Capsular polysaccharide of Streptococcus pneumoniae type 19F: Synthesis of the repeating unit

Article abstract of DOI:10.1016/S0008-6215(98)00218-3

A new and more versatile synthesis of β-d-ManpNAc-(1→4)-α-d-Glcp-(1→2)-α-l-Rhap, the trisaccharide repeating unit of the Streptococcus pneumoniae type 19F capsular polysaccharide, is described. The present approach allows a simple access to different fragments containing the trisaccharide and the conjugation of the product(s) to a protein through the selective manipulation of the anomeric position at the reducing end and of the HO-4 function at the nonreducing end. The synthetic scheme shows an efficient application of the sulfoxide method for the stereoselective and high yielding formation of the glycosidic linkages. Copyright (C) 1998 Elsevier Science Ltd. A new and more versatile synthesis of β-D-ManpNAc-(1→4)-α-D-Glcp-(1→2)-α-L-Rhap, the trisaccharide repeating unit of the Streptococcus pneumoniae type 19F capsular polysaccharide, is described. The present approach allows a simple access to different fragments containing the trisaccharide and the conjugation of the product(s) to a protein through the selective manipulation of the anomeric position at the reducing end and of the HO-4 function at the nonreducing end. The synthetic scheme shows an efficient application of the sulfoxide method for the stereoselective and high yielding formation of the glycosidic linkages.

Full text of DOI:10.1016/S0008-6215(98)00218-3

Carbohydrate Research 311 (1998) 171±181
Capsular polysaccharide of Streptococcus
pneumoniae type 19F: synthesis of the
repeating unit
Ennio Bousqueta, Malika Khitrib, Luigi Layb, Francesco Nicotrab,
Luigi Panzab, Giovanni Russob,*
a Istituto di Chimica Farmaceutica e Toss., v. le A. Doria 6, I-95125 Catania, Italy
b Dipartimento di Chimica Organica e Industriale, via Venezian 21, I-20133 Milano, Italy
Received 27 April 1998; accepted 27 July 1998
Abstract
A new and more versatile synthesis of ꢀ-d-ManpNAc-(1!4)-ꢁ-d-Glcp-(1!2)-ꢁ-l-Rhap, the
trisaccharide repeating unit of the Streptococcus pneumoniae type 19F capsular polysaccharide, is
described. The present approach allows a simple access to di€erent fragments containing the tri-
saccharide and the conjugation of the product(s) to a protein through the selective manipulation
of the anomeric position at the reducing end and of the HO-4 function at the nonreducing end.
The synthetic scheme shows an ecient application of the sulfoxide method for the stereoselective
and high yielding formation of the glycosidic linkages. # 1998 Elsevier Science Ltd. All rights
reserved
Keywords: Glycoconjugate vaccines; Streptococcus pneumoniae; Oligosaccharides, synthesis; Anomeric sulf-
oxides, glycosylation
1. Introduction
where carbohydrate antigens and haptens are
covalently linked to protein carriers working as T-
helper dependent antigens [1].
Recent investigations [2] have been devoted to
the assessment of the structure-activity relationship
of capsular polysaccharides for di€erent serotypes
of Streptococcus pneumoniae.
Pneumococcal capsular polysaccharides have
recently attracted considerable attention because of
their use in a multivalent vaccine against pneumo-
coccal infections. The highly puri®ed poly-
saccharide vaccines introduced in the seventies
have been demonstrated to be ecacious in adults
and older children but not signi®cantly immuno-
genic in young infants. A new strategy has been
then devised involving the use of glycoconjugates,
In a project aimed at designing new semi-syn-
thetic oligoglycoconjugate vaccines, we are inter-
ested in de®ning the minimal oligosaccharide
sequence required for the production of antibodies
against serotype 19F of S. pneumoniae. The syn-
thesis of a repeating unit (!4)-ꢀ-d-ManpNAc-
-
* Corresponding author.
(1!4)-ꢁ-d-Glcp-(1!2)-ꢁ-l-Rhap-(1-PO4 !) of the
0008-6215/98/$Ðsee front matter # 1998 Elsevier Science Ltd. All rights reserved
PII S0008-6215(98)00218-3

172
E. Bousquet et al./Carbohydrate Research 311 (1998) 171±181
capsular polysaccharide of this serotype has
already been described by us [3] and others [4].
In this paper we present a new and more versa-
tile synthetic approach to the fully protected tri-
saccharide 1, where the protecting groups strategy
allows the formation of dimers or trimers through
phosphate bridges and the introduction of a spacer
arm for the conjugation to protein carriers. Di€er-
ent strategies were adopted to assemble the tri-
saccharide 1 (Scheme 1).
phenyl thioglycoside, phenyl sulfoxide) were used.
These preliminary results suggested to change the
synthetic strategy by inverting the joining order
of the monosaccharide units. Thus, the formation
of disaccharide B-C ꢁ-d-Glcp-(1!2)-ꢁ-l-Rhap
followed by the glycosylation with a suitably
protected donor and inversion at C-200 using N3
-
as a nucleophile were planned to a€ord the tri-
saccharide 1 (Scheme 1).
The strategy reported in Scheme 1 gave excellent
results in the ®rst coupling (A+B!AB), but low
yields and stereoselectivity were obtained in the
second glycosylation (AB+C!ABC), although
di€erent glycosyl donors (trichloroacetimidate,
2. Results and discussion
Phenyl 4,6-O-benzylidene-1-thio-ꢀ-d-glucopyr-
anoside 2 [5] was used as a common precursor in
Scheme 1.

E. Bousquet et al./Carbohydrate Research 311 (1998) 171±181
173
the synthesis of the gluco and manno moieties.
Compound 2 was fully benzylated (!3), then
careful oxidation with m-chloroperbenzoic acid
gave sulfoxide donor 4. Condensation of 4 with
Zemplen deacetylation of 11 (Scheme 4) gave
trisaccharide 12 having the HO-200 function avail-
able for the introduction of the azido group.
Although the gluco±manno conversion through a
SN2 inversion was easily performed in the pre-
paration of the disaccharide AB (Scheme 1), the
same transformation on the trisaccharide 12 was
dicult. Usual procedures (Mitsunobu reaction,
tri¯ate or mesylate formation followed by treat-
allyl 3,4-di-O-benzyl-ꢁ-l-rhamnopyranoside
using tri¯uoromethanesulfonic anhydride as a pro-
5
moter and 2,6-di-tert-butyl-4-methyl pyridine as an
ꢀ
acid scavenger at 60 C [6] a€orded disaccharide
6 in a good yield (80%). Only the ꢁ product was
isolated, although small traces of by-products were
detected on TLC. The ꢁ con®guration of the newly
formed glycosidic linkage was ascertained through
13C NMR spectroscopy (C-1 and C-10 exhibited
signals at ꢂ 97.31 and 98.56).
The benzylidene ring of disaccharide 6 was
hydrolysed with aqueous 90% tri¯uoroacetic acid
and the resulting diol was regioselectively 60-O-
benzylated via a stannylene acetal to give com-
pound 7 in 80% yield (Scheme 2).
