A practical synthesis of capped 4-methylumbelliferyl hyaluronan disaccharides and tetrasaccharides as potential hyaluronidase substrates

Article abstract of DOI:10.1016/j.carres.2011.03.042

The synthesis of hyaluronan dimers and tetramers equipped with a 4-methylumbelliferyl group at the reducing end to potentially allow monitoring of hyaluronidase activities is described. The 4-OH at the non-reducing glucuronate in the presented series is either removed or methylated to prohibit transglycosylase reactions, leading to a total of four probes.

Full text of DOI:10.1016/j.carres.2011.03.042

Carbohydrate Research 346 (2011) 1467–1478
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
A practical synthesis of capped 4-methylumbelliferyl hyaluronan
disaccharides and tetrasaccharides as potential hyaluronidase substrates
Henrik Gold a, Stefan Munneke a, Jasper Dinkelaar a, Herman S. Overkleeft a, Johannes M. F. G. Aerts b,
a,
Jeroen D. C. Codée a, , Gijs A. van der Marel
⇑
⇑
a Leiden Institute of Chemistry, Leiden University, Gorlaeus Laboratories, Einsteinweg 55, 2300 RA Leiden, The Netherlands
b Department of Medical Biochemistry, Academic Medical Center, University of Amsterdam, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of hyaluronan dimers and tetramers equipped with a 4-methylumbelliferyl group at the
reducing end to potentially allow monitoring of hyaluronidase activities is described. The 4-OH at the
non-reducing glucuronate in the presented series is either removed or methylated to prohibit transgly-
cosylase reactions, leading to a total of four probes.
Received 11 February 2011
Received in revised form 24 March 2011
Accepted 29 March 2011
Available online 6 April 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Hyaluronan
Hyaluronidase
Fluorogenic substrate
Glycosylation
Barton deoxygenation
1. Introduction
in a single step. Obviously, fluorogenic substrates targeting
endoglycosidases are more challenging to synthesize and as a con-
sequence literature on these is relatively scarce. Endoglycosidases
may further have a considerable affinity for the natural aglycone, a
feature well exemplified by most cellulase binding grooves that
can accommodate a number of glucoside residues at both sides
of the scissile glycosidic linkages. Finally, and a feature we have
encountered in the past in our work on chitinase probes, endogly-
cosidases may possess considerable transglycosylase activity, by
which a released oligosaccharide fragment is condensed with the
next substrate, thus obscuring the interpretation of a fluorescence
assay.3 Our rationale and the subject of this work is that in order to
establish whether fluorogenic endoglycosidase substrates for a gi-
ven enzyme family are suitable probes, these first need be de-
signed and synthesized. In designing a probe and its synthesis
one should take into consideration both the number of monosac-
charide units comprising the non-reducing part, which should be
modifiable with relative ease, and the possible occurrence of trans-
glycosidation, which should be negated by blocking potential
transglycosylation sites. In this work we report on our synthetic ef-
forts towards the generation of potential fluorogenic hyaluronidase
probes.
4-Methylumbelliferyl glycosides are often used to monitor the
activity of glycosidases from different sources.1 They are stable un-
der physiological conditions and undergo enzyme-catalyzed
hydrolysis, after which the generated 4-methylumbelliferonate
has excellent fluorescence properties. As such, 4-methylumbellife-
ryl glycosides are ideal molecular probes for glycobiology studies
in which a glycosidase activity needs to be assessed in in vitro or
in situ research settings. Glycosidase substrate recognition is often
governed to a large extent by the nature of the non-reducing
fragment of a glycoconjugate and less so by the nature of the agly-
cone, enabling the design of glycosidase fluorogenic probes by
grafting the 4-methylumbelliferonate onto the non-reducing frag-
ment in the appropriate stereochemical form (alpha or beta). This
is true in particular for exoglycosidases, enzymes that recognize
and remove a single monosaccharide residue from a substrate gly-
coconjugate.2 Indeed, effective 4-methylumbelliferyl glycosides for
many exoglycosidase activities are now commercially available.
The situation is less optimal with respect to probes to monitor
the activity of endoglycosidases, enzymes that recognize and re-
move oligosaccharides composed of a number of monosaccharides
Hyaluronan, a member of the glycosaminoglycan polysaccha-
ride superfamily, is an important structural component of several
mammalian tissues.4 Hyaluronan is
a
linear polysaccharide
-glucuronic acid-(1?3)-N-acetyl-b-
-glucosamine repeating disaccharides and occurs in macromolecular
⇑
Corresponding authors.
E-mail addresses: jcodee@chem.leidenuniv.nl (J.D.C. Codée), marel_g@chem.
composed of (1?4)-linked b-
D
D
leidenuniv.nl (G.A. van der Marel).
0008-6215/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2011.03.042

1468
H. Gold et al. / Carbohydrate Research 346 (2011) 1467–1478
sizes up to 20 MDa.5 Several mammalian hyaluronidase activities
have been identified to date and the list is considerably longer
when considering hyaluronidases originating from other organ-
isms.6 The substrate preference of these enzymes appears to vary
to an extent that hyaluronan can be processed to macromolecules
of a size smaller than the aforementioned 20 MDa but also to oli-
gosaccharides numbering from four to about 40 monosaccharides,
with the latter oligomers commonly referred to as low molecular
weight hyaluronan. One intriguing recent finding is that low
molecular weight hyaluronan, but not the macromolecules, has
immunostimulating properties in mammalian systems and by
extension, hyaluronidases may partake in immune regulation pro-
cesses.7 In all, we reasoned that hyaluronidases are interesting tar-
gets for fluorogenic probe development, the more so since such
entities are unprecedented in the literature.
2. Results and discussion
Hyaluronidases process hyaluronan by cleavage of glycosidic
bonds between N-acetyl-b-D-glucosamine and b-D-glucuronic acid
residues, to reveal the glucosamine at the reducing end of one frag-
ment and the glucuronic acid on the non-reducing end of the other
fragment.6 We therefore based the design of our potential hyal-
uronidase fluorogenic substrates on a 4-methylumbelliferonate
b-linked to N-acetylglucosamine at the reducing end. We further
considered the likelihood that hyaluronidases of different nature
would require hyaluronan fragments of different size for recogni-
tion, and endeavoured to establish a flexible synthesis strategy to
accommodate this. Finally, and as said in our previous work on
endoglycosidase fluorogenic substrate development we encoun-
tered considerable transglycosylation activity, which complicated
Scheme 1. Reagents and conditions: (a) TBABr, CsOH, 4-methylumbelliferone, CH2Cl2/H2O, rt, 1 h, 72%; (b) (1) NaOMe, MeOH, rt, 4 h; (2) (tBu)2Si(OTf)2, pyridine, DMF,
ꢀ40 °C, 45 min, 94%; (c) (1) Im2CS, toluene, 90 °C, 20 h, 89%; (2) AIBN, n-Bu3SnH, toluene, 90 °C, 6 h, 83%; (d) (1) TBAF, THF, rt, 2 h, 94%; (2) TEMPO, BAIB, CH2Cl2/H2O, 0 °C, 1 h;
(3) TMSCHN2, CH2Cl2/MeOH, rt, 20 min, 90%; (e) NIS, TFA, CH2Cl2, 0 °C, 2.5 h, 96%; (f) (1) TEMPO, BAIB, CH2Cl2/H2O, rt, 2.5 h; (2) TMSCHN2, CH2Cl2/MeOH, rt, 20 min, 73% (over
two steps); (3) TMSCHN2, BF3OEt2, CH2Cl2, ꢀ40 °C to rt, 2 h, 49% (92% based on recovered starting material); (g) NIS, TFA, CH2Cl2, 0 °C, 2.5 h, 75%; (h) Ph2SO, Tf2O, CH2Cl2,
ꢀ20 °C, 11: 82%, 12: 53%; (i) (1) NaOMe (cat), MeOH, CH2Cl2; (2) HF-pyridine, 18 h; (3) Na2CO3 (aq); (4) RP-HPLC, 13: 77%, 14: 63%.

H. Gold et al. / Carbohydrate Research 346 (2011) 1467–1478
1469
assessment of glycosidase activity making use of fluorogenic sub-
strate assays. In these studies we had an interest in the study of hu-
man chitinases (chitotriosidase, acid mammalian chitinase) and we
found that both enzymes are capable of transglycosylation of the
commercial chitinase substrate, chitobiosyl-4-methylumbellifer-
one, at the 40 position. Blocking this position by either deoxygen-
ation or methylation obliterated this transglycosylation, leading
to efficient fluorogenic substrates that are clean in the sense that
they undergo a single enzymatic transformation, namely the de-
sired hydrolysis.3a,8 We therefore decided to target both the corre-
sponding 4-deoxy and 4-methoxy derivatives of our projected HA
probes, taking into account an even number of monosaccharide
residues and a GlcNAc moiety at the reducing end in our design.
Altogether this leads to the design of a set of potential hyaluronidase
2 in 72% yield. Global deprotection followed by installation of the
di-tert-butylsilylene protective group gave the desired acceptor
glycoside 3 in 94% yield over the two steps. We elected to employ
the di-tert-butylsilylene group12 instead of the benzylidene group
because we,13 and others,11 have previously shown the former to
confer desirable properties to the acceptor. In particular, GlcNAc-
derived donor/acceptor glycosides often suffer from poor solubility
in organic solvents, and introduction of the di-tert-butylsilylene
protective group, which withstands most acidic glycosylation con-
ditions, makes these glycosides better soluble in organic solvents.
Donor uronic acid derivatives 7 and 10 were readily prepared as
follows. Barton deoxygenation14 of the 4-hydroxyl in the partially
and orthogonally protected thioglucoside 4 gave 4-deoxyglucose
derivative 5. Fluoride-mediated desilylation followed by TEMPO-
BAIB oxidation15 and transformation of the carboxylate to the
methyl ester provided phenyl thiouronate 6, which was anomeri-
cally deprotected using conditions we previously described (N-
iodosuccinimide, trifluoroacetic acid)16 to give donor hemi-acetal
7. In a related sequence of events, the primary alcohol in phenyl
thioglucoside 817 was selectively oxidized using the TEMPO/BAIB
reagent combination, after which the carboxylate was methylated
with TMS-diazomethane. In a subsequent step the 4-OH group was
capped with the same reagent under the agency of BF3OEt2.18,19
Liberation of the anomeric hydroxyl provided donor hemiacetal 10.
Condensation of 7 and 10 with acceptor 3 under dehydrative
glycosylation conditions (Ph2SO, Tf2O)20 provided the fully pro-
tected disaccharides 11 and 12, respectively, which were globally
deprotected and purified to homogeneity to deliver the potential
hyaluronidase fluorogenic substrates 13 and 14 in good quantities
and good overall yield. It should be noted here that phenylthioglu-
fluorogenic substrates with the general structure R-[(?4)-b-
D-glu-
curonic acid-(1?3)-N-acetyl-b- -glucosamine-(1?)]n-4-methyl-
D
umbelliferone, with R = H or OMe. In this work we demonstrate
the synthetic feasibility of these substrates through the synthesis
of a set of dimeric and tetrameric potential hyaluronan substrates
of the above design and with n = 1 or 2.
The synthesis of the hyaluronan umbelliferonates composed of
a single b-D-glucuronic acid-(1?3)-N-acetyl-b-D-glucosamine moi-
ety is depicted in Scheme 1.9 In our experience,8 the formation of a
glycosidic linkage with the 4-methylumbelliferyl aglycone can be
troublesome and we decided to introduce this linkage at the onset
of our synthetic schemes.10 This leads to partially protected N-ace-
tylglucosamine derivative 3 as the common starting point and this
building block was prepared in three steps from the anomeric chlo-
ride 1.11 SN2 substitution of the anomeric chloride with the sodium
salt of 4-methylumbelliferone provided fully protected b-glycoside
tBu
tBu
tBu
Si
O
O
Si
O
tBu
O
MeO
Si
O
tBu
O
tBu
O
MeO
O
O
O
a
O
O
O
LevO
O
O
O
O
SPh
O
BzO
HO
LevO
O
CF3
Ph
TCAHN
BzO
AcHN
BzO
TCAHN
BzO
N
15
16
3
b
tBu
Si
tBu
O
Si
O
tBu
O
tBu
O
MeO
O
O
O
O
O
O
O
O
RO
O
BzO
TCAHN
AcHN
BzO
17 R = Lev
18 R = H
c
O
O
MeO
BzO
O
MeO
MeO
O
e
e
MeO
BzO
d
O
MeO
MeO
d
O
OBz
O
O
CF3
Ph
O
O
CF3
BzO
OH
OBz
OH
BzO
OBz
N
OBz
N
7
19
Ph
22
10
tBu
Si
tBu
Si
tBu
Si
tBu
Si
O
O
O
O
tBu
O
O
tBu
O
O
tBu
O
O
tBu
O
MeO
BzO
MeO
MeO
BzO
MeO
MeO
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
BzO
BzO
TCAHN
AcHN
TCAHN
AcHN
OBz
OBz
BzO
20
BzO
23
f
f
O
O
O
O
HO
HO
HO
MeO
HO
HO
HO
HO
HO
HO
HO
HO
HO
HO
HO
O
O
O
O
AcHN
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
HO
HO
AcHN
AcHN
AcHN
OH
OH
OH
OH
21
24
Scheme 2. Reagents and conditions: (a) (1) NIS, TFA, CH2Cl2, 0 °C, 2 h, 89%; (2) Cs2CO3, ClC(NPh)CF3, acetone, 0 °C, 3 h, 85%; (b) TfOH, CH2Cl2, 0 °C, 2 h, 60%; (c) hydrazine
acetate, pyridine/acetic acid, rt, 10 min, 99%; (d) Cs2CO3, ClC(NPh)CF3, acetone, 0 °C, 3 h, 19: 73%, 22: 65%; (e) TfOH, CH2Cl2, 0 °C, 4 h, 20: 78%, 23: 85%; (f) (1) zinc dust, AcOH,
rt, 40 h; (2) KI, acetone, rt, 20 h; (3) zinc dust, AcOH, rt, 20 h; (4) NaOMe (cat), MeOH, CH2Cl2, 6 h; (5) (HF)3ꢁEt3N, pyridine, 18 h; (6) Na2CO3 (aq), 4 h; (7) RP-HPLC, 21: 60%, 24:
43%.

