Synthesis and characterization of mannosyl mimetic derivatives based on a beta-cyclodextrin core.

Article abstract of DOI:10.1039/b301670f

The synthesis of branched beta-cyclodextrins substituted with mannosyl mimetic derivatives at one primary hydroxy group is described. It was shown that the self-inclusion phenomenon observed for the target compounds in water did not preclude the inclusion properties of the cyclodextrin moiety.

Full text of DOI:10.1039/b301670f

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Synthesis and characterization of mannosyl mimetic derivatives
based on a ꢀ-cyclodextrin core
Duplex Yockot, Vincent Moreau, Gilles Demailly and Florence Djedaïni-Pilard*
Laboratoire des glucides, Université Picardie Jules Verne, 33 rue St-Leu, 80039 Amiens, France
Received 12th February 2003, Accepted 31st March 2003
First published as an Advance Article on the web 10th April 2003
The synthesis of branched β-cyclodextrins substituted with mannosyl mimetic derivatives at one primary hydroxy
group is described. It was shown that the self-inclusion phenomenon observed for the target compounds in water did
not preclude the inclusion properties of the cyclodextrin moiety.
The route to the targeted glycosylated CDs 1 and 2 involved
the design and synthesis of three building blocks, namely,
2,3,4,6-tetra-O-benzoyl-β--mannopyranosyl amine 3, 2,4-di-
Introduction
Cyclodextrins (CDs) are hydrosoluble torus-like cyclic oligo-
saccharides composed of 6, 7 or 8 glucopyranose units. CDs are
able to accommodate small guest molecules in their hydro-
phobic cavity, which has enabled them to act as drug carriers.
O-benzoyl-3,6-di-O-(2,3,4,6-tetra-O-acetyl-α--mannopyrano-
syl)-β--mannopyranosyl amine 4 and N-6I-deoxy-(-tyrosinyl-
amido)-6I-succinylamidocyclomaltoheptaose 5 (Fig. 2).
Intensive efforts have been made to design modified CDs that
The route to the target compounds 1 and 2 involved pep-
are capable of delivering drugs to specific target sites. For
tide type coupling reactions between N-6I-deoxy-(-tyrosinyl-
example, CDs glycosylated on either primary hydroxy groups1–5
amido)-6I-succinylamidocyclomaltoheptaose 5 and the respect-
or both primary and secondary hydroxy groups6 have been pre-
ive glycosylamines 2,3,4,6-tetra-O-benzoyl-β--mannopyrano-
pared and investigated to assess their binding characteristics to
sugar-specific receptors.
syl amine
3 and 2,6-di-O-benzoyl-3,4-di-O-(2,3,4,6-tetra-
O-acetyl-α--mannopyranosyl)-β--mannopyranosyl amine 4.
All of the final products and building blocks were fully charac-
terized by NMR spectroscopy and high-resolution mass
spectrometry. The inclusion properties of 1 and 2 towards
hydrophobic guests were evaluated by NMR to establish that
the chemical substitution of a cyclodextrin by a “signal” mole-
cule should neither preclude the inclusion of a drug of interest
nor impair the recognition of a “signal” at the receptor level.
This would imply that the cavity of the modified cyclodextrin
should remain vacant for the transportation of a drug.
Sugar-specific receptors are known to be important in the
interactions between the HIV-1 envelope glycoprotein gp120
and a macrophage in the early stages of infection. A complex
multistep process is involved in which the gp120 binds to the
receptor CD4 and co-receptors CXCR4 and CCR5 of a
macrophage. Owing to the inhibition properties showed by
mannans in such interactions,7 N-glycans present on the highly
glycosylated gp1208 are believed to play an important part in
viral infection.
This work reports the synthesis of the novel glycosylated
CDs 1 and 2 as potential inhibitors of the recognition events
involved in HIV infection with a view to studying any struc-
ture–activity relationships which may emerge. Such glyco-
sylated CDs are envisaged to interfere with or completely
inhibit sugar–sugar interactions between gp120 and a macro-
phage (Fig. 1). The glycosidic component of the novel com-
pounds was specifically chosen to mimic the mannose type
oligosaccharides that are attached to the polypeptide backbone
of gp120. A further feature of the design of the new com-
pounds was to introduce a peptide spacer between the mannose
moiety and the CDs to mimic the gp120 polypeptide backbone.
We herein demonstrate that the self-inclusion phenomenon
occurs in aqueous solution and that such conditions do not
preclude the formation of an inclusion complex.
Results and discussion
The target compounds were designed with the intention that
chemical modification of the hydroxy groups of the cyclo-
dextrin moiety should preclude neither the recognition of the
signal at the receptor nor the inclusion for a drug into the cavity
of the cyclodextrin. Moreover the final compounds were
Fig. 1 Structures of glycosylated targets CDs 1 and 2.
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 1 8 1 0 – 1 8 1 8
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 3
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benzoyl-α--mannopyranosyl azide 8 in 43% and 37% yields
respectively (Scheme 1). Both compounds were readily separ-
ated by flash chromatography.
Scheme 1 Synthesis of acceptor 7. Reagents: (a) MeONa, MeOH;
(b) triethyl orthobenzoate, CSA, CH3CN, 2 h; (c) CF3CO2H 90%,
CH3CN, 10 min, 39% of 7 (3 steps).
Glycosylation of the two free hydroxy groups of the acceptor
7 was achieved in one step in the presence of a catalytic amount
of trimethylsilyl triflate (TMSOTf ). The primary hydroxy
group was first glycosylated at Ϫ40 ЊC by the addition of
1.5 equivalents of the known trichloroacetimidate donor 9.16 It
is notable that at this temperature, no glycosylation of the
secondary hydroxy group was detected even when two equiv-
alents of the donor were used. Without isolation, the disacchar-
ide thus formed was extended to a trisaccharide by the addition
of 1.5 equivalents of the donor 9 at room temperature
thus affording the desired 3,6-di-O-(α--mannopyranosyl)-α--
mannopyranosyl azide 10 in high yield (82%).
Unfortunately, attempts to reduce the azido group to
the desired amine by catalytic hydrogenation with Pd/C were
unsuccessful in our hands. Treatment of the trisaccharide 10
with 1,3-propanedithiol as reducing agent17 in the presence of
Hünig’s base afforded 3,6-di-O-(α--mannopyranosyl)-β--
mannopyranosyl amine 4 in 37% yield (Scheme 2).
Fig. 2 Structures of building blocks 3, 4 and 5.
required to bear a dedicated probe to assist future biological
evaluations. Therefore, the following synthesis strategy was
used:
Tyrosine was chosen as a classical probe since a specific
iodination process by 125I is well known;9
A hydrophobic spacer between the cyclodextrin and adduct
moieties was chosen to reduce possible steric hindrance effects
induced by attaching a glycosidic moiety onto the cyclo-
dextrin;10
Only monosubstitution of the cyclodextrin moiety by the 3
and 4 building blocks was considered important since it is
known that in some cases hyper-branched cyclodextrins exhibit
a substantial decrease in the inclusion capacity.5
Synthesis of mannopyranosylamine derivatives 3 and 4
2,3,4,6-Tetra-O-benzoyl-β--mannopyranosyl amine
3 was
prepared by catalytic hydrogenation of the known 2,3,4,6-tetra-
O-benzoyl-α--mannopyranosyl azide 6.11 In keeping with
the observations of Chida et al.,12 a rapid anomerization was
found to occur to give the desired β--mannopyranosyl amine
in 80% yield along with a 10% yield of the α-anomer. The
same result was obtained starting from the more difficult to
prepare β-azide analog.11 Complete inversion of configuration
at the 1-carbon atom of the mannose residue was observed by
NOESY.
