Carbasugar-thioether pseudodisaccharides related to N-glycan biosynthesis

Article abstract of DOI:10.1016/j.carres.2008.12.023

Analogues of the α-Glcp-(1→3)-α-Glcp and α-Glcp-(1→3)-α-Manp disaccharides (representing the two α-gluco linkages cleaved by α-Glucosidase II in N-glycan biosynthesis) in which the non-reducing-end sugar is replaced by a carbasugar and the inter-glycosidi

Full text of DOI:10.1016/j.carres.2008.12.023

Carbohydrate Research 344 (2009) 454–459
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Carbasugar–thioether pseudodisaccharides related to N-glycan biosynthesis
b
b
Ian Cumpstey a, , Dominic S. Alonzi , Terry D. Butters
*
a Department of Organic Chemistry, Stockholm University, Arrhenius Laboratory, 106 91 Stockholm, Sweden
b Department of Biochemistry, Oxford Glycobiology Institute, University of Oxford, South Parks Road, Oxford OX1 3QU, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Analogues of the
a
-Glcp-(1?3)-
a-Glcp and a-Glcp-(1?3)-a-Manp disaccharides (representing the two
Received 27 November 2008
Received in revised form 16 December 2008
Accepted 24 December 2008
Available online 3 January 2009
a
-gluco linkages cleaved by
a
-Glucosidase II in N-glycan biosynthesis) in which the non-reducing-end
sugar is replaced by a carbasugar and the inter-glycosidic oxygen by a sulfur were synthesised. The
key coupling step was an SN2 displacement of an equatorial triflate at C-1 of the carbasugar by C-3 gluco
or manno thiolates with inversion of configuration to give thioether pseudodisaccharides with axial sub-
stitution at C-1 of the carbasugar. The deprotected pseudodisaccharides failed to inhibit the action of
Keywords:
Thioethers
Pseudodisaccharides
Carbasugars
a
-Glucosidase II as measured both by an in vitro assay and by free oligosaccharide (FOS) analysis from
cell studies.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
glucose ring-oxygen with a methylene group to give a carbasug-
ar8,9 renders the pseudodisaccharide stable to the hydrolytic action
of the enzyme. In this paper, we report our results on the synthesis
of thioether-linked carbasugar pseudodisaccharides 1 and 2 mim-
During N-glycan biosynthesis, a Glc3Man9GlcNAc2 tetradeca-
saccharide is assembled with a dolichol phosphate aglycon and
transferred to newly synthesised polypeptides in the endoplasmic
reticulum before completion of folding to give the finished glyco-
proteins.1 After glycosyl transfer to the protein, the oligosaccharide
is remodelled by glycosidases and glycosyltransferases to give high
mannose, complex and hybrid structures, this process begins with
sequential cleavage of the terminal glucose residues by the two
icking the two linkages cleaved by a-Glucosidase II (Fig. 1b and c).
1-Thio-carbasugars have been shown to inhibit enzymes be-
fore.10–13 Only one example of a thioether-linked saturated carba-
sugar pseudodisaccharide appears to be known, mimicking a-Glcp-
(1?4)-Glcp.14 It acts as a glycosidase inhibitor, albeit a weak one.
It has been shown that the 1-amino-carbasugars valienamine
a
a
-Glucosidases I and II (Fig. 1a). The transient presence of the three
-glucosides has been proposed to be important for glycosyl trans-
(C-5@C-5a unsaturated) and validamine (saturated) inhibit
a-Glu-
cosidases I and II.15 A valienamine-based mimic of
a
-Glcp-(1?2)-
fer by oligosaccharyltransferase (OST) complex,2 and also the
monoglucosylated oligosaccharide is bound by the lectin chaper-
ones Calnexin and Calreticulin in a quality control process to en-
sure correct protein folding.3 A recently described lectin with
high selectivity for the diglucosylated oligosaccharide may also
be relevant in N-glycan biosynthesis.4
Glcp failed to inhibit
a
-Glucosidase I.16 A mimic of the
a
-Glcp-
α-Glucosidase II
OH
O
HO
HO
HO
HO
O
O
O
HO
It has been proposed that
tive sites, each responsible for the cleavage of one of the
(1?3)- -Glcp or -Glcp-(1?3)-
-Manp linkages.5 A difference in
a-Glucosidase II has two different ac-
HO
O
OR
a-Glcp-
O
HO
HO
HO
HO
HO
HO
a
a
a
the rate of cleavage of these two glucose residues suggests that
the enzyme recognises a larger part of the structure than just the
terminal monosaccharide. The two disaccharides are substrates
O
α-Glucosidase I
for the enzyme,6 and in designing inhibitors for
a-Glucosidase II,
OH
HO
HO
HO
O
HO
HO
HO
disaccharide-mimicking structures may give more enzyme-specific
inhibition than a monosaccharide-mimicking inhibitor, such as
N-butyl deoxynojirimycin (NB-DNJ).7 Formal replacement of the
S
OMe
HO
HO
OMe
HO
S
HO
2
1
HO
HO
O
HO
* Corresponding author. Tel.: +46 8 16 2481; fax: +46 8 15 4908.
E-mail address: cumpstey@organ.su.se (I. Cumpstey).
Figure 1.
0008-6215/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2008.12.023

I. Cumpstey et al. / Carbohydrate Research 344 (2009) 454–459
455
OH
OH
OTf
H2, Pd/C
BnO
BnO
BnO
BnO
BnO
BnO
Tf2O, py
OBn
OBn
OBn
EtOAc, Et3N
3 / 19%
+ ido 78%
CH2Cl2, 0 oC
OBn
OBn
OBn
4
3
7
TBDMSCl,
im, DMAP,
CH2Cl2, DMF,
95%
TBAF, THF
3 / 72%
+ ido 19%
OTBDMS
H2, Pd/C
OTBDMS
OBn
BnO
BnO
BnO
BnO
OBn
EtOAc, Et3N
OBn
OBn
5
6
Scheme 1.
