Synthesis of fluorescently labelled rhamnosides: Probes for the evaluation of rhamnogalacturonan II biosynthetic enzymes

Article abstract of DOI:10.1016/j.carres.2011.03.039

Three fluorescently labelled saccharides 10-12, representing structures found in pectic glycan rhamnogalacturonan II (RG-II), were synthesised by chemical glycosylation of O-6 of diacetone-d-galactose followed by deprotection and reductive amination with amino-substituted fluorophore APTS. This convenient method installs a common aminogalactitol-based tether in order to preserve the integrity of the reducing end of specific carbohydrates of interest. APTS-labelled glycans prepared in this manner were purified by carbohydrate gel electrophoresis and subjected to capillary electrophoresis analysis, as a basis for the subsequent development of high sensitivity assays for RG-II-active enzymes.

Full text of DOI:10.1016/j.carres.2011.03.039

Carbohydrate Research 346 (2011) 1617–1621
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Note
Synthesis of fluorescently labelled rhamnosides: probes for the evaluation
of rhamnogalacturonan II biosynthetic enzymes
⇑
Efthymia Prifti, Stephan Goetz, Sergey A. Nepogodiev, Robert A. Field
Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Three fluorescently labelled saccharides 10–12, representing structures found in pectic glycan rhamno-
Received 7 February 2011
Received in revised form 25 March 2011
Accepted 29 March 2011
Available online 6 April 2011
galacturonan II (RG-II), were synthesised by chemical glycosylation of O-6 of diacetone-D-galactose fol-
lowed by deprotection and reductive amination with amino-substituted fluorophore APTS. This
convenient method installs a common aminogalactitol-based tether in order to preserve the integrity
of the reducing end of specific carbohydrates of interest. APTS-labelled glycans prepared in this manner
were purified by carbohydrate gel electrophoresis and subjected to capillary electrophoresis analysis, as a
basis for the subsequent development of high sensitivity assays for RG-II-active enzymes.
Ó 2011 Elsevier Ltd. All rights reserved.
Dedicated to Professor András Lipták on the
occasion of his 75th birthday
Keywords:
Glycosyltransferase assay
Fluorescent oligosaccharide
Reductive amination
APTS labelling
Capillary electrophoresis
Despite the ever-expanding repertoire of plant genome
sequences and the essential role of carbohydrates in plant cell wall
architecture,1 detailed knowledge of the biosynthetic machinery
required for cell wall polysaccharide biosynthesis is at best patchy
and in places non-existent.2 Much remains to be done in order to
confirm the biological function of the ca. 2000 gene products2
required for cell wall assembly. In terms of polysaccharide biosyn-
thesis, in the model plant Arabidopsis thaliana an excess of 200
glycosyltransferases (GTs)3 are probably required. However, across
all plants GT substrate specificities have only been convincingly
demonstrated for a dozen or so to date.1d A major bottle-neck lies
in the lack of available oligosaccharide acceptor substrates and
radiolabelled sugar nucleotide donor substrates. Addressing this
issue, we have prepared a series for rhamnogalacturonan II (RG-II)
fragments as methyl glycosides,4 to enable assays with radiola-
belled donor substrates. However, only a few relevant sugar nucle-
otides are readily accessible in radiolabelled form. We therefore,
sought a complementary approach, based on acceptor substrates
fluorescently labelled with 8-aminopyrene-1,3,6-trisulfonic acid
(APTS), which can be employed in conjunction with non-radiola-
belled sugar nucleotides. Specifically, we opted for capillary elec-
trophoresis (CE)-compatible materials, given the established high
resolution and high sensitivity of CE coupled with laser-induced
fluorescence detection (CE-LIF) for carbohydrate analysis.5
Typical protocols for fluorescent labelling of reducing sugars6
make use of reductive amination reactions with amino-substituted
chromophores. In the case of APTS labelling, the procedure has been
shown to be highly reproducible and efficient (88% efficiency for
maltose).7 Inevitably the integrity of the reducing terminal sugar
ring is lost in the process (Scheme 1, route A). We therefore, devised
a means of installing an aldehyde-terminating tether to which the
amino-fluorophore could be attached. For practical reasons,
D-galactose was attractive since the readily available 1,2:3,4-di-O-
isopropylidene- -galactose (1) may serve initially as an acceptor
a-D
for glycosylation. Subsequent removal of the acetonide protecting
groups liberates a reducing terminus for conjugation to the fluoro-
phore, generating an aminogalactitol spacer between the sugar of
interest and the fluorophore without loss of the reducing terminal
ring of the sugar in question (Scheme 1, route 2).8
L-Rhamnose, which occurs in several different places in the
RG-II structure and is a putative glycosyl acceptor for a number
of sugar nucleotide donors in the biosynthesis of plant cell walls,
was chosen as a non-reducing end sugar for our initial targets.
