Synthesis of a cellobiosylated dimer and trimer and of cellobiose-coated polyamidoamine (PAMAM) dendrimers to study accessibility of an enzyme, cellodextrin phosphorylase

Article abstract of DOI:10.1002/ejoc.200300018

To examine the accessibility of the enzyme cellodextrin phosphorylase (CDP) towards multivalent cluster carbohydrates, the cellobiosylated dimer 10 and trimer 12, as well as the cellobiose-coated PAMAM dendrimers 14, 16, 18, 20 and 22, with four, eight, sixteen, thirty-two and sixty-four cellobiose units at the outer surface of PAMAM dendrimers, respectively, have been synthesized for the first time and used as acceptor substrates for the enzyme CDP. It was found that CDP was able to transfer a glucosyl moiety from glucose-1-phosphate (Glc-1-P) into these synthesized cluster cellobiosylated glycoconjugates and cellobiose-coated PAMAM dendrimers, which were thus acceptor substrates for CDP. It was found that the ability of CDP to interact with smaller cellobiosylated glyconjugates and with PAMAM dendrimers containing up to eight cellobiose units was similar to that seen with cellobiose. However, this capability of CDP was somewhat lessened with the PAMAM dendrimer containing sixteen cellobiose moieties and dramatically decreased towards PAMAM dendrimers with thirty-two and sixty-four cellobiose units. This might be due to their steric bulk, CDP enzyme no longer being able to hold them properly on its active site. ( Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003).

Full text of DOI:10.1002/ejoc.200300018

FULL PAPER
Synthesis of a Cellobiosylated Dimer and Trimer and of Cellobiose-Coated
Polyamidoamine (PAMAM) Dendrimers to Study Accessibility of an Enzyme,
Cellodextrin Phosphorylase
[a]
[a]
[a]
Ambar K. Choudhury, Mitomitsu Kitaoka,* and Kiyoshi Hayashi
Keywords: Dendrimers / Enzymes / Glycoconjugates / Substrate specificity
To examine the accessibility of the enzyme cellodextrin phos-
phorylase (CDP) towards multivalent cluster carbohydrates,
found that the ability of CDP to interact with smaller cellobi-
osylated glyconjugates and with PAMAM dendrimers con-
the cellobiosylated dimer 10 and trimer 12, as well as the taining up to eight cellobiose units was similar to that seen
cellobiose-coated PAMAM dendrimers 14, 16, 18, 20 and 22,
with four, eight, sixteen, thirty-two and sixty-four cellobiose
units at the outer surface of PAMAM dendrimers, respect-
ively, have been synthesized for the first time and used as
acceptor substrates for the enzyme CDP. It was found that
CDP was able to transfer a glucosyl moiety from glucose-1-
phosphate (Glc-1-P) into these synthesized cluster cellobios-
with cellobiose. However, this capability of CDP was some-
what lessened with the PAMAM dendrimer containing six-
teen cellobiose moieties and dramatically decreased towards
PAMAM dendrimers with thirty-two and sixty-four cellobi-
ose units. This might be due to their steric bulk, CDP enzyme
no longer being able to hold them properly on its active site.
ylated glycoconjugates and cellobiose-coated PAMAM den- ( Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
drimers, which were thus acceptor substrates for CDP. It was
Germany, 2003)
[
6]
Introduction
prove drug delivery systems. Similarly, sugar-persubsti-
[
8]
tuted PAMAM dendrimers exhibited binding affinities
Phosphorylase-mediated enzymatic synthesis in carbo- towards carbohydrate binding proteins several times higher
hydrates is well documented.[1] Cellodextrin phosphorylase than those of monovalent ligands.
(
CDP),
a
β-1,4-oligoglucan orthophosphate glucosyl-
Syntheses of persubstituted PAMAM dendritic sugar
[
2]
[9a,9b]
[9a,9b]
transferase, has been found to be very effective for the balls containing glucosyl,
synthesis of derivatives of cellooligosaccharides, requiring tones of lactose
cellobiose or various β-glucosides (laminaribiose, phenyl-β- scribed by Okada and co-workers. Lindhorst et al. and

glucoaminosyl,
lac-
[
9c]
[9c]
and maltose
moieties have been de-
[
9]
[10]
[
11]
-glucoside, etc.) as the smallest substrate for the elong- Cloninger et al.
have reported mannosyl-persubstituted
[
10,11]
[12a]
[8,12]
[8]
ation of the sugar chain to form a new β-1,4-glucosyl link- glycoPAMAM dendrimers,
whereas Roy et al.
age.[ Study on CDP to date is limited to the application have synthesized mannosyl-,
2]
lactosyl-,
[12b]
sialosyl-
[
12c]
of monovalent cellobiosylated substrate.
and T-antigen-persubstituted
glycoPAMAM dendri-
Multivalent carbohydrate ligands are expected to be very mers by coupling with the respective glycosyl donors and
promising in terms of carbohydrate-protein interactions. To the free amines of the PAMAM dendrimers. Glucosyl-, lac-
explore the effect of multivalency, carbohydrate-based den- tosyl-, and sialosyl-persubstituted aminoxy PAMAM den-
[
3,4]
[13]
drimers
have been synthesized and the importance of drimers have also been reported.
[4]
multivalent sugar residues has been described.
There is, however, no report on cellobiose-persubstituted
Poly(amidoamine) (PAMAM) dendrimers,[5] on the other PAMAM dendrimers. So far, only Lindhorst et al.
[10]
have
hand, are highly branched macromolecules containing am- reported a triantennary cellobioside, obtained by coupling
ino groups on their surfaces. Their unique structural shapes, of an isothiocyanate derivative of cellobiose with tris(amino-
as well as their cytotoxic natures,[ have encouraged scien- ethyl)amine.
6]
tists to produce PAMAM dendrimer-based synthetic
macromolecules for biochemical, biophysical and medicinal
The cluster effects of carbohydrates for the recognition
[4,8,12c]
of proteins are well established.
However, the effect
[
6Ϫ8]
applications.
It has been demonstrated that the syn-
of the population density of carbohydrates on recognition
of enzymes as substrates has not been well studied. Enzy-
matic chain elongation of carbohydrates on dendrimers is
considered to be useful in preparation of bioactive glyco-
conjugates, so it is important to examine the accessibility of
the enzyme towards the multivalent cluster carbohydrates.
thetic persubstituted PAMAM dendrimers are able to im-
[
a]
Enzyme Laboratory, Biological Function Division, National
Food Research Institute,
2
-1-12 Kanondai, Tsukuba, Ibaraki 305-8642, Japan
E-mail: mkitaoka@nfri.affrc.go.jp
2462
 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
DOI: 10.1002/ejoc.200300018
Eur. J. Org. Chem. 2003, 2462Ϫ2470

Cellobiosylated Dimer and Trimer and Cellobiose-Coated Polyamidoamine Dendrimers
FULL PAPER
Since CDP, obtained from Clostridium thermocellum,[14] drolysis of methyl ester 4 with 1.1 equivalents of NaOH
was found to be very useful for the synthesis of cellooligo- afforded the desired carboxylic acid 5 in 97% yield. Com-
[
14]
1
saccharides through the use of cellobiose as the smallest pounds 3, 4 and 5 were confirmed by their H NMR and
13
substrate, we focussed on synthesizing cellobiosylated di-
C NMR spectra as well as by their FAB and high-resolu-
mer, trimer and cellobiose-coated PAMAM dendrimers as tion ESI mass spectra. The assignment of compound 5 was
model substrates to examine the ability of the enzyme CDP based on its HMBC spectrum. The β-glycosidic linkages of
to interact with those multivalent cellobiosylated glycocon- compound 5 were confirmed by the presence of character-
jugate clusters.
