Chemoenzymatic synthesis of thio-nod factor intermediates - Enzymatic transfer of glucosamine on thiochitobiose derivatives

Article abstract of DOI:10.1139/V06-043

The chemoenzymatic syntheses of thioanalogues of nodulation factors in which the nonreducing end glucosamine residue is available for the introduction of the fatty acid moiety at the free NH² group are reported. We are describing the chemical s

Full text of DOI:10.1139/V06-043

587
Chemoenzymatic synthesis of thio-nod factor
intermediates — Enzymatic transfer of
glucosamine on thiochitobiose derivatives1
Latino Loureiro Morais, Hideya Yuasa, Khalil Bennis, Isabelle Ripoche, and
France-Isabelle Auzanneau
Abstract: The chemoenzymatic syntheses of thioanalogues of nodulation factors in which the nonreducing end
glucosamine residue is available for the introduction of the fatty acid moiety at the free NH2 group are reported. We
are describing the chemical synthesis of UDP-GlcNH2 and its use in the enzymatic transfer of GlcNH2 by the bovine
galactosyltransferase (EC 2.4.1.90) onto O-4 of the nonreducing end N-acetylglucosamine residues of chitobiose,
thiochitobiose, and allyl thiochitobioside. The enzymatic reactions on chitobiose and thiochitobiose were followed by
TLC and MALDI MS and showed about 50% conversion of the disaccharides to the desired products. However, these
reducing trisaccharides could not be obtained totally free of salts and degraded on ion exchange chromatography. Thus,
we investigated the enzymatic transfer on the nonreducing allyl thiochitobioside analogue. We describe here the chemi-
cal synthesis of this thiodisaccharide and the enzymatic transfer of GlcNH2 at O-4 of its nonreducing end glucosamine
residue to give the desired allyl thiotrisaccharide. This thiotrisaccharide was obtained pure in 41% yield and was char-
1
acterized by H NMR (HSQC) and HRMS.
Key words: nodulation factors, synthesis, enzymatic transfer, thiooligosaccharides, UDP-glucosamine.
Résumé : On a réalisé la synthèse chimioenzymatique de thioanalogues de facteurs de nodulation dans lesquels le ré-
sidu glucosamine de l’extrémité non réductrice est disponible pour l’introduction de portions d’acides gras au niveau
du groupe NH2. On décrit la synthèse chimique de UDP-GlcNH2 ainsi que son utilisation dans le transfert enzymatique
du GlcNH2 par la galactosyltransférase bovine (EC 2.4.1.90) sur le O-4 des résidus N-acétylglucosamine non réducteurs
du chitobiose, du thiochitobiose et du thiochitobioside d’allyle. Les réactions enzymatiques sur le chitobiose et le thio-
chitobiose ont été suivies par chromatographie sur couche mince et par spectrométrie de masse MALDI et elles mon-
trent qu’il se produit une conversion d’environ 50 % des disaccharides en produits désirés. Toutefois, on n’a pas pu
éliminer complètement le sel de ces trisaccharides réducteurs et ils se dégradent lors de la chromatographie par
échange d’ions. On a donc étudié le transfert enzymatique sur l’analogue thiochitobioside d’allyle non réducteur. On
décrit les synthèses chimiques de ce thiodisaccharide et le transfert enzymatique du GlcNH2 vers le O-4 du résidu glu-
cosamine de l’extrémité non réductrice pour obtenir le thiotrisaccharide d’allyle désiré. Ce thiotrisaccharide a été ob-
1
tenu à l’état pur avec un rendement de 41 % et on l’a caractérisé par RMN du H (HSQC) et par spectrométrie de
masse à haute résolution.
Mots clés : facteurs de nodulation, synthèse, transfert enzymatique, thiooligosaccharides, UDP-glucosamine.
[Traduit par la Rédaction] Loureiro Morais et al. 596
Introduction
Azorhizobium leads to the formation of nodules on the plant
roots. Within these nodules the bacteria can fix and metabo-
lize atmospheric nitrogen into ammonia and alanine and re-
lease it for use by the host plant, which in turn supplies the
Symbiotic association (1) of leguminous plants with soil
bacteria of the genera Rhizobium, Bradyrhizobium, and
Received 6 September 2005. Published on the NRC Research Press Web site at http://canjchem.nrc.ca on 5 May 2006.
This paper is dedicated to Professor W. Szarek on his 65th birthday.
L. Loureiro Morais, K. Bennis, and I. Ripoche.2 Laboratoire de Chimie des Hétérocycles et des Glucides, École Nationale
Supérieure de Chimie de Clermont-Ferrand, BP 187, F-63174 Aubière, France.
H. Yuasa.3 Department of Chemistry, University of Alberta, Edmonton, AB T6G 2G2, Canada.
F.-I. Auzanneau.4 Department of Chemistry, University of Guelph, Guelph, ON N1G 2W1, Canada.
1This article is part of a Special Issue dedicated to Professor Walter A. Szarek.
2Corresponding author (e-mail: ripoche@chimie.univ-bpclermont.fr).
3Present address: Department of Life Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology,
4259 Nagatsutacho, Midoriku, Yokohama 2268501, Japan.
4Corresponding author (e-mail: fauzanne@uoguelph.ca).
Can. J. Chem. 84: 587–596 (2006)
doi:10.1139/V06-043
© 2006 NRC Canada

588
Can. J. Chem. Vol. 84, 2006
Fig. 1. Schematic representation of nodulation factors.
bacteria with various nutrients. Nodulation, the key step
leading to this symbiotic association, is induced by the inter-
action with specific plant receptors of nodulation factors
(Nod factors) produced by the bacteria (2). These lipooligo-
saccharides (3) are comprised of a tetra- or penta-oligomer
of chitin with substitutions that determine the host specific-
ity of the bacteria (Fig. 1, ref. 3). Although only very small
concentrations (10–6 to 10–12 mol/L) of natural Nod factors
are required to initiate nodulation, their activity is limited by
the action of chitinases that cleave the glycosidic bond be-
tween the sugar units B and C (4). Therefore, we have de-
veloped a program (5) aimed at preparing thioanalogues of
Nod factors in which a sulphur atom replaces one or more
interglycosidic oxygen atoms, thus increasing the overall re-
sistance of the lipooligosaccharide to chitinases (6). The
synthesis of such oligosaccharide analogues with well-
defined glycosidic linkages remains a challenge for carbohy-
drate chemists despite the advances made over the past de-
cades. Therefore, we are investigating here the possibility of
introducing a glucosamine unit at the nonreducing end of
thioanalogues of Nod factors by enzymatic transfer (7). We
report the use of the well-studied β-(1,4)-galactosyltransferase
(7) isolated from bovine milk and involved in the biochemi-
cal synthesis of lactose and N-acetyllactosamine. The transfer
of a glucosamine unit to the nonreducing end of chitotriose
using the commercially available bovine galactosyltrans-
ferase (UDP-D-galactose:2-acetamido-2-deoxy-D-glucose 4-
β-D-galactosyl tranferase; EC 2.4.1.90) has already been
described (8). The work described here is, to our knowledge,
the first enzymatic transfer to chitobiose derivatives that
contain a sulphur atom in the glycosidic linkage and thus
constitutes the first enzymatic synthesis of thiochitotriose
derivatives. We describe herein the efficient chemical syn-
thesis of UDP-glucosamine as well as the chemoenzymatic
synthesis of new thiotrisaccharides precursors of thio-
analogues of nodulation factors using this nucleotide donor
and the bovine galactosyltranferase EC 2.4.1.90.
C (n = 1)
C-D (n = 2)
D (n = 1)
E (n = 2)
A
B
OH
OH
OH
OR1
O
HO
HO
O
O
O
O
O
O
HO
HO
N
OH
NHAc
HO
n
AcHN
AcHN
R3
R2
R1 = SO3H, Fuc, Ara, Me-Fuc, AcFuc...
R2 = acyl substituent (wide variety)
R3 = H or Me
in acetone and coupled with the bistrioctylammonium salt of
uridine 5′-monophophate dissolved in DMF. The crude adduct
was in turn treated with ammonium hydroxide in methanol
to remove the O-acetyl and N-trifluoroacetyl groups and the
desired UDP-GlcNH2 (6) was isolated pure in 51% yield
(from 4) following ion exchange chromatography.