(1?3)-Glcp disaccharide in which the non-reducing-end glucose is
carbasugar in the thioether-forming reaction.
replaced by an inositol and with an inter-residue amine bridge has
Deprotection of the pseudodisaccharides was carried out under
Birch conditions: the presence of the sulfur ruled out catalytic
hydrogenolysis for benzyl ether deprotection, but the Birch reduc-
tion worked well, and peracetylation facilitated purification of the
pseudodisaccharides 14 and 15. Deacetylation with sodium meth-
oxide then gave the free pseudodisaccharides 1 and 2 (Scheme 3).
been synthesised, but this compound failed to inhibit
a-Glucosi-
dase II.17,18
2. Results and discussion
Both pseudodisaccharides were non-inhibitory to
a-Glucosi-
Our synthetic strategy for pseudodisaccharide formation was
based on thioetherification under conditions favouring SN2 dis-
placement of an equatorial triflate at C-1 of a carbasugar by gluco
and manno C-3 thiolates to ensure formation of the axial thioether
linkages.19 Similar SN2 displacement of carbohydrate triflates by
anomeric thiolates is a well-known method for the synthesis of thi-
ooligosaccharides.20–22 The synthesis of the carbaglucose alcohol 3
has been described,11 but here we detail an alternative route from
dase II activity at concentrations of 100 M when measured using
l
both Glc1Man7GlcNAc2 and Glc2Man7GlcNAc2 oligosaccharides
labelled with anthranilic acid (2AA) in vitro. In contrast, NB-DNJ
had an IC50 of 9.7 0.7
lM and an IC90 of 110 0.9
lM for
Glc1Man7GlcNAc2, and was totally inhibitory at 100
l
M for
a-Glu-
cosidase-mediated hydrolysis of Glc2Man7GlcNAc2–2AA. Using
Glc2Man5GlcNAc1–2AA as a substrate, NB-DNJ had an IC50 value
of 10.8 1.13
The pseudodisaccharides were evaluated for
inhibition in HL60 cells for 24 h at a non-cytotoxic concentration
l
M.27
the unsaturated carbocycle 4, which is accessible from L-sorbose by
a ring-closing metathesis approach.23 Hydrogenation of the car-
bon–carbon double bond in 4 gave mainly the ido diastereomer
(ido/gluco, 4:1), but protection with a bulky TBDMS ether blocked
the top face, and hydrogenation of fully protected species 5 re-
versed the diastereoselectivity, and the required gluco diastereo-
mer was the major product (ido/gluco, 1:3). Silyl ether removal
and triflation gave the SN2 electrophile 7 (Scheme 1).
a-Glucosidase II
of 100 lM, and no inhibition was observed using the free oligosac-
charide (FOS) assay.27 NP-HPLC profiles of fluorescently labelled
oligosaccharides were identical to non-inhibitor treated FOS pro-
files (results not shown). The combination of a cell-free and a cel-
lular-based assay provides convincing evidence for the lack of
The required S-3 gluco 8 and manno 924 compounds were ob-
tained by thioacetate displacement of triflates of the OH-3 allo
1025 and altro 1126 alcohols. The moderate yields are explained
by competing elimination reactions (Scheme 2). For the formation
of the gluco compound 8, best results were found using THF as sol-
vent (8, 51%; elimination, 13%), while DMF (8, 30%; elimination,
42%) gave rather worse selectivity.
In contrast, in the formation of the manno compound 9, better
selectivity for substitution was found with DMF as solvent (9,
38%; elimination, 53%)24 than THF (5, 26%; elimination, 60%). The
origin of this difference in selectivity is not clear.
The manno thiol was formed by methanolysis of the thioester
protection in 9 using excess sodium methoxide, and then coupled
with the carbasugar triflate 7 to give the pseudodisaccharide 13.
For the gluco nucleophile, both the 3-thioacetate and the 2-benzo-
ate were removed from 8 with excess methoxide, and after cou-
pling with the carbasugar triflate 7, it was necessary to acetylate
the free OH-2 in the pseudodisaccharide product 12 to allow puri-
fication. Both pseudodisaccharides 12 and 13 were formed as sin-
gle diastereomers with inversion of configuration at C-1 of the
inhibition of a-Glucosidase II by the pseudodisaccharides synthes-
ised in this paper.
To conclude, thioether a-(1–3)-linked carbasugar pseudodisac-
charides are synthesised by an SN2 route by displacement of an
equatorial carbasugar C-1 triflate by carbohydrate thiolates. The
pseudodisaccharides failed to inhibit a-Glucosidase II. It is possible
that better inhibition would be obtained with C-5@C-5a unsatu-
rated carbasugars or analogues where the inter-glycosidic hetero-
atom is nitrogen rather than sulfur.14 Nitrogen may be protonated
when bound to the enzyme, and may bind more tightly than a neu-
tral ligand either by mimicking the charge build-up at the transi-
tion state of the cleavage reaction or because of favourable
electrostatic interactions with carboxylate side-chains in the active
site.28 We hope to report further results of our investigations in
this area shortly.
3. Experimental
3.1. General methods
Melting points were recorded on a Gallenkamp melting point
apparatus and are uncorrected. Proton nuclear magnetic reso-
nance (1H) spectra were recorded on Bruker Avance II 400
Acetonitrile (8, 42%; elimination, 32%) and butanone 80 °C (8, 42%; elimination,
48%) were also tested.

456
I. Cumpstey et al. / Carbohydrate Research 344 (2009) 454–459
BnO
BnO
OTf
BnO
BnO
BnO
i) Tf2O, py,
Ph
O
O
Ph
O
BnO
CH2Cl2, 0 oC;
O
7
O
Ph
O
BnO
O
AcS
O
O
BnO
BzO
S
OMe
BzO
i) NaOMe, MeOH
ii) 7, NaH, DMF, 50 oC
iii) Ac2O, py
10
HO
OMe
ii) KSAc,
THF, RT
AcO
8 / 51%
OMe
12 / 47%
BnO
BnO
BnO
i) Tf2O, py,
OBn
O
OTf
OBn
O
BnO
BnO
BnO
Ph
O
O
CH2Cl2, 0 oC;
OBn
Ph
O
Ph
O
BnO
7
O
O
O
BnO
AcS
S
11
ii) KSAc,
DMF, RT
OMe
OH OMe
i) NaOMe, MeOH;
9 / 38%
OMe
13 / 54%
ii) 7, NaH, DMF, 50 oC
Scheme 2.
i) Na, NH3o(l)
,
BnO
AcO
HO
HO
NaOMe,
MeOH
BnO
AcO
–
THF, 78 C
Ph
O
O
AcO
AcO
HO
BnO
AcO
HO
O
O
O
HO
S
BnO
S
AcO
S
HO
ii) Ac2O, py
AcO
OMe
AcO
HO
12
OMe
OMe
14
/ 83%
1 / 90%
BnO
i) Na, NH3o(l)
,
AcO
HO
HO
NaOMe,
BnO
AcO
–
OBn THF, 78 C
OAc MeOH
O
OH
O
AcO
AcO
HO
Ph
BnO
AcO
HO
O
O
O
HO
BnO
S
AcO
S
HO
S
ii) Ac2O, py
OMe
OMe
OMe
13
15 / 77%
2 / 99%
Scheme 3.
