A new preparation of the disaccharide β-D-ManNAcp-(1 → 4)-D-Glc from lactose through a highly stereoselective β-D-Galp to β-D-ManNAcp transformation

Article abstract of DOI:10.1081/CAR-200030011

A new method for the construction of the β-D-ManNAcp-(1 → 4)-D-Glc framework from lactose avoiding the β-mannosaminylation step was developed starting from 4-O-(2-acetamido-2-deoxy-3,4-O-isopropylidene-6-O- trityl-β-D-talopyranosyl)-2,3 : 5,6-di-O-isoprop

Full text of DOI:10.1081/CAR-200030011

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A New Preparation of the Disaccharide
β‐D‐ManNAcp‐(1 → 4)‐D‐Glc from
Lactose Through a Highly Stereoselective
β‐D‐Galp to β‐D‐ManNAcp Transformation
Emanuele Attolino a , Giorgio Catelani a , Felicia D'Andrea a & Maria
Nicolardi a
a Dipartimento di Chimica Bioorganica e Biofarmacia , Università di
Pisa , Via Bonanno, 33‐I‐56126, Pisa, Italy
Published online: 17 Aug 2006.
To cite this article: Emanuele Attolino , Giorgio Catelani , Felicia D'Andrea & Maria Nicolardi (2004)
A New Preparation of the Disaccharide β‐D‐ManNAcp‐(1 → 4)‐D‐Glc from Lactose Through a Highly
Stereoselective β‐D‐Galp to β‐D‐ManNAcp Transformation, Journal of Carbohydrate Chemistry, 23:4,
179-190, DOI: 10.1081/CAR-200030011
To link to this article: http://dx.doi.org/10.1081/CAR-200030011
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JOURNAL OF CARBOHYDRATE CHEMISTRY
Vol. 23, No. 4, pp. 179–190, 2004
A New Preparation of the Disaccharide
b-D-ManNAcp-(1!4)-D-Glc from Lactose Through
a Highly Stereoselective b-D-Galp
to b-D-ManNAcp Transformation#
*
Emanuele Attolino, Giorgio Catelani, Felicia D’Andrea,
and Maria Nicolardi
`
Dipartimento di Chimica Bioorganica e Biofarmacia, Universita di Pisa, Pisa, Italy
CONTENTS
ABSTRACT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180
I. INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180
II. RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . 181
III. EXPERIMENTAL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184
A. General Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184
ACKNOWLEDGMENT . . . . . . . . . . . . . . . . . . . . . . . . . . . 188
REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188
#Part 19 of the series, “Chemical Valorisation of Milk-Derived Carbohydrates”; for part 18, see Ref. [26]
.
Correspondence: Giorgio Catelani, Dipartimento di Chimica Bioorganica e Biofarmacia,
*
`
Universita di Pisa, Via Bonanno, 33-I-56126, Pisa, Italy; Fax: þ39 050 43321; E-mail: giocate@
farm.unipi.it.
179
DOI: 10.1081/CAR-200030011
0732-8303 (Print); 1532-2327 (Online)
www.dekker.com
Copyright # 2004 by Marcel Dekker, Inc.

180
Attolino et al.
ABSTRACT
A new method for the construction of the b-D-ManNAcp-(1 ! 4)-D-Glc framework
from lactose avoiding the b-mannosaminylation step was developed starting from
4-O-(2-acetamido-2-deoxy-3,4-O-isopropylidene-6-O-trityl-b-D-talopyranosyl)-2,3 :
5,6-di-O-isopropylidene-aldehydo-D-glucose dimethyl acetal 2, obtained from 60-O-
trityl-triacetonelactose dimethyl acetal, as previously reported [Barili, P.L.; Berti, G.;
Catelani, G.; D’Andrea, F.; Puccioni, L. Stereoselective synthesis of 4-O-(2-acet-
amido-2-deoxy-b-D-talopyranosyl)-D-glucose derivatives from lactose. J. Carbohydr.
Chem. 2000, 19, 79–81.] After a preliminary modification of the protecting groups on
the b-D-talosamine unit into the 30,60-di-O-benzyl derivative 6, a new stereoselective
protocol of C-40 epimerization was applied using (a) a regioselective dehydration
providing the 40-deoxy-hex-30-eno derivative 7 through a simultaneous activation–
elimination reaction with NaH-sulfuryldiimidazole system, (b) its regio- and
stereoselective hydroboration–oxidation to give the protected b-D-mannosamine
disaccharide 8, with an overall 64% yield. The completely deprotected b-D-
ManNAcp-(1 ! 4)-D-Glc disaccharide was, finally, obtained with high yield, as an
about 45 : 55 mixture of a- and b-pyranose forms, through catalytic debenzylation
followed by acid hydrolysis of 8.
Key Words: Mannosamine; Talosamine; Epimerization; 2-Acetamido-2-deoxy-b-D-
mannopyranosides; Lactose; Hex-3-enopyranosides.