-
ment with N3 , oxidation with pyridinium chlor-
with
ochromate
followed
by
treatment
hydroxylamine hydrochloride and subsequent
reduction of the resulting oxime) were unsuccess-
ful. Treating 12 with NaH and sulfonyl diimidazole
ꢀ
in N,N-dimethylformamide at 40 C [8], imida-
zoyl sulfate 13 was obtained in quantitative yield;
then, reaction with tetrabutylammonium azide in
toluene a€orded trisaccharide 14 in almost quanti-
tative yield.
The second glucosyl donor was synthesized
again from 2 through regioselective benzylation of
HO-3 (!8) [7] followed by acetylation of HO-2
(!9) and oxidation of the thiophenyl glycoside to
sulfoxide 10. Condensation of 10 with acceptor 7,
performed as previously described [6], gave tri-
saccharide 11 in 77% yield (Scheme 3). Again, the
stereochemistry of the newly synthesized glycosidic
linkage was deduced by NMR analysis [13C NMR,
signals at ꢂ 101.69 and 101.35 attributable to C-100
The reduction of the azido group of 14 was also
attempted in many di€erent conditions (hydrogen
sul®de±pyridine, dithiols, Staudinger reaction); the
best results were obtained using a slightly modi®ed
recent literature procedure [9] which allows to
obtain directly the acetamido group. Thus, com-
pound 14 was treated with a Zn±Cu couple in tetra-
hydrofuranÐacetic anhydrideÐacetic acid to a€ord
compound 1 (84%).
The trisaccharide repeating unit 1 was then
alternatively deprotected at positions O-1 or O-
400. Reductive opening of the benzylidene ring of 1
and to CHPh; 1H NMR, H-100ꢀ ꢂ 4.34 (J1 ,2
00 00
7.9 Hz)].
Scheme 2.

174
E. Bousquet et al./Carbohydrate Research 311 (1998) 171±181
Scheme 3.
Scheme 4.
[10] (borane trimethylamine complex, boron tri-
¯uoride ethyl etherate) gave compound 15 (82%
yield) with a HO-400 group. On the other hand,
the allyl group of 1 was isomerized to the corre-
sponding 1-propenyl glycoside [11] which was
®nally hydrolyzed with iodine in tetrahydrofuran±
water [12] to give 16 (96%) as an anomeric mixture
(Scheme 5).

E. Bousquet et al./Carbohydrate Research 311 (1998) 171±181
175
Scheme 5.
3. Experimental
(200 mL) under N2; the mixture was stirred for
10 min and after the dropwise addition of benzyl
bromide (16 mL, 134.6 mmol) boiled under re¯ux
overnight. The reaction was quenched with MeOH
and the solution was partitioned between CH2Cl2
and aq NH4Cl. The organic layer was washed with
water, dried (Na2SO4), and concentrated. The resi-
due was puri®ed by crystallization from 1:1 hex-
ane±EtOAc to give 3 (15.2 g, 96%); mp 155±156 ꢀC;
General methods.Ð1H NMR and 13C NMR
spectra were recorded on Bruker AC 300 and Bru-
ker AC 200 spectrometers for solutions in CDCl3.
Melting points were determined with a Buchi
apparatus and are not corrected. Optical rotations
were measured at room temperature with a Perkin±
Elmer 241 polarimeter. TLC was carried out on
Merck Silica-gel 60 F254 plates (0.25 mm thick-
ness), and spots were visualized by spraying with a
solution containing H2SO4 (31 mL), ammonium
molybdate (21 g) and Ce(SO4)2 (1 g) in 500 mL
[ꢁ]d 21.6ꢀ (c 1, CHCl3); H NMR (300 MHz,
1
CDCl3): ꢂ 7.70±7.20 (m, 20 H, ArH), 5.60 (s, 1 H,
CHPh), 4.98 (d, 1 H, Jgem 11.1 Hz, CHHPh), 4.92±
4.77 (m, 4 H, H-1, CH2Ph and CHHPh), 4.41 (dd,
1 H, J6a,6b 12.9, J6a,5 5.0 Hz, H-6a), 3.90±3.80 (m, 2
H, H-2,6b), 3.73 (t, 1 H, J4,3=J4,5=9.2 Hz, H-4),
3.58±3.46 (m, 2 H, H-3,5); 13C NMR (75.46 MHz,
CDCl3): ꢂ 101.79 (d, CHPh), 88.93 (d, C-1), 83.67,
82.11, 81.12, and 70.90 (4 d, C-2,3,4,5), 76.56,
75.98, and 69.35 (3 t, C-6 and 2 CH2Ph). Anal.
Calcd for C33H32O5S: C, 73.31; H, 5.97. Found : C,
73.54; H, 6.12.
ꢀ
water, followed by heating at 110 C for 5 min.
Column chromatography was performed by the
¯ash procedure using Merck Silica-gel 60 (230±400
mesh). IR spectra were recorded on a Perkin±
Elmer 681 spectrometer. Elemental analyses were
performed using the Carlo Erba elemental analyser
1108.