istic peaks at δ ϭ 4.44 ppm (J ϭ 7.9 Hz) for H-1 and 4.45
1
PAMAM dendrimers of generations 0, 1, 2, 3 and 4 con- ppm (J ϭ 7.9 Hz) for H-1Ј in the H NMR spectrum and
tain 4, 8, 16, 32 and 64 amino groups on their surfaces, peaks at δ ϭ 102.5 ppm for C-1 and 103.0 for C-1Ј in the
13
respectively. To examine the effectiveness of the enzyme
CDP, a cellobiosylated dimer and trimer, as well as cello-
biose-coated PAMAM dendrimers possessing 4, 8, 16, 32
and 64 cellobiose units at the PAMAM dendrimer outer
surface, were synthesized by conjugation of dendrimer free
amine with the free carboxylic acid moiety attached at the
C NMR spectrum.
Besides cellobiose-coated PAMAM dendrimers, the di-
valent cellobiose derivative 10, with a heteroatom attached
at the reducing end of cellobiose, and the triantennary cello-
biose derivative 12 were designed and synthesized as the
smallest possible multivalent acceptor substrates for the en-
zyme cellodextrin phosphorylase.
[
15]
reducing end of cellobiose in the presence of BOP
agent and used as acceptor substrates for that enzyme.
re-
The desired β--cellobiosyl dimer 10 was obtained in
four steps, starting from the known hepta-O-acetyl-α--cel-
To the best of our knowledge, this report describes for the
first time both the syntheses of multivalent cellobiosylated
glycoconjugates and the ability of the enzyme CDP to inter-
act with those synthesized cluster cellobiosylated glycocon-
[17]
lobiosyl bromide
(1) as shown in Scheme 2. Firstly, the
[17]
known hepta-O-acetyl-β--cellobiosyl azide (6) was syn-
thesized by treatment of 1 with sodium azide at 65Ϫ70 °C
in anhydrous DMF for 3 h, and was obtained in 67% yield.
[
16]
jugates.
1
13
Both the H and the C NMR spectra of 6 were similar to
[
17a]
those reported.
The azido group of compound 6 was
Results and Discussion
reduced to provide the desired amine 7 in 80% yield by cata-
lytic hydrogenation on 10% Pd-C in ethyl acetate as solvent.
The free amine 7 was then treated with succinoyl chloride
Synthesis
The strategy adopted for the syntheses of the desired cel- (8) in anhydrous pyridine at 0 °C for 2 h to afford the per-
lobiose-coated PAMAM dendrimers of generations 0, 1, 2, acetylated β--cellobiosylamine dimer 9 in 74% yield. De-
3
and 4 was firstly to synthesize carboxymethyl β--cello- acetylation of compound 9 by Zemplen’s method afforded
bioside (5), with a free carboxylic acid attached at the re- the desired cellobiosyl dimer 10 in 88% yield. Compounds
1
13
ducing end of cellobiose through the smallest possible 7, 8, 9 and 10 were characterized by their H and C NMR
spacer. Methoxycarbonylmethyl β--cellobioside 5 was thus spectra as well by their FAB and high-resolution ESI mass
synthesized for the first time, as described in Scheme 1.
spectra. The characteristic peaks at δ ϭ 4.43 ppm (J ϭ
Treatment of the known hepta-O-acetyl-α--cellobiosyl 7.9 Hz) for H-1 and 4.90 ppm (J ϭ 9.2 Hz) for H-1Ј in the
[
17]
1
bromide (1)
and the commercially available methyl gly-
H NMR spectrum of compound 10, as well as peaks at
colate (2) in the presence of silver triflate afforded the de- δ ϭ 79.1 ppm for C-1 (anomeric CϪNHϪCO) and 102.5
13
sired methoxycarbonylmethyl hepta-O-acetyl β--cellobio- ppm for C-1Ј in the C NMR spectrum, confirmed its β-
side (3) in 75% yield. Zemplen de-O-acetylation of com- glycosidic linkages. The 13C NMR spectroscopic data for
pound 3 afforded methyl ester 4 in 96% yield. Finally, hy- the anomeric β-C-NHCO in compound 10 were satisfac-
Scheme 1. Synthesis of carboxymethyl β--cellobioside (5)
Eur. J. Org. Chem. 2003, 2462Ϫ2470
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 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2463

A. K. Choudhury, M. Kitaoka, K. Hayashi
FULL PAPER
Scheme 2. Synthesis of N,N-bis(β--cellobiosyl)succinamide (10)
torily consistent with values reported for other sugar de- NMR and 13C NMR spectra and also by its high-resolution
[
18]
rivatives.
Compound 10 was further verified by gel-per- ESI mass spectra. Compound 12 was further checked by
meation HPLC, showing only a single peak with a retention gel-permeation HPLC and showed only a single peak with
time of 20.969 min, as shown in Table 1. The conditions a retention time of 20.490 min. The characteristic peaks at
for gel-permeation HPLC have been described in a general δ ϭ 4.51 ppm (J ϭ 8.0 Hz) for H-1 and 4.60 ppm (J ϭ
1
procedure for the synthesis of cellobiose-coated PAMAM 7.8 Hz) for H-1Ј in the H NMR spectrum as well as peaks
dendrimers of generations 0, 1, 2, 3 and 4.
at δ ϭ 102.7 ppm for C-1, 102.9 ppm for C-1Ј and 172.2
13
ppm for NHCOCH in the C NMR spectrum confirmed
2
Table 1. Retention times (min) of gel-permeation HPLC chromato-
grams
the presence both of the β-glycosidic linkages and of the
amide bond, respectively.
Cellobiose-coated PAMAM dendrimer of generations 0,
Compounds
Retention time of products (min)
1
, 2, 3, and 4 were each synthesized in one step, as outlined
Compound 10
Compound 12
Compound 14
Compound 16
Compound 18
Compound 20
Compound 22
20.97
20.49
in Schemes 4Ϫ8.