Since the minimum acceptor substrate structure required for GT
activities may be represented by a larger oligosaccharide acceptor,
we extended our choice of targets by selecting disaccharide b-Rha-
(1?30)-b-Api, which is a common fragment of two highly branched
side chains of RG-II. Both a- and b-(1?6)-linked disaccharides, 10
and 11, respectively, and trisaccharide 12 incorporating a Rha-
(1?30)-Api motif, were synthesised (Scheme 2) as compounds
ready for derivatisation with the fluorophore. Glycosylation of
⇑
Corresponding author. Tel.: +44 1603 450720.
E-mail address: rob.field@bbsrc.ac.uk (R.A. Field).
0008-6215/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2011.03.039

1618
E. Prifti et al. / Carbohydrate Research 346 (2011) 1617–1621
O
O
APTS, NaBH3CN
OH
H
N
O
O
O
Route A
X
SO3
OH
O
O
H2N
O
H2N
=
SO3
glycosylation
APTS =
O
O
1
SO3
Route B
O
O
O
O
O
O
i. deprotection
O
O
O
ii. APTS, NaBH3CN
HO
HO
H
N
O
OH
HO
O
O
O
Scheme 1. Strategy for the preparation of fluorescent glycans suitable for carbohydrate-active enzyme analysis CE-LIF.
STol
Br
STol
Me
AcO
STol
O
O
RO
O
O
b
Me
AcO
Me
AcO
O
O
O
+
O
53%
AcO
O
O
O
O
O
OAc
O
O
2
Ph
H
Ph
H
O
6
4 R = ClCH2CO
5 R = H
3
a, 94%
Me
Me
OH
O
O
O
O
O
Me
2
Me
c, 80%
6
c, 38%
1
3
b, 72%
O
O
Me
O
O
O
O
Me
O
Me
AcO
O
O
O
O
O
Me
O
Me
O
O
Me
AcO
O
Me
O
O
O
O
Me
AcO
O
O
O
Me
Me
O
O
Me
OAc
Me
O
O
O
O
Me
AcO
O
Me
O
Me
Ph
H
7
8
9
d, e, 78%
d, e, 95%
d, f, e, 39%
OH
OH
OH
OH
O
OH
O
O
HO
O
Me
HO
O
O
O
HO
Me
HO
O
O
OH
O
HO
Me
HO
HO
HO
O
OH
HO
HO
OH
HO
OH
OH
HO
OH
10
11
12
Scheme 2. Synthesis of oligosaccharides 10–12 using glycosylation of 1 with glycosyl donors 2, 3 and 6. Reagents and conditions: (a) Et3N–MeOH (95:5), 20 °C, 4 h; (b) Ag2O,
MS 4 Å, CH2Cl2, 20 °C, 20 h; (c) NIS, Me3SiOTf, CH2Cl2, ꢀ20 to 0 °C, 3 h; (d) 90% CF3CO2H, 20 °C; (e) 0.1 M NaOMe, MeOH; (f) H2-Pd/C, EtOH–EtOAc 1:1, 20 °C.
