH2), 62.2, 74.6, 76.2, 77.9, 80.3, 80.7, 82.9,
84.9 (8ꢃ d, C-2a, C-3a, C-4a, C-5a, C-2b, C-3b, C-4b, C-
5b), 67.7, 68.6, 69.1, 73.5, 74.8, 74.9, 75.6, 77.3 (8ꢃ t, C-
6a, C-6b, 5ꢃ PhCH2, OCH2CH2CH2), 97.6, 101.5,
102.5 (3ꢃ d, C-1a, C-1b, PhCH), 115.1 (t, CHÄCH2),
126.1, 127.4, 127.5, 127.7, 127.8, 127.8, 127.9, 128.0,
/5.08 (2 H,
/
/
/
/
/
/
/
/
/
/
/
/
2b), 79.7 (d, C-4a), 81.3 (d, C-3b), 96.9 (d, C-1b), 99.4 (d,
C-1a), 102.4 (d, PhCH), 126.2, 126.4, 127.1, 127.3, 127.4,
127.5, 127.6, 127.8, 127.9, 128.1, 128.2, 128.2, 128.3,
/
/
/
/
128.4, 129.2 (15ꢃ
/
d, ArÃ
/
CH), 137.4, 137.9, 138.0,
C); m/z (ESꢀ) 990 (55),
138.2, 138.5, 138.7 (6ꢃ
/
s, ArÃ
/
128.1, 128.1, 128.1, 128.2, 128.3, 128.3, 128.9, (15ꢃ
30ꢃ ArÃCH), 137.4, 138.2, 138.4, 138.7, 138.7 (5ꢃ
6ꢃ ArÃC), 137.9 (d, CHÄ
(Mꢀ
Naꢀ, 15), 966 (MꢀNHꢀ4 ; 28); 307 (39), 121
(100%), (APCIꢂ) 983 (MꢀClꢂ, 100), 893 (12), 857
(MꢂBn, 13), 317 (22%).
/
d,
984 (
MꢀNHꢀ4 ; 100%), HRMS Calcd. for C59H70O12N
/
/
/
/
s,
(MNHꢀ4 ) 984.4898. Found 984.4915.
/
/
/
CH2); m/z (APCIꢀ) 971
/
3.10. 5?-Hydroxypentyl 2,3,4,6-tetra-O-benzyl-a-D-
/
glucopyranosyl-(10
/
3)-2-O-benzyl-4,6-O-benzylidene-a-
/
D-glucopyranoside (13b)
3.9. 5?-Hydroxypentyl 2,3,4,6-tetra-O-benzyl-a-
glucopyranosyl-(103)-2-O-benzyl-4,6-O-benzylidene-a-
-mannopyranoside (13a)
D
-
Disaccharide 12a (39 mg, 0.041 mmol) was dissolved in
toluene (2 mL) and 9-BBN (0.5 M solution in THF)
(0.66 mL, 0.33 mmol) was added. The reaction mixture
was heated to 50 8C and stirred under Ar. After 18 h, the
reaction mixture was cooled to rt and ice-cold water (1
mL) was added. Then aq H2O2 (35% v/v, 0.5 mL) and aq
/
D
Disaccharide 7 (36 mg, 0.039 mmol) was dissolved in
toluene (2 mL) and 9-BBN (0.5 M solution in THF, 0.62

1946
I. Cumpstey et al. / Carbohydrate Research 338 (2003) 1937ꢁ1949
/
NaOH (2 M, 0.75 mL) were added dropwise. The
reaction mixture was stirred for a further 4 h at which
residue was dissolved in DMF (0.5 mL), and NaN3 (6.5
mg, 0.099 mmol) added. After 48 h, TLC (cyclohexaneꢁ
/
point TLC (cyclohexaneꢁ
/
EtOAc, 1:1) indicated no
EtOAc, 1:1) indicated the absence of starting material
(Rf 0.5) and the formation of a major product (Rf 0.7).