19.94 (22.21)[
18.78 (20.60)[
17.49 (19.38)[
a]
Commercially available methanolic solutions of PAMAM
dendrimers of generations 0 (13), 1 (15), 2 (17), 3 (19) and
4 (21) were coupled with 5 (2 equivalents per amine unit)
in anhydrous DMF at 25 °C for 2Ϫ21 h. The products were
purified by passage through a mixed ion (50Ϫ50 cation and
anion) exchange resin column and obtained in 53Ϫ76%
yields. The desired cellobiose-coated PAMAM dendrimers
a]
a]
[a]
16.51 (18.20)
15.02 (17.02)[
a]
[
a]
Values in parentheses are the retention times (min) for starting
PAMAM Dendrimers
14, 16, 18, 20 and 22 were further verified by gel-per-
Cellobiosyl trimer 12 was synthesized in one step meation HPLC, only a single peak being found in the gel-
Scheme 3) by coupling of 5 with commercially available tris- permeation HPLC chromatogram for each product. The re-
(
(
aminoethyl)amine (11) in anhydrous DMF at 25 °C for tention times of the products are summarized in Table 1
15]
1
6 h in the presence of BOP[ {[(benzotriazol-1-yloxy-tris- and were readily distinguishable from the retention times of
(dimethylamino)]phosphonium hexafluorophosphate}, and the corresponding starting PAMAM dendrimers. The syn-
was obtained in 64% yield. The selection of BOP for coup- thesized PAMAM dendrimers 14, 16, 18, 20 and 22 were
1
ling was based on its previous usefulness even in the syn- assigned by the presence of characteristic peaks in their H
thesis of complex carbohydrate derivatives.[ Compound NMR and C NMR spectra. MALDI-TOF mass spectra
19]
13
ϩ
1
2 was purified by column chromatography on silica gel by showed masses of 2046.6Ϯ1.0 [M ϩ H] for compound 14
use of 2:1 and 1:1 acetonitrile/water as eluent, followed by (molecular mass of 14: 2044.8), 4505Ϯ5 [M ϩ Na] for
passage of the solvent through a Sep-Pak Plus environmen- compound 16 (molecular mass of 16: 4485.9), 9397Ϯ7 [M
ϩ
1
ϩ
tal cartridge (Waters, USA), and was assigned by its H ϩ Na] for compound 18 (molecular mass of 18: 9368.1),
2464
 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Eur. J. Org. Chem. 2003, 2462Ϫ2470

Cellobiosylated Dimer and Trimer and Cellobiose-Coated Polyamidoamine Dendrimers
FULL PAPER
Scheme 3. Synthesis of tris(aminoethyl N-carbonylmethyl β--cellobiosyl)amine (12)
Scheme 7. Synthesis of 32-meric β--cellobiosyl PAMAM den-
drimer 20
Scheme 4. Synthesis of tetrameric β--cellobiosyl PAMAM den-
drimer 14
Scheme 5. Synthesis of octameric β--cellobiosyl PAMAM den-
drimer 16
Scheme 8. Synthesis of 64-meric β--cellobiosyl PAMAM den-
drimer 22
ϩ
and 19166Ϯ15 [M ϩ Na] for compound 20 (molecular
mass of 20: 19132). The MALDI-TOF method failed to
obtain the mass spectrum of compound 22 (molecular mass
of 22, C1518H2656O892N250: 38661).
Acceptor Activities of Cellobiosylated Dimer and Trimer
and Cellobiose-Coated PAMAM Dendrimers
The following experiment was designed for the determi-
nation of the substrate specificities of cellodextrin phos-
phorylase with use of the multivalent cellobiosylated glyco-
conjugates described above as acceptor substrates. The en-
Scheme 6. Synthesis of hexadecameric β--cellobiosyl PAMAM
dendrimer 18
Eur. J. Org. Chem. 2003, 2462Ϫ2470
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 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2465

A. K. Choudhury, M. Kitaoka, K. Hayashi
FULL PAPER
zymatic reaction in general is described in Scheme 9, where and 64 (22) cellobiose units, and hence that these were ac-
‘‘G’’ represents other groups present in the molecule.
ceptor substrates for that enzyme. It was found that the ac-
cessibility of CDP towards smaller cellobiosylated glyconju-
gates and up to eight cellobiose units containing PAMAM
dendrimers as substrates was not significantly different than
cellobiose. However, the accessibility of CDP was slowly
decreased toward sixteen cellobiose-coated PAMAM den-
drimer 16 and dramatically decreased towards thirty-two
20 and sixty-four 22 cellobiose units containing PAMAM
dendrimers. This might be due to their sterically hindered
sizes, enzyme CDP no longer being able to hold them prop-
erly on its active site. The rates for 32-cellobiose-coated PA-
MAM dendrimer 20 and 64-cellobiose-coated PAMAM
dendrimer 22 were practically identical, indicating that the
critical difference in the accessibility of CDP was found
only between 16 and 32 cellobiosyl units.
From this study, we conclude that the smaller cellobiosyl-
ated glyconjugates and the PAMAM dendrimers containing
up to eight cellobiose units are, thanks to their sterically
less bulky sizes, readily available to the active site of CDP
for transfer of the glucosyl residue from Glc-1-P, relative to
their sterically more bulky PAMAM dendrimer homol-
ogues with sixteen, thirty-two and sixty cellobiose units (18,
Scheme 9. Enzyme reaction
During the CDP reaction, Glc-1-P was quantified by the
phosphoglucomutase/glucose-6-phosphate dehydrogenase
[
16]
method.
The reaction mixture, containing aqueous glu-
cose-1-phosphate, aqueous substrate and a 50 m MOPS
buffer solution of CDP, was incubated at 37 °C. At certain
intervals from 0 h to 120 h, aliquots were removed and the
remaining Glc-1-P was quantified by the above method, fol-
lowed by measurement of the absorbance at 340 nm. The
percentage of remaining Glc-1-P was calculated in each case
and plotted against time (h), and the results are summar-
ized in Figure 1.
20 and 22). Further evaluation of the results should be
possible once the three-dimensional structure of CDP has
been determined.
Experimental Section
General Methods: All reactions were carried out under nitrogen in
oven-dried glassware. Methyl glycolate, silver triflate, and PAMAM
dendrimers of generations 0, 1, 2, 3 and 4 were purchased from
2 2
Sigma, Aldrich Chemicals (USA). Anhydrous CH Cl and anhy-
drous DMF were purchased from Wako Chemicals (Japan). Diiso-
propylethylamine was purchased from Nacalai Tesque Chemicals
(
Japan) and was used directly without further distillation. Anhy-
drous sodium chloride and sodium dihydrogen phosphate were
purchased from Nacalai Tesque Chemicals (Japan). Thin-layer
chromatography was performed on 60 F254 plates (E. Merck) and
results were viewed by charring with 5% sulfuric acid in methanol.
Silica gel 60 (230Ϫ400 mesh) was used for column chromatography.
Gel permeation HPLC was performed on a TSKGel G2000 SWXL
(
7.8 mm I.D. ϫ 30 cm) HPLC column purchased from TOSOH,
Japan. Detection was at 220 nm and 50 m sodium dihydrogen
phosphate buffer of pH 7.0 containing 0.3  NaCl per liter was
used as eluent, with a flow rate of 0.5 mL/min. Phosphoglucomut-
ase and glucose-6-phosphate dehydrogenase were purchased from
Sigma Chemicals (USA). Cellodextrin phosphorylase used in this
study was obtained from Clostridium thermocellum.[
14]
Figure 1. Time course of the CDP reaction with cellobiosylated
dimer and trimer and cellobiose-coated PAMAM dendrimers.
