Synthesis of carbosilane dendrimers having peripheral mannose and mannobiose

Article abstract of DOI:10.1016/j.tet.2005.01.090

The mannose monosaccharide derivative, acetylthiopropyl 2,3,4,6-tetra-O-acetyl-α-d-mannopyranoside (Man), and the mannobiose derivative, acetylthiopropyl 2,4,6-tri-O-acetyl-3-O-(2′,3′,4′, 6′-tetra-O-acetyl-α-d-mannopyranosyl)-α-d-mannopyranoside (α-1,3-Man), were synthesized respectively. These mannose derivatives were introduced into carbosilane dendrimer scaffolds of the zero and first generations. As a result, six carbosilane dendrimers were functionalized by Man and α-1,3-Man. Isothermal titration microcalorimetry was done to determine binding assay between mannose moieties of carbosilane dendrimer and concanavalin A. It was found that carbosilane dendrimers bound more efficiently to concanavalin A than free mannose (Me-α-Man) and mannobiose (Me-α-1,3-Man).

Full text of DOI:10.1016/j.tet.2005.01.090

Tetrahedron 61 (2005) 2751–2760
Synthesis of carbosilane dendrimers having peripheral mannose
and mannobiose
a
a
c
d
Tomonori Mori,a,b, Ken Hatano, Koji Matsuoka, Yasuaki Esumi, Eric J. Toone
*
and Daiyo Terunumaa,
*
aDepartment of Functional Materials Science, Saitama University, Shimo-ohkubo, Sakura-ku, Saitama 338-8570, Japan
bJapan Association for the Advancement of Medical Equipment, 3-42-6 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
cThe Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan
dDepartment of Chemistry, Duke University, B120 LSRC, Durham, NC 27708, USA
Received 28 December 2004; revised 20 January 2005; accepted 24 January 2005
Abstract—The mannose monosaccharide derivative, acetylthiopr0op0yl 0 2,3,4,6-tetra-O-acetyl-a-D-mannopyranoside (Man), and the
mannobiose derivative, acetylthiopropyl 2,4,6-tri-O-acetyl-3-O-(2 ,3 ,4 ,60-tetra-O-acetyl-a-D-mannopyranosyl)-a-D-mannopyranoside
(a-1,3-Man), were synthesized respectively. These mannose derivatives were introduced into carbosilane dendrimer scaffolds of the zero
and first generations. As a result, six carbosilane dendrimers were functionalized by Man and a-1,3-Man. Isothermal titration
microcalorimetry was done to determine binding assay between mannose moieties of carbosilane dendrimer and concanavalin A. It was
found that carbosilane dendrimers bound more efficiently to concanavalin A than free mannose (Me-a-Man) and mannobiose (Me-a-1,3-
Man).
q 2005 Elsevier Ltd. All rights reserved.
1. Introduction
carbohydrates are known to greatly enhance interaction
between the binding proteins (lectins) and the ligands
involving carbohydrates.9 This phenomenon, called ‘the
cluster effect’,10 enticed to generate a large number of
functional neoglyconconjugates to achieve superior binding
affinity.11 Dendrimers, one of the typical forms to manifest
the cluster effect, are targets of intensive investigation of the
cluster effect, because their structures are easy to control
and prepare.12 Glycodendrimers with peripheral mannosyl
group as one of the neoglycoconjugates have been shown to
mimic the structure and functions of the high mannose type
N-glycans.13–15
Oligosaccharide chains in natural glycoconjugates which
contain glycoproteins, glycolipids, and proteoglycans com-
ponents of extracellular matrixes and cell surfaces play
crucial roles in a variety of biological systems.1,2 Mannose
is one of the important and characteristic monosaccharides
in N-glycans (asparagine-linked oligosaccharides).3
A
group of N-glycans, which contain high levels of mannose
residues, is called a high-mannose type. The majority of
nascent peptides in the endoplasmic reticulum (ER) are
N-glycosylated with high-mannose type oligosaccharides.4,5
The functions of high-mannose type oligosaccharides in the
ER glycoprotein quality control have attracted recent
attention.6
Large number of dendrimers with mannose moieties have
been synthesized.13–15 However, only one case of glyco-
coating carbosilane dendrimer with mannose moiety has
been synthesized by Lindhorst et al.16 as far as we know.
They described the pathway to introduce mannose deriva-
tives into carbosilane dendrimer scaffold via a hydrosilyl-
ation reaction of a protected allyl mannoside with a
carbosilane containing Si–H end groups in the presence of
a platinum catalyst, thus leading to an Si–C linked structure.
The interactions between lectins (carbohydrate-binding
proteins),7 and carbohydrates in glycoconjugates play
principal roles in many cellular recognition processes.8 At
the monosaccharide level these interactions typically have
weak affinities (KD in mM). However, multivalent
Keywords: Carbosilane dendrimer; Mannose; Mannobiose; N-Glycan;
Glycocluster; Isothermal titration calorimetry.
* Corresponding authors. Tel./fax: C81 48 858 3535 (T.M.); tel./fax: C81
48 858 3532 (D.T.); e-mail addresses: tmori@fms.saitama-u.ac.jp;
teru@fms.saitama-u.ac.jp
The carbosilane dendrimer scaffold is easy to control the
number of branches at each generation and the chain length
between the terminal silicon. We have been preparing
0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2005.01.090

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T. Mori et al. / Tetrahedron 61 (2005) 2751–2760
carbosilane dendrimers with peripheral functional carbo-
hydrate moieties by a different route.17 Our approach to vary
the molecular design of carbohydrates containing carbo-
silane dendrimers is to control the methylene chain length
of dendrimer scaffolds and the aglycon moiety length of
carbohydrates. Peripheral globotriose clustered on carbo-
silane dendrimers were synthesized for the purpose of
neutralizing Shiga-toxin producing Escherichia coli
O157:H7.18
In this article, we describe syntheses of carbosilane
dendrimers with peripheral mannose, and their characteri-
zation by spectrometric methods. We also determined the
binding assay of concanavalin A (Con A), by the means of
isothermal titration microcalorimetry (ITC).
2. Results and discussion
Scheme 2. (a) HBr-AcOH, Ac2O, quant; (b) NaBH4/MeCN, 64%;
(c) NaOMe/MeOH, then PhCH(OMe)2, CSA/DMF, quant. (2 steps);
(d) AgOTf, MS4A/CH2Cl2, 66%; (e) 90% CF3COOH aq, then AcONa/Ac2O,
64% (2 steps); (f) allyl alcohol, BF3-OEt2/CH2Cl2, 43%; (g) AcSH,
AIBN/1,4-dioxane, 97%.
2.1. Preparation of mannose monosaccharide derivative
(Man)
Scheme 1 summarizes the synthetic steps of mannose
monosaccharide derivative, acetylthiopropyl 2,3,4,6-tetra-
O-acetyl-a-D-mannopyranoside (Man; 3). The treatment of
penta-O-acetylmannopyranose with allyl alcohol in the
presence of borone trifluoride diethyl ether complex as
Lewis acid produces allyl tetraacetylmannose (2).17b,19
Compound 3 was synthesized by the anti-Markovnikov
addition of the thio group to the allyl moiety of 2 although 3,
which is synthesized by another synthetic method of
activating 2,20-azobisisobutyronitrile (AIBN) irradiated in
a photochemical reactor.20 In this reaction, AIBN was
activated by heat at 80 8C.17d,e Each NMR signal of 3 was
was assigned by measurements of the high resolution mass
and NMR spectra to form the a-1,3-glycoside bond with
both mannose moieties. Next 7 was treated with aqueous
trifluoroacetic acid (90% v/v) to remove both 1,2-O-
ethylidene and 4,6-O-benzylidene groups, followed by
acetylation with acetic anhydride and sodium acetate to
provide 8.24 1-Allylation17b,19 and thioacetylation17d,e of the
allyl moiety were synthesized by the same method to
mannose monosaccharide to give 10.
2.3. Preparation of carbosilane dendrimders having
peripheral mannose
1
assigned by following measurements: H, 13C, DEPT, HH,
and HC COSY. Chemical shifts of 3 are described in
Section 4.
For the introduction of mannose derivatives, we used three
carbosilane dendrimer scaffolds: three-branched (Fan(0)3-
Br), four-branched (Ball(0)4-Br), and six-branched (Dumb-
bell(1)6-Br), as described in Figure 1. Fan(0)3-Br and
Ball(0)4-Br are the zero generation scaffolds which were
prepared with triallylphenylsilane and tetraallylsilane by
following three reaction steps: hydroxylation, mesylation,
and bromination.17b,25 On the other hand, Dumbbell(1)6-Br
is the first generation carbosilane dendrimer scaffold,
Scheme 1. (a) AcONa/Ac2O, then allyl alcohol, BF3-OEt2/CH2Cl2, 70% (2
steps); (b) AcSH, AIBN/1,4-dioxane, 73%.
2.2. Preparation of mannose disaccharide derivative
(a-1,3-Man)
Mannose disaccharide derivative, 1-O-(30-acetylthiopro-
pyl)-2,4,6-tri-O-acetyl-3-O-(2,3,4,6-tetra-O-acetyl-D-man-
nopyranosyl) D-mannopyranose (a-1,3-Man; 10), was
synthesized starting from D-mannose (Scheme 2). D-Man-
nose was converted to 1-bromo 2,3,4,6-tetra-O-acetyl
mannose (4)21 which will be used as a glycosyl acceptor
and will also lead to a donor. Compounds 5 and 6 were
synthesized by the method described in the literature:22,23
1,2-O-ethylidene protection of 4 was prepared by using
NaBH4 in acetonitrile at room temperature,22 then 4,6-O-
benzylidene protection of 5 using benzaldehyde dimethyl
acetal and 1,10-camphorsulfonic acid.23 Glycosylation of 6
with 4 in the presence in AgOTf, the reagent which was
used for the formation of a-glycoside, in dichloromethane at
K20 8C proceeded stereoselectively to give 7. Compound 7
Figure 1. Carbosilane dendrimer scaffolds.
