Synthesis, molecular dynamics simulations, and biology of a carba-analogue of the trisaccharide repeating unit of Streptococcus pneumoniae 19F capsular polysaccharide

Article abstract of DOI:10.1039/b911323a

The synthesis of a carba-analogue corresponding to the trisaccharide repeating unit of Streptococcus pneumoniae type 19F capsular polysaccharide, where a residue of carba-l-rhamnose has been inserted into the natural trisaccharide in place of l-rhamnose,

Full text of DOI:10.1039/b911323a

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis, molecular dynamics simulations, and biology of a carba-analogue
of the trisaccharide repeating unit of Streptococcus pneumoniae 19F capsular
polysaccharide†
Laura Legnani,‡a Silvia Ronchi,‡§b Silvia Fallarini,c Grazia Lombardi,c Federica Campo,c Luigi Panza,c
Luigi Lay,d Laura Poletti,d Lucio Toma,a Fiamma Ronchettib and Federica Compostella*b
Received 10th June 2009, Accepted 20th July 2009
First published as an Advance Article on the web 20th August 2009
DOI: 10.1039/b911323a
The synthesis of a carba-analogue corresponding to the trisaccharide repeating unit of Streptococcus
pneumoniae type 19F capsular polysaccharide, where a residue of carba-L-rhamnose has been inserted
into the natural trisaccharide in place of L-rhamnose, is described. The conformational properties of the
analogue were investigated with the aid of molecular dynamics simulations and were strictly analogous
to those of the natural compound. The biological activity of the carba-analogue was comparable to that
of the corresponding natural repeating unit, thus suggesting that this compound, more stable to
hydrolysis, is a good mimic of the natural structure.
the serotype coverage and to provide optimum immunological
protection in vaccinated individuals.
Recently, the intrinsic problems of manufacturing and safety
Introduction
Infections elicited by Streptococcus pneumoniae (pneumonia, otitis
media, sinusitis, meningitis) are frequently occurring diseases that
connected to conventional vaccines, obtained from bacterial
are associated with considerable morbidity and mortality even
cultures and traditionally conjugated to carriers, have led to
in developed countries. Pneumococcal diseases primarily affect
the development of synthetic vaccines that offer advantages in
elderly people and toddlers.1 The World Health Organization
terms of purity, lot-to-lot consistency, and a high specificity in
eliciting immune responses.6 Indeed, different studies in the field
of bacterial infections have shown the potential of synthetic
carbohydrate antigens as highly sensitive and specific detection
systems or for the development of glycoconjugate vaccines provid-
ing effective prophylaxis against the diseases. Successful examples
have been reported in the literature for Shigella dysenteriae type
1,7 Haemophilus influenzae type b,8 and Bacillus anthracis.9
In this context, we are currently involved in a project aimed
at a rational development of glycoconjugate vaccines,10 exploiting
chemical synthesis as a tool for obtaining new molecular entities
related to natural structures. The aim of the design is to identify
molecules that are endowed with the natural biological activity,
but superior in terms of stability, potency or efficacy. In particular
we have focused on the capsular polysaccharide of Streptococcus
pneumoniae (SP) type 19F,11 which is one of the most commonly
isolated serotypes causing pneumococcal disease, and is covered
up from all licensed vaccines.12
(WHO) estimates that every year up to one million children under
5 years of age die from pneumococcal infection.2 Vaccination
of infants and young children against pneumococcal diseases
has become a global health priority in order to reduce and/or
control antibiotic resistance to Streptococcus pneumoniae.3 A 23-
valent pneumococcal polysaccharide vaccine (PPV23), containing
purified capsular polysaccharides (CPS) from 23 serotypes, and a
pneumococcal 7-valent conjugate vaccine are now available and
have demonstrated effectiveness in older children and adults, or in
children younger than 2 years, respectively.4,5 Nevertheless, with
the need to prevent these diseases in the developing world an
urgent priority, there is increasing attention on the improvement
of vaccine formulations for maximum public-health impact.
Strategic decisions should consider the possibility of broadening
aDipartimento di Chimica Organica, Universita` di Pavia, Via Taramelli 10,
27100, Pavia, Italy
bDipartimento di Chimica, Biochimica e Biotecnologie per la Medicina,
Universita` di Milano, Via Saldini 50, 20133, Milano, Italy. E-mail: federica.
compostella@unimi.it; Fax: +39 02 50316036; Tel: +39 02 50316045
The capsular polysaccharide of SP type 19F comprises the
trisaccharide repeating unit 1, (→4)-b-D-ManpNAc-(1→4)-a-
-
cDipartimento di Scienze Chimiche, Alimentari, Farmaceutiche
e
D-Glcp-(1→2)-a-L-Rhap-(1-OPO3 →), with a phosphodiester
bridge connecting a residue of rhamnose at the reducing end of
one unit to the non-reducing end of the following unit (Fig. 1).
The lability of polysaccharide structures is one of the problems
associated with conventional vaccines that necessitate transport-
ing and storing in cold conditions due to their limited shelf-life at
room temperature. In this case, the presence of a phosphate group
at the anomeric position of rhamnose is the cause of the limited
stability in water of structure 1, due to the high propensity to
hydrolysis of this group at an acetalic position.13 The replacement
of the pyranose oxygen of L-rhamnose with a methylene group
Farmacologiche, Universita` del Piemonte Orientale, Via Bovio 6, 28100,
Novara, Italy
dDipartimento di Chimica Organica e Industriale and CISI, Universita` di
Milano, Via Venezian 21, 20133, Milano, Italy
† Electronic supplementary information (ESI) available: f2/y2 scatter
plots and history of f3 from the MD simulations of the mono- and
disaccharide substructures in vacuum and using explicit water as solvent
(Figs. S1 and S2); 13C and 1H spectra of compounds 9, 12, 6, 17 and 4. See
DOI: 10.1039/b911323a
‡ These authors contributed equally to this work.
§ Present address: Istituto di Scienze e Tecnologie Molecolari, C.N.R., c/o
Polo Scientifico Tecnologico, Via Fantoli 16/15, 20138 Milano, Italy.
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vacuum and using explicit water as solvent, on the monosaccharide
and disaccharide substructures, a-L-Rhap-1-OPO3H2 and a-D-
Glcp-(1→2)-a-L-Rhap-1-OPO3H2, and the corresponding carba-
analogues (see ESI†). The results in vacuum and in water were very
similar, so only vacuum simulations were performed for the larger
molecules 4 and 5. The corresponding torsion angle analyses are
given in Fig. 3, where scatter plots of the populated conformations
at the two inter-residue glycosidic linkages are presented as well
as the trajectory of the f3 torsional angle.
As expected,15 the conformational behavior of the two trisac-
charide structures at both the glycosidic linkages was quite
superimposable in consequence of the fact that the rhamnose
ring oxygen (or methylene) is far enough away not to affect the
f1/y1 and f2/y2 torsional angles. In the b-D-ManpNAc-(1→4)-
a-D-Glcp glycosidic linkage two major conformations are present.
They correspond for the f1 torsion to that of an exo-anomeric
effect (f1 > 0) and for the y1 torsion to those alternating between
positive and negative values. Population at the non-exo-anomeric
conformations where f1 < 0 is very limited for the region with y1
positive, whereas it is higher for the region with y1 negative. In the
a-D-Glcp-(1→2)-a-L-Rhap linkage two conformational regions
exist, also in this case corresponding to the exo-anomeric effect
(f2 < 0); in this case one is much more populated than the other,
and corresponds to values of f2 and y2 both negative. The second
region is centered at values of the y2 torsional angle shifted
towards positive values.