3,6-Di-O-(α--mannopyranosyl)-β--mannopyranosyl
amine 5 was synthesised according to a modified procedure
recently described by Bundle et al.:13 a 3,6-di-O-unprotected
mannoside acceptor was obtained simultaneously along with
its 3,4-di-O-unprotected analogue in one step from the corre-
sponding 1-O-protected mannopyranosyl derivative, which was
glycosylated using the trichloroacetimidate method14 to afford
a mixture of a mannotrioside and mannodioside.
Scheme 2 Synthesis of 4. Reagents: (a) TMSOTf, CH2Cl2, Ϫ40 ЊC
then rt, 82%; (b) N,N-diisopropylethylamine, 1,3-propanedithiol,
MeOH, 37%.
Synthesis of L-tyrosinyl-ꢀ-cyclodextrin 5
2,3,4,6-Tetra-O-benzoyl-α--mannopyranosyl azide 6 was
first deprotected by Zemplèn methanolysis to give the corre-
sponding α--mannopyranosyl azide, which was then reacted
with triethyl orthobenzoate in the presence of p-toluenesulfonic
acid as a catalyst. The 2,3–4,6-di-orthoester intermediate thus
obtained was hydrolysed with aqueous trifluoroacetic acid
(1 : 9). As expected, the opening of the 2,3-cyclic orthoester to
the axial ester–equatorial alcohol15 was highly regioselective
affording the 2-benzoate exclusively. In contrast, the 4,6-
orthoester opened without any pronounced regioselectivity to
give a mixture of the corresponding 4- and 6-benzoates, namely,
2,4-di-O-benzoyl-α--mannopyranosyl azide 7 and 2,6-di-O-
6I-Deoxy-6I-succinylamidocyclomaltoheptaose 12 was pre-
pared from the known 6I-deoxy-6I-aminocyclomaltoheptaose18
by nucleophilic addition on succinic anhydride as has been
already described.19 -Tyrosine methyl ester, freshly obtained
from the commercially available -tyrosine methyl ester hydro-
chloride, was attached to 12 by a peptide link using N,NЈ-diiso-
propylcarbodiimide (DIC) and N-hydroxysuccinimide (NHS)
to give 13 in high yield (97%). Transformation of the ester
group into the corresponding acid was performed in aqueous
NaOH to afford the desired -tyrosinyl-cyclomaltoheptaose
derivative 5 (Scheme 3). It should be pointed out that this
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 1 8 1 0 – 1 8 1 8
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Table 1 Solubilities in water of native β-cyclodextrin, thio-glucosylated and mannosylated derivatives
Compounds
Solubility at 20 ЊC/mmol LϪ1
β-Cyclodextrin
15
R =
327
23
R =
R =
549
23
R =
1
2
505
620
monomannosyl-β-CD 14 and trimannosyl-β-CD 15, which
were readily isolated from the reaction mixture by a “precipit-
ation” procedure. The monomannosyl-β-CD 14 was obtained
in high yield (99%) with sufficient purity to be used in the next
step without further purification. Nevertheless, high-perform-
ance liquid chromatography (HPLC: using a H2O/CH3CN
gradient elution; tr of 23.43 min) of an analytical sample
was undertaken to characterize 14. Zemplèn methanolysis of
crude 14 gave the desired final monoglycosylated β-CD 1 in
quantitative yield, which was purified by HPLC (H2O/CH3CN,
gradient elution, tr of 10.39 min). In contrast, a similar
“precipitation” procedure was unsuccessful in achieving
comparable purity of the protected trimannosyl-β-CD 15.
Therefore, purification of crude 15 was undertaken by HPLC
(H2O/CH3CN, gradient elution; tr of 23.84 min). Moreover,
deprotection of 15, following the Zemplèn procedure, proved
to be much longer than expected.
As depicted in Fig. 3a, total deprotection of 15 was not
achieved under standard conditions and gave a mixture of the
desired 2 and the monobenzoylated intermediate 16. Two main
peaks can be observed at m/z = 1904 and m/z = 2008 corre-
sponding to [M2 ϩ Na]ϩ and [M16 ϩ Na]ϩ respectively even
after ten days of reaction (Fig. 3b).
Despite monitoring by mass spectrometry, no reactions con-
ditions could be found to achieve complete deprotection. The
reaction mixture containing the target trimannosyl-β-CD 2
was contaminated by 10% of 16, probably benzoylated on the
fourth position of the mannopyranosyl unit A.
Purification of 2 (39% yield) was finally achieved by HPLC
(H2O/CH3CN, gradient elution; tr of 7.52 min). Structure
elucidation of 1 and 2 was achieved using NMR and ESI-MS
(high and low resolution).
Scheme 3 Synthesis of -tyrosinyl-β-cyclomaltoheptaose derivative 5.
Reagents: (a) DIC, NHS, -tyrosine methyl ester hydrochloride, N,N-
diisopropylethylamine, DMF, 72 h, rt, 97%; (b) NaOH, H2O, 3 h, rt,
97%.
Solubility in water of 1 and 2
The solubility in water at 20 ЊC was determined for compounds
1 and 2 (Table 1), and is compared with the solubility data for
native β-cyclodextrin and some 6-S-glycosyl-6-thio derivatives
of β-CD.20 In the latter case, it can be seen that branched deriv-
atives with the β-anomeric configuration exhibit a much lower
solubility in water compared with the corresponding α-anomer.
Conversely, the derivatives 1 and 2 with the β-anomeric con-
figuration exhibit a much higher solubility in water even when
they bear a hydrophobic spacer between the cyclodextrin and
glycosyl moieties.
basic treatment is required to be effected with no more than
5 equivalents of NaOH to avoid epimerization of the amino
acid.
Synthesis of target glycosylated cyclodextrins 1 and 2
The -tyrosinyl-β-CD 5 was reacted with each of the manno-
pyranosyl amine derivatives 3 and 4 under standard coupling
conditions (DIC and hydroxybenzotriazole (HOBt)) to give the
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 1 8 1 0 – 1 8 1 8
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Fig. 3 Partial ESI-MS spectra of crude 2 following 3 h (a) and 10 days (b) of deprotection reaction conditions and HPLC purified 2 (c). Isotope
profile measured by ESI-HRMS at m/z = 1904 (d) and calculated (e) for C75H115N3NaO53.
the several peptide type coupling reactions employed. The
NMR investigations of 1 and 2
mannosyl, amino acid and cyclodextrin moieties were
The structural elucidation of the final compounds by NMR
was performed in d6-DMSO and D2O and demonstrates that
the purified samples were free of any included by-products or
reagents. Since the spectra are relatively complex owing to the
lack of molecular symmetry of the cyclodextrin moiety, a com-
plete analysis was obtained by stepwise identification of both of
the final compounds by COSY and successive RELAY, HMQC,
HMBC and NOESY experiments. Being located in a highly
specific domain, anomeric and amide protons were used as
starting points for stepwise assignment. All the resonances of
the spacer and glycosylated moieties (A, B, and C) were identi-
fied. However since the resonances relating to the glycosidic
units (I–VII) show strong overlaps, only the substituted glyco-
sidic unit (I) was unambiguously assigned. It should be pointed
out that the NMR structural analysis revealed no evidence
of epimerisation of the H1A and Hα protons of the mannosyl
and amino acid moieties respectively, potentially induced by
sequenced by T-ROESY.21
Since the different compounds were intended to target bio-
logical receptors, we also investigated the conformation of 1
and 2 in water. Each of the compounds displayed a consider-
able spectral dispersion in D2O. Conversely when NMR spectra
were collected using d6-DMSO as solvent, the spectral disper-
sion collapsed. DMSO is well known to preclude the formation
of an inclusion complex. Also, the self-inclusion process in
water is a well-known phenomenon in cyclodextrin chemistry.