Phenyl 2,3-di-O-benzyl-4,6-O-benzylidene-1-thio-
ꢀ-d-glucopyranoside (3).ÐSodium hydride (10 g,
416.6 mmol) was added with stirring to a solution
of phenyl 4,6-O-benzylidene-1-thio-ꢀ-d-glucopyr-
anoside 2 (12.01 g, 33.32 mmol) in dry THF
Phenyl 2,3-di-O-benzyl-4,6-O-benzylidene-1-thio-
ꢀ-d-glucopyranoside sulfoxide (4).ÐA solution of 3
(7.0 g, 9.2 mmoꢀl) in dry CH2Cl2 (230 mL) was
cooled at 60 C, then 85% m-chloroperbenzoic

176
E. Bousquet et al./Carbohydrate Research 311 (1998) 171±181
acid (2.88 g, 10.88 mmol) was addedꢀ, and the mix-
ture was allowed to warm up to 0 C and stirred
for 5 h. The reaction was quenched with aq 5%
FeSO4 and after extraction with CH2Cl2, the
organic layer was washed with satd NaHCO3 and
water, dried (Na2SO4), and concentrated. The resi-
due was crystallized from hexane±EtOAc to give 4
(5.1 g, 98%) as a mixture of diastereoisomers. A
small amount (200 mg) was chromatographed (3:1
petroleum ether±EtOAc) to give pure analytical
samples; the diastereoisomer with the higher Rf
value was obtained as white crystals; mp 161±
162 ꢀC; [ꢁ]d 19.8ꢀ (c 1, CHCl3); 1H NMR
(300 MHz, CDCl3): ꢂ 7.58±7.08 (m, 20 H, ArH),
5.51 (s, 1 H, CHPh), 4.90 (d, 1 H, Jgem 11.0 Hz,
CHHPh), 4.88 (d, 1 H, Jgem 10.9 Hz, CHHPh), 4.75
(d, 1 H, CHHPh), 4.69 (d, 1 H, CHHPh), 4.57 (d, 1
H, J1,2 9.3 Hz, H-1), 4.40 (dd, 1 H, J6a,6b 9.9, J6a,5
3.7 Hz, H-6a), 3.92 (t, 1 H, J4,3=J4,5=8.1 Hz, H-
4), 3.81 (t, 1 H, J3,2 8.1 Hz, H-3), 3.68 (m, 1 H, H-
5), 3.62±3.55 (m, 2 H, H-2,6b); 13C NMR
(75.46 MHz, CDCl3): ꢂ 101.95 (d, CHPh), 96.48 (d,
C-1), 83.61, 81.77, 75.94, and 70.71 (4 d, C-2,3,4,5),
75.46 and 74.65 (2 t, 2 CH2Ph), 69.05 (t, C-6); fur-
ther elution gave the second diastereoisomer as
isolated as a syrup (2.44 g, 97%); [ꢁ]d 21.3ꢀ (c 1,
CHCl3); H NMR (300 MHz, CDCl3): ꢂ 7.40±7.20
1
(m, 10 H, ArH), 5.81 (m, 1 H, OCH2CH=CH2),
5.34 (t, 1 H, J2,3 1.6 Hz, H-2), 5.12±5.24 (m, 2 H,
OCH2CH=CH2), 4.86 (d, 1 H, Jgem 11.0 Hz,
CHHPh), 4.72 (d, 1 H, J1,2 1.6 Hz, H-1), 4.65 (d, 1
H, Jgem 11.0, CHHPh), 4.56 (d, 1 H, CHHPh), 4.48
(d, 1 H, CHHPh), 4.09 (m, 1 H, OCHHCH
=CH2), 3.94±3.87 (m, 2 H, OCHHCH=CH2 and
H-3), 3.72 (m, 1 H, H-5), 3.38 (t, 1 H, J4,3
=J4,5=9.8 Hz, H-4), 2.10 (s, 3 H, CH3CO), 1.28
(d, 3 H, J6,5 5.9 Hz, H-6). Anal. Calcd for C25H30O6:
C, 70.40; H, 7.09. Found: C, 70.35; H, 6.98.
Allyl glycoside (2.34 g, 5.48 mmol) was dissolved
in a solution of NaOH (440 mg, 11.0 mmol) in
EtOH (73 mL) and kept for 2 h under stirring at
room temperature. The mixture was concentrated
to a small volume, then diluted with CH2Cl2
(50 mL), washed with aq 5% HCl, satd NaHCO3,
and water, dried (Na2SO4), and concentrated to
a€ord 5, isolated as a syrup (1.93 g, 92%); [ꢁ]d
43.6ꢀ (c 1, CHCl3); 1H NMR (300 MHz, CDCl3):
ꢂ 7.42±7.28 (m, 10 H, ArH), 5.87 (m, 1 H,
OCH2CH=CH2), 5.29±5.15 (m, 2 H, OCH2CH
=CH2), 4.88 (d, 1 H, Jgem 10.9 Hz, CHHPh), 4.84
(brd, 1 H, H-1), 4.68 (brs, 2 H, CH2Ph), 4.63 (d, 1
H, CHHPh), 4.16 (m, 1 H, OCHHCH=CH2), 4.05
(brt, 1 H, H-2), 3.96 (m, 1 H, OCHHCH=CH2),
3.86 (dd, 1 H, J3,2 3.7 Hz, H-3), 3.74 (m, 1 H, H-5),
3.45 (t, 1 H, J4,3=J4,5=9.2 Hz, H-4), 2.48 (d, 1 H,
OH), 1.35 (d, 3 H, J6,5 6.0 Hz, H-6); 13C NMR
(75.46 MHz, CDCl3): ꢂ 134.46 (d, OCH2CH
=CH2), 117.97 (t, OCH2CH=CH2), 98.85 (d, C-1),
80.71, 80.62, 69.23, and 68.03 (4 d, C-2,3,4,5), 76.03,
72.67, and 68.48 (3 t, 2 CH2Ph and CH2CH=CH2),
18.52 (q, C-6); Anal. Calcd for C23H28O5: C, 71.85;
H, 7.34. Found: C, 72.00; H, 7.25.
Allyl (2,3-di-O-benzyl-4,6-O-benzylidene-ꢁ-d-
glucopyranosyl)-(1!2)-3,4-di-O-benzyl-ꢁ-l-rhamno-
pyranoside (6).ÐCompound 4 (1.96 g, 3.53 mmol)
was dissolved in 1:1 dry CH2Cl2±dry Et2O con-
taining freshly activated powdered 4 A molecular
sieves. After being cooled at 78 ꢀC, Tf2O (580 ꢃL,
3.53 mmol) was added with stirring for 30 min,
then a solution of 5 (905 mg, 2.353 mmol) and
2,6-di-tbutyl-4-methylpyridine (725 mg, 3.53 mmol)
in 1:1 CH2Cl2±Et2O (15 mL) was added unꢀder
N2 and the mixture was stirred for 5 h at 60 C.
The mixture was ®ltered through Celite, and the
®ltrate was washed with satd NaHCO3 and
water, dried (Na2SO4), and concentrated. The resi-
due was puri®ed by ¯ash chromatography with 8:2
ꢀ
white crystals; mp 148±149 C; [ꢁ]d 116.7ꢀ (c 1,
1
CHCl3); H NMR (300 MHz, CDCl3): ꢂ 7.65±7.20
(m, 20 H, ArH), 5.52 (s, 1 H, CHPh), 5.04 and 4.95
(2 d, 2 H, Jgem 10.7 Hz, CH2Ph), 4.98 and 4.82 (2 d,
2 H, Jgem 11.5 Hz, CH2Ph), 4.15 (t, 1 H, J2,3 9.2 Hz,
H-2), 4.02 (dd, 1 H, J6a,6b 11.6, J6a,5 4.8 Hz, H-6a),
4.01 (d, 1 H, J1,2 9.8 Hz, H-1), 3.91 (t, 1 H, J3,4
9.2 Hz, H-3), 3.79 (d, 1 H, H-6b), 3.74 (t, 1 H, H-
4), 3.28 (m, 1 H, H-5); 13C NMR (75.46 MHz,
CDCl3): ꢂ 101.92 (d, CHPh), 94.38 (d, C-1), 83.32,
81.83, 76.88, and 71.59 (4 d, C-2,3,4,5), 76.72 and
75.54 (2 t, 2 CH2Ph), 68.70 (t, C-6). Anal. Calcd
for C33H32O6S: C, 71.20; H, 5.79. Found: C, 70.85;
H, 5.98.