The enzymatic transfer of glucosamine by the β-1,4-
galactosyltransferase isolated from bovine milk (13) was
first attempted on the reducing disaccharides chitobiose (7)
and thiochitobiose (8) (Scheme 2) Although the products ob-
tained in these reactions could not be purified (see the fol-
lowing), these reactions were used to determine the best
reaction and purification conditions for the enzymatic trans-
fer and more importantly to establish MALDI-MS condi-
tions that can be used to follow such transfers. While
chitobiose is commercially available, the known (14)
thiodisaccharide (8) was prepared in one step through the
deacetylation of the previously reported (5b) peracetylated
thiochitobiose. The disaccharide (8) obtained as an anomeric
mixture could not be totally purified by conventional chro-
1
matography. However, since H NMR (Fig. 2C and ref. 14b)
showed a purity superior than 90% and HRMS confirmed its
structure, the α,β-thiochitobiose (8) was submitted to enzy-
matic transfer. The reducing chitobiose (7) and thiochito-
biose (8) were incubated with UDP-GlcNH2 (6, 1.2 equiv.)
and galactosyltransferase (5.6 U) at ambient temperature
with occasional gentle mixing. TLC analysis of the reaction
mixture showed in both cases the formation of a new
ninhydrin positive compound that was more polar than the
starting material. The formation of the trisaccharides 9 and
10 was also assessed by MALDI-MS of the reaction mix-
tures (Fig. 3). As can be seen in Fig. 3A, we observed for
the enzymatic transfer on 7 (+Na, m/z 448 1) the appear-
ance of a signal at m/z 608 1 (+Na) corresponding to the
desired adduct 9. Similarly, both the starting thiochitobiose
(8) and the adduct 10 were observed at m/z 464 1 (+Na)
and m/z 625 1 (+Na), respectively, by MALDI-MS for the
enzymatic transfer on disaccharide 8. After 2 weeks of incu-
bation, the conversion evaluated by TLC had reached ~50%
in both cases, and the reaction mixtures were applied to a
gel permeation chromatography column of Bio-Gel eluted
with a 0.25 mol/L ammonium acetate solution. While this
was an efficient way to separate the unreacted disaccharides
7 and 8 from the desired trisaccharides 9 and 10, respec-
tively, the products could not be isolated totally free of am-
monium acetate even after repeated freeze drying from
water. Additional attempts to remove the excess acetate ions
Results and discussion
As an alternative to the known (9) enzymatic preparation
of UDP-GlcNH2, the nucleotide donor 6 was synthesized
chemically (Scheme 1) from the trifluoroacetamido deriva-
tive (1) obtained as described by Wolform and Conigliaro
(10) from commercially available crystalline glucosamine
hydrochloride. The synthetic strategy followed to prepare
the key intermediate analogue (5) was similar to that em-
ployed by Sala et al. (11). Acetylation in pyridine – acetic
anhydride gave the crude peracetate (2) as an anomeric mix-
ture that contained the α anomer as the major compound.
The crude peracetate was treated with hydrazine acetate to
give the known (10, 11) α-hemiacetal (3) with an improved
yield of 74% from 1 upon purification on silica gel. The
hemiacetal 3 was treated with butyllithium to give the
anomeric alkoxide that was reacted with dibenzyl phosphoryl
chloride (12) in THF to give the anomeric dibenzylphos-
phate (4) in 54% yield, a yield similar to that described by
Sala et al. (11) for the synthesis of the analogous anomeric
diphenylphosphate (52%). The benzyl groups were removed
by hydrogenolysis and the crude triacetylated anomeric
phosphate (5) was activated with 1,1Ј-carbonyldiimidazole
© 2006 NRC Canada

Loureiro Morais et al.
589
Scheme 1. Reagents: (i) Ac2O–pyridine (crude, α/β = 94:6); (ii) NH2NH2-AcOH, DMF (74% pure α over i and ii); (iii) a. BuLi, THF;
b. ClPO(OBn)2, THF (59%); (iv) H2-Pd/C, MeOH (not isolated); (v) a. 5, 1,1′-carbonyldiimidazole, acetone; b. uridine 5′-
monophosphate bistrioctylammonium salt, DMF; c. MeOH–NH4OH (51% over iv and v).
OAc
O
OAc
O
OH
AcO
AcO
O
AcO
HO
i
ii
AcO
HO
OAc
OH
CF3COHN
CF3COHN
CF3COHN
OH
1
2
3
O
N
OH
OAc
OAc
O
HO
O
AcO
AcO
CF3COHN
OPO(OH)2
O
HN
iii AcO
iv
v
HO
3
AcO
CF3COHN
OPO(OBn)2
O
O
H2N
O
O
O P O P O
O-
4
5
O-
6
OH
OH
Scheme 2.
4.82 ppm for the anomeric H-1′′ and at 3.06 ppm for H-2′′.
1
The H NMR spectrum obtained for 10 (Fig. 2D) showed
OH
OH
the presence of the same contaminant already observed in 8,
which we concluded was also a substrate for the galactosyl-
transferase. However, it also clearly demonstrated that a
glucosamine unit giving typical signals at 4.83 and 3.09 ppm
for H-1′′ and H-2′′, respectively, had been transferred onto
the thiochitobiose. Finally, the structures of 9 and 10 were
unambiguously confirmed by HRMS that gave protonated
molecular ions at m/z 586.2463 for 9 (calcd. [M + H]
586.2459) and m/z 602.2237 for 10 (calcd. [M + H]
602.2231). Thus, these results showed that the transfer of a
glucosamine residue to thiochitobiose using UDP-GlNH2
and bovine β-(1,4)-galactosyltransferase can be used for the
preparation of thionodulation factor intermediate trisaccha-
rides. However, because trisaccharide 10 could not be iso-
lated pure and free of salt without degradation, we focused
our synthetic effort on the preparation of the nonreducing
allyl thiochitobioside derivative (20) (Scheme 3).
O
HO
HO
x
O
OH
NHAc
HO
NHAc
7 X = O
8 X = S
UDP-GlcNH2 (6) β-(1,4)-GalT
OH
OH
OH
O
HO
HO
O
O
O
x
HO
OH
NHAc
HO
NHAc
NH2
9 X = O
10 X = S
1
Fig. 2. H NMR (600 MHz) of disaccharide acceptor (A) 7,
(B) semi-pure trisaccharide 9, (C) disaccharide acceptor 8, and
(D) semi-pure trisaccharide 10.
The allyl group was chosen as a versatile aglycon that is
not only easily introduced and removed at the anomeric po-
sition, but can also be used to immobilize the oligosaccha-
rides on solid support (15) to further attempt to isolate the
specific plant receptor. Nucleophilic displacement of the
known (5c) 1,6-anhydro triflate 12 with the anomeric
thiolate obtained by treatment of the thiol 11 (5b) with so-
dium hydride, afforded the thiodisaccharide 13 in excellent
yield (90%). The 1,6-anhydro ring in disaccharide 13 was
opened with a mixture of Ac2O and TFA to give the
anomeric mixture of 14 (α/β, 65:35) in high yield (96%).
Considering the low stability of the allyl group in debenzyl-
ation conditions, we replaced the benzyl group at the C-3
hydroxyl group of the reducing end glucosamine with an
acetyl group prior to introducing the allyl aglycon. Thus, the
benzyl group in compound 14 was removed using iron
trichloride (16) in dichloromethane and the crude intermedi-
ate alcohol was acetylated to give the peracetate 15 as an
anomeric mixture (Scheme 3). The thiodisaccharide 15 was
subsequently converted to the hemiacetal 16 by selective
deacetylation of the anomeric hydroxyl group and the hemi-
acetal 16 was treated with DBU and trichloroacetonitrile to
give trichloroacetimidate 17 as an anomeric mixture in favor
of the α anomer (α/β, 85:15). Glycosylation of allyl alcohol
with the donor 17 was performed under BF3·Et2O catalysis
in dichloromethane and gave the desired thiodisaccharide 18
by applying the impure products to ion exchange columns
(AG1X-8/OH–) resulted in the degradation or adsorption and
subsequent loss of the reducing sugars. Although the prod-
ucts could not be obtained free of salts, NMR confirmed that
the products isolated were the desired trisaccharides. 1H
NMR of 9 (Fig. 2B) clearly showed signals that could be as-
signed to the newly introduced glucosamine residue at
© 2006 NRC Canada

590
Can. J. Chem. Vol. 84, 2006
Fig. 3. MALDI-MS (accuracy m/z: 1) of enzymatic transfer on (A) chitobiose (7) and (B) thiochitobiose (8).
in good yield (83%) and high selectivity in favor of the β
anomer (α/β, 1:9). The β anomer 18β was isolated
anomerically pure by chromatography on alumina and con-
verted to the N-acetate 19 in moderate yield through the
reduction of the azido group (Zn-AcOH, ref. 17) followed
by in situ N-acetylation. Finally, Zemplèn deacetylation of
disaccharide 19 gave the acceptor 20 in good yield.
Enzymatic transfer of the glucosamine residue (Scheme 4)
was accomplished using the same conditions as those de-
scribed for the preparation of trisaccharides 9 and 10. Thus,
thiodisaccharide 20 was incubated for 2 weeks at ambient
temperature with UDP-GlcNH2 6 (1.2 equiv.) and the bovine
galactosyltransferase (5.6 U). The thiotrisaccharide 21 was
obtained pure in 41% yield after chromatography on Bio-Gel
P-2 (AcONH4, 0.25 mol/L), followed by desalting on a
small column of AG MP-1 (OH–) eluted with water. The
pure compound 21 was fully characterized by 1H NMR
spectroscopy, COSY, and HSQC, and its structure was con-
firmed by HR-ESI MS.