Scheme 3. (a) NaOMe/MeOH, DMF, then Ac2O/pyridine, 66% (2 steps)
and (b) NaOMe/MeOH, then 0.1 mol/l NaOH aq, 61%.

T. Mori et al. / Tetrahedron 61 (2005) 2751–2760
2753
prepared with allylation of dichlorodimethylsilane followed
by hydrosilylation17d,25,26 with the first generation skeleton.
The resulting reactions were the same as for the zero
generation carbosilane dendrimer scaffolds.
Introduction of Man and Man-a-1,3-Man to carbosilane
dendrimer scaffolds was done concurrently with deacetyl-
ation, that is, using sodium methoxide/methanol and
N,N-dimethylformamide (Scheme 3). This reaction includes
de-O- and -S-acetylation, followed by SN2 replacement
reaction, and then acetylation for purification by silica gel
and gel permeation chromatography. After purification by
means of recycling GPC, products of mannose-coated
carbosilane dendrimers were obtained, and disulfide
byproducts (Man-SS-Man or a-1,3-Man-SS-a-1,3-Man)
was removed.
Figure 2. 1H NMR spectra (400 MHz, CDCl3 or D2O): (A) Fan(0)3-
Man(OAc) and (B) Fan(0)3-Man.
In summary, six carbosilane dendrimers were synthesized,
and functionalized with acetyl-protected derivatives of
mannose or mannose disaccharide (a-1,3-Man). The yields
of addition of mannose monosaccharide were 62–76%, and
those of a-1,3-Man were 30–35%. The difference in the
yields between the mannose and mannobiose derivatives
may be due to the bulkier structure of mannobioside.
1
All synthesized dendrimers were characterized by H and
13C NMR and high resolution mass spectrometry. From the
results of high resolution mass spectrometry, the proton or
sodium ion adduct peaks, [MCH]C or [MCNa]C, were
determined and these showed good agreement with the
calculated values, within the G5 ppm error margins.
1
Moreover, from the measurement of H NMR measure-
Figure 3. 13C NMR spectrum (100 MHz, D2O) of Fan(0)3-Man.
ments, we found the new signal at ca. 2.5 ppm (Fig. 2A)
which showed that a bond was formed between the sulfur
atom of saccharide moiety and the methylene carbon of the
corresponding carbosilane dendrimer scaffold. Thus, these
spectrometric results confirmed the structures of a carbo-
silane dendrimer with peripheral mannose and mannobiose.
The dendrimers with acetylated mannose moieties were
deacetylated by sodium methoxide/methanol, deacetylation
is saponification to yield the corresponding carbosilane
dendrimers with peripheral mannose and mannose di-
saccharide, and then purified by gel filtration. All six
types of carbosilane dendrimers functionalized by periph-
eral mannose moieties were synthesized and characterized
by the measurements of 1H and 13C NMR, and high
1
resolution mass spectrometry. Figures 2B and 3 show H
and 13C NMR spectra of Fan(0)3-Man, respectively. Signals
of methyl proton from the acetyl groups in mannose moiety
Figure 4. Carbosilane dendrimers having peripheral mannose and
mannobiose moieties.
Table 1. 13C NMR spectroscopic data (d values) of carbosilane dendrimers functionalized peripheral mannose moieties (I)
Mannose moieties
C-1
C-10
C-2
C-20
C-3
C-30
C-4
C-40
C-5
C-50
C-6
C-60
Fan(0)3-Man
Ball(0)4-Man
Dumbbell(1)6-Man
100.7
100.6
100.1
71.1
71.0
70.7
67.2
67.2
66.8
71.7
71.6
71.2
73.5
73.4
72.8
61.5
61.5
60.9
Fan(0)3-a-1,3-Man
100.5
102.9
100.6
103.0
100.7
103.1
70.5
70.8
70.6
70.9
70.7
71.0
79.3
71.0
79.4
71.1
79.6
71.3
66.7
67.1
66.9
67.3
67.0
67.5
73.8
73.4
73.8
73.5
74.0
73.6
61.1
61.4
61.3
61.5
61.5
61.8
Ball(0)4-a-1,3-Man
Dumbbell(1)6-a-1,3-Man

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T. Mori et al. / Tetrahedron 61 (2005) 2751–2760
Table 2. 13C NMR spectroscopic data (d values) of carbosilane dendrimers functionalized peripheral mannose moieties (II)
Dendrimer scaffolds
C-7
C-8
C-9
C-10
C-11
C-12
C-13
C-14
C-15
Fan(0)3-Man
Ball(0)4-Man
Dumbbell(1)6-Man
66.9
66.9
66.3
30.1
30.1
29.5
29.1
29.1
28.6
36.2
36.3
35.8
24.6
24.8
24.4
12.4
12.5
12.0
19.1
18.1
20.3
19.4
21.0
21.0
Fan(0)3-a-1,3-Man
Ball(0)4-a-1,3-Man
Dumbbell(1)6-a-1,3-Man
66.1
66.3
66.4
29.8
29.8
30.0
28.9
29.0
29.2
35.9
36.2
36.5
24.1
24.8
25.0
12.1
12.4
12.6
disappeared in Figure 2B, which is distinct from Figure 2A.
This is in agreement with the results of 13C NMR
measurements. Figure 4 shows all six carbosilane dendri-
mers. Signals of 13C NMR spectra are all assigned and
partly listed in Table 1 (mannose moieties) and Table 2
(carbosilane dendrimer scaffolds). Signals from mannose
moieties were assigned according to the published
results.24e,27 In Table 2, each signal displays good
agreement in spite of the differences between dendrimer
scaffolds and saccharides. Characteristic signals are
observed on Fan(0)3- and Dumbbell(1)6-scaffolds: these
were four signals of phenyl carbons at about 130 ppm on
Fan(0)3 types, and the signal of methyl carbon binding core
silicon atom at about K2 ppm on Dumbbell(1)6 types.
2.4. Binding affinity between carbosilane dendrimers
and concanavalin A
Con A is one of the most widely used lectins in biological
studies. Mannose residue is recognized specifically by Con
A. Recently, isothermal titration microcalorimetry (ITC)
has been utilized to study protein–carbohydrate inter-
action.10a A soluble protein is titrated with aliquots of a
soluble ligand in this measurement. The heat produced
during ligand addition serves as a reporter signal for
binding, which is yielded a binding constant, which, in turn
can be related to the free energy of binding. Since this
technique also directly measures binding enthalpies, an
entropy of binding can be readly calculated.
The bindings of tri-, tetra-, and hexavalent ligands Fan(0)3-
Man, Ball(0)4-Man, and Dumbbell(1)6-Man to dimeric Con
A (pH 5.2) were evaluated by titration microcalorimetry.
The titration microcalorimetry of all multivalent ligands
yielded curves indicative of simple reversible binding.
Figure 5 shows one of the titration curves for the carbosilane
dendrimer with peripheral mannose moieties (Fan(0)3-Man)
when bound to Con A in glutarate buffer (top) and the
resulting one-site fit of the integrated differential power
signal with respect to time (bottom). Table 3 lists the
calculated binding constant and other parameters of binding
between the carbosilane dendrimers and Con A. All of the
carbosilane dendrimers have higher binding constant values
with Con A, K, than the non-dendric mannose derivatives,
Me-a-Man and Me-a-1,3-Man,28 demonstrating cluster
glycoside effect. As for monosaccharide types of the
Figure 5. Calorimetric data for titration of concanavalin A, 0.21 mM, with
trivalent ligand Fan(0)3-Man, 2.1 mM. Both protein and ligand were
dissolved in buffer consisting of 50 mM 3,3-dimethylglutarate, 250 mM
NaCl, 1 mM CaCl2, and 1 mM MnCl2 adjusted pH 5.2. Top, raw (power vs
time) data; bottom integrated heat vs molar ratio of ligand. Solid line shows
best fit of data using a one-site model: nZ0.85; KZ2.09!104; DHZ
K9.2 kcal molK1. A fit to a two-site model does not provide a statistically
superior fit.
Table 3. Binding of carbosilane dendrimers to concanavalin A
K/MK1
DG
DH
TDSa
kcal molK1
Man
4.2!103
7.6!103
2.1!104
2.2!104
6.0!104
3.0!104
7.9!104
9.1!104
6.1!104
K4.9
K5.3
K5.9
K5.9
K6.5
K2.8
2.2
K1.5
K3.3
0.3
Me-a-Manb
Fan(0)3-Man
Ball(0)4-Man
Dumbbell(1)6-Man
K6.8
K9.2
K5.6
K3.6
3.0
Me-a-1,3-Manb
Fan(0)3-a-1,3-Man
Ball(0)4-a-1,3-Man
Dumbbell(1)6-a-1,3-Man
K6.0
K6.7
K6.8
K6.5
K7.4
K14.1
K9.8
K1.4
K7.4
K3.0
2.3
K4.2
a 298 K.
b See Ref. 28c.