To investigate these phenomena, a comparison was made of
T-ROESY spectra of 2 in D2O and d6-DMSO respectively
(Fig. 4). A large number of cross-peaks between the aromatic
protons of tyrosine and presumably the protons H3–H5 of
cyclodextrin are observed in water (Fig. 4b). A quite different
situation is encountered in DMSO (Fig. 4a) such that only
dipolar interactions are observed between aromatic protons
(Ha, Hb) and Hα and Hβ of tyrosine moiety.
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 1 8 1 0 – 1 8 1 8
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Table 2 Rotamer populations about the Cα–Cβ bond derived from
coupling constants, in the tyrosine moiety of 2 (in D2O, d6-DMSO and
D2O in the presence of ASANa respectively) and comparisons with
values obtained for the free amino acid in water
anthraquinone-2-sulfonate (ASANa) by NMR. The molecular
structure of ASANa is shown in Fig. 5. It is well established that
ASANa forms a 1 : 1 inclusion complex with β-CD and that it
can be used as a shift reagent for the NMR studies of cyclo-
dextrins.22 An apparent binding constant of 350 MϪ1 was esti-
mated. It should be pointed out that this value is lower than the
average value (490 MϪ1) extrapolated to 721 different guest :
β-CD inclusion complexes.23
One dimensional NMR spectra of 2 in the absence and in the
presence of ASANa as guest molecule were compared (Fig. 5).
It can be seen that ASANa induces shifts consistent with its
inclusion.
Rotamer (%)
Derivative
gt
tg
gg
Free tyrosine in D2O
2 in D2O
25
28
25.7
24.6
50
25
5.4
18.7
19.7
66.9
55.6
55.7
2 in DMSO
2 ϩ ASANa in D2O
Moreover, the rotamer populations of the Cα–Cβ bond of the
amino acid moiety of 2 in the presence of ASANa and water
are similar to those observed for 2 alone in DMSO (Table 2).
This result suggests that the phenol group is expelled from the
cyclodextrin cavity in either ASANa and water or in DMSO
alone.
From these results, it was concluded that 1 and 2 formed an
intramolecular inclusion complex in aqueous solution but the
interactions were clearly much weaker than for the N-6I-deoxy-
6I-tyrosinylamidocyclomaltoheptaose analogue.10 This suggests
a much looser fitting of the aromatic group in the cyclodextrin
cavity which does not inhibit the inclusion capacity of 1 and 2,
especially when the complex formation involves the secondary
hydroxy group rim of the cyclodextrin.
In conclusion, we report here the total synthesis of mannosyl
mimetic derivatives based on the β-cyclodextrin core. Both of
the compounds were fully characterized by NMR spectroscopy
and mass spectrometry. The NMR study showed that even in
the presence of a succinic spacer the cavity of the cyclodextrin
is occupied by the phenol group of the tyrosine moiety. How-
ever it was demonstrated that this self-inclusion process did not
preclude the inclusion properties of the branched β-cyclo-
dextrins. We are currently investigating the inhibition properties
of the target molecules on the interactions between gp120 and
receptors and co-receptors using dedicated biological models.
Fig. 4 Partial T-ROESY contour plots (200 ms of spin lock at 22 dB
attenuation) for 2 (10 mM, 300 K, 500.13 MHz) (a) in d6-DMSO and
(b) in D2O.
Experimental
General methods
The investigation was extended to study the conformation
about the Cα–Cβ bond of the amino acid moiety in water and in
DMSO. The pertinent coupling constants were collected in
both solvents, converted into rotamer populations10 and com-
pared to the values found for the free amino acid (Table 2). A
significant but relatively weak conformational strain is observed
for 2 in water. In DMSO solution, the conformational equi-
librium between the three rotamers is partially restored. At this
stage, it can be concluded that the phenol part of the amino
acid is included in the cyclodextrin cavity when in water but is
expelled from the same cavity when in DMSO. Similar results
were obtained from parallel experiments with the compound 1.
In keeping with the well-known self-inclusion phenomenon in
the cyclodextrin field, we have already shown that N-6I-deoxy-
6I-tyrosinylamidocyclomaltoheptaose forms a very strong self-
inclusion complex in water precluding formation of other
inclusion complexes.10 However, the self-inclusion process
seems to be weaker for 1 and 2 when compared with N-6I-
deoxy-6I-tyrosinylamido-cyclomaltoheptaose due to inhibition
caused by the spacer.
Optical rotations were measured with a JASCO DIP-370 digital
polarimeter, using a sodium lamp (λ = 589 nm) at 20 ЊC. All
NMR experiments were performed at 300.13 and 500.13 MHz
using Bruker DMX300 and DRX500 spectrometers equipped
with a Z-gradient unit for pulsed-field gradient spectroscopy.
Me4Si was used as an external standard and calibration was
performed using the signal of the residual protons or of the
carbon of the solvents as a secondary reference. Measurements
were performed at 300 K with careful temperature regulation.
The length of the 90Њ pulse was approximately 7 µs. 1D NMR
spectral data were collected using 16K data points. 2D experi-
ments were run using 1K data points and 256 time increments.
The phase sensitive (TTPI) sequence was used and processing
resulted in a 1K × 1K (real–real) matrix. Details concerning
experimental conditions are given in the figure captions.
Low resolution electrospray mass spectra were obtained
on a hybrid quadrupole/time-of-flight (Q-TOF) instrument,
equipped with a pneumatically assisted electrospray (Z-spray)
ion source (Micromass). High resolution mass spectra were
recorded in positive mode on a ZabSpec TOF (Micromass, UK)
tandem hydrid mass spectrometer with EBETOF geometry.
The compounds were individually dissolved in water–CH3CN
(1 : 1) at a concentration of 10 µg cmϪ3 and then infused into
the electrospray ion source at a flow rate of 10 mm3 minϪ1 at
60 ЊC. The mass spectrometer was operated at 4 kV whilst scan-
ning the magnet at a typical range of 4000–100 Da. The mass
spectra were collected as continuum profile data. Accurate mass
measurement was achieved using polyethylene glycol as internal
Investigation of inclusion properties of the glycosylated cyclo-
dextrins 1 and 2
It was therefore necessary to evaluate the inclusion properties
of 1 and 2 towards hydrophobic guests to confirm that the self-
inclusion process does not inhibit the inclusion capacity of
these derivatives. To demonstrate the inclusion properties of 2,
we studied the formation of the inclusion complex with sodium
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 1 8 1 0 – 1 8 1 8
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Fig. 5 Partial 1H NMR spectra (300 K, 500.13 MHz, 10 mM in D2O) of 2 (a) alone and (b) in the presence of ASANa (15 mM).
reference masses with a resolving power set to a minimum of
10 000 (10% valley).