The results reported herein show that the transfer of a
glucosamine unit to a thiochitobiose derivative by enzymatic
catalysis using the well-known β-(1,4)-galactosyltransferase
is applicable to the preparation of thiooligosaccharides. Fur-
ther reactions on the thiotrisaccharide 21, including the in-
troduction of the fatty acid moiety, should provide access to
a large series of thioanalogues of nodulation factors.
Experimental section
General procedure for the enzymatic reactions
The acceptors 7, 8, and 20 (~5 mg, ~11 µmol) and UDP-
glucosamine (~9 mg, 1.2 equiv.) were dissolved in 1 mL of a
pH 7.4 buffer (80 mmol/L sodium cacodylate, 5 mmol/L
MgCl2, 0.1 mmol/L DTT) containing ␤-1,4-galactosyl-
© 2006 NRC Canada

Loureiro Morais et al.
591
Scheme 3. Reagents: (i) a. 11, NaH, DMF, 0 °C; b. 12, 20 °C (90%); (ii) 9:1 Ac2O–TFA (96%, α/β = 65:35); (iii) a. FeCl3, CH2Cl2;
b. Ac2O, pyridine (62%, α/β = 8:2); (iv) H2NNH2–AcOH, DMF (75%, α/β = 7:3); (v) Cl3CCN, DBU, CH2Cl2 (95%, α/β = 85:15);
(vi) AllOH, BF3·Et2O, CH2Cl2 (83%, α/β = 1:9); (vii) a. Zn, AcOH, THF; b. Ac2O (45% from 18β); (viii) MeONa, MeOH (72%).
O
OBn
O
OAc
O
OAc
O
O
OAc
O
OAc
O
OBn
AcO
AcO
S
i
ii
AcO
AcO
OAc
O
AcO
AcO
RO
TfO
SH
S
N3
+
AcHN
iii
N3
AcHN
N3
12
AcHN
14 R = Bn
15 R = Ac
11
13
OAc
O
OAc
O
OAc
O
OAc
O
iv
AcO
AcO
S
AcO
AcO
S
15
v
OH
OR
AcO
AcO
AcHN
AcHN
N3
N3
16
17 R = C(NH)CCl3
18 R = All
vi
OH
O
OAc
O
OH
OAc
O
vii
viii
HO
O
S
AcO
AcO
S
18β
OAll
HO
OAll
AcO
HO
AcHN
AcHN
AcHN
AcHN
20
19
favor of the desired α anomer (94:6). Therefore, the crude
material was dissolved in DMF (50 mL) and the solution
was warmed to 50 °C. Hydrazine acetate (1.74 g,
18.9 mmol, 1.05 equiv.) was added to the solution and stir-
ring at 50 °C was maintained for 25 min. The reaction mix-
ture was diluted with EtOAc and washed three times with
brine. The organic layer was dried over MgSO4 and concen-
trated to give a syrup (8.55 g). Chromatography on silica gel
(hexane–EtOAc, 3:2) gave the title compound 3 (5.09 g,
74%) as a pure white powder. Compound 3 had characteris-
tics in excellent agreement with those described (10, 11).
[α]D +21° (c 1.6, CHCl3) (lit. value (10) +25° (c 1, CHCl3),
Scheme 4.
20
β-(1,4)-GalT
UDPGlcNH2 (6)
OH
OH
OH
O
HO
HO
O
O
O
S
OAll
NHAc
HO
HO
NH2
NHAc
21
transferase (5.6 U). Solutions of MnCl2 (1 mol/L, 25 µL,
5 mmol), α-lactalbumin (25 mg/mL, 45 µL), and alkaline
phosphatase (1 U) were added to the mixture, which was
then left at room temperature for 2 weeks. TLC monitoring
of the reactions (i-PrOH–NH4OH–H2O, 7:2:1 or CHCl3–
MeOH–H2O, 65:35:2) showed, in all three cases, the forma-
tion of new compounds more polar than the starting materi-
als in ~50% yield estimated from the TLC. The reaction
mixtures were then applied directly to a Bio-Gel P-2 column
1
lit. value (11) +20° (c 0.5, CHCl3)). H NMR (360 MHz,
CDCl3) δ: 6.75 (d, 1H, JNH,2 = 9.7 Hz, NH), 5.37 (dd, 1H,
J2,3 = 9.7 Hz, J3,4 = 9.5 Hz, H-3), 5.37–5.34 (m, 1H, H-1),
5.27 (t, 1H, J4,5 = 9.5 Hz, H-4), 4.32 (dt, 1H, J1,2 = 3.5 Hz,
H-2), 4.27–4.08 (m, 3H, H-5, H-6a, H-6b), 3.78 (bs, 1H,
OH), 2.11, 2.06, and 2.04 (3 s, 9H, 3 × CH3CO). 13C NMR
(75 MHz, CDCl3) δ: 171.4, 171.0, 169.4 (COCH3), 157.3 (q,
JC,F = 37.4 Hz, COCF3), 115.5 (q, JC-F = 290 Hz, CF3), 91.0
(C-1), 70.5, 67.9 (C-3, C-4, C-5), 61.9 (C-6), 52.8 (C-2),
20.8, 20.6, 20.5 (CH3CO).
+
(100 cm × 1 cm) eluted with a 0.25 mol/L NH4 AcO– solu-
tion. The fractions containing the new compounds were
combined and freeze dried repeatedly from MQ water. The
allyl thiotrisaccharide 21 was additionally deionized on a
column of AG MP-1(OH–) eluted with H2O.
3,4,6-Tri-O-acetyl-2-deoxy-2-trifluoroacetamido-␣-D-
glucopyranosyl dibenzyl phosphate (4)
A
solution of butyllithium (1.6 mol/L, 8.4 mL,
3,4,6-Tri-O-acetyl-2-deoxy-2-trifluoroacetamido-␣-D-
glucopyranose (3)
13.4 mmol) in hexane was added to a solution of hemiacetal
3 (5.31 g, 13.2 mmol) in THF (160 mL) stirred at –70 °C.
After 2 min, a solution of dibenzyl chlorophosphate (12)
(5.5 mL, 29.3 mmol) in THF (200 mL) was added slowly at
–70 °C under stirring and the mixture was stirred for a fur-
ther 30 min while the temperature was kept between –60 and
–50 °C. Triethylamine was added to pH 8 and the solvent
was evaporated. The residue was partitioned between chloro-
form and aqueous sodium chloride. The organic layer was
dried (MgSO4), concentrated, and chromatography on silica
gel (hexane–EtOAc, 3:2) gave the pure dibenzylphosphate 4
(5.17 g, 59%) as a colorless glass. [α]D +55° (c 1.2, CHCl3).
1H NMR (360 MHz, CDCl3) δ: 8.2 (d, 1H, JNH,2 = 9.0 Hz,
2-Deoxy-2-trifluoroacetamido-D-glucopyranose (4.72 g,
17.1 mmol) was prepared as described by Wolform and
Conigliaro (10) from commercially available crystalline D-
glucosamine hydrochloride and was dissolved in pyridine
(32 mL) and acetic anhydride (18 mL). The reaction mixture
was kept at room temperature for 16 h and poured into ice
water and stirred for 1 h. The mixture was extracted with di-
chloromethane and the organic layer was washed with aq.
NaHCO3 and dried on Na2SO4. The solution was concen-
trated to give a crude syrup (8.56 g) containing the peracety-
lated 2-deoxy-2-trifluoroacetamido-α/β-D-glucopyranose (2).
1H NMR analysis showed that the α/β ratio was largely in
© 2006 NRC Canada

592
Can. J. Chem. Vol. 84, 2006
NH), 7.40–7.20 (m, 10H, H-aromatics), 5.80 (dd, 1H, J1,2
=
and concentrated and the residue dissolved in water was
treated with Dowex 50-X8 (H+) until the pH was below 7
and CO2 gas had evolved. The resin was filtered off and the
pH of the filtrate was adjusted to 8 by adding aqueous so-
dium hydroxide. The solution was passed through a Millex
SR filter unit and the filtrate was lyophilized to give pure
UDP-GlcNH2 (6) as a white solid (2.43 g, 51%). H NMR
(400 MHz, D2O) δ: 7.96 (d, 1H, J5,6 = 8.0 Hz, H-6), 5.99 (d,
1H, J1′,2′ = 4.4 Hz, H-1′), 5.96 (d, 1H, H-5), 5.75 (dd, 1H,
3.2 Hz, J1,P = 6.7 Hz, H-1), 5.35 (dd, 1H, J2,3 = 10.5 Hz,
J3,4 = 10.0 Hz, H-3), 5.16 (t, 1H, J4,5 = 10.0 Hz, H-4), 5.28–
4.98 (m, 4H, 2 × CH2Ph), 4.41 (ddd, 1H, J2,P = 3.2 Hz, H-2),
4.25–3.98 (m, 3H, H-5, H-6a, H-6b), 2.03, 2.02, 1.99 (3s,
9H, 3 × CH3CO). 13C NMR (75 MHz, CDCl3) δ: 171.0,
170.5, 169.1 (COCH3), 157. 6 (q, JC,F = 37.7 Hz, COCF3),
135.2, 135.0, 129.0,128.8, 128.2 (C-aromatics), 115.5 (q,
JC-F = 286.8 Hz, CF3), 95.1 (d, JC-P = 5.7 Hz, C-1), 70.3,
70.2, 69.8, 67.2 (C-3, C-4, C-5, 2 × CH2Ph), 61.1 (C-6),
52.6 (d, J2,P = 7.5 Hz, C-2), 20.6, 20.5, 20.4 (CH3CO). Anal.
calcd. for C28H31NO12PF3: C 50.84, H 4.72, N 2.12; found:
C 50.90, H 4.67, N 1.97.
