Synthesis of the pentasaccharide repeating unit of the major O-antigen component from Pseudomonas syringae pv. ribicola NVPPB 1010

Article abstract of DOI:10.1016/j.carres.2003.10.002

The synthesis of the repeating unit of the major O-antigen component from Pseudomonas syringae pv. ribicola NVPPB 1010 is reported. The strategy used was based on the successive coupling of a trisaccharide rhamnosyl trichloroacetimidate with a rhamnosyl a

Full text of DOI:10.1016/j.carres.2003.10.002

Carbohydrate
RESEARCH
Carbohydrate Research 339 (2004) 393–400
Synthesis of the pentasaccharide repeating unit of the
major O-antigen component from Pseudomonas syringae
pv. ribicola NVPPB 1010
Emiliano Bedini,a Gaspare Barone,a Carlo Unverzagtb and Michelangelo Parrillia,*
aDipartimento di Chimica Organica e Biochimica, Universitꢀa di Napoli ‘‘Federico II’’, Complesso Universitario Monte Santangelo,
Via Cintia 4, 80126 Napoli, Italy
bBioorganische Chemie, Geb€aude NWI, Universit€at Bayreuth, 95440 Bayreuth, Germany
Received 29 May 2003; revised 3 October 2003; accepted 6 October 2003
Abstract—The synthesis of the repeating unit of the major O-antigen component from Pseudomonas syringae pv. ribicola NVPPB
1010 is reported. The strategy used was based on the successive coupling of a trisaccharide rhamnosyl trichloroacetimidate with a
rhamnosyl acceptor with a free hydroxyl group on C-2. The pentasaccharide was then obtained by coupling with a N-Troc-tri-O-
acetyl-glucosamine trichloroacetimidate. The synthesis allowed the oligomerisation of the repeating unit.
ꢀ 2003 Elsevier Ltd. All rights reserved.
Keywords: Pseudomonas ribicola; O-chain; Glycosylation; Oligosaccharides; Repeating unit
1. Introduction
backbone structure, whereas the LPS from phytopatho-
genic bacteria very often present the GlcNAc unit as
single monosaccharide side chain.
It has been recently reviewed1 that O-chains of lipo-
polysaccharides (LPS) from phytopathogenic bacteria
typically consist of repeating units composed of rham-
nan backbones bearing single monosaccharide branches.
Only a few types of sugars constitute these monosac-
charide side chains, for example, GlcNAc.
Glucosaminylated rhamnans, that occur in human
pathological bacteria, have been largely studied2;3 and
their repeating units have been synthesized.4;5 They very
often contain the GlcNAc residue as a component of the
Since the role of the O-chain structures in phyto-
pathogenic bacteria is much less known in comparison
with the O-chain from human pathological bacteria, the
synthesis of their repeating unit is strictly requested.
Therefore, in order to investigate the structure–bioac-
tivity relationship of the O-chain in plant infection, the
synthesis of the glucosaminylated rhamnan repeating
unit A (Scheme 1) of the phytopathogenic P. syringae
pv. ribicola NVPPB 1010 bacterium, the causative agent
-D-GlcpNAc
1
↓
3
→3)-α-L-Rhap-(1→3)-α-L-Rhap-(1→2)-α-L-Rhap-(1→2)-α-L-Rhap-(1→
A
Scheme 1. Repeating unit of the major O-antigen component from P. syringae pv. ribicola NVPPB 1010.
* Corresponding author. Tel.: +39-081-674147; fax: +39-081-674393; e-mail: parrilli@unina.it
0008-6215/$ - see front matter ꢀ 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2003.10.002

394
E. Bedini et al. / Carbohydrate Research 339 (2004) 393–400
OBn
OH
NH
O
HO
O
O
HO
HO
CCl3
O
OAc
O
NHAc
HO
O
AcO
AcO
O
O
BzO
HO
BzO
O
TeocNH
O
CCl3
O
O
NH
O
O
3
HO
O
BzO
O
OBn
OH
OBz
O
HO
BzO
ClAcO
O
BzO
HO
OH
OBz
AllO
OH
1
2
4
Scheme 2. Retrosynthetical analysis of structure A.
of defoliation of Ribes aurum,7 is, at the best of our
knowledge, for the first time reported.ꢀ The approach
described in this paper aims at the synthesis of a penta-
saccharide building block that can also be employed to
obtain higher oligomers of the repeating unit A.
coside 5 obtaining the trisaccharide 9 in excellent yield
(96%). Treating 9 with anhydrous FeCl3 in CH2Cl2,13 we
could selectively cleave the benzyl protecting group on
the anomeric position of 9, obtaining a hemiacetal that
was directly converted in the trisaccharide imidate 2 in
an overall yield of 44% over two steps.
The rhamnosyl acceptor 4 was obtained by selective
allylation of benzyl 4-O-benzoyl-a-L-rhamnopyranoside
2. Results and discussion
10,10 whose a-anomeric configuration was assigned by
1
As outlined in Scheme 1, the major repeating unit of the
O-chain from P. syringae pv. ribicola NVPPB 1010
consists of a rhamnan backbone tetrasaccharide with a
GlcNAc branch. This residue is attached to O-3 of a
rhamnosyl residue, which is elongated by the rhamnosyl
chain at C-2.
low-field H NMR chemical shift value (d 5.191). The
cis-diol 10 was activated as a stannylene acetal by re-
fluxing with Bu2SnO in 10:1 benzene–methanol. Sub-
sequent alkylation with allyl bromide–Bu4NBr in
toluene gave the desired 3-allylated compound 4 in 85%
yield (Scheme 4).
Retrosynthetic analysis of the benzylated pentasac-
charide repeating unit 1 suggested a trisaccharide and a
glucosaminyl donor and a rhamnosyl acceptor with a
free OH group at C-2 and a selectively removable pro-
tecting group at C-3. We chose at best the building
blocks 2, 39 and 4, respectively (Scheme 2).