Elemental analyses were performed at the Service de Micro-
analyse de l’Université de Champagne-Ardennes in Reims,
France. The samples were previously dried under vacuum for
one week.
Preparative HPLC was carried out with a Waters Prep LC
4000 System chromatograph fitted with an evaporative
light scattering detector PL-ELS 1000 (Polymer Laboratories)
and a Prevail C-18 column (5 µm, 22 × 250 mm). Thin-
layer chromatography was performed on E. Merck glass
plates silica gel sheets (silica gel F254) followed by charring with
vanillin. Column chromatography was performed on Kieselgel
(E. Merck 230–400 mesh).
was stirred overnight, neutralized with acidic resin (Amberlite
IR 120) and then filtered. After concentration in vacuo, the
residue was dissolved in water and washed twice with EtOAc.
Then, the aqueous layer was freeze-dried to afford the
α--mannopyranosyl azide (3 g, 90%). To a suspension of
α--mannopyranosyl azide (3 g, 14.6 mmol) in dry CH3CN
(150 ml) were added triethyl orthobenzoate (15.2 ml, 66.95
mmol) and camphor-10-sulfonic acid (640 mg, 2.75 mmol). The
reaction mixture was stirred for 2 h at room temperature. Tri-
ethylamine (1.5 ml) was added and the solution was evaporated
to dryness. The residue was dissolved in CH3CN (150 ml) and a
solution of 90% trifluoroacetic acid–water (9.4 ml) was added.
The mixture was stirred at room temperature for 10 min. The
solution was then diluted with toluene (100 ml) and evaporated.
The residue was dissolved in CH2Cl2 (50 ml) and the solu-
tion was successively washed with saturated aq. NaHCO3 (2 ×
20 ml), and water (20 ml). The organic layer was dried over
Na2SO4, filtered and concentrated in vacuo to give a syrup from
which the products were separated by flash chromatography
(EtOAc–hexane 3 : 7 v/v). Compound 7 (2.89 g, 43.5%) was the
first to be eluted followed by compound 8 (2.47 g, 37.1%). Data
for 7: (Found: C, 57.41; H, 4.69; N, 9.73 C20H19N3O7 requires C,
58.11; H, 4.60; N, 10.16%); [α]D ϩ68Њ (c 0.5, CHCl3); 1H NMR
(300 MHz, CDCl3): δ 8.12–7.19 (m, 10 H, OCOC6H5), 5.57 (t,
1 H, J3,4 = J4,5 = 9.9 Hz, H-4), 5.55 (d, 1 H, J1,2 1.7 Hz, H-1), 5.33
(dd, 1 H, J2,3 3.3 Hz, H-2), 4.34 (dd, 1 H, H-3), 4.06 (ddd, 1 H,
J5,6 2.7 Hz, J5,6Ј 3.9 Hz, H-5), 3.81 (dd, 1 H, J6,6Ј 11.5 Hz, H-6),
3.71 (dd, 1 H, H-6Ј), 3.49 (s, 1 H, OH), 3.01 (s, 1 H, OH); 13C
NMR (75 MHz, CDCl3): δ 166.89 and 165.74 (OCOC6H5),
133.54–128.34 (OCOC6H5), 87.44 (C-1), 72.56 (C-2), 72.22
(C-5), 69.48 (C-4), 67.54 (C-3), 61.00 (C-6); m/z (ES-HRMS)
436.1123 ([M ϩ Na]ϩ C20H19N3NaO7 requires 436.1121); data
for 8: (Found: C, 57.93; H, 4.51; N, 9.92 C20H19N3O7 requires C,
2,3,4,6-Tetra-O-benzoyl-ꢀ-D-mannopyranosyl amine 3
Pd/C (100 mg) was added to a solution of 2,3,4,6-tetra-O-
benzoyl-α--mannopyranosyl azide 611 (3.5 g, 5.63 mmol) in
EtOH (200 ml). The mixture was stirred in a hydrogen atmos-
phere (20 bar) for 24 h. The catalyst was removed by filtration
and the filtrate was evaporated to dryness. The residue was
purified by flash chromatography (EtOAc–hexane 1 : 1 v/v)
to give compound 3 (2.65 g, 80%) as a white powder (Found:
C, 68.29; H, 4.84; N, 2.42. C34H29NO9 requires C, 68.57;
1
H, 4.87; N, 2.35%); [α]D Ϫ116Њ (c 1, CHCl3); H NMR (300
MHz, CDCl3): δ 8.09–7.22 (m, 20 H, OCOC6H5), 5.96 (t, 1 H,
J3,4 = J4,5 10.0 Hz, H-4), 5.90 (dd, 1 H, J1,2 0.8 Hz, J2,3 3.18 Hz,
H-2), 5.63 (dd, 1 H, H-3), 4.77 (d, 1 H, H-1), 4.72 (dd, 1 H,
J5,6 2.7 Hz, J6,6Ј 11.8 Hz, H-6), 4.44 (dd, 1 H, J5,6Ј 4.4 Hz,
H-6Ј), 4.12 (ddd, 1 H, H-5), 2.18 (s, 2 H, NH2); 13C NMR
(75 MHz, CDCl3): δ 166.16, 165.53, 165.41 and 165.36
(OCOC6H5), 133.45–128.20 (OCOC6H5), 82.49 (C-1), 73.31,
72.84, 71.45 and 66.72 (C-2, C-3, C-4 and C-5), 63.08 (C-6);
m/z (ES-HRMS) 618.1747 ([M ϩ Na]ϩ C34H29NNaO9 requires
618.1740).
1
58.11; H, 4.60; N, 10.16%); [α]D ϩ59Њ (c 1, CHCl3); H NMR
(300 MHz, CDCl3): δ 8.09–7.22 (m, 10 H, OCOC6H5), 5.49 (d,
1 H, J1,2 1.7 Hz, H-1), 5.25 (dd, 1 H, J2,3 2.9 Hz, H-2), 4.78 (dd,
1 H, J6,6Ј 11.4 Hz, J5,6 3.3 Hz, H-6), 4.56 (dd, 1 H, H-6Ј), 4.10
(dd, 1 H, J3,4 10.09 Hz, H-3), 3.99 (m, 2 H, H-4, H-5); 13C NMR
(75 MHz, CDCl3): δ 166.84 and 165.83 (OCOC6H5), 133.36–
128.29 (OCOC6H5), 87.50 (C-1), 72.60 (C-5), 71.83 (C-2),
69.08 (C-3), 67.16 (C-4), 63.17 (C-6); m/z (ES-LRMS) 431
([M ϩ NH4]ϩ C20H23N4O7 requires 431).