1470
H. Gold et al. / Carbohydrate Research 346 (2011) 1467–1478
curonates 6 and 9 could, in principle, also serve as donors to gen-
erate disaccharides 11 and 12. We however found that in our
hands, and by making use of conditions advocated by us (Ph2SO,
Tf2O),21 and others (BSP, Tf2O),22 these thioglycosides failed to give
productive couplings.
methane and THF were freshly distilled, before use, over P2O5
and Na/benzophenone, respectively. Et3N was destilled over cal-
cium hydride and stored over potassium hydroxide. Trifluorometh-
anesulfonic anhydride was distilled from P2O5. All moisture
sensitive reactions were performed under an argon atmosphere.
Traces of water were removed from starting compounds by
co-evaporation with dichloroethane, dioxane and/or toluene.
Molecular sieves 3 Å were flamedried prior to use. Liquid column
chromatography was performed using forced flow of the indicated
The construction of tetramers 21 and 24 is depicted in Scheme 2
and is based on orthogonally protected hyaluronan disaccharide 15
we previously employed in the modular assembly of hyaluronan
oligomers of varying size.13 Hydrolysis of the thioacetal and ensu-
ing transformation into N-phenyl-trifluoroimidate23 donor 16 set
the stage for condensation with acceptor GlcNAc derivative 3. Con-
densation to give the fully protected 17 under the agency of triflic
acid in methylene chloride proceeded in 60% yield. Attempts to ef-
fect the same transformation but starting from phenylthiodisac-
charide 15 or the intermediate hemiacetal obtained after NIS/TFA
treatment under the appropriate glycosylation conditions proved
low yielding. Liberation of the 300-OH by hydrazine-mediated re-
moval of the Lev protective group gave acceptor trisaccharide 18,
which was effectively condensed with either donor imidate 19 or
donor imidate 22 to give the fully protected tetrasaccharides 20
and 23 in 78% and 85% yield, respectively. Hemiacetals 7 and 10,
the precursors of imidates 19 and 20, could also be condensed with
trisaccharide 18 under the agency of Ph2SO and Tf2O, however in
considerable reduced yield (around 40%). Global deprotection of
the tetrasaccharides proved to proceed with some difficulties, in
that reductive transformation of the trichloroacetamide moieties
in 20/23 into the corresponding N-acetyl groups did not go to com-
pletion even after prolonged exposure to zinc dust in acetic acid.24
Rather, the monochlorides were obtained as the major product. We
therefore had to resort to a Finkelstein transformation of the
monochloroacetamide intermediates to give the corresponding
mono-iodo acetamides and resubject these to the zinc dust medi-
ated reductive dehalogenation. This three-step dehalogenation
proceeded with good efficiency and removal of the remaining pro-
tective groups proceeded under standard conditions to give the
target tetrasaccharides 21 and 24 in 43% and 60% yield, respec-
tively, after HPLC purification.
solvent systems on Screening Devices Silica Gel 60 (40–63 lm
mesh). Size exclusion chromatography was performed on Sephadex
LH20 (eluent MeOH/CH2Cl2, 1:1). Analytical TLC was performed on
aluminium sheets, pre-coated with silica gel (Merck, Silica Gel 60,
F254). Compounds were visualized with UV absorption (245 nm),
by spraying with either 20% H2SO4 in ethanol, or ammonium
molybdate/cerium sulfate solution [(NH4)6Mo7O24ꢁ4H2O (25 g/L),
(NH4)4Ce(SO4)6ꢁ2H2O (10 g/L), 10% sulphuric acid in ethanol], or
phosphormolybdic acid in EtOH (150 g/L) followed by charring
(ꢂ150 °C) or by spraying with potassium permanganate (1.6% in
concd sulfuric acid). IR spectra were recorded on a Shimadzu
FTIR-8300 and are reported in cmꢀ1. Optical rotations were mea-
sured on
a Propol automatic polarimeter (Sodium D-line,
k = 589 nm). 1H and 13C NMR spectra were recorded on a Bruker
AV 400 MHz spectrometer at 400.2 (1H) and 100.6 (13C) MHz or
on a Bruker AV 500 MHz spectrometer at 500.0 (1H) and 125.1
(
13C) MHz respectively. Chemical shifts are reported as d values
(ppm) and directly referenced to TMS (0.00 ppm) in CDCl3 or via
the solvent residual peak (D2O). Coupling constants (J) are given
in Hertz and all 13C spectra are proton decoupled. NMR assign-
ments were made using COSY and HSQC and in some cases TOCSY
experiments. LC–MS analyses were performed on a LCQ Advantage
Max (Thermo Finnigan) equipped with a Gemini C18 column (Phe-
nomenex, 50 ꢃ 4.6 mm, 3
l), utilizing the following buffers: A:
H2O, B: acetonitrile and C: 1.0% TFA(aq). HPLC purifications were
performed on a Gilson GX-281 automated HPLC system, equipped
with a preparative Gemini C18 column (Phenomenex, 150 ꢃ 21.20, 5
l).
Products were eluted using the following buffers: A: ammonium
acetate (20 mM(aq)) or triethylammonium acetate (50 mM(aq)), B:
acetonitrile (HPLC-grade), 20 mL/min. Purified products were
lyophilized on a CHRIST ALPHA 2–4 LDPLUS to remove water and
traces of buffer salts. High resolution mass spectra were recorded
3. Conclusion
In summary, we have demonstrated the synthesis of a set of
four potential hyaluronidase fluorogenic substrates, equipped with
the 4-methylumbelliferonate fluorogenic leaving group. Our strat-
egy is flexible in that both dimeric and tetrameric glycoconjugates
can be assessed through the use of common intermediates, and
that the nature of the non-reducing cap (deoxy or methoxy) can
be determined in the final glycosylation step by selection of the
appropriate donor glucuronate. A protecting group strategy has
been developed, which allows the deprotection of the HA-saccha-
rides, leaving the coumarine moiety untouched. Our future work
will entail the assessment of the fluorogenic substrates against a
panel of hyaluronidases. Finally, if one considers the modular
assembly of the tetrameric derivatives making use of disaccharides
15/16 it is evident that our strategy can be readily adapted to gen-
erate hyaluronan oligomers composed of a larger number of mono-
saccharides. In this scheme, we envisage elongation of 18 with one
or more copies of imidate 16 prior to introducing the non-reducing
glucuronate moiety.
by direct injection (2 lL of a 2 lM solution in water/acetonitrile;
50/50; v/v and 0.1% formic acid) on a mass spectrometer equipped
with an electrospray ion source in positive mode (source voltage
3.5 kV, sheath gas flow 10, capillary temperature 250 °C) with
resolution R = 60000 at m/z 400 (mass range m/z = 150–2000) and
dioctylpthalate (m/z = 391.28428) as a ‘lock mass’. The high resolu-
tion mass spectrometer was calibrated prior to measurements with
a calibration mixture.
4.1.1. 4-Methylumbelliferyl 2-acetamido-2-deoxy-3,4,6-tri-O-
acetyl-b-D-glucopyranoside (2)
4-Methylumbelliferone (2.24 g, 12.7 mmol, 1.5 equiv) and tet-
rabutylammonium bromide (4.10 g, 12.7 mmol, 1.5 equiv) were
dissolved in CH2Cl2 (30 mL). To this solution cesium hydroxide
(3.56 g, 11.9 mmol, 1.4 equiv), dissolved in water (30 mL) was
added and the two-phase system was stirred vigorously for
10 min at room temperature. 2-Acetamido-2-deoxy-3,4,6-tri-O-
acetyl-b-D
-glucopyranosyl chloride11 (3.1 g, 8.48 mmol, 1.0 equiv),
dissolved in CH2Cl2 (10 mL) was then added dropwise over 10 min.
The mixture was stirred for an additional 45 min and then trans-
ferred to an extraction funnel with CH2Cl2 (20 mL) and washed
with water (2ꢃ 100 mL) and brine (80 mL). The aqueous layers
were extracted with CH2Cl2 (100 mL) and the combined organics
were dried (Na2SO4), filtered and concentrated in vacuo. Purifica-
4. Experimental
4.1. General methods and materials
Commercially available reagents and solvents (Acros, Fluka or
Merck) were used as received unless stated otherwise. Dichloro-

H. Gold et al. / Carbohydrate Research 346 (2011) 1467–1478
1471
tion by column chromatography (5–20% acetone in CH2Cl2)
1616, 1389, 1268, 1071, 827, 653 cmꢀ1
[C26H37NO8Si+H]+: 520.2361, found 520.2363.
;
HRMS calcd for
produced the title compound as a white solid (3.08 g, 6.09 mmol,
72%). 1H NMR (400 MHz, CDCl3): d = 7.56 (m, 1H, H-54-MU), 7.01–
6.96 (m, 2H, H-64-MU and H-84-MU), 6.18 (d, 1H, J = 1.1 Hz,
H-34-MU), 5.44–5.38 (m, 2H, H-1 and H-3), 5.13 (dd, 1H, J = 10.2,
9.4 Hz, H-2), 4.30 (dd, 1H, J = 12.3, 5.7 Hz, H-6a), 4.22 (dd, 1H,
J = 10.2, 8.3 Hz, H-4), 4.19 (dd, 1H, J = 12.3, 2.3 Hz, H-6b), 4.02
(ddd, 1H, J = 10.2, 5.7, 2.3 Hz, H-5), 3.95 (s, 1H, NH), 2.43 (s, 3H,
CH3-4-MU), 2.13 (s, 3H, CH3-OAc), 2.09 (s, 6H, CH3-OAc ꢃ2), 1.93 (s,
3H, CH3-NAc); 13C NMR (100 MHz, CDCl3): d = 171.5 (C@ONAc),
170.8, 170.6, 169.6 (C@OOAc ꢃ3), 161.4, 159.42, 154.35, 153.0
(C-24-MU, C-74-MU, C-94-MU and C-104-MU), 125.5 (C-54-MU), 114.9
(C-44-MU), 114.0 (C-64-MU), 112.2 (C-34-MU), 103.5 (C-84-MU), 97.7
(C-1), 72.0 (C-3), 71.8 (C-5), 68.4 (C-2), 61.9 (C-6), 53.8 (C-4),
22.3 (CH3-NAc), 20.3, 20.17, 20.16 (CH3-OAc ꢃ3), 18.3 (CH3-4-MU); IR
(neat): 2959, 2932, 2860, 1718, 1616, 1267, 1072, 1034, 827,
766 cmꢀ1; HRMS calcd for [C24H27NO11 +Na]+: 528.1476, found
528.1474.
4.1.3. Phenyl 2,3-di-O-benzoyl-6-(tert-butyldiphenylsilyl)-1-
thio-b- -glucopyranoside (4)
To a solution of phenyl 1-thio-2,3-di-O-benzoyl-b-
D
D-glucoside
(5.21 g, 10.8 mmol, 1.0 equiv) in DMF (20 mL), imidazole (1.47 g,
21.6 mmol, 2.0 equiv) was added followed by TBDPS-Cl (3.60 mL,
14.0 mmol, 1.3 equiv). The reaction was quenched after 20 h with
MeOH (5 mL) and then diluted with water (500 mL) and extracted
with ether (2ꢃ 400 mL). The organics were dried with MgSO4, fil-
tered and concentrated in vacuo. Purification by column chroma-
tography (5–25% EtOAc in petroleum ether) yielded the title
compound as a colourless oil (7.55 g, 10.5 mmol, 97%). Rf = 0.63
(30% acetone in petroleum ether); ½a D22
ꢄ
+56 (c 1.0 CHCl3); 1H
NMR (400 MHz, CDCl3) d = 8.03–7.99 (m, 4H, Harom), 7.80–7.75
(m, 4H, Harom), 7.54–7.38 (m, 15H, Harom), 7.31–7.25 (m, 4H, Harom),
5.52 (t, 1H, J = 9.2 Hz, H-3), 5.45 (t, 1H, J = 9.2 Hz, H-2), 4.97 (d, 1H,
J = 9.6 Hz, H-1), 4.04–4.10 (m, 3H, H-4, H-6a, H-6b), 3.71 (m, 1H,
H-5), 3.17
(br s, 1H, OH), 1.13 (s, 9H, CH3-tBu), 13C NMR
4.1.2. 4-Methylumbelliferyl 2-acetamido-2-deoxy-4,6-O- di-tert
(101 MHz, CDCl3) d = 167.4, 165.4 (C@OBz ꢃ2), 135.8 (ꢃ2), 133.6,
133.4 (CHarom ꢃ4), 132.9 (Cq-arom), 132.7(CHarom), 132.6 (Cq-arom),
130.1, 130.00, 129.95 (CHarom ꢃ3), 129.5, 129.1 (Cq-arom ꢃ2),
129.0, 128.5, 128.1, 128.0 (CHarom ꢃ4), 86.2 (C-1), 80.1 (C-5), 78.2
(C-3), 70.5, 70.2 (C-2, C-4), 64.2 (C-6), 27.0 (CH3-tBu), 19.4 (Cq-tBu);
-butylsilanediyl-b-D-glucopyranoside (3)
Peracetylated N-acetyl glucosamine
2
(4.0 g, 7.91 mmol,
1.0 equiv) was dissolved in MeOH/CH2Cl2 1:1 (250 mL) and so-
dium methoxide (30% in MeOH) (0.33 mL, 2.37 mmol, 0.3 equiv)
was added and the reaction was left stirring at room temperature
for 4 h. The reaction was then quenched with Amberlite-H+, fil-
tered and concentrated in vacuo. The product was obtained with-
out further purification in >95% purity according to 1H NMR
(2.88 g, 7.59 mmol, 96%). 1H NMR (400 MHz, CDCl3): d = 7.65 (d,
1H, J = 8.7 Hz, H-54-MU), 7.05–6.98 (m, 2H, H-64-MU and H-84-MU),
6.20 (d, 1H, J = 1.0 Hz, H-34-MU), 5.18 (d, 1H, J = 8.4 Hz, H-1),
4.00–3.91 (m, 2H, H-2 and H-3), 3.76 (dd, 1H, J = 12.0, 5.0 Hz,
H-6a), 3.61 (dd, 1H, J = 10.4, 8.3 Hz, H-4), 3.53–3.45 (m, 2H, H-5
and H-6b), 2.46 (d, 3H, J = 1.0 Hz, CH3-4-MU), 2.00 (s, 3H, CH3-
NAc); IR (neat): 3298, 2924, 2890, 1717, 1616, 1539, 1290, 1081,
IR (neat): 3494, 2930, 2858, 1728, 1067, 1275 734, 701, 502 cmꢀ1
HRMS calcd for [C42H42O7SSi+Na]+: 741.2313, found 741.2313.
;
4.1.4. Phenyl 2,3-di-O-benzoyl-6-(tert-butyldiphenylsilyl)-3-
deoxy-1-thio-b-D-glucopyranoside (5)
Phenyl thioglycoside 4 (6.7 g, 9.3 mmol, 1.0 equiv) and thi-
ocarbonyldiimidazole (2.49 g, 14.0 mmol, 1.5 equiv) were dis-
solved in dry toluene (120 mL). The mixture was heated for 5 h
at 90 °C and then cooled down to room temperature. The reaction
was washed with satd aq NaHCO3 (200 mL) and brine (200 mL).
The organics were dried with MgSO4, filtered and concentrated in
vacuo. Purification by column chromatography (30–50% EtOAc in
petroleum ether) yielded the 4-thiocarbonylimidazole compound
as an off-white solid (6.88 g, 8.3 mmol, 89%). 1H NMR (400 MHz,
CDCl3): d = 8.16 (s, 1H, H-2imidazole), 7.95 (d, 2H, J = 8.1 Hz, Harom),
7.80 (d, 2H, J = 8.1 Hz, Harom), 7.70 (dd, 2H, J = 8.0, 1.3 Hz, Harom),
7.62 (dd, 1H, J = 8.0, 1.3 Hz, Harom), 7.55–7.48 (m, 3H, J = 6.8 Hz,
H-5imidazole and Harom ꢃ2), 7.46–7.22 (m, 17H, Harom), 6.97 (dd,
1H, J = 1.6, 0.7 Hz, H-4imidazole), 6.21 (t, 1H, J = 9.6 Hz, H-4), 5.96
(t, 1H, J = 9.5 Hz, H-3), 5.54 (t, 1H, J = 9.8 Hz, H-2), 5.09 (d, 1H,
J = 10.0 Hz, H-1), 3.95 (m, 1H, H-5), 3.91 (dd, 1H, J = 11.8, 2.4 Hz,
H-6a) 3.85 (dd, 1H J = 11.8, 4.7 Hz, H-6b), 1.07 (s, 9H, CH3-tBu); 13C
NMR (101 MHz, CDCl3) d = 182.4 (C@S), 165.7, 164.9 (C@OBz ꢃ2),
137.0, 135.6, 135.4, 133.42, 133.35, 132.7 (CHarom ꢃ6), 132.5,
132.2 (Cq-arom ꢃ2), 130.9 (CHimidazole), 129.82, 129.78, 129.0,
128.4, 128.3, 128.2, 127.7 (CHarom ꢃ7), 118.0 (CHimidazole), 86.5
(C-1), 78.9 (C-5), 76.5 (C-4), 74.2 (C-3), 70.2 (C-2), 62.7 (C-6),
26.7 (CH3-tBu), 19.1 (Cq-tBu); IR (neat): 3070, 2934, 2860, 1734,
1393, 1270, 1221, 1068, 1026, 985, 703 cmꢀ1; HRMS calcd for
[C46H44N2O7S2Si+H]+: 829.2432, found 829.2439. The 4-thiocarb-
onylimidazole derivative (6.88 g, 8.3 mmol, 1.0 equiv) was coevap-
orated with dry toluene two times to remove traces of water and
then dissolved in anhydrous toluene (100 mL). Bu3SnH (5.54 mL,
20.8 mmol, 2.5 equiv) and AIBN (0.20 g, 1.2 mmol, 0.15 equiv)
were added at 90 °C. The reaction was stirred at this temperature
for 2 h and then cooled down, before being washed with satd aq
NaHCO3 (100 mL) and brine (100 mL). The organics were dried
with MgSO4, filtered and concentrated in vacuo. Purification by
column chromatography (30% EtOAc in petroleum ether) gave 4-
deoxy glucose derivate 5 as a colourless oil (4.85 g, 6.90 mmol,
1042, 853, 628 cmꢀ1
;
HRMS calcd for [C18H21NO8+H]+:
380.1340, found 380.1341. 4-Methylumbelliferyl glycoside
(2.8 g, 7.38 mmol, 1.05 equiv) was coevaporated once in anhy-
drous DMF and then dissolved in anhydrous DMF (140 mL). The
mixture was cooled to ꢀ40 °C, before drop-wise addition of di-
tert-butylsilanediyl bistriflate (2.28 mL, 7.03 mmol, 1.0 equiv).
The reaction was stirred for 30 min at ꢀ40 °C and pyridine was
added (1.7 mL, 21.1 mmol, 3.0 equiv). The reaction was stirred
an additional 15 min and then transferred to an extraction funnel
with diethyether (400 mL). The organics were washed with water
(2ꢃ 400 mL) and brine (350 mL) The aqueous layers were ex-
tracted with ether (400 mL) and the combined organics were
dried (Na2SO4), filtered and concentrated in vacuo. The residue
was purified by column chromatography (80–100% EtOAc in
petroleum ether), giving the title compound as a white solid
(3.58 g, 6.89 mmol, 98%). Rf = 0.41 (EtOAc); 1H NMR (400 MHz,
CDCl3): d = 7.41 (d, 1H, J = 8.8 Hz, H-54-MU), 7.23 (d, 1H,
J = 7.6 Hz, NH), 6.91 (dd, 1H, J = 8.8, 2.0 Hz, H-64-MU), 6.87 (d,
1H, J = 2.0 Hz, H-84-MU), 6.04 (s, 1H, H-34-MU), 5.48 (d, 1H,
J = 7.9 Hz, H-1), 4.20 (dd, 1H, J = 10.1, 4.9 Hz, H-6a), 4.06 (s, 1H,
3-OH), 4.04–3.98 (m, 3H, H-2, H-3, H-6b), 3.85 (dd, 1H, J = 9.5,
8.1 Hz, H-4), 3.60 (ddd, 1H, J = 9.7, 9.5, 5.3 Hz, H-5), 2.33 (s, 3H,
CH3-4-MU), 2.09 (s, 3H, CH3-NAc), 1.06 (s, 9H, CH3-tBu-Si), 0.99 (s,
9H, CH3-tBu-Si); 13C NMR (100 MHz, CDCl3): d = 13
C
NMR
(100 MHz, Aceton) d 171.8 (C@ONAc), 161.1, 159.7, 154.4, 152.6
(C-24-MU, C-74-MU, C-94-MU and C-104-MU), 125.4 (C-54-MU), 114.6
(C-44-MU), 114.0 (C-64-MU), 112.2 (C-34-MU), 103.5 (C-84-MU), 98.1
(C-1), 77.2 (C-4), 74.3 (C-3), 70.5 (C-5), 66.0 (C-6), 56.3(C-2),
27.3, 26.8 (CH3-tBu-Si ꢃ2), 23.3 (Cq-tBu-Si), 22.5 (CH3-NAc), 19.8
(CH3-4-MU), 18.4 (Cq-tBu-Si); IR (neat): 3308, 2936, 2861, 1718,
83%). ½a 2D2
ꢄ
+60 (c 1.0 CHCl3); 1H NMR (500 MHz, CDCl3) d = 7.99