The synthesis of the trisaccharide donor 2 has been
already reported in one of ours recent short communi-
cations.10 It begins with the coupling of the thioglyco-
side 510 and the acceptor 611 following the typical
conditions for the activation of disarmed thioglycoside
with NIS/TfOH12 (Scheme 3). The disaccharide 7 was
obtained in high yield (82%). A selective removal of the
chloroacetyl group on 7 with thiourea in ethanol (80%
yield) afforded 8, that, following the same activation
condition used before, was then coupled with thiogly-
The tetrasaccharide 11 was obtained after coupling of
the trisaccharide imidate 2 with the acceptor 4 in 79%
yield. The a-configuration of the new glycosidic bond in
11 was ascertained by a coupled HMQC-COSY exper-
0
0
iment (JC-1 ;H-1 ¼ 173 Hz). Subsequent deallylation of
compound 11 with PdCl2 in 5:2 methanol–dichlorome-
thane furnished the tetrasaccharide acceptor 12 in 94%
yield. Some dechloroacetylation of the starting material
11 was observed (5% detected by NMR and ESI-MS),
however, this side reaction stopped after the initial
phase. The following glycosylation of the tetrasaccha-
ride 12 with the glucosaminyl donor 39 afforded the
pentasaccharide 13 in 45% yield together with a 51%
recovery of unreacted 12. The b-configuration of the
new glycosidic bond was confirmed by a coupled
0
0
HMQC-COSY experiment (JC-1 ;H-1 ¼ 164 Hz).
The final steps to the target molecule 1 required the
deprotection of pentasaccharide 13. The initial depro-
tection strategy was based on the conversion of the
N-Troc group into an N-Ac group and a subsequent
O-deacylation. Surprisingly the N-Troc/N-Ac conversion
ꢀ
When this work was already completed we were acquainted with an
alternative synthesis of our target, in a paper where a general method
for the synthesis of glucosaminylated rhamnanic backbones was
reported.8

E. Bedini et al. / Carbohydrate Research 339 (2004) 393–400
395
OR
SEt
O
BzO
OBn
O
O
BzO
O
BzO
BzO
O
ClAcO
O
OBz
BzO
5
O
BzO
c
a
BzO
5 + 8
O
OBn
+
OBz
O
BzO
b
O
BzO
RO
BzO
OBz
ClAcO
c
OH
OBz
9 R=Bn
2 R=C(NH)CCl3
7
6
R=ClAc
8 R=H
ꢁ
Scheme 3. Reagents and conditions: (a) NIS, TfOH, molecular sieves 4 A, CH2Cl2, )20 ꢁC, 90 min; 82%; (b) NH2CSNH2, EtOH, rt, 16 h, 80%;
ꢁ
(c) NIS, TfOH, molecular sieves 4 A, CH2Cl2, )20 ꢁC, 15 min; 96%; (d) i: anhydrous FeCl3, CH2Cl2, rt, 45 min; ii: Cl3CCN, DBU, CH2Cl2, 0 ꢁC,
80 min; 44% over two steps.
OBn
OBn
OBn
OAc
O
O
O
A
O
a
BzO
HO
O
BzO
RO
O
AcO
AcO
E
O
O
HO
10
HO
TrocNH
BzO
O
O
B
BzO
BzO
BzO
e
d
b
O
3 + 12
1
2 + 4
O
O
C
OBn
BzO
BzO
O
O
OBz
OBz
O
O
O
D
BzO
BzO
BzO
ClAcO
ClAcO
AllO
OBz
OBz
OH
11 R = All
12 R = H
13
4
c
Scheme 4. Reagents and conditions: (a) i: Bu2SnO, 10:1 benzene–methanol, reflux, 90 min; ii: AllBr, Bu4NBr, toluene, 65 ꢁC, 90 min; 85% over two
ꢁ
steps; (b) BF3ꢀOEt2, molecular sieves 4 A, CH2Cl2, )50 ꢁC, 30 min; 79%; (c) PdCl2, 5:2 methanol–dichloromethane, rt, 4 h; 94%; (d) TMSOTf,
ꢁ
molecular sieves 4 A, CH2Cl2, )10 ꢁC, 3 h; 45%; (e) i: 2 M KOH, THF, 40 ꢁC, 19 h; ii: Ac2O, MeOH, rt, 2 h; 56% over two steps.
with Zn/Ac2O9 was ineffective with the pentasaccharide
13. Thus, a different deprotection strategy was envi-
sioned starting with the complete removal of all ester
groups and the subsequent replacement of the Troc
moiety by an N-acetyl group. This approach was also
not successful because the N-Troc function was con-
verted to a methyl carbamate during methanolysis with
NaOMe in MeOH. Finally, treatment of 13 with 2 M
KOH in THF assured global deacylation and basic re-
moval of the Troc moiety. The free amino group of the
intermediate pentasaccharide was selectively acetylated
with Ac2O in MeOH to obtain the desired repeating unit
the synthetic pentasaccharide 1 and the natural O-chain6
show very good accordance, except for few values; in
particular the 13C NMR C-3D signal is shifted highfield
(8.2 ppm) in comparison with the value of the natural O-
chain, due to the absence of the glycosylation shift for
our oligosaccharide structure. There are other differ-
1
ences related to some H and 13C NMR values of the
residue A, due to the effect of the benzyl group as a
glycon.
In conclusion, we have synthesized a rhamnanic
pentasaccharide containing a GlcNAc branch corre-
sponding to the major O-chain repeating unit from P.
syringae pv. ribicola NVPPB 1010. It is noteworthy that
1
1. In Table 1 the H and 13C NMR chemical shifts for

396
E. Bedini et al. / Carbohydrate Research 339 (2004) 393–400
Table 1. 1H and 13C NMR chemical shifts (d in ppm) of 1 (in parentheses the differences in ppm between 1 and the related natural O-polysaccharide6
are given)
Sugar residue
A
H-1
H-2
H-3
H-4
H-5
H-6
H-60
4.83
()0.27)
5.26
4.13
()0.18)
4.02
3.84
()0.13)
3.85
3.51
()0.03)
3.45
3.70
()0.14)
3.52
1.27
()0.04)
1.13
B
C
D
E
()0.07)
4.95
()0.04)
4.13
()0.05)
3.86
()0.04)
3.53
()0.17)
3.79
()0.15)
1.23
()0.11)
5.06
()0.01)
4.25
(0.01)
4.07
()0.06)
3.47
()0.01)
3.84
()0.11)
1.29
()0.01)
4.68
(0.10)
3.71
(0.16)
3.53
()0.12)
3.39
()0.03)
3.39
()0.03)
3.71
3.88
()0.06)
()0.07)
()0.03)
()0.05)
()0.03)
()0.06)
()0.04)
C-1
C-2
C-3
C-4
C-5
C-6
A
B
C
D
E
98.5
77.6
81.5
(0.01)
70.9
69.9
()2.5)
73.3
70.3
(0.3)
70.6
(0.1)
70.3
(0.3)
70.9
(0.3)
76.7
()0.3)
18.0
()3.6)
100.7
()0.1)
102.8
(0.2)
(0.3)
79.2
(0.2)
17.9
(0.1)
71.1
()0.4)
78.9
()0.4)
72.4
()0.1)
18.0
()0.2)
71.5
()0.2)
71.4
()0.4)
70.6
()0.4)
18.0
103.0
()0.1)
104.5
(0.4)
(0.2)
56.6
()8.2)
75.3
()2.1)
71.0
(0.1)
62.1
()0.7)
()0.1)
()0.7)
()0.6)
the synthetic approach used should also permit the
anomeric activation of the repeating unit to obtain
higher oligomers suitable for structure–activity studies.
