A new route for the synthesis of Streptococcus pneumoniae 19F and 19A capsular polysaccharide fragments avoiding the β-mannosamine glycosylation step

Article abstract of DOI:10.1016/j.carres.2009.04.012

The recently described [Attolino, E.; Bonaccorsi, F.; Catelani, G.; D'Andrea, F. Carbohydr. Res. 2008, 343, 2545-2556.] β-d-MaNAcp-(1→4)-β-d-Glcp thiophenyl glycosyl donor 3 was used in α-glycosylation reactions of OH-2 and OH-3 of the suitably protected

Full text of DOI:10.1016/j.carres.2009.04.012

Carbohydrate Research 344 (2009) 1442–1448
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
A new route for the synthesis of Streptococcus pneumoniae 19F and 19A
capsular polysaccharide fragments avoiding the b-mannosamine
glycosylation step q
b,
Filippo Bonaccorsi a, Giorgio Catelani a, , Stefan Oscarson
*
*
a Dipartimento di Chimica Bioorganica e Biofarmacia, Università di Pisa, Via Bonanno 33, I-56126 Pisa, Italy
b Centre for Synthesis and Chemical Biology, UCD School of Chemistry and Chemical Biology, University College Dublin, Belfield, Dublin 4, Ireland
a r t i c l e i n f o
a b s t r a c t
Article history:
The recently described [Attolino, E.; Bonaccorsi, F.; Catelani, G.; D’Andrea, F. Carbohydr. Res. 2008, 343,
Received 23 February 2009
Received in revised form 18 March 2009
Accepted 6 April 2009
2545–2556.] b-
tions of OH-2 and OH-3 of the suitably protected p-MeO-benzyl
Glycosylation of the axial OH-2 of 7 took place in high yield (76%) and with acceptable stereoselectivity
/b = 3.4) leading to the protected trisaccharide -11, corresponding to the repeating unit of Streptococ-
cus pneumoniae 19F. The same reaction on equatorial OH-3 of acceptor 8 gave the trisaccharide -15, a
constituent of the repeating unit of S. pneumoniae 19A, but in lower yield (41%) and without stereoselec-
tion (
/b = 1:1.3). Utilizing the introduced orthogonal protection of OH-1 and OH-400, the trisaccharide
D
-MaNAcp-(1?4)-b-
D
-Glcp thiophenyl glycosyl donor 3 was used in
a-glycosylation reac-
a-L
-rhamnopyranoside acceptors 7 and 8.
Available online 17 April 2009
(
a
a
a
Dedicated to Professor Hans Kamerling on
the occasion of his 65th birthday
a
a-
11 was transformed into a trisaccharide building block suitable for the synthesis of its phosphorylated
oligomers.
Keywords:
Glycoconjugate vaccines
Thioglycosides
Ó 2009 Elsevier Ltd. All rights reserved.
Oligosaccharide synthesis
1. Introduction
of biological contaminants.3,4 Streptococcus pneumoniae serotypes
19F (SP 19F) and 19A (SP 19A) are responsible for a large num-
Encapsulated bacteria present an external carbohydrate coat
for protection against the host’s immune system and osmotic
lysis, that is, in the pathogenic strains, responsible for their vir-
ulence. The capsular polysaccharide (CPS) structure defines the
serotype, and since the discovery that CPS fragments induce a
response of the host’s adaptive immune system, research has
been focused on developing CPS-based vaccines as an alternative
to antibiotic therapy.2 The first attempts to create such vaccines
were made by administration of CPS fragments obtained by puri-
fication after controlled lysis of the capsule, and there are still
commercial multivalent vaccines produced by this method. More
recently, there has been a development of most efficient glyco-
conjugate vaccines, where the CPS is conjugated to a carrier pro-
tein to allow a T-cell-dependent immune response. Also, large
efforts have been made to develop synthetic vaccines portrayed
by well-defined molecular structures and the complete absence
ber of infections of the upper respiratory system and meningitis,
especially in children and immunodeficient subjects. These infec-
tions are associated with a large number of deaths (1.2 million/
year just in developing countries). The repeating units5 of SP 19F
and SP 19A CPS (Fig. 1) are both made up of a trisaccharide
containing an N-acetyl-
a b-1?4 bond to a -glucose (B) residue that is linked to an
-rhamnose unit (C) through an -1?2 (SP 19F) or an -1?3
bond (SP 19A). The repeating units are linked to each other via
an -1?4 phosphodiester bridge (Fig. 1).
Since the elucidation of these structures, chemists have been
D-mannosamine unit (A) linked through
D
L
a
a
a
involved with their synthesis, especially with that of SP 19F. The
reported synthetic approaches involve, in all cases, two different
glycosidation reactions, and can be classified into two groups:
the first is based on the initial synthesis of the A–B fragment
and the successive coupling with the unit C,6 while the second
approach involves the coupling of unit A with the B–C frag-
ment.7 The most challenging task is the introduction of the
q
N-acetyl-b-D-mannosamine linkage, because of the difficulties
Part 27 of the series ‘Chemical Valourisation of Milk-Derived Carbohydrates’. For
part 26, see Ref. 1.
in stereoselective formation of b-
sides by direct glycosidation with
D
-mannosamine glycopyrano-
D
-mannosamine donors.8 The
* Corresponding authors. Tel.: +39 0502219700; fax: +39 0502219660 (G.C.); tel.:
+35 317162318; fax: +35 317162501 (S.O.).
E-mail addresses: giocate@farm.unipi.it (G. Catelani), stefan.oscarson@ucd.ie (S.
Oscarson).
strategy most employed is based on the initial formation of a
b- -glucopyranoside residue, followed by its transformation into
D
0008-6215/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2009.04.012

F. Bonaccorsi et al. / Carbohydrate Research 344 (2009) 1442–1448
1443
-
-
OPO3
OPO3
O
O
C
C
HO
HO
HO
HO
OH
HO
HO
O
OH
O
OH
NHAc
O
NHAc
O
HO
O
O
A
A
O
O
HO
HO
O
OH
O
OH
B
B
1
2
Figure 1. SP 19F (1) and SP 19A (2) CPS repeating units.
a b-D-mannosamine moiety by amination with inversion of con-
2. Results and discussion
figuration at C-20 through an oxidation–oximation, followed by
reduction of the oximino derivative6a or through an SN2 dis-
placement with sodium azide on a 2-O-sulfonyl intermediate
followed by reduction.7b,6d Other methods described are direct
Because of the poor donor properties experienced with
compounds 3 and 4,10 we initially thought that the best way to
obtain the CPS trisaccharide repeating units of SP 19F and 19A
was to glycosidate acceptors 711 and 812 with donor 5 and subse-
glycosidations with 2-azido-2-deoxy-D-mannopyranose donors,
either the glycosyl bromide activated by silver silicate7a,6b or
with a C-2 oximino glycosyl donor, followed by stereoselective
reduction.6c Still, most of these methods suffer from problems
related to low reaction yields and stereoselectivity reducing the
efficiency of the syntheses.
quently convert the
D-galactopyranoside units in the trisaccharides
so obtained into -mannosamine residues (Scheme 2).