2,4-Di-O-benzoyl-ꢁ-D-mannopyranosyl azide 7 and 2,6-di-O-
benzoyl-ꢁ-D-mannopyranosyl azide 8
Sodium methoxide (65 ml, 1 M in MeOH) was added to a
solution of 2,3,4,6-tetra-O-benzoyl-α--mannopyranosyl azide
611 (10 g, 16.1 mmol) in dry methanol (180 ml). The mixture
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 1 8 1 0 – 1 8 1 8
1815

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2,4-Di-O-benzoyl-3,6-di-O-(2,3,4,6-tetra-O-acetyl-ꢁ-D-manno-
pyranosyl)-ꢁ-D-mannopyranosyl azide 10
N-6I-Deoxy-(L-tyrosinylamido methyl ester)-6I-succinylamido-
cyclomaltoheptaose 13
TMSOTf (15 µl, 0.083 mmol) was added to a stirred solution of
A solution of compound 12 (1 g, 0.81 mmol), DIC (630 µl, 4.06
mmol) and NHS (470 mg, 4.08 mmol) in dry DMF (12 ml) was
stirred for 2 h at room temperature under argon. A solution of
-tyrosine methyl ester hydrochloride (290 mg, 1.25 mmol) and
N,N-diisopropylethylamine (700 µl, 4.02 mmol) in dry DMF
(3 ml) was prepared 30 min before use and then added to
the reaction mixture. After 72 h of stirring, DMF was removed
in vacuo. The residual syrup was dissolved in DMF (5 ml) and
stored in a refrigerator for 2 h to allow the precipitation of the
urea derivative. After filtration through a Millex-GS filter, the
filtrate was concentrated to dryness. The subsequent residue
was suspended in acetone (50 ml) and stirred for 20 min to give
a precipitate which was filtered and washed several times with
acetone to afford compound 13 (1.11 g, 97%) as a white powder.
[α]D ϩ109Њ (c 1, H2O); 1H NMR (300 MHz, DMSO-d6): δ 9.23
(s, 1 H, OH-Tyr), 8.24 (d, 1 H, JNH,α 7.4 Hz, NH-Tyr), 7.63 (t,
7
(192 mg, 0.46 mmol) and 2,3,4,6-tetra-O-acetyl-α--
mannopyranosyl trichloroacetimidate 916 (336 mg, 0.70 mmol)
in dry CH2Cl2 (10 ml) under argon at Ϫ40 ЊC. Stirring was
continued at Ϫ40 ЊC for 30 min. The mixture was allowed to
warm to room temperature, after which a solution of 2,3,4,6-
tetra-O-acetyl-α--mannopyranosyl trichloroacetimidate
9
(336 mg, 0.70 mmol) and TMSOTf (15 µl, 0.083 mmol) in
dry CH2Cl2 (5 ml) was added. The reaction mixture was
stirred at room temperature for 30 min, neutralized with tri-
ethylamine and concentrated in vacuo. The residue was puri-
fied by flash chromatography (EtOAc–hexane 5.5 : 4.5 v/v) to
afford the trisaccharide 10 (408 mg, 82%) (Found: C, 53.15;
H, 5.12; N, 3.56 C48H55N3O25 requires C, 53.68; H, 5.12; N,
3.91%); [α]D ϩ50Њ (c 0.73, CHCl3); 1H NMR (300 MHz,
CDCl3): δ 8.10–7.41 (m, 10 H, OCOC6H5), 5.63 (t, 1 H, J3,4
=
1
J4,5 9.9 Hz, H-4A), 5.59 (d, 1H, J1,2 1.8 Hz, H-1A), 5.35 (dd,
1 H, J2,3 3.2 Hz, H-2A), 5.30 (dd, 1 H, J2,3 3.4 Hz, J3,4 10.1
Hz, H-3C), 5.20 (dd, 1 H, J1,2 1.6 Hz, H-2C), 5.18 (t, 1 H,
H-4C), 5.04–5.01 (m, 2 H, H-3B, H-4B), 4.97 (d, 1 H, J1,2 1.7
Hz, H-1B), 4.82 (dd, 1 H, J2,3 3.0 Hz, H-2B), 4.76 (d, 1 H,
H-1C), 4.36 (dd, 1 H, H-3A), 4.26 (ddd, 1 H, J5,6 5.7 Hz, J5,6Ј 2.8
Hz, H-5A), 4.12–3.84 (m, 7 H, H-5B, H-5C, H-6A, H-6B,
H-6ЈB, H-6C, H-6ЈC), 3.62 (dd, 1 H, J6,6Ј 10.8 Hz, H-6ЈA), 2.06,
2.05, 2.03, 1.99, 1.92, 1.87, 1.81 and 1.77 (8 s, 24 H, OCOCH3);
13C NMR (75 MHz, CDCl3): δ 170.45, 169.74, 169.58, 169.45
and 168.99 (OCOCH3), 165.70 and 165.01 (OCOC6H5),
133.77–128.48 (OCOC6H5), 99.26 (C-1B), 97.26 (C-1C), 87.31
(C-1A), 74.36 (C-3A), 71.46 (C-5A), 71.17 (C-2A), 69.30
(C-5B), 69.14 (C-2C), 68.99 (C-2B), 68.90 (C-3C), 68.44
(C-5C), 68.06 (C-3B or C-4B), 67.96 (C-4B), 66.40 (C-6A),
65.74 (C-3B or C-4B, C-4C), 62.19 (C-6B, C-6C), 20.56 and
20.35 (OCOCH3); m/z (ES-LRMS) 1091 ([M ϩ NH4]ϩ
C48H59N4O25 requires 1091).
1 H, JNH,6I = JNH,6IЈ 6.1 Hz, NH-βCD); H NMR (300 MHz,
D2O): δ 6.90 (d, 2 H, Ja,b 8.2 Hz, H-a, H-aЈ), 6.74 (d, 2 H, H-b,
H-bЈ), 4.91 (m, 7 H, H-1I-VII), 4.22–4.17 (m, 2 H, H-6I, H-α),
3.95–3.19 (m, 43 H, H-2I-VII, H-3I-VII, H-4I-VII, H-5I-VII,
H-6II-VII, H-6ЈII-VII, OCH3), 2.72 (dd, 1 H, J5,6 9.5 Hz,
J6,6Ј 15.4 Hz, H-6ЈI), 2.68–2.61 (m, 2 H, H-β, H-βЈ), 2.57–2.18
(m, 4 H, 2 × CH2); 13C NMR (75 MHz, D2O): δ 175.12, 173.81
and 172.95 (NHCO), 155.65, 130.17, 128.42 and 115.62
(C6H4OH), 102.45, 102.20 and 102.04 (C-1I-VII), 84.01 (C-4I),
81.41, 81.10 and 80.76 (C-4II-VII), 73.39, 73.13, 72.47 and
72.26 (C-2I-VII, C-3I-VII, C-5I-VII), 60.25 and 60.02 (C-6II-
VII), 55.40 (C-α), 53.17 (OCH3), 40.54 (C-6I), 37.72 (C-β),
31.43 and 31.19 (2 × CH2 Succ); m/z (ES-LRMS) 1433
([M ϩ Na]ϩ C56H86N2NaO39 requires 1433).
N-6I-Deoxy-(L-tyrosinylamido)-6I-succinylamidocyclomalto-
heptaose 5
A solution of compound 13 (1 g, 0.7 mmol) and 1 M NaOH
(3.54 ml) in water was stirred at room temperature for 3 h.