1472
H. Gold et al. / Carbohydrate Research 346 (2011) 1467–1478
(d, 2H, J = 7.8 Hz, Harom), 7.94 (d, 2H, J = 7.8 Hz, Harom), 7.74–7.67
(m, 4H, Harom), 7.51–7.45 (m, 4H, Harom), 7.43–7.32 (m, 10H, Harom),
7.25–7.20 (m, 3H, Harom), 5.43–5.35 (m, 2H, H-2, H-3), 4.94 (d, 1H,
J = 9.4 Hz, H-1), 3.89–3.83 (m, 2H, H-5, H-6a), 3.76 (m, 1H, H-6b),
2.44 (dd, 1H, J = 12.4, 3.4 Hz, H-4eq), 1.86 (dd, 1H, J = 12.4,
11.5 Hz, H-4ax), 1.14 (s, 9H, CH3-tBu), 13C NMR (126 MHz, CDCl3)
d = 165.81, 165.37 (C@OBz ꢃ2), 135.60, 135.58, 133.12, 133.10
(CHarom ꢃ4), 133.01 (Cq-arom), 132.30, 129.74, 129.68 (CHarom ꢃ3),
129.57, 129.43 (Cq-arom ꢃ2), 128.80, 128.30, 127.72, 127.52 (CHarom
ꢃ4), 86.4 (C-1), 76.6 (C-5), 73.2 (C-3), 71.2 (C-2), 66.0 (C-6), 32.9
(C-4), 26.8 (CH3-tBu), 19.2 (Cq-tBu); IR (neat): 2931, 2857, 1718,
1451, 1428, 1274, 1070, 1027, 908, 733, 702, cmꢀ1; HRMS calcd
for [C42H42O6SSi+Na]+: 725.2364, found 725.2364.
(CH3-COOMe), 33.35 (C-4); IR (neat): 2954, 1718, 1451, 1272, 1026,
906, 728, 706 cmꢀ1; HRMS calcd for [C27H24O7S+Na]+: 515.1135,
found 515.1130.
4.1.6. Methyl (2,3-di-O-benzoyl-4-deoxy-a/b-D-glucopyranose)
uronate (7)
To a solution of methyl (phenyl thioglucopyranoside)uronate 6
(0.50 g, 1.02 mmol, 1.0 equiv) in CH2Cl2 (8 mL), N-iodosuccinimide
(0.25 g, 1.12 mmol, 1.1 equiv) and TFA (86 lL, 1.12 mmol,
1.1 equiv) were added at 0 °C. The reaction was left stirring at
0 °C (2.5 h) and then quenched with sodium thiosulphate (20%
aq) (10 mL). The reaction mixture was transferred to an extraction
funnel with EtOAc (40 mL) and washed with water (40 mL), satd aq
NaHCO3 (40 mL) and brine (40 mL). The aqueous layers were ex-
tracted with EtOAc (40 mL) and the combined organics were dried
with MgSO4, filtered and concentrated in vacuo. Purification by
column chromatography (20–40% EtOAc in petroleum ether)
yielded the title compound as a white solid (0.39 g, 0.98 mmol,
4.1.5. Methyl (phenyl 2,3-di-O-benzoyl-3-deoxy-1-thio-b-D-
glucopyranoside) uronate (6)
To
a solution of 4-deoxy phenyl thioglycoside 5 (3.66 g,
5.2 mmol, 1.0 equiv) in THF (40 mL) TBAF (1 M in THF) (10.4 mL,
10.4 mmol, 2.0 equiv) was added at room temperature. The reac-
tion was stirred for 2 h and then dissolved in EtOAc (200 mL) and
washed with satd aq NaHCO3 (250 mL) and brine (200 mL). The
aqueous layers were extracted with EtOAc (200 mL) and the com-
bined organics were dried (Na2SO4) and concentrated in vacuo.
Purification by column chromatography (20–50% EtOAc in petro-
96%, a a-isomer: d = 8.07–7.95
/b = 4:1) 1H NMR (400 MHz, CDCl3)
(m, 4H, Harom), 7.53–7.46 (m, 2H, Harom), 7.40–7.35 (m, 4H, Harom),
5.83 (ddd, 1H, J = 11.2, 9.9, 5.2 Hz, H-3), 5.80 (br t, 1H, J = 3.5 Hz, H-
1), 5.31 (dd, 1H, J = 9.9, 3.2 Hz, H-2), 4.89 (dd, 1H, J = 12.0, 2.7 Hz,
H-5), 4.42 (d, 1H, J = 3.5 Hz, OH), 3.76 (s, 3H, CH3-COOMe), 2.75
(ddd, 1H, J = 12.8, 5.0, 2.7 Hz, H-4eq), 2.00 (ddd, 1H, J = 12.8, 12.0,
leum ether) produced the title compound as
a
white solid
11.2 Hz, H-4ax); 13C NMR (101 MHz, CDCl3)
a-isomer: d = 170.8
(2.28 g, 4.91 mmol, 94%). ½a D22
ꢄ
+101 (c 1.0 CHCl3); 1H NMR
(C@OCOOMe), 165.9, 165.7 (C@OOac ꢃ2), 133.3, 133.2, 129.8, 129.6
(CHarom ꢃ4), 129.4, 129.2 (Cq-arom ꢃ2), 128.3 (CHarom), 91.2 (C-1),
72.1 (C-2), 67.8 (C-3), 66.2 (C-5), 52.5 (CH3-COOMe), 33.1 (C-4).
The b-isomer: 1H NMR (400 MHz, CDCl3) d = 8.13–7.95 (m, 4H,
(500 MHz, CDCl3) d = 7.99 (d, 2H, J = 7.4 Hz, Harom), 7.92 (d, 2H,
J = 7.4 Hz, Harom), 7.55–7.45 (m, 4H, Harom), 7.41–7.33 (m, 4H, Harom),
7.32–7.28 (m, 3H, Harom), 5.44–5.36 (m, 2H, H-2, H-3), 4.96
(d, 1H, J = 10.0 Hz, H-1), 3.85 (m, 1H, H-5), 3.76 (ddd, 1H, J = 11.4,
7.8, 3.2 Hz, H-6a), 3.68 (dt, 1H, J = 11.4, 5.9 Hz, H-6a), 2.33 (m, 1H,
H-4eq), 2.16 (dd, 1H, J = 7.8, 5.9 Hz, OH), 1.82 (m, 1H, H-4ax); 13C
NMR (126 MHz, CDCl3) d = 165.8, 165.4 (C@OBz ꢃ2), 132.6, 133.4
(CHarom ꢃ2), 132.3 (Cq-aorm), 129.8, 129.7 (CHarom ꢃ2), 129.5, 129.4
(Cq-aorm ꢃ2), 128.2, 128.5, 129.1 (CHaromꢃ3), 86.3 (C-1), 76.6 (C-5),
73.0 (C-3), 71.2 (C-2), 65.0 (C-6), 32.3 (C-4); IR (neat): 3513, 2931,
2872, 1718, 1451, 1274, 1068, 1026, 908, 748, 705 cmꢀ1; HRMS
calcd for [C26H24O6S+Na]+: 487.1186, found 487.1183. A solution of
the 6-hydroxyl phenyl thioglucoside (2.02 g, 4.35 mmol, 1.0 equiv),
TEMPO (136 mg, 0.87 mmol, 0.2 equiv) and BAIB (3.5 g, 10.9 mmol,
2.5 equiv), in CH2Cl2/H2O 2:1 (45 mL) was vigorously stirred for
1 h. The reaction was then quenched with Na2S2O3 (10% aq)
(150 mL) and the mixture was extracted with EtOAc (2ꢃ 150 mL).
The organics were washed with water (150 mL) and brine
(150 mL), before being dried (MgSO4), filtered and concentrated in
vacuo. The crude mixture was co-evaporated with toluene two times
to remove traces of water and acetic acid and dissolved in CH2Cl2/
MeOH 2:1 (45 mL). Trimethylsilyl diazomethane (2 M in diethyl
ether) (6.5 mL, 13.0 mmol, 3.0 equiv) was added until the solution
remained orange. The reaction was then quenched with AcOH
(5 mL), diluted with EtOAc (100 mL), washed with satd aq NaHCO3
(2ꢃ 100 mL) and brine (100 mL). The aqueous layers were extracted
with EtOAc (100 mL) and the combined organics were dried with
MgSO4, filtered and concentrated in vacuo. Purification by column
chromatography (20–40% EtOAc in petroleum ether) yielded the
title methyl uronate as a white solid (1.92 g, 4.06 mmol, 90%).
Harom), 7.53–7.46 (m, 2H, Harom), 7.40 - 7.35 (m, 4H, Harom), 5.48
(m, 1H, H-3), 5.30 (m, 1H, H-2), 4.97 (br t, 1H, J = 7.2 Hz, H-1),
4.54 (br d, 1H, J = 7.8 Hz, OH), 4.37 (dd, 1H, J = 11.9, 2.5 Hz, H-5),
3.76 (s, 3H, CH3-COOMe), 2.72 (ddd, 1H, J = 12.9, 5.3, 2.5 Hz, H-4eq),
2.04 (m, 1H, H-4ax); 13C NMR (101 MHz, CDCl3) d = 169.6
(C@OCOOMe), 166.4, 165.7 (C@OOac ꢃ2), 133.4, 133.3, 129.8, 129.7
(CHarom ꢃ4), 129.4, 129.0 (Cq-arom ꢃ2), 128.3 (CHarom), 95.9 (C-1),
74.2 (C-2), 70.4 (C-3), 70.3 (C-5), 52.7 (CH3-COOMe), 33.0 (C-4); IR
(neat): 3440, 2956, 1718, 1451, 1261, 1069, 909, 707 cmꢀ1; HRMS
calcd for [C21H20O8+Na]+: 423.1050, found 423.1048.
4.1.7. Methyl (phenyl 2,3-di-O-benzoyl-4-O-methyl-1-thio-b-D-
glucopyranoside) uronate (9)
A solution of diol 817 (1.64 g, 3.41 mmol, 1.0 equiv), TEMPO
(0.10 g, 0.66 mmol, 0.2 equiv) and BAIB (2.64 g, 8.2 mmol,
2.5 equiv) in CH2Cl2 (30 mL) and H2O (15 mL) was stirred vigor-
ously. The reaction was quenched after 2.5 h with Na2S2O3 (10%
aq) (100 mL). The mixture was diluted with EtOAc (50 mL) and
washed with brine (50 mL). The organics were dried with MgSO4,
filtered and concentrated in vacuo. The crude mixture was coevap-
orated with toluene two times to remove traces of water and acetic
acid, dissolved in CH2Cl2/MeOH 2:1 (50 mL). Trimethylsilyldia-
zomethane (2 M in diethyl ether) (4.9 mL, 9.84 mmol, 3.0 equiv)
was added until the solution remained orange. The reaction was
quenched with AcOH (2 mL), diluted with EtOAc (50 mL), washed
with aq satd NaHCO3 (2ꢃ 100 mL) and brine (100 mL). The organ-
ics were dried with MgSO4, filtered and concentrated in vacuo.
Purification by column chromatography (20–40% EtOAc in petro-
½
a 2D2 +78 (c 1.0 CHCl3); 1H NMR (400 MHz, CDCl3) d = 7.99
ꢄ
(d, 2H, J = 7,9 Hz, Harom), 7.93 (m, 2H, J = 7,9 Hz, Harom), 7.55–7.49
(m, 4H, Harom), 7.40 (t, 2H, J = 7.8 Hz, Harom), 7.36 (t, 2H, J = 7.8 Hz,
Harom), 7.32–7.29 (m, 3H, Harom), 5.42–5.37 (m, 2H, H-2, H-3),
4.93 (d, 1H, J = 9.4 Hz, H-1), 4.34 (dd, 1H, J = 12.1, 2.2 Hz, H-5),
3.82 (s, 3H, CH3-COOMe), 2.73 (m, 1H, H-4eq), 2.03 (m, 1H, H-4ax);
13C NMR (126 MHz, CDCl3) d 168.8 (C@OCOOMe), 165.7, 165.2
(C@OBz ꢃ2), 133.30, 133.25 (CHarom ꢃ2), 131.94 (Cq-arom), 129.77,
129.72 (CHarom ꢃ2), 129.4, 129.1 (Cq-arom ꢃ2), 128.9, 128.37, 128.32
(CHarom ꢃ3), 86.8 (C-1), 74.0 (C-5), 72.3 (C-2), 70.5 (C-3), 52.6
leum ether) produced the title compound as a white solid
(1.27 g, 2.50 mmol, 73% over two steps). Rf = 0.13 (20% EtOAc in
petroleum ether); NMR data are in accordance with literature pre-
cedence.15 IR (neat): 2463, 3058, 2959, 1718, 1451, 1258, 1216,
1064, 708 cmꢀ1; HRMS calcd for [C27H24O8S+Na]+: 531.1084, found
531.1081. To a solution of the methyl (glucopyranoside)uronate
(0.51 g, 1.0 mmol, 1.0 equiv) in dry CH2Cl2 (5 mL), BF3ꢁOEt2
(0.25 mL, 2.0 mmol, 2.0 equiv) and trimethylsilyldiazomethane
(2 M in diethyl ether) (1.0 mL, 2.0 mmol, 2.0 equiv) were added