D
Thus, acceptors 7 and 8 were coupled with donor 5 using MeO-
Tf13 (5 equiv) as promoter in 4:1 CH2Cl2–Et2O leading to anomeric
mixtures of 9 (75%,
(Scheme 2). In both cases, the mixtures obtained were easily sepa-
rated by chromatographic means, affording pure samples of -9
and -10, in 50% and 49% isolated yields, respectively. Successful
application of the sequence previously optimized for the analogous
isopropyl
-disaccharide10 to the 2-O-allyl-3-O-benzyl-4,6-O-ben-
zylidene-b- -galactopyranosyl unit of -9 and -10 would afford
a/b 2:1) and 10 (78%, a/b 1.7:1), respectively
A recently reported method9 for the synthesis of b-
minosides and b- -mannosides is based on the completely stereo-
selective elaboration in positions 2 (amination with inversion) and
D-mannosa-
D
a
a
4 (epimerization) of b-
possibility for obtaining
D
-galactopyranosides. This suggested the
-glycosides of the b- -ManNAcp-(1?4)-
a
D
a
D
D
-Glcp of type 6 from lactose by converting its nonreducing end
a
a
into the N-acetyl-D-mannosamine moiety. To this end a systematic
the protected trisaccharide repeating units of SP 19F and SP 19A
CPS. However, realizing the rather long sequence required for the
above procedure (20% overall yield over 12 steps in the reported
case10), we reconsidered the possibility of employing the mannosa-
mine disaccharide 3 as a donor to glycosylate the two rhamnoside
acceptors 7 and 8. Taking into account the completely negative re-
sults obtained in the preliminary study using NIS–TfOH or MeOTf
as promoter,10 we decided to explore the glycosidation properties
of 3 with other activating systems. Hence, glycosidation reactions
between donor 3 and rhamnoside acceptor 7 (Scheme 3) were
carried out with the most widely used activating systems of
thioglycoside donors,14 but, as in the case of the previously tried
NIS–TfOH and MeOTf, also NIS–TMSOTf, PhIO–TMSOTf, MeOTf–
investigation10 (Scheme 1) has been performed on the glycosida-
tion properties of three different disaccharide thiophenyl glycosyl
donors obtained from lactose, each carrying at the nonreducing
end a
D-mannosamine (3), a D-talosamine (4) or a D-galactopyra-
nose (5) unit, with a simple alcoholic acceptor.
Using NIS/TfOH or MeOTf as activators, donor 3 gave no glyco-
side product, whereas donor 4 afforded the desired glycosides but
only in a low yield and without any
a-stereoselectivity. The best
results were obtained with donor 5. As a continuation of these
results, we herein present an investigation for obtaining the trisac-
charides of the repeating units of SP 19 F and 19A from lactose
avoiding the b-mannosaminylation step.
BnO
NHAc
OBn
O
BnO
O
AllO
BnO
SPh
O
OBn
3
OBn
OR
BnO
NHAc
AllO
BnO
NHAc
O
OH
BnO
HO
HO
OBn
O
OH
O
O
BnO
O
BnO
HO
HO
BnO
SPh
O
O
O
OH
O
OBn
OH
OBn
OH
6
Lactose
4
Ph
O
O
OBn
SPh
O
BnO
O
BnO
O
OAll
OBn
5
Scheme 1. Complementary approaches to b-D-ManNAcp-(1?4)-a-D-Glcp glycosides from lactose.

1444
F. Bonaccorsi et al. / Carbohydrate Research 344 (2009) 1442–1448
OPMB
O
BnO
OPMB
Ph
O
BnO
O
O
O
BnO
OBn
BnO
O
BnO
O
1
BnO
OH
BnO
O
OBn
7
Ph
OAll
a
α-9 + β-9
O
O
O
BnO
O
SPh
BnO
O
a
OAll
OBn
OPMB
5
Ph
O
O
BnO
BnO
OPMB
O
O
OBn
O
O
BnO
BnO
O
2
BnO
O
HO
OBn
OAll
8
OBn
α-10 + β-10
Scheme 2. Glycosylation of acceptors 7 and 8 with the disaccharide donor 5. Reagents and conditions: (a) MeOTf, 4:1 CH2Cl2–Et2O, 0 °C, 30 min.
OPMB
OPMB
O
O
BnO
SPh
BnO
BnO
BnO
BnO
NHAc
O
BnO
OBn
O
OH
BnO
O
BnO
O
NHAc
O
7
AllO
BnO
BnO
AllO
BnO
O
O
OBn
3
OBn
α-11 + β-11
Scheme 3. Glycosidation of acceptor 7 with donor 3. Reagents and conditions:see Table 1.
collidine or DMTST gave disappointing results. Again no product
could be isolated, and only retrieved starting materials and decom-
position products were obtained from workup of the reaction
mixture.
However, an explorative reaction with NIS–AgOTf in CH2Cl2 car-
ried out without molecular sieves and temperature control gave a
the activating system at ꢀ35 °C and keeping the reaction for a longer
time (1.3 h), resulting in a total yield of 76% with a stereoselectivity
ratio of 3.4:1, which is equivalent to a 58% yield of the
(entry 4). Efforts were made to increase the /b ratio using a mixture
of CH2Cl2 and an
-directing15 ethereal solvent. However, using the
a anomer
a
a
suggested16 1:1 (v/v) mixture of CH2Cl2–Et2O (entry 5), the reaction
was slower (2 h), needing a triple amount of AgOTf compared to that
used for the reaction carried out in just CH2Cl2, and, surprisingly,
resulted in a complete loss of stereoselectivity. A preparative reac-
tion was performed using the conditions of entry 4, Table 1, and,
mostsatisfactorily, theresultswerereproducible, andthetargetpro-
mixture of trisaccharides
a-11 and b-11, although in minute
amounts. Still, encouraged by this result, we started a systematic
study on the reaction conditions to optimize the yield of the desired
a
anomer. Relevant results of this study are reported in Table 1. Run-
ning the reaction at ꢀ15 to ꢀ8 °C (entry 1) gave a 34% yield of prod-
uct with a 6.5 /b ratio, and an even lower temperature and using
two equivalents of acceptor resulted in an increased yield but with
lower stereoselectivity (52%, 4.6 /b ratio, entry 2). The nonreacted
a
tected trisaccharide
flash chromatographic purification.