The residue was concentrated in vacuo and purified by
further consumption of starting material (Rf 0.7) and
the formation of a major product (Rf 0.3). The mixture
was dissolved in ether (30 mL) and washed with water
(30 mL). The aqueous layer was re-extracted with ether
(30 mL) and the combined organics dried (MgSO4),
filtered and concentrated in vacuo. The residue was
purified by flash column chromatography (cy-
flash column chromatography (cyclohexaneꢁ
3:1) to afford azide 14a (27 mg, 81%) as a clear oil;
[a]2D5
64.3 (c, 1.52 in CHCl3); nmax (thin film) 2090 (N3)
cmꢂ1; dH (400 MHz, CDCl3) 1.43 (2 H, m, OCH2CH2-
CH2), 1.57ꢁ1.62 (4 H, m, OCH2CH2CH2CH2), 3.24ꢁ
/
EtOAc,
ꢀ
/
/
/
clohexaneꢁ
60%) as a clear oil; [a]D25
(thin film) 3418 (br, OH/NH) cmꢂ1; dH (400 MHz,
CDCl3) 1.48ꢁ1.53 (2 H, m, OCH2CH2CH2), 1.58ꢁ1.66,
1.67ꢁ1.74 (4 H, 2ꢃ m, OCH2CH2CH2CH2), 3.39ꢁ3.51
(4 H, m, H-2b, CH2CHH?O, CH2CH2O), 3.60ꢁ3.67 (4
H, m, H-2a, H-4b, H-6b, H-6?b); 3.70ꢁ3.77 (2 H, m,
CH2CHH?O, H-6a), 3.80 (1 H, at, J 9.5 Hz, H-4a), 3.90
(1 H, dd, J5,6 4.8 Hz, J 9.6 Hz, H-5a), 3.97 (1 H, at, J 9.3
Hz, H-3b), 4.21 (1 H, m, H-5b), 4.25 (1 H, dd, J6,6? 10.2
Hz, H-6a?); 4.30 (1 H, d, J 12.2 Hz, PhCHH?), 4.34 (1 H,
d, J 12.4 Hz, PhCHH?), 4.39, 4.79 (2 H, ABq, JAB 11.0
/
EtOAc, 3:1) to afford alcohol 13b (24 mg,
3.28 (2 H, at, J 6.8 Hz, CH2N3), 3.37 (1 H, m,
OCHH?CH2), 3.54 (1 H, dd, J1,2 3.5 Hz, J2,3 9.7 Hz,
ꢀ
/
58.9 (c, 3.0 in CHCl3); nmax
H-2b), 3.59ꢁ
OCHH?CH2), 3.81ꢁ
H-6a), 4.01 (1 H, at, J 9.3 Hz, H-3b), 4.22 (1 H, d, J5,6?