T. Mori et al. / Tetrahedron 61 (2005) 2751–2760
2755
synthesized carbosilane dendrimers, the magnitude of the
effects depend on the amount of mannose in a dendrimer. In
the case of three-branched dendrimer scaffolds having
peripheral mannose, K value of the carbosilane dendrimer
was higher than that of the non-carbosilane dendrimer, and
other thermodynamic parameters were similar values.28d
However, the multivalency effect was not clearly measured
in the mannobiose-type of carbosilane dendrimers, because
these dendrimers became highly aggregated during titration
and the orientation of the saccharides could not match
tightly to the binding pockets of Con A.
mixture of sodium acetate (2.51 g, 30.62 mmol) and acetic
anhydride (25.0 mL, 263 mmol). Under an argon atmos-
phere, penta-O-acetyl-a-D-mannopyranose was dissolved in
dry-dichloromethane (123 mL) and allyl alcohol (9.50 mL,
139 mmol) was added, then the mixture was cooled to
K5 8C. Boron trifluoride diethyl ether complex (94 mL,
742 mmol) was dropped into the solution. The reaction
solution was stirred for 30 min at 0 8C, then stirred for 54 h
at room temperature. After the reaction, the solution was
poured into ice-water, washed with water, saturated aqueous
sodium hydrogen carbonate, brine, and dried over anhy-
drous magnesium sulfate. The solution was filtered through
a celite bed and concentrated. The residue was purified by
silica gel column chromatography with toluene–ethyl
acetate (5:1 (v/v)) as eluent to yield pure 2 (7.53 g, 70%
(2 steps)).
3. Conclusion
We synthesized six carbosilane dendrimers with peripheral
mannose and mannobiose. The structures of these dendri-
mers were characterized by measurements of NMR and
mass spectrometry. Isothermal titration microcalorimetry
(ITC) was done for determining the binding assay between
the carbosilane dendrimer and concanavalin A (Con A).
It was found that the carbosilane dendrimers bound to Con
A more frequently than to free mannose (Me-a-Man) and
mannobiose (Me-a-1,3-Man), thus showing the cluster
effect.
To a stirred solution of 2 (3.65 g, 9.40 mmol) and thioacetic
acid (13.4 mL, 188 mmol) in 1,4-dioxane (2.0 mL), 2,20-
azobisisobutyronitrile (AIBN; 7.72 g, 47.0 mmol) was
added at 50 8C under an argon atmosphere. The mixture
was stirred for 2.5 h at 80 8C, then cooled to room
temperature. Cyclohexene (5.0 mL, 49.3 mmol) was
added, and the mixture was stirred at room temperature
for 30 min. After evaporation, silica gel chromatography of
the residual syrup (toluene–ethyl acetate 10:1–5:1–3:1)
yielded sulfide 3 (3.16 g, 73%): 1H NMR (400 MHz,
CDCl3, TMS) d (ppm); 5.33 (1H, m, H-3), 5.28 (1H, m,
H-4), 5.24 (1H, dd, H-2, J1,2Z1.61 Hz, J2,3Z3.21 Hz), 4.81
4. Experimental
4.1. Analyses and GPC
(1H, H-1), 4.28 (1H, dd, H-6a, J5,6aZ5.35 Hz, J6a,6b
12.31 Hz), 4.11 (1H, dd, H-6b, J5,6bZ2.14 Hz, J6a,6b
Z
Z
NMR spectra were recorded with a Bruker DRX-400,
AM-400, and a Valian Gemini-2000 spectrometer. Fast
atom bombardment (FAB) and electron spray ionization
(ESI) mass spectra were obtained with a JEOL JMS-
HX110A spectrometer and a JEOL JMS-T100LC spectro-
meter, respectively. Optical rotations were measured with a
JASCO DIP-1000 digital polarimeter. Isothermal titration
microcalorimetry was performed using the MicroCal
Omega titration microcalorimeter. High resolution mass
spectrometry (HRMS) measurements were valid to
G5 ppm. Recycling preparative GPC was performed with
a LC-908W (Japan Analytical Industry Co., Ltd) connected
to an RI detector RI-5 (column, JAIGEL-1H-A and
JAIGEL-2H-A; solvent, chloroform).
12.31 Hz), 3.98 (1H, m, H-5), 3.77 (1H, m, OCH2CH2CH2-
S), 3.52 (1H, m, OCH2CH2CH2S), 2.97 (2H, t, OCH2CH2-
CH2S, JZ6.96 Hz), 2.34 (3H, s, CH3(SAc)), 2.16, 2.12,
2.06, 2.00 (12H, s, CH3(OAc)), 1.91 (2H, m, OCH2CH2-
CH2S); 13C NMR (100 MHz, CDCl3) d (ppm); 195.2 (C,
C]O(SAc)), 170.3, 169.7, 169.5, 169.4 (C, C]O(Ac)),
97.4 (CH, C-1), 69.2 (CH, C-2), 68.8 (CH, C-3), 68.3 (CH,
C-5), 66.5 (CH2, OCH2CH2CH2S), 65.8 (CH, C-4), 62.2
(CH2, C-6), 30.3 (CH3, CH3(SAc)), 28.9 (CH2, OCH2CH2-
CH2S), 25.5 (CH2, OCH2CH2CH2S), 20.6, 20.5, 20.41,
20.39 (CH3, CH3(OAc)).
4.3.2. 4,6-O-Benzylidene-1,2-ethylidene-b-D-manno-pyr-
anoside (6).23 Under an argon atmosphere, 1-bromo 2,3,4,6-
tetra-O-acetyl mannose ((4)21; 22.9 g, 55.6 mmol) was
dissolved in acetonitrile (130 mL), and sodium borohydrate
(10.5 g, 278 mmol) was added, then the mixture was stirred
for 22 h at room temperature. After the reaction, the solution
was diluted with ethyl acetate, washed with water and brine,
and dried over anhydrous magnesium sulfate. The solution
was filtered through a celite bed and concentrated. The
residue was purified by silica gel column chromatography
with n-hexane–ethyl acetate (5:1–3:1–2:1) yielded pure 522
(11.8 g, 64%).
4.2. Materials
Concanavalin A (Type IV, lot No. 102K7044) was
purchased from Sigma Chemical Company and dialyzed
with a glutarate buffer. Protein concentration was deter-
mined by the method of Edelhoch.29 Carbohydrate concen-
trations were determined by phenol-sulfuric acid method.30
For calorimetric measurements, water was purified with a
Millpore purification system that involved passage through
reverse osmosis, charcoal, and two ion exchange filters to
attain resistance of O10 MU cmK1
.
Under an argon atmosphere, 5 (5.73 g, 17.3 mmol) was
dissolved in methanol (5.0 mL), and sodium methoxide
(0.14 g, 2.60 mmol) was added, then the mixture was stirred
for 1 h at room temperature. After the reaction, IR120B
(HC) resin was added to neutralize the reaction solution,
and the suspension was filtered and evaporated. The residue
was dissolved in N,N-dimethylformamide (15.0 mL).
4.3. Reactions
4.3.1. Acetylthiopropyl 2,3,4,6-tetra-O-acetyl-a-D-man-
nopyranoside (3). D-Mannose (5.00 g, 27.8 mmol) was
acetylated to yield penta-O-acetyl-a-D-mannose by using a

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T. Mori et al. / Tetrahedron 61 (2005) 2751–2760
Benzaldehyde dimethylacetal (3.70 mL, 24.6 mmol) and
(C)-10-camphorsulfonic acid (379 mg, 1.63 mmol) was
added, and the mixture was stirred over evaporation for 6 h
at 30 8C. The solution was cooled to room temperature, and
triethylamine (0.45 mL, 3.34 mmol) added to neutralize.
The solution was evaporated, and purified by silica gel
column chromatography with n-hexane–ethyl acetate
(10:1–5:1–3:1–1:1) as eluent to yield pure 6 (5.08 g,
quant. (2 steps)).
aqueous sodium hydrogen carbonate, brine, and dried over
anhydrous magnesium sulfate. The solution was filtered
through a celite bed and concentrated. The residue was
purified by silica gel column chromatography with n-hex-
ane–ethyl acetate (1:1–1:2) as eluent to yield pure 8
(598 mg, 64% (2 steps)).
Under an argon atmosphere, 8 (4.08 g, 6.01 mmol) was
dissolved in dry-dichloromethane (27 mL) and allyl alcohol
(2.1 mL, 30.7 mmol) was added, then cooled to K5 8C.