The trisaccharide -11, carrying two orthogonal protecting
a-11 was obtained in 59% yield after a simple
a
a
excess of the acceptor is easily retrieved from the reaction mixture
by simple flash chromatography. Changing the donor molar concen-
tration (entry 3) decreased the yield to 31% without any change in
stereoselectivity (4.9). The best results were obtained when adding
groups on C-1 and C-400, is designed to be suitable for the synthesis
of oligomers of the SP 19F repeating unit. A successful synthesis of
hexa- and nonasaccharide phosphorylated fragments of SP 19F CPS
has already been described by Nilsson and Norberg6d using the key
Table 1
Glycosidation of acceptor 7 with disaccharide glycosyl donor 3a
Entry
[3]/[7]
Activating system (equiv)
T (°C)
t (h)
Solvent (v/v)
Isolated yield (%)
a/b
1b
1
NIS (1.2)/AgOTf (0.5)
NIS (1.4) AgOTf (0.55)
NIS (1.5) AgOTf (0.5)
NIS (1.5) AgOTf (0.5)
NIS (1.5) AgOTf (1.67)
ꢀ15?ꢀ8
0.5
1
1
1.3
2
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
34
52
31
76
73
6.5
4.6
4.9
3.4
1
2b
0.47
0.52
0.52
0.44
ꢀ40?ꢀ15
ꢀ35?ꢀ12
ꢀ35?ꢀ10
ꢀ35?ꢀ10
3c
4b
5b,d
CH2Cl2–Et2O (1:1)
a
All the reactions were conducted in the presence of 4 Å MS (about 150 mg per 0.1 mmol of 3).
[Donor] = 0.028 M.
[Donor] = 0.056 M.
Reaction starts at ꢀ15 °C.
b
c
d

F. Bonaccorsi et al. / Carbohydrate Research 344 (2009) 1442–1448
1445
OPMB
O
OPMB
O
BnO
BnO
BnO
BnO
BnO
BnO
O
O
BnO
HO
BnO
AllO
NHAc
O
NHAc
O
a
BnO
O
BnO
O
O
BnO
O
BnO
OBn
OBn
12
α-11
b
OPMB
OH
O
O
BnO
BnO
BnO
BnO
BnO
BnO
O
O
BnO
AcO
BnO
AcO
NHAc
O
NHAc
O
c
BnO
O
BnO
O
O
BnO
O
BnO
OBn
OBn
14
13
Scheme 4. Synthesis of the key intermediate for the preparation of the phosphorylated oligomers of the SP 19F CPS repeating unit. Reagents and conditions: (a) PdCl2, 1:1
MeOH–EtOH, room temperature (94%); (b) 1:2 Ac2O–pyridine, room temperature, 16 h (92%); (c) DDQ, 9:1 CH3CN–H2O, room temperature, 30 h (83%).
intermediate 14, which
a
-11 is easily transformed into (Scheme 4).
ing block 14 for the synthesis of phosphorylated oligomers of the
CPS repeating unit of SP 19F.
Exchange of the allyl protecting group at O-400 to an acetyl group
was accomplished by deprotection with PdCl2 in EtOH–MeOH,17
to obtain alcohol 12, followed by acetylation to give 13 (90% yield
over two steps). Removal of the p-methoxybenzyl protection group
with DDQ in CH3CN–H2O then afforded 14 in 85% yield.
3. Experimental
3.1. General methods
In light of these positive results, the synthesis of the trisaccharide
repeating unit of SP 19A CPS was also attempted by submitting the
rhamnosyl acceptor 8 to a glycosylation with disaccharide donor 3
Melting points were determined with a Kofler hot-stage appara-
tus and are uncorrected. Optical rotations were measured on a
Perkin–Elmer 241 polarimeter at 20 2 °C. NMR spectra were
under the same conditions employed for the preparation of
a-11
(Scheme 5). However, in this case the reaction outcome was less sat-
isfactory both in terms of chemical yield (41%) and stereoselectivity
recorded with
a
Bruker Avance II250 (250.15 MHz for 1H,
63.0 MHz for 13C, respectively) and with a Varian INOVA 500
(499 MHz for 1H, 125 MHz for 13C) using Me4Si as internal reference.
Assignments were made, when possible, with the aid of DEPT, HET-
COR and COSY experiments, and by comparison of values for known
compounds. In the case of mixtures, assignments were made by
referring to the differences in peak intensities. HRMS were deter-
mined with an LCT (Liquid Chromatography Time-of-flight) mass
spectrometer (Waters Ltd, Micromass MS Technology Centre, Man-
chester, UK). All reactions were monitored by TLC on Kieselgel 60
F254, with detection by UV light and/or with ethanolic 10% phospho-
molybdic or sulfuric acid, and heating. Kieselgel 60 (E. Merck, 70–
230 and 230–400 mesh, respectively) was used for column and flash
chromatography. Solvents were dried and purified by distillation
according to standard procedure18 and stored over 4 Å molecular
sieves activated for at least 24 h at 200 °C. MgSO4 was used as the
(a/b 1:1.3). After chromatography, the pure trisaccharide a-15 was
isolated, although in a modest 18% yield. This result is rather surpris-
ing considering the assumed greater reactivity of the equatorial OH-
3 of 8 with respect to that of the axial OH-2 of 7.