5.4 Hz, H-6?a); 4.31ꢁ4.33 (2 H, m, H-3a, H-4a) 4.33, 4.58
/
3.72 (4 H, m, H-4b, H-6b, H-6b?;
/
/
/
3.87 (4 H, m, H-5b, H-5a, H-2a,
/
/
/
/
/
/
(2 H, ABq, JAB 12.2 Hz, PhCH2), 4.44, 4.61 (2 H, ABq,
JAB 12.0 Hz, PhCH2), 4.44, 4.86 (2 H, ABq, JAB 10.8
Hz, PhCH2), 4.75, 4.98 (2 H, ABq, JAB 11.0 Hz,
PhCH2), 4.78, 4.95 (2 H, ABq, JAB 12.4 Hz, PhCH2),
4.82 (1 H, d, J1,2 1.6 Hz, H-1a), 5.48 (1 H, s, PhCH),
5.56 (1 H, d, H-1b), 7.01 (2 H, ad, J 6.7 Hz, ArÃ
7.14ꢁ7.37 (26 H, m, ArÃH), 7.47 (2 H, ad, J 6.7 Hz,
ArÃH); dC (100.6 MHz, CDCl3) 23.3 (t, OCH2CH2-
CH2), 28.6, 28.9 (2ꢃ t, OCH2CH2CH2CH2), 51.2, 67.5
(2ꢃ t, CH2N3, CH2CH2O), 64.1 (d, C-5a), 68.5, 68.9
(2ꢃ t, C-6b, C-6a), 70.6, 73.3, 73.8, 74.9, 75.5 (5ꢃ t,
5ꢃ
(2ꢃ
/
H),
Hz, PhCH2), 4.41 (1 H, at, J 9.3 Hz, H-3a), 4.56ꢁ
/4.61 (3
/
/
H, m, 3ꢃ PhCHH?), 4.64 (1 H, d, J 11.1 Hz, PhCHH?),
/
/
4.81, 5.01 (2 H, ABq, JAB 10.8 Hz, PhCH2), 4.86 (1 H, d,
J1,2 3.5 Hz, H-1a), 5.49 (1 H, s, PhCH), 5.59 (1 H, d, J1,2
/
/
3.6 Hz, H-1b), 6.92ꢁ
7.08ꢁ7.43 (28 H, m, ArÃ
22.4 (t, OCH2CH2CH2), 29.0, 32.4 (2ꢃ
CH2CH2CH2), 61.9 (d, C-5a), 62.6 (t, C-6b), 68.0,
68.4, 69.2 (3ꢃ t, C-6a, 2ꢃ CH2CH2O), 69.6 (d, C-
5b), 71.0 (t, PhCH2), 72.7 (d, C-3a), 72.9, 73.2, 74.7, 75.5
(4ꢃ t, 4ꢃ PhCH2), 77.5, 78.1 (2ꢃ d, C-2a, C-4b), 78.7
/
6.94 (2 H, ad, J 7.1 Hz, ArÃ
H); dC (100.6 MHz, CDCl3)
t, OCH2-
/
H),
/
/
/
/
/
CH2Ar), 70.8 (d, C-5b), 72.8 (d, C-3a), 77.3, 77.5
/
/
d, C-2a, C-4b), 78.9 (d, C-2b), 79.7 (d, C-4a), 81.3
(d, C-3b), 96.9 (d, C-1b), 99.5 (d, C-1a), 102.3 (d, PhCH),
126.4, 127.1, 127.2, 127.5, 127.5, 127.7, 127.8, 127.8,
/
/
127.9, 128.1, 128.2, 128.3, 128.4, 129.2 (14ꢃ
CH), 137.9, 137.9, 138.2, 138.5, 138.7 (5ꢃ s, ArÃ
Naꢀ, 55), 1009 (Mꢀ
/
d, ArÃ
/
/
/
/
/
/
C);
(d, C-2b), 81.7 (d, C-3b), 82.9 (d, C-4a), 96.1 (d, C-1b),
97.4 (d, C-1a), 102.1 (s, CHAr), 126.4, 127.3, 127.4,
127.5. 127.6, 127.9, 128.0, 128.1, 128.2, 128.3, 128.4,
m/z (ESꢀ) 1037 (30), 1013 (Mꢀ
/
NHꢀ4 ; 18); 153 (100%); Isotope distribution Calcd. for
C59H65N3NaO11 (MNaꢀ) 1017.5 (7), 1016.5 (25), 1015.5
(68), 1014.5 (100%). Found 1017.5 (5), 1016.6 (24),
1015.5 (71), 1014.5 (100%).
128.5, 129.4 (16ꢃ
138.1, 138.8, 138.9 (6ꢃ
Naꢀ, 100), 984 (MꢀNHꢀ4 ; 75%); HRMS Calcd. for
C59H70NO12 (MNHꢀ4 ) 984.4898. Found 984.4902.