1H NMR and 13C NMR spectra were recorded at 25 °C with a
Bruker DRX 600 NMR instrument. Proton NMR shifts are re-
ported in ppm with use of TMS (δ ϭ 0 ppm) as reference standard
(
᭜) 64-mer (22); (᭿) 32-mer (20); (᭡) 16-mer (18); (᭺) octamer (16);
*) tetramer (14); (᭹) trimer (12); (᭝) dimer (10) and (ᮀ) cellobiose
(
for CDCl
3 2
solutions and δ ϭ 4.70 ppm for D O solutions. Carbon
NMR shifts were reported in ppm with use of δ ϭ 77.0 ppm as
Figure 1 clearly indicates that the enzyme cellodextrin
phosphorylase was able to transfer glucosyl moieties from
glucose-1-phosphate to the synthesized cellobiosylated di-
13
3 2
reference standard for CDCl solutions. C NMR spectra for D O
solutions were measured by fixing of the HOD peak at δ ϭ
13
4
.70 ppm followed by measurement of the C NMR spectra, so
mer 10 and trimer 12 and to the cellobiose-coated PAMAM carbon NMR shifts for D O solutions are reported in ppm without
2
dendrimers containing four (14), eight (16), 16 (18), 32 (20) any reference standards. FAB and ESI mass spectra were measured
2466
 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Eur. J. Org. Chem. 2003, 2462Ϫ2470

Cellobiosylated Dimer and Trimer and Cellobiose-Coated Polyamidoamine Dendrimers
with a Bruker APEX II 70e mass instrument. MALDI-TOF mass H, H-2Ј), 3.31Ϫ3.38 (m, 2 H, H-2, H-5Ј), 3.39Ϫ3.47 (m, 2 H, H-
FULL PAPER
spectra were recorded with a Bruker REFLUX II mass instrument
using retinoic acid as matrix. A Beckman DU 650 Spectrophoto-
meter, USA, was used for spectrophotometric measurements.
5, H-6Ј), 3.48Ϫ3.54 (m, 1 H, H-6Ј), 3.55Ϫ3.63 (m, 2 H, H-6, H-4),
3.64Ϫ3.70 (m, 1 H, H-6), 3.72Ϫ3.78 (m, 1 H, H-4Ј), 3.82Ϫ3.87 (m,
1 H, H-3), 3.88Ϫ3.92 (m, 1 H, H-3Ј), 4.06 (d, J ϭ 15.6 Hz, 1 H,
OCH
J ϭ 7.9 Hz, 1 H, H-1), 4.45 (d, J ϭ 7.9 Hz, 1 H, H-1Ј) ppm.
NMR (150 MHz, D O, 25 °C): δ ϭ 60.3 (C-6Ј), 60.9 (C-6), 68.7
OCH COOH), 69.8 (C-4), 73.3, 73.5, 74.5, 75.2, 75.9, 76.4, 78.9,
2 2
COOH), 4.24 (d, J ϭ 15.6 Hz, 1 H, OCH COOH); 4.44 (d,
Methoxycarbonylmethyl Hepta-O-acetyl-β-
mixture of hepta-O-acetyl β--cellobiosyl bromide (1, 1.5 g,
D
-cellobioside (3):
A
13
C
2
2
.15 mmol), methyl glycolate (2, 0.18 mL, 2.33 mmol) and molecu-
(
2
˚
lar sieves (4 A, 1 g) in CH
2
Cl
2
(20 mL) was stirred at 0Ϫ4 °C for
1
02.5 (C-1), 103.0 (C-1Ј) and 177.4 (COOH) ppm. MS (FAB):
3
0 min. Silver triflate (600 mg, 2.33 mmol) was then added. The
reaction mixture was stirred at 0Ϫ4 °C for 48 h and then diluted
with CH Cl (20 mL). The insoluble solids were separated by fil-
tration through a Celite bed and washed twice with CH Cl (10 mL Hepta-O-acetyl-β-
ϫ 2). The combined organic solution was washed with saturated
NaHCO solution and brine. The organic layer was dried
Na SO ), filtered and concentrated under reduced pressure. The
ϩ
ϩ
m/z ϭ 423.11 [M ϩ Na] . ESI-MS: m/z ϭ 423.1116 [M ϩ Na]
13Na requires 423.1109).
14 24
(C H O
2
2
2
2
D-cellobiosyl Azide (6): NaN
3
(184 mg,
2.83 mmol) was added to a solution of 1 (1.8 g, 2.57 mmol) in anhy-
drous DMF (10 mL). The reaction mixture was stirred at 65Ϫ70
°C for 3 h. The insoluble materials were filtered through a Celite
bed and washed twice with ethyl acetate (10 mL ϫ 2). The com-
3
(
2
4
residue was purified by column chromatography on a silica gel col-
umn (22 ϫ 3 cm). Elution with ethyl acetate/hexane (1:1, 2:1 and
bined organic solution was washed with saturated NaHCO
SO ), filtered and
concentrated under reduced pressure. The residual DMF was re-
), 3.54Ϫ3.62 (m, 1 H), moved by co-evaporation with toluene (10 mL ϫ 3). The residue
), 3.80 (t, J ϭ 9.8 Hz, was purified by column chromatography on a silica gel column (20
H), 3.99Ϫ4.13 (m, 2 H), 4.25 (d, J ϭ 1.0 Hz, 2 H, OCH2- ϫ 3 cm). Elution with ethyl acetate/hexane (1:1 and 2:1) afforded
), 4.36 (dd, J ϭ 12.5, J ϭ 4.5 Hz, 1 H), 4.51 (d, J ϭ 6 as white solid: yield 1.14 g (67%); silica gel TLC R ϭ 0.66 (ethyl
3
solu-
4
R
2
3
1
:1) afforded 3 as white solid. Yield 1.15 g (75%); silica gel TLC tion and brine. The organic layer was dried (Na
2
4
1
f
ϭ 0.38 (ethyl acetate/hexane, 2:1). H NMR (600 MHz, CDCl
5 °C): δ ϭ 1.98Ϫ2.13 (7s, 21 H, OCOCH
.63Ϫ3.69 (m, 1 H), 3.74 (s, 3 H, COOCH
3
,
3
3
COOCH
.0 Hz, 1 H, H-1), 4.53 (dd, J ϭ 12.0, J ϭ 2.1 Hz, 1 H), 4.61 (d,
3
f
1
13
8
acetate/hexane, 2:1). The H and C NMR spectra of compound
6 were identical with those reported.[
17a]
J ϭ 7.8 Hz, 1 H, H-1Ј), 4.88Ϫ4.98 (m, 2 H), 5.06 (t, J ϭ 9.9 Hz, 1
H), 5.14 (t, J ϭ 9.4 Hz, 1 H) and 5.21 (t, J ϭ 9.3 Hz, 1 H) ppm.