The orientation of the phosphate group is described by the
f3 torsional angle. Toma et al.14 had shown that at the B3LYP/
6-31G(d) DFT level of theory the mG conformation (f3 ª -60◦) is
the most populated (80%) in 2 because of the exo-anomeric effect,
the remaining percentage being attributable to the T conformation
(f3 ª 180 ◦), whereas the percentage of the G conformation (f3 ª
60 ◦) is negligible. In 3, in spite of the absence of such an effect, the
mG conformation remains the most populated (61%), followed
by the T conformation, indicating that the oxygen/methylene
replacement exerts only a slight influence on the orientation of
the phosphate group.
Though the computational approach used herein is very differ-
ent, similar results are obtained by the MD simulation on 4 and 5.
In fact, an analysis of the f3 trajectories reported in Fig. 3 shows
that only the mG and T conformations are populated, whereas
the G conformation was never visited. The analysis allows one
to determine percentages of the mG conformations of 61 and
43% for 5 and 4, respectively, confirming that the effect of the
oxygen/methylene replacement consists in a small lowering of
the conformation corresponding to the exo-anomeric effect that,
however, remains highly populated also in 4. Moreover, a closer
inspection of the trajectories shows that the mG/T transition is
easier for the carba-analogue than for the natural compound; in
fact, it occurs much more frequently in 4 than in 5. This seems to
indicate a lowering of the energy barrier between mG and T in 4
as a consequence of the oxygen/methylene replacement.
Fig. 1 Streptococcus pneumoniae 19F polysaccharide.
would transform the natural sugar in a more stable carba-sugar
analogue, i.e. carba-L-rhamnose, where the instability associated
with the acetalic anomeric phosphate has been removed. Recently,
within a theoretical quantum mechanical study on the confor-
mational behavior of a-L-rhamnose-1-phosphate analogues, a
series of stable mimics of the minimum substructure representing
the rhamnose moiety in the trisaccharide unit, i.e. methyl(2-O-
methyl-a-L-rhamnopyranosyl)-phosphate (2, Fig. 2), have been
compared.14 Carba-L-rhamnose analogue 3 emerged as the best
candidate to mimic the rhamnosyl phosphate 2, as it preserves the
geometrical properties of the reference compound. In fact, in spite
of the lack of the anomeric and exo-anomeric effects, calculations
showed that compound 3 maintains (as does 2) a strong preference
for the 1C4 conformation, every 4C1 conformation being almost 5
kcal/mol higher in energy than the global minimum. This suggests
that the carbocycle 3, when inserted into a trisaccharidic unit in
the place of a rhamnose residue, could produce an oligosaccharide
analogue having immunogenic activity. For these reasons, we
have focused our attention on trisaccharide 4, where a residue of
carba-L-rhamnose replaces the L-rhamnose present in the natural
repeating unit. Compound 4, together with the corresponding
natural trisaccharide repeating unit 5, was first submitted to
a modeling study to confirm that the carba-L-rhamnose, also
when embedded in the trisaccharide, maintains the geometrical
preference reported for compound 3. Then, compound 4 was
prepared and subjected to ELISA tests to assess its activity in
comparison with 511b and the native SP 19F polysaccharide.
Fig. 2
Synthesis of the carba-analogue repeating unit 4
Results and discussion
Protected trisaccharide 6 (Fig. 4) is the intermediate planned
for the synthesis of the target compound 4, namely the carba-
L-rhamnose analogue of the repeating unit of Streptococcus
pneumoniae capsular polysaccharide type 19F. The hydroxyl
Modeling
In a stepwise approach to the modeling of trisaccharide 5 and
its carba-analogue 4, we first carried out MD simulations, both in
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Fig. 3 f1/y1 and f2/y2 scatter plots and history of f3 from the MD simulations of (A) trisaccharide 5 and (B) its carba-analogue 4. (C) Significant
conformations of compounds 5 and 4 with their glycosidic torsional angles shown on the plots: 5A, f1/y1 19/-51 (ꢀ), f2/y2 -35/-38 (᭹), f3 -168 (᭜);
5B, f1/y1 43/5 (★), f2/y2 -36/-37 (᭹), f3 -69 (᭢); 4A, f1/y1 24/-55 (ꢀ), f2/y2 -39/-43 (᭹), f3 -166 (᭜); 4B, f1/y1 41/9 (★), f2/y2 -33/-37 (᭹),
f3 -73 (᭢).
stereoselectivity of the reaction of a-glucosylation is a function of
the protecting groups on glucose, and 4,6-O-benzylidene glucosyl
donors, protected with no participating groups at the 2-position,
are usually a-selective.11a,16,17 Mannosamine unit A is in turn
conjugated to disaccharide B–C through b-glucosylation followed
by epimerization of b-glucopyranoside to b-mannopyranoside.
Previously obtained carba-L-rhamnose 7 (Scheme 1),18 suitably
protected for later modifications, was glycosylated at position
2 with 4,6-O-benzylidene glucosyl trichloroacetimidate donor
Fig. 4 The trisaccharide intermediate 6.
8.19 The reaction was carried out in dichloromethane under the
catalysis of triethylsilyl triflate and gave product 9, with the desired
stereochemistry, in acceptable yield. The a-configuration of the
protecting groups of intermediate 6 are all removable by catalytic
hydrogenolysis but possess the required orthogonality to have
preferential access to the points of chain elongation for further
manipulations (e.g. conjugation and oligomerization). It is in fact
possible to selectively deprotect either the anomeric position at
the reducing end by removal of the 2-naphthylmethyl (NAP)
ether, or the 4-OH of mannosamine by regioselective opening
of the benzylidene acetal. The disconnection strategy adopted
for the synthesis of the trisaccharide consists in the initial
formation of the a-linked disaccharide B–C by the use of a 4,6-O-
benzylidene glucosyl donor. It is in fact well established that the
1
new glycosidic bond was confirmed through H-NMR analysis
by the value of 3.8 Hz for the J1¢,2¢ coupling constant. Reductive
opening of the benzylidene acetal to the corresponding 6-O-benzyl
ether was in turn accomplished by treatment of 9 with triethylsilane
in the presence of boron trifluoride–diethyl ether complex, which
gave the disaccharide acceptor 10 in good yield.20
Trimethylsilyl triflate-promoted coupling of donor 11,21 with
acceptor 10 in dichloromethane at low temperature gave trisac-
charide 12 in high yield. The b-configuration of the glycosidic
bond was demonstrated by the characteristic 8.2 Hz value of the
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Scheme 2 Reagents and conditions: (a) DDQ, DCM/MeOH, 0–25 ◦C,
78%; (b) PCl3, imidazole, TEA, BnOH, mCPBA, DCM/CH3CN, 73%; (c)
H2, Pd(OH)2, EtOAc/MeOH/H2O, 60%; (d) HOPO(OBn)2, DCM, 87%;
(e) see ref. 11b.
acid to give compound 17 in good yield. Final removal of
the protecting groups by hydrogenolysis gave the desired carba-
analogue trisaccharide 4.
The natural trisaccharide repeating unit 5, necessary for the
biological evaluation as reference compound, was prepared as
described previously.11b We wish to report an alternative to the for-
mer procedure in the phosphorylation step to give compound 19,
which was obtained after glycosylation between trichloroacetimi-
date donor 187b and commercially available dibenzyl phosphate
at room temperature.23 The reaction afforded stereoselectively
a-phosphate 19 in high yield and was highly reproducible.