/
d, CHÃ
/
Ar), 137.1, 137.6, 137.8,
/
s, ArÃ
/
C); m/z (ESꢀ) 989 (Mꢀ
/
/
3.12. 5?-Azidopentyl 2,3,4,6-tetra-O-benzyl-a-
glucopyranosyl-(103)-2-O-benzyl-4,6-O-benzylidene-a-
D-glucopyranoside (14b)
D-
/
3.11. 5?-Azidopentyl 2,3,4,6-tetra-O-benzyl-a-
glucopyranosyl-(103)-2-O-benzyl-4,6-O-benzylidene-a-
-mannopyranoside (14a)
D-
/
D
Methanesulfonyl chloride (3 mL, 0.035 mmol) was added
dropwise to a stirred solution of alcohol 13b (28 mg,
0.029 mmol) and Et3N (6 mL, 0.044 mmol) in an DCM
(1 mL) at 0 8C under Ar. After 20 min, TLC (cy-
Methanesulfonyl chloride (3 mL, 0.040 mmol) was added
dropwise to a stirred solution of alcohol 13a (32 mg,
0.033 mmol) and Et3N (7 mL, 0.050 mmol) in an DCM
(1 mL) at 0 8C under Ar. After 20 min, TLC (cy-
clohexaneꢁEtOAc, 1:1) indicated the absence of starting
/
material (Rf 0.2) and the formation of a major product
(Rf 0.3). The reaction mixture was diluted with DCM
(20 mL) and washed with distilled water (20 mL), 1 M
HCl (20 mL), NaHCO3 (20 mL of a saturated aqueous
solution) and brine (20 mL). The organic phase was
dried (MgSO4), filtered and concentrated in vacuo. The
residue was dissolved in DMF (0.5 mL), and NaN3 (6
clohexaneꢁEtOAc, 1:1) indicated the absence of starting
/
material (Rf 0.4) and the formation of a major product
(Rf 0.5). The reaction mixture was diluted with DCM
(40 mL) and washed with distilled water (40 mL), 1 M
HCl (40 mL), NaHCO3 (40 mL of a saturated aqueous
solution) and brine (40 mL). The organic phase was
dried (MgSO4), filtered and concentrated in vacuo. The
mg, 0.087 mmol) added. After 48 h, TLC (cyclohexaneꢁ
/

I. Cumpstey et al. / Carbohydrate Research 338 (2003) 1937ꢁ
/1949
1947
EtOAc, 1:1) indicated the absence of starting material
(Rf 0.3) and the formation of a major product (Rf 0.7).
The residue was concentrated in vacuo and purified by
1.64 (4 H, m, OCH2CH2CH2CH2), 2.16ꢁ
NC(O)CH2CH2), 2.36ꢁ
NC(O)CH2), 3.19ꢁ3.23 (2 H, m, CH2N), 3.25 (1 H,
dd, J 10.0 Hz, J 9.0 Hz), 3.37ꢁ3.45 (4 H, m, H-2b,