In conclusion, a new and effective strategy for obtaining a
protected derivative of the b-
-Rhap trisaccharide has been developed, using the disaccharide
thiophenyl glycosyl donor under appropriate activating
D-ManNAcp-(1?4)-a-D-Glcp-(1?2)-
a-L
3
conditions. The major novelty of this method, with respect to the
previously reported ones,6,7 is that it avoids the difficult b-manno-
samine glycosylation step exploiting the pre-formed b-interglycos-
idic bond naturally present in lactose. Furthermore, an easy
orthogonalization of protecting groups on key positions of the
disaccharide donor 3 and the rhamnoside acceptor 7 has been
achieved, leading to the previously reported6d trisaccharide build-
OPMB
OPMB
O
BnO
BnO
O
BnO
SPh
BnO
O
BnO
NHAc
O
OBn
NHAc
O
OBn
HO
OBn
BnO
8
BnO
AllO
BnO
AllO
BnO
O
O
O
O
a
3
OBn
OBn
α-15 + β-15
Scheme 5. Glycosidation of acceptor 8 with donor 3. Reagents and conditions: (a) NIS–AgOTf, CH2Cl2, 4 Å MS, ꢀ35 to 10 °C (1:1.3
a/b ratio, 43% combined yield).

1446
F. Bonaccorsi et al. / Carbohydrate Research 344 (2009) 1442–1448
drying agent for solutions. Donors 310 and 510 and acceptors 711 and
12 were prepared according to literature procedures.
3.3. p-Methoxybenzyl (2-O-allyl-3-O-benzyl-4,6-O-benzylidene-
b- -galactopyranosyl)-(1?4)-(2,3,6-tri-O-benzyl-
glucopyranosyl)-(1?3)-2,4-di-O-benzyl- -rhamnopyranoside
-10) and p-methoxybenzyl (2-O-allyl-3-O-benzyl-4,6-O-
benzylidene-b- -galactopyranosyl)-(1?4)-(2,3,6-tri-O-benzyl-b-
-glucopyranosyl)-(1?3)-2,4-di-O-benzyl-
rhamnopyranoside (b-10)
8
D
a-D-
a
-L
3.2. p-Methoxybenzyl (2-O-allyl-3-O-benzyl-4,6-O-benzylidene-
b- -galactopyranosyl)-(1?4)-(2,3,6-tri-O-benzyl-
glucopyranosyl)-(1?2)-3,4-di-O-benzyl- -rhamnopyranoside
-9) and p-methoxybenzyl (2-O-allyl-3-O-benzyl-4,6-O-
benzylidene-b- -galactopyranosyl)-(1?4)-(2,3,6-tri-O-benzyl-b-
-glucopyranosyl)-(1?2)-3,4-di-O-benzyl-
rhamnopyranoside (b-9)
(a
D
a
-D
-
D
a-
L
D
a-L-
(a
D
D
a
-L
-
A mixture of 5 (498 mg, 0.537 mmol), 8 (300 mg, 0.645 mmol)
and 4 Å molecular sieves (1.500 g) in 4:1 (v/v) CH2Cl2–Et2O
(15 mL) was stirred for 30 min at room temperature, then cooled
A mixture of 5 (150 mg, 0.162 mmol), 7 (91 mg, 0.194 mmol)
and 4 Å molecular sieves (500 mg) in 4:1 (v/v) CH2Cl2–Et2O
(5 mL) was stirred for 30 min at room temperature. The suspension
to 0 °C, and MeOTf (267 lL, 2.43 mmol) was added. The reaction
mixture was allowed to slowly attain room temp with stirring over-
night, and it was then cooled again to 0 °C, and Et3N (5 mL) was
added, followed by stirring for 30 min. After filtration of the mixture
through a short pad of Celite and concentration under reduced pres-
sure, the residue was purified by silica gel flash chromatography
(13:7 n-hexane–EtOAc) to give two fractions. The first fraction con-
sisted of b-10 and unreacted 8 (396 mg total mass), and the second
was then cooled to 0 °C, and MeOTf (89 lL, 0.81 mmol) was added.
The reaction mixture was allowed to slowly attain room tempera-
ture with stirring overnight, then it was again cooled to 0 °C, and
Et3N (2 mL) was added. After 30 min the mixture was filtered
through a short pad of Celite and concentrated. The residue was
purified by silica gel flash chromatography (49:1 CH2Cl2–Me2CO)
one was pure a-10 (333 mg, 49%). The first fraction was subjected
to give
a
-9 (103 mg, 50%) and b-9 (52 mg, 25%).
to acetylation by treatment with a 1:2 Ac2O–pyridine mixture
(18 mL). After 17 h the solution was repeatedly co-evaporated with