Allyl 3,4-di-O-benzyl-ꢁ-l-rhamnopyranoside (5).Ð
3,4-Di-O-benzyl-1,2-O-(1-methoxyethylidene)-ꢀ-l-
rhamnopyranose [13] (2.37 g, 5.91 mmol) was dis-
solved in dry CH2Cl2 (45 mL) and powdered 4 A
molecular sieves, allyl alcohol (8.9 mL, 130 mmol)
and TMSOTf (244 ꢃL, 1.353 mmol) were added at
room temperature. After stirring for 15 min, the
mixture was diluted with CH2Cl2 and ®ltered over
a Celite pad. The ®ltrate was successively washed
with satd NaHCO3 and water, dried (Na2SO4), and
concentrated. The residue was puri®ed by ¯ash
chromatography (9:1 hexane±EtOAc) to give allyl
2-O-acetyl-3,4-di-O-benzyl-ꢁ-l-rhamnopyranoside,

E. Bousquet et al./Carbohydrate Research 311 (1998) 171±181
177
0
0
0
hexane±EtOAc to give 6, isolated as a syrup
(1.61 g, 84%); [ꢁ]d +34.0ꢀ (c 1, CHCl3); H NMR
J2 ,3 9.8 Hz, H-20), 3.44 (dd, 1 H, J5,6 a 4.1 Hz, H-
60a), 3.36 (dd, 1 H, J6 a,6 b 10.0, J5,6 b 3.1 Hz, H-
60b), 2.30 (brs, 1 H, OH), 1.35 (d, 3 H, J6,5 6.0 Hz,
H-6); 13C NMR (75.46 MHz, CDCl3): ꢂ 134.57 (d,
OCH2CH=CH2), 117.74 (t, OCH2CH=CH2),
96.94 (d, C-1,10), 81.47, 80.90, 80.25, 79.56, 74.66,
71.43, 70.90, and 68.93 (8 d, C-2,3,4,5,20,30,40,50),
75.81, 75.63, 74.14, 72.88, 72.52, 69.89, and 68.46
(7 t, 5 CH2Ph, C-60, and OCH2CH=CH2), 18.68
(q, C-6). Anal. Calcd for C50H56O10: C, 73.51; H,
6.91. Found: C, 73.12; H, 6.98.
1
0
0
0
(300 MHz, CDCl3): ꢂ 7.60±7.10 (m, 25 H, ArH),
5.86 (m, 1 H, OCH2CH=CH2), 5.53 (s, 1 H,
CHPh), 5.28±5.14 (m, 2 H, OCH2CH=CH2),
4.95±4.61 (m, 10 H, 4 CH2Ph and H-1,10), 4.23 (m,
1 H, H-50), 4.20±3.85 (m, 6 H, H-2,3,30,60a and
OCH2CH=CH2), 3.74 (m, 1 H, H-5), 3.66±3.51
(m, 4 H, H-20,4,40,60b), 1.35 (d, 3 H, J6,5 6.0 Hz,
H-6); 13C NMR (75.46 MHz, CDCl3): ꢂ 134.66
(d, OCH2CH=CH2), 117.70 (t, OCH2CH=CH2),
101.88 (d, CHPh), 98.56 and 97.31 (2 d, C-1,10),
83.14, 80.84, 80.15, 79.66, 78.93, 75.89, 69.00, and
63.42 (8 d, C-2,3,4,5,20,30,40,50), 75.78, 75.65, 73.50,
72.92, 69.71, and 68.44 (6 t, 4 CH2Ph, C-60 and
OCH2CH=CH2), 18.63 (q, C-6). Anal. Calcd for
C50H54O10: C, 73.69; H, 6.68. Found: C, 73.31; H,
6.82.
Phenyl 3-O-benzyl-4,6-O-benzylidene-1-thio-ꢀ-
d-glucopyranoside (8).ÐCompound
2
(5.0 g,
13.87 mmol) was stirred with sodium hydride
(1.5 g, 36.5 mmol) in dry Me2NCHO (50 mL) at
room temperature. When the evolution of hydro-
gen was ceased, anhydrous cupric chloride (2.25 g,
18.26 mmol) was added and the mixture was stirred
for 10 min. Then, benzyl bromide (5.0 mL,
41.5 mmoꢀl) was added. The reaction was heated for
4 h at 70 C, then MeOH was added and the mix-
ture was diluted with EtOAc, washed with water,
dried (Na2SO4), and concentrated. The residue was
puri®ed on a short silica gel column (8:2 hexane±
EtOAc) a€ording compound 8 (4.5 g, 72%) as a
Allyl (2,3,6-tri-O-benzyl-ꢁ-d-glucopyranosyl)-
(1!2)-3,4-di-O-benzyl-ꢁ-l-rhamnopyranoside (7).Ð
To a stirred solution of 6 (500 mg, 0.6 mmol) in
ꢀ
dry CH2Cl2 (20 mL) cooled at 0 C was added aq
90% CF3CO2H. The reaction was complete within
ꢀ
1 h at 0 C. The reaction was quenched with satd
NaHCO3, and the mixture extracted with CH2Cl2.
The organic phase was washed with satd NaHCO3
ꢀ
powder; mp 136±137 C; [ꢁ]d 46.8ꢀ (c 1, CHCl3);
1
and brine, dried (Na2SO4), and concentrated. H
1H NMR (200 MHz, CDCl3): ꢂ 7.57±7.26 (m, 15 H,
ArH), 5.57 (s, 1 H, CHPh), 4.95 and 4.78 (2 d, each
1 H, Jgem 11.5 Hz, CH2Ph), 4.64 (d, 1 H, J1,2
9.5 Hz, H-1), 4.39 (dd, 1 H, J6a,5 4.8, J6a,6b 10.4 Hz,
H-6a), 3.85±3.61 (m, 4 H, H-2,3,4,6b), 3.53 (m, 1
H, H-5), 2.55 (d, 1 H, OH); 13C NMR (50.29 MHz,
CDCl3): ꢂ 101.95 (d, CHPh), 89.20 (d, C-1), 82.36,
81.77, 73.02, and 71.40 (4 d, C-2,3,4,5), 75.40 and
69.27 (2 t, C-6 and CH2Ph). Anal. Calcd for
C26H26O5S: C, 69.31; H, 5.82. Found: C, 69.14; H,
5.98.
NMR analysis of the crude residue showed the
disappearance of the signal at ꢂ 5.53 corresponding
to the benzylidene acetal group. The crude residue,
dibutyltin oxide (224 mg, 0.9 mmol) and benzene
(60 mL) were placed in a two-necked ¯ask equip-
ped with a Dean±Stark separator. The mixture was
boiled under re¯ux for 3 h, then concentrated to
ꢀ
half its initial volume and cooled at 50 C. Then
Bu4NBr (290 mg, 0.28 mmol) and benzyl bromide
(1.8 mmol, 214 ꢃL) were added, and the mixture
was stirred for 18 h at 90 ꢀC. After completion, the
solvent was evaporated, and the residue was pur-
i®ed by ¯ash chromatography (8:2 hexane±EtOAc)
yielding compound 7, isolated as an oil (392 mg,
80%); [ꢁ]d +13.4ꢀ (c 1, CHCl3); 1H NMR
(300 MHz, CDCl3): ꢂ 7.40±7.20 (m, 25 H, ArH),
5.86 (m, 1 H, OCH2CH=CH2), 5.26±5.13 (m, 2 H,
OCH2CH=CH2), 4.93 (brd, 1 H, H-10), 4.81 (d, 1
H, J1,2 2.2 Hz, H-1), 4.98±4.52 (m, 8 H, 4 CH2Ph),
4.45 and 4.36 (2 d, 2 H, Jgem 12.2 Hz, CH2Ph), 4.13
(m, 1 H, OCHHCH=CH2), 4.09±4.03 (m, 2 H, H-
2,50), 3.96±3.85 (m, 3 H, H-3,30 and OCHHCH
Phenyl 2-O-acetyl-3-O-benzyl-4,6-O-benzylidene-
1-thio-ꢀ-d-glucopyranoside (9).ÐTo a solution of 8
(2.03 g, 4.5 mmol) in dry CH2Cl2 (60 mL) were
added dry pyridine (4.5 mL) and Ac2O (3.0 mL).