Then, the mixture was neutralized with acidic resin (Amberlite
IR 120), filtered and evaporated to dryness. The residue was
dissolved in water (10 ml) and freeze-dried to afford compound
2,4-Di-O-benzoyl-3,6-di-O-(2,3,4,6-tetra-O-acetyl-ꢁ-D-manno-
pyranosyl)-ꢀ-D-mannopyranosyl amine 4
A solution of the trisaccharide 10 (3.5 g, 3.26 mmol),
N,N-diisopropylethyl amine (7 ml, 41 mmol) and 1,3-propane-
dithiol (15 ml, 148 mmol) in dry MeOH (100 ml) was stirred
at room temperature for 4.5 h. The mixture was concentrated
in vacuo and the residue purified by flash chromatography
(EtOAc–hexane 4 : 1 v/v) to afford the desired amine 4
(1.23 g, 37%) (Found: C, 54.34; H, 5.40; N, 1.36 C48H57NO25
requires C, 55.01; H, 5.44; N, 1.33%); [α]D Ϫ16Њ (c 0.95,
CHCl3); 1H NMR (300 MHz, CDCl3): δ 8.20–7.40 (m,
10 H, OCOC6H5), 5.75 (dd, 1 H, J1,2 0.9 Hz, J2,3 2.5 Hz, H-2A),
5.55 (t, 1 H, J3,4 = J4,5 9.8 Hz, H-4A), 5.39 (dd, 1 H,
J2,3 3.4 Hz, J3,4 10.1 Hz, H-3C), 5.27 (dd, 1 H, J1,2 1.7 Hz, J2,3 3.5
Hz, H-2C), 5.24 (t, 1 H, J3,4 = J4,5 9.9 Hz, H-4C), 5.15
(t, 1 H, J4,5 10.1 Hz, H-4B), 5.03 (dd, 1 H, J2,3 3.5 Hz,
H-3B), 4.94 (d, 1 H, J1,2 1.9 Hz, H-1B), 4.85 (dd, 1 H, H-2B),
4.80 (d, 1 H, H-1C), 4.39 (m, 1 H, H-5B), 4.33 (dd, 1 H, J5,6
4.8 Hz, J6,6Ј 15.7 Hz, H-6B or H-6C), 4.24 (dd, 1 H, H-3A),
4.19–3.98 (m, 4 H, H-5C, H-6B or H-6C, H-6ЈB, H-6ЈC),
3.91 (dd, 1 H, J5,6 6.12 Hz, J6,6Ј 10.6 Hz, H-6A), 3.81 (ddd,
1 H, J5,6Ј 2.2 Hz, H-5A), 3.60 (dd, 1 H, H-6ЈA), 2.13, 2.05,
2.02, 2.00, 1.99, 1.80 and 1.77 (7 s, 24 H, OCOCH3); 13C
NMR (75 MHz, CDCl3): δ 170.58, 169.74 and 168.96
(OCOCH3), 166.16 and 165.07 (OCOC6H5), 133.56–128.50
(OCOC6H5), 99.37 (C-1B), 97.57 (C-1C), 82.73 (C-1A), 77.03
(C-3A), 73.82 (C-5A), 72.67 (C-2A), 69.33, 69.03, 68.45
and 68.14 (C-4A, C-2B, C-3B, C-5B, C-2C and C-5C) 67.20
(C-6A), 65.97 and 65.90 (C-4B and C-4C), 62.35 and 62. 19
(C-6B and C-6C), 20.72, 20.41 and 20.28 (OCOCH3); m/z
(ES-HRMS) 1070.3120 ([M ϩ Na]ϩ C48H57NNaO25 requires
1070.3117).
1
5 (959 mg, 97%). [α]D ϩ117Њ (c 1, H2O); H NMR (300 MHz,
DMSO-d6): δ 9.21 (s, 1 H, OH-Tyr), 8.11 (d, 1 H, JNH,α 7.6 Hz,
1
NH-Tyr), 7.64 (t, 1 H, JNH,6I = JNH,6IЈ 6.1 Hz, NH-βCD); H
NMR (300 MHz, D2O): δ 6.99 (d, 2 H, Ja,b 8.2 Hz, H-a, H-aЈ),
6.83 (d, 2 H, H-b, H-bЈ), 5.00–4.66 (m, 7 H, H-1I-VII), 4.33 (dd,
1 H, J5,6 < 1 Hz, J6,6Ј 13.4 Hz, H-6I), 4.15 (t, 1 H, Jα,β
=
Jα,βЈ 6.8 Hz, H-α), 4.06–3.41 (m, 39 H, H-2I-VII, H-3I-VII,
H-4II-VII, H-5I-VII, H-6II-VII, H-6ЈII-VII), 3.32 (t, 1 H,
J3,4 = J4,5 9.0 Hz, H-4I), 2.84–2.76 (m, 2 H, H-6ЈI and H-β),
2.68–2.45 (m, 4 H, 3 CH2 Succ and H-βЈ), 2.31 (m, 1 H, 1 CH2
Succ); 13C NMR (75 MHz, D2O): δ 174.87, 174.61 and 173.98
(NHCO and COOH), 155.57, 130.09, 128.77 and 115.69
(C6H4OH), 102.61 and 102.19 (C-1I-VII), 84.13 (C-4I), 81.96,
80.99 and 80.85 (C-4II-VII), 73.48, 73.03 and 72.35 (C-2I-VII,
C-3I-VII, C-5I-VII), 60.58 and 60.29 (C-6II-VII), 56.32 (C-α),
40.62 (C-6I), 38.03 (C-β), 31.75 and 31.56 (2 × CH2 Succ);
ES-LRMS [M ϩ H]ϩ m/z 1397 calcd. for C55H85N2O39, found:
1397.