toluene and then purified by silica gel flash chromatography (7:3 n-
hexane–EtOAc) to give b-10 (195 mg, 29%).
Data for
a
-9: Colourless syrup; [
a]
D +43.5 (c 1.1, CHCl3); 1H NMR
(250 MHz, CD3CN): d 7.59–7.16 (m, 37H, Ar-H), 6.92 (m, 2H, Ar-H),
5.78 (ddt, 1H, J 5.4, Jcis 10.5, Jtrans 17.3, @CH), 5.55 (s, 1H, PhCH),
5.16 (dq, 1H J 1.7 Hz, Jtrans 17.3 Hz, @CH2), 5.16–4.71 (AB system,
Data for a-10: Colourless syrup; [a]D +19.1 (c 1.1, CHCl3); Rf 0.24
2H, JA,B 10.9 Hz PhCH2), 5.00 (d, 1H, J1 ,2 3.4 Hz, H-10), 4.93 (dq, 1H,
J1.3, Jtrans 10.5, @CH2), 4.88 (d, 1H, J1,2 1.9 Hz, H-1), 4.87–4.70(ABsys-
tem, 2H, JA,B 11.1 Hz, PhCH2), 4.74–4.55 (AB system, 2H, JA,B 12.4 Hz,
PhCH2), 4.71–4.64 (m, 4H, 2 ꢁ CH2), 4.49–4.31 (AB system, 2H, JA,B
(13:7 n-hexane–EtOAc); 1H NMR (250 MHz, CD3CN): d7.59–7.16 (m,
0
0
37H, Ar-H), 6.89(m, 2H, Ar-H), 5.744 (ddt, 1H, J5.3, Jcis 10.3, Jtrans 17.4,
0
0
0
@CH), 5.54 (s, 1H, PhCH), 5.24 (d, 1H, J1 ,2 3.5 Hz, H-1 ), 5.17–4.83 (AB
system, 2H, JA,B 11.4 Hz PhCH2), 5.12 (dq, 1H J 1.6 Hz, Jtrans 17.5 Hz,
@CH2), 4.99 (dq, 1H, J 1.4, Jtrans 10.5, @CH2), 4.93–4.58 (AB system,
2H, JA,B 11.2 Hz, PhCH2), 4.89 (d, 1H, J1,2 1.8 Hz, H-1), 4.69–4.53 (AB
system, 2H, JA,B 11.5 Hz, PhCH2), 4.68–4.47 (AB system, 2H, JA,B
12.2 Hz, PhCH2), 4.67–4.59 (AB system, 2H, JA,B 12.0 Hz, PhCH2),
4.63–4.56 (AB system, 2H, JA,B 11.5 Hz, PhCH2), 4.49–4.37 (AB sys-
11.8 Hz, PhCH2), 4.40 (d, 1H, J1 ,2 7.6 Hz, H-100), 4.22 (m, 1H, H-400),
00 00
4.21–4.13 (m, 3H, CH2O and H-50), 4.06 (dd, 1H, J2,3 3.0 Hz, H-2),
4.03 (m, 1H, H-6a00), 3.98 (m, 1H, H-6b00), 3.92–3.80 (m, 3H, H-6a0,
H-6b0, H-40), 3.76 (dd, 1H, J3,4 9.0, H-3), 3.74 (s, 3H, OCH3), 3.60 (q,
1H, J5,6 6.1 Hz, H-5), 3.50 (dd, 1H, J4,5 9.3 Hz, H-4), 3.48–3.38 (m,
3H, H-30, H-300, H-200), 3.34 (dd, 1H, J2 ,3 9.7 Hz, H-20), 3.16 (m, 1H,
H-500), 1.22 (d, 3H, H-6). 13C NMR (63 MHz, CD3CN): d 160.2
(MeOArC), 140.7–139.5 (Ar-C), 136.6 (@CH), 130.6–127.3 (ArCH),
116.3 (@CH2), 114.6 (MeOArCHCH2), 103.6 (C-100), 101.6 (PhCH),
97.8 (C-1), 97.7 (C-10), 80.6, 80.2, 79.6, 78.9 (C-20, C-30, C-200, C-300),
80.5 (C-4), 79.6 (C-3), 78.0 (C-40), 75.9 (C-2), 75.6, 75.4, 73.6, 73.1,
72.0, 71.9, 69.3 (PhCH2, MeOPhCH2), 74.2 (CH2O), 73.9 (C-400), 71.6
(C-50), 69.7 (C-600), 69.2 (C-5), 68.9 (C-60), 67.2 (C-500), 55.8 (OCH3),
18.4 (C-6). Anal. Calcd for C78H84O16: C, 73.33; H, 6.63. Found: C,
73.29; H, 6.66.
tem, 2H, JA,B 12.0 Hz, PhCH2), 4.44 (d, 1H, J1 ,2 7.8 Hz, H-100), 4.21
0
0
00 00
(m, 1H, H-40), 4.08 (dd, 1H, J5 ,6 b 2.9 Hz, J6 a,6 b 9.4 Hz, H-60b), 4.06–
3.82 (m, 9H, CH2O, H-600a, H-600b, H-30, H-40, H-50, H-60a, H-2), 3.78
(s, 3H, CH3O), 3.67 (q, 1H, J5,6 6.1 Hz, H-5), 3.55 (dd, 1H, J2,3 3.5 Hz,
0
0
0
0
0
0
H-3), 3.53 (t, 1H, J3,4 9.6 Hz, J4,5 9.6 Hz, H-4), 3.52 (dd, 1H, J2 ,3
9.6 Hz, H-20), 3.39 (m, 2H, H-200, H-300), 3.17 (m, H-500), 1.23 (d, 3H,
H-6). 13C NMR (63 MHz, CD3CN): d 160.2 (MeOArC), 140.5–139.4
(Ar-C), 136.4 (@CH), 130.6–127.3 (ArCH), 116.1 (@CH2), 114.6
(MeOArCHCH2), 104.1 (C-100), 101.5 (PhCH), 97.7 (C-1), 94.1 (C-10),
80.9 (C-30), 80.7 (C-4), 80.2–79.1 (C-20, C-3, C-200, C-300), 76.4 (C-40),
75.9, 75.8, 75.6, 73.8, 73.4, 71.9, 69.4 (PhCH2, MeOPhCH2), 74.2
(CH2O), 73.8 (C-400), 71.5 (C-50), 69.6 (C-600), 69.1, 69.0 (C-60, C-5),
67.2 (C-500), 58.8 (CH3O), 18.4 (C-6). Anal. Calcd for C78H84O16: C,
73.33; H, 6.63. Found: C, 73.29; H, 6.68.
Data for b-9: Colourless syrup; [a]
D +11.0 (c 1.0, CHCl3); 1H NMR
(250 MHz, CD3CN): d 7.58–7.13 (m, 37H, Ar-H), 6.89 (m, 2H, Ar-H),