Compound 19 was deprotected according to ref. 11b.
Biology
In order to compare the biological activity of the newly synthesized
carba-L-rhamnose trisaccharide 4 with that of the corresponding
natural trisaccharide 5 and the native polysaccharide 19F (positive
control), classical competitive ELISA assays were performed. In
particular we evaluated the abilities of increasing concentrations
(10-8–100 mg/mL) of each compound to inhibit the binding
between the 19F polysaccharide, coated onto plates, and the anti-
19F human polyclonal antibody.
Fig. 5 shows the inhibition curves obtained with the compounds
under evaluation. The relative efficacy of each compound was
calculated by measuring the maximum effect elicited in this system
(relative efficacy), while the concentration that produces the 50%
of the maximum effect (EC50) was taken as indirect index of its
relative potency. Table 1 shows the calculated data. As expected,
the chain length of the saccharide molecules seems to be important
for their biological activity. The 19F native polysaccharide,
indeed, exhibited both an higher efficacy (100% of inhibition at
10-2 mg/mL) and affinity (EC50 = 8.9 ¥ 10-5 mg/mL) than the
synthesized compounds, confirming that high molecular weight
polysaccharides have a conformational specificity (conformational
epitopes) more suitable for antibody binding.24 Herein we show
that even the single repeating unit has inhibitory properties. In
fact, the analysis of the results demonstrates that both compound
4 and the corresponding natural trisaccharide 5 are recognized
by the anti-19F antibody. The carba-L-rhamnose trisaccharide
4 was slightly more effective than the natural trisaccharide 5,
Scheme 1 Reagents and conditions: (a) TESOTf, DCM, -20 ◦C, 65%; (b)
Et3SiH, BF3·Et2O, DCM, 0 ◦C, 85%; (c) TMSOTf, DCM, -20 ◦C, 90%;
(d) MeONa, DCM/MeOH, 88%; (e) Im2SO2, NaH, DMF, -40 ◦C, 93%;
(f) NaN3, DMF, 110 ◦C, 70%; (g) NiCl2, NaBH4, DCM/MeOH, 0–10 ◦C,
then Ac2O, 74%.
1
trans-diaxial J1¢¢,2¢¢ coupling constant in the H-NMR spectrum.
After removal of the acetate with Ze´mplen transesterification,
the 2-OH of compound 13 was activated in high yield as its
imidazylate and subjected to azide displacement with sodium azide
to give mannoside 15. Final conversion of the azido function
into the acetamido group, accomplished through reduction fol-
lowed by acetylation, afforded the desired orthogonally protected
intermediate 6. In order to introduce the phosphate, the NAP
group of compound 6 was removed by treatment with DDQ22
to yield 16 (Scheme 2), which was subjected to phosphorylation
using phosphorus trichloride–imidazole, followed by treatment
with benzyl alcohol and in situ oxidation by m-chloroperbenzoic
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Table 1 Results of the competitive ELISA assays
longer involves an anomeric position, thus reducing the chem-
ical problems associated with the preparation of pure a-linked
anomeric phosphates.
Compound
EC50 [mg/mL]
Maximum inhibition values [%]a
19F
4
5
8.9 ¥ 10-5
2.7 ¥ 10-2
5.2 ¥ 10-3
100
48
33
3
3
Experimental
Computational methods
a Measured at 1 mg/mL.
Molecular dynamics (MD) simulations were performed with the
HyperChemTM 7.5 software from Hypercube, Inc. employing
the MM+ force field. The simulations on compounds 4 and 5
were carried out in vacuum whereas those on their substructures
a-L-Rhap-1-OPO3H2,
a-D-Glcp-(1→2)-a-L-Rhap-1-OPO3H2,
and the corresponding carba-analogues were carried out both
in vacuum and using explicit water as solvent. For the vacuum
simulations, following a 20 ps equilibration of the molecule,
the production run was carried out at 300 K for 1000 ps (4
and 5) and for 500 ps (substructures). A time step of 1 fs was
used in the integration algorithm and the trajectories were saved
every 50 fs for further analysis. For the water simulations initial
conditions were prepared by placing the mono- and disaccharides
in a previously equilibrated cubic water box of approximately 19
˚
and 23 A, respectively, following minimization and heating. This
procedure resulted in systems with the mono and disaccharides
and either 198 and 382 TIP3P water molecules, respectively.
Following a 20 ps equilibration of the system the production run
was carried out for 500 ps at 300 K with a switched cutoff for
Fig. 5 Concentration–response curves of saccharides on the inhibition of
the binding between the 19F polysaccharide, coated onto the plates, and
the anti-19F human polyclonal antibody were evaluated by a competitive
ELISA method (see the Experimental section). Values are means of at least
four experiments run in triplicate.
˚
non-bond interactions set from 5.35 to 9.35 A and from 7.55 to
˚
11.55 A for the mono and disaccharides, respectively. Also for
water simulations a time step of 1 fs was used in the integration
algorithm and the trajectories were saved every 50 fs. Trajectory
analyses were carried out on the glycosidic angles of compounds
4 and 5, defined as f1 H1–C1¢¢–O–C4¢, y1 C1¢¢–O–C4¢–H4¢, f2
H1¢–C1¢–O–C2, y2 C1¢–O–C2–H2, f3 X–C1–O–P (5: X = O; 4:
X = CH2), and on the corresponding torsional angles of their
mono- and disaccharide substructures.
though less potent (see Table 1), in inhibiting the binding to
the specific antibody. This suggests that the replacement of the
pyranose oxygen of L-rhamnose with a methylene group, which
confers advantages in term of chemical stability, does not strongly
affect the biological properties of the compound.
General
Conclusions
All reagents were the highest commercial quality and used without
further purification except where noted. Air- and moisture-
sensitive liquids and solutions were transferred via oven-dried
syringe or stainless steel cannula through septa. Organic solu-
tions were concentrated by rotary evaporation below 45 ◦C at
approximately 20 mmHg. All reactions were carried out under
anhydrous conditions using oven-dried glassware within an argon
atmosphere. Dry solvents and liquid reagents were distilled prior
to use. Dichloromethane (DCM) was distilled from calcium
hydride; N,N-dimethylformamide (DMF), acetonitrile (CH3CN),
and methanol (MeOH) were dried on activated molecular sieves;
triethylamine (TEA) was distilled from calcium hydride, triethylsi-
lane (Et3SiH) was distilled at 20 torr. NaH was washed three times
with hexane prior to use. All reactions were monitored by TLC
on silica gel 60 F-254 plates (Merck), spots being developed with
5% H2SO4 in MeOH/H2O (1:1) or with a phosphomolybdate-
In conclusion, we have reported a concise synthesis of compound
4, the carba-analogue of the CPS repeating unit of Streptococcus
pneumoniae type 19F, where the residue of L-rhamnose has been
replaced with a carba-L-rhamnose. Analogue 4 appears to be
a good mimic of the natural compound as supported both by
molecular dynamics simulations, which show a conformational
behavior nearly identical for the two compounds, and by biological
assays, which evidence a slightly different but comparable potency.