CHH?O, CH2Ar), 3.58 (1 H, ddd, J 2.6 Hz, J 5.7 Hz, J
8.1 Hz), 3.65 (1 H, dd, J 6.4 Hz, J 11.6 Hz), 3.69ꢁ3.76 (3
H, m, CHH?O, H-3b), 3.78 (1 H, dd, J2,3 3.2 Hz, J3,4 9.6
Hz, H-3a), 3.82ꢁ3.87 (3 H, m), 3.89 (1 H, m), 4.07 (1 H,
dd, J1,2 1.9 Hz, H-2a), 4.73 (1 H, d, H-1a), 5.09 (1 H, d,
J1,2 4.3 Hz, H-1b), 7.92 (1 H, d, J 7.8 Hz, ArÃH), 8.01 (1
H, t, J 7.7 Hz, ArÃH), 8.05, 8.06 (2 H, 2ꢃ s, ArÃH),
8.15ꢁ8.22 (4 H, m, ArÃH), 8.36 (1 H, d, J 9.5 Hz, Ar);
dC (100.6 MHz, CD3OD) 23.8 (t, OCH2CH2CH2), 28.1,
29.2 (2ꢃ t, NC(O)CH2CH2, OCH2CH2CH2CH2), 32.8
(t, CH2Ar), 35.8 (t, NC(O)CH2), 39.3 (t, CH2N), 61.7,
61.8 (2ꢃ t, C-6b, C-6a), 67.5 (t, CH2CH2O), 66.4, 70.4,
70.8, 73.0, 73.1, 73.6, 74.2 (7ꢃ d, C-2b, C-3b, C-4b, C-
/
2.19 (2 H, m,
/
2.39 (2 H, at, J 7.3 Hz,
/
flash column chromatography (cyclohexaneꢁ
3:1) to afford azide 14b (23 mg, 80%) as a clear oil;
[a]2D5
64.9 (c, 2.32 in CHCl3); nmax (thin film) 2096 (N3)
cmꢂ1; dH (400 MHz, CDCl3) 1.47ꢁ
1.53 (2 H, m,
OCH2CH2CH2), 1.62ꢁ1.71 (4 H, m, OCH2CH2-
CH2CH2), 3.26 (2 H, at, J 6.8 Hz, CH2N3), 3.39ꢁ3.47
(3 H, m, H-6b, H-6b?; OCHH?CH2), 3.49 (1 H, dd, J1,2
3.7 Hz, J2,3 9.7 Hz, H-2b), 3.61ꢁ3.68 (2 H, m, H-2a, H-
4b), 3.70ꢁ3.76 (2 H, m, H-6a OCHH?CH2), 3.80 (1 H, at,
J 9.3 Hz, H-4a), 3.89 (1 H, dd, J5,6 4.5 Hz, H-5a), 3.97 (1
H, at, J 9.3 Hz, H-3b), 4.20ꢁ4.27 (2 H, m, H-5b, H-6?a);
4.29, 4.59 (2 H, ABq, JAB 12.0 Hz, PhCH2), 4.34, 4.57 (2
H, ABq, JAB 12.3 Hz, PhCH2), 4.36ꢁ4.43 (2 H, m, H-3a,
/EtOAc,
/
ꢀ
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
PhCHH?), 4.57, 4.63 (2 H, ABq, JAB 11.2 Hz, PhCH2),
4.78 (1 H, d, J 11.2 Hz, PhCHH?), 4.81, 5.01 (2 H, ABq,
JAB 10.8 Hz, PhCH2), 4.87 (1 H, d, J1,2 3.7 Hz, H-1a),
5.48 (1 H, s, PhCH), 5.59 (1 H, d, H-1b), 6.92 (2 H, ad, J
/
5b, C-3a, C-4a, C-5a), 81.2 (d, C-2a),100.6 (d, C-1a), 101.1
(d, C-1b), 123.5, 124.9, 125.0, 126.0, 126.7, 127.4, 127.6
(7ꢃ
174.8 (7ꢃ
HRMS Calcd. for C37H46NO12 (Mꢂ
Found 696.3038.
/
d, ArÃ
/
CH), 135.1, 128.9, 130.4, 131.3, 131.8, 136.4,
C); m/z (ESꢂ) 696.46 (MꢂHꢀ, 52%);
Hꢀ) 696.3020.