N-6I-Deoxy-(2,3,4,6-tetra-O-benzoyl-ꢀ-D-mannopyranosyl-L-
tyrosinylamido)-6I-succinylamidocyclomaltoheptaose 14
A solution of compound 5 (1 g, 0.71 mmol), DIC (554 µl,
3.57 mmol) and HOBt (484 mg, 3.58 mmol) in dry DMF was
stirred for 2 h at 0 ЊC. Then, a solution of 2,3,4,6-tetra-O-
benzoyl-β--mannopyranosyl amine 3 (555 mg, 0.93 mmol) in
dry DMF (5 ml) was added to the reaction mixture and stirred
at room temperature for 72 h. DMF was removed in vacuo and
the residue precipitated in diethyl ether. The precipitate was
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 1 8 1 0 – 1 8 1 8
1816

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removed by filtration and washed several times with diethyl
ether to afford compound 14 (1.4 g, 99%) as a white powder
(Found C, 49.65; H, 5.31; N, 2.42 C89H111N3O47ؒ10H2O requires
added and the reaction mixture was stirred for 72 h at room
temperature. DMF was removed in vacuo and the residue added
to water (200 ml) to give a suspension, which was stirred for 30
min. The mixture was filtered and the filtrate freeze-dried to
afford the crude “high-mannosyl”-β-CD 15 (1.25 g, 90%) as a
white powder. Purification was effected by high performance
liquid chromatography (water–CH3CN, gradient elution from
100 to 0 in 40 min, tr of 23.84 min) to give the pure β-CD
derivative 15 (290 mg, 22%) (Found C, 46.92; H, 5.52; N, 2.50
C103H139N3O63ؒ10H2O requires C, 47.12; H, 6.06; N, 1.60%);
1
C, 49.60; H, 6.08; N, 1.95%); [α]D ϩ52Њ (c 1, DMF); H NMR
(500 MHz, DMSO-d6): δ 9.06 (s, 1 H, OH-Tyr), 8.82 (d, 1 H,
JNH,1 8.6 Hz, NH-Sugar), 8.02–7.28 (m, 22 H, OCOC6H5, NH-
Tyr (8.00 ppm), NH-βCD (7.60)); 1H NMR (500 MHz, DMSO-
d6–D2O 9 : 1 v/v) δ 6.94 (d, 2 H, Ja,b 8.6 Hz, H-a, H-aЈ), 6.88
(d, 2 H, H-b, H-bЈ), 5.84 (t, 1 H, J3,4 = J4,5 9.6 Hz, H-4), 5.80
(s, 1 H, H-1), 5.75 (dd, 1 H, J2,3 3.3 Hz, H-3), 5.65 (d, 1 H,
H-2), 4.79–4.76 (m, 7 H, H-1I-VII), 4.55–4.50 (m, 2 H, H-6,
H-6Ј), 4.45–4.40 (m, 2 H, H-5, H-α), 3.65–3.30 (m, 40 H,
H-2I-VII, H-3I-VII, H-4II-VII, H-5I-VII, H-6I-VII, H-6ЈII-
VII), 3.20 (t, 1 H, J3,4 = J4,5 8.8 Hz, H-4I), 3.15 (dd, 1 H, J5,6Ј
6.6 Hz, J6,6Ј 14.0 Hz, H-6ЈI), 2.80 (dd, 1 H, Jα,βЈ 4.8 Hz, Jβ,βЈ
13.9 Hz, H-β), 2.60 (dd, 1 H, H-βЈ), 2.25–2.15 (m, 4 H, 2 ×
CH2 Succ); 13C NMR (75 MHz, DMSO-d6): δ 172.71, 172.48
and 172.24 (NHCO), 166.11, 165.79 and 165.40 (OCOC6H5),
156.60, 134.55, 131.13, 130.31, 130.02, 129.69 and 115.59
(OCOC6H5 and C6H4OH), 102.80 (C-1I-VII), 84.14 (C-4I),
82.56 and 82.38 (C-4II-VII), 77.1 (C-1), 73.85, 73.26, 72.85,
71.38, 70.47 and 67.00 (C-2I-VII, C-3I-VII, C-5I-VII, C-2,
C-3, C-4, C-5), 63.27 (C-6), 60.69 (C-6II-VII), 54.44
(C-α), 40.68 (C-6I), 37.69 (C-β), 31.42 (2 × CH2 Succ); m/z
(ES-HRMS) 1996.6256 ([M ϩ Na]ϩ C89H111N3NaO47 requires
1996.6286).
1
[α]D ϩ77Њ (c 1, H2O–CH3CN 4 : 6 v/v); H NMR (500 MHz,
DMSO-d6): δ 9.11 (s, 1 H, OH-Tyr), 8.85 (d, 1 H, JNH,1A 8.7 Hz,
NH-Sugar), 8.14–7.50 (m, 12 H, OCOC6H5, NH-Tyr (7.98
ppm), NH-βCD (7.58 ppm)), 7.00 (d, 2 H, Ja,b 8.0 Hz, H-a, H-
aЈ), 6.60 (d, 2 H, H-b, H-bЈ), 5.80–3.25 (m, 91 H, OH-2 βCD,
OH-3 βCD, OH-6 βCD, H-1I-VII, H-2I-VII, H-3I-VII, H-4I-
VII, H-5I-VII, H-6I (3.58 ppm), H-6II-VII, H-6ЈI (3.28 ppm),
H-6ЈII-VII, H-1A (5.74 ppm), H-1B-C, H-2A-C, H-3A-C,
H-4A-C, H-5A-C, H-6A-C, H-6ЈA-C, H-α (4.53 ppm)), 2.80
(dd, 1 H, Jα,β 3.3 Hz, Jβ,βЈ 14.0 Hz, H-β), 2.59 (dd, 1 H, H-βЈ),
2.25–1.75 (m, 28 H, 2 × CH2 Succ, OCOCH3); 13C NMR
(75 MHz, DMSO-d6): δ 172.66, 172.50 and 172.00 (NHCO),
170.96, 170.57, 170.42 and 169.70 (OCOCH3), 166.47 and
165.67 (OCOC5H6), 156.54, 134.61, 130.62, 130.28, 129.67,
129.50, 128.46 and 115.61 (OCOC6H5 and C6H4OH), 102.83
(C-1I-VII), 98.99 and 97.55 (C-1B, C-1C), 84.09 (C-4I), 82.55
and 82.39 (C-4II-VII), 77.59 (C-1A), 76.82, 73.85, 73.26,
72.86, 70.48, 69.75, 69.61, 69.23, 68.91, 68.64, 66.39 and 65.94
(C-2I-VII, C-3I-VII, C-5I-VII, C-2A-C, C-3A-C, C-4A-C,
C-5A-C, C-6A (66.39 ppm)), 62.62 and 62.38 (C-6B, C-6C),
60.71 (C-6II-VII), 54.43 (C-α), 40.67 (C-6I), 37.81 (C-β),
31.54 and 31.38 (2 × CH2 Succ), 21.39, 21.29, 21.08, 21.02 and
20.82 (OCOCH3); m/z (ES-HRMS) 2448.7692 ([M ϩ Na]ϩ
C103H139N3NaO63 requires 2448.7663).