1
3
Hepta-O-acetyl-β-
.66 mmol) and Pd-C (10%, 165 mg) in anhydrous ethyl acetate
10 mL) was stirred under H at 25 °C for 4 h. The reaction mixture
D-cellobiosylamine (7): A mixture of 6 (1.1 g,
C NMR (150 MHz, CDCl
3
, 25 °C): δ ϭ 20.5Ϫ20.8 (OCOCH
COOCH
3
),
),
1
5
6
2.0 (COOCH ), 61.5 (C-6Ј), 61.6 (C-6), 65.1 (OCH
3
2
3
(
2
7.8 (C-4), 71.2, 71.6, 72.0, 72.1, 72.8, 72.9, 75.3, 100.0 (C-1), 100.7
) ppm. MS (FAB): m/z ϭ 709.29
20 requires 709.2185), ESI-MS: m/z ϭ 731.1997
20Na requires 731.2005).
was filtered through a Celite bed and washed twice with ethyl acet-
ate/ethanol (4:1, 10 mL ϫ 2). The combined organic solution was
evaporated under reduced pressure. The residue was purified by
column chromatography on a silica gel column (15 ϫ 3 cm). Elu-
tion with ethyl acetate followed by ethyl acetate/acetone (10:1 and
(C-1Ј) and 169.0Ϫ170.5 (OCOCH
3
ϩ
[
[
41
M ϩ H] (C29H O
M ϩ Na] (C29
ϩ
H
40
O
Methoxycarbonylmethyl β-D-Cellobioside (4): NaOMe solution (0.1

, 5 mL) was added to a solution of 3 (1 g, 1.41 mmol) in anhy-
1:1) afforded 7 as white powder: yield 850 mg (80%); silica gel TLC
1
drous MeOH/CHCl
at 25 °C for 3 h and neutralized with Dowex 50-WX8 (H ) cation- CDCl
3
(5:1, 6 mL). The reaction mixture was stirred
R
f
ϭ 0.35 (ethyl acetate/acetone, 10:1). H NMR (600 MHz,
ϩ
3
2
, 25 °C): δ ϭ 1.88Ϫ1.96 (m, 2 H, NH ), 1.98Ϫ2.13 (7s, 21
exchange resin. The solution was filtered and the solvents were eva-
porated under reduced pressure. The residue was purified by col-
umn chromatography on silica gel (15 ϫ 3 cm). Elution with meth-
anol/ethyl acetate (1:1 and 2:1) afforded 4 as a white foam. Yield
H, OCOCH ), 3.56Ϫ3.61 (m, 1 H), 3.63Ϫ3.68 (m, 1 H), 3.71 (t,
3
J ϭ 9.9 Hz, 1 H), 4.01Ϫ4.10 (m, 2 H), 4.11Ϫ4.20 (m, 1 H, H-1),
4.36 (dd, J ϭ 12.5, J ϭ 4.4 Hz, 1 H), 4.46 (dd, J ϭ 12.6, J ϭ
2.0 Hz, 1 H), 4.50 (d, J ϭ 8.0 Hz, 1 H, H-1Ј), 4.74 (dd, J ϭ 9.8,
5
2
3
3
3
60 mg (96%); silica gel TLC R
f
ϭ 0.58 (methanol/ethyl acetate, J ϭ 9.0 Hz, 1 H), 4.92 (dd, J ϭ 9.4, J ϭ 8.0 Hz, 1 H), 5.07 (t, J ϭ
O, 25 °C): δ ϭ 3.19Ϫ3.26 (m, 1 H), 12.5 Hz, 1 H), 5.14 (t, J ϭ 9.3 Hz, 1 H), 5.22 (t, J ϭ 9.2 Hz, 1 H)
.29Ϫ3.35 (m, 2 H), 3.37Ϫ3.44 (m, 2 H), 3.46Ϫ3.51 (m, 1 H), ppm. C NMR (150 MHz, CDCl
.52Ϫ3.61 (m, 2 H), 3.62Ϫ3.67 (m, 1 H), 3.69 (s, 3 H, COOCH ), OCH ), 61.6 (C-6Ј), 62.2 (C-6), 67.8 (C-4), 71.6, 71.9, 72.4, 72.7,
.70Ϫ3.76 (m, 1 H), 3.80Ϫ3.90 (m, 2 H), 4.23Ϫ4.40 (m, 2 H), 4.42 73.0, 73.7, 76.8, 84.7 (C-1), 100.8 (C-1Ј) and 169.0Ϫ170.5 (OC-
1
:1). H NMR (600 MHz, D
2
1
3
3
, 25 °C): δ ϭ 20.5Ϫ20.9 (OC-
3
3
ϩ
(
3
d, J ϭ 7.9 Hz, 1 H, H-1) and 4.46 (d, J ϭ 9.5 Hz, 1 H, H-1Ј) ppm. OCH ) ppm. MS (FAB): m/z ϭ 636.28 [M ϩ H] , ESI-MS: m/z ϭ
1
3
ϩ
C NMR (150 MHz, D
Ј), 59.5 (C-6), 65.1 (OCH
3.8, 74.4, 74.9, 77.3, 100.9 (C-1), 101.5 (C-1Ј) and 171.3 (CO-
2
O, 25 °C): δ ϭ 51.5 (COOCH
3
), 58.8 (C-
636.2128 [M ϩ H] (C26
H
38
O17N requires 636.2134).
6
7
2
COOCH ), 68.4 (C-4), 71.7, 72.1, 73.1,
3
N,N-Bis(hepta-O-acetyl-β-D-cellobiosyl)succinamide (9): Com-
ϩ
pound 7 (465 mg, 0.73 mmol) was dissolved in anhydrous pyridine
(3 mL) and the mixture was stirred at 0Ϫ4 °C for 15 min. A solu-
tion of succinoyl chloride (8, 44 mL, 0.4 mmol) in anhydrous
OCH
quires 415.1452), ESI-MS: m/z
13Na requires 437.1265).
3
) ppm. MS (FAB): m/z ϭ 415.15 [M ϩ H] (C15
H
ϩ
27
O
13 re-
Na]
ϩ
ϭ
437.1267 [M
15 26
(C H O
CHCl
-Cellobioside (5): NaOH (16 mg, 0.4 mmol) in was stirred at 0Ϫ4 °C for 2 h. The solution was evaporated to dry-
ness and the residual pyridine was removed by co-evaporation with
3
(1 mL) was then added dropwise and the reaction mixture
Carboxymethyl β-
O (100 mL) was added to a solution of 4 (160 mg, 0.386 mmol)
in MeOH/H O (3:1, 3 mL). The reaction mixture was stirred at 25 toluene (5 mL ϫ 3). The residue was dissolved in ethyl acetate
C for 2 h and then neutralized with Dowex 50-WX8 (H ) cation-
D
H
2
2
ϩ
°
3
(20 mL) and washed with saturated NaHCO solution and brine.
exchange resin. The solution was filtered and the solvents were eva-
porated under reduced pressure. The residue was purified by col-
umn chromatography on silica gel (6 ϫ 3 cm). Elution with meth-
anol/ethyl acetate (2:1) and methanol afforded 5 as a white solid.