Boron trifluoride diethyl ether complex (8.0 mL,
63.1 mmol) was dropped into the solution. The reaction
solution was stirred for 30 min at 0 8C, then stirred for 71 h
at room temperature. After the reaction, the solution was
poured onto ice-water, washed with water, saturated
aqueous sodium hydrogen carbonate, brine, and dried over
anhydrous magnesium sulfate. The solution was filtered
through a celite bed and concentrated. The residue was
purified by silica gel column chromatography with toluene–
ethyl acetate (5:1–3:1–2:1–1:1–0:1) as eluent to yield pure 9
(1.73 g, 43%): 1H NMR (400 MHz, CDCl3, TMS) d (ppm);
5.87 (1H, m, OCH2CH]CH2), 5.34–5.18 (6H, m, H-2, 3, 4,
4.3.3. 4,6-O-Benzylidene-1,2-ethylidene-3-O-(20,30,40,60-
tetra-O-acetyl-a-D-mannopyranosyl)-b-D-mannopyrano-
side (7). 4,6-O-Benzylidene-1,2-ethylidene-3-O-(20,30,40,60-
tetra-O-acetyl-a-D-mannopyranosyl)-b-D-mannopyranoside
(7). A solution of 2,3,4,6-tetra-O-acetyl-a-D-mannopyrano-
syl bromide (4) (298 mg, 0.72 mmol) and 4,6-O-benzyl-
idene-1,2-ethylidene-b-D-mannopyranoside (6) (100 mg,
0.34 mmol) in anhydrous dichloromethane (8.0 mL) was
stirred in the presence of activated MS4A (1.0 g) and silver
trifluoromethanesulfonate (228 mg, 0.89 mmol) was added
under an argon atmosphere. The reaction mixture was
stirred for 2 h at K20 8C. Further, silver trifluoromethane-
sulfonate (113 mg, 0.44 mmol) was added to the mixture
under an argon atmosphere, and the mixture was stirred for
40 min at K20 8C. Sodium carbonate (302 mg, 2.85 mmol)
was added to the reaction solution, then the solution was
filtered through a celite bed, diluted with chloroform,
washed with saturated aqueous sodium hydrogen carbonate,
brine, and dried over anhydrous magnesium sulfate. Then
the solution was filtered through a celite bed and
concentrated. The residue was purified by silica gel column
chromatography with toluene–ethyl acetate (5:1) as eluent
to yield pure 7 (139 mg, 66%): HRMS (ESI); calcd for
C29H36O15Na [MCNa]C 647.1952, found 647.1936.
[a]3D3ZK16.28 (cZ1.0 in CHCl3). 1H NMR (400 MHz,
CDCl3, TMS) d (ppm); 7.45–7.33 (5H, m, Ph), 5.59 (1H, s,
CH(4,6-bndn)), 5.45 (1H, m, H-30), 5.40 (1H, m, H-40), 5.34
30, OCH2CH]CH2), 5.01 (1H, m, H-20), 5.00 (1H, d, H-10
0
0
0
J1 ,2 Z1.61 Hz), 4.88 (1H, H-1), 4.30–3.98 (8H, m, H-6, 4 ,
50, 60, OCH2CH]CH2), 3.90 (1H, ddd, H-5, J4,5
Z
10.17 Hz, J5,6aZ5.35 Hz, J5,6bZ2.68 Hz), 2.21, 2.14,
2.13, 2.113, 2.106, 2.06, 1.99 (21H, s, CH3(OAc)); 13C
NMR (100 MHz, CDCl3) d (ppm); 170.65, 170.62, 170.4,
170.0, 169.9, 169.8, 169.5 (C, C]O(Ac)), 132.8 (CH,
OCH2CH]CH2), 118.5 (CH2, OCH2CH]CH2), 98.8 (CH,
C-10), 96.5 (CH, C-1), 74.6 (CH, C-3), 70.9 (CH, C-2), 69.9
(CH, C-20), 69.3 (CH, C-50), 68.7 (CH, C-5), 68.5 (CH2,
OCH2CH]CH2), 68.2 (CH, C-30), 67.7 (CH, C-40), 65.9
(CH, C-4), 62.5, 62.4 (CH2, C-6, 60), 20.9, 20.8, 20.73,
20.71, 20.63, 20.60, 20.59 (CH3, CH3(Ac)).
4.3.5. Acetylthiopropyl 2,4,6-tri-O-acetyl-3-O-(20,30,40,60-
tetra-O-acetyl-a-D-mannopyranosyl)-a-D-mannopyrano-
side (10). AIBN (2.11 g, 12.8 mmol) was added to a stirred
solution of 9 (1.73 g, 2.56 mmol) and thioacetic acid
(3.7 mL, 52.0 mmol) in 1,4-dioxane (1.5 mL) at 50 8C
under an argon atmosphere. The mixture was stirred for 3 h
at 80 8C, then cooled to room temperature. Cyclohexene
(1.5 mL, 14.8 mmol) was added, and the mixture was stirred
for 30 min at room temperature. After evaporation, the
residue was purified by silica gel column chromatography
with toluene–ethyl acetate (10:1–5:1–3:1–2:1) and size
exclusion chromatography (Sephadex LH-20; eluent:
methanol) as eluent to yield pure 10 (1.87 g, 97%):
HRMS (ESI); calcd for C31H44O19SNa [MCNa]C
775.2095, found 775.2065. [a]3D3ZC33.88 (cZ1.0 in
CHCl3). 1H NMR (400 MHz, CDCl3, TMS) d (ppm);
5.29, 5.26 (2H, m, H-3, 4), 5.23–5.19 (2H, m, H-2, 30), 5.02
(1H, q, JZ5.35 Hz, CH-(1,2-etdn)), 5.30–5.25 (2H, m, H-1,
0
20), 5.19 (1H, d, J1 ,2 Z1.61 Hz, H-1 ), 4.33–4.22 (4H, m,
H-2, 3, 6a, 60a), 4.11–4.04 (3H, m, H-4, 50, 60b), 3.78 (1H,
m, H-6b), 3.39 (1H, m, H-5), 2.11, 2.09, 2.05, 1.99 (12H, s,
Ac), 1.54 (3H, d, JZ5.35 Hz, CH3-(1,2-etdn)); 13C NMR
(100 MHz, CDCl3) d (ppm); 170.5, 169.8, 169.7, 169.6
(C, C]O (Ac)), 136.9 (C, C (Ph)), 128.9, 128.2, 125.9 (CH,
CH(Ph)), 104.7 (CH, CH (1,2-etdn)), 101.1 (CH, CH (4,6-
bndn)), 99.5 (CH, C-10), 96.8 (CH, C-1), 79.6 (C0H, C-3),
76.9 (CH,0 C-4), 75.9 (CH, C-50), 69.3 (CH, C-4 ), 68.80
(CH, C-3 ), 68.77 (CH, C-2), 68.4 (CH2, C-6), 66.4
(CH, C-20), 65.7 (CH, C-5), 62.6 (CH2, C-60), 21.8 (CH3,
CH3-(etdn)), 20.77, 20.75, 20.70, 20.66 (CH3, CH3-(Ac)).
0
0
4.3.4. Allyl 2,4,6-tri-O-acetyl-3-O-(20,30,40,60-tetra-O-
acetyl-a-D-mannopyranosyl)-a-D-mannopyranoside (9).
A solution of 7 (860 mg, 1.38 mmol) in 90% (v/v) aqueous
trifluoroacetic acid (10 mL) was stirred for 22 h at room
temperature. The solution was cooled in an ice-water bath
and neutralized with sodium carbonate. Then the solution
was evaporated and dried with a vacuum pump. Sodium
acetate (229 mg, 2.79 mmol) and acetic anhydride (15 mL,
158 mmol) were added to the residue, and the reaction
mixture was stirred for 1 h at 110 8C. To the reaction
mixture was added ice-water, and the mixture was extracted
with chloroform. The extract was washed with saturated
(1H, dd, H-20, J1 ,2 Z1.61 Hz, J2 ,3 Z2.14 Hz), 5.01 (1H, d,
0
0
0
0
H-10, J1 ,2 Z1.61 Hz), 4.82 (1H, d, H-1, J1,2Z1.60 Hz),
0
0
4.31–4.21 (2H, m, H-6a, 60a), 4.16 (1H, dd, H40, J3 ,40 Z
0
0
0
0
0
3.75 Hz, J4 ,5 Z10.17 Hz), 4.13–4.04 (3H, m, H-6b, 5 , 6 b),
3.86 (3H, m, H-5), 3.73 (1H, m, OCH2CH2CH2S), 3.50 (1H,
m, OCH2CH2CH2S), 2.94 (2H, t, OCH2CH2CH2S, JZ
6.96 Hz), 2.34 (3H, s, CH3(SAc)), 2.21, 2.14, 2.13, 2.12,
2.11, 2.06, 2.00 (21H, s, CH3(OAc)), 1.88 (2H, m,
OCH2CH2CH2S); 13C NMR (100 MHz, CDCl3) d (ppm);
195.2 (C, C]O(SAc)), 170.5, 170.4, 170.2, 169.8, 169.7,

T. Mori et al. / Tetrahedron 61 (2005) 2751–2760
2757
169.6, 169.4 (C, C]O(OAc)), 98.8 (CH, C-10), 97.3 (CH,
C-1), 74.8 (CH, C-3), 70.7 (CH, C-2), 69.8 (CH, C-20), 69.2
(CH, C-50), 68.7 (CH, C-5), 68.1 (CH, C-30), 67.5 (CH,
C-40), 66.4 (CH2, OCH2CH2CH2S), 65.7 (CH, C-4), 62.4,
62.2 (CH2, C-6, 60), 30.4 (CH3, CH3(SAc)), 29.1 (CH2,
OCH2CH2CH2S), 25.6 (CH2, OCH2CH2CH2S), 20.8, 20.7,
20.60, 20.56, 20.49, 20.45 (CH3, CH3(OAc)).
(1H, dd, H-6b, J5,6bZ2.14 Hz, J6a,6bZ12.32 Hz), 3.99 (1H,
ddd, H-5, J4,5Z9.63 Hz, J5,6aZ5.35 Hz, J5,6bZ2.14 Hz),
3.83 (1H, m, H-7a), 3.56 (1H, m, H-7b), 2.60 (2H, t, H-9,
J8,9Z6.96 Hz), 2.53 (2H, t, H-10, J10,11Z6.96 Hz), 2.16
(3H, s, CH3(Ac)), 2.11 (3H, s, CH3(Ac)), 2.05 (3H, s,
CH3(Ac)), 2.00 (3H, s, CH3(Ac)), 1.90 (2H, m, H-8), 1.58
(2H, m, H-11), 0.67 (2H, m, H-12); 13C NMR (100 MHz,
CDCl3) d (ppm);170.4, 169.8, 169.6, 169.5 (C, C]O(Ac)),
97.4 (CH, C-1), 69.3 (CH, C-2), 68.9 (CH, C-3), 68.3 (CH,
C-5), 66.4 (CH2, C-7), 65.9 (CH, C-4), 62.2 (CH2, C-6),
35.7 (CH2, C-10), 28.9 (CH2, C-8), 28.4 (CH2, C-9), 23.9
(CH2, C-11), 20.7, 20.54, 20.48, 20.45 (CH3, CH3(Ac)),
11.7 (CH2, C-12).