1
J1′′,2′′ = 3.3 Hz, J
= 6.8 Hz, H-1′′), 4.36 (m, 2H, H-5a′,
1′′-P
H-5b′), 4.31–4.16 (m, 3H, H-2′, H-3′, H-4′), 3.78–3.96 (m,
4H, H-3′′, H-5′′, H-6a′′, H-6b′′), 3.53 (t, 1H, J3′′,4′′
J4′′,5′′ = 9.6 Hz, H-4′′), 3.16 (dd, 1H, J2′′,3′′ = 10.4 Hz, J2′′,P
=
=
3.5 Hz, H-2′′). 13C NMR (75 MHz, D2O) δ: 167.1 (C-4),
153.7 (C-2), 142.5 (C-6), 103.5 (C-5), 93.7 (d, JC,P = 5.3 Hz,
C-1′′), 89.5 (C-1′), 83.9 (d, JC,P = 8.7 Hz, C-4′), 74.6, 74.1,
70.8, 70.4, 69.9 (C-2′, C-3′, C-3′′, C-4′′, C-5′′), 65.9 (d,
JC,P = 5.3 Hz, C-5′), 60.9 (C-6′′), 55.0 (d, JC,P = 9.3 Hz, C-2′′).
31P NMR (145 MHz, D2O) δ: –10.9 (d, JP,P = 22.0 Hz,
P-O-uridine), –13.5 (d, P-O-Glc). HRMS calcd. for
C15H23O16N3Na2P2S m/z: 632.0247 [M + Na]; found:
632.0245.
Uridine 5′-(2-amino-2-deoxy-␣-D-glucopyranosyl)-
diphosphate (6)
A solution of dibenzylphosphate 4 (5.17 g, 7.82 mmol) in
MeOH (400 mL) containing 5% Pd/C (2.14 g) was stirred
under hydrogen for 2.5 h. The catalyst was filtered off and
the filtrate concentrated to dryness to give 3,4,6-tri-O-acetyl-
2-deoxy-2-trifluoroacetamido-α-D-glucopyranosyl phosphate
(5) as a white solid. Rf 0.28 (60:40:3, CH2Cl2–MeOH–H2O).
1H NMR (360 MHz, CDCl3–CD3OD, 2:1) δ: 5.64 (dd, 1H,
4-S-(2-Acetamido-2-deoxy-␤-D-glucopyranosyl)-2-
J1,2 = 3.0 Hz, J1,P = 6.0 Hz, H-1), 5.42 (dd, 1H, J2,3
=
acetamido-2-deoxy-4-thio-␣,␤-D-glucopyranose (8)
The peracetylated thiochitobiose (5b) (37.2 mg,
0.040 mmol) was dissolved in anhydr. MeOH (4 mL) and a
solution of sodium methoxide in MeOH (1 mol/L, 40 µL)
was added. The reaction mixture was stirred for 3 days at
room temperature and deionized with Amberlite® IR120
resin. The resin was filtered off, rinsed with MeOH, and the
combined filtrate and washings were concentrated to dry-
ness. The anomeric mixture of thiochitobiose (8) was ob-
tained as a white precipitate (11.2 mg) from a mixture of
10.0 Hz, J3,4 = 9.5 Hz, H-3), 5.17 (t, 1H, J4,5 = 10.0 Hz, H-
4), 4.42 (dt, 1H, J2,P = 3.0 Hz, H-2), 4.33–4.10 (m, 3H, H-5,
H-6a, H-6b), 2.11, 2.06, 2.02 (3s, 9H, 3 × CH3CO). Crude 5
was dissolved in acetone (130 mL) and 80% 1,1′-
carbonyldiimidazole (3.3 g, 16.3 mmol) was added and the
mixture was stirred for 15 h at room temperature. After
completion of the reaction (CHCl3–MeOH, 3:1, Rf 0.28),
methanol (170 µL) was added and the mixture was stirred
for another 0.5 h. The reaction mixture was concentrated and
dried under reduced pressure over P2O5 for 6 h. While the
crude imidazolate was drying, uridine 5′-monophosphate di-
sodium salt (3.5 g, 9.51 mmol) was passed slowly through a
column of Dowex 50-5X8 (H+) resin and the column was
thoroughly washed with water. The eluate was concentrated
and trioctylamine (8.8 mL, 20 mmol) was added. The mix-
ture was coevaporated three times with DMF and a solution
of the aforementioned dry imidazolate in DMF (100 mL)
was added. The mixture was stirred for 24 h at room temper-
ature and the formation of the adduct was followed by TLC
(CH2Cl2–MeOH–H2O, 60:40:3, Rf 0.35). The solvent was
evaporated and the dry residue was dissolved in methanol
(150 mL) and ammonium hydroxide (60 mL). The solution
was stirred at room temperature for 24 h and concentrated.
The crude deacylated product was dissolved in 40 mmol/L
aqueous ammonium bicarbonate and slowly passed through
1
MeOH and acetone. H NMR was reported previously (14b)
for the two anomers and showed the presence of less than
10% of an impurity (Fig. 2C) that could not be removed nor
identified. Nevertheless, HRMS confirmed that the wanted
anomeric mixture of thiochitobiose was the main product
isolated. HRMS calcd. for C16H28O10 N2S m/z: 463.1362
[M + Na]; found: 463.1364.
Enzymatic transfer on chitobiose (7) and thiochitobiose
(8)
MALDI-MS conditions (Kratos Kompact MALDI-TOF,
external calibration on insulin): 10 µL of the reaction mix-
ture were diluted with 90 µL of water or 0.25 mol/L AcONH4
and 0.5 µL of these solutions were deposited on the target
with 0.5 µL of THAP as the matrix. The trisaccharides 9
(5.7 mg) and 10 (10 mg) were isolated as amorphous white
powders showing residual acetate salts by NMR (Supple-
mentary material).5 Compound 10 also showed the impurity
–
a Dowex 1X2-200 (HCO2 ) column (3 cm × 30 cm). The
column was kept at 23 °C and was first washed with
40 mmol/L aq. NH4HCO2, then it was eluted (1.6 mL/min)
with a linear gradient of ammonium bicarbonate (mixing
vessel 550 mL of 40 mmol/L NH4HCO2; reservoir 550 mL
of 700 mmol/L NH4HCO2). The UV absorption of the eluate
at 280 nm was monitored and 20 mL fractions were col-
lected. Those fractions containing the product were pooled
1
seen in 8. Nevertheless, HRMS and H NMR showed that
these compounds were the desired trisaccharides. HRMS for
9 calcd. for C22H39O15N3 m/z: 586.2459 [M + H]; found:
586.2463. HRMS for 10 calcd. for C22H39O14N3S m/z:
602.2231 [M + H]; found: 602.2237.
5 Supplementary data for this article are available on the journal Web site (http://canjchem.nrc.ca) or may be purchased from the Depository
of Unpublished Data, Document Delivery, CISTI, National Research Council Canada, Ottawa, ON K1A 0R6, Canada. DUD 5022. For more
information on obtaining material refer to http://cisti-icist.nrc-cnrc.gc.ca/irm/unpub_e.shtml.
© 2006 NRC Canada

Loureiro Morais et al.
593
CH3COα and 4 × CH3COβ), 1.71 (s, 3Hα, CH3CONHα),
1.68 (s, 3Hβ, CH3CONHβ). 13C NMR (100 MHz, CDCl3)
for the α anomer δ: 170.5, 168.7 (COCH3), 128.6, 128.2,
127.6, 127.5 (C-aromatics), 90.4 (C-1), 83.6 (C-1′), 77.0 (C-
3), 75.6 (CH2Ph), 75.2 (C-5′), 72.8 (C-3′), 72.5 (C-5), 68.2
(C-4′), 63.9 (C-2), 63.2 (C-6), 62.1 (C-6′), 54.5 (C-2′), 46.8
(C-4), 23.1, 21.0, 20.6 (CH3CO). HRMS calcd. for
C31H40N4O14S m/z: 747.2159 [M + Na]; found: 747.2158.