N-6I-Deoxy-(ꢀ-D-mannopyranosyl-L-tyrosinylamido)-6I-succinyl-
amidocyclomaltoheptaose 1
A solution of sodium methoxide (730 µl, 1 M in methanol) was
added to a solution of compound 14 (110 mg, 0.055 mmol) in
methanol/DMF (20 ml 1 : 1 v/v). The reaction mixture was
stirred at room temperature for 24 h, neutralized with acidic
resin (Amberlite IR 120) and filtered. Solvents were removed
in vacuo and the resulting residue was dissolved in water. The
aqueous layer was washed with CH2Cl2 (4 × 20 ml) and finally
freeze-dried to afford the crude “mannosyl”-β-CD 1 (83 mg,
96%). Purification by high-performance liquid chromatography
(water–CH3CN, gradient elution from 100 to 0 in 40 min, tr of
10.39 min) gave the final product 1 (42 mg, 48%) (Found C,
42.21; H, 6.35; N, 3.01 C61H95N3O43ؒ10H2O requires C, 42.14;
H, 6.62; N, 2.41%); 1H NMR (500 MHz, DMSO-d6): δ 9.12 (s,
1 H, OH-Tyr), 8.12 (d, 1 H, JNH,1 9.0 Hz, NH-Sugar), 8.08 (d,
1 H, JNH,α 8.4 Hz, NH-Tyr), 7.61 (t, 1 H, JNH,6I = JNH,6ЈI 5.1 Hz,
N-6I-Deoxy-(3,6-di-O-ꢁ-D-mannopyranosyl-ꢀ-D-mannopyrano-
syl-L-tyrosinylamido)-6I-succinylamidocyclomaltoheptaose 2
A solution of compound 15 (280 mg, 0.11 mmol) and sodium
methoxide (87 mg, 1.61 mmol) in methanol (10 ml) and dry
DMF (10 ml) was stirred at RT for ten days, then neutralized
with acidic resin (Amberlite IR 120) and filtered. Solvents were
removed in vacuo. The residue was dissolved in water and freeze
dried. High-performance liquid chromatography (water–
CH3CN, gradient elution from 100 to 0 in 40 min, tr 7.52 mn) of
the crude β-CD derivative thus obtained was performed to give
final material 2 (85 mg, 39%) (Found C, 41.14; H, 6.55; N, 2.53
C73H115N3O53ؒ13H2O requires C, 41.41; H, 6.66; N, 1.98%); 1H
NMR (500 MHz, DMSO-d6): δ 9.10 (s, 1 H, OH-Tyr), 8.26
(d, 1 H, JNH,1A 9.1 Hz, NH-Sugar), 8.01 (d, 1 H, JNH,α 8.9 Hz,
1
NH-βCD); H NMR (500 MHz, D2O): δ 7.04 (d, 2 H, Ja,b
8.1 Hz, H-a, H-aЈ), 6.82 (d, 2 H, H-b, H-bЈ), 5.19 (s, 1 H, H-1),
5.05–4.97 (7 d, 7 H, H-1I-VII), 4.30 (dd, 1 H, J5I,6I < 1 Hz, J6I,6ЈI
14.0 Hz, H-6I), 4.32 (dd, 1 H, Jα,β 4.1 Hz, Jα,βЈ 8.4 Hz, H-α),
4.07–3.27 (m, 44 H, H-2I-VII, H-3I (t, 4.02 ppm, J2I-3I = J3I-4I
9.5 Hz), H-3II-VII, H-4I (t, 3.36 ppm), H-4II-VII, H-5I-VII,
H-6II-VII, H-6ЈII-VII, H-2, H-3, H-4, H-5, H-6, H-6Ј), 2.97
(dd, 1 H, Jβ,βЈ 13.9 Hz, H-β), 2.84 (dd, 1 H, J5I,-6ЈI 9.8 Hz, H-6ЈI)
2.69–2.54 (m, 4 H, H-βЈ, 3 CH2 Succ), 2.35 (ddd, 1 H, CH2
Succ); 13C NMR (75 MHz, D2O): δ 175.53, 173.95 and 173.25
(NHCO), 155.47, 130.17, 129.42 and 115.55 (C6H4OH), 102.51
and 102.20 (C-1I-VII), 84.21 (C-4I), 81.39–80.72 (C-4II-VII),
78.07 (C-1), 73.42–66.65 (C-2I-VII, C-3I-VII, C-5I-VII, C-2,
C-3, C-4, C-5), 61.19 and 60.43 (C-6II-VII, C-6), 56.54 (C-α),
40.63 (C-6I), 36.77 (C-β), 31.30 and 30.75 (2 × CH2 Succ); m/z
(ES-HRMS) 1580.5300 ([M ϩ Na]ϩ C61H95N3NaO43 requires
1580.5237).
1
NH-Tyr), 7.58 (t, 1 H, JNH,6I = JNH,6ЈI 5.5 Hz, NH-βCD); H
NMR (500 MHz, D2O): δ 7.12 (d, 2 H, Ja,b 8.3 Hz, H-a,
H-aЈ), 6.89 (d, 2 H, H-b, H-bЈ), 5.30 (s, 1 H, H-1A), 5.20 (d,
1 H, J1,2 1.3 Hz, H-1B or H-1C), 5.13–5.05 (7 d, 7 H, H-1I-
VII), 4.92 (d, 1 H, J1,2 1.5 Hz, H-1B or H-1C), 4.37 (dd, 1 H,
J5I,6I 1.7 Hz, J6I,6ЈI 14.3 Hz, H-6I), 4.32 (dd, 1 H, Jα,β 3.8 Hz,
Jα,βЈ 8.3 Hz, H-α), 4.15–3.37 (m, 56 H, H-2I-VII, H-3I-VII,
H-4I (t, 3.44 ppm), H-4II-VII, H-5I-VII, H-6II-VII, H-6ЈII-
VII, H-2A-C, H-3A-C, H-4A-C, H-5A-C, H-6A-C, H-6ЈA-C),
3.06 (dd, 1 H, Jβ,βЈ 14.3 Hz, H-β), 2.93 (dd, 1 H, J5I,6ЈI 10.3
Hz, H-6ЈI) 2.76–2.58 (m, 4 H, H-βЈ, 3 CH2 Succ), 2.48 (ddd,
1 H, CH2 Succ); 13C NMR (75 MHz, D2O): δ 175.55, 174.00
and 173.20 (NHCO), 155.60, 130.22, 129.46 and 115.61
(C6H4OH), 102.65 (C-1I-VII, C-1B or C-1C), 98.81 (C-1B or
C-1C), 84.26 (C-4I), 82.44–65.82 (C-2I-VII, C-3I-VII, C-4II-
VII, C-5I-VII, C-1A (78.21 ppm), C-2A-C, C-3A-C, C-4A-C,
C-5A-C), 65.41 (C-6A), 61.43–60.00 (C-6II-VII, C-6A-C),
56.51 (C-α), 40.67 (C-6I), 36.89 (C-β), 31.39 and 30.78
(2 × CH2 Succ); m/z (ES-HRMS) 1904.6300 ([M ϩ Na]ϩ
C73H115N3NaO53 requires 1904.6294).
N-6I-Deoxy-(2,4-di-O-benzoyl-3,6-di-O-(2,3,4,6-tetra-O-acetyl-
ꢁ-D-mannopyranosyl)-ꢀ-D-mannopyranosyl-L-tyrosinylamido)-
6I-succinylamidocyclomaltoheptaose 15
A solution of the β-CD derivative 5 (800 mg, 0.57 mmol), DIC
(455 µl, 2.93 mmol) and HOBt (396 mg, 2.93 mmol) in dry
DMF (15 ml) was stirred for 2 h at 0 ЊC. Then, a solution of the
trisaccharide 4 (560 mg, 0.53 mmol) in dry DMF (10 ml) was
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 1 8 1 0 – 1 8 1 8
1817

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9 C. Péan, A. Wijkhuisen, F. Djedaïni-Pilard, J. Fischer, S. Doly,
M. Conrath, J-Y. Couraud, J. Grassi, B. Perly and C. Créminon,
Biochim. Biophys. Acta, 2001, 1541, 150–160.
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1999, 40, 2573–2576.
Acknowledgments
The authors gratefully acknowledge Pierre Guenot (CRMPO,
Université de Rennes I, France) and Serge Pilard (Université
Picardie Jules Verne, France) for mass spectrometry. This work
was supported by the Conseil Régional de Picardie under
the scientific IBFBio program. D. Yockot is grateful to the
“Ministère Gabonais des Finances et des Bourses et Stages” for
a doctoral fellowship.
13 N. H. Yu, C-C. Ling and D. R. Bundle, J. Chem. Soc., Perkin Trans.
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