(400 MHz) or 500 (500 MHz) or Varian Mercury 400 (400 MHz)
spectrometers; multiplicities are quoted as singlet (s), broad sin-
glet (br s), doublet (d), doublet of doublets (dd), triplet (t), appar-
ent triplet (at), apparent triplet of doublets (atd), doublet of
apparent triplets (dat), quartet (q), apparent quartet (aq), AB
quartet (ABq) or multiplet (m). Carbon nuclear magnetic reso-
nance (13C) spectra were recorded on Bruker Avance II 400
(100 MHz) or 500 (125 MHz) or Varian Mercury 400 (100 MHz)
spectrometers, and multiplicities assigned by DEPT. Spectra were
assigned using COSY, HSQC and DEPT experiments. All chemical
shifts are quoted on the d scale in parts per million (ppm). Resid-
ual solvent signals were used as an internal reference (CDCl3:
7.26 and 77.16 ppm); spectra in D2O were calibrated with either
MeOH (3.34 and 49.50 ppm) or AcOH (2.08 and 21.03 ppm). Low-
and high-resolution (HRMS) electrospray (ESI) mass spectra were
recorded using a Bruker Microtof instrument. Infra-red spectra
were recorded on a Perkin–Elmer Spectrum One FT-IR spectrom-
eter using the thin film method on a NaCl plate. Optical rotations
were measured on a Perkin–Elmer 241 polarimeter with a path
length of 1 dm; concentrations are given in g/100 mL. Thin layer
chromatography (TLC) was carried out on Merck Kieselgel sheets,
pre-coated with 60F254 silica. Plates were visualised with UV light
and developed using 10% sulfuric acid, or an ammonium molyb-
date (10% w/v) and cerium (IV) sulfate (2% w/v) solution in 10%
sulfuric acid. Flash column chromatography was carried out on
3.2. 2,3,4,6-Tetra-O-benzyl-1-O-(tert-butyldimethylsilyl)-5a-
carba-b- -xylo-hex-5(5a)-enopyranose (5)
D
Alcohol 4 (278 mg, 0.52 mmol) was dissolved in a mixture of
CH2Cl2 (5 mL) and DMF (5 mL). Imidazole (106 mg, 1.56 mmol)
and TBDMSCl (233 mg, 1.55 mmol) were added, and the mixture
stirred at rt. After 20 h, TLC (pentane–EtOAc 3:1) showed forma-
tion of a single product (Rf = 0.9) and no remaining starting mate-
rial (Rf = 0.3). The mixture was added to brine (50 mL) and
extracted with Et2O (2 ꢀ 50 mL). The organic phase was dried
(Na2SO4), filtered and concentrated in vacuo. The residue was puri-
fied by flash column chromatography (CH2Cl2) to give the silyl
ether 5 (320 mg, 95%) as a colourless oil; ½a D21
ꢂ47.4 (c 1.0, CHCl3);
ꢁ
1H NMR (400 MHz, CDCl3): d 0.12, 0.14 (6H, 2 ꢀ s, 2 ꢀ CH3), 0.95
(9H, s, C(CH3)3), 3.62 (1H, dd, J2,3 = 10.5 Hz, J = 7.8 Hz, H-2 or H-3),
0
3.81 (1H, dd, J = 7.8 Hz, H-2 or H-3), 3.93 (1H, d, J6,6 = 12.3 Hz, H-
6), 4.24 (1H, dd, H-60), 4.35 (1H, d, J = 7.8 Hz, H-1 or H-4), 4.41
(1H, d, J = 7.8 Hz, H-1 or H-4), 4.50, 4.55 (2H, ABq, JAB = 11.9 Hz,
PhCH2), 4.73, 4.85 (2H, ABq, JAB = 10.9 Hz, PhCH2), 4.80, 4.98 (2H,
ABq, JAB = 11.0 Hz, PhCH2), 4.89, 4.92 (2H, ABq, JAB = 11.3 Hz,
PhCH2), 5.63 (1H, s, H-5a), 7.26–7.38 (20H, m, Ar-H); 13C NMR
(100 MHz, CDCl3): dꢂ4.4, ꢂ4.4 (2 ꢀ q, Si(CH3)2), 18.2 (s, SiC(CH3)3),
26.0 (q, SiC(CH3)3), 70.0 (t, C-6), 72.3, 74.9, 75.6, 75.6 (4 ꢀ t,
4 ꢀ PhCH2), 73.2, 80.5 (2 ꢀ d, C-1, C-4), 84.7, 85.1 (2 ꢀ d, C-2, C-
3), 127.5, 127.6, 127.7, 127.9, 128.0, 128.4, 128.5, 128.5, 128.5,
129.2 (10 ꢀ d, Ar-CH, C-5a), 135.3 (s, C-5), 138.4, 138.8, 138.8,
139.0 (4 ꢀ s, 4 ꢀ Ar-C); m/z (ESI+) 673 ([M + Na]+, 100%); HRMS
(ESI+) calcd for C41H50O5SiNa [M+Na]+: 673.3320, found 673.3320.
silica gel (35–70 lm, Grace). Reactions that were performed un-
der an atmosphere of hydrogen or nitrogen were maintained by
an inflated balloon.

I. Cumpstey et al. / Carbohydrate Research 344 (2009) 454–459
457
3.3. 2,3,4,6-Tetra-O-benzyl-5a-carba-b-
D
-glucopyranose (3)
0.48 mmol) and Tf2O (39
lL, 0.23 mmol) were added, and the mix-
ture was stirred at 0 °C. After 30 min, TLC (pentane–EtOAc 4:1)
Silyl ether 5 (314 mg, 0.49 mmol) was dissolved in EtOAc
(10 mL), and Et3N (0.1 mL, 0.74 mmol) was added. Palladium
(10% on carbon, 30 mg) was added, and the mixture was degassed
and stirred under H2. After 18 h, the mixture was filtered through
Celite and concentrated in vacuo.
showedproductformation(Rf = 0.8)and remainingstartingmaterial
(Rf = 0.1). Further pyridine (39 lL, 0.48 mmol) and Tf2O (39 lL,
0.23 mmol)wereadded.Afterafurther30 min,TLCshowedthatonly
traces of starting material remained. The reaction mixture was
poured into ice-water (25 mL) and extracted with CH2Cl2 (25 mL).
The organic phase was dried (Na2SO4), filtered and concentrated in
vacuotogivethetriflate7thatwasusedwithoutfurtherpurification.