7.0 Hz, ArÃ
MHz, CDCl3) 21.0 (t, OCH2CH2CH2), 28.6, 28.9 (2ꢃ
t, OCH2CH2CH2CH2), 62.0 (d, C-5a), 51.3, 68.0, 68.2
(3ꢃ t, C-6b, OCH2CH2, CH2N3), 69.2 (t, C-6a), 69.6 (d,
C-5b), 71.1, 72.9, 73.2, 74.7, 75.5 (5ꢃ t, 5ꢃ PhCH2),
/
H), 7.09ꢁ
/
7.41 (28 H, m, ArÃ
/
H); dC (100.6
/
s, ArÃ
/
/
/
/
/
/
/
3.14. 5?-(1ƒ-Pyr-1§enylbutyramido)pentyl a-
D-
72.7 (d, C-3a), 76.7 (d, C-4b), 77.0 (d, C-2a), 78.1 (d, C-
2b), 81.7 (d, C-3b), 82.9 (d, C-4a), 96.1 (d, C-1b), 97.4 (d,
C-1a), 102.1 (d, PhCH), 126.4, 127.3, 127.4, 127.5, 127.6,
glucopyranosyl-(103)-a- -glucopyranoside (15b)
/
D
Azide 14b (47 mg, 0.047 mmol) and palladium on
carbon (10% Pd/C, 60 mg) were stirred in MeOHꢁ
127.9, 128.0, 128.1, 128.2, 128.3, 128.4, 129.4 (12ꢃ
ArÃCH), 137.1, 137.6, 137.8, 138.1, 138.8, 138.9 (6ꢃ
ArÃ
/
d,
s,
/
/
/
AcOH (99:1, 4 mL) under an atmosphere of hydrogen.
After 63 h, TLC (CMAW) indicated complete conver-
sion of starting material (Rf 0.95) to a single product (Rf
0.05). The mixture was filtered through Celite† (MeOH)
and concentrated in vacuo. The residue was dissolved in
DMF (2 mL) and PFP ester 16 (23 mg, 0.047 mmol)
added. After 28 h Et3N (65 mL, 0.474 mmol) was added.
After a further 16 h TLC (CMAW) indicated complete
conversion of starting material (Rf 0.05) to a major
product (Rf 0.2). The reaction mixture was concentrated
in vacuo and purified by flash column chromatography
/
C); m/z (ESꢀ) 1036 (65), 1009 (MꢀNHꢀ4 ; 100%);
Isotope distribution Calcd. for C59H65N3NaO11
(MNaꢀ) 1017.5 (7), 1016.5 (25), 1015.5 (68), 1014.5
(100%). Found 1017.5 (6), 1016.5 (21), 1015.5 (68),
1014.5 (100%).
3.13. 5?-(1ƒ-Pyr-1§enylbutyramido)pentyl a-
D-
glucopyranosyl-(103)-a- -mannopyranoside (15a)
/
D
Azide 14a (29 mg, 0.029 mmol) and palladium on
carbon (10% Pd/C, 45 mg) were stirred in MeOHꢁ
/
(MeOHꢁ
(26 mg, 79%) as a clear oil; [a]D25
MeOH); nmax (thin film) 3359 (br, OH), 1652 (CÄ
cmꢂ1; dH (400 MHz, CD3OD) 1.40ꢁ
1.46 (2 H, m,
OCH2CH2CH2), 1.49ꢁ1.55, 1.60ꢁ1.67 (4 H, 2ꢃ m,
OCH2CH2CH2CH2), 2.14 (2 H, at, 7.7 Hz,
NC(O)CH2CH2), 2.36 (2 H, at, J 7.1 Hz, NC(O)CH2),
3.19ꢁ3.21 (2 H, m, CH2N), 3.27 (1 H, dd, J 10.1 Hz, J
8.9 Hz), 3.36 (2 H, s, PhCH2), 3.43ꢁ3.55 (4 H, m, H-2b,
H-2a, OCHH?CH2), 3.58 (1 H, dd, J 2.1 Hz, J 5.2 Hz),
3.62ꢁ3.77 (5 H, m, OCHH?CH2), 3.79 (1 H, dd, J 11.9
/
CHCl3, 1:4) to afford labelled disaccharide 15b
73.0 (c, 1.45 in
O)
AcOH (99:1, 3 mL) under an atmosphere of hydrogen.