4-S-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-␤-D-
glucopyranosyl)-1,6-anhydro-2-azido-3-O-benzyl-2-
deoxy-4-thio-␤-D-glucopyranose (13)
A solution of thiol 11 (5b) (17.6 g, 43 mmol) in DMF
(300 mL) was added to a suspension of NaH (55% in oil,
2.33 g, 53 mmol, 1.2 equiv.) in DMF (10 mL) cooled to
0 °C. The mixture was stirred under Ar for 10 min at 0 °C,
10 min at 20 °C and was, in turn, added to triflate 12 (5c)
(18.6 g, 51 mmol, 1.2 equiv.). The reaction mixture was
stirred for 1 h at 20 °C and the reaction was quenched by ad-
dition of AcOH (20 mL), followed by stirring for a further
30 min at room temperature. The solvent was removed in
vacuo and the residue was purified by chromatography on
silica gel (1:0 to 1:4 toluene–EtOAc) to give thiodi-
1,3,6-Tri-O-acetyl-4-S-(2-acetamido-3,4,6-tri-O-acetyl-2-
deoxy-␤-D-glucopyranosyl)-2-azido-2-deoxy-4-thio-␣,␤-D-
glucopyranose (15)
Anhydrous FeCl3 (20.4 g, 126 mmol, 4 equiv.) was added
to a solution of benzyl 14 (22.7 g, 31 mmol) in freshly dis-
tilled anhydr. CH2Cl2 (220 mL) and the mixture was stirred
for 1.5 h under Ar at 20 °C. The solvent was evaporated then
pyridine (210 mL) and Ac2O (105 mL) were added. The re-
action mixture was stirred for 1.5 h at 20 °C and concd.
EtOAc (350 mL) was added to the residue and the resulting
solution was washed successively with 1 mol/L HCl
(250 mL), satd. NaHCO3 (250 mL), and brine (250 mL).
The aqueous layers were reextracted with EtOAc (2 ×
250 mL) and the combined organic layers were dried and
concentrated. The crude product was filtered off from EtOH
and further purified by chromatography on silica gel (0:1 to
9:1 EtOAc–cyclohexane) to afford acetate 15 (13.1 g, 62%)
as a white powder. 1H NMR spectra gave an α/β ratio of 8:2.
1H NMR (400 MHz, CDCl3) δ: 6.33 (d, 1Hα, J1,2 = 3.3 Hz,
H-1α), 5.77 (d, 1Hα, JNH,2 = 9.8 Hz, NHα), 5.69 (d, 1Hβ,
JNH,2 = 10.1 Hz, NHβ), 5.49 (d, 1Hβ, J1,2 = 8.6 Hz, H-1β),
5.43 (t, 1Hα, J2,3 = J3,4 = 10.5 Hz, H-3α), 5.11 (t, 1Hα,
J2′,3′ = J3′,4′ = 9.7 Hz, H-3′α), 5.08–4.91 (m, 1Hα and 3Hβ,
H-4′α, H-4′β, H-3′β and H-3β), 4.74 (d, 1Hα and 1Hβ,
1
saccharide 13 (24.5 g, 90%) as a white solid; mp 183 °C. H
NMR (400 MHz, CDCl3) δ: 7.41–7.36 (m, 5H, H-aromatics),
5.54 (d, 1H, JNH,2 = 9.1 Hz, NH), 5.44 (s, 1H, H-1), 5.26 (t,
1H, J2,3 = J3,4 = 9.9 Hz, H-3′), 5.09 (t, 1H, J3′ ,4′ = J4′,5′
=
9.8 Hz, H-4′), 4.93 (d, 1H, J1′,2′ = 10.4 Hz, H-1′), 4.76–4.70
(m, 2H, CHPh, H-5), 4.67 (d, 1H, J = 11.9 Hz, CHPh), 4.27
(d, 1H, J6a,6b = 7.4 Hz, H-6a), 4.19 (dd, 1H, J6a′ ,6b′
12.4 Hz, J5′ ,6a′ = 4.7 Hz, H-6a′), 4.14 (dd, 1H, J5′ ,6b′
=
=
2.3 Hz, H-6b′), 4.00 (td, 1H, J2′ ,3′ = 10.4 Hz, H-2′), 3.82
(m, 1H, H-4), 3.77 (dd, 1H, J5,6b = 5.6 Hz, H-6b), 3.68 (m,
1H, H-5′), 3.52 (s, 1H, H-3), 3.23 (s, 1H, H-2), 2.06, 2.05,
2.03, 1.95 (4s, 12H, 4 × CH3CO). 13C NMR (100 MHz,
CDCl3) δ: 170.9, 170.6, 170.2, 169.4 (COCH3), 137.5 (C-
ipso), 128.6, 128.0, 127.7 (C-aromatics), 100.1 (C-1), 83.4
(C-1′), 78.6 (C-4), 76.1 (C-5′ and C-5), 73.3 (C-3′), 72.3
(CH2Ph), 68.3 (C-4′), 67.8 (C-6), 62.0 (C-6′), 60.2 (C-3),
53.8 (C-2′), 44.0 (C-2), 23.3, 20.7, 20.6 (CH3CO). HRMS
calcd. for C27H34N4O11S m/z: 645.1843 [M + Na]; found:
645.1845.
J1′,2′ = 10.4 Hz, H-1′α and H-1′β), 4.60 (dd, 1Hα, J6a,6b
12.5 Hz, J5,6a = 3.9 Hz, H-6aα), 4.55 (dd, 1Hβ, J6a,6b
=
=
1,6-Di-O-acetyl-4-S-(2-acetamido-3,4,6-tri-O-acetyl-2-
deoxy-␤-D-glucopyranosyl)-2-azido-3-O-benzyl-2-deoxy-
4-thio-␣,␤-D-glucopyranose (14)
Thiodisaccharide 13 (24.5 g, 39 mmol) was dissolved in
Ac2O–TFA (9:1, 166 mL), and the solution was stirred for
6.5 h at 40 °C. Solvents were evaporated under reduced
pressure and residual Ac2O was coevaporated with toluene.
The residue was submitted to chromatography (3:2 to 0:1 to-
luene–EtOAc) to furnish compound 14 (23 g, 96%). 1H
12.5 Hz, J5,6a = 4.6 Hz, H-6aβ), 4.46 (m, 1Hβ, H-6bβ),
4.40–4.29 (m, 2Hα, H-5α and H-6bα), 4.28–4.13 (m, 2Hα
and 2Hβ, H-2′α, H-2′β, H-6a′α and H-6a′β), 4.10–4.00 (m,
1Hα and 1Hβ, H-6b′α and H-5β), 3.95 (dd, 1Hβ, J6a′,6b′
=
12.1 Hz, J5′,6b′ = 7.9 Hz, H-6b′β), 3.76–3.63 (m, 2Hα and
2Hβ, H-2α, H-2β, H-5′α and H-5′β), 2.95 (t, 1Hα, J =
11.2 Hz, H-4α), 2.86 (t, 1Hβ, J3,4 = J4,5 = 10.5 Hz, H-4β),
2.22 (s, 3Hα, CH3COα), 2.19 (s, 3Hα, CH3COα), 2.19–2.18
(2s, 6Hβ, 2 × CH3COβ), 2.13 (s, 3Hβ, CH3COβ), 2.07 (s,
3Hα, CH3COα), 2.06–2.01 (m, 9Hα and 9Hβ, 3 × CH3COα
and 3 × CH3COβ), 1.95 (s, 3Hα, CH3CONHα) and 1.92 (s,
3Hβ, CH3CONHβ). 13C NMR (100 MHz, CDCl3) for the α
anomer δ: 171.3, 170.6, 170.3 (COCH3), 90.3 (C-1), 82.3
(C-1′), 75.9 (C-5′), 74.2 (C-3′), 71.1 (C-5), 68.5 (C-4′), 67.8
(C-3), 63.0, 62.9 (C-6 and C-6′), 61.8 (C-2), 51.9 (C-2′),
45.8 (C-4), 23.0, 20.9, 20.7, 20.6 (CH3CO). HRMS calcd.
1
NMR spectroscopy showed an α/β ratio of 65:35. H NMR
(400 MHz, CDCl3) δ: 7.50–7.30 (m, 5Hα and 5Hβ, H-
aromatics), 6.29 (d, 1Hα, J1,2 = 3.2 Hz, H-1α), 5.58 (d, 1Hα,
J1′,2′ = 8.6 Hz, H-1′α), 5.47 (m, 2Hβ, H-1β and H-1′β),
5.30 (t, 1Hα and 1Hβ, J2′,3′+3′,4′ = 19.7 Hz, H-3′α and H-
3′β), 5.08–4.95 (m, 2Hα and 2Hβ, CHPhα, CHPhβ, H-4′α
and H-4′β), 4.95–4.85 (m, 2Hα and 2Hβ, CHPhα, CHPhβ,
NHα and NHβ), 4.57–4.0 (m, 2Hα and 2Hβ, H-6aα, H-
6aβ, H-6bα and H-6bβ), 4.19 (dd, 1Hα, J6a′,6b′ = 12.5 Hz,
J5′,6a′ = 4.9 Hz, H-6a′α), 4.11–3.96 (m, 2Hα and 2Hβ, H-
5α, H-6b′α, H-6b′β and H-6a′β), 3.89–3.77 (m, 1Hα and
2Hβ, H-2′α, H-2β and H-5β), 3.73 (t, 1Hα, J = 10.1 Hz, H-
3α), 3.69–3.63 (m, 2Hα and 1Hβ, H-5′α, H-5′β and H-2α),
for C26H36N4O15S m/z: 699.1796 [M
+ Na]: found:
699.1796.