3.2. Benzyl (2,4-di-O-benzoyl-3-O-chloroacetyl-a-L-
rhamnopyranosyl)-(1 fi 2)-3,4-di-O-benzoyl-a-
pyranoside (7)
L-rhamno-
To a stirred solution of thioglycoside 59 (1.658 g,
3.37 mmol), acceptor 610 (1.208 g, 2.61 mmol) and NIS
(1.665 g, 7.41 mmol) in CH2Cl2 (50 mL) containing
3. Experimental
ꢁ
powdered 4 A molecular sieves, at )20 ꢁC was added
3.1. General methods
TfOH (130 lL, 1.50 mmol) and the mixture was stirred
until TLC showed that the reaction was complete
(90 min). The brown solution was then filtered on Celite;
the filtrate was diluted with CH2Cl2 (150 mL) and then
extracted with Na2S2O3 10% (200 mL), and then with
KHCO3 2 M (200 mL). The organic phase was dried and
concentrated. Silica gel chromatography (14:1 cyclo-
hexane–ethyl acetate) of the residue afforded 7 (1.915 g,
1H and 13C NMR spectra were recorded on a Bruker
DRX-400 NMR (400 MHz) and Bruker Avance-360
NMR (360 MHz) in DMSO-d6 (internal standard, for
1H: (CH3)2SO at d 2.49; for 13C: (CH3)2SO at d 39.5), in
1
CDCl3 (internal standard, for H: CHCl3 at d 7.26; for
13C: CDCl3 at d 77.0) and in D2O (internal standard, for
1H: (CH3)2CO at d 2.22; for 13C: (CH3)2CO at d 31.5,
respectively). Assignment of proton and carbon chemi-
cal shifts were based on DQF-COSY, TOCSY, ROESY,
HSQC and HMQC-COSY experiments. Positive ESI-
MS spectra were recorded on a Finnigan LCQ-DECA
ion trap mass spectrometer. Optical rotations were
measured on a JASCO P-1010 polarimeter. Elementary
analysis were performed on a Carlo Erba 1108 instru-
ment. Analytical thin layer chromatography (TLC) was
performed on aluminium plates precoated with Merck
Silica Gel 60 F254 as the adsorbent. The plates were
developed with 5% H2SO4 ethanolic solution and then
heated to 130 ꢁC. Column chromatography was per-
formed on Kieselgel 60 (63–200 mesh). Gel filtration
chromatography were performed on a Biogel P2 column
(1.0 · 20 cm) with H2O as eluant.
1
82%) as a white foam. ½aꢁ +79.4 (c 0.5, CH2Cl2); H
D
NMR (360 MHz, DMSO-d6): d 7.99–7.30 (m, 25H, 5Ph),
5.686 (dd, J3;4 ¼ 9:8 Hz, J3;2 ¼ 3:0 Hz, 1H, H-3A), 5.672
(br s, 1H, H-2B), 5.606 (dd, J3;4 ¼ 9:9 Hz, J3;2 ¼ 3:3 Hz,
1H, H-3B), 5.441 (t, J4;3 ¼ J4;5 ¼ 9:8 Hz, 1H, H-4A), 5.360
(t, J4;3 ¼ J4;5 ¼ 9:9 Hz, 1H, H-4B), 5.285 (br s, 1H, H-1B),
5.121 (br s, 1H, H-1A), 4.813 (d, Jgem ¼ 11:9 Hz, 1H,
OCH2U), 4.665 (d, Jgem ¼ 11:9 Hz, 1H, OCH2U), 4.407
(br s, 1H, H-2A), 4.236 (d, Jgem ¼ 15:2 Hz, 1H, CH2Cl),
4.11–4.21 (m, 3H, CH2Cl, H-5A, H-5B), 1.261 (d,
J6;5 ¼ 6:2 Hz, 3H, H-6A), 1.131 (d, J6;5 ¼ 6:1 Hz, 3H, H-
6B). 13C NMR (90 MHz, DMSO-d6): d 166.8–164.7 (5C,
5C@O), 137.1–127.9 (C–Ar), 98.1 (1C, C-1A), 97.0 (1C,
1JC;H ¼ 173 Hz, C-1B), 75.8 (1C, C-2A), 71.9 (1C, C-4A),
70.8 (1C, C-4B), 70.4 (1C, C-3B), 70.2 (1C, C-3A), 69.6
(1C, C-2B), 68.8 (1C, OCH2U), 66.8 (1C, C-5B), 66.2 (1C,

E. Bedini et al. / Carbohydrate Research 339 (2004) 393–400
397
1
C-5A), 40.7 (1C, CH2Cl), 17.6 (1C, C-6A), 17.2 (1C, C-
6B). ESI-MS for C49H45ClO14 (m=z): Mr (calcd) 892.2, Mr
(found) 915.1 (M+Na)þ. Anal. calcd: C, 65.88; H, 5.08.