INTRODUCTION
One of the more biologically relevant members of the 2-amino-2-deoxyhexose family[1]
is 2-acetamido-2-deoxy-D-mannose (D-mannosamine, ManNAc), frequently occurring in its
pyranose form in the capsular polysaccharide repeating unit of some pathogens, including
Haemophilus influenzae and Pneumococcus strains.[2,3] The b-D-ManNAcp residue is
a component of the natural spacer unit linking teichoic acids to the peptidoglycan chain
in some Gram-positive bacteria.[4] More recently, Kren and co-workers[5] demonstrated
that ManNAc is the strongest 2-acetamido-2-deoxyhexose ligand for the natural killer cell
activating protein (NKR-P1).[5] Furthermore, disaccharides containing ManNAc unit at
the reducing end were proved to have the highest affinity for the NKR-P1 receptors.[5]
Syntheses of b-D-GlcNAcp- and b-D-GalNAcp-(1 ! 4)-D-ManNAc disaccharides were
performed[6,7] by the Lobry de Bruyn–Alberda van Ekenstein epimerization[8] of the
GlcNAc unit of pertinent disaccharide, followed by separation of the resulting disaccharide
mixtures by chromatographic[6] or enzymatic means.[7] The synthesis of disaccharides
having a b-D-ManNAcp unit at the non-reducing end, potentially active as NKR-P1
ligands,[5] has constituted until now one of the most challenging problems in
synthetic carbohydrate chemistry.[9,10] Only one[11] of the five reported syntheses[12–15] of
b-D-ManNAcp-(1 ! 4)-D-Glcp disaccharide derivatives employed a direct glycosylation
between a 2-azido-2-deoxy-D-mannopyranosyl donor and a glucopyranosyl acceptor
leading to a b-enriched mixture of diastereoisomeric disaccharides.[11] Other constructions
of the b-D-ManNAcp-(1 ! 4)-D-Glcp framework[12–15] were made indirectly by amination
with inversion at C-2 of b-D-Glcp units[12,14] or by reduction of a 20-oximino group of dis-
accharides obtained by glycosylation with 2-deoxy-2-oximino-a-D-arabino-hexopyranosyl

A New Preparation of Disaccharide
181
donors.[15,16] In one case[11] the complete deprotection leading to b-D-ManNAcp-(1 ! 4)-
D-Glcp (1), has been described.
Owing to the biological interest of 1[5] and its rather complicated reported preparation,[11]
we have envisaged a new synthetic way to 1 starting from lactose through a recently disclosed
stereoselective protocol for the transformation of b-D-Galp into b-D-ManNAcp units[17]
based (Sch. 1) on the first amination with configurational inversion at C-2 followed by an
epimerization at C-4, thus avoiding the difficult b-mannosaminylation step. Described here
is the synthesis of 1 starting from 4-O-(2-acetamido-2-deoxy-3,4-O-isopropylidene-
6-O-trityl-b-D-talopyranosyl)-2,3: 5,6-di-O-isopropylidene-aldheydo-D-glucose dimethyl
acetal (2) (Sch. 2), previously obtained by us in overall 32% yield from lactose.[18]
RESULTS AND DISCUSSION
In order to realize the TalNAcp ! ManNAcp epimerization we needed a talosamino
derivative having exclusively the 4-OH group in the free form,[17] and, consequently, a
preliminary procedure to perform a regioselective 30,40-de-O-isopropylidenation of 2.
This task, however, is not directly obtainable. We have, in fact, previously found[18]
that a partial de-O-isopropylidenation of 2 through mild acid hydrolysis with 80%
aqueous AcOH determined the contemporary removal of 60-O-trityl protecting
group.[18] Any attempt to operate a more selective deprotection by acting on the AcOH
concentration as well as other reaction parameters (temperature and time) gave only mix-
tures of partially deprotected derivatives not viable for preparative purposes. This problem
could be circumvented by a change of protection of the OH-60 group from the acid labile
trityl ether to the more acid stable benzyl ether. The de-O-tritylation step, however, turned
to be more complicated than expected; catalytic removal of trityl group, in fact, failed
under different reaction conditions with several lots of Pd/C and Pd(OH)2/C catalysts.
A satisfactory selective de-O-tritylation was made in an acceptable yield (77%) taking
.
advantage of the Kong’s methodology employing FeCl3 6H2O as acid catalyst in
CH2Cl2.[19] The known[18] intermediate alcohol 3, previously obtained as side product iso-
lated in minute amounts during the synthesis of 2,[18] was then 60-O-benzylated (BnBr,
KOH, 18-crown-6, wet THF, room temperature, 1.5 hr, 87% yield) to give derivative 4.
The following de-O-isopropylidenation step with 80% aq. AcOH gave, as
expected,[18] the tetraol 10 with good yield (79%). As previously found for the same reac-
tion of 2,[18] the 5,6- and the 30,40-O-isopropylidene groups, involving respectively a
primary hydroxyl group and an annular strain, showed a relative reactivity very similar
to each other and much higher with respect to the remaining 2,3-O-isopropylidene group.
A highly regioselective re-acetonation of the 5,6-diol function of 10 was performed by
treatment with one equivalent of 2-methoxypropene (2-MP) under mild acid catalysis
Scheme 1. Retrosynthetic approach for the b-D-Galp to b-D-ManNAcp transformation.

182
Attolino et al.
.
Scheme 2. (a) FeCl3 6H2O, CH2Cl2, 3 hr, 77%; (b) KOH, 18-crown-6, BnBr, THF, 1.5 hr, 87%;
(c) i. 80% aq. AcOH, 408C, 1.45 hr; ii. 2-MP, PyHOTs, CH2Cl2, 1.5 hr, 82% for two steps; (d)
Bu2SnO, toluene, reflux, overnight then BnBr, Bu4NBr, reflux, 5.5 hr, 95%; (e) NaH, Im2SO2,
.
DMF, 2308C ! rt, 4 hr; (f) BH3 Me2S, Et2O, 5 hr then NaOH, H2O2, 2 hr, 64% from 6;
(g) Pd(OH)2/CH2, 5 hr; 93%; (h) 80% aq. AcOH, 808C, 3.5 hr, 89%.