3,6-Di-O-acetyl-4-S-(2-acetamido-3,4,6-tri-O-acetyl-2-
deoxy-␤-D-glucopyranosyl)-2-azido-2-deoxy-4-thio-␣,␤-D-
glucopyranose (16)
Hydrazine acetate (2.1 g, 23 mmol, 1.2 equiv.) was added
to a solution of compound 15 (12.8 g, 19 mmol) in DMF
(110 mL). The mixture was stirred for 1 h under Ar at room
temperature and the solvent was removed under reduced
3.60 (t, 1Hβ, J1,2 = J2,3 = 8.9 Hz, H-2β), 3.41 (t, 1Hβ, J3,4
=
10.0 Hz, H-3β), 2.98 (t, 1Hα, J3,4 = J4,5 = 10.5 Hz, H-4α),
2.93 (t, 1Hβ, J4,5 = 10.5 Hz, H-4β), 2.20–2.17 (2s, 3Hα and
3Hβ, CH3COα and CH3COβ), 2.12 (s, 3Hα, CH3COα),
2.09 (s, 3Hα, CH3COα), 2.05–2.00 (m, 6Hα and 12Hβ, 2 ×
© 2006 NRC Canada

594
Can. J. Chem. Vol. 84, 2006
pressure. Residual traces of DMF were coevaporated with
toluene and the residue was dissolved in EtOAc (200 mL)
and washed with water (150 mL) and brine (150 mL). The
aqueous layers were reextracted with EtOAc (2 × 250 mL)
and the combined organic solvents were dried and concen-
trated. The crude product was purified by chromatography
on silica gel (1:0 to 95:5 CHCl3–MeOH) to yield hemiacetal
J6a′,6b′ = 12.4 Hz, J5′,6a′ = 2.8 Hz, H-6a′α), 4.06–3.98 (m,
1Hα and 1Hβ, H-6b α and H-5β), 3.94 (dd, 1Hβ, J
=
′
6a′,6b′
12.0 Hz, J5′,6b′ = 8.4 Hz, H-6b′β), 3.88–3.78 (m, 1Hα and
1Hβ, H-2α and H-2β), 3.74 (m, 1Hβ, H-5′β), 3.54 (m,
1Hα, H-5′α), 3.01 (t, 1Hα, J4,5 = 11.0 Hz, H-4α), 2.93 (t,
1Hβ, J3,4 = J4,5 = 11.0 Hz, H-4β), 2.20 (s, 3Hβ, CH3COβ),
2.19 (s, 3Hα, CH3COα), 2.14 (s, 3Hβ, CH3COβ), 2.06 (s,
3Hα, CH3COα), 2.05–2.03 (m, 6Hα and 6Hβ, 2 × CH3COα
and 2 × CH3COβ), 2.02 (s, 3Hα and 3Hβ, CH3COα and
CH3COβ), 1.95 (s, 3Hα, CH3COα), 1.93 (s, 3Hβ, CH3COβ).
1
16 (8.9 g, 75%). The H NMR spectra showed an α/β ratio
1
of 7:3. H NMR (400 MHz, CDCl3) δ: 5.90 (d, 1Hα, J1′,2′
=
9.9 Hz, H-1′α), 5.81 (d, 1Hβ, J1′,2′ = 9.9 Hz, H-1′β), 5.55 (t,
1Hα, J2,3 = J3,4 = 10.5 Hz, H-3α), 5.40 (d, 1Hα, J1,2
=
3.1 Hz, H-1α), 5.09 (t, 1Hα, J2′,3′ = J3′,4′ = 9.5 Hz, H-3′α),
Allyl 3,6-di-O-acetyl-4-S-(2-acetamido-3,4,6-tri-O-acetyl-
2-deoxy-␤-D-glucopyranosyl)-2-azido-2-deoxy-4-thio-␤-D-
glucopyranoside (18␤)
5.08–4.97 (m, 1Hα, 3Hβ, H-3′β, H-3β, H-4′α and H-4′β),
4.82 (d, 1Hα, JNH,2 = 10.4 Hz, NHα), 4.78 (d, 1Hβ, JNH,2
=
10.5 Hz, NHβ), 4.69 (d, 1Hβ, J1,2 = 7.9 Hz, H-1β), 4.57 (dd,
A suspension of imidate 17 (8.87 g, 11 mmol) in anhydr.
CH2Cl2 (140 mL) containing 4 Å activated molecular sieves
(8.0 g) was stirred under Ar at room temperature for 1 h and
was then cooled down to – 45 °C. Allyl alcohol (1.55 mL,
23 mmol, 2 equiv.) and BF3·Et2O (920 µL, 3.4 mmol,
3.2 equiv.) were added to the suspension and the mixture
was allowed to reach –10 °C over 2 h. Triethylamine
(200 µL) was added and after additional stirring (20 min) at
room temperature, the molecular sieves were filtered off.
The filtrate was diluted with CH2Cl2 (250 mL) and washed
with brine (250 mL). The aqueous layer was reextracted
with CH2Cl2 (3 × 100 mL) and the combined organic solu-
tions were dried and concentrated. The residue was submit-
ted to chromatography on neutral alumina (1:2, 1:1, 3:1, 5:1,
then 1:0, EtOAc–cyclohexane) to give the thiodisaccharide
1Hα, J6a,6b = 11.8 Hz, J5,6a = 3.7 Hz, H-6aα), 4.55 (m, 1Hα
and 2Hβ, H-5α, H-6aβ and H-6bβ), 4.44 (dd, 1Hα, J5,6b
=
1.6 Hz, H-6bα), 4.26–4.16 (m, 2Hα and 2Hβ, H-2′α, H-2′β,
H-6a′α and H-6a′β), 4.01 (dd, 1Hα and 1Hβ, J6a′,6b′
=
12.3 Hz, J5′,6b′ = 6.0 Hz, H-6b′α and H-6b′β), 3.92 (m, 1Hβ,
H-5β), 3.73 (m, 1Hα and 1Hβ, H-5′α and H-5′β), 3.50 (m,
1Hα and 1Hβ, H-2α and H-2β), 2.91–2.82 (m, 1Hα and
1Hβ, H-4α and H-4β), 2.17 (s, 3Hα and 3Hβ, CH3COα and
CH3COβ), 2.10 (s, 3Hα, CH3COα), 2.09 (s, 3Hβ, CH3COβ),
2.07 (s, 3Hα, CH3COα), 2.06 (s, 3Hβ, CH3CO β), 2.03 (s,
3Hα and 3Hβ, CH3COα and CH3COβ), 2.01 (s, 3Hα and
3Hβ, CH3COα and CH3COβ), 1.94 (s, 3Hα, CH3COα) and
1.92 (s, 3Hβ, CH3COβ). 13C NMR (100 MHz, CDCl3) δ:
170.8, 170.7, 170.1, 169.4 (COCH3), 96.1 (C-1β), 91.8 (C-1
α), 81.9 (C-1′α), 81.7 (C-1′β), 75.5 (C-5′α), 75.4 (C-5′β),
74.3 (C-3′α), 74.1 (C-3′β), 73.6 (C-5β), 70.1 (C-3β), 68.6
(C-5α), 68.4 (C-4′α), 68.3 (C-4′β), 67.9 (C-3α), 66.2 (C-
2β), 63.5 (C-6α and C-6β), 63.2 (C-2α), 62.5 (C-6′α and C-
6′β), 52.0 (C-2′α and C-2′β), 46.3 (C-4α), 46.1 (C-4β),
22.9, 21.1, 20.9, 20.7, 20.6 (CH3CO). HRMS calcd. for
C24H34N4O14S m/z: 657.1690 [M + Na]; found: 657.1714.