Our studies support that the trisaccharide carba-analogue is a
good candidate for further investigations. Since the biological
activity at the level of the repeating unit monomers is comparable,
further developments may involve the study of the activity of
longer oligomers, in order to eventually find new molecules for
the development of synthetic vaccines against pneumococcal
infection or address important aspects of the immune response
to analogues of antigenic structures. With respect to the natural
compound, the carba-analogue offers advantages in terms of
chemical stability. Furthermore, from a synthetic point of view, it
would be easier to oligomerize the monomeric carba-trisaccharide
since the phosphodiester bridge among the repeating units no
◦
based reagent and subsequently heated at 110 C. Flash column
chromatography was performed on silica gel 60 (230–400 mesh,
Merck) Optical rotations were measured with a Perkin-Elmer
◦
1
241 polarimeter at 20 C. H, 13C and 31P NMR spectra were
recorded with a Bruker AVANCE-500 spectrometer at a sample
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temperature of 298 K. Chemical shifts are reported on the d
(ppm) scale and are relative to TMS as internal reference. 31P
NMR spectrum for compound 17 was calibrated by using the
H3PO4 signal (d = 0.0 ppm). Resonances were assigned by means
of 2D COSY experiment (standard Bruker pulse program). The
mass spectrometric analyses were performed in positive or negative
electrospray (ESI-MS). MS spectra were recorded on a Thermo
Quest Finnigan LCQTM Deca ion trap mass spectrometer equipped
with a Finnigan ESI interface; data were processed by Finnigan
Xcalibur software system.
4.33–5.01 (m, 12 H, 6 CH2Ph), 4.78 (d, J1¢,2¢ = 3.3 Hz, 1 H, H-1¢),
7.15–7.90 (m, 32 H, arom.); dC(125 MHz, CDCl3) 18.2, 31.9, 32.0,
69.0, 69.9, 70.8, 70.9, 72.2, 73.0, 73.5, 74.3, 75.0, 75.2, 75.4, 79.8,
80.7, 80.8, 83.0, 96.6, 125.5–128.2 (40 C); ESI-MS (positive-ion
mode) m/z 937.6 [M + Na]+ (100%), Calcd for C59H62O9 + Na+
937.4.
2-Naphthylmethyl 2-O-acetyl-3-O-benzyl-4,6-O-benzylidene-b-D-
glucopyranosyl-(1→4)-2,3,6-tri-O-benzyl-a-D-glucopyranosyl-
(1→2)-3,4-di-O-benzyl-5a-carba-a-L-rhamnopyranoside (12)
A
solution of 2-O-acetyl-glucosyltrichloroacetimidate21 11
2-Naphthylmethyl 2,3-di-O-benzyl-4,6-O-benzylidene-a-D-
glucopyranosyl-(1→2)-3,4-di-O-benzyl-5a-carba-a-L-
rhamnopyranoside (9)
(375 mg, 0.688 mmol) and 4¢-O-deprotected disaccharide 10
(252 mg, 0.275 mmol) in dry DCM (10 mL) was cooled to
-20 ◦C, then trimethylsilyl trifluoromethanesulfonate (0.1 M
solution in dry DCM, 1.38 mL) was added. After 2 h, the reaction
mixture was quenched with triethylamine and the solvent evapo-
rated. The crude product was purified by flash chromatography
(toluene/acetone 96:4, v/v) to give 12 (320 mg, 90%) as colourless
oil (Found: C, 74.89; H, 6.34. Calc. for C81H84O15: C, 74.98; H,
6.53%); [a]D +30.7 (c 1 in CHCl3); dH(500 MHz, CDCl3) 1.08
(d, J5,6 = 6.5 Hz, 3 H, 3 H-6), 1.66–1.79 (m, 5 H, OCOCH3 and 2 H-
A
solution of glucosyltrichloroacetimidate19
1.408 mmol) and 2-OH carba-rhamnoside18
8
7
(835 mg,
(340 mg,
0.704 mmol) in dry DCM (14 mL) was cooled to -20 ◦C,
then triethylsilyl trifluoromethanesulfonate (0.1 M solution in dry
DCM, 1.41 mL) was added dropwise. After 45 min the reaction
was quenched by the addition of saturated NaHCO3 solution
and diluted with DCM. After separation, the aqueous layer was
extracted three times with DCM and the combined organic layers
were dried and concentrated. The crude product was purified
twice by flash chromatography (first hexane/EtOAc 85:15, then
toluene/EtOAc 97:3, v/v) to give pure 9 (420 mg, 65%) as an
amorphous powder (Found: C, 77.70; H, 6.43. Calc. for C59H60O9:
C, 77.61; H, 6.62%); [a]D +44.5 (c 1 in CHCl3); dH(500 MHz,
CDCl3) 1.07 (d, J5,6 = 6.5 Hz, 3 H, 3 H-6), 1.65–1.81 (m, 2 H, 2
H-5a), 1.91–2.03 (m, 1 H, H-5), 3.42 (dd, J3,4 = J4,5 = 10.0 Hz, 1
H, H-4), 3.52 (dd, J1¢,2¢ = 3.8, J2¢,3¢ = 9.5 Hz, 1 H, H-2¢), 3.56–3.65
(m, 3 H, H-1, H-4¢ and H-6¢a), 3.86 (dd, J2,3 = 2.9, J3,4 = 9.5 Hz,
1 H, H-3), 3.96 (dd, J5¢,6¢b = 5.0, J6¢a,6¢b = 10.5 Hz, 1 H, H-6¢b),
4.06 (dd, J2¢,3¢ = J3¢,4¢ = 9.5 Hz, 1 H, H-3¢), 4.09–4.12 (m, 1 H, H-
2), 4.29–4.36 (m, 1 H, H-5¢), 4.47–5.02 (m, 10 H, 5 CH2Ph), 4.73
(d, J1¢,2¢ = 3.5 Hz, 1 H, H-1¢), 5.55 (s, 1 H, CHPh), 7.18–7.94 (m, 32
H, arom.); dC(125 MHz, CDCl3) 18.2, 31.8, 32.0, 62.6, 69.1, 70.7,
72.5, 73.7, 74.4, 75.0, 75.2, 76.4, 78.4, 79.4, 80.7, 82.6, 82.8, 97.8,
101.2, 125.5–139.0 (40C arom.); ESI-MS (positive-ion mode) m/z
935.5 [M + Na]+ (100%), Calcd for C59H60O9 + Na+ 935.4.
5a), 1.92–2.10 (m, 1 H, H-5), 3.11 (ddd, J5¢¢,6¢¢b = 5.0, J4¢¢,5¢¢ = J5¢¢,6¢¢a
=
10.0 Hz, 1 H, H-5¢¢), 3.19 (dd, J5¢,6¢a = 1.5, J6¢a,6¢b = 11.0 Hz, 1
H, H-6¢a), 3.36–3.49 (m, 4 H, H-4, H-2¢, H-3¢¢ and H-6¢¢a), 3.52
(dd, J5¢,6¢b = 1.7, J6¢a,6¢b = 11.0 Hz, 1 H, H-6¢b), 3.58–3.61 (m,
1 H, H-1), 3.64 (dd, J3¢¢,4¢¢ = J4¢¢,5¢¢ = 10.0 Hz, 1 H, H-4¢¢), 3.77
(dd, J2¢,3¢ = J3¢,4¢ = 9.5 Hz, 1 H, H-3¢), 3.85 (dd, J2,3 = 2.6, J3,4
=
9.6 Hz, 1 H, H-3), 3.92 (dd, J3¢,4¢ = J4¢,5¢ = 9.5 Hz, 1 H, H-4¢),
4.04–4.11 (m, 2 H, H-2 and H-5¢), 4.15–4.21 (m, 2 H, H-6¢¢b and
CHHPh), 4.31 (d, J1¢¢,2¢¢ = 8.2 Hz, 1 H, H-1¢¢), 4.45–5.03 (m, 13 H,
CH2Ph), 4.72 (d, 1 H, J1¢,2¢ = 3.5 Hz, H-1¢), 4.91 (dd, J1¢¢,2¢¢ = J2¢¢,3¢¢
=
8.2 Hz, 1 H, H-2¢¢), 5.50 (s, 1 H, CHPh), 7.10–7.90 (m, 42H, arom.);
dC(125 MHz, CDCl3) 18.2, 20.6, 31.8, 32.2, 65.9, 67.1, 68.7, 70.1,
70.8, 72.5, 73.2, 73.4, 73.5, 73.9, 74.8, 75.1, 76.7, 77.1, 77.2, 78.5,
79.1, 79.7, 81.4, 81.7, 82.8, 97.8, 100.6, 101.2, 125.5–139.4 (52C
arom.), 168.9. ESI-MS (positive-ion mode) m/z 1319.9 [M + Na]+
(100%), Calcd for C81H84O15+ Na+ 1319.6.