alcohol 19 with thioglycoside 210 in the presence of NIS-Me3SiOTf
afforded
-rhamnoside 711 in 80% yield (Scheme 2). b-Rhamnosy-
lation of 1, which was carried out with bromide 312 under hetero-
a
geneous conditions4a,12 involving insoluble Ag2O as a promoter, led

E. Prifti et al. / Carbohydrate Research 346 (2011) 1617–1621
1619
to disaccharide 812 in 72% yield (Scheme 2). NMR spectroscopic
characteristics of 7 and 8 were consistent with literature data, thus
confirming the a- and b-configuration, respectively, for rhamnosi-
Gel 60 F254, Merck). Column chromatography was performed on
Biotage SP4 purification system using silica gel pre-packed car-
tridges. Optical rotations were measured using a Perkin–Elmer
141 polarimeter. 1H and 13C NMR spectra were recorded at 22 °C
with a Bruker Avance III spectrometer at 400 and 100 MHz, respec-
tively. ESI-MS were obtained on the DecaXPplus ion trap mass spec-
trometer (Thermo). Purification of APTS-labelled carbohydrates was
performed using the carbohydrate gel electrophoresis (PACE) meth-
od.17 Desalting of aqueous solutions of APTS-labelled oligosaccha-
rides was carried out on PD-10 desalting columns (GE Healthcare).
dic linkages in these compounds. Thioapiofuranoside derivative
5,13 prepared in 94% yield by selective deprotection (Et3N–MeOH)
of the 30-position of chloroacetate 4, was glycosylated with bro-
mide 3 using the b-rhamnosylation approach (used to prepare
disaccharide 8) to yield disaccharide 6 (53%) (Scheme 2). This
disaccharide was reacted with alcohol 1 in the presence of NIS
and HClO4 adsorbed on silica14 to afford target trisaccharide 9 in
38% yield.15 The b-configuration of the newly formed apiofuranos-
idic bond in 9 was confirmed by the appearance of the anomeric
proton as a singlet in 1H spectra (d 5.26 ppm) and the position of
the anomeric carbon in 13C NMR spectra (d 106.8 ppm), which
are characteristic for 1,2-trans-furanosidic linkages.16
1.2. p-Tolyl 2,3-O-(R)-benzylidene-3-C-hydroxymethyl-1-thio-b-
D-erythrofuranoside (5)
A
solution of p-tolyl 2,3-O-(R)-benzylidene-3-C-(2-chloro-
Deprotection of compounds 7 and 8 was achieved by hydrolysis
of isopropylidene groups in 90% CF3CO2H and subsequent trans-
esterification with NaOMe in MeOH to give unprotected glycans
10 and 11, which were used without further purification in the
fluorescent labelling step. Trisaccharide 12 was synthesised from
protected derivative 9 in a similar way, but Pd-catalysed hydroge-
nation was included as an additional step to ensure complete re-
moval of the benzylidene acetal group. Purification of 12 was
performed by gel permeation chromatography in water. NMR spec-
tra of deprotected oligosaccharides 10–12 indicated the presence
of two species corresponding to two anomeric forms of the reduc-
ing end galactopyranose residue: signals of H-1 Gal and C-1 Gal ap-
acetoxy)methyl-1-thio-b-
D
-erythrofuranoside (4)13 (400 mg, 0.95
mmol) in MeOH–Et3N (95:5, 10 mL) was stirred for 4 h at 20 °C,
the concentrated residue was purified by crystallisation (hexane–
Et2O) to give alcohol 7, 308 mg (94%), mp 101–102 °C; ½a D20
ꢀ246
ꢂ
(c 1.0, CHCl3); 1H NMR (CDCl3, 400 MHz): d 7.53–7.45 (m, 2H,
aromatics), 7.43–7.31 (m, 5H, aromatics), 7.11 (d, 2H, J 7.9 Hz, aro-
matics), 5.91 (s, 1H, PhCH), 5.76 (s, 1H, H-1), 4.58 (s, 1H, H-2),
4.16 (m, 2H, H-4a, H-4b), 3.90 (m, 2H, H-30a, H-30b), 2.47 (t, 1H, J
5.9 Hz, OH), 2.30 (s, 3H, Me); 13C NMR (100 MHz, CDCl3) d 138.1,
136.2, 132.9, 130.1, 130.0, 129.2, 128.5, 127.2 (aromatics), 106.6
(PHCH), 93.0 (C-3), 92.6 (C-1), 87.4 (C-2), 73.0 (C-4), 63.6 (C-30),
21.2 (Me). ESI MS: m/z 344.9 [M+H]+; calcd for C19H20O4S 345.12.
peared as distinct peaks (
spectra of these compounds.
a
/b ꢁ1.5:1) both in 1H and 13C NMR
1.3. p-Tolyl 3-C-(4-O-acetyl-2,3-O-carbonyl-b-
L-
Reductive amination is a commonly used two-step, one-pot
method to introduce a chromophore into a carbohydrate molecule.