1
18β (5.71 g, 74%) as a white powder; mp 187 °C. H NMR
(400 MHz, CDCl3) δ: 5.94 (m, 1H, OCH2CHcCH2), 5.70 (d,
1H, JNH,2 = 9.7 Hz, NH), 5.34 (dd, 1H, Jc,e = 17.2 Hz, Jd,e
=
1.5 Hz, OCH2CHCHdHe), 5.26 (dd, 1H, Jc,d = 10.4 Hz,
OCH2CHCHdHe), 5.08–4.96 (m, 2H, H-3′ and H-4′), 4.93
(dd, 1H, J2,3 = 9.9 Hz, J3,4 = 11.0 Hz, H-3), 4.72 (d, 1H,
J1′,2′ = 10.7 Hz, H-1′), 4.54–4.49 (m, 2H, H-6a and H-6b),
4.43–4.35 (m, 2H, H-1 and OCHaHbCHCH2), 4.25–4.09 (m,
3H, H-2′, H-6a′ and OCHaHbCHCH2), 4.01 (dd, 1H, J6a′,6b′
= 12.3 Hz, J5′,6b′ = 6.4 Hz, H-6b′), 3.84 (m, 1H, H-5), 3.69
(m, 1H, H-5′), 3.52 (dd, 1H, J1,2 = 8.1 Hz, J2,3 = 9.7 Hz, H-
2), 2.83 (t, 1H, J4,5 = 10.9 Hz, H-4), 2.17, 2.09, 2.07, 2.04,
2.02, 1.92 (6s, 6 × CH3CO). 13C NMR (100 MHz, CDCl3) δ:
171.3, 170.9, 170.5, 170.2, 169.7, 169.3 (COCH3), 133.0
(OCH2CH=CH2), 118.1 (OCH2CH=CH2), 100.8 (C-1), 81.7
(C-1′), 75.7 (C-5′), 74.1 (C-3′), 73.5 (C-5), 70.3 (C-6), 69.8
(C-3), 68.3 (C-4′), 65.0 (C-2), 63.5 (OCH2CH=CH2), 62.7
(C-6′), 51.9 (C-2′), 46.2 (C-4), 23.0, 21.1, 20.8, 20.6
(CH3CO). HRMS calcd. for C27H38N4O14S m/z: 697.2003
[M + Na]; found: 697.1996.
3,6-Di-O-acetyl-4-S-(2-acetamido-3,4,6-tri-O-acetyl-2-
deoxy-␤-D-glucopyranosyl)-2-azido-3-O-benzyl-2-deoxy-
4-thio-␣,␤-D-glucopyranosyl trichloroacetimidate (17)
Trichloroacetonitrile (27.5 mL, 275 mmol, 19 equiv.) and
DBU (532 µL, 3.6 mmol, 0.26 equiv.) were added to a solu-
tion of hemiacetal 16 (8.74 g, 14 mmol) in anhydr. CH2Cl2
(130 mL). The reaction mixture was stirred under Ar at
room temperature for 45 min. The reaction mixture was con-
centrated and the residue submitted to chromatography on
silica gel (silica gel was previously washed with a 5% solu-
tion of Et3N in EtOAc) (1:0 to 95:5 CHCl3–MeOH) to give
1
the unstable imidate 17 (10.2 g, 95%) as a glass. H NMR
1
spectra showed an α/β ratio of 85:15. H NMR (400 MHz,
Allyl 3,6-di-O-acetyl-4-S-(2-acetamido-3,4,6-tri-O-acetyl-
2-deoxy-␤-D-gluco-pyranosyl)-2-acetamido-2-deoxy-4-
thio-␤-D-glucopyranoside (19)
CDCl3) δ: 8.81 (s, 1Hα, NHCCl3α), 8.73 (s, 1Hβ,
NHCCl3β), 6.52 (d, 1Hα, J1,2 = 3.4 Hz, H-1α), 5.78 (d,
1Hα, JNH,2 = 9.9 Hz, NHα), 5.70 (d, 1Hβ, JNH,2 = 10.2 Hz,
Zinc (52.4 g) and AcOH (23 mL) were added to a solution
of compound 18β (5.4 g, 8 mmol) in anhydr. THF (260 mL).
The mixture was stirred under Ar at room temperature for
1.5 h and Ac2O (140 mL) was added. The reaction mixture
was stirred for 1.5 h at room temperature and filtered
through Celite. The filtrate was concentrated to dryness and
the residue dissolved in EtOAc (300 mL), which was washed
successively with water (200 mL), satd. NaHCO3 (200 mL),
NHβ), 5.66 (d, 1Hβ, J1,2 = 8.5 Hz, H-1β), 5.52 (t, 1Hα, J2,3
=
J3,4 = 10.5 Hz, H-3α), 5.11–5.00 (m, 2Hα and 2Hβ, H-3′α,
H-3′β, H-3β and H-4′α), 4.95 (t, 1Hβ, J3′ ,4′ = J4′,5′ = 9.6 Hz,
H-4′β), 4.78–4.68 (m, 1Hα and 1Hβ, H-1′β and H-1′α),
4.66–4.55 (m, 1Hα and 1Hβ, H-6aα and H-6aβ), 4.50–4.38
(m, 2Hα and 1Hβ, H-5α, H-6bα and H-6bβ), 4.28–4.17 (m,
1Hα and 2Hβ, H-2′α, H-2′β and H-6a′β), 4.09 (dd, 1Hα,
© 2006 NRC Canada

Loureiro Morais et al.
595
and brine (200 mL). The aqueous layers were reextracted
with EtOAc (6 × 100 mL) and the combined organic solu-
tions were dried and concentrated. The crude product was
submitted to chromatography on silica gel (1:0 and 95:5
CHCl3–MeOH) to give the title compound 19 (2.5 g, 45%)
D2O) δ: 5.88 (m, 1H, OCH2CHcCH2), 5.35–5.19 (m, 2H,
OCH2CHCHdHe and OCH2CHCHdHe), 4.71 (d, 1H, J1′,2′
=
10.1 Hz, H-1′), 4.52 (d, 1H, J1,2 = 8.4 Hz, H-1), 4.46 (d, 1H,
J1′′ ,2′′ = 8.0 Hz, H-1′′), 4.30 (dd, 1H, Ja,b = 13.1 Hz, Ja,c
6.3 Hz, OCHaHbCHCH2), 4.12 (dd, 1H, Jb,c = 6.4 Hz,
OCHaHbCHCH2), 4.05 (dd, 1H, J6a,6b = 12.0 Hz, J6a,5
=
1
as a white powder; mp 238 °C. H NMR (400 MHz, CDCl3)
=
δ: 5.87 (m, 1H, OCH2CHcCH2), 5.78 (d, 1H, JNH,2 = 9.4 Hz,
1.4 Hz, H-6a), 3.96–3.82 (m, 3H, H-6a′, H-6b and H-3′ or
H-4′ or H-5′), 3.82–3.64 (m, 6H, H-2, H-2′, H-6b′, H-6a′′,
H-6b′′ and H-3′ or H-4′ or H-5′), 3.63–3.52 (m, 3H, H-5,
H-3 and H-3′ or H-4′ or H-5′), 3.46 (m, 1H, H-4′′ or H-
5′′), 3.42–3.32 (m, 2H, H-3′′ and H-4′′ or H-5′′), 2.85 (t,
1H, J3,4 = J4,5 = 10.8 Hz, H-4), 2.66 (m, 1H, H-2′′), 2.03,
2.02 (2s, 2 × 3H, CH3CO). 13C NMR (D2O, measured from
HSQC at 400 MHz) δ: 132.0 (CH2CH=CH2), 117.0
(CH2CH=CH2), 102.0 (C-1′′), 99.0 (C-1′), 83.0 (C-1), 78.0,
77.2, 76.1, 76.0, 75.5, 72.8, 71.0, 68.9 (not assigned), 69.0
(CH2CH=CH2), 61.0 (C-6), 59.7, 59.0, 56.5 (not assigned),
56.0 (C-2′′), 54.0 (not assigned), 47.3 (C-4), 21.8 (CH3CO).
HRMS calcd.for C25H43N3O14S m/z: 642.2544 [M + H];
found: 642.2563.
NH′), 5.74–5.65 (m, 2H, H-3 and NH), 5.27 (dd, 1H, Jc,e
=
=
17.2 Hz, Jd,e = 1.6 Hz, OCH2CHCHdHe), 5.21 (dd, 1H, Jc,d
10.3 Hz, OCH2CHCHdHe), 5.12–4.98 (m, 2H, H-3′ and
H-4′), 4.91 (d, 1H, J1,2 = 8.3 Hz, H-1), 4.74 (d, 1H, J1′,2′
=
10.5 Hz, H-1′), 4.53 (m, 2H, H-6a and H-6b), 4.32 (m, 1H,
OCHaHbCHCH2), 4.23–4.12 (m, 2H, H-2′ and H-6a′), 4.10–
4.02 (m, 2H, H-6b′ and OCHaHbCHCH2), 3.90 (m, 1H,
H-5), 3.74 (m, 1H, H-5′), 3.46 (m, 1H, H-2), 2.83 (t, 1H,
J3,4 = J4,5 = 10.8 Hz, H-4), 2.11, 2.10, 2.07, 2.04, 2.02, 1.99,
1.92 (7s, 7 × 3H, CH3CO). 13C NMR (100 MHz, CDCl3) δ:
170.7, 169.8 (COCH3), 133.6 (OCH2CH=CH2), 117.8
(OCH2CH=CH2), 98.8 (C-1), 82.2 (C-1′), 75.7 (C-5′), 74.3
(C-3′), 73.4 (C-5), 70.0 (OCH2CH=CH2), 68.9 (C-3), 68.2
(C-4′), 63.6 (C-6), 62.6 (C-6′), 57.3 (C-2), 52.2 (C-2′), 46.7
(C-4), 23.5, 23.0, 21.0, 20.9, 20.6 (CH3CO). HRMS calcd.