4.3.6. Introduction of mannose and mannobiose into
carbosilane dendrimer scaffolds: Fan(0)3-Man(OAc).
Under an argon atmosphere, a mixture of 3 (348 mg,
0.75 mmol) and a dendrimer scaffold (for example, Fan(0)-
Br: 56.4 mg, 0.12 mmol) was dissolved in N,N-dimethyl-
formamide (0.5 mL) and methanol (0.5 mL), and stirred at
room temperature for 20 min. Sodium methoxide in
methanol solution (1.0 M, 0.75 mL, 0.75 mmol) was
added to the reaction solution and stirred at room
temperature over night. Acetic acid (0.1 mL) was added to
the reaction solution, and stirred at room temperature
for 10 min, then evaporated in vacuo. The residue was
suspended in a mixture of pyridine (0.5 mL) and acetic
anhydride (1.0 mL, 10.5 mmol), and stirred at room
temperature over night. The reaction mixture was evapo-
rated in vacuo, added to ice-water and chloroform, then
washed with 1 M hydrochloric acid, saturated aqueous
sodium hydrogen carbonate, brine, and dried over anhy-
drous magnesium sulfate. The solution was filtered through
a celite bed and concentrated. The residue was purified by
silica gel column chromatography with hexane–ethyl
acetate (1:1–1:2–0:1) as the eluent to produce pure
Fan(0)3-Man(OAc): Yield 136 mg (76% (2 steps)).
HRMS (ESI): Calcd for C66H98O30S3SiNa [MCNa]C
1517.4972, found 1517.4990. [a]3D2ZC42.38 (cZ1.0 in
CHCl3). 1H NMR (400 MHz, CDCl3, TMS) d (ppm); 7.50–
7.34 (5H, Ph), 5.34–5.24 (6H, m, H-3, 4), 5.23 (3H, dd, H-2,
Dumbbell(1)6-Man(OAc). Yield 141.6 mg (62% (2 steps)).
HRMS (FAB): Calcd for C128H205O60S6Si3 [MCH]C
2978.0622, found 2978.0669. [a]2D9ZC40.18 (cZ1.0 in
1
CHCl3). H NMR (400 MHz, CDCl3, TMS) d (ppm); 5.32
(3H, m, H-3), 5.28 (3H, m, H-4), 5.23 (3H, dd, H-2, J1,2
Z
1.61 Hz, J2,3Z2.14 Hz), 4.82 (3H, d, J1,2Z1.61 Hz, H-1),
4.29 (3H, dd, H-6a, J5,6aZ4.82 Hz, J6a,6bZ12.32 Hz), 4.12
(3H, dd, H-6b, J5,6bZ2.14 Hz, J6a,6bZ12.32 Hz), 3.99 (3H,
ddd, H-5, J4,5Z9.63 Hz, J5,6aZ4.82 Hz, J5,6bZ2.14 Hz),
3.83 (3H, m, H-7a), 3.55 (3H, m, H-7b), 2.60 (6H, t, H-9,
J8,9Z6.96 Hz), 2.53 (6H, t, H-10, J10,11Z6.96 Hz), 2.16
(9H, s, CH3(Ac)), 2.11 (9H, s, CH3(Ac)), 2.05 (9H, s,
CH3(Ac)), 2.00 (9H, s, CH3(Ac)), 1.90 (6H, m, H-8), 1.57
(6H, m, H-11), 1.31 (2H, m, H-14), 0.67–0.62 (8H, m, H-12,
13), 0.56 (2H, m, H-15), K0.04 (3H, s, CH3(Si–Me)); 13C
NMR (100 MHz, CDCl3) d (ppm);170.4, 169.8, 169.6,
169.5 (C, C]O(Ac)), 97.4 (CH, C-1), 69.3 (CH, C-2), 68.9
(CH, C-3), 68.3 (CH, C-5), 66.4 (CH2, C-7), 65.9 (CH, C-4),
62.2 (CH2, C-6), 35.8 (CH2, C-10), 28.9 (CH2, C-8), 28.4
(CH2, C-9), 24.0 (CH2, C-11), 20.7, 20.55, 20.49, 20.46
(CH3, CH3(Ac)), 20.2 (CH2, C-15), 18.1 (CH2, C-14), 16.9
(CH2, C-13), 11.9 (CH2, C-12), K3.4 (CH3, CH3(Si–Me)).
J1,2Z1.60 Hz, J2,3Z2.14 Hz), 4.81 (3H, d, H-1, J1,2
1.60 Hz), 4.28 (3H, dd, H-6a, J5,6aZ5.35 Hz, J6a,6b
12.32 Hz), 4.11 (3H, dd, H-6b, J5,6bZ2.14 Hz, J6a,6b
Z
Z
Z
Fan(0)3-a-1,3-Man(OAc). Yield 61.2 mg (30% (2 steps)).
HRMS (ESI): Calcd for C102H146O54S3SiNa [MCNa]C
2381.7508, found 2381.7485. [a]3D2ZC32.38 (cZ1.0 in
CHCl3). 1H NMR (400 MHz, CDCl3, TMS) d (ppm); 7.47–
7.34 (50H, Ph), 5.29, 5.26 (6H, m, H-3, 4), 5.23–5.19 (6H, m,
H-2, 3 ), 5.02 (3H, m, H-20), 4.99 (3H, H-10), 4.82 (3H,
H-1), 4.29–4.21 (6H, m, H-6a, 60a), 4.13 (3H, dd, H-40,
12.32 Hz), 3.98 (3H, m, H-5), 3.80 (3H, m, H-7a), 3.52 (3H,
m, H-7b), 2.55 (6H, t, H-9, J8,9Z6.96 Hz), 2.53 (6H, t,
H-10, J10,11Z6.96 Hz), 2.16 (9H, s, CH3(Ac)), 2.10 (9H, s,
CH3(Ac)), 2.04 (9H, s, CH3(Ac)), 1.99 (9H, s, CH3(Ac)),
1.86 (6H, m, H-8), 1.60 (6H, m, H-11), 0.94 (6H, m, H-12);
13C NMR (100 MHz, CDCl3) d (ppm);170.4, 169.8, 169.7,
169.5 (C, C]O(Ac)), 136.0 (C, Ph), 133.8, 129.0, 127.8
(CH, Ph), 97.4 (CH, C-1), 69.4 (CH, C-2), 68.9 (CH, C-3),
68.3 (CH, C-5), 66.4 (CH2, C-7), 66.0 (CH, C-4), 62.2 (CH2,
C-6), 35.6 (CH2, C-10), 28.9 (CH2, C-8), 28.4 (CH2, C-9),
23.8 (CH2, C-11), 20.7, 20.6, 20.53, 20.51 (CH3, CH3(Ac)),
11.7 (CH2, C-12).
0
0
0
0
J3 ,4 Z3.75 Hz, J4 ,5 Z10.17 Hz), 4.12–4.03 (9H, m, H-6b,
50, 60b), 3.86 (3H, ddd, H-5, J4,5Z10.17 Hz, J5,6aZ5.36 Hz,
J5,6bZ2.14 Hz), 3.75 (3H, m, H-7a), 3.51 (3H, m, H-7b),
2.51 (12H, t, H-9, 10, J8,9Z J10,11Z6.96 Hz), 2.21, 2.14,
2.13, 2.102, 2.099, 2.05, 1.99 (63H, s, CH3(Ac)), 1.84 (6H,
m, H-8), 1.58 (6H, m, H-11), 0.92 (6H, m, H-12); 13C NMR
(100 MHz, CDCl3) d (ppm);170.4, 170.3, 170.2, 169.8,
169.65, 169.59, 169.4 (C, C]O(Ac)), 135.9 (C, Ph), 133.8,
129.1, 127.8 (CH, Ph), 98.8 (CH, C-10), 97.2 (CH, C-1),
74.9 (CH, C-3), 70.7 (CH, C-2), 69.7 (CH, C-20), 69.2 (CH,
C-50), 68.5 (CH, C-5), 68.0 (CH, C-30), 67.4 (CH, C-400),
66.3 (CH2, C-7), 65.7 (CH, C-4), 62.3, 62.1 (CH2, C-6, 6 ),
35.6 (CH2, C-10), 28.8 (CH2, C-8), 28.4 (CH2, C-9), 23.7
(CH2, C-11), 20.7, 20.61, 20.58, 20.52, 20.46, 20.42 (CH3,
CH3(Ac)), 11.7 (CH2, C-12).
Another carbosilane dendrimer with peripheral mannose or
mannobiose acetate was prepared by the same method as
Fan(0)3-Man. The mannobiose-bearing carbosilane dendri-
mers were synthesized using compound 10.
Ball(0)4-Man(OAc). Yield 120.3 mg (66% (2 steps)).