During the first step, a chromophore with a primary amino group
rhamnopyranosyloxymethyl)-2,3-O-(R)-benzylidene-1-thio-b-
D-erythrofuranoside (6)
´
reacts reversibly with the reducing carbohydrate to form a Schiffs
A mixture of bromide 3 (240 mg, 0.81 mmol), alcohol 7 (85 mg,
base. A stable chromophore–carbohydrate derivative is subse-
quently obtained by reduction of the Schiff’s base with sodium cya-
noborohydride. To purify the resulting APTS-derivatised glycan
samples, the PACE (polysaccharide analysis using carbohydrate
gel electrophoresis) method was used.17 After electrophoretic sep-
aration, bands on the gel corresponding to the APTS-derivatised
glycan were identified under a UV-light lamp, cut from the gel,
extracted with water and desalted with a PD-10 desalting column.
Quantification of materials purified in this manner was carried out
using the previously reported molar extinction coefficient for
APTS-derivatised maltoheptaose,5a with compound purity being
assessed by CE-LIF, calibrated against an APTS-glucose standard
[retention times of reductively aminated APTS adducts: Glc
0.25 mmol), MS 4 Å (500 mg) and Ag2O (280 mg, 1.21 mmol) in
CH2Cl2 (10 ml) was stirred for 20 h at 24 °C. The solids were re-
moved by filtration, the filtrate was concentrated and the residue
was purified by column chromatography (hexane–EtOAc,
4:1?2:3) to give disaccharide 6 (73 mg 53%): ½a D20
ꢀ112 (c 1.0,
ꢂ
CHCl3); 1H NMR (CDCl3, 400 MHz): d 7.56 (m, 2H, aromatics),
7.38 (m, 5H, aromatics), 7.13 (m, 2H, aromatics), 5.99 (s, 1H, PhCH),
5.77 (s, 1H, H-1 Api), 5.45 (dd, 1H, J 8.7, 5.5 Hz, H-4 Rha), 5.12 (d,
1H, J 2.6 Hz, H-1 Rha), 4.82 (m, 2H, H-2 and H-3 Rha), 4.62 (s,
1H, H-2 Api), 4.29–4.18 (m, 3H, H-30a, H-30b and H-4a Api), 3.92
(d, 1H, J 10.9 Hz, H-4b Api), 3.81 (dq, 1H, J 8.6, 6.3 Hz, H-5 Rha),
2.33 (s, 3H, C6H4CH3), 2.11 (s, 3H, Ac), 1.33 (d, 3H, J 6.3 Hz, H-6
Rha); 13C NMR (100 MHz, CDCl3): d 168.9 (CH3CO), 153.4 (C@O),
138.1, 136.2, 133.0, 129. 9, 128.3, 127.3 (aromatics), 106.7 (PhCH),
95.3 (C-1 Rha), 92.65 (C-3 Api), 91.6 (C-1 Api), 87.3 (C-2 Api), 76.1,
72.9 (C-2 and C-3 Rha), 72.2 (C-4 Rha), 71.2 (C-4 Api), 70.5 (C-5
Rha), 69.1 (C-30 Api), 21.1 (Me), 20.8 (Ac), 19.6 (C-6 Rha); ESI MS:
m/z 581.4 [M+Na]+; calcd for C28H30O10SNa 581.59.
3.89 min;
a-Rha-Gal 10: 4.46 min; b-Rha-Gal 11: 4.33 min;
b-Rha-b-Apif-Gal 12: 4.77 min].
In summary, herein we report a convenient strategy for the gen-
eration of fluorescently-labelled glycans related to rhamnogalactu-
ronan II that are suitable for use in glycosyltransferase assay
development. Details of the biological evaluation of these materials
will be reported in due course.