Acknowledgements
for C27H38N4O14S m/z: 713.2204 [M
+ Na]; found:
713.2200.
L. Loureiro Morais was supported by a grant from the
French Ministry in charge of higher education. The authors
are thankful for a grant from the France–Canada Research
Foundation to support the collaborative exchange between
the French and Canadian (F-IA) groups. LLM, KB, IR, and
F-IA are thankful to Professor J. Gelas (École Nationale
Supérieure de Chimie de Clermont-Ferrand (ENSCCF)) for
his support of this work. UDP-GlcNH2 was synthesized by
HY in Professor O. Hindsgaul’s group (University of
Alberta, Edmonton, Alberta) and the isolation of the
galactosyltransferase, as well as the transfers on 7 and 8,
were carried out by F-IA in Professor M. Palcic’s group
(University of Alberta). The authors are grateful to both O.
Hindgsaul and M. Palcic (now at the Carlsberg Laboratories,
Valby, Denmark) for their support of this work.
Allyl 4-S-(2-acetamido-2-deoxy-␤-D-glucopyranosyl)-2-
acetamido-2-deoxy-4-thio-␤-D-glucopyranoside (20)
MeONa (75 mg, 1.4 mmol) was added to a solution of
allyl glycoside 19 (300 mg, 434 µmol) in anhydr. MeOH
(3 mL). The mixture was stirred for 4 h under Ar at room
temperature and then diluted with water. The pH was ad-
justed to 7 by adding Amberlite® IR120 resin, the resin was
filtered off, and the filtrate was concentrated to dryness. The
residue was precipitated from a mixture of acetone–MeOH
(2:1) to afford the pure thiodisaccharide 20 (150 mg, 72%)
1
as a white powder. H NMR (400 MHz, D2O) δ: 5.92 (m,
1H, OCH2CHcCH2), 5.40–5.22 (m, 2H, OCH2CHCHdHe and
OCH2CHCHdHe), 4.74 (d, 1H, J1′,2′ = 10.4 Hz, H-1′), 4.55
(d, 1H, J1,2 = 8.5 Hz, H-1), 4.35 (dd, 1H, Ja,b = 12.9 Hz,
Ja,c = 4.5 Hz, OCHaHbCHCH2), 4.17 (dd, 1H, Jb,c = 6.0 Hz,
OCHaHbCHCH2), 4.09 (d, 1H, J6a,6b = 11.8 Hz, H-6a), 3.98–
3.85 (m, 2H, H-6a′ and H-6b), 3.82–3.54 (m, 6H, H-2′,
H-6b′, H-2, H-5, H-3, and H-3′), 3.54–3.40 (m, 2H, H-4′
and H-5′), 2.89 (t, 1H, J3,4 = J4,5 = 10.5 Hz, H-4), 2.04 (s,
6H, 2 × CH3CO). 13C NMR (100 MHz, D2O) δ: 172.6, 172.5
(COCH3), 131.3 (OCH2CH=CH2), 116.2 (OCH2CH=CH2),
97.7 (C-1), 81.8 (C-1′), 77.8 (C-4′ or C-5′), 74.4 (C-5), 72.9
(C-3′), 69.4 (C-3), 68.4 (OCH2CH=CH2), 67.6 (C-5′ or
C-4′), 59.4 (C-6), 58.7 (C-6′), 54.8 (C-2), 52.9 (C-2′), 46.1
(C-4), 20.1 (CH3CO). HRMS calcd. for C19H32N2O10S m/z:
503.1675 [M + Na]; found: 503.1672.
References
1. P. Van Rhin and J. Venderleyden. Microbiol. Rev. 59, 124
(1995).
2. P. Lerouge, P. Roche, C. Faucher, F. Maillet, G. Truchet, J.-C.
Promé, and J. Denarié. Nature, 344, 781 (1990); G. Truchet, P.
Roche, P. Lerouge, J. Vasse, S. Camut, F. de Billy, J.-C.
Promé, and J. Dénarié. Nature, 351, 670 (1991).
3. For reviews, see: J.-C. Promé and N. Demont. Plant-microbe
interactions. Edited by G. Stacey and N.T. Keen. Chapman and
Hally, New York. 1996. p. 272; J. Denarié, F. Debelle, and J.-
C. Promé. Annu. Rev. Biochem. 65, 503 (1996); W. D’Haeze
and M. Holsters. Glycobiology, 12, 79R (2002).
4. M. Schultze, B. Quiclet-Sire, E. Kondorosi, H. Virelizier, J.N.
Glushka, G. Endre, S.D. Gero, and A. Kondorosi. Proc. Natl.
Acad. Sci. U.S.A. 89, 192 (1992); A.O. Ovtsyna, M. Schultze,
I.A. Tikhonovich, H.P. Spaink, E. Kondorosi, A. Kondorosi,
and C. Staehelin. Mol. Plant. Microbe Interact. 13, 799 (2000);
A.O. Ovtsyna, E.A. Dolgikh, A.S. Kilanova, V.E. Tsyganov,
A.Y. Borisov, I.A. Tikhonovich, and C. Staehelin. Plant
Physiol. 139, 1051 (2005).
Allyl 4-S-(2-acetamido-4-O-(2-amino-2-deoxy-␤-D-
glucopyranosyl)-2-deoxy-␤-D-glucopyranosyl)-2-
acetamido-2-deoxy-4-thio-␤-D-glucopyranoside (21)
Thiodisaccharide 20 (5.45 mg, 11.35 µmol) was incubated
for 14 days with UDP-GlcNH2 (6) (8.9 mg, 13.7 µmol,
1.2 equiv.) and β-1,4-galactosyltransferase (5.6 U) as de-
scribed in the general method for the enzymatic reactions.
The pure trisaccharide 21 (3 mg, 41%) was isolated as a
5. (a) F.-I. Auzanneau, M. Mialon, D. Promé, J.-C. Promé, and J.
1
glass after freeze-drying from water. H NMR (400 MHz,
Gelas. J. Org. Chem. 63, 6460 (1998); (b) F.-I. Auzanneau, K.
© 2006 NRC Canada

596
Can. J. Chem. Vol. 84, 2006
12. F.R. Atherton. Biochem. Prep. 5, 1 (1957).
13. R. Barker, K.W. Olsen, J.H. Shaper, and R.L. Hill. J. Biol.
Chem. 247, 7135 (1972); T.A. Beyer, J.E. Sadler, J.I. Rearick,
J.C. Paulson, and R.L. Hill. Adv. Enzymol. Relat. Areas Mol.
Biol. 52, 23 (1981).
14. (a) L.-X. Wang and Y.C. Lee. J. Chem. Soc. Perkin Tran. 1,
581 (1996); (b) J.L. Muñoz, A. Gracía-Herrero, J.L. Asensio,
F.-I. Auzanneau, F.J. Cañada, and J. Jiménez-Barbero. J.
Chem. Soc. Perkin Tran. 1, 867 (2001).
Bennis, E. Fanton, D. Promé, J. Defaye, and J. Gelas. J. Chem.
Soc. Perkin Trans. 1, 3629 (1998); (c) L. Loureiro Morais, K.
Bennis, I. Ripoche, L. Liao, F.-I. Auzanneau, and J. Gelas.
Carbohydr. Res. 338, 1369 (2003).
6. J. Defaye and J. Gelas. In Studies in natural products chemis-
try. Vol. 8. Edited by Atta-ur-Rhaman. Elsevier Science Pub-
lishers B.V., Amsterdam. 1991. p. 315.
7. X. Qian, K. Sujino, M.M. Palcic, and R.M. Ratcliffe. J.
Carbohydr. Chem. 21, 911 (2002).
8. M. Atkinson, M.M. Palcic, O. Hindsgaul, and S. Long. Proc.
Natl. Acad. Sci. U.S.A. 91, 8418 (1994).
9. (a) P.A. Ropp and P.-W. Cheng. Anal. Biochem. 187, 104
(1990); (b) J.E. Heidlas, W.J. Lees, P. Pale, and G.M.
Whitesides. J. Org. Chem. 57, 146 (1992).
15. F.-I. Auzanneau, M.K. Christensen, S.L. Harris, M. Meldal,
and B.M. Pinto. Can. J. Chem. 76, 1109 (1998).
16. J.I. Padrón and J.T. Vásquez. Tetrahedron: Asymmetry, 6, 857
(1995).
17. D. Qui and R.D. Koganty. Tetrahedron Lett. 38, 45 (1997).
10. M.L. Wolform and P.J. Conigliaro. Carbohyd. Res. 11, 63 (1969).
11. R.F. Sala, S. MacKinnon, M.M. Palcic, and M.E. Tanner.
Carbohydr. Res. 306, 127 (1998).
© 2006 NRC Canada