H. Gold et al. / Carbohydrate Research 346 (2011) 1467–1478
1473
at ꢀ40 °C. The reaction was allowed to warm to ambient tempera-
ture in 2 h. The reaction was then quenched with acetic acid
(0.5 mL), diluted with CH2Cl2 (20 mL) and washed with aq satd
NaHCO3 (2ꢃ 50 mL) and brine (50 mL). The organics were dried
(MgSO4), filtered and concentrated in vacuo. Purification by col-
umn chromatography (20% EtOAc in petroleum ether) afforded
the title compound 9 as a white solid (254 mg, 0.49 mmol, 49%,
92% based on recovered starting material). Rf = 0.45 (30% EtOAc
in petroleum ether); 1H NMR (400 MHz, CDCl3): d = 7.99–7.91 (m,
4H, Harom), 7.54–7.49 (m, 5H, Harom), 7.42–7.31 (m, 6H, Harom),
5.68 (t, 1H, J = 9.2 Hz, H-3), 5.38 (t, 1H, J = 9.6 Hz, H-2), 4.98 (d,
1H, J = 9.6 Hz, H-1), 4.10 (d, 1H, J = 9.6 Hz, H-5), 3.92 (t, 1H,
J = 9.2 Hz, H-4), 3.89 (s, 3H, CH3-COOMe), 3.42 (s, 3H, CH3-OMe); 13C
NMR (100 MHz, CDCl3): d = 168.0 (C@OCOOMe), 165.5 (C@OBz),
165.1 (C@OBz), 133.3, 133.2 (CHarom ꢃ2), 131.8 (C Cq-arom), 130.0,
129.8, 129.7 (CHarom ꢃ3), 129.1 (Cq-arom), 128.9, 128.3 (CHarom
ꢃ2), 86.8 (C-1), 78.9 (C-4), 77.8 (C-5), 75.5 (C-3), 70.2 (C-2), 52.7
(CH3-COOMe), 60.4 (CH3-OMe); IR (neat): 3070, 2951, 1747, 1733,
1718, 1452, 1264, 1065, 1022, 709, 686 cmꢀ1; HRMS calcd for
[C28H26O8S+Na]+: 545.1241, found 545.1236.
with EtOAc (40 mL) and washed with satd aq NaHCO3 (40 mL) and
brine (40 mL). The aqeous layers were extracted with EtOAc
(40 mL) and the combined organics were dried (Na2SO4), filtered
and concentrated in vacuo. Purification by column chromatogra-
phy (10–50% EtOAc in petroleum ether) produced the title com-
pound as a white solid (0.30 g, 0.34 mmol, 82%). Rf = 0.50 (30%
EtOAc in CH2Cl2); ½a D22
ꢄ
+61 (c 0.5 CHCl3); 1H NMR (500 MHz, CDCl3)
d = 7.99–7.96 (m, 2H, Harom), 7.94–7.89 (m, 2H, Harom), 7.53–7.48
(m, 2H, Harom), 7.43 (d, 1H, J = 8.5 Hz, H-54-MU), 7.41–7.34 (m, 4H,
Harom), 6.85 (dd, 1H, J = 8.5, 2.4 Hz, H-64-MU), 6.84 (d, 1H,
J = 2.4 Hz, H-84-MU), 6.12 (d, 1H, J = 1.1 Hz, H-34-MU), 5.98 (d, 1H,
J = 7.2 Hz, NH), 5.82 (d, 1H, J = 8.2 Hz, H-1), 5.44 (dd, 1H, J = 9.4,
7.5 Hz, H-20), 5.34 (ddd, 1H, J = 11.4, 9.4, 5.2 Hz, H-30), 5.20 (d,
1H, J = 7.5 Hz, H-10), 4.45 (dd, 1H, J = 9.8, 8.8 Hz, H-3), 4.31 (dd,
1H, J = 12.1, 2.2 Hz, C-50), 4.19 (dd, 1H, J = 10.3, 4.9 Hz, C-6eq),
4.10 (t, 1H, J = 9.0 Hz, H-4), 3.93 (dd, 1H, J = 10.3, 9.8 Hz, H-6ax),
3.79 (s, 3H, CH3-COOMe), 3.65 (ddd, 1H, J = 9.8, 9.0, 4.9 Hz, H-5),
3.47 (ddd, 1H, J = 9.8, 8.2, 7.2 Hz, H-2), 2.70 (ddd, 1H, J = 12.6, 5.2,
2.2 Hz, H-40eq), 2.35 (d, 3H, J = 1.1 Hz, CH3-4MU), 2.05 (ddd, 1H,
J = 12.6, 12.1, 11.4 Hz, H-40ax), 1.56 (s, 3H, CH3-NAc), 1.05 (s, 9H,
CH3-tBu-Si), 1.03 (s, 9H, CH3-tBu-Si); 13C NMR (126 MHz, CDCl3)
d = 170.8 (C@ONAc), 169.2 (C@OCOOMe), 165.7, 165.4 (C@OOBz ꢃ2),
160.9, 159.6, 154.6, 152.5 (C-24-MU, C-74-MU, C-94-MU and C-
104-MU), 133.4, 133.3, 129.7, 129.6 (CHarom ꢃ4), 129.2, 129.0
(Cq-arom ꢃ2), 128.5, 128.3 (CHarom ꢃ2), 125.5 (C-54-MU), 115.1
(C-44-MU), 113.5 (C-64-MU), 112.4 (C-34-MU), 104.2 (C-84-MU), 99.4
(C-10), 97.0 (C-1), 79.7 (C-3), 76.4 (C-4), 73.3 (C-20), 71.4 (C-30),
70.6 (C-5), 70.3v (C-50), 66.0 (C-6), 57.4 (C-2), 52.5 (CH3-COOMe),
33.2 (C-40), 27.3, 26.9 (CH3-tBu-Si ꢃ2), 23.0 (CH3-NAc), 22.5, 19.9
(Cq-tBu-Si ꢃ2), 18.6 (CH3-4MU); HRMS calcd for [C47H55NO15Si+Na]+:
924.3233, found 924.3237.
4.1.8. Methyl (2,3-di-O-benzoyl-4-O-methyl-a/b-D-
glucopyranose) uronate (10)
To a solution of methyl (phenyl thioglucoside)uronate 9 (0.23 g,
0.43 mmol, 1.0 equiv) in CH2Cl2 (8 mL), N-iodosuccinimide (0.10 g,
0.45 mmol, 1.05 equiv) and TFA (35 lL, 0.45 mmol, 1.05 equiv)
were added at 0 °C. The reaction was left stirring at room temper-
ature (2.5 h) and then quenched with sodium thiosulphate (20%
aq) (2.0 mL). The reaction mixture was transferred to an extraction
funnel with EtOAc (40 mL) and washed with satd aq NaHCO3
(40 mL) and brine (30 mL). The aqueous layers were extracted with
EtOAc (40 mL) and the combined organics were dried with MgSO4,
filtered and concentrated in vacuo. Purification by column chroma-
tography (10–30% EtOAc in petroleum ether) yielded the title com-
4.1.10. 4-Methylumbelliferyl 2-acetamido-2-deoxy-3-O-(methyl
2,3-di-O-benzoyl-4-O-methyl-b-D-glucopyranosyl uronate)-4,6-
pound in a 10:1
Rf = 0.45 (30% EtOAc in petroleum ether); NMR assignment for
major isomer (
1H NMR (400 MHz, CDCl3): d = 8.04–8.00 (m,
a
/b-ratio as a white solid (0.14 g, 0.32 mmol, 75%).
O-di-tert-butylsilanediyl-b- -glucopyranoside (12)
D
Hemiacetal 10 (0.14 g, 0.32 mmol, 1.2 equiv) and diphenyl sulf-
oxide (0.15 g, 0.75 mmol, 2.8 equiv) were coevaporated two times
with anhydrous toluene, dissolved in anhydrous CH2Cl2 (6 mL) and
stirred with flame dried molecular sieves (3 Å) for 30 min. The
mixture was then cooled to ꢀ60 °C and trifluoromethanesulfonic
a)
2H, Harom), 7.99–7.95 (m, 2H, Harom), 7.56–7.47 (m, 2H, Harom),
7.43–7.33 (m, 4H, Harom), 6.00 (t, 1H, J = 9.5 Hz, H-3), 5.70 (dd,
1H, J = 3.7, 3.4 Hz, H-1), 5.18 (dd, 1H, J = 9.5, 3.4 Hz, H-2), 4.61
(d, 1H, J = 9.7 Hz, H-5), 3.88 (dd, 1H, J = 9.7, 9.5 Hz, H-4), 3.83
(s, 3H, CH3-COOMe), 3.43 (s, 3H, CH3-OMe); 13C NMR (101 MHz, CDCl3)
d 169.5 (C@OCOOMe), 165.9, 165.5 (C@OBz ꢃ2), 133.4, 133.3, 129.9,
129.7 (CHarom ꢃ4), 129.5 (Cq-arom), 128.4 (CHarom), 90.7 (C-1),
79.1 (C-4), 71.8 (C-2), 71.4 (C-3), 67.1 (C-5), 60.3 (CH3-OMe), 52.8
(CH3-COOMe); IR (neat): 3440, 2955, 2849, 1725, 1452, 1265, 1108,
1069, 709 cmꢀ1; HRMS calcd for [C22H22O9+Na]+: 453.1156, found
453.1152.
anhydride (65 lL, 0.39 mmol, 1.44 equiv) was added. The reaction
mixture was allowed to warm to ꢀ20 °C and left stirring at this
temp for 1 h. N-Acetylglucosamine acceptor 3 (0.14 g, 0.27 mmol,
1.0 equiv) was coevaporated two times with anhydrous toluene
(with a few drop of anhydrous CH2Cl2) and then dissolved in anhy-
drous CH2Cl2 (3 mL) before addition to the activated donor. The
reaction was warmed to ꢂ0 °C and left stirring at this temperature
over night. The reaction was then quenched with Et3N (0.19 mL,
1.35 mmol, 5.0 equiv), transferred to an extraction funnel with
EtOAc (40 mL) and washed with satd aq NaHCO3 (40 mL) and brine
(40 mL). The aqeous layers were extracted with EtOAc (40 mL) and
the combined organics were dried (Na2SO4), filtered and concen-
trated in vacuo. Purification by column chromatography (10–50%
EtOAc in petroleum ether) produced the title compound as a white
solid (132 mg, 0.14 mmol, 53%). Rf = 0.54 (30% EtOAc in CH2Cl2);
4.1.9. 4-Methylumbelliferyl 2-acetamido-2-deoxy-3-O-(methyl
2,3-di-O-benzoyl-4-deoxy-b-D-glucopyranosyl uronate)-4,6-O-
di-tert-butylsilanediyl-b- -glucopyranoside (11)
D
Hemiacetal 7 (0.20 g, 0.50 mmol, 1.2 equiv) and diphenyl sulf-
oxide (0.23 g, 1.15 mmol, 2.8 equiv) were coevaporated two times
with anhydrous toluene, dissolved in anhydrous CH2Cl2 (10 mL)
and stirred with flame dried molecular sieves (3 Å) for 30 min.
The mixture was then cooled to ꢀ60 °C and trifluoromethanesulfo-
nic anhydride (0.10 mL, 0.62 mmol, 1.44 equiv) was added. The
reaction mixture was allowed to warm to ꢀ20 °C and left stirring
at this temp for 1 h. N-Acetylglucosamine acceptor 3 (0.21 g,
0.41 mmol, 1.0 equiv) was coevaporated two times with anhydrous
toluene (with a drop of anhydrous CH2Cl2) and then dissolved in
anhydrous CH2Cl2 (5 mL) before addition to the activated donor.
The reaction was warmed to ꢂ0 °C and left stirring at this temper-
ature over night. The reaction was then quenched with Et3N
(0.29 mL, 2.1 mmol, 5.0 equiv), transferred to an extraction funnel
½
a 2D2 +45 (c 1.0 CHCl3); 1H NMR (400 MHz, CDCl3) d = 7.98–7.93
ꢄ
(m, 4H, Harom), 7.56 - 7.50 (m, 2H, Harom), 7.44 (d, 1H, J = 8.3 Hz,
H-54-MU), 7.42 - 7.36 (m, 4H, Harom), 6.84 (dd, 1H, J = 8.3, 2.3 Hz,
H-64-MU), 6.82 (d, 1H, J = 2.3 Hz, H-84-MU), 6.13 (d, 1H, J = 1.1 Hz,
H-34-MU), 5.74 (d, 1H, J = 8.2 Hz, H-1), 5.68 (d, 1H, J = 7.1 Hz, NH),
5.60 (t, 1H, J = 9.1 Hz, H-30), 5.40 (dd, 1H, J = 9.1, 7.5 Hz, H-20),
5.11 (d, 1H, J = 7.4 Hz, H-10), 4.41 (dd, 1H, J = 9.9, 8.6 Hz, H-3),
4.18 (dd, 1H, J = 10.3, 5.0 Hz, H-6eq), 4.10 (d, 1H, J = 9.1 Hz, C-50),
4.00–3.86 (m, 3H, C-4, C-6ax and C-40), 3.80 (s, 3H, CH3-COOMe),
3.61 (ddd, 1H, J = 9.9, 9.7, 5.0 Hz, H-5), 3.39 (s, 3H, CH3-OMe), 3.30
(ddd, 1H, J = 9.9, 8.2, 7.1 Hz, H-2), 2.36 (d, 3H, J = 1.1 Hz,