Found: C, 66.01; H, 5.03.
a white foam. ½aꢁ +120.8 (c 1.9, CH2Cl2); H NMR
D
(360 MHz, DMSO-d6): d 8.07–7.33 (m, 35H, 7Ph), 5.676
(dd, J3;4 ¼ 10:0 Hz, J3;2 ¼ 3:0 Hz, 1H, H-3A), 5.662 (br s,
1H, H-2B), 5.441 (t, J4;3 ¼ J4;5 ¼ 9:9 Hz, 1H, H-4A),
5.376 (t, J4;3 ¼ J4;5 ¼ 9:9 Hz, 1H, H-4B), 5.337 (s, 1H, H-
1C), 5.309 (s, 1H, H-1B), 5.274 (dd, J3;4 ¼ 10:1 Hz,
J3;2 ¼ 3:1 Hz, 1H, H-3C), 5.216 (t, J4;5 ¼ J4;3 ¼ 9:9 Hz,
1H, H-4C), 5.077 (br s, 1H, H-2C), 5.054 (s, 1H, H-1A),
4.806 (d, Jgem ¼ 11:9 Hz, 1H, OCH2U), 4.655 (bd, 2H,
OCH2U, H-3B), 4.409 (br s, 1H, H-2A), 4.144 (m, 3H, H-
5A, H-5B, H-5C), d ¼ 4:069 (d, Jgem ¼ 15:1 Hz, 1H,
CH2Cl), d ¼ 3:995 (d, Jgem ¼ 15:1 Hz, 1H, CH2Cl),
d ¼ 1:248 (d, J6;5 ¼ 6:2 Hz, 3H, H-6A), d ¼ 1:131 (t, 6H,
H-6B, H-6C). 13C NMR (90 MHz, DMSO-d6): d 166.4–
164.3 (7C, C@O), 137.0–127.9 (C–Ar), 98.6 (1C, C-1B),
3.3. Benzyl (2,4-di-O-benzoyl-a-L-rhamnopyranosyl)-
(1 fi 2)-3,4-di-O-benzoyl-a- -rhamnopyranoside (8)
L
A solution of 7 (1.860 g, 2,08 mmol) and thiourea
(1.425 g, 18.72 mmol) in absolute EtOH (45 mL) was
stirred at 25 ꢁC until TLC showed that the reaction was
complete (16 h). The solution was then concentrated and
the residue was triturated with CH2Cl2 (150 mL) and
then filtrated. The filtrate was extracted firstly with HCl
1 N (150 mL), then with KHCO3 2 M (150 mL) and fi-
nally with water (150 mL). The organic phase was dried
and concentrated. Silica gel chromatography (11:1 cy-
clohexane–ethyl acetate) of the residue afforded 8
98.1 (1C, C-1C, JC;H ¼ 176:1 Hz), 97.1 (1C, C-1A), 76.5
1
(1C, C-2A), 73.8 (1C, C-3B), 72.9 (1C, C-4B), 72.0 (1C,
C-4A), 71.7 (1C, C-2B), 70.8 (1C, C-4C), 70.5 (1C, C-3A),
69.8 (1C, C-3C), 69.5 (1C, C-2C), 68.8 (1C, OCH2U),
66.8 (2C, C-5B, C-5C), 66.3 (1C, C-5A), 40.6 (1C,
CH2Cl), 17.6 (1C, C-6A), 17.2 (2C, C-6B, C-6C). ESI-MS
for C69H63ClO20 (m=z): Mr (calcd) 1246.4, Mr (found)
1269.3 (M+Na)þ. Anal. calcd: C, 66.42; H, 5.09. Found:
C, 66.54; H, 4.88.
(1.367 g, 80%) as a white foam. ½aꢁ +39 (c 0.3, CH2Cl2);
D
1H NMR (360 MHz, DMSO-d6): d 8.03–7.30 (m, 25H,
5Ph), 5.711 (d, JH;OH ¼ 5:6 Hz, 1H, OH-3B), 5.679 (dd,
J3;4 ¼ 9:9 Hz, J3;2 ¼ 3:0 Hz, 1H, H-3A), 5.525 (br s, 1H,
H-2B), 5.423 (t, J4;3 ¼ J4;5 ¼ 9:9 Hz, 1H, H-4A), 5.184 (t,
J4;3 ¼ J4;5 ¼ 9:8 Hz, 1H, H-4B), 5.130 (br s, 1H, H-1B),
5.033 (br s, 1H, H-1A), 4.807 (d, Jgem ¼ 11:9 Hz, 1H,
OCH2U), 4.659 (d, Jgem ¼ 11:9 Hz, 1H, OCH2U), 4.357
(br s, 1H, H-2A), 4.240 (m, 1H, H-3B), 4.128 (m, 1H, H-
5A), 4.016 (m, 1H, H-5B), 1.255 (d, J6;5 ¼ 6:1 Hz, 3H, H-
6A), 1.067 (d, J6;5 ¼ 6:1 Hz, 3H, H-6B). 13C NMR
(90 MHz, DMSO-d6): d 166.8–164.7 (4C, C@O), 137.0–
128.0 (C–Ar), 98.8 (1C, C-1B), 97.2 (1C, C-1A), 75.8 (1C,
C-2A), 74.2 (1C, C-4B), 72.6 (1C, C-2B), 72.0 (1C, C-4A),
70.6 (1C, C-3A), 68.8 (1C, O–CH2U), 66.9 (1C, C-5B),
66.4 (1C, C-3B), 66.3 (1C, C-5A), 17.7 (1C, C-6A), 17.4
(1C, C-6B). ESI-MS for C47H44O13 (m=z): Mr (calcd)
816.3, Mr (found) 839.3 (M+Na)þ. Anal. calcd: C, 69.11;
H, 5.43. Found: C, 69.00; H, 5.45.
3.5. (2,4-Di-O-benzoyl-3-O-chloroacetyl-a-
-rhamnopyranosyl)-
-rhamnopyranosyl trichloro-
L-rhamnopyr-
anosyl)-(1 fi 3)-(2,4-di-O-benzoyl-a-
L
(1 fi 2)-3,4-di-O-benzoyl-a-
L
acetimidate (2)
To a solution of 9 (730 mg, 0.585 mmol) in CH2Cl2
(50 mL) was added anhydrous FeCl3 (2.2 g, 13.6 mmol)
and the green mixture so obtained was stirred at room
temperature. When TLC (2:1 cyclohexane–ethyl acetate)
showed complete conversion of 9 in a new product
(45 min), CH2Cl2 was added (750 mL) and the solution
was extracted with water (750 mL) and then with
KHCO3 2 M (750 mL). The organic phase was dried and
concentrated. Silica gel chromatography (9:2 cyclo-
hexane–ethyl acetate) of the residue afforded an amor-
phous solid (352 mg, hemiacetal) that was solved in
freshly distilled CH2Cl2 (6 mL). To the solution cooled
at 0 ꢁC were added Cl3CCN (115 lL, 1.15 mmol) and
DBU (14 lL, 0.11 mmol). When TLC (2:1 cyclohexane–
ethyl acetate) showed that the reaction was completed
(80 min), the solution was concentrated at 20 ꢁC. Silica
gel chromatography (12:1 cyclohexane–ethyl acetate) of
the residue afforded 2 (335 mg, 44%) as a white foam.