After 46 h, TLC (CMAW) indicated complete conver-
sion of starting material (Rf 0.95) to a single product (Rf
0.05). The mixture was filtered through Celite† (MeOH)
and concentrated in vacuo. The residue was dissolved in
DMF (1 mL) and PFP ester 16 (14 mg, 0.047 mmol) and
Et3N (41 mL, 0.293 mmol) were added. After 23 h TLC
(CMAW) indicated complete conversion of starting
material (Rf 0.05) to a major product (Rf 0.2). The
reaction mixture was concentrated in vacuo and purified
ꢀ
/
/
/
/
/
/
J
/
/
/
by flash column chromatography (MeOHꢁ
to afford labelled disaccharide 15a (9 mg, 44%) as a clear
oil; [a]2D5
46.0 (c, 0.4 in MeOH); nmax (thin film) 3349
(br, OH), 1642 (CÄ
O) cmꢂ1; dH (400 MHz, CD3OD)
1.41ꢁ1.46 (2 H, m, OCH2CH2CH2), 1.51ꢁ1.55, 1.60ꢁ
/
CHCl3, 1:4)
Hz, J 2.0 Hz), 3.86 (1 H, dd, J 2.2 Hz, J 11.6 Hz), 3.95
(1 H, m), 4.79 (1 H, d, J1,2 4.0 Hz, H-1a), 5.15 (1 H, d,
ꢀ
/
J1,2 3.7 Hz, H-1b), 7.87 (1 H, d, J 7.8 Hz, ArÃ
8.02 (3 H, m, ArÃH), 8.12ꢁ8.18 (4 H, m, ArÃH), 8.31 (1
H, d, J 9.2 Hz, ArÃH); dC (100.6 MHz, CD3OD) 25.1 (t,
/
H), 7.96ꢁ
/
/
/
/
/
/
/
/
/

1948
I. Cumpstey et al. / Carbohydrate Research 338 (2003) 1937ꢁ1949
/
OCH2CH2CH2), 29.6 (t, NC(O)CH2CH2), 30.6 (2ꢃ
OCH2CH2CH2CH2), 34.2 (t, CH2Ar), 37.2 (t,
NC(O)CH2), 40.7 (t, CH2N), 62.9, 63.2 (2ꢃ t, C-6b,
C-6a), 69.4 (t, OCH2CH2), 71.9, 72.2, 72.6, 73.8, 74.4,
74.6, 75.6, 85.3 (8ꢃ d, C-2b, C-3b, C-4b, C-5b, C-2a, C-
3a, C-4a, C-5a), 100.6, 102.2 (2ꢃ d, C-1b, C-1a), 124.8,
126.2, 126.4, 127.4, 128.1, 128.8, 128.9, 129.0 (8ꢃ
ArÃCH), 130.3, 131.7, 132.7, 133.2, 137.8, 176.2 (6ꢃ
ArÃ
C); m/z (ESꢀ) 720 (MꢀNaꢀ, 12), 698 (Mꢀ
/
t,
David Neville for his advice and use of HPLC equip-
ment.
/
/
References
/
/
d,
s,
1. Kornfield, R.; Kornfield, S. Annu. Rev. Biochem. 1985, 54,
/
/
631ꢁ
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/
664.
/
/
/
Hꢀ,
3), 152.91 (100%); HRMS Calcd. for C37H47NNaO12
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/
3. Pelletier, M. F.; Marcil, A.; Sevigny, G.; Jakob, C. A.;
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827.