1474
H. Gold et al. / Carbohydrate Research 346 (2011) 1467–1478
CH3-4MU), 1.64 (s, 3H, CH3-NAc), 1.06 (s, 9H, CH3-tBu-Si), 1.01 (s, 9H,
CH3-tBu-Si); 13C NMR (101 MHz, CDCl3) d = 170.9 (C@ONAc), 168.5
(C@OCOOMe), 165.4, 165.2 (C@OOBz ꢃ2), 160.9 , 159.5, 154.6, 152.3
(C-24-MU, C-74-MU, C-94-MU and C-104-MU), 133.6, 133.3, 129.7,
129.7 (CHarom ꢃ4), 129.1, 129.0 (Cq-arom ꢃ2), 128.6, 128.4 (CHarom
ꢃ2), 125.6 (C-54-MU), 115.2 (C-44-MU), 113.4 (C-64-MU), 112.8
(C-34-MU), 104.3 (C-84-MU), 100.6 (C-10), 96.7 (C-1), 80.1 (C-3),
79.1 (C-40), 76.1 (C-4), 74.4 (C-50), 74.2 (C-30), 73.0 (C-20), 70.4
(C-5), 66.1 (C-6), 60.3 (CH3-OMe), 57.7 (C-2), 52.6 (CH3-COOMe),
27.3, 26.9 (CH3-tBu-Si ꢃ2), 23.2 (CH3-NAc), 22.5, 19.9 (Cq-tBu-Si ꢃ2),
18.6 (CH3-4MU); IR (neat): 2936, 2860, 1734, 1616, 1390, 1272,
according to the general procedure. Repeated lyofilization followed
by filtration over wet Amberlite-Na+ (2 mL) gave the title com-
pound as a white solid (19 mg, 32 l
mol, 63%). 1H NMR (500 MHz,
MeOD-d4) d = 7.72 (d, 1H, J = 8.7 Hz, H-54-MU), 7.07–7.02 (m, 2H,
H-64-MU and H-84-MU), 6.00 (d, 1H, J = 1.2 Hz, H-34-MU), 5.24 (d, 1H,
J = 8.5 Hz, H-1), 4.38 (d, 1H, J = 7.6 Hz, H-10), 4.10 (dd, 1H, J = 10.3,
8.5 Hz, H-2), 3.94 (dd, 1H, J = 12.2, 1.8 Hz, H-6a), 3.81 (dd, 1H,
J = 10.3, 7.9 Hz, H-3), 3.76 (dd, 1H, J = 12.2, 4.8 Hz, H-6b), 3.66
(d, 1H, J = 9.6 Hz, H-50), 3.58–3.51 (m, 5H, H-4, H-5 and CH3-OMe),
3.45 (t, 1H, J = 9.0 Hz, H-30), 3.36 (dd, 1H, J = 9.2, 7.6 Hz, H-20),
3.28 (t, 1H, J = 9.0 Hz, H-40), 2.46 (d, 3H, J = 1.2 Hz, CH3-4-MU),
2.00 (s, 3H, CH3-NAc). 13C NMR (126 MHz, MeOD-d4) d = 176.1
1090, 832, 709 cmꢀ1
;
HRMS calcd for [C48H57NO16Si+Na]+:
954.3339, found 954.3345.
(C@OCOO-Na+), 174.5 (C@ONAc), 163.2, 161.8, 156.0, 155.4 (C-24-MU
,
C-74-MU, C-94-MU and C-104-MU), 127.4 (C-54-MU), 116.2 (C-44-MU),
114.9 (C-64-MU), 113.0 (C-34-MU), 104.9 (C-84-MU), 104.8 (C-10),
100.1 (C-1), 83.93 (C-3), 83.85 (C-40), 78.2 (C-50), 78.1 (C-5), 77.2
(C-30), 74.4 (C-20), 70.3 (C-4), 62.4 (C-6), 60.7 (CH3-OMe), 55.9 (C-2),
23.2 (CH3-4MU), 18.6 (CH3-NAc); HRMS calcd for [C25H31NO14+Na]+:
592.1637, found 592.1634.
4.1.11. 4-Methylumbelliferyl 2-acetamido-2-deoxy-3-O-(4-
deoxy-b- -glucopyranosyl uronicacid)-b- -glucopyranoside
sodium salt (13)
Protected dimer 11 (130 mg, 0.14 mmol, 1.0 equiv) was dis-
solved in anhydrous MeOH (4 mL) and sodium methoxide (30%
D
D
in MeOH) (40 lL, 0.14 mmol, 1.0 equiv) was added under an atmo-
sphere of argon. The reaction was allowed to run over night at
ambient temperature and monitored by HPLC-MS. The reaction
was quenched with acetic acid (0.3 mL) and then coevaporated
with toluene. The residue was dissolved in pyridine (2 mL) and
4.1.13. Methyl (2,3-di-O-benzoyl-4-O-(4,6-O-di-tert-
butylsilanediyl-2-deoxy-3-O-levulinoyl-2-trichloroacetamido-
b-D-glucopyranosyl)-a/b-D-glucopyranoside N-Phenyl-2,2,2-
trifluoroacetimidate) uronate (16)
Et3N tris-hydrogenfluoride (47
lL, 0.29 mmol, 2.0 equiv) was
To a solution of disaccharide 1513 (4.50 g, 4.28 mmol, 1.0 equiv)
in CH2Cl2 (150 mL) N-iodosuccinimide (1.93 g, 8.56 mmol,
2.0 equiv) and trifluoroacetic acid (0.32 mL, 4.28 mmol, 1.0 equiv)
were added at 0 °C. After 2 h the mixture was quenched by addi-
tion of Et3N (1 mL) and transferred to an extraction funnel with
CH2Cl2 (100 mL). The organics were washed with satd aq NaHCO3
(300 mL) and brine (300 mL). The aqueous layers were then ex-
tracted with EtOAc (300 mL) and the combined organics were
dried (MgSO4), filtered and concentrated in vacuo. Purification by
column chromatography (10–40% EtOAc in petroleum ether)
added. The mixture was stirred for 6 h at ambient temperature,
while the reaction progress was monitored by HPLC-MS. Upon
completion, water (2 mL) and satd aq Na2CO3 (1 mL) were added
and the mixture stirred over night at ambient temperature. The
reaction progress was monitored by HPLC-MS and then concen-
trated in vacuo. The residue was purified by HPLC according to
the general procedure. Repeated lyofilization followed by filtration
over wet Amberlite-Na+ (3 mL) gave the title compound as a white
solid (62 mg, 0.11 mmol, 77%). 1H NMR (500 MHz, D2O) d = 7.42 (d,
1H, J = 8.9 Hz, H-54-MU), 6.88 (dd, 1H, J = 8.8, 2.1 Hz, H-64-MU), 6.79
(d, 1H, J = 2.3 Hz, H-84-MU), 6.00 (s, 1H, H-34-MU), 5.20 (d, 1H,
J = 8.5 Hz, H-1), 4.41 (d, 1H, J = 7.8 Hz, H-10), 4.11 (dd, 1H,
J = 10.4, 8.5 Hz, H-2), 3.98 (dd, 1H, J = 12.2, 2.2 Hz, H-50), 3.92 (dd,
1H, J = 12.6, 1.7 Hz, H-6a), 3.84 (dd, 1H, J = 10.5, 7.8 Hz, H-3), 3.79
(m, 1H, H-6b), 3.71 (m, 1H, H-5), 3.63 (m, 1H, H-4), 3.20 (dd, 1H,
J = 9.2, 7.9 Hz, H-20), 2.29–2.21 (m, 4H, H-40eq and CH3-4MU), 1.99
(s, 3H, CH3-NAc), 1.52 (q, 1H, J = 12.2 Hz, H-40ax). 13C NMR
(126 MHz, D2O) d = 178.3 (C@OCOO-Na+), 176.2 (C@ONAc), 165.4,
160.6, 157.2, 154.9 (C-24-MU, C-74-MU, C-94-MU and C-104-MU),
127.9 (C-54-MU), 116.4 (C-44-MU), 115.1 (C-64-MU), 112.6 (C-34-MU),
104.9 (C-84-MU), 104.4 (C-10), 99.8 (C-1), 83.9 (C-3), 77.1 (C-5),
75.7 (C-20), 73.7 (C-50), 71.7 (C-30), 69.8 (C-4), 61.8 (C-6), 55.6 (C-
2), 37.5 (C-40), 23.6 (CH3-4MU), 19.2 (CH3-NAc).
yielded the disaccharide hemiacetal as a white solid in a 4:1
ratio (3.68 g, 3.83 mmol, 89%). Rf = 0.19 (50% EtOAc in petroleum
ether); NMR assignment for the major isomer (
): 1H NMR
(400 MHz, CDCl3): d = 7.97–7.93 (m, 4H, Harom), 7.56–7.45 (m, 2H,
arom), 7.43–7.37 (m, 2H, Harom), 7.35–7.30 (m, 2H, Harom), 6.85
a/b-
a
H
(d, 1H, J = 8.9 Hz, NH), 5.97 (dd, 1H, J = 10.0, 8.9 Hz, H-3), 5.68 (d,
1H, J = 3.4 Hz, H-1), 5.21 (dd, 1H, J = 10.0, 3.4 Hz, H-2), 5.02 (dd,
1H, J = 10.7, 9.1 Hz, H-30), 4.97 (d, 1H, J = 8.3 Hz, H-10), 4.61 (d,
1H, J = 9.5 Hz, H-5), 4.21 (t, 1H, J = 9.2 Hz, H-4), 3.86–3.81 (m, 4H,
H-20 and CH3-COOMe), 3.61 (br s, 1H, OH), 3.56 (t, 1H, J = 9.2 Hz, H-
40), 3.49 (dd, 1H, J = 10.4, 5.0 Hz, H-60a), 3.25 (ddd, 1H, J = 9.9, 9.8,
5.0 Hz, H-50), 2.71–2.66 (m, 3H, H-6b0 and CH2-Lev), 2.57–2.54 (m, 2H,
CH2-Lev), 2.04 (s, 3H, CH3-Lev), 0.88 (s, 9H, CH3-tBu-Si), 0.87 (s, 9H,
CH3-tBu-Si); 13C NMR (100 MHz, CDCl3): d = 205.9 (C@OLev-ketone),
172.4 (C@OLev-ester), 170.1 (C@OCOOMe), 165.8, 165.1 (C@OBz ꢃ2),
161.7 (C@OTCA), 133.4, 133.1 (CHarom ꢃ2), 130.1 (Cq-arom), 129.9,
129.6 (CHarom ꢃ2), 128.9 (Cq-arom), 128.5, 128.4 (CHarom ꢃ2),
100.5 (C-10), 92.3 (Cq-CCl3), 90.5 (C-1), 76.7 (C-4), 74.4 (C-30), 74.2
(C-40), 71.0 (C-50), 70.6 (C-2), 69.7 (C-3), 69.5 (C-5), 65.0 (C-60),
55.8 (C-20), 53.1 (CH3-COOMe), 38.8 (CH2-Lev), 29.7 (CH3-Lev), 27.8
(CH2-Lev), 27.2, 26.7 (CH3-tBu-Si ꢃ2), 22.4, 19.7 (Cq-tBu-Si ꢃ2); IR
4.1.12. 4-Methylumbelliferyl 2-acetamido-2-deoxy-3-O-(4-O-
methyl-b-
D-glucopyranosyl uronicacid)-b-
D
-glucopyranoside
sodium salt (14)
Protected dimer 12 (48 mg, 51
lmol, 1.0 equiv) was dissolved in
anhydrous MeOH (2 mL) and sodium methoxide (30% in MeOH)
(7 L, 51 mol, 1.0 equiv) was added under an atmosphere of ar-
l
l
gon. The reaction mixture was stirred over night at ambient tem-
perature and the reaction progress was monitored by HPLC-MS.
The reaction was quenched with acetic acid (0.2 mL) and then
coevaporated with toluene. The residue was dissolved in pyridine
(neat): 3362, 2935, 2860, 1722, 1526, 1276, 1070, 827, 710 cmꢀ1
;
HRMS calcd for [C42H52Cl3NO16Si+Na]+: 982.2013, found
928.2018. To a solution of the disaccharide hemiacetal (3.68 g,
3.84 mmol, 1.0 equiv) in acetone (10 mL) cesium carbonate
(1.870 g, 5.76 mmol, 1.5 equiv) and N-phenyl-2,2,2-trifluoroace-
timidoyl chloride (0.88 mL, 5.76 mmol, 1.5 equiv) were added at
0 °C. After 1.5 h the mixture filtered was over Celite and concen-
trated in vacuo. Purification by column chromatography (0–15%
acetone in petroleum ether) produced the title disaccharide imi-
(2 mL) and Et3N tris-hydrogenfluoride (17 lL, 0.10 mmol,
2.0 equiv) was added. The reaction mixture was stirred for 6 h at
ambient temperature, while the reaction progress was monitored
by HPLC-MS. Upon completion water (2 mL) and satd aq Na2CO3
(1 mL) were added and the mixture was stirred over night at ambi-
ent temperature. The reaction was monitored by HPLC-MS and
then concentrated in vacuo. The residue was purified by HPLC
date as a colourless oil in a 1:3
a/b-ratio (3.712 g, 3.28 mmol,
85%). Rf = 0.22 (50% EtOAc in petroleum ether); 1H NMR