The organic layer was dried (Na SO ), filtered and concentrated
2
4
under reduced pressure. The residue was purified by column chro-
matography on a silica gel column (18 ϫ 3 cm). Elution with ethyl
acetate followed by ethyl acetate/acetone (10:1 and 1:1) afforded 9
Yield 150 mg (97%); silica gel TLC R
f
ϭ 0.21 (methanol/ethyl acet-
f
as white foam. Yield 400 mg (40.5%); silica gel TLC R ϭ 0.45
1
1
ate, 2:1). H NMR (600 MHz, D
2
O, 25 °C): δ ϭ 3.21Ϫ3.27 (m, 1 (ethyl acetate/acetone, 10:1). H NMR (600 MHz, CDCl
3
, 25 °C):
Eur. J. Org. Chem. 2003, 2462Ϫ2470
www.eurjoc.org
 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2467

A. K. Choudhury, M. Kitaoka, K. Hayashi
FULL PAPER
δ ϭ 1.98Ϫ2.13 (7s, 42 H, OCOCH
3
), 2.33Ϫ2.42 (m, 2 H,
General Procedure for the Synthesis of Cellobiose-Coated PAMAM
2 2
NHCOCH ), 2.46Ϫ2.57 (m, 2 H, NHCOCH ), 3.62Ϫ3.71 (m, 4 Dendrimers of Generations 0, 1, 2, 3 and 4: A mixture of a meth-
H), 3.74 (t, J ϭ 9.8 Hz, 2 H), 4.04 (dd, J ϭ 12.4, J ϭ 2.3 Hz, 2 H, anolic solution of the PAMAM dendrimer of generation 0, 1, 2, 3
H-1), 4.08Ϫ4.16 (m, 2 H), 4.37 (dd, J ϭ 12.6, J ϭ 4.5 Hz, 2 H), or 4, carboxymethyl β--cellobioside 5 (2 equivalents per free am-
4
1
5
2
.45 (dd, J ϭ 12.3, J ϭ 1.9 Hz, 2 H), 4.50 (d, J ϭ 7.9 Hz, 2 H, H-
Ј), 4.83 (t, J ϭ 9.6 Hz, 2 H), 4.92 (dd, J ϭ 9.3, J ϭ 7.9 Hz, 2 H),
ine) and diisopropylamine (2 equivalents per carboxylic acid) in
anhydrous DMF was stirred at 25 °C for 30 min, and BOP reagent
.06 (t, J ϭ 9.5 Hz, 2 H), 5.11Ϫ5.18 (m, 4 H), 5.26 (t, J ϭ 9.2 Hz, (1 equivalent per carboxylic acid) was then added. The reaction
H), 6.32 (d, J ϭ 9.2 Hz, 2 H, NHCOCH
, 25 °C): δ ϭ 20.5Ϫ20.9 (OCOCH
2
) ppm. 13C NMR
mixture was stirred at 25 °C for 2Ϫ21 h and during that time Ϫ
(
150 MHz, CDCl
NHCOCH
2.1, 72.9, 74.5, 76.3, 78.1 (C-1), 100.7 (C-1Ј) and 169.0Ϫ171.7 tates were separated by filtration, washed twice with ethyl acetate/
3
3
), 30.6 for generations 2, 3 and 4 Ϫ the products were precipitated. The
(
7
2
), 61.6 (C-6), 61.8 (C-6Ј), 67.8 (C-4), 70.7, 71.6, 72.0, mixture was diluted with ethyl acetate/methanol (3:1). The precipi-
(
1
OCOCH
353.4248 [M ϩ H] (C56
3
) ppm. MS (FAB): m/z ϭ 1353.32; ESI-MS: m/z ϭ
methanol (3:1) and dissolved in water. The aqueous solution was
concentrated under reduced pressure. The residue was dissolved in
water and passed through a mixed ion (50Ϫ50 cation and anion)
exchange resin column (8 ϫ 1 cm) to remove the free amine-con-
ϩ
77 36 2
H O N requires 1353.4250).
N,N-Bis(β-
D-cellobiosyl)succinamide (10): NaOMe solution (0.1 ,
2
mL) was added to a solution of 9 (200 mg, 0.148 mmol) in anhy- taining by-products, unchanged PAMAM dendrimer and un-
drous MeOH/CHCl (4:1, 2.5 mL). The reaction mixture was changed free carboxylic acid. Elution with water, followed by evap-
3
stirred at 25 °C for 2 h and was then neutralized with Dowex 50-
WX8 (H ) cation-exchange resin. The solution was diluted with
water and filtered to remove the cation-exchange resin. The filtrate
was concentrated under reduced pressure and the residue was dis-
solved in water and re-precipitated with MeOH and filtered. The and 22 were further verified by gel-permeation HPLC on a TSKGel
oration of the desired fractions (confirmed by spotting on TLC
plates and charring of those spots with 5% sulfuric acid in meth-
anol) and finally lyophilization afforded 14, 16, 18, 20 and 22 as
white foam: yields 45Ϫ60 mg (53Ϫ76%). Compounds 14, 16, 18, 20
ϩ
precipitates were dissolved in water and lyophilized to afford 10 as
white foam. Gel-permeation HPLC of the product 10 showed only
G2000 SWXL (7.8 mm I.D. ϫ 30 cm) HPLC column purchased
from TOSOH, Japan and detection at 220 nm. 50 m sodium dihy-
drogen phosphate buffer of pH 7.0 containing 0.3  ₄ NaCl was
a single peak, with a retention time of 20.969 min: Detection at
1
2
20 nm. Yield 100 mg (88%). H NMR (600 MHz, D
2
O, 25 °C): used as eluent, with a flow rate of 0.5 mL/min. Gel-permeation
δ ϭ 2.50Ϫ2.64 (m, 4 H), 3.20Ϫ3.27 (m, 2 H), 3.30Ϫ3.37 (m, 4 HPLC showed only a single peak for each product. The retention
H), 3.37Ϫ3.45 (m, 4 H), 3.53Ϫ3.62 (m, 6 H), 3.63Ϫ3.68 (m, 2 H),
times of the products are summarized in Table 1 and are readily
.69Ϫ3.77 (m, 2 H), 3.79Ϫ3.89 (m, 4 H), 4.43 (d, J ϭ 7.9 Hz, 1 H, distinguishable from the retention times of the corresponding start-
3
13
H-1), 4.90 (d, J ϭ 9.2 Hz, 1 H, H-1Ј) ppm. C NMR (150 MHz, ing PAMAM dendrimers.
O, 25 °C): δ ϭ 30.3 (NHCOCH ), 59.8 (C-6), 60.6 (C-6Ј), 69.4
C-4), 71.6, 73.1, 75.0, 75.5, 76.0, 76.3, 78.1, 79.1 (C-1), 102.5 (C-
Ј) and 176.0 (NHCOCH ) ppm. MS (FAB): m/z ϭ 765.30, ESI-
MS: m/z ϭ 765.2755 [M ϩ H] (C28
D
2
2
(
Tetrameric β-D-Cellobiosyl-PAMAM Dendrimer (14): To a mixture
1
2
of methanolic solution of PAMAM dendrimer, generation 0, 13
(20% wt. in methanol, 80µL, 0.031 mmol), 5 (100 mg, 0.25 mmol)
in 4 mL of DMF, diisopropylethylamine (88 µL, 0.5 mmol) and
BOP reagent (110 mg, 0.25 mmol) were added and the reaction
ϩ
49 22 2
H O N requires 765.2771).