1.4. 1,2:3,4-Di-O-isopropylidene-6-O-(2,3,4-tri-O-acetyl-
a-L-
rhamnopyranosyl)- -galactopyranose (7)
a-D
1. Experimental
Me3SiOTf (21
lL, 0.11 mmol) was added to a stirred mixture of
alcohol (266 mg, 1.02 mmol), thioglycoside
1
2
(450 mg,
1.1. General methods
1.14 mmol), NIS (276 mg, 1.23 mmol) and MS 4 Å (500 mg) in
CH2Cl2 (10 mL) at ꢀ20 °C. The stirring continued for 3 h at ꢀ20 to
10 °C, Et3N (0.5 mL) was added, the mixture was diluted with
CH2Cl2 and solids were filtered off. The filtrate was extracted with
10% aqueous Na2S2O3, the organic extract was washed with satd
NaHCO3 solution and H2O, dried and concentrated in vacuo. The
residue was purified by column chromatography (hexane–EtOAc,
4:1?3:2) to give the title compound 4 (440 mg, 80%): mp 112–
All reagents, including 8-aminopyrene-1,3,6-trisulfonic acid
(APTS), were used as purchased without further purification. Reac-
tions were carried out in dry solvents using septa and syringes for
addition of reagents. The SiO2-supported HClO4-catalyst
(ꢁ0.5 mmol of the acid per 1 g of powder) was prepared using
Merck silica gel (15–40 lm) according to procedure reported in
114 °C (hexane), ½a D20
ꢂ
ꢀ89.5 (c 1.0 CHCl3) (lit.11 mp 114–115, ½a D20
ꢂ
Ref. 14b. TLC was performed on pre-coated aluminium plates (Silica

1620
E. Prifti et al. / Carbohydrate Research 346 (2011) 1617–1621
ꢀ89); 1H NMR (400 MHz, CDCl3): d 5.51 (d, 1H, J 5.0 Hz, H-1 Gal),
5.32–5.21 (m, 2H, H-2 and H-3 Rha), 5.07 (pt, 1H, J 7.9 Hz, H-4
Rha), 4.81 (d, 2H, J 0.9 Hz, H-1 Rha), 4.63 (dd, 2H, J 2.4, 7.9 Hz, H-
3 Gal), 4.36–4.26 (m, 2H, H-2 and H-4 Gal), 4.06–3.95 (m, 2H, H-5
Rha, H-5 Gal), 3.86 (dd, 3H, J 7.4, 9.6 Hz, H-6a Gal), 3.59 (dd. 3H, J
6.2, 9.6 Hz, H-6b Gal), 2.16 (s, 3H, Ac), 2.04 (s, 3H, Ac), 2.00 (s, 3H,
Ac), 1.56 (s, 3H, C(CH3)2), 1.44 (s, 3H, C(CH3)2), 1.34 (m, 6H,
C(CH3)2), 1.21 (d, 3H, J 6.3 Hz, H-6 Rha); 13C NMR (100 MHz, CDCl3):
d 109.2, 108.7 (C(CH3)2), 97.3 (C-1 Rha), 96.2 (C-1 Gal), 71.2, 70.8,
70.62, 70.58, 69.8, 69.2 (C-2, C-3, C-4 Rha and C-2, C-3, C-4 Gal),
66.3, 66.2 (C-5 Rha, C-5 Gal), 65.3 (C-6 Gal), 26.1, 26.0, 25.0, 24.4
(C(CH3)2), 21.0, 20.9, 20.8, 20.75 (CH3CO), 17.2 (C-6 Rha).
Rha), 91.1 (C-3 Api), 86.5 (C-2 Api), 76.2, 72.2 (C-2 and C-3 Rha),
73.5 (C-30 Api), 71.3 (C-4 Rha), 71.1 (C-4 Gal), 70.7 (C-3 Gal), 70.4
(C-2 Gal), 69.4 (C-4 Api), 66.9 (C-5 Gal), 66.3 (C-5 Rha), 60.4 (C-6
Gal), 26.0, 24.9, 24.5 (C(CH3)2), 21.1 (CH3CO), 20.8 (Ac), 19.4 (C-6
Rha); ESI MS: m/z 712.2 [M+NH4]+; calcd for C33H42O16ꢃNH4 712.72.