(PyHOTs) in dry CH2Cl2 leading in good yield (92%) to the diol 5. The overall result of
this two-steps de-acetonation/re-acetonation procedure was, thus, the selective deprotec-
tion of the 30,40diol function. A closely similar result, taking advantage of the higher reac-
tivity of the primary hydroxyl function compared to the secondary ones,[20] had been used
for the preparation of 20,60-di-O-benzyl-2,3 : 5,6-di-O-isopropylidenelactose dimethyl
acetal from its relative triacetonide.[21] The efficiency of the two step 30,40-de-O-isopro-
pylidenation of 4 was further improved, raising to an overall 82% yield, avoiding the
chromatographic purification of 10 and subjecting the crude product obtained by acid
hydrolysis of 4 to the acetonation with 2-MP.
The protection of the 30-OH group of 5 was efficiently achieved through a regioselec-
tive stannylidene acetal mediated benzylation[22] (Bu2SnO/toluene under azeotropic

A New Preparation of Disaccharide
183
anhydrification followed by BnBr and Bu4NBr) leading with excellent yield (95%) to the
derivative 6 as the sole reaction product. Although we have not found previous reports on
the opening of 3,4-O-stannylidene acetals of the talo series, the complete regioselectivity
observed is quite expected on the basis of the well known results for analogous galacto
derivatives, in which the electrophilic attack was inferred exclusively at the equatorial
O-3 of the intermediate 3,4-O-stannylidene acetal.[22]
The C-40 epimerization of the derivative 6 was based on a two step procedure,[17]
requiring first its regioselective dehydration to the hex-30-enopyranoside 7 through an
activation–elimination reaction by treatment with sulfuryldiimidazole and an excess
NaH. The complete regioselectivity in the elimination of the putative intermediate
40-O-imidazole-1-sulfonate (40-O-imidazylate) was ascribed[23] to the stereoelectronic
assistance of the C-20 axial substituent favoring the anti elimination of the axial H-30
to a greater extent than to the axial H-50. The specific role of the axial orientation of
the C-20 substituent of the talo derivatives, was demonstrated by the complete lack of
regioselectivity observed in the same reaction of galacto derivatives.[23] The second
step of the epimerization procedure was the regio- and stereoselective hydroboration–
.
oxidation (BH3 Me2S/Et2O, room temperature 5 hr, followed by aq. H2O2, room temp-
erature 2 hr) of the enol ether 7 leading to the b-D-mannosamine derivative 8, in which
the equatorial position of the 40-OH was nicely demonstrated by the NMR signal of H-40
0
0
0
0
at d 3.88 with vicinal coupling constants (J3 ,4 ¼ 9.4 Hz, J4 ,5 ¼ 9.6 Hz) compatible
only with a trans-diaxial disposition of H-30, H-40, and H-50. The exclusive formation
of the manno derivative 8 was attributed to the polarization of the enol ether double
bond ensuring the complete regioselective attack at C-40 of the electrophilic boron
atom while the complete diastereoselection of the reaction was evidently due to
the steric shielding of the b face exerted by the three substituents at C-10, C-20,
and C-50. The application of this new protocol for the C-40 epimerization of a
b-D-TalNAcp derivative to compound 6 was directly performed without purification
of the intermediate enol ether 7 leading with good overall yield (64%) to the manno-
samine derivative 8.
The complete deprotection of 8 was finally made with high yield by classical pro-
cedures providing for its catalytic debenzylation (H2, 5% Pd(OH)2/C-EtOAc, room temp-
erature, 5 hr, 93%) followed by hydrolysis of the triol 9 (80% aq. AcOH, 808C 3.5 hr, 89%
yield) involving de-O-isopropylidenation, exposition of the C-1 aldehyde group and its
spontaneous pyranylization to 1.
The structure of 1 and its anomeric composition as an about 45 : 55 a- and b-pyranose
mixture were easily established on the basis of 13C spectra. The chemical shift of the
b-mannosamine carbons were, in fact, coincident for the two anomers (see Experimental)
and strictly similar to those reported for other 2-acetamido-2-deoxy-b-D-mannopyranose
units.[24] Furthermore the D-glucosyl reducing unit of 1 showed two well separated sets of
signals very closely correspondent to those of a- and b-4-O-glycosyl-D-glucopyranose
anomers.[25]
In conclusion, we have usefully applied our recently disclosed stereoselective pro-
cedure for the b-D-Galp to b-D-ManNAcp transformation[17] to the definition of a new
simple and effective synthesis of the biologically relevant disaccharide 1 starting from
lactose, a readily available and cheap natural reducing disaccharide. Further studies
directed to the utilization of some protected derivatives of 1 for the synthesis of more
complex biologically active glycides are now planned.

184
Attolino et al.
EXPERIMENTAL
General Methods
Melting points were determined with a Kofler hot-stage apparatus and are uncor-
rected. Optical rotations were measured on a Perkin-Elmer 241 polarimeter at
20 + 28C. 1H NMR spectra were recorded with a Bruker AC 200 instrument at
200 MHz in the stated solvent (Me4Si was used as the internal standard).13C NMR
spectra were recorded with the same spectrometer at 50 MHz. Assignments were made
with the aid of DEPT and HETCOR experiments and by comparison with those of
known compounds. All reactions were followed by TLC on Kieselgel 60 F254 with detec-
tion by UV light and/or with ethanolic 10% phosphomolybdic or sulphuric acid, and
heating. Kieselgel 60 (Merck, 70–230 and 230–400 mesh, respectively) was used for
column and flash chromatography. Tetrahydrofuran (THF) and diethyl ether were distilled
from sodium using benzophenone radical as an indicator, and stored under argon before
˚
use. Other solvents were distilled and stored over 4 A molecular sieves activated at
least 24 hr at 4008C. MgSO4 was used as the drying agent for solutions.