Sodium (9 mg, 0.39 mmol) was dissolved in MeOH (2 mL) and
added to the thioacetate 8 (55 mg, 0.12 mmol). The solution was
degassed and stirred at 50 °C under N2. After 25 min, TLC (pen-
tane–EtOAc 4:1) showed the complete consumption of starting
material (Rf = 0.7) and the formation of two products (Rf = 0.8 and
0.3). After a further 35 min, the less polar component had been
completely converted into the more polar component. The reaction
mixture was added to NH4Cl (satd aq, 30 mL) and extracted with
CH2Cl2 (25 mL). The organic phase was dried (Na2SO4), filtered
and concentrated in vacuo to give the thiol, which was used with-
out further purification.
Crude triflate 7 was dissolved in DMF (2 ꢀ 1.5 mL) and added to
the thiol. The mixture was degassed and left under N2. Sodium hy-
dride (60% in oil, 20 mg, 0.50 mmol) was added, and the mixture
was stirred at 50 °C. After 30 min, TLC (pentane–EtOAc 2:1) showed
the presence of a major component (Rf = 0.3). The mixture was
added to NH4Cl (satd aq, 30 mL) and extracted with Et2O
(2 ꢀ 30 mL). The organic phase was dried (Na2SO4), filtered and con-
centrated in vacuo. The residue was purified by flash column chro-
matography (pentane–EtOAc 2:1). Product-containing fractions
were concentrated in vacuo, and the residue was dissolved in pyri-
dine (1.5 mL) and Ac2O (1.5 mL) and stirred at rt. After 18 h, the mix-
ture was diluted with EtOAc (30 mL) and washed with H2SO4 (1 M,
30 mL) and NaHCO3 (satd aq, 30 mL). The organic phase was dried
(Na2SO4), filtered and concentrated in vacuo. The residue was puri-
fied by flash column chromatography (pentane–EtOAc 3:1) to give
the thioether pseudodisaccharide 12 (50 mg, 47%) as a colourless
The residue was dissolved in THF (3 mL), TBAF (1 M in THF,
1.5 mL, 1.5 mmol) was added, and the mixture was stirred at
50 °C. After 2 h 30 min, TLC (pentane–EtOAc, 9:1) showed forma-
tion of product (Rf = 0) and no remaining starting material
(Rf = 0.8). The mixture was concentrated in vacuo, and the residue
was purified by flash column chromatography (CH2Cl2–Et2O 30:1)
to give the ido alcohol (49 mg, 19%) and the gluco alcohol 3
(186 mg, 72%)11 as white crystals, mp 97–99 °C (EtOAc–pentane);
1H NMR (400 MHz, CDCl3): d 1.53 (1H, aq, J = 12.6 Hz, H-5a), 1.76
(1H, m, H-5), 2.04 (1H, dat J5a,5a = 13.2 Hz, Jat = 4.2 Hz, H-5a0),
0
3.33 (1H, ddd, J = 2.2 Hz, J = 6.9 Hz, J = 9.1 Hz), 3.51 (1H, dd,
0
J5,6 = 2.6 Hz, J6,6 = 8.9 Hz, H-6), 3.55–3.60 (3H, m), 3.63 (1H, dd,
J5,6 = 5.0 Hz, H-60), 4.47 (2H, s, PhCH2), 4.56 (1H, d, J = 10.8 Hz,
0
PhCHH0), 4.71, 5.01 (2H, ABq, JAB = 11.3 Hz, PhCH2), 4.88–4.96
(3H, m, PhCH2, PhCHH0), 7.22–7.37 (20H, m, Ar-H).
3.4. Methyl 3-S-acetyl-2-O-benzoyl-4,6-O-benzylidene-3-deoxy-
3-thio-a-D-glucopyranoside (8)
Alcohol 10 (1.00 g, 2.59 mmol) was dissolved in CH2Cl2 and
cooled to 0 °C under N2. Pyridine (0.46 mL, 5.72 mmol) and Tf2O
(0.47 mL, 2.78 mmol) were added. After 5 min, the ice-bath was re-
moved. After 30 min, TLC (pentane–EtOAc 3:1) showed the pres-
ence of a single reaction product (Rf = 0.3). The mixture was
poured into ice-water (100 mL) and extracted with CH2Cl2
(100 + 30 mL). The organic phases were dried (Na2SO4), filtered
and concentrated in vacuo to give crude triflate (1.34 g), which
was used without further purification.
Triflate (497 mg, 0.96 mmol) was dissolved in THF. KSAc
(2.19 mg, 1.92 mmol) was added, and the mixture stirred at rt under
N2. After 72 h, brine (50 mL) was added, and the mixture was ex-
tracted with Et2O (2 ꢀ 50 mL). The organic phase was dried
(Na2SO4), filtered and concentrated in vacuo. The residue was puri-
fied by flash column chromatography (pentane–EtOAc 5:1) to give
the thioacetate 8 (219 mg, 51%) as an oil, which was recrystallised
oil; ½a 2D0
ꢁ
+67.5 (c 1.0, CHCl3); m ;
1746 (C@O) cmꢂ1 1H NMR
(400 MHz, CDCl3):
d
1.71 (1H, m, H-5aII), 2.03 (1H, dat,
II
J5a,5a = 14.5 Hz, J = 3.3 Hz, H-5a0 ), 2.11 (3H, s, C(O)CH3), 2.35 (1H,
m, H-5II), 3.36 (1H, at, J = 11.1 Hz, H-3I), 3.40–3.53 (3H, m, H-2II,
H-4II, H-6II), 3.43 (3H, s, OCH3), 3.62 (1H, dd, J3,4 = 10.5 Hz,
0
J4,5 = 9.3 Hz, H-4I), 3.45–3.53 (3H, m, H-6I, H-3II, H-60 ), 3.84 (1H,
II
to give white crystals, mp 129–132 °C (EtOAc–pentane); ½a D22
ꢁ
+107
aq, J = 4.2 Hz, H-1II), 3.89 (1H, atd, J5,6 = 4.8 Hz, J = 9.8 Hz, H-5I),
0
(c 1.0, CHCl3);
m
1703, 1722 (2 ꢀ C@O) cmꢂ1
;
1H NMR (400 MHz,
4.20, 4.57 (2H, ABq, JAB = 12.2 Hz, PhCH2), 4.27 (1H, dd,
I