1.7. 6-O-(a-L-Rhamnopyranosyl)-D-galactopyranose (10)
Compound 7 (120 mg, 0.23 mmol) was dissolved in 90% aque-
ous CF3CO2H (1.5 mL), the solution was stirred for 15 min at
22 °C, toluene (5 mL) was added and solvents were evaporated in
vacuum. The residue was dissolved in toluene and concentrated
to dryness; this operation was repeated twice. The product
(95 mg), Rf = 0.44 (CH2Cl2–MeOH 9:1) was dissolved in dry MeOH
(1.0 mL), then 0.02 M NaOMe (1.0 mL) was added and after 1 h at
22 °C the reaction mixture was neutralised by addition of Amber-
lite IR120 (H+). The resin was removed by filtration, the filtrates
were concentrated in vacuum and the residue was freeze-dried
from water to give disaccharide 10 as white powder (59 mg,
1.5. 6-O-(4-O-Acetyl-2,3-O-carbonyl-b-
L-rhamnopyranosyl)-
1,2:3,4-di-O-isopropylidene- -galactopyranose (8)
a-D
Reaction of glycosyl acceptor 1 (300 mg, 1.15 mmol) with glyco-
syl bromide 3 (500 mg, 1.69 mmol) was carried out in the presence
of Ag2O (500 mg, 2.16 mmol) and MS 4 Å (3.0 g) in essentially the
same way as for the synthesis of 6. The product was purified by
column chromatography (toluene–acetone, 4:1) to give disaccha-
78%); 1H NMR (400 MHz, D2O): d 5.17 (d, J 3.6 Hz, H-1
a-Gal),
4.74 (br s, H-1 Rha), 4.48 (d, J 7.9 Hz, H-1 b-Gal), 1.21 (d, J 6.2 Hz,
ride 8 (390 mg, 72%); ½a D20
ꢂ
ꢀ16 (c 1.0, CHCl3) (lit.12
[
a]
D
ꢀ10.2);
H-6 Rha); 13C NMR (100 MHz, D2O) d 100.6, 100.4 (JC–H 175.8 Hz,
1H NMR (CDCl3, 400 MHz): d 5.51 (d, 1H, J 5.0 Hz, H-1 Gal), 5.42
(m, 1H, H-4 Rha), 4.97 (br s, 1H, H-1 Rha), 4.78 (m, 2H, H-2 and
H3 Rha), 4.62 (dd, 1H, J 7.9, 2.4 Hz, H-3 Gal), 4.31 (dd, 1H, J 5.0,
2.4 Hz, H-2 Gal), 4.29 (dd, 2H, J 7.9, 2.0 Hz, H-4 Gal), 4.02 (m, 1H,
H-5 Gal), 3.94 (dd, 1H, J 9.8, 6.1 Hz, H-6a Gal), 3.79–3.72 (m, 2H,
H-5 Rha, H-6b Gal), 2.11 (s, 3H, Ac), 1.54 (s, 3H, C(CH3)2), 1.43 (s,
3H, C(CH3)2), 1.35 (s, 3H, C(CH3)2), 1.33 (s, 3H, C(CH3)2), 1.29 (d,
3H, J 6.3 Hz, H-6 Rha); 13C NMR (100 MHz, CDCl3): d 169.0 (CH3CO),
153.6 (C@O), 109.2, 108.8 (C(CH3)2), 96.2 (C-1 Gal), 95.5 (C-1 Rha),
76.3, 72.5 (C-2 and C-3 Rha), 71.4 (C-4 Rha), 70.7, 70.55, 70.5 (C-2,
C-3 and C-4 Gal), 70.2 (C-5 Rha), 67.4 (C-6 Gal), 66.1 (C-5 Gal), 26.1,
26.0, 25.0, 24.4 (C(CH3)2), 20.8 (CH3CO), 19.4 (C-6 Rha); ESI MS: m/z
497.4 [M+Na]+; calcd for C21H30O12Na 497.44.