2-Naphthylmethyl 3-O-benzyl-4,6-O-benzylidene-b-D-
glucopyranosyl-(1→4)-2,3,6-tri-O-benzyl-a-D-glucopyranosyl-
(1→2)-3,4-di-O-benzyl-5a-carba-a-L-rhamnopyranoside (13)
2-Naphthylmethyl 2,3,6-tri-O-benzyl-a-D-glucopyranosyl-(1→2)-
3,4-di-O-benzyl-5a-carba-a-L-rhamnopyranoside (10)
To a stirred solution of 12 (293 mg, 0.226 mm) in dry DCM
(4.5 mL) sodium methoxide in dry methanol (0.5 M solution,
0.23 mL) was added. The reaction was stirred for 60 h at room
temperature, then it was neutralized with an ion exchange resin
(Dowex 50 ¥ 8, H+ form), filtered and concentrated. The crude
product was subjected to flash chromatography (toluene/acetone
97:3, v/v) to give pure 13 (250 mg, 88%) as colourless oil (Found:
C, 75.71; H, 6.78. Calc. for C79H82O14: C, 75.58; H, 6.58%); [a]D
+42.8 (c 1 in CHCl3); dH(500 MHz, CDCl3) 1.10 (d, J5,6 = 6.5 Hz,
3 H, 3 H-6), 1.68–1.81 (m, 2 H, 2 H-5a), 1.95–2.05 (m, 1 H, H-5),
Compound 9 (311 mg, 0.341 mmol) was dissolved in dry DCM
(6.5 mL), and triethylsilane (0.54 mL, 3.410 mmol) was added.
The solution was cooled to 0 ◦C followed by the addition of
BF3·Et2O (0.086 mL, 0.682 mmol). The reaction was stirred at
0
◦C for 3 h, then quenched with triethylamine, diluted with
DCM and concentrated in vacuo. The residue was purified by flash
chromatography (hexane/EtOAc 85:15, v/v) to afford compound
10 (265 mg, 85%). (Found: C, 77.61; H, 6.90. Calc. for C59H62O9: C,
77.44; H, 6.83%); [a]D +48.2 (c 1 in CHCl3); dH(500 MHz, CDCl3)
1.08 (d, J5,6 = 6.5 Hz, 3 H, 3 H-6), 1.66–1.80 (m, 2 H, 2 H-5a),
3.05–3.11 (m, 2 H, H-5¢¢ and OH), 3.19 (dd, J5¢,6¢a = 1.8, J6¢a,6¢b
=
1.92–2.02 (m, 1 H, H-5), 2.33–2.40 (m, 1 H, OH), 3.28 (dd, J5¢,6¢a
=
11.6 Hz, 1 H, H-6¢a), 3.41–3.62 (m, 7 H, H-1, H-4, H-2¢, H-2¢¢,
H-3¢¢, H-4¢¢ and H-6¢¢a), 3.65 (dd, J5¢,6¢b = 2.7, J6¢a,6¢b = 11.6 Hz,
1 H, H-6¢b), 3.85 (dd, J2,3 = 3.0, J3,4 = 9.5 Hz, 1 H, H-3), 3.91
(dd, J2¢,3¢ = J3¢,4¢ = 9.5 Hz, 1 H, H-3¢), 3.96–4.01 (m, 2 H, H-4¢
and H-6¢¢b), 4.13–4.15 (m, 1 H, H-2), 4.21–4.26 (m, 1 H, H-5¢),
3.7, J6¢a,6¢b = 10.5 Hz, 1 H, H-6¢a), 3.38 (m, 2 H, H-4 and H- 6¢b),
3.49 (dd, J1¢,2¢ = 3.5, J2¢,3¢ = 9.5 Hz, 1 H, H-2¢), 3.56–3.60 (m, 1 H,
H-1), 3.72 (dd, J2¢,3¢ = J3¢,4¢ = 9.5 Hz, 1 H, H-3¢), 3.79–3.86 (m, 2 H,
H-3 and H- 4¢), 4.13–4.16 (m, 1 H, H-2), 4.16–4.21 (m, 1 H, H-5¢),
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4.31–5.02 (m, 14 H, CH2Ph), 4.53 (d, J1¢¢,2¢¢ = 8.0 Hz, 1 H, H-1¢¢),
4.75 (d, J1¢,2¢ = 3.6 Hz, 1 H, H-1¢), 5.49 (s, 1H, CHPh), 7.10–7.90
(m, 42 H, arom.); dC(125 MHz, CDCl3) 18.2, 31.9, 32.0, 66.3, 68.2,
68.7, 69.8, 70.9, 72.4, 73.4, 73.7, 74.4, 74.5, 74.9, 75.2, 75.6, 75.9,
77.3, 79.8, 80.3, 80.6, 80.9, 81.2, 83.1, 96.7, 101.2, 103.8, 125.3–
139.3 (52 C arom.); ESI-MS (positive-ion mode) m/z 1277.7 [M +
Na]+ (100%), Calcd for C79H82O14 + Na+ 1277.6.
2.05 (m, 1 H, H-5), 2.92–2.99 (m, 1 H, H-5¢¢), 3.01–3.11 (m, 2 H,
2 H-6¢), 3.31 (dd, J2¢¢,3¢¢ = 3.5, J3¢¢,4¢¢ = 9.5 Hz, 1 H, H-3¢¢), 3.42–3.48
(m, 2 H, H-4 and H-2¢), 3.53–3.63 (m, 3 H, H-1, H-2¢¢ and H-6¢¢a),
3.84–3.93 (m, 4 H, H-3, H-3¢, H-4¢ and H-4¢¢), 4.01 (dd, J5,6¢¢b
=
4.8, J6¢¢a,6¢¢b = 10.5 Hz, 1 H, H-6¢¢b), 4.08 (d, Jgem = 12.2 Hz, 1 H,
CHHPh), 4.13–4.16 (m, 1 H, H-2), 4.20–4.25 (m, 1 H, H-5¢), 4.37
(d, J1¢¢,2¢¢ = 1 Hz, 1 H, H-1¢¢), 4.46–5.05 (m, 13 H, CH2Ph), 4.72
(d, J1¢.2¢ = 3.5 Hz, 1 H, H-1¢), 5.52 (s, 1 H, CHPh), 7.15–7.90 (m, 42
H, arom.); dC(125 MHz, CDCl3) 18.1, 32.0, 32.1, 63.6, 67.1, 67.9,
68.4, 69.3, 70.9, 72.3, 72.6, 73.3, 73.5, 74.5, 75.1, 75.4, 75.6, 76.3,
77.5, 78.5, 79.4, 80.1, 81.0, 83.2, 96.6, 100.4, 101.5, 125.5–139.3
(52 C arom.); ESI-MS (positive-ion mode) m/z 1302.6 [M + Na]+
(100%), Calcd for C79H81N3O13 + Na+ 1302.6.