The mixture was kept for 5 h at room temperature,
then diluted with CH2Cl2 (50 mL), washed with aq
5% HCl, satd NaHCO3, and water, dried
(Na2SO4), and concentrated to a€ord 9 (2.04 g,
93%)ꢀ as a white solid; mp 177±179 ꢀC; [ꢁ]d
1
+1.4 (c 1, CHCl3); H NMR (200 MHz, CDCl3):
ꢂ 7.54±7.20 (m, 10 H, ArH), 5.60 (s, 1 H, CHPh),
5.05 (m, 1 H, H-2), 4.88 (d, 1 H, Jgem 11.9 Hz,
CHHPh), 4.73 (d, 1 H, J1,2 10.6 Hz, H-1), 4.67 (d, 1
H, CHHPh), 4.42 (dd, 1 H, J6a,6b 10.8, J6a,5
5.0 Hz, H-6a), 3.88±3.74 (m, 3 H, H-3,4,6b), 3.57
=CH2), 3.72 (dq, 1 H, J5,4 9.2, J5,6 6.1 Hz, H-5),
0
0
3.64 (t, 1 H, J4 ,3 =J4 ,5 =9.5 Hz, H-4 ), 3.57 (t, 1
0
0
0
0
H, J4,3=J4,5=9.2 Hz, H-4), 3.54 (dd, 1 H, J2 ,1 3.5,
0

178
E. Bousquet et al./Carbohydrate Research 311 (1998) 171±181
(m, 1 H, H-5), 2.10 (s, 3 H, CH3CO); 13C NMR
(75.46 MHz, CDCl3): ꢂ 169.19 (s, CO), 101.20 (d,
CHPh), 86.76 (d, C-1), 81.27, 79.77, 71.39, and
70.49 (4 d, C-2,3,4,5), 74.29 and 68.51 (2 t, C-6 and
CH2Ph), 20.91 (q, CH3CO). Anal. Calcd for
C28H28O6S: C, 68.27; H, 5.73. Found: C, 68.54; H,
5.92.
and the mixture was stirred for 3 h at 60 ꢀC. Then
the mixture was neutralized with satd NaHCO3,
diluted with CH2Cl2, ®ltered through Celite, and
the ®ltrate was washed with water, dried (Na2SO4),
and concentrated. The residue was puri®ed by ¯ash
chromatography (95:5 toluene±EtOAc) to a€ord
ꢀ
11 (1.34 g, 77%) as a powder; mp 122±125 C; [ꢁ]d
Phenyl 2-O-acetyl-3-O-benzyl-4,6-O-benzylidene-
1-thio-ꢀ-d-glucopyranoside sulfoxide (10).ÐThis
compound was prepared as described for com-
pound 4. Compound 9 (3.18 g, 6.45 mmol) gave
sulfoxide 10 (3.12 g, 95%) as a mixture of diaster-
eoisomers. A small amount (100 mg) was chroma-
tographed (7:3 petroleum ether±EtOAc) to give
pure analytical samples. The diastereoisomer with
the higher Rf value was obtained as an amorphous
mass; [ꢁ]d 11.6ꢀ (c 1, CHCl3); 1H NMR (300 MHz,
CDCl3): ꢂ 7.70±7.20 (m, 15 H, ArH), 5.50 (s, 1 H,
CHPh), 5.18 (dd, 1 H, J2,3 8.3 Hz, H-2), 4.83 and
4.63 (2 d, each 1 H, Jgem 11.8 Hz, CH2Ph), 4.50 (d,
1 H, J1,2 9.7 Hz, H-1), 4.33 (dd, 1 H, J6a,6b 8.9, J6a,5
2.5 Hz, H-6a), 3.80 (t, 1 H, J3,4 8.3 Hz, H-3), 3.65±
+32.8ꢀ (c 1, CHCl3); 1H NMR (300 MHz, CDCl3):
ꢂ 7.50±7.11 (m, 35 H, ArH), 5.87 (m, 1 H,
OCH2CH=CH2), 5.45 (s, 1 H, CHPh), 5.26±5.13
(m, 2 H, OCH2CH=CH2), 4.95±4.56 (m, 15 H, H-
1,10,200 and 6 CH2Ph), 4.33 (d, 1 H, J1 ,2 7.9 Hz, H-
00 00
100), 4.18±4.09 (m, 3 H, OCHHCH=CH2 and H-
600a,50), 3.99±3.82 (m, 5 H, H-2,3,30,60a and OCH
HCH=CH2), 3.78±3.35 (m, 7 H, H-20,300,4,40,400,
5,600b), 3.19 (brdd, 1 H, H-60b), 3.07 (dt, 1 H, J5 ,6 a
00 00
00
00 00
4.8, J5 ,6 b=J5 ,4 =9.7 Hz, H-5 ), 1.70 (s, 3 H,
00 00
CH3CO), 1.35 (d,
3
H, H-6); 13C NMR
(75.46 MHz, CDCl3): ꢂ 169.51 (s, CO), 134.57 (d,
OCH2CH=CH2), 117.80 (t, OCH2CH=CH2),
101.69, 101.35, 100.70, and 98.70 (4 d, C-1,10,100
and CHPh), 82.30, 80.85, 80.12, 79.70, 79.19,
77.49, 76.75, 73.93, 71.04, 69.00, and 66.54 (11 d,
C-2,20,200,3,30,300,4,40,400,5,50,500), 75.82, 75.66, 74.54,
74.15, 73.41, 72.61, 69.29, 68.35, and 67.80 (9 t,
C-60,600, 6 CH2Ph, and OCH2CH=CH2), 21.28 (q,
CH3CO), 18.62 (q, C-6). Anal. Calcd for C72H78
O16: C, 72.10; H, 6.55. Found: C, 72.31; H, 6.39.