3.4. Benzyl (2,4-di-O-benzoyl-3-O-chloroacetyl-a-L-
rhamnopyranosyl)-(1 fi 3)-(2,4-di-O-benzoyl-a-
L-
L-rhamno-
rhamnopyranosyl)-(1 fi 2)-3,4-di-O-benzoyl-a-
pyranoside (9)
A
stirred solution of thioglycoside 59 (1.033 g,
2.10 mmol), acceptor 8 (1.318 g, 1.61 mmol) and NIS
(1.039 g, 4.62 mmol) in CH2Cl2 (45 mL) containing
ꢁ
freshly powdered 4 A molecular sieves, was cooled at
)20 ꢁC. TfOH (80 lL, 0.92 mmol) was added and the
mixture was stirred until TLC (3:1 cyclohexane–ethyl
acetate) showed that the reaction was complete (15 min).
The brown solution was then filtered on Celite, the fil-
trate diluted with CH2Cl2 (150 mL) and then extracted
with Na2S2O3 10% (200 mL) and then with KHCO3 2 M
(200 mL). The organic phase was dried and concen-
trated. Silica gel chromatography (25:2 cyclohexane–
ethyl acetate) of the residue afforded 9 (1.921 g, 96%) as
½aꢁ +120.8 (c 1.5, CH2Cl2); 1H NMR (360 MHz,
D
DMSO-d6): d 8.10–7.35 (m, 30H, 6Ph), 6.414 (s, 1H, H-
1A), 5.738 (d, J3;4 ¼ 9:6 Hz, 1H, H-3A), 5.658 (br s, 1H,
H-2B), 5.549 (t, J4;3 ¼ J4;5 ¼ 9:8 Hz, 1H, H-4A), 5.414 (t,
3H, H-4B, H-1C, H-1B), 5.271 (dd, J3;4 ¼ 10:1 Hz,
J3;2 ¼ 2:6 Hz, 1H, H-3C), 5.208 (t, J4;3 ¼ J4;5 ¼ 9:8 Hz,
1H, H-4C), 5.083 (br s, 1H, H-2C), 4.731 (d,
J3;4 ¼ 9:7 Hz, 1H, H-3B), 4.653 (br s, 1H, H-2A), 4.277

398
E. Bedini et al. / Carbohydrate Research 339 (2004) 393–400
(m, 2H, H-5A, H-5B), 4.160 (m, 1H, H-5C), 4.070 (d,
Jgem ¼ 15:1 Hz, 1H, CH2Cl), 3.998 (d, Jgem ¼ 15:1 Hz,
1H, OCH2Cl), 1.283 (t, 6H, H-6A, H-6B), 1.127 (d,
J6;5 ¼ 6:2 Hz, 3H, H-6C). 13C NMR (90 MHz, DMSO-
d6): d 166.4–164.2 (7C, C@O), 157.3 (1C, C@NH),
134.1–128.6 (C–Ar), 98.2 (2C, C-1B, C-1C), 95.0 (1C,
1JC;H ¼ 179 Hz, C-1A), 74.4 (C-2A), 73.9 (C-3B), 72.6 (C-
4B), 71.6 (C-2B), 71.1 (C-4A), 70.7 (C-4C), 70.0 (C-3A),
69.7 (C-3C), 69.3 (C-2C), 68.9 (C-5A), 67.0 (C-5B), 66.8
(C-5C), 40.5 (OCH2Cl), 17.4 (C-6A, C-6B), 17.0 (C-6C).
ESI-MS for C64H57Cl4NO20 (m=z): Mr (calcd) 1299.2, Mr
(found) 1322.3 (M+Na)þ. Anal. calcd: C, 59.04; H, 4.41;
N, 1.08. Found: C, 59.54; H, 4.55; N, 1.07.
mixture was stirred at )50 ꢁC and after 30 min BF3ÆOEt2
(34 lL, 0.271 mmol) was added. When TLC (2:1 cy-
clohexane–ethyl acetate) showed that the reaction was
complete (30 min), the mixture was diluted with CH2Cl2
(10 mL), filtered over Celite, and the filtrate extracted
with NaHCO3 1 M. The organic layer was collected,
dried, and concentrated. The residue was purified by
silica gel chromatography (6:1 petroleum ether–ethyl
acetate) to give tetrasaccharide 11 (237 mg, 79%) as a
white foam: ½aꢁ +88.4 (c 1.0, CH2Cl2); 1H NMR
D
(400 MHz, CDCl3): d 8.20–7.23 (m, 40H, 8Ph), 5.837
(dd, 1H, J3;4 9.7 Hz, J3;2 3.2 Hz, H-3B), 5.76–5.66 (m, 1H,
CH@CH2), 5.717 (br s, 1H, H-2C), 5.552 (t, 1H,
J4;5 ¼ J4;3 9.7 Hz, H-4B), 5.544 (t, 1H, J4;5 ¼ J4;3 9.7 Hz,
H-4C), 5.439 (dd, 1H, J3;4 9.6 Hz, J3;2 3.3 Hz, H-3D),
5.382 (t, 1H, J4;5 ¼ J4;3 9.7 Hz, H-4A), 5.366 (br s, 1H,
H-1B), 5.323 (t, 1H, J4;5 ¼ J4;3 9.6 Hz, H-4D), 5.236 (br s,
1H, H-1C), 5.200 (br s, 1H, H-1D), 5.183 (br s, 1H,
H-2D), 5.108 (dd, 1H, Jvic 17.6 Hz, Jgem 1.6 Hz, CH@CH2
trans), 4.944 (br s, 1H, H-1A), 4.935 (dd, 1H, Jvic 9.9 Hz,
Jgem 1.6 Hz, CH@CH2 cis), 4.793 (d, 1H, Jgem 12.2 Hz,
OCH2Ph), 4.596 (d, 1H, Jgem 12.2 Hz, OCH2Ph), 4.588
(dd, 1H, J3;4 9.7 Hz, J3;2 3.3 Hz, H-3C), 4.500 (br s, 1H,
H-2B), 4.25–4.18 (m, 2H, H-2A, H-5D), 4.17–4.03 (m,
3H, H-5B, H-5C, OCH2CH@CH2), 4.01–3.95 (m, 3H,
H-3A, H-5A, OCH2CH@CH2), 3.730 (d, 1H, Jgem 14.9 Hz,
COCH2Cl), 3.682 (d, 1H, Jgem 14.9 Hz, COCH2Cl),
1.304 (d, 3H, J6;5 6.4 Hz, H-6A), 1.286 (d, 3H, J6;5 6.4 Hz,
H-6C), 1.228 (d, 3H, J6;5 6.4 Hz, H-6B), 1.200 (d, 3H, J6;5
6.4 Hz, H-6D); 13C NMR (100 MHz, CDCl3): d 167.1
(1C, COCH2Cl), 165.3–164.6 (7C, COPh), 134.1 (1C,
CH2CH@CH2), 133.7–127.5 (C–Ar), 117.1 (1C,
CH2CH@CH2), 99.9 (1C, JC;H 173 Hz, C-1B), 98.7 (1C,
C-1C), 98.6 (1C, C-1D, 1JC;H ¼ 173 Hz), 97.8 (1C, C-1A),
77.0 (1C, C-3A), 75.8 (1C, C-2B), 74.6 (1C, C-3C), 74.3
(1C, C-2A), 73.3 (1C, C-4A), 73.2 (1C, C-4C), 71.9 (1C,
C-4B), 71.8 (1C, C-5B), 71.6 (2C, C-2C, C-5A), 71.1 (1C,
C-4D), 70.3 (1C, C-3B), 70.2 (1C, C-3D), 69.7 (1C, C-2D),
68.8 (1C, OCH2Ph), 67.4 (2C, C-5C, C-5D), 67.0 (1C,
CH2CH@CH2), 40.0 (1C, COCH2Cl), 18.7–18.4 (4C,
C-6A, C-6B, C-6C, C-6D). ESI-MS for C85H81ClO25 (m=z):
Mr (calcd) 1536.5, Mr (found) 1559.6 (M+Na)þ. Anal.