J6,6 = 10.2 Hz, H-60 ), 4.42, 4.47 (2H, ABq, JAB = 12.0 Hz, PhCH2),
0
CDCl3): d 2.24 (3H, s, C(O)CH3), 3.43 (3H, s, OCH3), 3.66 (1H, dd,
J3,4 = 11.3 Hz, J4,5 = 9.2 Hz, H-4), 3.77 (1H, at J = 10.4 Hz, H-6), 4.02
4.48, 4.86 (2H, ABq, JAB = 10.7 Hz, PhCH2), 4.67, 4.92 (2H, ABq,
JAB = 10.7 Hz, PhCH2), 4.78 (1H, dd, J1,2 = 3.6 Hz, J2,3 = 11.5 Hz, H-
2I), 4.89 (1H, d, H-1I), 5.56 (1H, s, PhCH), 7.02–7.46 (25H, m, Ar-H);
13C NMR (100 MHz, CDCl3): d 21.0 (q, C(O)CH3), 29.6 (t, C-5aII),
37.8 (d, C-5II), 44.8 (d, C-3I), 45.2 (d, C-1II), 55.3 (q, OCH3), 64.2 (d,
0
0
(1H, atd, J = 9.7 Hz, J5,6 = 4.8 Hz, H-5), 4.32 (1H, dd, J6,6 = 10.4 Hz,
H-60), 4.40 (1H, at J = 11.3 Hz, H-3), 5.04 (1H, d, J1,2 = 3.6 Hz, H-1),
5.14 (1H, dd, J2,3 = 11.4 Hz, H-2), 5.54 (1H, s, PhCH), 7.34–7.49 (7H,
m, Ar-H), 7.57 (1H, m, A-H), 8.03–8.06 (2H, m, Ar-H); 13C NMR
(125 MHz, CDCl3): d 31.0 (q, C(O)CH3), 44.3 (d, C-3), 55.5 (q, OCH3),
64.6 (d, C-5), 69.1 (t, C-6), 71.8 (d, C-2), 78.2 (d, C-4), 97.5 (d, C-1),
102.0 (d, PhCH), 126.4, 128.4, 128.6, 129.2, 130.2, 133.6 (6 ꢀ d, Ar-
CH), 129.3, 137.2 (2 ꢀ s, 2 ꢀ Ar-C), 166.0 (s, OC@O), 193.8 (s, SC@O);
m/z(ESI+)911([2M+Na]+, 20), 467([M+Na]+, 100%);HRMS(ESI+)calcd
for C23H24O7SNa [M+Na]+: 467.1135, found 467.1128. Anal. Calcd for
C23H24O7S: C, 62.15; H, 5.44; S, 7.21. Found: C, 62.17; H, 5.60; S, 7.07.
Elimination products (46 mg, 13%), two isomers that were
inseparable were also produced.
C-5I), 69.2 (t, C-6I), 70.3 (t, C-6II PhCH2), 71.4 (d, C-2I), 73.2, 75.4,
,
75.7 (3 ꢀ t, 3 ꢀ PhCH2), 80.8 (d, C-4II), 81.3 (d, C-2II), 83.1 (d, C-4I),
83.8 (d, C-3II), 97.3 (d, C-1I), 102.8 (d, PhCH), 126.6, 127.3, 127.5,
127.6, 127.6, 127.8, 128.0, 128.2, 128.4, 128.5, 128.5, 129.5
(12 ꢀ d, Ar-CH), 137.4, 138.2, 138.7, 139.0, 139.2 (5 ꢀ s, 5 ꢀ Ar-C),
170.3 (s, C@O); m/z (ESI+) 1744 ([2M+Na]+, 5), 899 ([M+K]+, 10),
883 ([M+Na]+, 100), 878 ([M+NH4]+, 45), 861 ([M+H]+, 25%). HRMS
(ESI+) calcd for C51H57O10S [M+H]+: 861.3667, found 861.3691.
3.6. Methyl 2,3,4,6-tetra-O-benzyl-5a-carba-a-D-glucopyran-
3.5. Methyl 2,3,4,6-tetra-O-benzyl-5a-carba-
anosyl-(1?3)-2-O-acetyl-4,6-O-benzylidene-3-deoxy-3-thio-
-glucopyranoside (12)
a
-
D
-glucopyr-
osyl-(1?3)-2-O-benzyl-4,6-O-benzylidene-3-deoxy-3-thio-a-D-
mannopyranoside (13)
a
-
D
As described above, Triflate 7 was prepared from alcohol 3
(83 mg, 0.15 mmol), pyridine (54 L, 66 mmol), and Tf2O (54 L,
32 mmol) in CH2Cl2 (1 mL), and was used crude.
Carbasugar alcohol 3 (100 mg, 0.19 mmol) was dissolved in
CH2Cl2 (2 mL) under N2 and cooled to 0 °C. Pyridine (39 L,
l
l
l

458
I. Cumpstey et al. / Carbohydrate Research 344 (2009) 454–459
Sodium (10 mg, 0.43 mmol) was dissolved in MeOH (1.5 mL)
allowed to warm to rt. The resulting solid was extracted with
MeOH/CHCl3, 1:1, and filtered, and the filtrate was concentrated
in vacuo.
The residue was suspended in pyridine (2 mL) and Ac2O (2 mL),
and was stirred at rt. After 18 h, the mixture was diluted with
EtOAc (30 mL) and washed with H2SO4 (1 M, 30 mL) and NaHCO3
(satd aq, 30 mL). The organic phase was dried (Na2SO4), filtered
and concentrated in vacuo. The residue was purified by flash col-
umn chromatography (pentane–EtOAc 1:2; 1% Et3N) to give the
and added to a solution of thioacetate 9 (108 mg, 0.25 mmol) in
MeOH (1.5 mL). The solution was degassed and stirred at rt under
N2. After 1 h, TLC (pentane–EtOAc 3:1) showed the complete
consumption of starting material (Rf = 0.5) and the formation of a
major product (Rf = 0.6) along with traces of
a by-product
(Rf = 0.4). The reaction mixture was added to NH4Cl (satd aq,
50 mL) and extracted with CH2Cl2 (2 ꢀ 50 mL). The organic phase
was dried (Na2SO4), filtered and concentrated in vacuo. The residue
was purified by flash column chromatography to give the thiol
heptaacetate 14 (18 mg, 93%) as a colourless oil; ½a D21
ꢁ
+61.5 (c
(83 mg, 85%); 1H NMR (500 MHz, CDCl3):
d
2.04 (1H, d,
0.5, CHCl3); 1H NMR (400 MHz, CDCl3):
d 1.88 (1H, atd,
II
JSH,3 = 8.3 Hz, SH-3), 3.38 (3H, s, OCH3), 3.42 (1H, m, H-3), 3.72
(1H, dd, J1,2 = 1.4 Hz, J2,3 = 3.2 Hz, H-2), 3.78–3.82 (3H, m, H-4,
H-5, H-6), 4.24 (1H, m, H-60), 4.68, 4.73 (2H, ABq, JAB = 11.5 Hz,
PhCH2), 4.71 (1H, d, H-1), 5.60 (1H, s, PhCH), 7.32–7.53 (10H, m,
Ar-H); 13C NMR (100 MHz, CDCl3): d 40.7 (d, C-3), 55.0 (q, OCH3),
65.7, 79.6 (2 ꢀ d, C-4, C-5), 68.9 (t, C-6), 74.0 (t, PhCH2), 79.8 (d,
C-2), 98.3 (d, C-1), 102.1 (d, PhCH), 126.2, 128.2, 128.4, 128.4,
128.6, 129.1 (6 ꢀ d, Ar-CH), 137.5, 137.6 (2 ꢀ s, Ar-C).