Allyl (3-O-benzyl-4,6-O-benzylidene-ꢀ-d-gluco-
pyranosyl)-(1!4)-(2,3,6-tri-O-benzyl-ꢁ-d-glucopyr-
anosyl)-(1!2)-3,4-di-O-benzyl-ꢁ-l-rhamnopyrano-
side (12).ÐCompound 11 (846 mg, 0.705 mmol)
was dissolved in dry MeOH (40 mL) and treated
with a catalytic amount of NaOMe, then stirred
overnight at room temperature. The mixture was
concentrated, diluted with CH2Cl2 and washed
with aq 5% HCl, satd NaHCO3, and water. The
organic layer was dried (Na2SO4) and con-
centrated. Flash chromatography of the residue
(8:2 hexane±EtOAc) gave 12 as an amorphous
3.48 (m, 3 H, H-4,5,6b), 1.90 (s, 3 H, CH3CO); 13
C
NMR (75.46 MHz, CDCl3): ꢂ 169.25 (s, CO),
101.32 (d, CHPh), 93.30 (d, C-1), 80.72, 79.44,
70.48, and 68.48 (4 d, C-2,3,4,5), 74.35 and 68.06 (2
t, C-6 and CH2Ph), 20.72 (q, CH3CO). Further
elution gave the second diꢀastereoisomer as an
1
amorphous mass; [ꢁ]d 63.2 (c 0.5, CHCl3); H
NMR (300 MHz, CDCl3): ꢂ 7.70±7.20 (m, 15 H,
ArH), 5.52 (s, 1 H, CHPh), 5.39 (t, 1 H, J2,3 9.0 Hz,
H-2), 4.85 and 4.64 (2 d, each 1 H, Jgem 12.0 Hz,
CH2Ph), 4.21 (d, 1 H, J1,2 9.0 Hz, H-1), 4.17 (dd, 1
H, J6a,6b 10.6, J6a,5 5.0 Hz, H-6a), 3.83±3.71 (m, 3
H, H-3,4,6b), 3.42 (dt, 1 H, J5,6b=J5,4=9.2 Hz, H-
5), 2.00 (s, 3 H, CH3CO); 13C NMR (75.46 MHz,
CDCl3): ꢂ 169.78 (s, CO), 101.24 (d, CHPh), 91.39
(d, C-1), 80.62, 79.37, 70.76, and 68.34 (4 d, C-
2,3,4,5), 74.10 and 67.97 (2 t, C-6 and CH2Ph),
20.69 (q, CH3). Anal. Calcd for C28H28O7S: C,
66.13; H, 5.55. Found: C, 65.90; H, 5.71.
mass (800 mg, 98%); [ꢁ]d +41.8ꢀ (c 1, CHCl3); H
1
Allyl (2-O-acetyl-3-O-benzyl-4,6-O-benzylidene-
ꢀ-d-glucopyranosyl)-(1!4)-(2,3,6-tri-O-benzyl-ꢁ-
d-glucopyranosyl)-(1!2)-3,4-di-O-benzyl-ꢁ-l-rham-
nopyranoside (11).ÐTo a mixture of 10 (1.108 g,
2.179 mmol) in dry 1:1 CH2Cl2±Et2O (15 mL)
containing freshly activated powdered 4 A mole-
NMR (300 MHz, CDCl3): ꢂ 7.50±7.20 (m, 35 H,
ArH), 5.87 (m, 1 H, OCH2CH=CH2), 5.45 (s, 1 H,
CHPh), 5.27±5.14 (m, 2 H, OCH2CH=CH2),
4.92±4.49 (m, 14 H, H-1,10,100, 5 CH2Ph and
CHHPh), 4.31 (d, 1 H, Jgem 12.1 Hz, CHHPh),
4.16±3.85 (m,
8
H, H-2,3,30,50,60a,600a and
ꢀ
cular sieves, cooled at 78 C, was added Tf2O
OCH2CH=CH2), 3.76±3.40 (m, 8 H, H-20,200,
(357 ꢃL, 2.179 mmol). After stirring for 30 min, a
solution of 7 (1.187 g, 1.452 mmol) and 2,6-di-
tbutyl-4-methylpyridine (447 mg, 2.179 mmol) in
dry 1:1 CH2Cl2±Et2O (27 mL) was added under N2
300,4,40,400,5,600b), 3.26 (brd, 1 H, H-60b), 3.05 (dt, 1
00
00 00
00 00
00 00
H, J5 ,6 a 4.8, J5 ,6 b=J5 ,4 =9.6 Hz, H-5 ), 2.95
(d, 1 H, J 1.5 Hz, OH), 1.35 (d, 3 H, J6,5 6.0 Hz,
H-6); 13C NMR (75.46 MHz, CDCl3): ꢂ 134.67 (d,

E. Bousquet et al./Carbohydrate Research 311 (1998) 171±181
179
CH2CH=CH2), 117.84 (t, OCH2CH=CH2),
104.50, 101.90, 97.33, and 97.22 (4 d, CHPh and C-
1,10,100), 81.92, 81.09, 80.46, 79.83, 77.99, 76.23,
75.38, 70.71, and 67.03 (9 d, C-2,20,200,3,30,300,
4,40,400,5,50,500), 75.92, 75.51, 75.16, 74.31, 73.37,
72.79, 69.43, and 68.52 (8 t, C-60,600, 6 CH2Ph, and
OCH2CH=CH2), 18.72 (q, C-6). Anal. Calcd for
C70H76O15: C, 72.65; H, 6.62. Found: C, 72.48; H,
6.83.
boiled under re¯ux for 3 h at 130 ꢀC using a Dean±
Stark apparatus, then cooled at room temperature,
diluted with water and extracted with CH2Cl2. The
extract was washed with water, dried (Na2SO4),
and concentrated. Flash chromatography of the
residue (8:2 hexane±EtOAc) gave 14 as a glass
(620 mg, 97%); [ꢁ]d +21.8ꢀ (c 1, CHCl3); IR
(KBr):
ꢄ
2110 (N=N+=N ); 1H NMR
(300 MHz, CDCl3): ꢂ 7.50±7.10 (m, 35 H, ArH),
5.86 (m, 1 H, OCH2=CHCH2), 5.40 (s, 1 H,
CHPh), 5.28±5.15 (m, 2 H, OCH2CH=CH2),
5.01±4.51 (m, 14 H, H-1,10 and 6 CH2Ph), 4.39
(brs, 1 H, H-100), 4.18±3.48 (m, 13 H, H-
2,20,200,3,30,4,40,400,50,600a,600b and OCH2CH=CH2),
3.73 (dq, 1 H, J5,6 6.0, J5,4 9.1 Hz, H-5), 3.31 (dd, 1
Allyl (3-O-benzyl-2-O-(N-imidazolyl-sulfonyl)-
4,6-O-benzylidene-ꢀ-d-glucopyranosyl)-(1!4)-
(2,3,6-tri-O-benzyl-ꢁ-d-glucopyranosyl)-(1!2)-
3,4-di-O-benzyl-ꢁ-l-rhamnopyranoside (13).ÐA
mixture of 12 (152 mg, 0.131 mmol) and 60% NaH
in oil (32 mg, 0.8 mmol) in dry Me2NCHO (10 mL)
ꢀ
H, J3 ,2 3.6, J3 ,4 9.5 Hz, H-300), 3.15 and 3.08 (2
00 00 00 00
was stirred for 1 h, then cooled at 40 C. Sulfo-
nyldiimidazole (266 mg, 134 mmol) was added, and
brd, each 1 H, J6 a,6 b 10.7 Hz, H-60a,60b), 2.93 (dt,
0 0
ꢀ
1 H, J5 ,6 a=J5 ,4 =9.5, J5 ,6 b 4.6 Hz, H-500), 1.35
(d, 3 H, H-6); 13C NMR (75.46 MHz, CDCl3): ꢂ
134.50 (d, OCH2=CHCH2), 117.84 (t, OCH2CH
=CH2), 102.07 (d, CHPh), 100.93, 97.54, and 97.17
(3 d, C-1,10,100), 80.93, 80.55, 79.94, 79.83, 79.09,
77.93, 77.05, 75.48, 70.13, 67.71, and 64.11 (11 d,
C-2,20,200,3,30,300,4,40,400,5,50,500), 75.97, 75.62, 74.07,
73.31, 73.19, 72.73, 69.05, 68.53, and 68.36 (9 t,
C-6,60, OCH2CH=CH2, and 6 CH2Ph), 18.55 (q,
C-6). Anal. Calcd for C70H75N3O14: C, 71.11; H,
6.39; N, 3.55. Found: C, 71.23; H, 6.54; N, 3.71.