2-Naphthylmethyl 3-O-benzyl-4,6-O-benzylidene-2-O-(N-
imidazole-1-sulfonyl)-b-D-glucopyranosyl-(1→4)-2,3,6-tri-O-
benzyl-a-D-glucopyranosyl-(1→2)-3,4-di-O-benzyl-5a-carba-a-L-
rhamnopyranoside (14)
NaH (45 mg, 1.87 mmol) was added to a stirred solution of 13
(240 mg, 0.191 mmol) in dry DMF (3.9 mL) at room temperature.
After 1 h, the suspension was cooled to -40 ◦C and 1,1¢-sulfonyl-
diimidazole (379 mg, 1.91 mmol) in dry DMF (1 mL) was
added. After 5 h the reaction mixture was quenched with MeOH
and allowed to warm to room temperature, then diluted with
DCM. The mixture was washed with H2O, then the aqueous
layer was extracted three times with DCM. The combined organic
layers were dried over Na2SO4, filtered and evaporated. Flash
chromatography (toluene/acetone 96:4, v/v) of the crude product
gave 14 (246 mg, 93%) as colourless oil (Found: C, 71.01; H, 6.28;
N, 2.05; S, 2.42. Calc. for C82H84N2O16S: C, 71.08; H, 6.11; N, 2.02;
S, 2.31%); [a]D +27.8 (c 1 in CHCl3); dH(500 MHz, CDCl3) 1.10
(d, J5,6 = 6.5 Hz, 3 H, 3 H-6), 1.72–1.77 (m, 2 H, 2 H-5a), 1.93–
2-Naphthylmethyl 2-acetamido-3-O-benzyl-4,6-O-benzylidene-
2-deoxy-b-D-mannopyranosyl-(1→4)-2,3,6-tri-O-benzyl-a-D-
glucopyranosyl-(1→2)-3,4-di-O-benzyl-5a-carba-a-L-
rhamnopyranoside (6)
To a stirred solution of 15 (140 mg, 0.109 mmol) in DCM/MeOH
1:1.5 (5 mL) cooled at 0 ◦C NiCl2·6H2O (156 mg, 0.656 mmol)
and NaBH4 (41 mg, 1.09 mmol) were added. The colour of the
solution changed from green to black. The reaction mixture was
maintained at 10 ◦C for 2 h, then Ac2O (0.11 mL) was added.
After 1 h the reaction was quenched by the addition of saturated
aqueous NaHCO3 and diluted with DCM. After separation,
the aqueous phase was extracted twice with DCM, twice with
EtOAc and twice with CHCl3. The combined organic layers were
dried over Na2SO4, filtered and evaporated. Flash chromatography
(hexane/AcOEt 7:3, v/v) of the crude product gave pure 6 (104 mg,
74%) as colourless oil (Found: C, 74.84; H, 6.56; N, 1.20. Calc.
2.04 (m, 1 H, H-5), 2.93–3.00 (m, 1 H, H-5¢¢), 3.24 (br d, J6¢a,6¢b
=
11.0 Hz, 1 H, H-6¢a), 3.34 (dd, J2¢¢,3¢¢ = J3¢¢,4¢¢ = 9.0 Hz, 1 H, H-3¢¢),
3.40–3.61 (m, 6 H, H-1, H-4, H-2¢, H-6¢b, H-4¢¢ and H-6¢¢a,), 3.74
(dd, J2¢,3¢ = J3¢,4¢ = 9.5 Hz, 1 H, H-3¢), 3.87 (dd, J2,3 = 2.5, J3,4
=
25
9.5 Hz, 1 H, H-3), 3.93 (dd, J3¢,4¢ = J4¢,5¢ = 9.5 Hz, 1 H, H-4¢), 4.10–
4.15 (m, 2 H, H-2 and CHHPh), 4.20–4.26 (m, 3 H, H-5¢, H-1¢¢ and
H-6¢¢b), 4.36 (dd, J1¢¢,2¢¢ = 8.2, J2¢¢,3¢¢ = 9.0 Hz, 1 H, H-2¢¢), 4.44–5.10
(m, 13 H, CH2Ph), 4.73 m, 1 H, H-1¢), 5.45 (s, 1 H, CHPh), 7.00–
7.02 (m, 1 H, imidazole), 7.08–7.10 (m, 1 H, imidazole), 7.15–7.90
(m, 43 H, arom. and imidazole); dC(125 MHz, CDCl3) 18.2, 32.0,
32.2, 65.7, 67.4, 68.5, 69.3, 70.8, 72.7, 73.4, 73.5, 74.2, 74.7, 75.1,
75.2, 75.8, 76.8, 77.2, 79.2, 79.4, 80.8, 82.0, 83.2, 84.9, 97.6, 98.5,
101.4, 118.3–139.3 (55 C arom.); ESI-MS (positive-ion mode) m/z
1407.4 [M + Na]+ (100%), Calcd for C82H84N2O16S + Na+ 1407.5.
for C81H85NO14: C, 75.04; H, 6.61; N, 1.08%); [a]D +16.1 (c 1
in CHCl3); dH(500 MHz, CDCl3) 1.10 (d, J5,6 = 6.5 Hz, 3 H, 3
H-6), 1.67–1.81 (m, 5 H, 2 H-5a and COCH3), 1.96–2.06 (m, 1
H, H-5), 3.03–3.16 (m, 2 H, H-6¢a and H-5¢¢), 3.27–3.34 (m, 2 H,
H-6¢b and H-3¢¢), 3.46 (dd, J3,4 = J4,5 = 9.8 Hz, 1 H, H-4), 3.49
(dd, J1¢,2¢ = 3.5, J2¢,3¢ = 9.5 Hz, 1 H, H-2¢), 3.52–3.60 (m, 2 H, H-1
and H-4¢¢), 3.65 (dd, J5¢¢,6¢¢a = J6¢¢a,6¢¢b = 10.3 Hz, 1 H, H-6¢¢a), 3.81
(dd, J2¢,3¢ = J3¢,4¢ = 9.5 Hz, 1 H, H-3¢), 3.86 (dd, J2,3 = 2.8, J3,4
=
9.8 Hz, 1 H, H-3), 3.99 (dd, J3¢,4¢ = J4¢,5¢ = 9.5 Hz, 1 H, H-4¢), 4.10
(dd, J5¢¢,6¢¢b = 4.7, J6¢¢a,6¢¢b = 10.3 Hz, 1 H, H-6¢¢b), 4.13–4.20 (m, 2
H, H-2 and H-5¢), 4.25 (d, Jgem = 12.0 Hz, 1 H, CHHPh), 4.43–
4.78 (m, 13 H, H-1¢, H-1¢¢, H-2¢¢ and CH2Ph), 4.82–5.05 (m, 3 H,
CH2Ph), 5.49–5.53 (m, 2 H, CHPh and NH), 7.05–7.95 (m, 42
H, arom.); dC(125 MHz, CDCl3) 18.2, 23.2, 29.7, 31.9, 50.6, 67.1,
67.9, 68.7, 69.5, 70.8, 71.1, 72.4, 73.3, 73.4, 74.4, 74.7, 75.0, 75.8,
75.9 (2C), 78.5, 79.6, 80.5, 80.7, 82.8, 96.8, 99.8, 101.7, 125.5–139.5
(52C arom.), 170.4; ESI-MS (positive-ion mode) m/z 1318.8 [M +
Na]+ (100%), Calcd for C81H85NO14 + Na+ 1318.6.