HRMS (FAB): Calcd for C80H125O40S4Si [MCH]C
1881.6399, found 1881.6445. [a]2D9ZC45.18 (cZ1.0 in
1
CHCl3). H NMR (400 MHz, CDCl3, TMS) d (ppm); 5.32
(1H, m, H-3), 5.28 (1H, m, H-4), 5.23 (1H, dd, H-2, J1,2Z
Ball(0)4-a-1,3-Man(OAc). Yield 81.1 mg (35% (2 steps)).
HRMS (FAB): Calcd for C128H189O72S4Si [MCH]C
3033.9780, found 3033.9751. [a]3D2ZC33.98 (cZ1.0 in
1.60 Hz, J2,3Z2.14 Hz), 4.82 (1H, d, J1,2Z1.60 Hz, H-1),
4.29 (1H, dd, H-6a, J5,6aZ5.35 Hz, J6a,6bZ12.32 Hz), 4.12

2758
T. Mori et al. / Tetrahedron 61 (2005) 2751–2760
CHCl3). 1H NMR (400 MHz, CDCl3, TMS) d (ppm); 5.30,
5.26 (2H0 , m, H-3, 4), 5.23–5.19 (2H, m, H-2, 30), 5.02 (1H,
m, H-2 ), 5.00 (1H, H-10), 4.83 (1H, H-1), 4.29–4.21 (2H, m,
H-6a, 60a), 4.14 (1H, m, H40), 4.13–4.03 (3H, m, H-6b, 50,
60b), 3.88 (1H, m, H-5), 3.79 (1H, m, H-7a), 3.54 (1H, m,
H-7b), 2.56 (2H, t, H-9, J8,9Z6.96 Hz), 2.51 (2H, t, H-10,
J10,11Z6.96 Hz), 2.21, 2.14, 2.13, 2.11, 2.05, 1.99 (21H, s,
CH3(Ac)), 1.87 (2H, m, H-8), 1.57 (2H, m, H-11), 0.65 (2H,
m, H-12); 13C NMR (100 MHz, CDCl3) d (ppm); 170.5,
170.4, 170.3, 169.9, 169.71, 169.65, 169.4 (C, C]O(Ac)),
98.8 (CH, C-10), 97.3 (CH, C-1), 75.0 (CH, C-3), 70.8 (CH,
C-2), 69.8 0(CH, C-20), 69.2 (CH, C-50), 68.6 (CH, C-5), 68.1
(CH, C-3 ), 67.4 (CH, C-40), 66.4 (CH2, C-7), 65.7
(CH, C-4), 62.4, 62.2 (CH2, C-6, 60), 35.9 (CH2, C-10),
28.9 (CH2, C-8), 28.6 (CH2, C-9), 24.0 (CH2, C-11), 20.8,
20.68, 20.65, 20.58, 20.53, 20.47 (CH3, CH3(Ac)), 11.9
(CH2, C-12).
(C, Ph), 134.8, 129.8, 128.8 (CH, Ph), 100.7 (CH, C-1), 73.5
(CH, C-5), 71.7 (CH, C-4), 71.1 (CH, C-2), 67.2 (CH, C-3),
66.9 (CH2, C-7), 61.5 (CH2, C-6), 36.2 (CH2, C-10), 30.1
(CH2, C-8), 29.1 (CH2, C-9), 24.6 (CH2, C-11), 12.4 (CH2,
C-12).
Another carbosilane dendrimer with peripheral mannose or
mannobiose acetate was deacetylated by the same method
as Fan(0)3-Man. Carbosilane dendrimers with peripheral
mannose or mannobiose which have no protective group of
saccharide moieties were synthesized.
Ball(0)4-Man. Yield 64.8 mg (82%). HRMS (ESI): Calcd
for C48H92O24S4SiNa [MCNa]C 1231.4528, found
1231.4581. [a]D24ZC52.78 (cZ1.0 in H2O). 1H NMR
(400 MHz, D2O) d (ppm); 4.87 (1H, H-1), 3.95 (1H, m,
H-2), 3.89–3.78 (4H, m, H-3, 6a, 7a), 3.74 (1H, m, H-6b),
3.61 (2H, m, H-5, 7b), 2.65 (4H, m, H-9, 10), 1.93 (2H, m,
H-8), 1.66 (2H, m, H-11), 0.76 (2H, m, H-12); 13C NMR
(50 MHz, D2O) d (ppm); 100.6 (CH, C-1), 73.4 (CH, C-5),
71.6 (CH, C-4), 71.0 (CH, C-2), 67.2 (CH, C-3), 66.9 (CH2,
C-7), 61.5 (CH2, C-6), 36.3 (CH2, C-10), 30.1 (CH2, C-8),
29.1 (CH2, C-9), 24.8 (CH2, C-11), 12.5 (CH2, C-12).
Dumbbell(1)6-a-1,3-Man(OAc). Yield 61.3 mg (31% (2
steps)). HRMS (FAB): Calcd for C200H301O108S6Si3 [MC
H]C 4706.5693, found 4706.5679. [a]3D3ZC33.08 (cZ1.0
1
in CHCl3). H NMR (400 MHz, CDCl3, TMS) d (ppm);
5.30, 5.26 (6H, m, H-3, 4), 5.23–5.19 (6H, m, H-2, 30), 5.02
(3H, m, H-20), 4.99 (3H, d, H-10, J1 ,2 Z1.61 Hz), 4.83 (3H,
0
0
H-1), 4.29–4.21 (6H, m, H-6a, 60a), 4.14 (3H, dd, H-40,
Dumbbell(1)6-Man. Yield 33.6 mg (81%). HRMS (FAB):
Calcd for C80H156O36S6Si3Na [MCNa]C 1991.7906, found
1991.7937. [a]D30ZC46.38 (cZ1.0 in H2O). 1H NMR
(400 MHz, D2O) d (ppm); 4.90 (3H, d, J1,2Z1.0 Hz, H-1),
3.99 (3H, m, H-2), 3.92–3.75 (15H, m, H-3, 4, 6, 7a), 3.68–
3.55 (6H, m, H-5, 7b), 2.66 (6H, m, H-9), 2.62 (6H, m,
H-10), 1.93 (6H, m, H-8), 1.66 (6H, m, H-11), 1.48 (2H, m,
H-14), 0.82–0.65 (10H, m, H-12, 13, 15), 0.06 (3H, s, Si–
CH3); 13C NMR (50 MHz, D2O) d (ppm); 100.1 (CH, C-1),
72.8 (CH, C-5), 71.2 (CH, C-4), 70.7 (CH, C-2), 66.8 (CH,
C-3), 66.3 (CH2, C-7), 60.9 (CH2, C-6), 35.8 (CH2, C-10),
29.5 (CH2, C-8), 28.6 (CH2, C-9), 24.4 (CH2, C-11), 21.0
(CH2, C-15), 20.3 (CH2, C-14), 19.1 (CH2, C-13), 12.0
(CH2, C-12), K2.7 (CH3, Si–CH3).
0
0
0
0
J3 ,4 Z3.75 Hz, J4 ,5 Z10.17 Hz), 4.13–4.03 (9H, m, H-6b,
50, 60b), 3.87 (3H, m, H-5), 3.79 (3H, m, H-7a), 3.54 (3H, m,
H-7b), 2.56 (6H, t, H-9, J8,9Z6.96 Hz), 2.51 (6H, t, H-10,
J10,11Z6.96 Hz), 2.21, 2.14, 2.13, 2.11, 2.05, 1.99 (63H, s,
CH3(Ac)), 1.87 (6H, m, H-8), 1.56 (6H, m, H-11), 1.29 (2H,
m, H-14), 0.65–0.60 (8H, m, H-12, 13), 0.54 (2H, m, H-15),
K0.05 (3H, s, CH3(Si–Me)); 13C NMR (100 MHz, CDCl3)
d (ppm); 170.5, 170.4, 170.3, 169.9, 169.75, 169.70, 169.5
(C, C]O(Ac)), 98.9 (CH, C-10), 97.3 (CH, C-1), 75.01 (CH,
C-3), 70.8 (CH, C-2), 69.8 (CH, C-20), 69.30(CH, C-5 ), 68.6
(CH, C-5), 68.1 (CH, C-30), 67.5 (CH, C-4 ), 66.5 (CH2, C-
7), 65.8 (CH, C-4), 62.4, 62.2 (CH2, C-6, 60), 35.9 (CH2, C-
10), 29.0 (CH2, C-8), 28.6 (CH2, C-9), 24.1 (CH2, C-11),
20.8, 20.72, 20.70, 20.62, 20.57, 20.52 (CH3, CH3(Ac)),
20.4 (CH2, C-15), 18.2 (CH2, C-14), 17.0 (CH2, C-13), 12.0
(CH2, C-12), K3.4 (CH3, CH3(Si–Me)).
Fan(0)3-a-1,3-Man. Yield 44.6 mg (quant.). HRMS (FAB):
Calcd for C60H104O33S3SiNa [MCNa]C 1499.5289, found
1499.5278. [a]D22ZC78.78 (cZ0.87 in H2O). 1H NMR
(400 MHz, D2O) d (ppm); 7.52–7.25 (5H, m, Ph), 5.14 (3H,
H-10), 4.82 (3H, H-1), 4.08 (6H,0 m, H-2, 20), 3.92–3.50
(36H, m, H-3, 4, 5, 6, 30, 40, 50, 6 , 7), 2.52 (12H, m, H-9,
10), 1.85 (6H, m, H-8), 1.57 (6H, m, H-11), 0.91 (6H, m,
H-12); 13C NMR (50 MHz, D2O) d (ppm); 137.1 (C, Ph),
134.6, 129.8, 128.6 (CH, Ph), 102.9 (CH, C-10), 100.5 (CH,
C-1), 79.3 (CH, C-3), 73.8 (CH, C-5), 73.4 (CH, C-50), 71.0
(CH, C-30), 70.8 (CH, C-20), 70.5 (CH, C-2), 67.1 (CH,
C-40), 66.7 (CH, C-4), 66.1 (CH2, C-7), 61.4 (CH2, C-60),
61.1 (CH2, C-6), 35.9 (CH2, C-10), 29.8 (CH2, C-8), 28.9
(CH2, C-9), 24.1 (CH2, C-11), 12.1 (CH2, C-12).