4-O-(2-Acetamido-2-deoxy-3,4-O-isopropylidene-b-D-talopyranosyl)-2,3 : 5,6-di-
O-isopropylidene-aldehydo-D-glucose dimethyl acetal (3). To a solution of trityl
derivative 2[18] (2.00 g, 2.52 mmol) in CH2Cl2 (40 mL) was added solid FeCl3 6H2O
.
(140 mg, 0.50 mmol). The mixture was stirred at room temperature until the starting
material was completely reacted [3 hr, TLC (EtOAc)]. The reaction mixture was treated
with water (50 mL) and diluted with CH2Cl2 (70 mL). The organic phase was separated,
the aqueous layer repeatedly extracted with CH2Cl2 (4 ꢀ 50 mL), and the collected
organic phases, after drying, were concentrated at reduced pressure. The crude residue
was subjected to a flash chromatographic purification (93 : 7 CHCl3–iPrOH) to give
pure 3 (1.07 g, 77% yield) as a white solid having physico-chemical properties identical
to those of an authentic sample previously obtained by us.[18]
4-O-(2-Acetamido-6-O-benzyl-2-deoxy-3,4-O-isopropylidene-b-D-talopyranosyl)-
2,3 : 5,6-di-O-isopropylidene-aldehydo-D-glucose dimethyl acetal (4). A stirred sol-
ution of 3 (0.85 g, 1.55 mmol) in wet THF (10 mL) was treated at room temperature
with powdered KOH (0.35 g, 6.21 mmol) followed by 18-crown-6 (4 mg, 0.8 mmol).
After 0.5 hr at room temperature the mixture was treated with neat BnBr (0.37 mL,
2.15 mmol). The starting material was completely reacted (TLC, 93 : 7 CH2Cl2–iPrOH)
after 1.5 hr, the reaction mixture was treated with MeOH (3 mL) and further stirred for
10 min, concentrated at reduced pressure to give a crude residue that was partitioned
between CH2Cl2 (50 mL) and brine (30 mL). The aqueous phase was repeatedly extracted
with CH2Cl2 (3 ꢀ 50 mL). The organic phases were collected, dried and concentrated at
reduced pressure to give, after a flash chromatography (3 : 7 hexane–EtOAc) the title com-
pound 4 (0.870 g, 87% yield) as a syrup; Rf 0.20 (3 : 7 hexane–EtOAc); [a]D ¼ 26.8 (c
1
1.1, CHCl3); H NMR (CD3CN) d 1.27, 1.28, 1.30, 1.31, 1.37, 1.43 [6s, each 3H,
3 ꢀ C(CH3)2], 1.88 (s, 3H, CH3CO), 3.30, 3.31 (2s, each 3H, 2 ꢀ OCH3), 3.63 (dd, 1H,
J6 a,6 b ¼ 10.1 Hz, J5 ,6 b ¼ 6.7 Hz, H-60b), 3.70 (dd, 1H, J5 ,6 a ¼ 5.4 Hz, H-60a), 3.88–
4.00 (m, 4H, H-50, H-30, H-6a, H-6b), 3.98 (dd, 1H, J3,4 ¼ 1.4 Hz, J2,3 ¼ 7.0 Hz, H-3),
0
0
0
0
0
0
0
0
4.20 (ddd, 1H, J5,6a ¼ 6.1 Hz, J5,6b ¼ 6.6 Hz, H-5), 4.22 (dd, 1H, J3 ,4 ¼ 7.0 Hz,
J4 ,5 ¼ 3.8 Hz, H-40), 4.30 (d, 1H, J1,2 ¼ 6.1 Hz, H-1), 4.33 (dd, 1H, J4,5 ¼ 2.6 Hz,
0
0
H-4), 4.37 (m, 1H, H-20), 4.43 (dd, 1H, H-2), 4.51 and 4.58 (AB system, 2H,

A New Preparation of Disaccharide
185
JA,B ¼ 12.1 Hz, CH2Ph), 4.87 (d, 1H, J1 ,2 ¼ 2.5 Hz, H-10), 6.36 (d, 1H, J2 ,NH ¼ 9.0 Hz,
NH), 7.28–7.39 (m, 5H, aromatic H); 13C NMR (CD3CN) d 23.5 (CH3CO), 25.3, 26.4,
26.7, 26.9, 27.7, 28.1 [3 ꢀ C(CH3)2], 48.6 (C-20), 54.1, 55.9 (2 ꢀ OCH3), 65.9 (C-6),
69.8 (C-60), 72.5, 72.8, 73.7 (C-2, C-40, C-50), 73.7 (CH2Ph), 76.2, 76.4, 78.4, 78.9 (C-
30, C-3, C-4, C-5), 100.1 (C-10), 106.2 (C-1), 108.7, 110.0, 110.8 [3 ꢀ C(CH3)2],
128.5–129.6 (aromatic CH), 139.6 (aromatic C), 170.4 (C55O).
0
0
0
Anal. Calcd for C32H49NO12 (639.75): C, 60.08; H, 7.72; N, 2.19; Found: C, 60.17; H,
7.73; N, 2.10.