Allyl (2-acetamido-3-O-benzyl-4,6-O-benzylidene-
2-deoxy-ꢀ-d-mannopyranosyl)-(1!4)-(2,3,6-tri-O-
benzyl-ꢁ-d-glucopyranosyl)-(1!2)-3,4-di-O-benzyl-
ꢁ-l-rhamnopyranoside (1).ÐA mixture of 14 (1.5 g,
1.2 mmol) and zinc (5.0 g) activated with aq 2%
CuSO4 in 6:4:2 THF±Ac2O±HOAc (48 mL) was
00 00
00 00
00 00
the stirring was continued for 5 h at 40 C. The
reaction was quenched with MeOH, then the mix-
ture was extracted with CH2Cl2, and the organic
layer was washed with water, dried (Na2SO4), and
concentrated. The residue was puri®ed by ¯ash
chromatography (8:2 hexane±EtOAc) to give 13
(168 mg, 99%); [ꢁ]d +28.0ꢀ (c 1, CHCl3); 1H NMR
(300 MHz, CDCl3): ꢂ 7.80 (s, 1 H, H-a imidazole),
7.50±7.10 (m, 35 H, ArH), 7.07 and 7.00 (2 s, each
1 H, H-b,c imidazole), 5.87 (m, 1 H, OCH2CH
=CH2), 5.43 (s, 1 H, CHPh), 5.27±5.15 (m, 2 H,
OCH2CH=CH2), 5.00 (d, 1 H, Jgem 11.2 Hz,
CHHPh), 4.90±4.59 (m, 13 H, H-1,10,100 and 5
CH2Ph), 4.50 (d, 1 H, Jgem 11.4 Hz, CHHPh), 4.36
00
00 00
00 00
(t, 1 H, J2 ,3 =J2 ,1 =8.3 Hz, H-2 ), 4.26±3.30 (m,
15 H, H-2,20,3,30,300,4,40,400,5,50,60a,600a,600b and
OCH2CH=CH2), 3.25 (brdd, 1 H, H-60b), 2.96 (m,
1 H, H-500), 1.35 (d, 3 H, H-6); 13C NMR
(75.46 MHz, CDCl3): ꢂ 134.57 (d, OCH2CH
=CH2), 117.74 (t, OCH2CH=CH2), 102.02 (d,
CHPh), 99.10, 98.90, and 97.81 (3 d, C-1,10,100),
85.58, 82.57, 81.14, 79.84, 77.52, 77.32, 76.41,
70.29, and 66.35 (9 d, C-2,20,200,3,30,300,4,40,400,
5,50,500), 75.78, 74.84, 74.13, 73.50, 72.77, 68.34,
and 67.96 (7 t, C-60,600, 6 CH2Ph, and OCH2CH
=CH2), 18.63 (q, C-6). Anal. Calcd for C73H78
N2O17S: C, 68.04; H, 6.05; N, 2.17; S, 2.48. Found:
C, 68.32; H, 6.13, N, 2.01; S, 2.25.
ꢀ
stirred for 2 h at 35 C. After dilution with EtOAc
and ®ltration through Celite, the solution was
washed with satd NaHCO3 and water, dried
(Na2SO4), and concentrated. The residue was pur-
i®ed by ¯ash chromatography (7:3 hexane±EtOAc)
to give 1 isolated as a glass (1.2 g, 84%); [ꢁ]d
+14.4ꢀ (c 0.7, CHCl3); 1H NMR (300 MHz,
CDCl3): ꢂ 7.55±7.15 (m, 35 H, ArH), 5.85 (m, 1 H,
OCH2CH=CH2), 5.50 (d, 1 H, J 9.5 Hz, NH), 5.46
(s, 1 H, CHPh), 5.27±5.14 (m, 2 H, OCH2CH
=CH2), 4.99±4.45 (m, 14 H, H-1,10,100, 5 CH2Ph,
and CHHPh), 4.24 (d, 1 H, Jgem 12.0 Hz, CHHPh),
4.18±3.47 (m, 13 H, H-2,20,200,3,30,4,40,400,50,600a,600b
and OCH2CH=CH2), 3.73 (dq, 1 H, J5,6 6.0, J5,4
9.1 Hz, H-5), 3.35±3.29 (m, 2 H, H-60a,300), 3.19
Allyl (2-azido-3-O-benzyl-4,6-O-benzylidene-2-
deoxy-ꢀ-d-mannopyranosyl)-(1!4)-(2,3,6-tri-O-
benzyl-ꢁ-d-glucopyranosyl)-(1!2)-3,4-di-O-benzyl-
ꢁ-l-rhamnopyranoside (14).ÐTo a stirred solution
of 13 (700 mg, 0.54 mmol) in dry toluene (80 mL)
under N2, was added 0.42 M Bu4NN3 in dry
toluene (6.6 mL, 0.138 mmol). The mixture was
(brd, 1 H, J6 a,6 b 10.8 Hz, H-60b), 3.05 (dt, 1 H,
0
0
J5 ,6 a=J5 ,4 =9.5, J5 ,6 b 4.7 Hz, H-500), 1.75 (s, 3
00 00
00 00
00 00
H, CH3CO), 1.35 (d, 3 H, H-6); 13C NMR

180
E. Bousquet et al./Carbohydrate Research 311 (1998) 171±181
(75.46 MHz, CDCl3): ꢂ 171.00 (s, CO), 134.54 (d,
OCH2CH=CH2), 117.95 (t, OCH2CH=CH2),
102.33 (d, CHPh), 100.34, 97.82, and 97.36 (3 d,
C-1,10,100), 80.95, 80.24, 79.70, 79.24, 76.60, 76.36,
70.54, 69.03, and 67.80 (9 d, C-2,20,3,30,300,
4,40,400,5,50,500), 75.39, 74.11, 73.54, 72.87, 71.88,
69.35, and 68.53 (7 t, C-60,600, 6 CH2Ph and OCH2
CH=CH2), 51.14 (d, C-200), 23.84 (q, CH3CO), 18.66
(q, C-6). Anal. Calcd for C72H79NO15: C, 72.16; H,
6.64; N, 1.17. Found: C, 72.24; H, 6.84; N, 1.30.