J1,5a = 3.2 Hz, J = 13.7 Hz, H-5aII), 1.98 (1H, m (obs), H-5a0 ), 1.99,
2.01, 2.05, 2.09, 2.11, 2.20 (21H, 6 ꢀ s, 7 ꢀ C(O)CH3), 2.25 (1H, m,
H-5II), 3.38 (3H, s, OCH3), 3.40 (1H, m (obs), H-3I), 3.72 (1H, m,
H-1II), 3.86 (1H, m, H-5I), 3.88 (1H, dd, J5,6 = 2.8 Hz, J6,6 = 12.6 Hz,
0
II
I
II
H-6 ), 4.07–4.10 (2H, m, H-6 , H-60 ), 4.19 (1H, dd, J5,6 = 4.8 Hz,
0
J6,6 = 12.2 Hz, H-6’I), 4.74 (1H, dd, J1,2 = 2.9 Hz, J2,3 = 11.3 Hz, H-
0
2I), 4.89–4.93 (2H, m, H-1I, H-4I), 4.95 (1H, at J = 10.0 Hz, H-4II),
5.03 (1H, dd, J1,2 = 4.0 Hz, J2,3 = 10.2 Hz, H-2II), 5.45 (1H, at,
J = 9.6 Hz, H-3II); 13C NMR (100 MHz, CDCl3): d 20.8, 20.8, 20.9,
20.9, 21.0 (5 ꢀ q, 7 ꢀ C(O)CH3), 30.6 (t, C-5aII), 35.9 (d, C-5II), 44.8
(d, C-1II), 46.9 (d, C-3I), 55.5 (q, OCH3), 62.5 (t, C-6I), 63.1 (t,
C-6II), 67.8 (d, C-4I), 68.7 (d, C-5I), 71.6 (d, C-4II), 71.8 (d, C-3II),
74.0 (d, C-2II), 74.6 (d, C-2I), 96.3 (d, C-1I), 169.8, 169.9, 170.1,
170.2, 170.9 (5 ꢀ s, 7 ꢀ C@O); m/z (ESI+) 687 (M+Na+, 100%); HRMS
(ESI+) calcd for C28H40O16SNa [M+Na]+: 687.1929, found 687.1918.
Thiol (83 mg, 0.19 mmol) was dissolved in DMF (2 ꢀ 1.5 mL)
and added to the crude triflate 7. The mixture was degassed and
left under N2. Sodium hydride (60% in oil, 20 mg, 0.50 mmol)
was added, and the mixture was stirred at 50 °C. After 40 min,
TLC (pentane–EtOAc, 3:1) showed the presence of a major compo-
nent (Rf = 0.4), no remaining triflate (Rf = 0.6), some thiol remain-
ing (Rf 0.5) and some more polar by-products. The mixture was
added to NH4Cl (satd aq, 30 mL) and extracted with Et2O
(2 ꢀ 30 mL). The organic phase was dried (Na2SO4), filtered and
concentrated in vacuo. The residue was purified by flash column
chromatography (pentane–EtOAc 4:1) to give the thioether pseu-
3.8. Methyl 2,3,4,6-tetra-O-acetyl-5a-carba-a-D-
glucopyranosyl-(1?3)-2,4,6-tri-O-acetyl-3-deoxy-3-thio-a-D-
mannopyranoside (15)
dodisaccharide 13 (72 mg, 54%) as a colourless oil; ½a D22
ꢁ
+20.0
(c 1.0, CHCl3); 1H NMR (400 MHz, CDCl3): d 1.65 (1H, ddd,
Ammonia (ca. 10 mL) was condensed into a flask at –78 °C. So-
dium (ca. 70 mg, 3 mmol) was added, and the mixture turned deep
blue. The pseudodisaccharide 13 (35 mg, 0.039 mmol) was dis-
solved in THF (2 mL) and transferred into the reaction vessel by
J1,5a = 3.0 Hz, J5,5a = 12.2 Hz, J5a,5a = 14.9 Hz, H-5aII), 2.01 (1H, dat,
0
II
J = 3.3 Hz, H-5a0 ), 2.43 (1H, m, H-5II), 3.23 (1H, dd, J2,3 = 3.1 Hz,
J3,4 = 11.0 Hz, H-3I), 3.35 (3H, s, OCH3), 3.39 (1H, dd, J3,4 = 9.0 Hz,
J4,5 = 10.7 Hz, H-4II), 3.49–3.53 (2H, m, H-2II, H-6II), 3.69 (1H, dd,
pipette. After 5 min, MeOH (20 lL, 0.49 mmol) was added, and
II
J5,6 = 5.0 Hz, J6,6 = 9.1 Hz, H-60 ), 3.71 (1H, dd, J1,2 = 2.0 Hz, H-2I),
the reaction mixture was stirred for a further 20 min. The blue col-
our persisted throughout this sequence. After this time, NH4Cl was
added until the blue colour disappeared, and the mixture was al-
lowed to warm to rt. The resulting solid was extracted with
MeOH–CHCl3, 1:1, and filtered, and the filtrate was concentrated
in vacuo.