5.94 (ddt, 1H, J 5.4, Jcis 10.5, Jtrans 17.3, @CH), 5.55 (s, 1H, PhCH),
5.29 (dq, 1H J 1.7 Hz, Jtrans 17.3 Hz, @CH2), 5.16–5.09 (AB system,
2H, JA,B 11.0 Hz PhCH2), 5.13 (dq, 1H, J 1.4, Jtrans 10.5, @CH2), 4.96
(d, 1H, J1,2 1.7 Hz, H-1), 4.81–4.51 (m, 4H, PhCH2), 4.78–4.69 (AB sys-
Data for b-10: [
a
]
ꢀ0.75 (c 1.0, CHCl3); Rf 0.33 (7:3 n-hexane–
D
EtOAc); 1H NMR (250 MHz, CD3CN): d 7.45–7.16 (m, 37H, Ar-H),
6.85 (m, 2H, Ar-H), 5.92 (ddt, 1H, J 5.3, Jcis 10.5 Hz, Jtrans 17.2 Hz,
@CH), 5.44 (s, 1H, PhCH), 5.27 (dq, 1H J 1.8 Hz, Jtrans 17.2 Hz,
@CH2), 5.15–4.73 (AB system, 2H, JA,B 11.1 Hz PhCH2), 5.13 (dq,
1H, J 1.3, Jtrans 10.5, @CH2), 4.93–4.79 (AB system, 2H, JA,B 11.4 Hz,
PhCH2), 4.87–4.38 (m, 10H, 4 ꢁ PhCH2), 4.83 (d, 1H, J1,2 1.4 Hz, H-
tem, 2H, JA,B 10.9 Hz, PhCH2), 4.67 (d, 1H, J1 ,2 7.9 Hz, H-10), 4.49 (d,
0
0
00
00 00
1H, J1 ,2 8.2 Hz, H-1 ), 4.43–4.35 (AB system, 2H, JA,B 12.0 Hz, PhCH2),
4.25 (m, 2H, CH2O), 4.23 (m, 1H, H-400), 4.10 (dd, 1H, J2,3 2.9 Hz, H-2),
4.10–3.98 (2 m, each 1H, H-6a00, H-6b00), 3.93 (m, 1H, H-40), 3.88 (m,
2H, H-6a0, H-6b0), 3.83 (dd, 1H, J3,4 9.3, H-3), 3.64 (q, 1H, J5,6 6.1 Hz,
H-5), 3.55 (dd, 1H, J3 ,4 8.9 Hz, H-30), 3.50 (t, 1H, J4,5 9.3, H-4), 3.44
1), 4.48 (d, 1H, J1 ,2 7.8 Hz, H-10), 4.42 (d, 1H, J1 ,2 8.2 Hz, H-100),
4.28–4.18 (m. 3H, CH2O, H-400), 4.22 (dd, 1H, J2,3 3.5 Hz, H-2),
4.10 (dd, 1H, J3,4 9.6 Hz, H-3), 4.05 (m, 5H, H-600a, H-600b, H-60a,
0
0
0
0
00 00
(m, 3H, H-50, H-400, H-500), 3.29 (dd, 1H, J2 3 9.1 Hz, H-20), 3.19 (m,
1H, H-500), 1.23 (d, 3H, H-6). 13C NMR (63 MHz, CD3CN): d 160.2
(MeOArC), 140.4–139.6 (Ar-C), 136.6 (@CH), 130.7–127.3 (ArCH),
116.4 (@CH2), 114.6 (MeOArCHCH2), 105.1 (C-10), 103.6 (C-100),
101.6 (PhCH), 99.2 (C-1), 83.4 (C-30), 82.3 (C-20), 81.1 (C-4), 80.3
(C-3), 80.2 (C-200), 79.0 (C-300), 78.0 (C-40), 77.1 (C-2), 75.6 (C-50),
75.6, 75.5, 75.0, 73.7, 72.4, 71.9, 69.1 (PhCH2, MeOPhCH2), 74.0
(CH2O), 73.9 (C-400), 69.7 (C-600), 69.2 (C-60), 68.8 (C-5), 67.2 (C-500),
55.8 (CH3O), 18.4 (C-6). Anal. Calcd for C78H84O16: C, 73.33; H,
6.63. Found: C, 73.30; H, 6.67.
0
0
H-60b, H-40), 3.68 (q, 1H, J5,6 6.1 Hz, H-5), 3.59 (dd, J3 ,4 8.8 Hz, H-
0
0
30), 3.51–3.39 (m, 4H, H-4, H-300, H-200, H-50), 3.38 (dd, 1H, J2 ,3
0
0
9.1 Hz, H-20), 3.18 (m, 1H, H-500), 1.24 (d, 3H, H-6). 13C NMR
(63 MHz, CD3CN): d 160.2 (MeOArC), 140.2–139.7 (Ar-C), 136.6
(@CH), 131.5–127.3 (ArCH), 116.5 (@CH2), 114.6 (MeOArCHCH2),
104.0 (C-10), 103.7 (C-10), 101.5 (PhCH), 98.5 (C-1), 84.2 (C-30),
82.9 (C-20), 81.4, 79.0 (C-200, C-300), 80.2 (C-4), 79.8 (C-40), 79.3 (C-
3), 75.9 (C-50), 75.4, 75.1, 74.1, 73.9, 73.6, 71.9, 69.3 (PhCH2,

F. Bonaccorsi et al. / Carbohydrate Research 344 (2009) 1442–1448
1447
MeOPhCH2), 74.4 (CH2O), 73.7 (C-2, C-400), 69.4 (C-600), 69.1 (C-60),
68.4 (C-5), 67.2 (C-500), 55.8 (CH3O), 18.4 (C-6). Anal. Calcd for
C78H84O16: C, 73.33; H, 6.63. Found: C, 73.29; H, 6.68.
7.84–7.17 (m, 37H, ArH), 6.85–6.84 (m, 2H, ArHOMe), 5.58 (d,
1H, J2 ,NH 9.69 Hz, NH), 4.84 (m, 2H, H-1, H-10), 4.48 (m, 1H, H-
0
100), 3.78 (s, 3H, OCH3); 13C NMR (125 MHz, CDCl3): d 170.4 (CO),
159.31 (ArCOMe), 139.7–137.7 (ArC), 129.6–126.5 (ArCH), 99.8
(C-100), 96.9 (C-10), 96.3 (C-1), 80.6, 80.3, 80.2, 79.5, 79.1, 75.8,
75.1, 75.0 (C-20, C-3, C-30, C-4, C-300, C-2, C-40, C-500), 69.7, 68.4,
67.3(C-5, C-50, C-400), 69.2, 68.5, 68.2 (C-60, C-600, CH2 p-MeOBn),
55.2 (OMe), 49.4 (C-20), 23.1 (CH3CO), 18.0 (C-6). HRMS: Calcd for
C77H86NO16 [M+H]+: 1280.5947. Found: 1280.6001.