H. Gold et al. / Carbohydrate Research 346 (2011) 1467–1478
1475
(400 MHz, CDCl3): d = 8.01–7.93 (m, 4H, Harom), 7.56–7.50 (m, 2H,
(dd, 1H, J = 10.3, 10.0, H-6b), 3.84–3.79 (m, 4H, CH3-COOMe and
H-200), 3.67–3.50 (m, 3H, H-5, H-6a00 and H-400), 3.44 (ddd, 1H,
J = 10.1, 8.2, 7.5 Hz, H-2), 3.26 (td, 1H, J = 9.9, 4.9 Hz, H-500),
2.72–2.65 (m, 3H, CH2-Lev and H-6b00), 2.56 (t, 2H, J = 6.8 Hz,
CH2-Lev), 2.37 (d, 3H, J = 1.1 Hz, CH3-4-MU), 2.13 (s, 3H, CH3-Lev),
1.79 (s, 3H, CH3-NAc), 1.05 (s, 9H, CH3-tBu-Si), 0.98 (s, 9H,
CH3-tBu-Si), 0.89 (s, 9H, CH3-tBu-Si), 0.87 (s, 9H, CH3-tBu-Si); 13C NMR
(100 MHz, CDCl3): d = 205.8 (C@OLev-ketone), 172.3 (C@OLev-ester),
170.7 (C@ONAc), 169.3 (C@OCOOMe), 165.3, 165.1 (C@OBz ꢃ2),
Harom), 7.44–7.35 (m, 4H, Harom), 7.27 (t, 0.3H, J = 7.8 Hz, Harom-
), 7.16–7.07 (m, 2H, H-1, Harom-NPh- /b), 7.01 (t, 0.7H,
NPh-a
a
J = 7.4 Hz, Harom-NPh-b), 6.93 (d, 0.3H, J = 8.9 Hz, NH ), 6.92 (d,
a
0.7H, J = 8.7 Hz, NHb), 6.79 (br d, 0.7H, J = 8.0 Hz, H-1b), 6.77 (br s,
0.6H, Harom-NPh- ), 6.43 (br d, 1.4H, J = 6.2 Hz, Harom-NPh-b), 6.23
a
(br s, 0.3H, H-1 ), 5.95 (t, 0.7H, J = 9.6 Hz, H-3b), 5.74 (t, 0.3H,
a
J = 7.6 Hz, H-3 ), 5.56 (m, 1H, H-2b and H-2 ), 5.11 (dd, 0.7H,
a
a
J = 10.7, 9.0 Hz, H-30b), 5.06 (dd, 0.7H, J = 10.5, 8.8 Hz, H-30a), 5.03
(d, 0.7H, J = 8.2 Hz, H-10b), 4.98 (d, 0.3H, J = 8.2 Hz, H-10a), 4.54–
4.48 (m, 1H, H-5 and H-5b), 4.44 (t, 0.3H, J = 7.9 Hz, H-4 ), 4.38
161.7 (C-24-MU), 160.9 (C@OTCA), 159.5, 154.7, 152.3 (C-74-MU
C-94-MU and C-104-MU), 133.7, 133.2 (CHarom ꢃ2), 129.9 (Cq-arom),
129.7, 129.6 (CH
ꢃ2), 129.0 (Cq-arom), 128.6, 128.4 (CHarom
,
a
a
(t, 0.7H, J = 9.4 Hz, H-4b), 3.87 (s, 2.1H, CH3COOMe-b), 3.86–3.79 (m,
arom
1H, H-20a and H-20 ), 3.78 (s, 0.9H, CH3COOMe- ), 3.64–3.55 (m,
ꢃ2), 125.6 (C-54-MU), 115.2 (C-44-MU), 113.5 (C-64-MU), 112.9
(C-34-MU), 104.1 (C-84-MU), 100.5 (C-10), 100.4 (C-100), 97.2 (C-1),
92.4 (Cq-CCl3), 79.2 (C-3), 76.4 (C-4), 76.2 (C-40), 74.4 (C-400),
74.3 (C-300), 74.0 (C-50), 72.8 (C-20), 72.6 (C-30), 70.6 (C-500), 70.4
(C-5), 66.1 (C-6), 65.0 (C-600), 57.1 (C-2), 55.8 (C-200), 53.1
(CH3-COOMe), 38.0 (CH2-Lev), 29.8 (CH3-Lev), 28.0 (CH2-Lev), 27.3,
27.2, 26.9, 26.7 (CH3-tBu-Si ꢃ4), 23.3 (CH3-NAc), 22.6, 22.4, 19.9,
19.7 (Cq-tBu-Si ꢃ4), 18.6 (CH3-4-MU); IR (neat): 2934, 2860, 1718,
a
b
1.7H, H-40b; H-6a0 - and H-60a-b), 3.51 (t, 0.3H, J = 9.3 Hz, H-40a), 3.31
a
(td, 0.3H, J = 9.7, 4.8 Hz, H-5a0 ), 3.27 (td, 0.7H, J = 9.9, 4.9 Hz,
H-50b), 2.83–2.66 (m, 3H, H-6b0 -a, H-6b0 -b and CH2-Lev- /b), 2.60–2.54
a
(m, 2H, CH2-Lev- /b), 2.13 (s, 2.1H, CH3-Lev-b), 2.12 (s, 0.9H, CH3-Lev- ),
a
a
0.91–0.89 (m, 9H, CH3-tBu-Si- /b), 0.88 (s, 2.7H, CH3-tBu-Si- ), 0.87
a
a
(s, 6.3H, CH3-tBu-Si-b); 13C NMR (101 MHz, CDCl3) d 205.7, 205.6
(C@OLev-ketone- /b), 172.2 (C@OLev-ester- ), 172.0 (C@OLev-ester-b),
a
a
168.7 (C@OCOOMe- ), 168.5 (C@OCOOMe-b), 165.1 (C@OBz-b), 164.9
1270, 1069, 827, 709 cmꢀ1
; HRMS calcd for [C68H87Cl3N2
a
(C@OBz- ), 164.8 (C@OBz-b), 164.7 (C@OBz- ), 161.6 (C@OTCA-b),
O32Si2+H]+: 1461.4377, found 1461.4393.
a
a
161.5 (C@OTCA- ), 142.7 (Cq-arom-NPh- ), 142.5 (Cq-arom-NPh-b), 133.6,
a
a
133.4, 133.2, 133.1, 129.7 (CHarom-
ꢃ5), 129.61 (Cq-arom- /b),
4.1.15. 4-Methylumbelliferyl 2-acetamido-4,6-O-di-tert-
butylsilanediyl-2-deoxy-3-O-(methyl 2,3-di-O-benzoyl-4-O-
(4,6-O-di-tert-butylsilanediyl-2-deoxy-2-trichloroacetamido-b-
a
/b
a
129.59 , 129.5 (CHarom-
128.4, 128.3, 128.2 (CHarom-
124.3, 119.1, 118.8 (CHarom-NPh-
ꢃ2), 129.4 (Cq-arom- /b), 128.6, 128.5,
a
/b
a
ꢃ5), 128.0 (Cq-arom- /b), 124.5,
a
/b
a
ꢃ4), 100.8 (C-10a), 100.0 (C-10b),
D
-glucopyranosyl)-b-
glucopyranoside (18)
To solution of trisaccharide 17 (1.902 g, 1.30 mmol,
D-glucopyranosyl uronate)-b-D-
a
/b
94.4 (C-1 ), 92.6 (Cq-CCl3- ), 92.3 (Cq-CCl3-b), 91.7 (C-1b), 75.8
a
a
(C-4b), 75.6 (C-4 ), 74.4 (C-40 ), 74.3 (C-40 ), 74.2 (C-30 ), 73.8
a
a
b
a
a
(C-30 ), 71.6 (C-5 /b), 70.5 (C-5 /b ), 70.4 (C-3 ), 70.2 (C-2 ), 69.6
1.0 equiv) in pyridine/acetic acid 4:1 (60 mL), hydrazine
monohydrate (0.32 g, 6.50 mmol, 5.0 equiv) was added at room
temperature. Upon full conversion of the starting material
(TLC) the mixture was diluted with EtOAc (100 mL) and washed
with satd aq NaHCO3 (200 mL) and brine (100 mL). The aqueous
layers were extracted with EtOAc (100 mL) and the combined
organics were dried (MgSO4), filtered and concentrated in vacuo.
Purification by column chromatography (20–50% EtOAc in
petroleum ether) yielded the title compound as a white solid
0
a
a
a
a
b
(C-3b), 69.3 (C-2b), 64.9 (C-60a=b), 55.9 (C-2b0 ), 55.7 (C-20a), 53.3
(CH3-COOMe-b), 53.0(CH3-COOMe-b), 37.9 (CH2-Lev- /b), 29.5 (CH3-Lev- /b),
a
a
29.2, 27.9 (CH2-Lev- /b), 27.1, 26.6 (CH3-tBu-Si-
ꢃ2), 22.3, 19.6
a
a
/b
(Cq-tBu-Si-
ꢃ2); IR (neat): 2935, 2861, 2360, 1718, 1526, 1268,
a
/b
1070, 907, 827, 729 cmꢀ1
.
4.1.14. 4-Methylumbelliferyl 2-acetamido-4,6-O-di-tert-
butylsilanediyl-2-deoxy-3-O-(methyl 2,3-di-O-benzoyl-4-O-
(4,6-O-di-tert-butylsilanediyl-2-deoxy-3-O-levulinoyl-2-
(1.76 g, 1.29 mmol, 99%).
(400 MHz, CDCl3): d = 7.97–7.91 (m, 4H, Harom), 7.56–7.50 (m,
½
a 2D2 +7 (c 1.0, CHCl3); 1H NMR
ꢄ
trichloro-acetamido-b-
uronate)-b- -glucopyranoside (17)
A mixture of imidate donor 16 (3.031 g, 2.67 mmol, 1.2 equiv)
and N-acetyl glucosamine acceptor (1.161 g, 2.23 mmol,
D-glucopyranosyl)-b-
D
-glucopyranosyl
D
2H, Harom), 7.45 (d, 1H, J = 9.6 Hz, H-54-MU), 7.42–7.37 (m, 4H,
Harom), 7.06 (d, 1H, J = 7.5 Hz, NHTCA), 6.85 (dd, 1H, J = 9.6,
3
2.5 Hz, H-64-MU), 6.84 (d, 1H, J = 2.5 Hz, H-84-MU), 6.13 (d, 1H,
J = 1.1 Hz, H-34-MU), 5.82 (d, 1H, J = 7.5 Hz, NHNAc), 5.64 (d, 1H,
J = 8.2 Hz, H-1), 5.58 (t, 1H, J = 9.1 Hz, H-30), 5.38 (dd, 1H,
J = 9.2, 6.6 Hz, H-20), 5.21 (d, 1H, J = 6.5 Hz, H-10), 4.94 (d, 1H,
J = 8.3 Hz, H-100), 4.41–4.32 (m, 2H, H-3 and H-40), 4.19 (dd, 1H,
J = 10.3, 5.1 Hz, H-6a), 4.15 (d, 1H, J = 9.3 Hz, H-50), 3.97 (dd,
1H, J = 9.5, 8.5 Hz, H-4), 3.90 (t, 1H, J = 10.2, H-6b), 3.83 (s, 3H,
CH3-COOMe), 3.78 (dd, 1H, J = 10.4, 8.5 Hz, H-300), 3.61 (td, 1H,
J = 9.9, 5.1 Hz, H-5), 3.56 (dd, 1H, J = 10.4, 5.1 Hz, H-6a00), 3.51–
3.43 (m, 2H, H-2 and H-200), 3.36 (dd, 1H, J = 9.4, 8.5 Hz, H-400),
1.0 equiv) was co-evaporated two times with anhydrous toluene,
dissolved in anhydrous CH2Cl2 (10 mL) and stirred over activated
molsieves (3 Å) for 1 h. Trifluoromethanesulfonic acid (20 lL,
0.23 mmol, 0.1 equiv) was added at 0 °C and the reaction mix-
ture was stirred overnight at 4 °C. Anhydrous Et3N (0.2 mL)
was added and the reaction was transferred to an extraction fun-
nel with EtOAc (40 mL) and washed with satd aq NaHCO3
(50 mL) and brine (50 mL). The aqueous layers were extracted
with EtOAc (50 mL) and the combined organics were dried
(MgSO4), filtered and concentrated in vacuo. Purification by size
exclusion chromatography followed by column chromatography
(20–40% EtOAc in petroleum ether) yielded trisaccharide 17 as
3.26 (td, 1H, J = 9.9, 5.1 Hz, H-500), 2.72
- 2.65 (t, 1H,
J = 10.3 Hz, H-6b00), 2.36 (d, 3H, J = 1.1 Hz, CH3-4-MU), 1.77 (s,
3H, CH3-NAc), 1.06 (s, 9H, CH3-tBu-Si), 0.98 (s, 9H, CH3-tBu-Si), 0.90
(s, 9H, CH3-tBu-Si), 0.89 (s, 9H, CH3-tBu-Si); 13C NMR (100 MHz,
CDCl3): d = 170.7 (C@ONAc), 169.3 (C@OCOOMe), 165.2, 165.1
(C@OBz ꢃ2), 162.3 (C-24-MU), 160.9 (C@OTCA), 159.5, 154.7,
a white solid (1.961 g, 1.34 mmol, 60%). ½a D22
ꢀ5 (c 0.43, CHCl3);
ꢄ
1H NMR (400 MHz, CDCl3): d = 7.97–7.92 (m, 4H, Harom), 7.57–
7.52 (m, 2H, Harom), 7.45 (d, 1H, J = 9.4 Hz, H-54-MU), 7.43–7.38
(m, 4H, Harom), 6.89 (d, 1H, J = 8.8 Hz, NHTCA), 6.86–6.82 (m,
2H, H-64-MU and H-84-MU), 6.14 (d, 1H, J = 1.1 Hz, H-34-MU), 5.75
(d, 1H, J = 7.5 Hz, NHNAc), 5.65 (d, 1H, J = 8.2 Hz, H-1), 5.59 (t,
1H, J = 9.1 Hz, H-30), 5.36 (dd, 1H, J = 9.1, 6.5 Hz, H-20), 5.19 (d,
1H, J = 6.5 Hz, H-10), 5.02 (dd, 1H, J = 10.6, 9.2 Hz, H-300), 4.89
(d, 1H, J = 8.3 Hz, H-100), 4.38 (dd, 1H, J = 10.1, 8.4 Hz, H-3), 4.33
(t, 1H, J = 9.2 Hz, H-40), 4.19 (dd, 1H, J = 10.3, 5.0 Hz, H-6a), 4.11
(d, 1H, J = 9.2 Hz, H-50), 3.96 (dd, 1H, J = 9.5, 8.4 Hz, H-4), 3.88
152.3 (C-74-MU, C-94-MU and C-104-MU), 133.6, 133.1 (CHarom
ꢃ2), 129.8 (Cq-arom), 129.7, 129.6 (CHarom ꢃ2), 129.0 (Cq-arom),
128.6, 128.4 (CHarom ꢃ2), 125.6 (C-54-MU), 115.2 (C-44-MU),
113.5 (C-64-MU), 112.8 (C-34-MU), 104.1 (C-84-MU), 100.4 (C-10),
99.3 (C-100), 97.2 (C-1), 92.6 (Cq-CCl3), 79.3 (C-3), 77.4 (C-400),
76.4 (C-4), 75.7 (C-40), 74.1 (C-50), 73.7 (C-300), 72.8 (C-20), 72.7
(C-30), 70.4 (C-500), 70.1 (C-5), 66.1 (C-6), 65.0 (C-600), 58.2
(C-2), 57.0 (C-200), 53.1 (CH3-COOMe), 27.34, 27.29, 26.9, 26.8