4-O-(2-Acetamido-6-O-benzyl-2-deoxy-b-D-talopyranosyl)-2,3-O-isopropyl-
idene-aldehydo-D-glucose dimethyl acetal (10). A solution of 4 (800 mg, 1.25 mmol) in
80% aq. AcOH (25 mL) was stirred at 408C until the starting compound was completely
reacted [1.45 hr, TLC (85 : 15 CHCl3–CH3OH)] with formation of a slower moving
product. The reaction mixture was concentrated at reduced pressure and repeatedly co-
evaporated with toluene (4 ꢀ 20 mL). The crude residue was directly applied to a flash
chromatography column eluting with 85 : 15 CHCl3–CH3OH to give 10 (550 mg, 79%
yield) as a solid foam; Rf ¼ 0.24 (85 : 15 CHCl3–CH3OH); [a]D ¼ 267.1 (c 0.9,
1
CHCl3); H NMR (CD3CN–D2O) d 1.30, 1.31 [2s, each 3H, C(CH3)2], 1.89 (s, 3H,
CH3CO), 3.28 (s, 6H, 2 ꢀ OCH3), 3.52–3.75 (m, 9H, H-30, H-40, H-50, H-60a, H-60b,
H-3, H-4, H-6a, H-6b), 4.13 (d, 1H, J1,2 ¼ 6.8 Hz, H-1), 4.35 (m, 3H, H-2, H-5, H-20),
4.52 (s, 2H, CH2Ph), 4.61 (d, 1H, J1 ,2 ¼ 1.5 Hz, H-10), 7.27–7.36 (m, 5H, aromatic
0
0
13
H); C NMR (CD3CN–D2O) d 23.9 (CH3CO), 26.9, 27.7 [C(CH3)2], 53.3 (C-20), 53.9,
55.9 (2 ꢀ OCH3), 63.4 (C-6), 68.7, 69.1 (C-40, C-5), 69.9 (C-60), 73.2, 74.8 (C-30,
C-50), 73.8 (CH2Ph), 76.2 (C-4), 78.2, 79.2 (C-2, C-3), 101.5 (C-10), 105.9 (C-1), 110.5
[C(CH3)2], 128.5–129.2 (aromatic CH), 139.2 (aromatic C), 172.7 (C55O).
Anal. Calcd for C26H41NO12 (559.62): C, 55.80; H, 7.38; N, 2.50. Found: C, 55.91; H,
7.40; N, 2.65.
4-O-(2-Acetamido-6-O-benzyl-2-deoxy-b-D-talopyranosyl)-2,3:5,6-di-O-isopropyl-
idene-aldehydo-D-glucose dimethyl acetal (5). To a solution of 10 (500 mg, 0.89 mmol)
and PyHOTs (23 mg, 0.09 mmol) in dry CH2Cl2 (10 mL) was dropwise added a 1 : 10 solu-
tion of 2-MP in dry CH2Cl2 (0.89 mL, 0.89 mmol). The reaction mixture was stirred at
room temperature until 10 was completely reacted [1.5 hr, TLC (9 : 1 CHCl3–CH3OH)],
diluted with CH2Cl2 (30 mL) and treated with saturated aq. NaHCO3. The organic
phase was separated, the aqueous layer repeatedly extracted with CH2Cl2 (3 ꢀ 20 mL)
and the collected organic phases, after drying were concentrated at reduced pressure.
The crude residue was subjected to a flash chromatographic purification (9 : 1 CHCl3–
CH3OH) to give 5 (491 mg, 92%) as a solid foam; mp ¼ 30–328C; Rf 0.24 (9 : 1
CHCl3–CH3OH); [a]D ¼ 232.9 (c 0.9, CHCl3); 1H NMR (CD3CN) d 1.29 [s, 6H,
C(CH3)2], 1.31, 1.38 [2s, each 3H, C(CH3)2], 1.84 (s, 3H, CH3CO), 3.29, 3.31 (2s, each
3H, 2 ꢀ OCH3), 3.50–3.71 (m, 6H, H-30, H-40, H-60a, H-60b, OH-30, OH-40), 3.73
(m, 1H, H-50), 3.89 (dd, 1H, J3,4 ¼ 1.5 Hz, J4,5 ¼ 4.1 Hz, H-4), 3.91 (m, 2H, H-6a,
H-6b), 3.98 (dd, 1H, J2,3 ¼ 6.6 Hz, H-3), 4.15 (ddd, 1H, J5,6a ¼ 6.8 Hz, J5,6b ¼ 7.1 Hz,
H-5), 4.30 (d, 1H, J1,2 ¼ 6.2 Hz, H-1), 4.40 (m, 2H, H-2, H-20), 4.53 (s, 2H, CH2Ph),
4.71 (d, 1H, J1 ,2 ¼ 1.7 Hz, H-10), 7.32–7.38 (m, 5H, aromatic H); 13C NMR (CD3CN)
d 23.9 (CH3CO), 25.7, 26.8, 26.9, 27.7 [2 ꢀ C(CH3)2], 52.9 (C-20), 54.2, 55.8
(2 ꢀ OCH3), 66.1 (C-6), 69.1 (C-60), 69.4 (C-40), 70.1 (CH2Ph), 73.9, 74.8 (C-30, C-50),
76.4, 76.5 (C-4, C-3), 78.4, 78.8 (C-2, C-5), 101.5 (C-10), 106.1 (C-1), 108.7, 110.9
[2 ꢀ C(CH3)2], 128.5–129.3 (aromatic CH), 139.5 (aromatic C), 171.3 (C55O).
0
0

186
Attolino et al.
Anal. Calcd for C29H45NO12 (599.68): C, 58.08; H, 7.56; N, 2.34. Found: C, 58.13; H,
7.60; N, 2.28.