3.15. a-Glucosidase hydrolysis of pyrene labelled
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/
38;
(b) Rodan, A. R.; Simons, J. F.; Trombetta, S. E.;
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as described.20 Rat liver a-glucosidase II was extracted
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strate. Rates of hydrolysis were similar to previously
published data21 and no cross contamination of gluco-
sidase activity could be detected. Pyrene disaccharides
were solubilised in MeOH and a 1 mL aliquot (2 mM
final concentration) taken for hydrolysis in a final
volume of 20 mL of a-glucosidase I in 0.1 M sodium
phosphate buffer, pH 6.8 containing 0.8% Lubrol PX
and 1 mg/mL bovine serum albumin, or 20 mL of a-
glucosidase II in 80 mM Et3N buffer, pH 7 containing
0.5% Triton X-100, 0.1 M NaCl, 10% glycerol and 1 mg/
mL bovine serum albumin. Control digestions were
perormed using glucosidase buffers and disaccharides
alone. Following incubation at 37 8C for 1 h, MeCN (80
mL) was added and protein removed by filtration using
Millipore 5000 NMWL Ultrafree filters. Aliquots were
analysed by HPLC using a Waters Alliance 2695
separations module and an in-line fluorescence detector
set at Ex l 345, Ex l 380 nm. Fluorescently derivatised
disaccharides were separated isocratically at 0.5 mL/min
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/
5. Tessier, D. C.; Dignadr, D.; Zapun, A.; Radominska, A.;
Parodi, A. J.; Bergeron, J. J. M.; Thomas, D. Y.
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6. Lellgaard, L.; Molinari, M.; Helenius, A. Science 1999,
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7. Alonso, J. M.; Santa-Cecilia, A.; Calvo, P. Biochem. J.
1991, 278, 721ꢁ727.
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412.
/
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8. Petrescu, A. J.; Butters, T. D.; Reinkensmeier, G.;
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/
9. Shibaev, V. N.; Veselovsky, V. V.; Lozanova, A. V.;
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Cheung, H. C.; Jedrzejas, M. J. Bioorg. Med. Chem. Lett.
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(c) Watanabe, T.; Inoue, N.; Kutsukake, T.; Matsuki, S.;
Takeuchi, M. Biol. Pharm. Bull. 2000, 23, 269ꢁ
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192.
/
/
/
273.
11. For some examples of the use of n-pentenyl glycosides for
the attachment of linkers or labels see:(a) Iyer, S.S.; Rele,
S.M.; Baskaran, S.; Chaikof, E.L. Tetrahedron, 2003, 59,
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/
638;
(b) Buskas, T.; Soderberg, E.; Konradsson, P.; Fraser-
¨
Reid, B. J. Org. Chem., 2000, 65, 958ꢁ963.
/
12. France, R. R.; Cumpstey, I.; Butters, T. D.; Fairbanks, A.
J.; Wormald, M. R. Tetrahedron: Asymmetry 2000, 11,
on a 4.6ꢃ
/
250 mm TSK gel amide-80 column at 30 8C
using a solvent composed of 80% MeCN, 20% ammo-
4985ꢁ4994.
/
13. For the synthesis of a fluorescence labelled substrate for
glucosidase I see: Scaman, C. H.; Hindsgaul, O.; Palcic, M.
nium acetate (100 mM), pH 3.85.
M.; Srivastava, O. P. Carbohydr. Res. 1996, 296, 203ꢁ
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/
Acknowledgements
/
We gratefully acknowledge financial support from the
Leverhulme Trust (Postdoctoral Fellowship to R.J.T.E.)
and the use of the EPSRC Mass Spectrometry Service
(Swansea, UK) and the Chemical Database Service
(CDS) at Daresbury, UK. We are also grateful to Dr
/
17. Fraser-Reid, B.; Udodong, U. E.; Wu, Z.; Ottosson, H.;
Merritt, J. R.; Rao, C. S.; Roberts, C.; Madsen, R. Synlett
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I. Cumpstey et al. / Carbohydrate Research 338 (2003) 1937ꢁ
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1949
18. Madsen, R.; Udodong, U. E.; Roberts, C.; Mootoo, D.
R.; Konradsson, P.; Fraser-Reid, B. J. Am. Chem. Soc.
20. Butters, T. D.; van den Broek, L. A. G. M.; Fleet, G. W.
J.; Krulle, T. M.; Wormald, M. R.; Dwek, R. A.; Platt, F.
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19. Andrews, C. W.; Rodebaugh, R.; Fraser-Reid, B. J. Org.
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/