4.3.7. Deprotection of carbosilane dendrimers with
mannose and mannobiose: Fan(0)3-Man. A solution of
sodium methoxide in methanol (12.7 mg, 235 mmol) was
added to a solution of Fan(0)3-Man(OAc) (135.8 mg,
90.8 mmol) in methanol (1.5 mL) at room temperature
under an argon atmosphere. The solution was stirred for 1 h,
then the aqueous solution of sodium hydroxide (0.1 M) was
added and was stirred at room temperature over night. After
neutralizing with acetic acid, the solution was evaporated in
vacuo. The residue was subjected to Sephadex G-25 size
exclusion chromatography eluting with 5% (v/v) aqueous
solution of acetic acid. The fractions containing carbosilane
dendrimer were combined and lyophilized to yield Fan(0)3-
Man as a white solid (54.8 mg (61%)): HRMS (ESI): Calcd
for C42H74O18S3SiNa [MCNa]C 1013.3704, found
1013.3696. [a]D27ZC49.08 (cZ1.0 in H2O). 1H NMR
(400 MHz, D2O) d (ppm); 7.52–7.19 (5H, m, Ph), 4.84
(3H, H-1), 3.93 (3H, m, H-2), 3.89–3.64 (15H, m, H-3, 4, 6,
7a), 3.64–3.45 (6H, m, H-5, 7b), 2.55 (6H, m, H-9), 2.48
(6H, m, H-10), 1.84 (6H, m, H-8), 1.56 (6H, m, H-11), 0.89
(6H, m, H-12); 13C NMR (100 MHz, D2O) d (ppm); 137.3
Ball(0)4-a-1,3-Man. Yield 75.5 mg (quant.). HRMS (ESI):
Calcd for C72H132O44S4SiNa [MCNa]C 1879.6641, found
1879.6622. [a]D30ZC100.48 (cZ1.0 in H2O). 1H NMR
(200 MHz, D2O) d (ppm); 5.14 (H-10), 4.92 (1H, H-1), 4.15
(2H, m, H-2, 20), 4.08–3.60 (12H, m, H-3, 4, 5, 6, 30, 40, 50,
60, 7), 2.71 (4H, m, H-9, 10), 2.02 (2H, m, H-8), 1.73 (2H,
m, H-11), 0.82 (2H, m,0 H-12); 13C NMR (50 MHz, D2O) d
(ppm); 103.0 (CH, C-1 ), 100.6 (CH, C-1), 79.4 (CH, C-3),
73.8 (CH, C-5), 73.5 (CH, C-50), 71.1 (CH, C-30), 70.9 (CH,

T. Mori et al. / Tetrahedron 61 (2005) 2751–2760
2759
C-20), 70.6 (CH, C-2), 67.3 (CH, C-40), 66.9 (CH, C-4), 66.3
(CH2, C-7), 61.5 (CH2, C-60), 61.3 (CH2, C-6), 36.2 (CH2,
C-10), 29.8 (CH2, C-8), 29.0 (CH2, C-9), 24.8 (CH2, C-11),
12.4 (CH2, C-12).
Freeze, H., Hart, G., Marth, J., Eds.; Cold Spring Harbor
Laboratory: Cold Spring Harbor, 1999.
3. Kuo, C.-C.; Takahashi, N.; Swanson, A. F.; Dzeki, Y.;
Hakomori, S.-I. J. Clin. Invest. 1996, 98, 2813–2818.
4. Helenius, A.; Trombetta, E. S.; Herbert, D. N.; Simons, J. F.
Trends Cell Biol. 1997, 7, 193–200.
Dumbbell(1)6-a-1,3-Man. Yield: 33.8 mg (90%). HRMS
(ESI): Calcd for C116H216O66S6Si3Na2/2 [MC2Na]2C/2
1493.5487, found 1493.5482. [a]2D9ZC48.38 (cZ1.0 in
H2O). 1H NMR (400 MHz, D2O) d (ppm); 5.15 (3H, H-10),
4.86 (3H, H-1), 4.09 (6H, m, H-2, 20), 4.00–3.65 (33H, m,
H-3, 4, 5, 6, 30, 40, 50, 60, 7a), 3.63 (3H, m, H-7b), 2.65 (12H,
m, H-9, 10), 1.94 (6H, m, H-8), 1.65 (6H, m, H-11), 1.46
(2H, m, H-14), 0.75 (8H, m, H-12, 13), 0.69 (2H, m, H-15),
0.05 (3H, s, Si–CH3); 13C NMR (50 MHz, D2O) d (ppm);
103.1 (CH, C-10), 100.7 (CH, C-1), 79.6 (CH, C-3), 74.0
(CH, C-5), 73.6 (CH, C-50), 71.3 (CH, C-30), 71.0 (CH,
C-20), 70.7 (CH, C-2), 67.5 (CH, C-40), 67.0 (CH, C-4), 66.4
(CH2, C-7), 61.8 (CH2, C-60), 61.5 (CH2, C-6), 36.5 (CH2,
C-10), 30.0 (CH2, C-8), 29.2 (CH2, C-9), 25.0 (CH2, C-11),
21.0 (CH2, C-15), 19.4 (CH2, C-14), 18.1 (CH2, C-13), 12.6
(CH2, C-12), K1.6 (CH3, Si–CH3).
5. Wada, I.; Kai, M.; Imai, S.; Kanoh, H. EMBO J. 1997, 16,
5420–5432.
6. Ellgaad, L.; Helenius, A. Nat. Rev. Mol. Cell Biol. 2003, 4,
181–191.
7. Lis, H.; Sharon, N. Chem. Rev. 1998, 98, 637–674.
8. (a) Dwek, R. A. Chem. Rev. 1996, 96, 683–720. (b) Toone,
E. J. Curr. Opin. Struct. Biol. 1994, 4, 719–728.
9. (a) Lindhorst, Th. K. Top. Curr. Chem. 2002, 218, 201–235.
(b) Kiessling, L. L.; Gestwicki, J. E.; Strong, L. E. Curr. Opin.
Chem. Biol. 2000, 4, 696–703. (c) Jayaraman, N.; Nepogodiev,
S. A.; Stoddart, J. F. Chem. Eur. J. 1997, 3, 1193–1199.
(d) Roy, R. Top. Curr. Chem. 1997, 187, 241–274.
10. (a) Lundquist, J. J.; Toone, E. J. Chem. Rev. 2002, 102,
555–578. (b) Lee, R. T.; Lee, Y. C. Glycoconjugate J. 2000,
17, 543–551. (c) Mammen, M.; Choi, S.-K.; Whitesides, G. M.
Angew. Chem., Int. Ed. 1998, 37, 2755–2794.
11. (a) Kawaguchi, K.; Lee, Y. C. Proteins Nucleic Acids Enzymes
1980, 25, 707–724. (b) Stowell, C. P.; Lee, Y. C. Adv.
Carbohydr. Chem. Biochem. 1980, 37, 225–281. (c) Connolly,
D. T.; Townsend, R. R.; Kawaguchi, K.; Bell, W. R.; Lee,
Y. C. J. Biol. Chem. 1982, 257, 939–945. (d) Lee, R. T.; Lin,
P.; Lee, Y. C. Biochemistry 1984, 23, 4255–4261. (e) Lee,
Y. C.; Lee, R. T. Acc. Chem. Res. 1995, 28, 321–327.
12. (a) Davis, B. G. J. Chem. Soc., Perkin Trans. 1 1999,
3215–3237. (b) Cloninger, M. J. Curr. Opin. Chem. Biol. 2002,
6, 742–748.
4.4. Calorimetry
Isothermal titration microcalorimetry was performed using
the MicroCal Omega titration microcalorimeter. Details of
instrument design and data analysis are described by
Wiseman et al.31 A solution of concanavalin A (0.21 mM)
in a buffer of 50 mM 3,3-dimethylglutarate, 250 mM NaCl,
and 1 mM each of CaCl2 and MnCl2 at pH 5.2 were placed
in the sample cell. Carbosilane dendrimer solutions
([mannose]Z2.1 mM) in a buffer identical to that used for
protein solutions were added in 10 mL increments during
30 s, with 3 min intervals between injections. Each calori-
metric titration was performed at a sample cell temperature
of 298 K. Protein concentrations were determined spectro-
metrically using an extinction coefficient of 3280Z1.24 for a
1 mg/mL of solution.
´
13. (a) Page, D.; Zanini, D.; Roy, R. Bioorg. Med. Chem. 1996, 4,
´
1949–1961. (b) Page, D.; Aravind, S.; Roy, R. Chem.
´
Commun. 1996, 1913–1914. (c) Page, D.; Roy, R. Bioorg.
´
Med. Chem. Lett. 1996, 6, 1765–1770. (d) Page, D.; Roy, R.
´
Bioconjugete Chem. 1997, 8, 714–723. (e) Page, D.; Roy, R.