4-O-(2-Acetamido-3,6-di-O-benzyl-2-deoxy-b-D-talopyranosyl)-2,3:5,6-di-O-iso-
propylidene-aldehydo-D-glucose dimethyl acetal (6). A solution of 5 (1.35 g,
2.25 mmol) and Bu2SnO (675 mg, 2.70 mmol) in toluene (135 mL) was refluxed in a
Dean–Stark apparatus overnight. The solution was cooled to room temperature, treated
with Bu4NBr (369 mg, 1.13 mmol) and neat BnBr (0.36 mL, 3.09 mmol) and newly
refluxed until the starting material was completely reacted (5.5 hr, TLC, 9 : 1 CHCl3–
CH3OH). The reaction solution was concentrated at reduced pressure, and the resulting
residue was directly subjected to flash chromatography, eluting in the order with
hexane and 2 : 8 hexane–EtOAc, to give 6 (1.47 g, 95% yield) as a solid foam; Rf 0.23
(2 : 8 hexane–EtOAc); mp ¼ 30–348C; [a]D ¼ 259.3 (c 0.9, CHCl3); 1H NMR
(CD3CN) d 1.30, 1.31, 1.33, 1.39 [4s, each 3H, 2 ꢀ C(CH3)2], 1.84(s, 3H, CH3CO),
3.29, 3.31 (2s, each 3H, 2 ꢀ OCH3), 3.47 (dd, 1H, J2 ,3 ¼ 4.4 Hz, J3 ,4 ¼ 2.4 Hz, H-30),
0
0
0
0
3.54 (m, 1H, H-50), 3.63 (dd, 1H, J6 a,6 b ¼ 9.6 Hz, J5 ,6 b ¼ 6.1 Hz, H-60b), 3.70 (dd, 1H,
0
0
0
0
J5 ,6 a ¼ 5.4 Hz, H-60a), 3.92 (m, 1H, H-4), 3.94 (m, 1H, H-40), 3.96 (m, 2H, H-6a,
H-6b), 3.99 (dd, 1H, J2,3 ¼ 6.8 Hz, J3,4 ¼ 1.5 Hz, H-3), 4.18 (ddd, 1H,
J5,6a ¼ J5,6b ¼ 6.7 Hz, J4,5 ¼ 3.8 Hz, H-5), 4.31 (d, 1H, J1,2 ¼ 6.2 Hz, H-1), 4.44 (dd,
1H, H-2), 4.50 and 4.63 (AB system, 2H, JA,B ¼ 11.6 Hz, CH2Ph), 4.53 (s, 2H,
0
0
CH2Ph), 4.72 (d, 1H, J1 ,2 ¼ 1.4 Hz, H-10), 4.76 (m, 1H, H-20), 6.52 (d, 1H,
0
0
J2 ,NH ¼ 9.5 Hz, NH), 7.31–7.38 (m, 10H, aromatic H); 13C NMR (CD3CN) d 23.9
(CH3CO), 25.8, 26.8, 27.1, 27.8 [2 ꢀ C(CH3)2], 49.8 (C-20), 54.1, 55.8 (2 ꢀ OCH3),
66.0 (C-6), 67.6 (C-40), 70.0 (C-60), 70.4, 73.9 (2 ꢀ CH2Ph), 74.9 (C-50), 75.7 (C-30);
76.4, 76.5 (C-2, C-4), 78.5 (C-5), 78.8 (C-3), 101.5 (C-10), 106.1 (C-1), 108.6, 110.9
[2 ꢀ C(CH3)2], 128.5–129.3 (aromatic CH), 139.2, 139.5 (aromatic C), 170.5 (C55O).
Anal. Calcd for C36H51NO12 (689.81): C, 62.68; H, 7.45; N, 2.03. Found: C, 62.71; H,
7.51; N, 2.10.
0
0
4-O-(2-Acetamido-3,6-di-O-benzyl-2,4-dideoxy-b-D-threo-hex-3-enopyranosyl)-
2,3:5,6-di-O-isopropylidene-aldehydo-D-glucose dimethyl acetal (7). A suspension of
60% NaH in mineral oil (304 mg, 7.66 mmol) was washed with hexane under an argon
atmosphere and slowly treated at room temperature with a solution of 6 (1.05 g,
1.52 mmol) in dry DMF (60 mL). The mixture was stirred at room temperature for
30 min, cooled at 2308C, treated with Im2SO2 (442 mg, 2.22 mmol) and was further
stirred at 2308C for 1 hr. The mixture was allowed to reach room temperature and was
further stirred for 3 hr when TLC analysis (2 : 8 hexane–EtOAc) revealed the complete
formation of a faster moving product. The reaction mixture was cooled to 08C, excess
of NaH was destroyed by addition of MeOH (1 mL) followed by 10 min stirring, and parti-
tioned between ethyl ether (120 mL) and crushed iced-water. The organic phase was sep-
arated, and the aqueous layer extracted with Et2O (100 mL). The organic phases were
collected, dried and concentrated at reduced pressure to give a syrup (900 mg) constituted
mainly by 7, sufficiently pure for further transformations. The flash chromatographic puri-
fication of a portion of the above crude reaction product (300 mg) eluting with 2 : 3
hexane–EtOAc þ 0.1% Et3N gave an analytically pure sample of 7 (269 mg, 79%
1