1476
H. Gold et al. / Carbohydrate Research 346 (2011) 1467–1478
(CH3-tBu-Si ꢃ4), 23.3 (CH3-NAc), 22.6, 22.4, 19.9, 19.7 (Cq-tBu-Si ꢃ4),
18.6 (CH3-4-MU); IR (neat): 3351, 2934, 2859, 1718, 1270, 1069,
827, 731, 709 cmꢀ1; HRMS calcd for [C63H81Cl3N2O21Si2+Na]+:
1385.3828, found 1385.3839.
anhydrous CH2Cl2 (4 mL). Activated molsieves (3 Å, 1.0 g) were
added and the reaction mixture was stirred at room temperature
for 1 h. The reaction was cooled to 0 °C and trifluoromethanesulfo-
nic acid (2.0 lL, 22 lmol, 0.15 equiv) was added. The reaction was
stirred for 4 h and then quenched with Et3N (0.2 mL, 1.4 mmol,
10 equiv). The reaction mixture was transferred to an extraction
funnel with CH2Cl2 (40 mL) and washed with satd aq NaHCO3
(40 mL) and brine (30 mL). The aqueous layers were extracted with
CH2Cl2 (40 mL) and the combined organics were dried MgSO4, fil-
tered and concentrated in vacuo. The residue was purified by size
exclusion followed by column chromatography (30–50% EtOAc in
petroleum ether) to give the title compound (200 mg, 0.114 mmol,
4.1.16. Methyl (2,3-di-O-benzoyl-4-deoxy-a/b-D-
glucopyranoside N-Phenyl-2,2,2-trifluoroacetimidate) uronate
(19)
To a solution of 4-deoxy hemiacetal 7 (361 mg, 0.73 mmol,
1.0 equiv) in acetone (5 mL) Cs2CO3 (474 mg, 1.46 mmol, 2.0 equiv)
and N-phenyl-2,2,2-trifluoroacetimidoyl chloride (0.26 mL,
1.46 mmol, 2.0 equiv) were added at 0 °C. After 2 h at 0 °C was
the mixture filtered over Celite and concentrated in vacuo. Purifi-
cation by column chromatography (0–5% acetone in petroleum
78% yield). Rf = 0.24 (50% EtOAc in petroleum ether); ½a D22
ꢄ
: +21
(C = 1.0, CHCl3); 1H NMR (400 MHz, CDCl3) d = 7.98–7.89 (m, 8H,
ether) produced the title imidate as a colourless oil in a 2:3
a/b-ra-
Harom), 7.57–7.48 (m, 4H, Harom), 7.45–7.33 (m, 9H, H-54-MU and
tio (306 mg, 0.54 mmol, 73%). NMR assignment for the major iso-
mer (b): 1H NMR (400 MHz, CDCl3) d = 8.06–7.95 (m, 4H, Harom),
7.59–7.49 (m, 2H, Harom), 7.46–7.36 (m, 4H, Harom), 7.27 (m, 1H,
Harom-NPh), 7.14–7.06 (m, 2H, Harom-NPh), 6.81 (d, 1H, J = 7.7 Hz,
H-1), 6.39 (br d, 2H, J = 5.0 Hz, Harom), 5.83 (ddd, 1H, J = 11.0,
10.8, 5.0 Hz, H-3), 5.60 (m, 1H, H-2), 4.82 (br d, 1H, J = 11.8 Hz,
H-5), 3.81 (s, 3H, CH3-COOMe), 2.89 (m, 1H, H-4eq), 2.13 (m, 1H,
H-4ax); 13C NMR (101 MHz, CDCl3) d = 169.0 (C@OCOOMe), 165.6,
165.3 (C@OBz ꢃ2), 142.8 (Cq-arom-NPh), 133.6, 133.4, 129.8, 129.74,
129.65 (CHarom ꢃ5), 129.1, 128.8 (Cq-arom ꢃ2), 128.6, 128.53,
128.48, 128.45, 128.41, 128.38 (CHarom ꢃ6), 124.3, 124.2, 119.0
(CHarom-NPh ꢃ3), 92.8 (C-1), 70.4 (C-2), 68.9 (C-5), 67.6 (C-3), 52.7
(CH3-COOMe), 32.9 (C-4); IR (neat): 2959, 2925, 1718, 1452, 1272,
1208, 1070, 1027, 777, 695 cmꢀ1; HRMS calcd for [C29H24F3NO8+
Na]+: 594.1346, found 594.1348.
H
arom), 7.04 (d, 1H, J = 7.5 Hz, NHTCA), 6.84 (dd, 1H, J = 7.5, 2.1 Hz,
H-64-MU), 6.83 (d, 1H, J = 2.1 Hz, H-84-MU), 6.14 (d, 1H, J = 1.1 Hz,
H-34-MU), 5.74 (d, 1H, J = 7.3 Hz, NHNAc), 5.70 (d, 1H, J = 8.2 Hz, H-
1), 5.54 (t, 1H, J = 8.9 Hz, H-30), 5.38 (dd, 1H, J = 8.9, 6.6 Hz, H-20),
5.31 (dd, 1H, J = 9.2, 6.6 Hz, H-2000), 5.26 (m, 1H, H-3000), 5.17 (d,
1H, J = 6.7 Hz, H-10), 5.08 (d, 1H, J = 6.8 Hz, H-1000), 5.07 (d, 1H,
J = 8.1 Hz, H-100), 4.42 (dd, 1H, J = 10.1, 8.5 Hz, H-40), 4.40 (t, 1H,
J = 9.1 Hz, H-3), 4.25 (dd, 1H, J = 11.9, 2.8 Hz, H-5000), 4.24–4.15
(m, 2H, H-300 and H-6a), 4.10 (d, 1H, J = 9.3 Hz, H-50), 3.94 (t, 1H,
J = 9.0 Hz, H-4), 3.88 (t, 1H, J = 10.3 Hz, H-6b), 3.80 (s, 3H,
CH3-COOMe), 3.79 (s, 3H, CH3-COOMe), 3.66–3.57 (m, 3H, H-5, H-400 and
H-60a0), 3.41–3.31 (m, 2H, H-2 and H-200), 3.16 (ddd, 1H, J = 9.8, 9.7,
4.8 Hz, H-500), 2.77 (t,00 1H, J = 10.3 Hz, H-60b0), 2.66 (ddd, 1H,
J = 12.9, 5.2,002.8 Hz, H-4eq), 2.36 (d, 3 H, J = 1.1 Hz, CH3-4-MU), 2.02
(m, 1H, H-4ax), 1.78 (s, 3H, CH3-NAc), 1.04 (s, 9H, CH3-tBu-Si), 0.97
(s, 9H, CH3-tBu-Si), 0.88 (s, 9H, CH3-tBu-Si), 0.81 (s, 9H, CH3-tBu-Si);
13C NMR (101 MHz, CDCl3) d = 170.8 (C@ONAc), 169.1, 168.6
(C@OCOOMe ꢃ2), 165.7, 165.3, 165.2, 165.1 (C@OBz ꢃ4), 161.6 (C-
4.1.17. Methyl (2,3-di-O-benzoyl-4-O-methyl-a/b-D-
glucopyranoside N-phenyl-2,2,2-trifluoroacetimidate) uronate
(22)
24-MU), 160.9 (C@OTCA), 159.5, 154.7, 152.2 (C-74-MU, C-94-MU and
To a solution of 4-methoxy hemiacetal 10 (0.754 g, 1.75 mmol,
1.0 equiv) in acetone (8 mL) Cs2CO3 (1.140 g, 3.50 mmol, 2.0 equiv)
and N-phenyl-2,2,2-trifluoroacetimidoyl chloride (0.54 mL,
3.50 mmol, 2.0 equiv) were added at 0 °C. After 1.5 h at 0 °C the
mixture was filtered over Celite and concentrated in vacuo. Purifi-
cation by column chromatography (0–10% EtOAc in petroleum
C-104-MU), 133.6, 133.3, 133.14, 133.12 (CHarom ꢃ4), 129.77
(Cq-arom), 129.75, 129.68 129.56 (CHarom ꢃ3), 129.3, 129.05,
129.04 (Cq-arom ꢃ3), 128.6, 128.42, 128.35, 128.27 (CHarom ꢃ4),
125.6 (C-54-MU), 115.2 (C-44-MU), 113.4 (C-64-MU), 112.8 (C-34-MU),
104.2 (C-84-MU), 100.3 (C-10), 98.5 (C-100), 98.3 (C-1000), 96.9 (C-1),
92.5 (Cq-CCl3), 79.1 (C-3), 76.7 (C-300), 76.0 (C-4), 75.1 (C-40), 74.5
(C-400), 74.1 (C-50), 73.0 (C-30), 72.9 (C-2000), 72.6 (C-20), 71.2 (C-300),
70.4 (C-5), 70.28 (C-5000), 70.25 (C-500), 66.1 (C-6), 65.2 (C-600), 58.8
(C-2), 57.5 (C-200), 53.1, 52.5 (CH3-COOMe ꢃ2), 32.4 (C-4000), 27.3,
27.3, 26.9, 26.7 (CH3-tBu-Si ꢃ4), 23.3 (CH3-NAc), 22.6, 22.4, 19.9,
19.6 (Cq-tBu-Si ꢃ4), 18.6 (CH3-4-MU); HRMS calcd for
[C84H99Cl3N2O28Si2+H]+: 1745.5061, found 1745.5077.
ether) yielded the title imidate as a colourless oil in a 1:5 a/b-ratio
(0.679 g, 1.13 mmol, 65%). NMR assignment for the major isomer
(b): Rf = 0.45 (20% EtOAc in petroleum ether); 1H NMR (400 MHz,
CDCl3): d = 8.04–8.01 (m, 2H, Harom), 7.98–7.94 (m, 2H, Harom),
7.56–7.50 (m, 2H, Harom), 7.14–7.08 (m, 2H, Harom-NPh), 6.99 (t,
1H, J = 7.4 Hz, Harom-NPh), 6.78 (d, 1H, J = 7.5 Hz, H-1), 6.40 (br d,
2H, J = 6.1 Hz, Harom-NPh), 6.03 (t, 1H, J = 9.8 Hz, H-3), 5.47 (dd,
1H, J = 10.1, 3.3 Hz, H-2), 4.52 (d, 1H, J = 10.0 Hz, H-5), 3.97 (t,
1H, J = 9.7 Hz, H-4), 3.87 (s, 3H, CH3-COOMe), 3.46 (s, 3H, CH3-OMe);
13C NMR (101 MHz, CDCl3) d 168.4 (C@OCOOMe), 165.31, 165.25
(C@OBz ꢃ2), 142.7 (Cq-arom-NPh), 133.6, 133.4, 129.9, 129.7 (CHarom
ꢃ4), 129.1 (Cq-arom), 128.54, 128.51, 128.48, 128.43 (CHarom ꢃ4),
124.5, 124.3, 119.0 (CHarom-NPh ꢃ3), 92.1 (C-1), 78.7 (C-4), 72.2
(C-5), 71.3 (C-3), 70.2 (C-2), 60.6 (CH3-OMe), 52.9 (CH3-COOMe); IR
(neat): 2959, 2925, 1718, 1451, 1261, 909, 707 cmꢀ1; HRMS calcd
for [C30H26F3NO9+Na]+: 624.1452, found 624.1451.
4.1.19. 4-Methylumbelliferyl 2-acetamido-4,6-O-di-tert-
butylsilanediyl-2-deoxy-3-O-[methyl 2,3-di-O-benzoyl-4-O-
{4,6-O-di-tert-butylsilanediyl-2-deoxy-3-O-(methyl 2,3-di-O-
benzoyl-4-O-methyl-b-
trichloroacetamido-b-
uronate]-b- -glucopyranoside (23)
D
-glucopyranosyl uronate)-2-
D-glucopyranosyl}-b- -glucopyranosyl
D
D
Trisaccharide acceptor 18 (100 mg, 0.073 mmol, 1.0 equiv) and
imidate donor 22 (88 mg, 0.15 mmol, 2.0 equiv) were coevaporated
two times with anhydrous toluene and then dissolved in anhydrous
CH2Cl2 (2 mL). Activated molsieves (3å, 0.5 g) were added and the
reaction mixture was stirred at room temperature for 1 h, before
cooling to 0 °C and addition of trifluoromethanesulfonic acid
4.1.18. 4-Methylumbelliferyl 2-acetamido-4,6-O-di-tert-
butylsilanediyl-2-deoxy-3-O-[methyl 2,3-di-O-benzoyl-4-O-
{4,6-O-di-tert-butylsilanediyl-2-deoxy-3-O-(methyl 2,3-di-O-
(ꢂ1.0
lL, 11 lmol, 0.15 equiv). The reaction was stirred for 4 h
benzoyl-4-deoxy-b-
trichloroacetamido-b-
uronate]-b- -glucopyranoside (20)
D
-glucopyranosyl uronate)-2-
and then quenched with Et3N (0.1 mL, 0.73 mmol, 10 equiv). The
reaction mixture was transferred to an extraction funnel with
CH2Cl2 (20 mL) and washed with satd aq NaHCO3 (20 mL) and brine
(20 mL). The aqueous layers were extracted with CH2Cl2 (20 mL)
and the combined organics were dried (MgSO4), filtered and
concentrated in vacuo. The residue was purified by size exclusion
D
-glucopyranosyl}-b- -glucopyranosyl
D
D
Trisaccharide acceptor 18 (201 mg, 0.147 mmol, 1.0 equiv) and
imidate donor 19 (168 mg, 0.29 mmol, 2.0 equiv) were co-evapo-
rated two times with anhydrous toluene and then dissolved in