C-1 Rha), 96.4 (C-1 b-Gal), 92.3 (C-1 a-Gal), 73.4, 72.7, 72.0, 71.8,
70.2, 70.0, 69.9, 69.4, 69.1, 69.0, 68.8, 68.6, 68.2; HRESI MS: m/z
349.1104 [M+Na]+; calcd for C12H22O10Na 349.1105.
1.8. 6-O-(b-L-Rhamnopyranosyl)-D-galactopyranose (11)
Treatment of compound 8 (103 mg, 0.22 mmol) as described for
synthesis of 10, afforded disaccharide 11 as white powder (68 mg,
95%); 1H NMR (D2O, 400 MHz): d 5.14 (br s, H-1
a-Gal), 4.56 (br s,
H-1 Rha), 4.47 (d, 2H, J 7.9 Hz, H-1 b-Gal), 1.18 (m, H-6 Rha); 13C
NMR (100 MHz, D2O) d 99.9, 99.7 (C-1 Rha), 96.5 (C-1 b-Gal),
92.4 (C-1
12H22O10Na 349.1105.
a
-Gal), HRESI MS: m/z 349.1105 [M+Na]+; calcd for
C
1.6. 6-O-(3-C-(4-O-Acetyl-2,3-O-carbonyl-b-
oxymethyl)-2,3-O-(R)-benzylidene-b- -erythrofuranosyl)-
1,2:3,4-di-O-isopropylidene- -galactopyranose (9)
L
-rhamnopyranosyl-
1.9. 6-O-(3-C-(b-
erythrofuranosyl)-
L
-Rhamnopyranosyloxymethyl)-b-
-galactopyranose (12)
D-
D
D
a-D
A soln of disaccharide 9 (35 mg, 50 lmol) in 90% aqueous
A mixture of glycosyl acceptor 1 (33.9 mg, 0.13 mmol), disac-
charide donor 2 (72.8 mg, 0.13 mmol), NIS 35.2 mg (0.16 mmol)
and MS 4 Å (100 mg) in CH2Cl2 (4 mL) was stirred for 30 min at
ꢀ20 °C, then HClO4/SiO2 (7 mg, prepared according to14b) was
added and the stirring continued for 30 min at ꢀ20 to 0 °C. The
reaction was quenched with addition of Et3N (0.1 ml), the mixture
was diluted with CH2Cl2 and filtered. The filtrates were washed
with 10% aqueous Na2S2O3, satd NaHCO3 and water, organic layer
was dried and concentrated. TLC analysis of the residue indicated
the presence of two products, the first having Rf = 0.51 and the sec-
ond having Rf = 0.32 (hexane–EtOAc 3:7), which were separated by
column chromatography (hexane–EtOAc 7:3?3:7). The first com-
CF3CO2H (1.8 mL) was stirred for 15 min at 22 °C and then concen-
trated in vacuo several times with toluene (5 mL). The residue was
dissolved in EtOH–EtOAc (1:1), cat. 10% Pd/C was added and the
mixture was stirred under H2 for 17 h at 22 °C. The catalyst was fil-
tered off, solvents were evaporated under vacuum and the residue
was treated with 0.1 M NaOMe in MeOH for 2 h at 22 °C. The mix-
ture was neutralised with Amberlite IR-120 (H+), the resin was fil-
tered off and filtrates were concentrated. The residue was purified
using gel permeation chromatography (Toyopearl HW-40S,
1.5 ꢄ 80 cm) in H2O and the product was freeze-dried to give the
title compound 12 as a white powder (9 mg, 39%): 1H NMR
(400 MHz, CDCl3): d (D2O, 600 MHz): d 5.10 (H-1
a-Gal), 4.95,
pound was identified as trisaccharide 12 (34.3 mg, 38%), ½a D20
ꢂ
4.88 (H-1 Api), 4.51 (H-1 Rha), 4.49 (H-1 b-Gal), 1.15 (H-6 Rha);
ꢀ45 (c 1.0, CHCl3); 1H NMR (400 MHz, CDCl3): d 7.56 (m, 2H, Ph),
7.37 (m, 3H, Ph), 5.95 (s, 1H, CHPh), 5.55 (d, 1H, J 5.1 Hz, H-1
Gal), 5.41 (m, 1H, H-4 Rha), 5.28 (s, 1H, H-1 Api), 5.12 (br s, 1H,
H-1 Rha), 4.77 (m, 2H, H-2 and H-3 Rha), 4.61 (dd, 1H, J 7.9,
2.4 Hz, H-3 Gal), 4.49 (s, 1H, H-2 Api), 4.32 (dd, 1H, J 5.1, 2.4 Hz,