2-Naphthylmethyl 2-azido-3-O-benzyl-4,6-O-benzylidene-
2-deoxy-b-D-mannopyranosyl-(1→4)-2,3,6-tri-O-benzyl-a-D-
glucopyranosyl-(1→2)-3,4-di-O-benzyl-5a-carba-a-L-
rhamnopyranoside (15)
To a stirred solution of 14 (240 mg, 0.173 mmol) in dry DMF
(3.5 mL), sodium azide (112 mg, 1.73 mmol) was added and the
resulting solution was heated at 110 ◦C. After 5 h, the reaction
was allowed to cool to room temperature and then diluted with
H2O and DCM. After separation, the aqueous phase was extracted
twice with DCM and twice with EtOAc, and the combined organic
layers were dried over Na2SO4, filtered and evaporated. The crude
product was purified by flash chromatography (toluene/acetone
96:4, v/v) to give 15 (155 mg, 70%) as colourless oil (Found: C,
74.22; H, 6.58; N, 3.21. Calc. for C79H81N3O13: C, 74.10; H, 6.38;
N, 3.28%); [a]D +20.2 (c 1 in CHCl3); dH(500 MHz, CDCl3) 1.10
(d, J5,6 = 6.5 Hz, 3 H, 3 H-6), 1.69–1.83 (m, 2 H, 2 H-5a), 1.95–
2-Acetamido-3-O-benzyl-4,6-O-benzylidene-2-deoxy-b-D-
mannopyranosyl-(1→4)-2,3,6-tri-O-benzyl-a-D-glucopyranosyl-
(1→2)-3,4-di-O-benzyl-5a-carba-a-L-rhamnopyranose (16)
To a solution of 6 (90 mg, 0.069 mmol) in DCM/MeOH
2:1 (4.5 mL) 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (48 mg,
0.208 mmol) was added at 0 ◦C. The reaction was allowed to warm
to room temperature and, after 3 h, saturated aqueous NaHCO3
was added. The reaction was diluted with DCM and washed
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three times with saturated aq. NaHCO3. The aqueous layers were
then extracted three times with DCM. The combined organic
layers were dried over Na2SO4, filtered and evaporated. The crude
product was purified by flash chromatography (hexane/EtOAc
from 6:4 to 1:1 v/v) to give 16 (63 mg, 78%) as colourless oil
(Found: C, 72.89; H, 6.83; N, 1.30. Calc. for C70H77NO14: C, 72.71;
H, 6.71; N, 1.21%); [a]D +16.3 (c 1 in CHCl3); dH(500 MHz, CDCl3)
1.12 (d, J5,6 = 7.0 Hz, 3 H, 3 H-6), 1.60–1.87 (m, 6 H, 2 H-5a,
COCH3 and OH), 2.02–2.11 (m, 1 H, H-5), 3.05–3.12 (m, 1H, H-
5¢¢), 3.23–3.28 (m, 1 H, H-6¢a), 3.36 (dd, J2¢¢,3¢¢ = 4.2, J3¢¢,4¢¢ = 9.5 Hz,
1 H, H-3¢¢), 3.39–3.44 (m, 1 H, H-6¢b), 3.49–3.59 (m, 3 H, H-4,
H-2¢ and H-4¢¢), 3.63 (dd, J5¢¢,6¢¢a = J6¢¢a,6¢¢b = 10.3 Hz, 1 H, H-6¢¢a),
79.3, 78.0, 80.5, 82.2, 97.2, 99.8, 101.6, 125.8–139.4 (54C arom.),
170.3; dP(202 MHz, CDCl3) -1.70; ESI-MS (positive-ion mode)
m/z 1438.7 (83%), 1439.6 (100%) 1440.4 (40%) [M + Na]+, Calcd
for C84H90NO17P + Na+ 1438.6.
2-Acetamido-2-deoxy-b-D-mannopyranosyl-(1→4)-
a-D-glucopyranosyl-(1→2)-5a-carba-a-L-rhamnopyranosyl
dihydrogen phosphate (4)
To a solution of compound 17 (50 mg, 0.035 mmol) in
EtOAc/MeOH/H2O = 1:1:1 (3 mL), Pd(OH)2/C (1:5, w/wsubstrate
)
and 3 drops of acetic acid were added. The mixture was shaken
under hydrogen atmosphere for 48 h and then filtered over Celite.
After evaporation of the solvent, the crude was purified by flash
chromatography (EtOAc/iPrOH/H2O = 1:1:1) to give compound
4 (13 mg, 60%) as an amorphous white solid. (Found: C, 41.44; H,
6.47; N, 2.38. Calc. for C21H38NO17P: C, 41.52; H, 6.30; N, 2.31%);
[a]D +11.9 (c 1 in H2O); dH(500 MHz, D2O) 0.94 (d, J5,6 = 6.2 Hz, 3
H, 3 H-6), 1.52–1.60 (m, 1 H, 1 H-5a), 1.70–1.79 (m, 2 H, H-5 and
1 H-5a), 2.00 (s, 3 H, COCH3), 3.30 (dd, J3,4 = J4,5 = 9.7 Hz, 1 H,
H-4), 3.35–3.41 (m, 1 H, H-5¢¢), 3.45 (dd, J3¢¢,4¢¢ = J4¢¢,5¢¢ = 9.7 Hz, 1
H, H-4¢¢), 3.52 (dd, J1¢,2¢ = 3.5, J2¢,3¢ = 9.7 Hz, 1 H, H-2¢), 3.59–3.69
(m, 2 H, H-4¢ and H-6¢a), 3.70–3.78 (m, 4 H, H-3, H-6¢b, H-3¢¢
3.85–3.93 (m, 3 H, H-2, H-3 and H-3¢), 3.99 (dd, J3¢,4¢ = J4¢,5¢
=
9.5 Hz, 1 H, H-4¢), 4.01–4.12 (m, 4 H, H-1, H-4¢, H-5¢, H-6¢¢b),
4.32 (d, Jgem = 12.2 Hz, 1 H, CHHPh), 4.49–4.89 (m, 12 H, H-1¢¢,
H-2¢¢ and CH2Ph), 4.91 (d, J1¢,2¢ = 3.5 Hz, 1 H, H-1¢), 4.97 (d, Jgem
=
12.0 Hz, 1 H, CHHPh), 5.47–5.54 (m, 2 H, CHPh and NH), 7.10–
7.55 (m, 35 H, arom.); dC(125 MHz, CDCl3) 18.0, 23.2, 31.5, 34.5,
50.5, 66.9, 67.1, 68.0, 68.7, 70.0, 71.2, 72.6, 73.5, 73.6, 73.7, 74.8,
75.9, 76.1, 77.2, 78.6, 79.6, 80.4, 80.6, 81.0, 98.4, 99.8, 101.6, 126.1–
139.4 (42C arom.), 170.3; ESI-MS (positive-ion mode) m/z 1178.8
[M + Na]+ (100%), Calcd for C70H77NO14 + Na+ 1178.5.
and H-6¢¢a), 3.81 (dd, J2¢,3¢ = J3¢,4¢ = 9.7 Hz, H-3¢), 3.86 (dd, J5¢¢,6¢¢
=
2-Acetamido-3-O-benzyl-4,6-O-benzylidene-2-deoxy-b-D-
mannopyranosyl-(1→4)-2,3,6-tri-O-benzyl-a-D-glucopyranosyl-
(1→2)-3,4-di-O-benzyl-5a-carba-a-L-rhamnopyranosyl dibenzyl
phosphate (17)
2.0, J6¢¢a,6¢¢b = 12.2 Hz, 1 H, H-6¢¢b), 3.95–4.01 (m, 2 H, H-2 and
H-5¢), 4.25–4.30 (m, 1 H, H-1), 4.46–4.50 (m, 1 H, H-2¢¢), 4.82
(d, J1¢¢,2¢¢ = 1.2 Hz, 1 H, H-1¢¢), 4.94 (d, J1¢,2¢ = 3.5 Hz, 1 H, H-1¢);
dC(125 MHz, D2O) 17.0, 22.0, 31.6, 33.0, 53.3, 59.8, 60.3, 66.6,
69.7 (br s, C-1), 70.0, 71.0, 71.1, 71.6, 72.0, 75.0, 76.5, 78.8, 96.6,
99.3, 175.4; ESI-MS (negative-ion mode) m/z 606.3 [M-1]-, Calcd
for C21H38NO17P 607.19.