0
0
3.78 (1H, at, J = 9.4 Hz, H-3II), 3.82–3.85 (3H, m, H-5I, H-6I, H-1II),
4.07–4.12 (2H, m, H-4I, PhCHH0), 4.22 (1H, m, H-60 ), 4.42, 4.46
I
(2H, ABq, JAB = 11.9 Hz, PhCH2), 4.47, 4.87 (2H, ABq, JAB = 10.8 Hz,
PhCH2), 4.55–4.58 (2H, m, PhCHH0, H-1I), 4.64, 4.75 (2H, ABq,
JAB = 11.7 Hz, PhCH2), 4.71, 4.97 (2H, ABq, JAB = 10.8 Hz, PhCH2),
5.60 (1H, s, PhCH), 6.98–7.45 (30H, m, Ar-H); 13C NMR (100 MHz,
CDCl3): d 29.3 (t, C-5aII), 37.3 (d, C-5II), 44.7 (d, C-1II), 45.4 (d, C-
3I), 55.0 (q, OCH3), 65.7 (d, C-5I), 69.2 (t, C-6I), 69.7, 73.1, 74.3,
75.5, 75.7 (5 ꢀ t, 5 ꢀ PhCH2), 70.4 (t, C-6II), 80.6, 80.7, 81.0, 81.1
(4 ꢀ d, C-2I, C-4I, C-2II, C-4II), 83.6 (d, C-3II), 98.8 (d, C-1I), 102.9
(d, PhCH), 126.5, 127.2, 127.4, 127.6, 127.7, 127.9, 128.0, 128.1,
128.1, 128.2, 128.3, 128.3, 128.4, 128.5, 129.3 (15 ꢀ d, Ar-CH),
137.8, 137.8, 138.3, 138.7, 139.0, 139.3 (6 ꢀ s, 6 ꢀ Ar-C); m/z
(ESI+) 947 ([M+K]+, 40), 931 ([M+Na]+, 50), 926 ([M+NH4]+,
100%); HRMS (ESI+) calcd for C56H60O9SNa [M+Na]+: 931.3850,
found 931.3843.
The residue was suspended in pyridine (3 mL) and Ac2O (3 mL),
and was stirred at rt. After 3 h, the mixture was diluted with EtOAc
(30 mL) and washed with HCl (1 M, 30 mL) and NaHCO3 (satd aq,
30 mL). The organic phase was dried (Na2SO4), filtered and concen-
trated in vacuo. The residue was purified by flash column chroma-
tography (pentane–EtOAc 1:1; 1% Et3N) to give the heptaacetate 15
(19 mg, 77%) as a colourless oil; ½a D21
ꢁ
+47.5 (c 1.0, CHCl3); 1H NMR
(400 MHz, CDCl3): d 1.83 (1H, ddd, J1,5a = 3.6 Hz, J5,5a = 12.8 Hz,
J5a,5a = 14.7 Hz, H-5aII), 1.94 (1H, dat (obs), J = 3.3 Hz, H-5a’II),
0
1.98, 2.01, 2.04, 2.07, 2.09, 2.14, 2.17 (21H, 7 ꢀ s, 7 ꢀ C(O)CH3),
2.25 (1H, m, H-5II), 3.35 (1H, dd, J2,3 = 3.1 Hz, J3,4 = 11.2 Hz, H-3I),
3.38 (3H, s, OCH3), 3.57 (1H, aq, J = 3.5 Hz, H-1II), 3.85–3.91 (2H,
II
m, H-5I, H-6II), 4.04 (1H, dd, J5,6 = 5.1 Hz, J6,6 = 11.3 Hz, H-60 ),
0
0
3.7. Methyl 2,3,4,6-tetra-O-acetyl-5a-carba-a-D-
glucopyranosyl-(1?3)-2,4,6-tri-O-acetyl-3-deoxy-3-thio-
a-
D
-
4.08 (1H, dd, J5,6 = 2.4 Hz, J6,6’ = 12.3 Hz, H-6I), 4.21 (1H, dd,
I
J5,6 = 5.6 Hz, H-60 ), 4.65 (1H, d, J1,2 = 1.3 Hz, H-1I), 4.92 (1H, dd,
0
glucopyranoside (14)
J4,5 = 9.5 Hz, H-4II), 4.97–5.06 (3H, m, H-2I, H-4I, H-2II), 5.42 (1H,
at, J = 9.8 Hz, H-3II); 13C NMR (100 MHz, CDCl3): d 20.7, 20.8,
20.8, 20.9, 21.0 (5 ꢀ q, C(O)CH3), 30.2 (t, C-5aII), 35.6 (d, C-5II),
43.5 (d, C-1II), 46.5 (d, C-3I), 55.3 (q, OCH3), 63.0 (t, C-6I), 63.2
(t, C-6II), 66.6 (d, C-4I), 69.5 (d, C-5I), 70.5 (d, C-2I), 71.6 (d, C-4II),
71.9 (d, C-3II), 73.9 (d, C-2II), 97.8 (d, C-1I), 169.9, 170.1, 170.1,
170.2, 170.3, 170.8, 170.9 (7 ꢀ s, 7 ꢀ C@O); m/z (ESI+) 687
([M+Na]+, 100), 682 ([M+NH4]+, 45%); HRMS (ESI+) calcd for
C28H40O16SNa [M+Na]+: 687.1929, found 687.1911.