3.4. p-Methoxybenzyl (2-acetamido-4-O-allyl-3,6-di-O-benzyl-
2-deoxy-b-
glucopyranosyl)-(1?2)-3,4-di-O-benzyl-
-11) and p-methoxybenzyl (2-acetamido-4-O-allyl-3,6-di-O-
benzyl-2-deoxy-b- -mannopyranosyl)-(1?4)-(2,3,6-tri-O-
benzyl-b- -glucopyranosyl)-(1?2)-3,4-di-O-benzyl-
rhamnopyranoside (b-11)
D
-mannopyranosyl)-(1?4)-(2,3,6-tri-O-benzyl-
a-D-
a
-L
-rhamnopyranoside
(a
D
D
a-L-
3.6. p-Methoxybenzyl (2-acetamido-4-O-acetyl-3,6-di-O-
benzyl-2-deoxy-b-
benzyl- -glucopyranosyl)-(1?2)-3,4-di-O-benzyl-
rhamnopyranoside (13)
D-mannopyranosyl)-(1?4)-(2,3,6-tri-O-
A
solution of
3
(62 mg, 0.0641 mmol) and
7
(56 mg,
a-
D
a-L-
0.124 mmol) in anhyd CH2Cl2 (2 mL) containing 4 Å MS (500 mg)
was stirred under an argon atmosphere at room temperature for
30 min. The solution was cooled to ꢀ35 °C, and then NIS (22 mg,
0.0978 mmol, 1.52 equiv) followed by AgOTf (8 mg, 0.031 mmol,
0.5 equiv) were added. The reaction mixture was allowed to slowly
attain ꢀ10 °C and was stirred for an additional 1 h at that temper-
ature, when TLC (1:1 cyclohexane–EtOAc) showed the complete
disappearance of the donor (Rf 0.40) and the formation of two spots
at Rf 0.67 and 0.49. The reaction mixture was filtered through a
short pad of Celite, diluted with CH2Cl2, and washed with 10% aq
Na2S2O3 (5 mL) and satd aq Na2HCO3 (5 mL). The aqueous phases
were extracted with CH2Cl2, and the combined organic layers were
dried over MgSO4, filtered and concentrated under reduced pres-
sure. The residue (135 mg) was purified by silica gel flash chroma-
Compound 12 (45 mg, 0.035 mmol) was dissolved in pyridine
(2 mL) and Ac2O (1 mL) was added. The reaction mixture was mon-
itored by TLC (7:3 cylcohexane–EtOAc) until, after 16 h the starting
material (Rf 0.16) had completely disappeared with concomitant
formation of a compound with Rf 0.24. The solution was co-evapo-
rated with toluene, and the residue was purified by silica gel flash
chromatography (7:3 cylcohexane–EtOAc) to give 13 (42 mg, 92%)
as a colourless syrup; [a]
D +12.0 (c 0.99, CHCl3); 1H NMR (500 MHz,
CDCl3): d 7.37–714 (m, 37H, ArCH), 6.85–6.84 (m, 2H, ArCHOMe),
5.63 (d, 1H, J2 ,NH 9.6 Hz, NH), 4.83 (m, 2H, H-1, H-10), 4.53 (m,
0
1H, H-100); 13C NMR (125 MHz, CDCl3): d 170.5 (CH3CONH), 169.6
(CH3CO), 159.2 (ArCOMe), 139.6–138.7 (ArC), 129.6–126.6 (ArCH),
113.8 (ArCHOMe), 99.4 (C-100), 96.9 (C-10), 96.4 (C-1), 80.8, 80.5,
79.7, 79.3 (C-20, C-3, C-30, C-4), 77.4, 76.0, 75.6, 74.1 (C-300, C-2, C-
40, C-500), 70.0, 68.6, 68.3 (C-5, C-50, C-400), 69.2, 68.8, 68.4 (C-60, C-
600, CH2 p-MeOBn), 55.5 (OMe), 49.7 (C-200), 23.5 (CH3CONH), 21.1
(CH3CO), 18.3 (C-6). HRMS: Calcd for C79H87NO17 [M+H]+:
1322.6052. Found: 1322.6123.
tography (4:1 to 7:3 cyclohexane–EtOAc) to give first a-11 (44 mg,
59%), followed by b-11 (13 mg, 17%).
Data for
a-11: Colourless syrup; [
a]
ꢀ22.0 (c 0.92, CHCl3); 1H
D
NMR (500 MHz, CDCl3): d 7.37–7.16 (m, 37H, ArH); 6.85–6.84 (m,
2H, ArHOMe); 5.79 (m, 1H, CH2@CH); 5.65 (d, 1H, J2 ,NH 9.6 Hz,
0
NH); 5.16–5.07 (m, 2H, CH2@CH); 4.83 (m, 2H, H-1, H-10); 4.50
(m, 1H, H-100). 13C NMR (125 MHz, CDCl3): d 170.4 (CO), 159.6 (Ar-
COMe), 135.0 (CH2@CH), 139.8–138.1 (ArC), 129.6–126.5 (ArCH),
116.3 (CH2@CH), 99.9 (C-100), 96.8 (C-10), 96.3 (C-1), 80.9, 80,67,
80.3, 79.5, 79.1 (C-4, C-400, C-20, C-3, C-30), 76.0, 75.4, 75.1, 73.7
(C-2, C-300, C-40, C-500), 69.7, 68.4 (C-5, C-50), 68.5, 68.3, 68.2 (C-60,
C-600), 55.3 (OMe), 49.9 (C-200), 23.3 (CH3CONH), 18.04 (C-6). HRMS:
Calcd for C80H90NO16 [M+H]+: 1320.6260. Found: 1320.6234.
3.7. (2-Acetamido-4-O-acetyl-3,6-di-O-benzyl-2-deoxy-b-
mannopyranosyl)-(1?4)-(2,3,6-tri-O-benzyl- -gluco-
pyranosyl)-(1?2)-3,4-di-O-benzyl- ,b- -rhamnopyranose (14)
D-
a-D
a
L
A solution of 13 (40 mg, 0.0302 mmol) in 9:1 CH3CN–H2O
(0.5 mL) was portion-wise treated over 29 h with DDQ (88 mg,
0.387 mmol). The reaction was cooled to 0 °C before every addition
of DDQ, followed by an immediate warming to room temperature.
When TLC analysis (1:1 cyclohexane–EtOAc) showed the disap-
pearance of the starting material (30 h) and the formation of two
spots at Rf 0.52 and 0.59, the reaction was cooled to 0 °C, and
Et3N (1 mL) was added. The mixture was stirred for an additional
10 min, then diluted with CH2Cl2 and washed with satd aq NaH-
CO3. The aqueous phase was extracted with CH2Cl2, and the col-
lected organic layers were dried over MgSO4, filtered and
concentrated under reduced pressure to give a residue that was
subjected to silica gel flash chromatography (7:3 cyclohexane–
EtOAc) to give 14 (30 mg, 83% yield) as a colourless syrup. The
physicochemical and spectral data were in full agreement with
those reported in the literature.6d HRMS: Calcd for C71H79NO16Na
[M+Na]+: 1224.5297. Found: 1224.5261.