calcd: C, 66.38; H, 5.31. Found: C, 66.60; H, 5.27.
3.6. Benzyl 3-O-allyl-4-O-benzoyl-a-L-rhamnopyranoside
(4)
To a solution of 109 (252 mg, 0.703 mmol) in 10:1 ben-
zene–methanol (5 mL), Bu2SnO (215 mg, 0.861 mmol)
was added and the mixture was stirred at reflux for
90 min, until the Bu2SnO was completely dissolved. The
mixture was dried in vacuo, and the white foamy residue
was dissolved in toluene (2.5 mL). Bu4NBr (230 mg,
0.714 mmol) and subsequently AllBr (610 lL, 7.4 mmol)
were added and the solution was stirred at 65 ꢁC. When
the TLC (2:1 cyclohexane–ethyl acetate) showed that all
the starting material 10 was consumed (90 min), the
solution was dried in vacuo. Silica gel chromatography
(6:1 petroleum ether–ethyl acetate) of the residue affor-
ded 4 (237 mg, 85%) as a white foam: ½aꢁ )33.8 (c 0.9,
D
1
CH2Cl2); H NMR (400 MHz, CDCl3): d8.09–7.30 (m,
10 H, 2Ph), 5.82–5.64 (m, 1H, CH@CH2), 5.311 (t, 1H,
J4;5 ¼ J4;3 ¼ 9:7 Hz, H-4), 5.160 (dd, 1H, Jvic ¼ 17:2 Hz,
Jgem ¼ 1:6 Hz, CH@CH2 trans), 5.063 (dd, 1H,
Jvic ¼ 10:2 Hz, Jgem ¼ 1:4 Hz, CH@CH2 cis), 4.987 (br s,
1H, H-1), 4.757 (d, 1H, Jgem ¼ 12:0 Hz, OCH2Ph), 4.556
(d, 1H, Jgem ¼ 12:0 Hz, OCH2Ph), 4.16–3.84 (m, 5H,
H-2, H-3, H-5, OCH2CH@CH2), 1.255 (d, 3H,
J6;5 ¼ 6:2 Hz, H-6); 13C NMR (100 MHz, CDCl3):
d165.7 (1C, COPh), 134.2–128.0 (C–Ar), 133.1 (1C,
CH@CH2), 117.5 (1C, CH@CH2), 98.4 (1C, C-1), 76.8
(1C, C-3), 73.2 (1C, C-4), 71.0 (1C, C-2), 69.3 (1C,
OCH2CH@CH2), 68.8 (1C, OCH2Ph), 66.5 (1C, C-5),
17.4 (1C, C-6). ESI-MS for C23H26O6 (m=z): Mr (calcd)
398.2, Mr (found) 421.0 (M+Na)þ. Anal. calcd: C, 69.33;
H, 6.58. Found: C, 69.55; H, 6.56.
3.8. Benzyl (2,4-di-O-benzoyl-3-O-chloroacetyl-a-L-
rhamnopyranosyl)-(1 fi 3)-(2,4-di-O-benzoyl-a-
rhamnopyranosyl)-(1 fi 2)-(3,4-di-O-benzoyl-a-
rhamnopyranosyl)-(1 fi 2)-4-O-benzoyl-a-
anoside (12)
L
-
L
-
L-rhamnopyr-
3.7. Benzyl (2,4-di-O-benzoyl-3-O-chloroacetyl-a-
L
-
rhamnopyranosyl)-(1 fi 3)-(2,4-di-O-benzoyl-a-
L
-
-
rhamnopyranosyl)-(1 fi 2)-(3,4-di-O-benzoyl-a-
L
rhamnopyranosyl)-(1 fi 2)-3-O-allyl-4-O-benzoyl-a-
rhamnopyranoside (11)
L
-
To
a
solution of tetrasaccharide 11 (115 mg,
0.075 mmol) in 5:2 methanol–dichloromethane, anhy-
drous PdCl2 (1.3 mg, 7.5 lmol) was added and the
mixture was stirred at room temperature until TLC (2:1
cyclohexane–ethyl acetate) indicated (4 h) that the
starting material was consumed. The mixture was fil-
A mixture of imidate 2 (352 mg, 0.271 mmol), acceptor 4
ꢁ
(77 mg, 0.194 mmol) and freshly powdered 4 A molecu-
lar sieves was suspended in absolute CH2Cl2 (9 mL). The

E. Bedini et al. / Carbohydrate Research 339 (2004) 393–400
399
1
tered over Celite diluted with dichloromethane (50 mL)
and extracted with saturated NaCl solution (50 mL).