H. Gold et al. / Carbohydrate Research 346 (2011) 1467–1478
1477
chromatography, followed by column chromatography (30–50%
EtOAc in petroleum ether) to give the title tetrasaccharide 23
(111 mg, 0.062 mmol, 85% yield). Rf = 0.22 (50% EtOAc in petroleum
residue was dissolved in pyridine (3 mL), before addition of hydro-
gen fluoride/Et3N (29 L, 17 mmol, 4 equiv). The reaction was stir-
l
red at ambient temperature for 3 h (the completion of the reaction
was confirmed by HPLC-MS) and water (3 mL) was added followed
by satd aq sodium carbonate (1.5 mL). The reaction was stirred at
ambient temperature over night and then concentrated in vacuo.
The product was purified by HPLC according to the general proce-
dure. The title compound was obtained as the bis ammonium salt
ether); ½a 2D2
ꢄ
: + 8.3 (C = 0.157, CHCl3); 1H NMR (400 MHz, CDCl3):
d = 7.96–7.90 (m, 8H, Harom), 7.53–7.49 (m, 4H, Harom), 7.45 (d, 1H,
J = 9.5 Hz, H-54-MU), 7.41–7.35 (m, 8H, Harom), 6.86 (d, 1H,
J = 9.5 Hz, H-64-MU), 6.84 (d, 1H, J = 7.5 Hz, NHTCA), 6.82 (s, 1H,
H-84-MU), 6.13 (d, 1H, J = 1.0 Hz, H-34-MU), 5.71 (d, 1H, J = 7.4 Hz,
NHNAc), 5.67 (d, 1H, J = 8.2 Hz, H-1), 5.52 (t, 1H, J = 9.1 Hz, H-3000),
5.50 (t, 1H, J = 9.1 Hz, H-30), 5.37 (dd, 1H, J = 9.1, 6.7 Hz, H-20),
5.25 (dd, 1H, J = 8.9, 6.5 Hz, H-2000), 5.13 (d, 1H, J = 6.7 Hz, H-10), 5.07
(d, 1H, J = 6.5 Hz, H-1000), 4.99 (d, 1H, J = 8.2 Hz, H-100), 4.41 (dd,
1H, J = 10.1, 8.5 Hz, H-3), 4.35 (t, 1H, J = 9.1 Hz, H-40), 4.20–4.14
(m, 2H, H-6a and H-300), 4.06 (d, 1H, J = 9.2 Hz, H-50), 4.02 (d, 1H,
J = 9.0 Hz, H-5000), 3.95 (t, 1H, J = 9.0 Hz, H-4000), 3.94 (dd, 1H, J = 9.5,
8.6 Hz, H-4), 3.88 (t, 1H, J = 10.3 Hz, H-6b), 3.81 (s, 3H, CH3-COOMe),
(24 mg, 26 l
mol, 60%). 1H NMR (600 MHz, D2O) d = 7.60 (d, 1H,
J = 8.8 Hz, H-54-MU), 6.99 (dd, 1H, J = 8.8, 2.5 Hz, H-64-MU), 6.93 (d,
1H, J = 2.5 Hz, H-84-MU), 6.16 (s, 1H, H-34-MU), 5.25 (d,
1H, J = 8.5 Hz, H-1), 4.62 (d, 1H, J = 7.9 Hz, H-10), 4.58
(d, 1H, J = 8.4 Hz, H-100), 4.42 (d, 1H, J = 7.8 Hz, H-1000), 4.30 (dd, 1H,
J = 12.3, 2.4 Hz, H-5000), 4.16 (t, 1H, J = 9.4 Hz, H-2), 4.02 (d, 1H,
J = 9.7 Hz, H-50), 3.95 (d, 1H, J = 12.1 Hz, H-6a), 3.93–3.87 (m, 2H,
00
H-6a and H-3), 3.86–3.80 (m, 3H, H-200, H-40 and H-6b), 3.79–3.72
3.79 (s, 3H, CH3-COOMe), 3.64–3.56 (m, 2H, H-5 and H-600), 3.51
(m, 3H, H-3000, H-6b and H-300), 3.71–3.67 (m, 2H, H-4 and H-5),
00
a
(t, 1H, J = 9.2 Hz, H-400), 3.39 (s, 3H, CH3-OMe), 3.35 (m, 1H, H-2),
3.27–3.13 (m, 2H, H-200 and H-500), 2.69 (t, 1H, J = 10.4 Hz,
3.65 (m, 1H, H-30), 3.56 (dd, 1H, J = 11.9, 6.4 Hz, H-400), 3.49
(m, 1H, H-500), 3.40 (t, 1H, J = 8.7 Hz, H-20), 3.21 (t, 1H, J = 8.5 Hz,
H-2000), 2.39–2.34 (m, 4H, H-4e00q0 and CH3-4MU), 2.01 (s, 3H, CH3-NAc),
1.99 (s, 3H, CH3-NAc), 1.64 (q, 1H, H-40a0x0 ); 13C NMR (151 MHz, CDCl3)
d = 175.82, 175.76 (C@ONAc ꢃ2), 174.4, 172.3 (C@OCOO-NH4+ ꢃ2),
H-600), 2.36 (s, 3H, CH3-4MU), 1.76 (s, 3H, CH3-NAc), 1.04 (s, 9H,
b
CH3-tBu-Si), 0.97 (s, 9H, CH3-tBu-Si), 0.90 (s, 9H, CH3-tBu-Si), 0.82 (s,
9H, CH3-tBu-Si); 13C NMR (101 MHz, CDCl3) d = 170.7 (C@ONAc),
168.6, 168.5 (C@OCOOMe ꢃ2), 165.4, 165.34, 165.29, 165.1 (C@OBz
165.4, 160.3, 157.0, 154.7 (C-24-MU
, C-74-MU, C-94-MU and
ꢃ4), 161.5 (C-24-MU), 160.9 (C@OTCA), 159.5, 154.7, 152.2 (C-74-MU
,
C-104-MU), 127.6 (C-54-MU), 116.3 (C-44-MU), 114.9 (C-64-MU), 112.3
(C-34-MU), 104.6 (C-84-MU), 104.0 (C-1000), 103.8 (C-10), 102.1 (C-100),
99.5 (C-1), 84.1 (C-300), 83.0 (C-3), 81.1 (C-40), 76.7 (C-5), 76.3
(C-500), 75.0 (C-2000), 74.6 (C-30), 74.5 (C-50), 73.1 (C-20), 71.2
(C-5000), 70.7 (C-3000), 69.4 (C-400), 69.0 (C-4), 61.5 (C-600), 61.3 (C-6),
55.20 (C-2), 55.16 (C-200), 36.2 (C-4000), 23.3, 23.2 (CH3-NAc ꢃ2), 18.9
(CH3-4-MU).
C-94-MU and C-104-MU), 133.6, 133.3, 133.1, 129.9 (CHarom ꢃ4),
129.8 (Cq-arom), 129.70, 129.67, 129.58 (CHarom ꢃ3), 129.2, 129.08,
129.03 (Cq-arom ꢃ3), 128.6, 128.42, 128.37, 128.33 (CHarom ꢃ4),
125.6 (C-54-MU), 115.2 (C-44-MU), 113.4 (C-64-MU), 112.9 (C-34-MU),
104.3 (C-84-MU), 100.4 (C-10), 99.6 (C-1000), 98.4 (C-100), 97.0 (C-1),
92.5 (Cq-CCl3), 79.2 (C-3), 78.4 (C-4000), 77.6 (C-300), 76.2 (C-4),
75.11 (C-400), 75.08 (C-40), 74.5 (C-50), 74.2 (C-5000), 74.1 (C-3000),
73.1 (C-2000), 72.8 (C-30), 72.6 (C-20), 70.4 (C-5), 70.2 (C-500), 66.1
(C-6), 65.2 (C-600), 60.4 (CH3-OMe), 58.7 (C-200), 57.4 (C-2), 53.1,
52.6 (CH3-COOMe ꢃ2), 27.3 (ꢃ2), 26.9, 26.7 (CH3-tBu-Si ꢃ4), 23.3
(CH3-NAc), 22.6, 22.4, 19.9, 19.6 (Cq-tBu-Si ꢃ4), 18.6 (CH3-4-MU);
IR (neat): 3352, 2935, 2860, 1730, 1616, 1269, 1070, 828,
710 cmꢀ1; HRMS calcd for [C85H101Cl3N2O29Si2+H]+: 1775.5167,
found 1775.5180.
4.1.21. 4-Methylumbelliferyl 2-acetamido-2-deoxy-3-O-[4-O-{2-
acetamido-2-deoxy-3-O-(4-O-methyl-b-D-glucopyranosyl
uronate)-b- -glucopyranosyl}-b- -glucopyranosyl uronate]-b-D-
D
D
glucopyranoside bis ammonium salt (24)
Protected tetrasaccharide 23 (18 mg, 10 lmol, 1.0 equiv) was
dissolved in acetic acid (2 mL) and activated zinc dust (33 mg,
0.50 mmol, 50 equiv) was added three times with 2 h interval. The
reaction was stirred at ambient temperature over night, after which
HPLC-MS indicated a ꢂ4:1 ratio of mono-chloro residue and the
fully dehalogenated product. The residue was diluted with toluene
and filtered through a plug of Celite. The solids were washed with
some additional toluene and the residue was then concentrated in
vacuo and dissolved in anhydrous acetone (5 mL). Potassium iodide
(50 mg, 0.30 mmol, 30 equiv) was added and the reaction mixure
was stirred over night at ambient temperature giving full conver-
sion to the monoiodo derivate as measured by HPLC-MS. The mix-
ture was filtered, concentrated in vacuo and the remaining
residue was dissolved in acetic acid (2 mL) and activated zinc
(33 mg, 0.5 mmol, 50 equiv) was added. The reaction was stirred
over night giving full conversion to the fully dehalogenated product.
The solids were iltered, washed and the eluent was concentrated in
vacuo. The residue was dissolved in MeOH (2 mL) and sodium
4.1.20. 4-Methylumbelliferyl 2-acetamido-2-deoxy-3-O-[4-O-{2-
acetamido-2-deoxy-3-O-(4-deoxy-b-
D-glucopyranosyl uronate)-
b- -glucopyranosyl}-b- -glucopyranosyl uronate]-b-D-
D
D
glucopyranoside bis ammonium salt (21)
Protected tetrasaccharide 20 (75 mg, 42 lmol, 1.0 equiv) was
dissolved in acetic acid (5 mL) and activated zinc dust (138 mg,
2.1 mmol, 50 equiv) was added three times with 2 h interval. The
reaction was then stirred at ambient temperature over night.
HPLC-MS indicated a ꢂ5:2 ratio of mono-chloro residue and the
fully dehalogenated product. The residue was diluted with toluene
and filtered through a plug of Celite. The solids were washed with
some additional toluene and the residue was then concentrated
in vacuo and dissolved in anhydrous acetone (5 mL). Potassium io-
dide (210 mg, 1.30 mmol, 30 equiv) was added and the reaction
mixure was stirred over night at ambient temperature giving full
conversion to the monoiodo derivate as measured by HPLC-MS.
The mixture was filtered, concentrated in vacuo and the remaining
residue was dissolved in acetic acid (5 mL) and activated zinc
(138 mg, 2.1 mmol, 50 equiv) was added. The reaction was stirred
over night giving full conversion to the fully dehalogenated prod-
uct. The solids were filtered, washed and the eluent was concen-
trated in vacuo. The residue was dissolved in MeOH (5 mL) and
methoxide (1.5 lL, 10 lmol, 1.0 equiv) was added and the reaction
was stirred under argon at room temperature over night. After com-
pletion of the reaction, as monitored by HPLC-MS, the reaction was
quenched with acetic acid (0.2 mL). The solvents were removed in
vacuo by coevaporation with toluene (5 mL) and the residue was
dissolved in pyridine (1 mL), before addition of hydrogenfluoride/
Et3N (6.6 lL, 40 lmol, 4 equiv). The reaction was stirred for 3 h at
ambient temperature (the completion of the reaction was con-
firmed by HPLC-MS) and water (1 mL) was added followed by satd
aq sodium carbonate (0.5 mL). The reaction was stirred at ambient
temperature over night and then concentrated in vacuo. The prod-
uct was purified by HPLC according to the general procedure. The ti-
tle compound was obtained as the bis ammonium salt (4.3 mg,
sodium methoxide (29 lL, 44 lmol, 1.0 equiv) was added and the
reaction was stirred under argon at room temperature over night.
Upon completion of the reaction, as monitored by HPLC-MS, the
reaction was quenched with acetic acid (0.2 mL). The solvents were
removed in vacuo by coevaporation with toluene (5 mL) and the

1478
H. Gold et al. / Carbohydrate Research 346 (2011) 1467–1478
l
mol, 43%). 1H NMR (600 MHz, D2O) d = 7.74 (d, 1H, J = 9.4 Hz,
G. E.; Debowski, A. W.; Chin, D.; Vocadlo, D. J. J. Biol. Chem. 2005, 280, 25313–
25322; (c) Wang, L.-X.; Keyani, N. O.; Roseman, S.; Lee, Y. C. Glycobiology 1997, 7,
855–860.
4.4
H-54-MU), 7.08–7.05 (m, 2H, H-64-MU and H-84-MU), 6.28 (s, 1H, H-
4-MU), 5.29 (d, 1H, J = 8.5 Hz, H-1), 4.57 (d, 1H, J = 7.9 Hz, H-10),
3
2. Sinnot, M. L. Chem. Rev. 1990, 90, 1171–1202.
4.55 (d, 1H, J = 8.3 Hz, H-100),4.47 (d, 1H, J = 7.8 Hz, H-1000), 4.16 (dd,
1H, J = 10.5, 8.5 Hz, H-2), 3.95 (dd, 1H, J = 12.1, 2.3 Hz, H-6a), 3.93–
3.89 (m, 2H, H-60a0 and H-50), 3.88–3.83 (m, 3H, H-3, H-5000 and H-
200),3.83–3.78 (m, 2H, H-6a and H-40), 3.76 (dd, 1H, J = 12.4, 5.2 Hz,
H-60a0), 3.73–3.69 (m, 2H, H-4 and H-5), 3.67 (m, 1H, H-300), 3.65
(m, 1H, H-30), 3.55 (t, 2H, J = 9.3 Hz, H-400 and H-3000), 3.50–3.46 (m,
4H, H-500 and CH3-OMe), 3.39 (dd, 1H, J = 9.5, 7.9 Hz, H-20), 3.33 (dd,
J = 9.5, 7.9 Hz, 1H, H-2000), 3.30 (t, J = 9.5 Hz, 1H, H-4000), 2.45 (s, 3H,
CH3-4MU), 2.00 (s, 3H, CH3-NAc), 1.99 (s, 3H, CH3-NAc); 13C NMR
3. (a) Aguilera, B.; Ghauharali-van der Vlugt, K.; Helmond, M. T. J.; Out, J. M. M.;
Donker-Koopman, W. E.; Groener, J. E. M.; Boot, R. G.; Renkema, G. H.; van der
Marel, G. A.; van Boom, J. H.; Overkleeft, H. S.; Aerts, J. M. F. G. J. Biol. Chem.
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Aerts, J. M. F. G. J. Clin. Invest. 1994, 93, 1288–1292.
4. (a) Meyer, K.; Palmer, J. W. J. Biol. Chem. 1934, 107, 629–634; (b) Laurent, T. C.;
Fraser, J. R. E. FASEB J. 1992, 6, 2397–2404; (c) Knudson, C. B.; Knudson, W.
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(151 MHz, D2O)
d
175.9, 175.8 (C@ONAc ꢃ2), 174.7, 173.4
(C@OCOO-NH4+ ꢃ2), 165.7, 160.4, 157.3, 155.0 (C-24-MU, C-74-MU
,
C-94-MU and C-104-MU), 127.7 (C-54-MU), 116.6 (C-44-MU), 114.9
(C-64-MU), 112.4 (C-34-MU), 104.7 (C-84-MU), 104.0 (C-1000), 103.6
(C-10), 101.9 (C-100), 99.6 (C-1), 83.5 (C-5), 82.9 (C-3), 82.7 (C-400),
81.0 (C-40), 76.7 (C-5), 76.3 (C-500), 75.69 (C-3000), 75.65 (C-50), 75.1
(C-5000), 74.6 (C-30), 73.5 (C-2000), 73.2 (C-20), 69.2 (C-400), 69.1 (C-4),
61.43 (C-600), 61.35 (C-6), 61.1 (CH3-OMe), 55.24 (C-2), 55.19 (C-200),
23.4, 23.1 (CH3-NAc ꢃ2), 19.0 (CH3-4-MU).
8. Dinkelaar, J.; Duivenvoorden, B. A.; Wennekes, T.; Overkleeft, H. S.; Boot, R. G.;
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Acknowledgements
This research was supported by The Netherlands Ministry of
Economic Affairs and the B-Basic partner organizations (www.b-
basic.nl) through B-Basic, a public-private NWO-ACTS program
(ACTS) Advanced Chemical Technologies for Sustainability) and
The Netherlands Organization of Scientific Research (NWO, Vidi
grant).
Supplementary data
22. Crich, D.; Smith, M. J. Am. Chem. Soc. 2001, 123, 9015–9020.
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1869.
Supplementary data (1H and 13C NMR spectra) associated with
this article can be found, in the online version, at doi:10.1016/
j.carres.2011.03.042.
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