Ammonia (ca. 25 mL) was condensed into a flask at ꢂ78 °C. So-
dium (ca. 200 mg, 8 mmol) was added, and the mixture turned
deep blue. The pseudodisaccharide 12 (25 mg, 0.029 mmol) was
dissolved in THF (1.5 mL) and transferred into the reaction vessel
by pipette. Then, MeOH (2 ꢀ 50
lL, 2.5 mmol) was added, and
the reaction mixture was stirred for a further 5 min. The blue col-
our persisted throughout this sequence. After this time, NH4Cl was
added until the blue colour disappeared, and the mixture was

I. Cumpstey et al. / Carbohydrate Research 344 (2009) 454–459
459
3.9. Methyl 5a-carba-
a
-
D
-glucopyranosyl-(1?3)-3-deoxy-3-
had been pre-washed with 150
l
L of water) to remove protein be-
thio- -glucopyranoside (1)
a-
D
fore NP-HPLC analysis as described previously.27
Sodium (6 mg, 0.26 mmol) was dissolved in MeOH (1.5 mL) and
added to the pseudodisaccharide heptaacetate 14 (16 mg,
0.024 mmol). After 4 h, TLC (CMAW) showed the presence of a sin-
gle component (Rf = 0.2). Dowex 50X8 resin (H+) was added, and
the mixture was filtered and concentrated in vacuo. The residue
was purified by flash column chromatography (EtOAc–MeOH–
water 7:2:1) to give the deprotected pseudodisaccharide 2 (8 mg,
3.12. Free oligosaccharides (FOS) analysis in cells following
compound treatment
HL60 Cells were cultured in RPMI media containing 10% foetal
calf serum, 2 mM
(Invitrogen), before the medium was replaced with fresh medium
containing compounds at 100 M. After 24 h incubation, the med-
L-glutamine and 1% penicillin-streptomycin
l
90%) as a colourless oil; ½a D22
ꢁ
+101 (c 0.5, MeOH); 1H NMR
ium was removed, and the cells were washed three times with PBS
by centrifugation. Washed cells were stored at ꢂ20 °C for a short
time before thawing and dounce homogenisation in water. The
FOS were labelled with 2-anthranilic acid, purified using Conca-
0
(500 MHz, D2O): d 1.67 (1H, ddd, J5a,5a = 14.3 Hz, J5,5a = 13.1 Hz,
J1,5a = 3.5 Hz, H-5aII), 1.88 (1H, m, H-5II), 2.08 (1H, dat, J = 3.3 Hz,
H-5a0 ), 2.93 (1H, at, J = 10.9 Hz, H-3I), 3.24 (1H, dd, J = 9.1 Hz,
II
J = 10.7 Hz, H-4II), 3.43 (3H, s, OCH3), 3.45–3.48 (2H, m, H-4I,
H-1II), 3.51 (1H, at, J 9.5 Hz, H-3II), 3.55 (1H, dd, J1,2 = 3.6 Hz,
J2,3 = 11.2 Hz, H-2I), 3.62–3.68 (3H, m, H-5I, H-2II, H-6II), 3.72–
navalin A (Con A)-Sepharose 4B beads (100 lL packed resin) and
analysed by NP-HPLC using a 4.6 ꢀ 250 mM TSK gel Amide-80 col-
umn (Anachem, Luton, UK) as described previously.27
I
II
3.76 (2H, m, H-6 , H-60 ), 3.85 (1H, dd, J5,6 = 2.2 Hz, J6,6 = 12.2 Hz,
0
0
H-60 ), 4.8 (1H, obs, H-1I); 13C NMR (125 MHz, D2O): d 31.2 (t,
I
C-5aII), 40.3 (d, C-5II), 49.2 (c, C-1II), 54.5 (d, C-3I), 55.5 (q, OCH3),
61.4 (t, C-6I), 62.9 (t, C-6II), 69.5 (d, C-4I), 70.1 (d, C-2I), 73.2, 74.2
(2 ꢀ d, C-5I, C-2II), 73.6 (d, C-4II), 76.3 (d, C-3II), 99.3 (d, C-1I); m/z
(ESI+) 763 ([2M+Na]+, 10), 393 ([M+Na]+, 100%); HRMS (ESI+) calcd
for C14H26O9SNa [M+Na]+: 393.1190, found 393.1207.
Acknowledgement
IC thanks Vetenskapsrådet and the Stockholm University Ivar
Bendixsons fond for financial support.
Supplementary data
3.10. Methyl 5a-carba-a-D-glucopyranosyl-(1?3)-3-deoxy-3-
thio- -mannopyranoside (2)
a
-D
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.carres.2008.12.023.
Sodium (6 mg, 0.26 mmol) was dissolved in MeOH (2 mL) and
added to the pseudodisaccharide heptaacetate 15 (19 mg,
0.029 mmol). After 4 h, TLC (CMAW) showed the presence of a sin-
gle component (Rf = 0.2). Dowex 50X8 resin (H+) was added, and
the mixture was filtered and concentrated in vacuo. The residue
was purified by flash column chromatography (EtOAc–MeOH–
water, 7:2:1) and freeze dried to give the deprotected pseudodisac-
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108, 42–46.
charide 2 (11 mg, 99%) as a white solid; ½a D22
ꢁ
+45 (c, 0.25, MeOH);
1H NMR (500 MHz, D2O):
d
1.85 (1H, ddd, J5a,5a = 14.3 Hz,
0
J5,5a = 12.9 Hz, J1,5a = 3.2 Hz, H-5aII), 2.11 (1H, m, H-5II), 2.19 (1H,
II
dat, J = 3.4 Hz, H-5a0 ), 3.29 (1H, dd, J2,3 = 2.9 Hz, J3,4 = 10.6 Hz,
H-3I), 3.40 (1H, dd, J = 9.0 Hz, J = 10.7 Hz, H-4II), 3.59 (1H, s,
OCH3), 3.68–3.73 (2H, m, H-1II, H-3II), 3.79–3.94 (6H, m, H-4I, H-
5I, H-6I, H-2II, H-6II, H-60 ), 4.05 (1H, dd, J5,6 = 1.8 Hz,
II
0
I
J6,6 = 12.2 Hz, H-60 ), 4.12 (1H, dd, J1,2 = 1.7 Hz, H-2I), 4.88 (1H, d,
0
H-1I); 13C NMR (125 MHz, D2O): d 29.1 (t, C-5aII), 38.0 (d, C-5II),
47.6 (d, C-1II), 49.9 (d, C-3I), 53.2 (q, OCH3), 59.7 (t, C-6I), 0.8 (t,
C-6II), 65.3 (d, C-4I), 69.5 (d, C-2I), 71.7, 72.2, 72.2 (3 ꢀ d, C-2II,
C-4II, C-5I), 74.0 (d, C-3II), 98.5 (d, C-1I); m/z (ESI+) 393 ([M+Na]+,
100%); HRMS (ESI+) calcd for C14H26O9SNa [M+Na]+: 393.1190,
found 393.1198.
3.11. In vitro glucosidase inhibition
16. Ogawa, S.; Ashiura, M.; Uchida, C. Carbohydr. Res. 1998, 370, 83–95.
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19. Cumpstey, I. Synlett 2006, 1711–1714.
Fluorescently labelled substrates Glc1Man7GlcNAc2 and
Glc2Man7GlcNAc2 were prepared using anthranilic acid (2AA) and
added to separate 1.5 mL centrifuge tubes with varying concentra-
20. Driguez, H. ChemBioChem 2001, 2, 311–318.
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tions of the compounds (0–100
glucosidase II was added to generate 25% hydrolysis of
Glc1Man7GlcNAc2–2AA in 90 min reaction time or 2 h for
lM). Sufficient purified rat liver a-
a
Glc2Man7GlcNAc2–2AA. Linear degradation of substrate occurred
over the time of incubation. The reactions were stopped by the
addition of 3 lL glacial acetic acid and 30 lL acetonitrile. Following
enzyme digestion, samples were centrifuged through a 10,000
molecular weight cut-off filter at 7000 rpm for 45 min (which
28. Lillelund, V. H.; Jensen, H. H.; Liang, X.; Bols, M. Chem. Rev. 2002, 102, 515–553.