yield) as a syrup; Rf 0.44 (1 : 4 hexane–EtOAc); [a]D ¼ þ18.6 (c 1.1, CHCl3); H
NMR (CD3CN) d 1.30, 1.31, 1.32, 1.39 [4s, each 3H, 2 ꢀ C(CH3)2], 1.77 (s, 3H,
0
0
CH3CO), 3.32, 3.33 (2s, each 3H, 2 ꢀ OCH3), 3.47 (dd, 1H, J6 a,6 b ¼ 9.9 Hz,
J5 ,6 a ¼ 5.2 Hz, H-60a), 3.58 (dd, 1H, J5 ,6 b ¼ 5.4 Hz, H-60b), 3.93 (dd, 1H,
0
0
0
0

A New Preparation of Disaccharide
187
J3,4 ¼ 1.4 Hz, J4,5 ¼ 4.3 Hz, H-4), 4.01 (dd, 1H, J2,3 ¼ 6.9 Hz, H-3), 4.05 (m, 2H, H-6a,
H-6b), 4.16 (ddd, 1H, J5,6a ¼ J5,6b ¼ 7.5 Hz, H-5), 4.32 (d, 1H, J1,2 ¼ 6.2 Hz, H-1),
4.37 (m, 1H, H-50), 4.48 (dd, 1H, H-2), 4.55 (s, 2H, CH2Ph); 4.72 and 4.80 (AB
0
0
0
0
system, 2H, JA,B ¼ 12.0 Hz, CH2Ph), 4.67 (ddd, 1H, J2 ,NH ¼ 9.8 Hz, J2 ,5 ¼ 1.5 Hz,
H-20), 4.89 (d, 1H, J4 ,5 ¼ 1.7 Hz, H-40), 4.91 (d, 1H, J1 ,2 ¼ 2.3 Hz, H-10), 6.45 (d, 1H,
NH), 7.30–7.39 (m, 10H, aromatic H); 13C NMR (CD3CN)d 23.2 (CH3CO), 25.9, 26.9,
27.0, 27.7 [2 ꢀ C(CH3)2], 49.3 (C-20), 54.4, 55.7 (2 ꢀ OCH3), 66.0 (C-6), 70.1 (C-60),
72.9 (C-50), 73.7, 73.9 (2 ꢀ CH2Ph), 76.3, 76.4 (C-2, C-4); 78.5, 78.6 (C-3, C-5), 98.8
(C-40), 100.3 (C-10), 106.2 (C-1), 108.7, 110.9 [2 ꢀ C(CH3)2], 128.4–129.4 (aromatic
CH), 137.8, 139.6 (aromatic C), 153.5 (C-30), 170.4 (C55O).
0
0
0
0
Anal. Calcd for C36H49NO11 (671.79): C, 64.37; H, 7.35; N, 2.08. Found: C, 64.41; H,
7.31; N, 2.12.
4-O-(2-Acetamido-3,6-di-O-benzyl-2-deoxy-b-D-mannopyranosyl)-2,3:5,6-di-O-
isopropylidene-aldehydo-D-glucose dimethyl acetal (8). A solution of crude 7 (600 mg)
in dry Et2O (19 mL) was treated at 08C with a 96% solution of borane dimethylsulfide
complex (0.14 mL, 1.33 mmol), warmed to room temperature and stirred until the starting
material was completely reacted [TLC (1 : 4 hexane–EtOAc), 5 hr]. The mixture was
cooled to 08C and treated in the order with H2O (2.5 mL), 10% aq. NaOH (5 mL) and,
finally, 35% aq. H2O2 (10 mL). The biphasic mixture was stirred for 2 hr, diluted with
Et2O (10 mL), and the organic phase was separated. The aqueous layer was repeatedly
extracted with Et2O (4 ꢀ 40 mL), and the collected organic phases, after drying, were
concentrated at reduced pressure. The crude residue was subjected to a flash chromato-
graphic purification (1 : 4 hexane–EtOAc) to give pure 8 (447 mg, 64% yield over two
steps from 6) as a white solid; Rf 0.18 (1 : 4 hexane–EtOAc); mp ¼ 187–1908C
1
(dec.); [a]D ¼ 250.1 (c 1.5, CHCl3); H NMR (C6D6) d 1.33, 1.38, 1.40, 1.51 [4s,
each 3H, 2 ꢀ C(CH3)2], 1.74 (s, 3H, CH3CO), 3.20, 3.28 (2s, each 3H, 2 ꢀ OCH3),
3.14 (m, 1H, H-50), 3.38 (dd, 1H, J3 ,4 ¼ 9.4 Hz, J2 ,3 ¼ 4.0 Hz, H-30), 3.70 (dd, 1H,
0
0
0
0
J6 a,6 b ¼ 10.6 Hz, J5 ,6 a ¼ 2.3 Hz, H-60a), 3.80 (dd, 1H, J5 ,6 b ¼ 3.9 Hz, H-60b), 3.88
0
0
0
0
0
0
(dd, 1H, J4 ,5 ¼ 9.6 Hz, J3 ,4 ¼ 9.4 Hz, H-40), 4.07–4.27 (m, 5H, H-3, H-4, H-5, H-6a,
H-6b), 4.31 (d, 1H, J1,2 ¼ 6.1 Hz, H-1), 4.45 (s, 2H, CH2Ph), 4.47, and 5.07 (AB
system, 2H, JA,B ¼ 11.1 Hz, CH2Ph), 4.73 (t, 1H, J2,3 ¼ 6.1 Hz, H-2), 4.96 (d, 1H,
0
0
0
0
J1 ,2 ¼ 1.0 Hz, H-10), 5.18 (m, 1H, H-20), 6.32 (d, 1H, J2 ,NH ¼ 9.8 Hz, NH), 7.08–7.46
(m, 10H, aromatic H); 13C NMR (C6D6) d 22.9 (CH3CO), 25.7, 26.6, 26.9, 27.9
[2 ꢀ C(CH3)2], 49.3 (C-20), 54.2, 55.3 (2 ꢀ OCH3), 65.5 (C-6), 67.0 (C-40), 69.7 (C-60),
70.9, 73.8 (2 ꢀ CH2Ph), 75.9, 76.1, 76.4 (C-4, C-5, C-50), 78.4, 78.6 (C-2, C-3), 80.6
(C-30), 100.7 (C-10), 106.0 (C-1), 108.0, 110.8 [2 ꢀ C(CH3)2], 128.0–128.8 (aromatic
CH), 138.7, 138.9 (aromatic C); 170.0 (C55O).