´
Glycoconjugete J. 1997, 14, 345–356. (f) Roy, R.; Page, D.;
Perez, S. F.; Bencomo, V. V. Glycoconjugete J. 1998, 15,
251–263. (g) Das, S. K.; Trono, M. C.; Roy, R. Methods
Enzymol. 2003, 362, 3–17.
The heat evolved upon each injection was digitally
recorded, and the data were integrated to generate a titration
curve upon completion of the experiment. The stoichio-
metry of the association, n, binding constant, K, and the
change in enthalpy, DH, were obtained from a nonlinear
least-squares fit using the Origin software program. All data
are presented on a valency-corrected basis.
14. (a) Lindhorst, Th. K. Nachr. Chem. Tech. Lab. 1996, 44,
1073–1079. (b) Lindhorst, Th. K.; Kieburg, C.; Krallmann-Wenzel,
¨
U. Glycoconjugate J. 1998, 15, 605–613. (c) Konig, B.; Fricke,
T.; Waßmann, A.; Krallmann-Wensel, U.; Lindhorst, Th. K.
¨
Tetrahedron Lett. 1998, 39, 2307–2310. (d) Kotter, S.;
Krallmann-Wensel, U.; Ethlers, S.; Lindhorst, Th. K.
J. Chem. Soc., Perkin Trans. 1 1998, 2193–2200. (e) Dubber,
M.; Lindhorst, Th. K. J. Org. Chem. 2000, 65, 5275–5281. (f)
Lindhorst, Th. K.; Dubber, M.; Krallmann-Wenzel, U.; Ehlers,
S. Eur. J. Org. Chem. 2000, 2027–2034. (g) Horst, A. K.;
Acknowledgements
This work was supported by a Health and Labour Sciences
Research Grant for Research on Advanced Medical
Technology (14-N-015) from the Ministry of Health,
Labour, and Welfare, Japan.
¨
Kotter, S.; Lindhorst, Th. K.; Ludwig, A.; Brandt, E.;
Wagener, C. Med. Microbiol. Immunol. 2001, 190, 145–149.
(h) Dubber, M.; Lindhorst, Th. K. Synthesis 2001, 327–330. (i)
Patel, A.; Lindhorst, Th. K. J. Org. Chem. 2001, 66,
¨
2674–2680. (j) Rockendorf, N.; Sperling, O.; Lindhorst,
Th. K. Aust. J. Chem. 2002, 55, 87–93. (k) Boysen,
M. M. K.; Elsner, K.; Sperling, O.; Lindhorst, Th. K. Eur.
¨
J. Org. Chem. 2003, 4376–4386. (l) Rochendorf, N.;
Lindhorst, Th. K. J. Org. Chem. 2004, 69, 4441–4445. (m)
References and notes
¨
Kohn, M.; Benito, J. M.; Ortiz Mellet, C.; Lindhorst, Th. K.;
´ ´
Garcıa Fernandez, J. M. ChemBioChem 2004, 5, 771–777.
1. Varki, A. Glycobiology 1993, 3, 97–130.
2. Essentials of Glycobiology; Varki, A., Cummings, R., Esko, J.,
15. (a) Ashton, P. R.; Hounsell, E. F.; Jayaraman, N.; Nilsen,

2760
T. Mori et al. / Tetrahedron 61 (2005) 2751–2760
T. M.; Spencer, N.; Fraser Stoddart, J.; Young, M. J. Org.
J. Org. Chem. 1982, 47, 2701–2707. (d) Itoh, Y.; Tejima, S.
Chem. Pharm. Bull. 1984, 32, 957–966. (e) Szurmai, Z.;
Janossy, L.; Szlagyi, Z.; Vekey, K. J. Carbohydr. Chem. 1998,
17, 417–437.
Chem. 1998, 63, 3429–3437. (b) Nakata, E.; Nagase, T.;
Shinkai, S.; Hamachi, I. J. Am. Chem. Soc. 2004, 126,
490–495.
16. Boysen, M. M. K.; Lindhorst, Th. K. Tetrahedron 2003, 59,
3895–3898.
25. (a) Terunuma, D.; Kato, T.; Nishio, R.; Matsuoka, K.;
Kuzuhara, H.; Aoki, Y.; Nohira, H. Chem. Lett. 1998,
59–60. (b) Terunuma, D.; Kato, T.; Nishio, R.; Aoki, Y.;
Nohira, H.; Matsuoka, K.; Kuzuhara, H. Bull. Chem. Soc. Jpn.
1999, 72, 2129–2134. (c) Tsuchida, T.; Shimazaki, C.; Hatano,
K.; Matsuoka, K.; Aoki, Y.; Nohira, H.; Esumi, Y.; Terunuma,
D. Kobunshi Ronbunshu 2003, 60, 561–568.
17. (a) Matsuoka, K.; Terabatake, M.; Saito, Y.; Hagihara, C.;
Esumi, Y.; Terunuma, D.; Kuzuhara, H. Bull. Chem. Soc. Jpn.
1998, 71, 2709–2713. (b) Matsuoka, K.; Terabatake, M.;
Esumi, Y.; Terunuma, D.; Kuzuhara, H. Tetrahedron Lett.
1999, 40, 7839–7842. (c) Matsuoka, K.; Saito, Y.; Terunuma,
D.; Kuzuhara, H. Kobunshi Ronbunshu 2000, 57, 691–695.
(d) Matsuoka, K.; Kurosawa, H.; Esumi, Y.; Terunuma, D.;
Kuzuhara, H. Carbohydr. Res. 2000, 329, 765–772.
(e) Matsuoka, K.; Oka, H.; Koyama, T.; Esumi, Y.; Terunuma,
D.; Kuzuhara, H. Tetrahedron Lett. 2001, 42, 3327–3330.
(f) Matsuoka, K.; Ohtawa, T.; Hinou, H.; Koyama, T.; Esumi,
Y.; Nishimura, S.-I.; Hatano, K.; Terunuma, D. Tetrahedron
Lett. 2003, 44, 3617–3620.
26. (a) van der Made, A. W.; van Leeuwen, P. W. N. M. J. Chem.
Soc., Chem. Commun. 1992, 1400–1401. (b) van der Made,
A. W.; van Leeuwen, P. W. N. M.; de Wilde, J. C.; Brandes,
R. A. C. Adv. Mater. 1993, 5, 466–468. (c) Muzafarov, A. M.;
Gorbatsevich, O. B.; Rebrov, E. A.; Ignat’eva, G. M.;
Chenskaya, T. B.; Myakushev, V. D.; Bulkin, A. F.; Papkov,
V. S. Vysokomol. Soedin., Ser. A Ser. B 1993, 35, 1867–1872.
(d) Seyferth, D.; Son, D. Y.; Rheingold, A. L.; Ostrander, R. L.
Organometallics 1994, 13, 2682–2690.
18. Nishikawa, K.; Matsuoka, K.; Kita, E.; Okabe, N.; Mizuguchi,
M.; Hino, K.; Miyazawa, S.; Yamasaki, C.; Aoki, J.;
Takashima, S.; Yamakawa, Y.; Nishijima, M.; Terunuma,
D.; Kuzuhara, H.; Natori, Y. Proc. Natl Acad. Sci. U.S.A.
2002, 99, 7669–7674.
27. (a) Winnik, F. M.; Brisson, J.-R.; Carver, J. P.; Krepinsky, J. J.
Carbohydr. Res. 1982, 103, 15–28. (b) Madiyalakan, R.;
Chowdhary, M. S.; Rana, S. S.; Matta, K. L. Carbohydr. Res.
1986, 152, 183–194.
19. Takano, T.; Nakatsubo, F.; Murakami, K. Carbohydr. Res.
1990, 203, 341–342.
28. (a) Williams, B. A.; Chervenak, M. C.; Toone, E. J. J. Biol.
Chem. 1992, 267, 22907–22911. (b) Chervenak, M. C.; Toone,
E. J. J. Am. Chem. Soc. 1994, 116, 10533–10539.
(c) Chervenak, M. C.; Toone, E. J. Bioorg. Med. Chem.
1996, 4, 1963–1977. (d) Dimick, S. M.; Powell, S. C.;
McMahon, S. A.; Moothoo, D. N.; Naismith, J. H.; Toone, E. J.
J. Am. Chem. Soc. 1999, 121, 10286–10296.
20. Houseman, B. T.; Gawalt, E. S.; Mrksich, M. Langmuir 2003,
19, 1522–1531.
21. Ravindranathan Karcha, K. P.; Jennings, H. J. J. Carbohydr.
Chem. 1990, 9, 777–781.
22. Betaneli, V. I.; Ovchinnikov, M. V.; Backinowsky, L. V.;
Kochetkov, N. K. Carbohydr. Res. 1982, 107, 285–291.
23. Zhang, J.; Kong, F. Tetrahedron: Asymmetry 2002, 13,
243–252.
29. Edelhoch, H. Biochemistry 1967, 6, 1948–1954.
30. Dubois, M.; Gilles, K. A.; Hamilton, J. K.; Robers, P. A.;
Smith, F. Anal. Chem. 1956, 28, 350–356.
24. (a) Pompipom, M. M. Carbohydr. Res. 1977, 59, 311–317.
(b) Lee, E. E.; Wood, J. O. Carbohydr. Res. 1979, 75,
322–324. (c) Winnik, F. M.; Carver, J. P.; Krepinsky, J. J.
31. Wiseman, T.; Williston, S.; Brandts, J. F.; Lin, L.-N. Anal.
Biochem. 1989, 179, 131–137.