Tris[2-({[(β-cellobiosyl)methyl]carbonyl}amino)ethyl]amine (12): A
mixture of 5 (70 mg, 0.175 mmol), tris(aminoethyl)amine (11, mixture was stirred at 25 °C for 2 h. Purification followed by ly-
7
0
3
.3 mL, 0.035 mmol) and diisopropylethylamine (61 mL,
.35 mmol) in anhydrous DMF (3 mL) was stirred at 25 °C for meation HPLC of the product 14 showed only a single peak, with
0 min, and BOP reagent (77 mg, 0.175 mmol) was then added.
a retention time of 19.939 min: detection at 220 nm (Table 1). Yield
ophilization afforded dendrimer 14 as a white foam. Gel-per-
1
The reaction mixture was stirred at 25 °C for 16 h and was then 60 mg (53%). H NMR (600 MHz, D O, 25 °C): δ ϭ 2.32Ϫ2.44
diluted with ethyl acetate/methanol (3:1). The precipitates were
separated by filtration, washed twice with ethyl acetate/methanol
2
(m, 7 H), 2.51Ϫ2.65 (m, 5 H), 2.71Ϫ2.84 (m, 10 H), 2.93Ϫ2.96 (d,
J ϭ 0.5 Hz, 3 H), 3.22Ϫ3.27 (m, 5 H), 3.31Ϫ3.38 (m, 16 H),
(3:1) and dissolved in water. The aqueous solution was concen- 3.39Ϫ3.47 (m, 9 H), 3.49Ϫ3.55 (m, 4 H), 3.56Ϫ3.62 (m, 8 H),
trated under reduced pressure. The residue was purified by column
chromatography on a silica gel column (8 ϫ 1 cm) by elution with
acetonitrile/water (2:1 and 1:1). The solvent was evaporated and
the residue was dissolved in water, passed through a Sep-Pak Plus
C18 environmental cartridge (Waters, USA) to remove insoluble
solids, and finally lyophilized to afford 12 as a white foam. Gel-
permeation HPLC of the product 12 showed only a single peak
3.63Ϫ3.70 (m, 4 H), 3.73Ϫ3.79 (m, 4 H), 3.82Ϫ3.95 (m, 9 H), 4.18
(d, J ϭ 15.6 Hz, 4 H), 4.31 (d, J ϭ 15.6 Hz, 4 H), 4.44 (d, J ϭ
1
3
7.9 Hz, 4 H, H-1) and 4.46 (d, J ϭ 7.8 Hz, 4 H, H-1Ј) ppm.
C
NMR (150 MHz, D O, 25 °C): δ ϭ 32.0, 36.2, 37.9, 38.0, 48.6,
2
49.4, 59.6 (C-6), 60.2 (C-6Ј), 67.6 (OCH CO), 69.1 (C-4), 72.3, 72.4,
2
72.7, 73.7, 73.9, 74.4, 75.1, 75.3, 75.5, 78.2, 101.8 (C-1), 102.0 (C-
1Ј) and 171.4, 174.3 and 174.6 (CH CONH) ppm. MS MALDI-
2
ϩ
with a retention time of 20.490 min: detection at 220 nm. Yield TOF: m/z ϭ 2046.6Ϯ1.0 [M ϩ H] (C H O N requires
7
8
137 52 10
1
3
0 mg (66%); silica gel TLC R
f
ϭ 0.38 (2:1 acetonitrile-water). H
2045.8).
NMR (600 MHz, D
2
O, 25 °C): δ ϭ 2.62Ϫ2.72 (m, 6 H), 3.20Ϫ3.25
(m, 3 H), 3.26Ϫ3.37 (m, 12 H), 3.38Ϫ3.45 (m, 6 H), 3.48Ϫ3.52 (m,
Octameric β-D-Cellobiosyl-PAMAM Dendrimer (16): To a mixture
3
H), 3.53Ϫ3.61 (m, 6 H), 3.62Ϫ3.68 (m, 3 H), 3.71Ϫ3.76 (m, 3
of methanolic solution of PAMAM dendrimer, generation 1, 15
(100 mg,
H), 4.29 (d, J ϭ 15.7 Hz, 3 H), 4.42 (d, J ϭ 7.9 Hz, 3 H, H-1) 0.25 mmol) in 4 mL of DMF, diisopropylethylamine (88 µL,
H), 3.81Ϫ3.84 (m, 3 H), 3.85Ϫ3.91 (m, 3 H), 4.18 (d, J ϭ 15.6 Hz, (20% wt. in methanol) (115 µL, 0.016 mmol),
3
5
13
and 4.43 (d, J ϭ 8.0 Hz, 3 H, H-1Ј) ppm. C NMR (150 MHz,
O, 25 °C): δ ϭ 36.9 (NCH CH NH), 52.5 (CONHCH CH NH),
0.3 (C-6), 61.0 (C-6Ј), 68.6 (OCH
5.3, 75.9, 76.4, 78.8, 102.7 (C-1), 103.0 (C-1Ј) and 172.3
0.5 mmol) and BOP reagent (110 mg, 0.25 mmol) were added and
the reaction mixture was stirred at 25 °C for 12 h. Purification fol-
D
6
7
2
2
2
2
2
2
CO), 69.8 (C-4), 73.1, 73.5, 74.5, lowed by lyophilization afforded dendrimer 16 as a white foam.
Gel-permeation HPLC of the product 16 showed only a single
ϩ
(CH
2
CONH) ppm. MS ESI-MS: m/z ϭ 1293.4932 [M ϩ H]
requires 1293.4938).
peak, with a retention time of 18.776 min: detection at 220 nm
1
(C
48
H
85
O
36
N
4
(Table 1). Yield 47 mg (65%). H NMR (600 MHz, D
2
O, 25 °C):
2468
 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
Eur. J. Org. Chem. 2003, 2462Ϫ2470

Cellobiosylated Dimer and Trimer and Cellobiose-Coated Polyamidoamine Dendrimers
δ ϭ 2.23Ϫ2.42 (m), 2.47Ϫ2.60 (m), 2.65Ϫ2.79 (m), 2.90Ϫ2.94 (m), peak, with a retention time of 15.016 min: detection at 220 nm
FULL PAPER
1
3
3
3
.11Ϫ3.28 (m), 3.28Ϫ3.36 (m), 3.36Ϫ3.45 (m), 3.46Ϫ3.53 (m),
.54Ϫ3.60 (m), 3.61Ϫ3.67 (m), 3.71Ϫ3.76 (m), 3.79Ϫ3.85 (m),
.86Ϫ3.91 (m), 4.16 (d, J ϭ 15.6 Hz, 8 H), 4.27 (d, J ϭ 15.6 Hz, 8
2
(Table 1). Yield 50 mg (65%). H NMR (600 MHz, D O, 25 °C):
δ ϭ 2.26Ϫ2.39 (m), 2.48Ϫ2.59 (m), 2.66Ϫ2.80 (m), 2.92 (s),
3.02Ϫ3.07 (m), 3.13Ϫ3.17 (m), 3.17Ϫ3.27 (m), 3.28Ϫ3.36 (m),
3.37Ϫ3.45 (m), 3.47Ϫ3.53 (m), 3.54Ϫ3.60 (m), 3.61Ϫ3.67 (m),
H), 4.42 (d, J ϭ 7.9 Hz, 8 H, H-1) and 4.43 (d, J ϭ 8.0 Hz, 8 H,
H-1Ј) ppm. 13C NMR (600 MHz, D
O, 25 °C): δ ϭ 32.5, 32.6, 3.70Ϫ3.76 (m), 3.79Ϫ3.91 (m), 4.16 (d, 64 H, J ϭ 15.7 Hz), 4.28
2
3
6.7, 36.9, 38.4, 38.6, 49.9, 51.2, 59.9 (C-6), 60.6 (C-6Ј), 68.1
(d, 64 H, J ϭ 15.7 Hz), 4.415 (d, 64 H, J ϭ 7.9 Hz, H-1) and 4.425
(d, 64 H, J ϭ 7.9 Hz, H-1Ј) ppm. C NMR (150 MHz, D O, 25
2
1
3
(OCH
2
CO), 69.5 (C-4), 72.7, 73.1, 74.1, 74.9, 75.5, 76.0, 78.5, 102.3
(
C-1), 102.6 (C-1Ј) and 172.0, 174.4 and 175.0 (CH CONH) ppm. °C): δ ϭ 32.7, 36.7, 38.5, 38.6, 39.7, 49.0, 51.3, 59.9 (C-6), 60.6 (C-
2
ϩ
MS MALDI-TOF: m/z
108Na requires 4509).