The organic layer was collected and dried. Silica gel
chromatography (11:2 cyclohexane–ethyl acetate) of the
(c 1.0, CH2Cl2); H NMR (400 MHz, CDCl3): d 8.18–
7.24 (m, 40H, 8Ph), 5.812 (dd, J3;4 ¼ 9:9 Hz,
J3;2 ¼ 3:2 Hz, 1H, H-3B), 5.644 (br s, 1H, H-2C), 5.619
(t, J4;3 ¼ J4;5 ¼ 10:0 Hz, 1H-4C), 5.594 (t, J4;3
¼
residue afforded 12 (105 mg, 94%) as a white foam. ½aꢁ
J4;5 ¼ 9:9 Hz, 1H, H-4B), 5.522 (br s, 1H, H-1C), 5.467
(dd, J3;4 ¼ 9:9 Hz, J3;2 ¼ 3:2 Hz, 1H, H-3D), 5.419 (br s,
1H, H-1B), 5.376 (t, J4;3 ¼ J4;5 ¼ 9:8 Hz, 1H, H-4A),
5.321 (t, J4;3 ¼ J4;5 ¼ 9:8 Hz, 1H, H-4D), 5.211 (br s, 2H,
H-1D, H-2D), 5.114 (t, J3;4 ¼ J3;2 ¼ 9:5 Hz, 1H, H-3E),
4.96–4.88 (m, 2H, H-1A, H-4E), 4.80–4.73 (m, 3H, H-1E,
NH, OCH2Ph), 4.663 (dd, J3;4 ¼ 10:0 Hz, J3;2 ¼ 3:3 Hz,
1H, H-3C), 4.599 (d, 1H, Jgem ¼ 12:0 Hz), 4.585 (br s, 1H,
H-2B), 4.338 (dq, 1H, J5;4 ¼ 10:0 Hz, J5;6 ¼ 6:2 Hz, 1H,
H-5C), 4.31–4.07 (m, 8H, H-2A, H-3A, H-5B, H-5D,
2 · H-6E, 2 · OCH2CCl3), 3.983 (dq, 1H, J5;4 ¼ 9:8 Hz,
J5;6 ¼ 6:2 Hz, 1H, H-5A), 3.745 (d, Jgem ¼ 15:0 Hz, 1H,
COCH2Cl), 3.698 (d, Jgem ¼ 15:0 Hz, 1H, COCH2Cl),
3.654 (m, 1H, H-5E), 3.523 (q, J2;1 ¼ J2;3 ¼ 9:4 Hz, 1H,
H-2E), 2.02–1.92 (3s, 9H, 3 · Ac), 1.457 (d, J6;5 ¼ 6:2 Hz,
1H, H-6D), 1.295 (d, J6;5 ¼ 6:2 Hz, 1H, H-6A), 1.256
(d, J6;5 ¼ 6:2 Hz, 1H, H-6B), 1.148 (d, J6;5 ¼ 6:2 Hz, 1H,
H-6C); 13C NMR (100 MHz, CDCl3): d 167.1 (1C,
COCH2Cl), 165.3–164.6 (8C, COPh, COCH2CCl3),
D
+80.2 (c 1.0, CH2Cl2); 1H NMR (400 MHz, CDCl3):
d 8.16–7.23 (m, 40H, 8Ph), 5.795 (dd, 1H, J3;4 ¼ 9:7 Hz,
J3;2 ¼ 3:3 Hz, H-3B), 5.697 (br s, 1H, H-2C), 5.569 (t, 1H,
J4;3 ¼ J4;5 ¼ 9:7 Hz, H-4B), 5.556 (t, 1H, J4;3 ¼ J4;5
¼
9:8 Hz, H-4C), 5.437 (dd, 1H, J3;4 ¼ 9:9 Hz, J3;2 ¼ 3:2 Hz,
H-3D), 5.325 (t, 1H, J4;3 ¼ J4;5 ¼ 9:9 Hz, H-4D), 5.279
(br s, 1H, H-1B), 5.215 (br s, 1H, H-1C), 5.197 (br s, 1H,
H-1D), 5.185 (br s, 1H, H-2D), 5.138 (t, 1H,
J4;3 ¼ J4;5 ¼ 9:9 Hz, H-4A), 5.003 (br s, 1H, H-1A),
4.789 (d, 1H, Jgem ¼ 12:2 Hz, OCH2Ph), 4.593 (d, 1H,
Jgem ¼ 12:2 Hz),
4.552
(dd,
1H,
J3;4 ¼ 9:8 Hz,
J3;2 ¼ 3:3 Hz, H-3C), 4.493 (br s, 1H, H-2B), 4.23–4.12
(m, 4H, H-5A, H-5B, H-5C, H-5D), 4.115 (br s, 1H,
H-2A), 4.052 (dd, J3;4 ¼ 9:6 Hz, J3;2 ¼ 3:2 Hz, H-3A),
3.728 (d, 1H, Jgem ¼ 14:6 Hz, COCH2Cl), 3.687 (d, 1H,
Jgem ¼ 14:6 Hz, COCH2Cl), 1.348 (d, 3H, J6;5 ¼ 6:2 Hz,
H-4A), 1.313 (d, 3H, J6;5 ¼ 6:2 Hz, H-4C), 1.204 (d, 6H,
J6;5 ¼ 6:2 Hz, H-4B, H-4D); 13C NMR (100 MHz,
CDCl3): d 167.1 (1C, COCH2Cl), 165.3–164.6 (7C,
COPh), 133.7–127.5 (C–Ar), 101.3 (C-1B), 98.9 (2C, C-
1C, C-1D), 97.4 (1C, C-1A), 78.7 (1C, C-2A), 75.9 (1C,
C-2B), 75.6 (1C, C-4A), 74.6 (1C, C-3C), 72.7 (1C, C-4C),
71.5 (1C, C-4B), 71.4 (1C, C-4C), 71.1 (1C, C-4D), 70.4
(1C, C-3B), 70.3 (1C, C-3D), 70.1 (2C, C-5A, C-5D), 69.5
(1C, C-2D), 69.0 (1C, OCH2Ph), 67.4 (2C, C-5B, C-5C),
66.2 (1C, C-3A), 40.0 (1C, COCH2Cl), 18.8 (1C, C-6A),
18.6 (1C, C-6C), 18.5 (2C, C-6B, C-6D). ESI-MS for
C85H81ClO25 (m=z): Mr (calcd) 1496.4, Mr (found) 1519.3
(M+Na)þ. Anal. calcd: C, 65.75; H, 5.18. Found: C,
65.90; H, 5.18.