H-2 Gal), 4.21 (dd, 1H, J 7.9, 1.9 Hz, H-4 Gal), 4.18 (d, 1H, J
11.1 Hz, H-4a Api), 4.14–4.09 (m, 3H, H-30a Api, H-6a and H-6b
Gal), 4.03 (m, 1H, H-5 Gal), 4.00 (d, 1H, J 10.3 Hz, H-30b Api), 3.89
(d, 1H, J 11.1 Hz, H-4b Api), 3.76 (m, 1H, H-5 Rha), 2.04 (s, 1H,
Ac), 1.55 (s, 3H, C(CH3)2), 1.45 (s, 3H, C(CH3)2), 1.33 (s, 6H,
C(CH3)2), 1.30 (d, 2H, J 6.3 Hz, H-6 Rha); 13C NMR (100 MHz,
CDCl3): d 168.9 (CH3CO), 153.4 (C@O), 136.4, 129.8, 128.3, 127.4
(Ph), 106.8 (C-1 Api), 106.4 (PhCH), 96.3 (C-1 Gal), 95.1 (C-1
13C NMR (150 MHz, D2O) d 108.7 (C-1 Api), 100.3 (C-1 Rha), 96.4
(C-1
a-Gal), 92.2 (C-1 b-Gal), 16.6 (C-6 Rha); HRESI MS: m/z
481.1526 [M+Na]+; calcd for C17H30O14Na 481.1528.
1.10. Typical procedure for derivatisation of oligosaccharide
samples with APTS
An oligosaccharide (ꢁ0.2
ring in a solution prepared by mixing of 0.2 M APTS in 15% AcOH
(5 l) and 1 M NaBH3CN in THF (5 l). The resulting solution was
lmol) was solubilised by vigorous stir-
l
l
incubated for 20 h at 37 °C and APTS-derivatised product was sep-
arated from unreacted oligosaccharide using gel electrophoresis.17
A band corresponding to the APTS-derivatised oligosaccharide

E. Prifti et al. / Carbohydrate Research 346 (2011) 1617–1621
1621
Deconstructing the Plant Cell Wall for Bioenergy; Himmel, M. E., Ed.; Wiley-
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was identified under UV-light, cut out from the gel and labelled sac-
charide was extracted with water. The aqueous extracts were de-
salted using a PD-10 column and concentrated to dryness. The
labelled product was quantified spectrophotometrically using the
previously reported molar extinction coefficient for APTS-deriva-
tised maltoheptaose (17160 Mꢀ1 cmꢀ1 at 455 nm).5a
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1.11. Typical procedure for the CE-LIF analysis of
oligosaccharide samples labelled with APTS
Analysis of APTS-labelled glycan samples was performed on a
PA800 ProteomeLab instrument (Beckman Coulter), equipped with
an N–CHO coated capillary (50 lm I.D, length to detector 40 cm,
total length 50.2 cm) and laser-induced fluorescence detection sys-
tem (excitation at 488 nm, emission at 520 nm). Samples were
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A related multi-step approach to generate a range of O-glycosides of p-
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Acknowledgements
Dr. Shirley Fairhurst and Dr. Lionel Hill are acknowledged for
invaluable assistance with NMR spectroscopy and mass spectrom-
etry, respectively. These studies were supported by the BBSRC, the
Nuffield Foundation and the Gatsby Foundation through the JIC/SL
Undergraduate Summer Research Training Programme: http://
opportunities.jic.ac.uk/summerprogramme.
15. The second component in reaction of 1 and 6 was isolated in 13% and identified
by NMR spectra as an isomeric trisaccharide with a-apiofuranosyl linkage.
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