0
0
0
0
Anal. Calcd for C36H51NO12 (689.81): C, 62.68; H, 7.45; N, 2.03. Found: C, 62.71; H,
7.42; N, 2.09.
4-O-(2-Acetamido-2-deoxy-b-D-mannopyranosyl)-2,3 : 5,6-di-O-isopropylidene-
aldehydo-D-glucose dimethyl acetal (9). A solution of 8 (396 mg, 0.57 mmol) in EtOAc
(40 mL) containing 5% Pd(OH)2 on charcoal (260 mg) was stirred at room temperature
under an H2 atmosphere for 5 hr. The solution was diluted with MeOH (30 mL), filtered
over celite, washed with MeOH, and the combined organic phases were concentrated at
reduced pressure. The crude residue was subjected to a chromatographic purification
(85 : 15 CHCl3–MeOH) to give pure 9 (272 mg, 93% yield) as a white solid; Rf 0.31
1
(85 : 15 CHCl3–MeOH); mp ¼ 222–2258C (dec.); [a]D ¼ 0 (c 1.0, CHCl3); H NMR

188
Attolino et al.
(CD3CN–D2O) d 1.28, 1.29, 1.30, 1.37 [4s, each 3H, 2 ꢀ C(CH3)2], 1.89 (s, 3H, CH3CO),
3.22 (m, 1H, H-50), 3.33 (dd, 1H, J4 ,5 ¼ 9.5 Hz, J3 ,4 ¼ 9.3 Hz, H-40), 3.40, 3.41 (2s, each
0
0
0
0
3H, 2 ꢀ OCH3), 3.59 (dd, 1H, J2 ,3 ¼ 4.2 Hz, H-30), 3.67–4.04 (m, 6H, H-3, H-4, H6a,
0
0
H-6b, H-60a, H-60b), 4.20 (m, 1H, H-5), 4.35 (d, 1H, J1,2 ¼ 6.8 Hz, H-1), 4.39 (m, 1H,
H-20), 4.43 (dd, 1H, J2,3 ¼ 7.0 Hz, H-2), 4.76 (d, 1H, J1 ,2 ¼ 1.1 Hz, H-10); 13C NMR
0
(CD3CN-D2O) d 23.1 (CH3CO), 25.4, 26.5, 26.7, 27.4 [2 ꢀ0 C(CH3)2], 53.8 (C-20), 55.1,
57.3 (2 ꢀ OCH3), 62.1 (C-60), 65.7 (C-6), 68.0 (C-40), 73.5 (C-30), 75.9, 76.3 (C-4,
C-5), 77.5 (C-50), 78.1, 78.7 (C-2, C-3), 100.5 (C-10), 107.2 (C-1), 109.2, 111.1
[2 ꢀ C(CH3)2], 174.1 (C55O).
Anal. Calcd for C22H39NO12 (509.56): C, 51.86; H, 7.71; N, 2.75. Found: C, 51.91; H,
7.74; N, 2.80.
4-O-(2-Acetamido-2-deoxy-b-D-mannopyranosyl)-a,b-D-glucopyranose (1). A
solution of 9 (211 mg, 0.41 mmol) in 80% aq. AcOH (7 mL) was stirred at 808C for
3.5 hr, then concentrated at reduced pressure and coevaporated with toluene
(3 ꢀ 10 mL). The residue was tritured with EtOAc to give an amorphous white solid
(140 mg, 89% yield) constituted (13C NMR, D2O) exclusively by a 45 : 55 a/b anomeric
mixture of 1, as established on the basis of the integration of the anomeric carbon signals;
Rf 0.09 (7 : 3 EtOAc–MeOH); [a]D ¼ 24.8 (c 1.0, H2O); lit.[11] [a]D 2 7.3 (c 1.0, H2O);
NMR data for a 2 1: 1H NMR (CD3OD) d 2.02 (s, 3H, CH3CO), 4.52 (m, 1H, H-20), 4.78
(m, 1H, H-10), 5.08 (d, 1H, J1,2 ¼ 4.0 Hz, H-1); 13C NMR (D2O) d 23.2 (CH3CO), 54.4 (C-
20), 61.3 (C-6), 61.5 (C-60), 67.8 (C-40), 71.0 (C-5), 72.5 (C-30), 73.2 (C-2, C-3), 77.7 (C-
50), 80.1 (C-4), 93.0 (C-1), 100.5 (C-10), 176.6 (C55O). NMR data for b-1:1H NMR
(CD3OD) d 2.02 (s, 3H, CH3CO), 3.18 (dd, 1H, J1,2 ¼ 7.9 Hz, J2,3 ¼ 8.7 Hz, H-2), 4.48
(d, 1H, H-1), 4.52 (m, 1H, H-20), 4.78 (m, 1H, H-10); 13C NMR (D2O) d 23.2 (CH3CO),
54.4 (C-20), 61.3 (C-6), 61.5 (C-60), 67.8 (C-40), 72.5 (C-30), 75.1, 75.4, 75.7 (C-2, C-3,
C-5), 77.7 (C-50), 79.9 (C-4), 97.0 (C-1), 100.5 (C-10), 176.6 (C55O).
ACKNOWLEDGMENT
This work was supported by a grant from MIUR (Ministero dell’Istruzione, dell’-
`
Universita e della Ricerca, Roma) in the frame of the COFIN 2002 program.
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Received October 3, 2003
Accepted January 12, 2004