133.8–127.5 (C–Ar), 101.8 (C-1E, JC;H ¼ 164 Hz), 100.5
1
(C-1B), 99.3 (C-1C), 99.0 (C-1D), 98.1 (C-1A), 95.3
(OCH2CCl3), 78.5 (C-2B), 78.1 (C-2A), 77.0 (C-3A), 74.7
(C-3C), 73.8 (OCH2CCl3), 73.3 (C-4A, C-4C), 72.2 (C-
2C), 72.0 (C-4B), 71.6 (C-5E), 71.5 (C-4D), 71.0 (C-3E),
70.5 (C-3D), 70.4 (C-3B), 69.9 (C-2D), 69.3 (OCH2Ph),
69.0 (C-4E), 67.6 (C-5C), 67.3 (C-5D), 67.2 (C-5B), 66.9
(C-5A), 62.3 (C-6E), 56.1 (C-2E), 40.2 (COCH2Cl), 21.0
(Ac), 18.1 (C-6C), 17.5 (C-6A, C-6B), 17.3 (C-6D). ESI-
MS for C85H81ClO25 (m=z): Mr (calcd) 1957.5, Mr
(found) 1958.65 (M+H)þ. Anal. calcd: C, 59.42; H, 4.88;
N, 0.71. Found: C, 59.49; H, 4.80; N, 0.70.
3.9. Benzyl (2,4-di-O-benzoyl-3-O-chloroacetyl-a-
L
-
3.10. Benzyl a-
pyranosyl-(1 fi 2)-a-
oxy-2-acetamido-b-
rhamnopyranoside (1)
L
-rhamnopyranosyl-(1 fi 3)-a-
-rhamnopyranosyl)-(1 fi 2)-[2-de-
-glucopyranosyl-(1 fi 3)]-a-
L-rhamno-
rhamnopyranosyl)-(1 fi 3)-(2,4-di-O-benzoyl-a-
rhamnopyranosyl)-(1 fi 2)-(3,4-di-O-benzoyl-a-
L
-
-
L
L
D
L-
rhamnopyranosyl)-(1 fi 2)-[3,4,6-tri-O-acetyl-2-deoxy-2-
(2,2,2-trichloroethoxycarbonylamino)-b- -glucopyrano-
syl-(1 fi 3)]-4-O-benzoyl-a- -rhamnopyranoside (13)
D
L
To a solution of 13 (39 mg, 0.020 mmol) in THF
(4.0 mL) was added 2 M aqueous KOH (400 lL) and the
mixture was stirred at 40 ꢁC. After 19 h the TLC (5:1
isopropanol–water) showed that the reaction was com-
plete. The mixture was diluted with MeOH (10 mL),
neutralized with Amberlyst-15 Hþ, filtered and dried.
The residue was dissolved in methanol (3.0 mL) and
Ac2O (300 lL) was added. After 2 h the solution was
dried to obtain and the residue was purified by gel fil-
tration on a P-2 (Biorad) column using water as eluant,
A suspension of acceptor 12 (100 mg, 0.067 mmol), im-
8
ꢁ
idate 3 (81 mg, 0.134 mmol) and freshly powdered 4 A
molecular sieves in absolute dichloromethane (2 mL)
was stirred at )10 ꢁC. After 30 min TMSOTf (0.47 lL,
2.7 lmol) was added and the mixture was kept at )10 ꢁC
until TLC (2:1 cyclohexane–ethyl acetate) showed that 3
was completely consumed (3 h). The reaction was
quenched with Et3N (18 lL), the mixture was filtered
over Celite, extracted with water, and dried. Silica gel
chromatography (9:2 cyclohexane–ethyl acetate) gave
two fractions, one affording unreacted acceptor 12
(52 mg, 51%), the other contained the desired penta-
to obtain 1 (10 mg, 56%) as a white foamy solid: ½aꢁ
D
1
)45 (c 0.5, H2O); H NMR (400 MHz, D2O) (see also
Table 1): d 7.47–7.42 (m, 5H, Ph), 4.775 (d, 1H,
Jgem ¼ 12:0 Hz, OCH2Ph), 4.629 (d, 1H, Jgem ¼ 12:0 Hz,
OCH2Ph), 2.05 (s, 3H, Ac); 13C NMR (100 MHz, D2O)
saccharide 13 (59 mg, 45%) as a white foam: ½aꢁ +53.0
D

400
E. Bedini et al. / Carbohydrate Research 339 (2004) 393–400
3. Brade, H.; Opal, S. M.; Vogel, S. N.; Morrison, D. C.
Endotoxin in Health and Disease; Marcel Dekker: New
York, 1999.
4. Mulard, L. A.; Clement, M.-J.; Imberty, A.; Delepierre,
M. Eur. J. Org. Chem. 2002, 2486–2498.
(see also Table 1): d 129.3 (Ar), 70.4 (OCH2Ph), 21.1
(Ac). ESI-MS for C39H61NO22 (m=z): Mr (calcd) 895.4,
Mr (found) 918.4 (M+Na)þ. Anal. calcd: C, 52.28; H,
6.86; N, 1.56. Found: C, 52.45; H, 6.80; N, 1.55.
€€
5. Hoog, C.; Rotondo, A.; Johnston, B. D.; Pinto, M.
Carbohydr. Res. 2002, 337, 2023–2036.
6. Ovod, V. V.; Zdorovenko, E. L.; Shashkov, A. S.;
Kocharova, N. A.; Knirel, Y. A. Eur. J. Biochem. 2000,
267, 2372–2379.
7. Young, J. M.; Saddler, G. S.; Takikawa, Y.; De Boer, S.
H.; Vauterin, L.; Gardan, L.; Gvozdyak, R. I.; Stead, D.
E. Rev. Plant Pathol. 1996, 75, 721–763.
8. Zhang, J.; Kong, F. Tetrahedron 2003, 1429–1441.
9. Dullenkopf, W.; Castro-Palomino, J. C.; Manzoni, L.;
Schmidt, R. R. Carbohydr. Res. 1996, 296, 35–147.
10. Bedini, E.; Parrilli, M.; Unverzagt, C. Tetrahedron Lett.
2002, 43, 8879–8882.
Acknowledgements
We thank Dr. Vincenzo Piscopo from Centro di Met-
odologie Chmico-Fisiche of the University Federico II
of Naples for the NMR spectra, and MIUR, Rome,
(Progetti di Ricerca di Interesse Nazionale 2002 M.P.)
for the financial support.
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