Data for b-11: Colourless syrup; [
NMR (500 MHz, CDCl3): d 7.34–7.13 (m, 37H, ArH), 6.84–6.82 (m,
2H, ArHOMe), 5.83–5.75 (m, 1H, CH2@CH), 5.70 (d, 1H, J2 ,NH
a]
ꢀ8.0 (c 0.84, CHCl3); 1H
D
0
9.7 Hz, NH), 5.00 (d, 1H, J1,2 1.43 Hz, H-1), 4.66 (m, 1H, H-100),
4.62 (d, 1H, J1,2 7.38, H-10), 3.76 (s, 3H, OCH3); 13C NMR
(125 MHz, CDCl3): d 170.5 (CO), 159.7 (ArCOMe), 135.1 (CH2@CH),
139.8–138.1 (ArC), 129.6–126.5 (ArCH), 116.4 (CH2@CH), 113.8
(ArCHOMe), 104.7 (C-10), 99.8 (C-100), 98.6 (C-1), 55.2 (OMe), 49.7
(C-200), 23.4 (CH3CONH), 18.04 (C-6). HRMS: Calcd for C80H90NO16
[M+H]+: 1320.6260. Found: 1320.6234.
3.5. p-Methoxybenzyl (2-acetamido-3,6-di-O-benzyl-2-deoxy-b-
-mannopyranosyl)-(1?4)-(2,3,6-tri-O-benzyl-
pyranosyl)-(1?2)-3,4-di-O-benzyl- -rhamnopyranoside (12)
D
a-D-gluco-
a-L
To a solution of
a-11 (50.0 mg, 0.0379 mmol) in 1:1 EtOH–
MeOH (7.5 mL) PdCl2 (16 mg, 0.0902 mmol) was added, and the
reaction mixture was stirred. After 45 min, another portion of
PdCl2 (21 mg, 0.118 mmol) was added. After 1 h and 45 min stir-
ring at room temperature, TLC (2:3 cylcohexane–EtOAc) showed
the disappearance of the starting material (Rf 0.9) and the forma-
tion of a spot at Rf 0.73. The reaction mixture was filtered through
Celite, diluted with CH2Cl2 and concentrated to give a crude resi-
due (51 mg) that was purified by silica gel flash chromatography
(7:3 cylcohexane–EtOAc) to give 12 (45 mg, 94%) as a colourless
3.8. p-Methoxybenzyl (2-acetamido-4-O-allyl-3,6-di-O-benzyl-
2-deoxy-b-
glucopyranosyl)-(1?3)-2,4-di-O-benzyl-
-15) and p-methoxybenzyl (2-acetamido-4-O-allyl-3,6-di-O-
benzyl-2-deoxy-b- -mannopyranosyl)-(1?4)-(2,3,6-tri-O-
benzyl-b- -glucopyranosyl)-(1?3)-2,4-di-O-benzyl-
rhamnopyranoside (b-15)
D
-mannopyranosyl)-(1?4)-(2,3,6-tri-O-benzyl-
a-D-
a-L-rhamnopyranoside
(a
D
D
a-L-
A solution of 3 (58 mg, 0.06 mmol) and 8 (55 mg, 0.118 mmol)
in anhyd CH2Cl2 (2 mL) containing 4 Å MS (330 mg) was treated
syrup; [
a]
+8.9 (c 0.88, CHCl3); 1H NMR (500 MHz, CDCl3): d
D

1448
F. Bonaccorsi et al. / Carbohydrate Research 344 (2009) 1442–1448
as described above for the synthesis of
a- and b-11, employing NIS
Università di Pisa for partly supporting a six-month stage of FB
(18.12 mg, 0.0806 mmol) and AgOTf (8 mg, 0.031 mmol). After 1 h
TLC analysis (1:1 cyclohexane–EtOAc) showed the complete disap-
pearance of the donor and the formation of two spots with Rf 0.60
and 0.71. The reaction mixture was filtered through a short pad of
Celite, diluted with CH2Cl2 and washed with 10% aq Na2S2O3
(7 mL), and satd aq NaHCO3 (6 mL). The aqueous phases were
repeatedly extracted with CH2Cl2, and the combined organic
phases were dried (MgSO4) and concentrated under reduced pres-
sure. The residue (120 mg) was purified by silica gel flash column
in Dublin.
References
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8815–8818.
chromatography (4:1 to 7:3 cyclohexane–EtOAc) to give
a-15
(14 mg, 18%) and b-15 (18 mg, 23%).
Data for
a-15: Colourless syrup; [
a
]
D
ꢀ6.0 (c 1.5, CHCl3); 1H
NMR (500 MHz, CDCl3): d 7.39–7.11 (m, 37H, ArH), 6.85–6.84 (m,
0
2H, ArCHOMe), 5.86–5.78 (m, 1H, CH2@CH), 5.58 (d, 1H, J2 ,NH
9.59 Hz, NH), 4.87 (d, 1H, J1 ,2 3.25 Hz, H-10), 4.77 (d, 1H, J1,2
4.08 Hz, H-1); 13C NMR (125 MHz, CDCl3): d 170.3 (CO), 159.3 (Ar-
COMe), 139.5–137.9 (ArC), 135.0 (CH2@CH), 129.5–126.6 (ArC),
116.2 (CH2@CH), 113.8 (ArCHOMe), 99.6 (C-100), 97.2 (C-10), 96.3
(C-1), 55.3 (OMe), 49.8 (C-200), 23.1 (CH3CO), 18.1 (C-6). HRMS:
Calcd for C80H90NO16 [M+H]+:1320.6260. Found: 1320.6234.
0
0
Data for b-15: Colourless syrup; [
NMR (500 MHz, CDCl3): d 7.38–7.12 (m, 37H, ArH), 6.81–6.79 (m,
a
]
ꢀ23.8 (c 1.6, CHCl3); 1H
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0
2H, ArHOMe), 5.83–5.75 (m, 1H, CH2@CH), 5.66 (d, 1H, J2 ,NH
9.66 Hz, NH). 13C NMR (125 MHz, CDCl3): d 170.5 (CO), 159.3 (Ar-
COMe), 139.2–138.0 (ArC), 134.9 (CH2@CH), 129.3–126.7 (ArC),
116.4 (CH2@CH), 113.7 (ArCHOMe), 103.6 (C-10), 99.9 (C-100), 97.7
(C-1), 55.3 (OMe), 49.7 (C-200), 23.3 (CH3CO), 17.9 (C-6). HRMS:
Calcd for C80H90NO16 [M+H]+: 1320.6260; Found 1320.6234.
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Acknowledgements
This work was supported by Ministero dell0Università e della
Ricerca (MIUR, Rome, Italy) in the frame of the COFIN national
programme and Science Foundation Ireland. Thanks are due to