ϭ
4505Ϯ5 [M
ϩ
Na] (C174
H
304
6Ј), 68.1 (OCH
78.6, 102.3 (C-1), 102.6 (C-1Ј) and 172.0, 174.6, 174.6, 175.0 and
75.1 (CH CONH) ppm.
2
CO), 69.5 (C-4), 72.7, 73.2, 74.1, 74.9, 75.5, 76.0,
26
N O
1
2
Hexadecameric β- -Cellobiosyl-PAMAM Dendrimer (18): To a
D
mixture of methanolic solution of PAMAM dendrimer, generation
Measurement of CDP Reaction on the Cellobiosyl Dendrimers: A
mixture containing glucose-1-phosphate solution (40 m, 25µL),
2
0
0
, 17 (20% wt. in methanol) (127 µL, 0.0078 mmol), 5 (100 mg,
.25 mmol) in 4 mL of DMF, diisopropylethylamine (88 µL, cellobiose (40 m, 25µL) solution and cellobiosylated glycoconju-
.5 mmol) and BOP reagent (110 mg, 0.25 mmol) were added and
gates (20 m of compound 10, 13.3 m of compound 12, 10 m
of compound 14, 5 m of compound 16, 2.5 m of compound 18,
1.25 m of compound 20 and 0.625 m of compound 22) and a
the reaction mixture was stirred at 25 °C for 14 h. Purification fol-
lowed by lyophilization afforded dendrimer 18 as a white foam.
Gel-permeation HPLC of the product 18 showed only a single solution of the enzyme cellodextrin phosphorylase (0.15 U/mL, 50
peak, with a retention time of 17.489 min: detection at 220 nm
µL) in MOPS buffer (pH 7.5, 50 m) was incubated at 37 °C. Ali-
quots were removed (2 µL) at certain intervals from 0 h to 120 h
1
2
(Table 1). Yield 56 mg (76%). H NMR (600 MHz, D O, 25 °C):
δ ϭ 2.24Ϫ2.44 (m), 2.47Ϫ2.61 (m), 2.65Ϫ2.81 (m), 2.92 (s), and were diluted 50 times with MOPS buffer (pH 7.5, 50 m⁾ and
3
.12Ϫ3.27 (m), 3.28Ϫ3.36 (m), 3.37Ϫ3.45 (m), 3.46Ϫ3.53 (m), heated at 100 °C for 10 min to stop the reaction. After keeping at
[16]
3.54Ϫ3.60 (m), 3.61Ϫ3.66 (m), 3.67Ϫ3.78 (m), 3.79Ϫ3.92 (m), 4.16
25 °C for 10 min, a solution of Glc-1-P quantification reagent
(
d, 16 H, J ϭ 15.6 Hz), 4.28 (d, 16 H, J ϭ 15.6 Hz), 4.41 (d, 16 H, (100 µL) was added. The reaction mixture was incubated at 25 °C
13
J ϭ 7.9 Hz, H-1) and 4.42 (d, 16 H, J ϭ 7.9 Hz, H-1Ј) ppm.
C
for 30 min and absorbance was measured at 340 nm to quantify
Glc-1-P. A standard curve was obtained by measurement of ab-
NMR (150 MHz, D
3
4
2
O, 25 °C): δ ϭ 31.3, 32.6, 36.7, 36.9, 38.4,
8.6, 48.9, 51.2, 59.9 (C-6), 60.6 (C-6Ј), 68.1 (OCH
), 72.7, 73.1, 74.1, 74.9, 75.5, 76.0, 78.5, 102.3 (C-1), 102.6 (C-1Ј) µ and 300 µ solutions of Glc-1-P. The percentage of remaining
2
CO), 69.5 (C- sorbance at 340 nm using 50 µ, 100 µ, 150 µ, 200 µ, 250
and 164.9, 172.0, 174.5 and 175.0 (CH
TOF: m/z ϭ 9397Ϯ7 [M ϩ Na] (C366 O N58Na requires and the results are summarized in Figure 1.
2
CONH) ppm. MS MALDI-
glucose-1-phosphate was calculated and plotted against time (h),
ϩ
H
640 220
9391).
3
2-Meric β-D-Cellobiosyl-PAMAM Dendrimer (20): To a mixture
Acknowledgments
of methanolic solution of PAMAM dendrimer, generation 3, 19
(20% wt. in methanol) (135 µL, 0.0039 mmol), 5 (100 mg,
The authors thank Dr. Masahiko Okada and Dr. Kaname Tsutsu-
0
0
.25 mmol) in 4 mL of DMF, diisopropylethylamine (88 µL, miuchi of Chubu University for helpful suggestions in the early
.5 mmol) and BOP reagent (110 mg, 0.25 mmol) were added and stage of this research. Thanks are also due to Dr. Mayumi O. Ka-
the reaction mixture was stirred at 25 °C for 21 h. Purification fol-
lowed by lyophilization afforded dendrimer 20 as a white foam.
Gel-permeation HPLC of the product 20 showed only a single
meyama and Dr. Takashi Murata of the National Food Research
Institute for mass spectrometry. The Japan Society for the Pro-
motion of Science is gratefully acknowledged for financial support
and for a JSPS Postdoctoral Fellowship to A.K.C. This work was
supported in part by a grant from the Program for the Promotion
peak, with a retention time of 16.510 min: detection at 220 nm
1
(
Table 1). Yield 40 mg (53%). H NMR (600 MHz, D
2
O, 25 °C):
δ ϭ 2.20Ϫ2.41 (m), 2.48Ϫ2.59 (m), 2.60Ϫ2.90 (m), 2.92 (d, J ϭ of Basic Research Activities for Innovative Bioscience.
.4 Hz), 3.17Ϫ3.28 (m), 3.29Ϫ3.36 (m), 3.37Ϫ3.45 (m), 3.46Ϫ3.53
m), 3.54Ϫ3.60 (m), 3.61Ϫ3.67 (m), 3.70Ϫ3.77 (m), 3.79Ϫ3.85 (m),
0
(
3
1
8
3
.86Ϫ3.92 (m), 4.14 (d, 32 H, J ϭ 15.6 Hz), 4.28 (d, 32 H, J ϭ
[1]
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3
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[2]
2
[3] [3a]
2.7, 36.7, 36.9, 38.4, 38.6, 39.5, 48.9, 49.0, 51.3, 59.9 (C-6), 60.6
CO), 69.5 (C-4), 72.7, 73.2, 74.1, 74.9, 75.5,
6.0, 78.5, 102.3 (C-1), 102.6 (C-1Ј) and 164.9, 172.0, 174.5, 174.6,
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(C-6Ј), 68.1 (OCH
2
3b]
7
1
3c]
2
ϩ
1312 444
m/z ϭ 19166Ϯ15 [M ϩ Na] (C750H O N122Na requires
3d]
19155).
64-Meric β-D-Cellobiosyl-PAMAM Dendrimer (22): To a mixture
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Eur. J. Org. Chem. 2003, 2462Ϫ2470