Allyl (2-acetamido-3,6-di-O-benzyl-2-deoxy-ꢀ-d-
mannopyranosyl)-(1!4)-(2,3,6-tri-O-benzyl-ꢁ-d-
glucopyranosyl)-(1!2)-3,4-di-O-benzyl-ꢁ-l-rhamno-
pyranoside (15).ÐTo a stirred solution of 1 (33 mg,
0.027 mmol) in dry CH3CN (5 mL) was added
(30 mg, 0.025 mmol) in dry THF (5 mL) was added
a catalytic amount of 1,5-cyclooctadiene-bis-
[methyldiphenylphosphine]iridium hexa¯uoropho-
sphate. The stirred solution was degassed by freez-
ing the solvent and allowing it to warm to room
temperature under vacuum and then placed under
H2 to activate the catalyst (the slightly red suspen-
sion became pale yellow). After 2 min, the solution
was degassed once more and stirred at room tem-
perature under N2. After 2 h, TLC analysis showed
a complete conversion of the allyl ether into the 1-
propenyl ether. The hydrolysis was accomplished
by adding water (2 mL) and I2 (5 mg), and stirring
the mixture for 5 min at room temperature. The
mixture was diluted with water and extracted with
CH2Cl2. The excess of iodine was destroyed by
washing the organic phase with freshly prepared aq
5% NaHSO3. After drying (Na2SO4) and con-
centration, the residue was puri®ed by ¯ash chro-
matography (7:3 petroleum ether±EtOAc) to give
16 (28 mg, 96%) as an oil (ꢁ,ꢀ mixture); [ꢁ]d +3.0ꢀ
(c 2.5, CHCl3); 1H NMR (300 MHz, CDCl3),
major anomer: ꢂ 7.60±7.00 (m, 35 H, ArH), 5.80
(d, 1 H, J 12.9 Hz, OH), 5.47 (s, 1 H, CHPh), 5.43
(d, 1 H, J 9.7 Hz, NH), 5.02±4.42 (m, 16 H, H-
.
BH3 Me3N (15 mg, 0.2 mmol). The reaction was
ꢀ
.
cooled at 0 C, then freshly distilled BF3 Et2O
(60 ꢃL) was added under N2. The reaction was
completed within 30 min, and the mixture was
concentrated, diluted with CH2Cl2 (10 mL),
washed with satd NaHCO3 and water, and con-
centrated. The residue was puri®ed on a short
silica-gel column (7:4 petroleum ether±EtOAc)
a€ording trisaccharide ꢀ 15 isolated as a glass
(27 mg, 82%); [ꢁ]d +2.8 (c 2.5, CHCl3); 1H NMR
(300 MHz, CDCl3): ꢂ 7.50±7.15 (m, 35 H, ArH),
5.86 (m, 1 H, OCH2CH=CH2), 5.59 (d, 1 H, J
9.6 Hz, NH), 5.26±5.13 (m, 2 H, OCH2CH=CH2),
4.96±4.48 (m, 16 H, H-1,10,100,200, 2 CHHPh, and 5
CH2Ph), 4.37 (d, 1 H, Jgem 11.9 Hz, CHHPh), 4.24
(d, 1 H, Jgem 11.1 Hz, CHHPh), 4.16±3.85 (m, 7 H,
H-2,3,30,40,50 and OCH2CH=CH2), 3.72 (dq, 1 H,
J5,6 6.0 Hz, H-5), 3.60 (t, 1 H, J4,5=J4,3=9.3 Hz,
H-4), 3.56±3.50 (m, 4 H, H-20,400,600a,600b), 3.40 (dd,
1,10,100,200 and 6 CH2Ph), 4.12 (dd, 1 H, J6 a,6 b
00
00
10.7, J6 a,5 4.7 Hz, H-600a), 4.08±3.85 (m, 4 H, H-
00
00
2,30,40,600b), 3.64±3.32 (m, 7 H, H-3,4,5,20,50,400,60a),
3.28 (dd, 1 H, J3 ,2 4.3, J3 ,4 9.8 Hz, H-300), 3.09±
3.01 (m, 2 H, H-60b,500), 1.80 (s, 3 H, CH3CO), 1.45
(d, 3 H, J6,5 6.0 Hz, H-6); 13C NMR (75.46 MHz,
CDCl3), major anomer: ꢂ 170.45 (s, CO), 102.47,
101.70, 99.41, and 93.96 (4 d, CHPh and C-1,10,100),
81.51, 81.27, 81.11, 79.78, 78.83, 78.51, 75.84,
71.44, 70.47, 67.17, and 50.53 (11 d, C-2,20,200,
3,30,300,4,40,400,5,50,500), 74.86, 74.46, 73.39, 71.67,
71.08, 68.63, and 67.52 (7 t, C-60,600 and 6 CH2Ph),
23.21 and 18.07 (2 q, CH3CO and C-6). Anal.
Calcd for C69H75NO15: C, 71.55; H, 6.53; N, 1.21.
Found: C, 71.82; H, 6.78; N, 1.42.
00 00
00 00
1 H, J6 a,5 2.1, J6 a,6 b 11.1 Hz, H-60a), 3.27 (brd, 1
H, H-60b), 3.15±3.07 (m, 2 H, H-300,500), 2.40 (brs, 1
H, OH), 1.75 (s, 3 H, CH3CONH), 1.35 (d, 3 H, H-
6); 13C NMR (75.46 MHz, CDCl3): ꢂ 170.40 (s,
CO), 139.91 (d, OCH2CH=CH2), 117.15 (t,
OCH2CH=CH2), 99.77, 96.92, and 96.58, (3 d, C-
1,10,100), 80.64, 80.24, 79.67, 79.01, 75.83, 75.02,
69.77, 68.32, and 67.35 (9 d, C-2,20,3,30,300,
4,40,400,5,50,500), 75.27, 74.73, 73.53, 73.18, 72.62,
72.05, 70.99, 69.28, 68.20, and 67.76 (10 t, C-60,600,
OCH2CH=CH2, and 7 CH2Ph), 49.37 (d, C-200),
23.14 and 18.01 (2 q, CH3CO and C-6). Anal.
Calcd for C72H81NO15: C, 72.04; H, 6.80; N, 1.17.
Found: C, 72.21; H, 6.64; N, 1.22.
0
0
0
0
Acknowledgements
The authors thank EU (contract BIO CT96 0158)
for ®nancial support.
(2-Acetamido-3-O-benzyl-4,6-O-benzylidene-2-
deoxy-ꢀ-d-mannopyranosyl)-(1!4)-(2,3,6-tri-O-
benzyl-ꢁ-d-glucopyranosyl)-(1!2)-3,4-di-O-benzyl-
ꢁ,ꢀ-l-rhamnopyranose (16).ÐTo a solution of 1
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