A solution of PCl3 (0.036 mL, 0.41 mmol) in dry DCM (6 mL) was
added at 0◦ C to a solution of imidazole (85 mg, 1.25 mmol) in dry
◦
DCM (6 mL). After stirring at 0 C for 10 min, TEA (0.17 mL,
1.25 mmol) was added and the mixture was stirred for further
10 min at 0 ◦C. A solution of 16 (60 mg, 0.052 mmol) in dry
DCM/CH3CN 1:1 (3 mL) was then added and, after stirring for
30 min at 0 ◦C, a solution of benzyl alcohol (0.13 mL, 1.25 mmol)
in dry DCM (6 mL) was added and the reaction mixture was
allowed to warm to room temperature. After 1 h the solution
was cooled again to 0 ◦C and m-chloroperbenzoic acid (90 mg,
0.52 mmol) was added. After an additional hour the reaction was
quenched with saturated aqueous Na2S2O3 and saturated aqueous
NaHCO3. The aqueous layer was extracted three times with DCM,
then the combined organics were dried over Na2SO4, filtered and
evaporated. Flash chromatography (hexane/AcOEt from 7:3 to
1:1, v/v) of the crude product gave 17 (54 mg, 73%) as colourless
oil (Found: C, 71.04; H, 6.52; N, 1.11. Calc. for C84H90NO17P: C,
71.22; H, 6.40; N, 0.99%); [a]D +25.4 (c 1 in CHCl3); dH(500 MHz,
CDCl3) 1.00 (d, J5,6 = 6.2 Hz, 3 H, 3 H-6), 1.50–1.88 (m, 6 H, H-5,
2 H-5a and COCH3), 3.04–3.14 (m, 2 H, H-5¢¢ and H-6¢a), 3.29–
2-Acetamido-3-O-benzyl-4,6-O-benzylidene-2-deoxy-b-D-
mannopyranosyl-(1→4)-2,3,6-tri-O-benzyl-a-D-glucopyranosyl-
(1→2)-3,4-di-O-benzyl-a-L-rhamnopyranosyl dibenzyl
phosphate (19)
Trichloroacetimidate donor 1811b (40 mg, 0.031 mmol) and diben-
zylphosphate (9 mg, 0.031 mmol) were dissolved in DCM (0.5 mL)
and allowed to react at room temperature for 1 h. The solvent was
removed under reduced pressure and the crude purified by flash
chromatography (toluene/acetone = 9:1) to give compound 19
(38 mg, 88%) as a colourless oil. Physical data were in agreement
with those reported in ref. 11b.
Competitive ELISA assay
◦
96-Well flat-bottomed plates were incubated overnight at 4–8 C
with a mixture of S. pneumoniae 19F CPS (Sanofi-Aventis, France)
(1 mg/mL) and methylated human serum albumin (1 mg/mL). A
solution of foetal calf serum (5%) in phosphate-buffered saline
supplemented with Brij-35 (0.1%) and sodium azide (0.05%) was
applied to the plates for blocking of nonspecific binding sites.
The plates were incubated overnight at 4–8 ◦C with a solution
(1:200) of rabbit anti-19F, used as reference serum (Statens Serum
Institut, Artillerivej, Denmark). When compounds 4 and 5 were
tested, they were added to each well immediately before the
addition of the reference serum. The plates were then incubated
with alkaline phosphatase conjugate goat anti-rabbit IgG (Sigma-
Aldrich, Milan, Italy), stained with p-nitrophenylphosphate, and
3.42 (m, 3 H, H-4, H-6¢b and H-3¢¢), 3.50 (dd, J1¢,2¢ = 3.5, J2¢,3¢
9.5 Hz, 1 H, H-2¢), 3.55 (dd, J3¢¢,4¢¢ = J4¢¢,5¢¢ = 9.5 Hz, 1 H, H-
4¢¢), 3.61–3.72 (m, 2 H, H-3 and H-6¢¢a), 3.76 (d, J2¢,3¢ = J3¢,4¢
=
=
9.5 Hz, 1 H, H-3¢), 4.01 (dd, J3¢,4¢ = J4¢,5¢ = 9.5 Hz, 1 H, H-4¢),
4.06–4.17 (m, 3 H, H-2, H-5¢ and H-6¢¢b), 4.27 (d, Jgem = 12.0 Hz,
1 H, CHHPh), 4.48–4.70 (m, 10 H, H-1, H-1¢¢, H-2¢¢ and CH2Ph),
4.71–5.06 (m, 8 H, CH2Ph), 4.78 (d, J1¢,2¢ = 3.5 Hz, 1 H, H-1¢),
5.48–5.54 (m, 2 H, CHPh and NH), 7.10–7.55 (m, 45 H, arom.);
dC(125 MHz, CDCl3) 17.8, 23.2, 31.5, 32.9, 50.6, 67.2, 67.9, 68.7,
69.4 (d, JC,P = 5.5 Hz), 69.4 (d, JC,P = 5.6 Hz), 69.8, 71.2, 72.2,
73.4 (2C), 74.1 (d, JC,P = 6.0 Hz), 74.6, 75.0, 75.8, 75.9 (2 C), 78.6,
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the absorbance was measured at 405 nm with an Ultramark
microplate reader (Bio-Rad Laboratories S.r.l., Milan, Italy).
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Acknowledgements
We thank Dr Monique Moreau from Sanofi-Aventis for sup-
plying the native S. pneumoniae 19F CPS. The Italian Ministry
of University and Research (MIUR-Italy, grants: PRIN 2006–
Prot. 2006039903 and FIRB RETI–Prot. RBPR05NWWC) and
Regione Piemonte–Ricerca Sanitaria Finalizzata–Bando 2006 are
gratefully acknowledged for financial support.
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17 Refs. 11a and 16 reported examples regarding the a-selectivity of
4,6-O-benzylidene-glucopyranosyl triflates, which is independent of
the nature of the triflate precursor. A similar behavior was observed
by us in the glycosylation of glucosyl trichloroacetimidates, pro-
tected with nonpartecipating groups at the 2-position, promoted by
boron trifluoride–diethyl ether complex. In the absence of the 4,6-O-
benzylidene protecting group on the glucosyl donor, an a/b ratio near
to 1:1 was always observed (unpublished results).
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20 The downfield shift of the H-4¢ signal (from 3.82 to 5.17 ppm)
after acetylation of a small amount of compound 9 in Ac2O/Py
unequivocally confirmed the regioselectivity of the reductive opening
of the benzylidene.
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