Synthesis of a new 2-amino-glycan, poly-(1→6)-α- D -mannosamine, by ring-opening polymerization of 1,6-anhydro-mannosamine derivatives

Article abstract of DOI:10.1002/pola.26262

A stereoregular 2-amino-glycan composed of a mannosamine residue was prepared by ring-opening polymerization of anhydro sugars. Two different monomers, 1,6-anhydro-2-azido-mannose derivative (3) and 1,6-anhydro-2-(N, N-dibenzylamino)-mannose derivative (6), were synthesized and polymerized. Although 3 gave merely oligomers, 6 was promptly polymerized into high polymers of the number-average molecular weight (M_n) of 2.3 × 10 ⁴ to 2.9 × 10⁴ with 1,6-α stereoregularity. The differences of polymerizability of 3 and 6 from those of the corresponding glucose homologs were discussed. It was found that an N-benzyl group is exceedingly suitable for protecting an amino group in the polymerization of anhydro sugars of a mannosamine type. The simultaneous removal of O- and N-benzyl groups of the resulting polymers was achieved by using sodium in liquid ammonia to produce the first 2-amino-glycan, poly-(1→6)-α-D- mannosamine, having high molecular weight through ring-opening polymerization of anhydro sugars.

Full text of DOI:10.1002/pola.26262

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Synthesis of a New 2-Amino-Glycan, Poly-(1fi6)-a-D-mannosamine, by
Ring-Opening Polymerization of 1,6-Anhydro-mannosamine Derivatives
Kazuyuki Hattori, Takashi Yoshida
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INTRODUCTION The 2-amino-glycans composed of 2-amino-
2-deoxy-hexose (hexosamine) are frequently derived from
natural sources and exhibit various biological activities. Poly-
(1!4)-b-D-glucosamine (chitosan), usually obtained by de-N-
acetylation of chitin, is the most well-known and possesses
numerous functions such as antibacterial activity,1,2 antitu-
mor activity,3,4 wound-healing effect,5,6 nontoxicity,6,7 and
biocompatibility.5,7 Poly-(1!4)-a-D-galactosamine was first
discovered in the fluid portion of the culture of fungus As-
pergillus parasiticus,8 and it is also produced by the micro-
organism Paecilomyces sp. I-1 strain.9 This 2-amino-glycan
shows significantly higher antitumor effects in vivo compared
to chitosan.10 Its action mechanism is believed to be similar
to that of chitosan and is ascribed to the activation of macro-
phage lineage cells and/or interferon produced from T lym-
phocytes.11 However, it is unclear whether the higher activity
originates from the structural differences of the repeating
hexosamine unit, glycosidic linkage, and molecular weight or
a difference in the essential action mechanism. Interestingly,
among three biologically important hexosamines correspond-
ing to glucosamine, galactosamine, and mannosamine, only
the polymer of mannosamine has not yet been found. On the
other hand, a unique linear 2-amino-glycan composed of
N- and partially O-acetylated D-mannosamines linked via a
(1!6)-a phosphodiester bond occurs in the capsule of
meningococcal,12 which plays a role in the infection of men-
ingitis in humans. Although, such phosphoglycans are gener-
ally not classified as "polysaccharides, " they are attractive
for some different biological properties from those of normal
polysaccharides.13 The amino or acetylated amino group of
the sugar moiety is responsible for all the aforementioned
biological functions. However, the structure of natural
2-amino-glycans is not quite homogeneous. The existence of
a few branches and/or partial modifications of amino and
hydroxyl groups greatly affects the biological activities and
complicates structure–activity relationship studies. Therefore,
the preparation of 2-amino-glycans having a definite repeat-
ing unit and glycosidic linkage, and high molecular weights
is desired.
Several reports have been published on the chemical synthe-
sis of 2-amino-glycans. Repeating the stepwise or blockwise
coupling of monosaccharides or oligosaccharides is suitable
for the elongation of multicomponent sugars.14–17 Recently, a
(1!6)-b-D-glucosamine undecamer18 and N-Ac-(1!4)-b-D-
glucosamine tetramer19 were synthesized using this method-
ology. Apparently, this method is not appropriate for prepar-
ing large glycans with a single repeating unit, because a long
complicated process is involved for obtaining high molecular
weights. Kadokawa et al. devised the novel synthetic method
of 2-amino-glycans by polyaddition of the oxazoline deriva-
tives of N-Ac-D-glucosamine in the presence of acid catalysts
to give poly-N-Ac-(1!4)-b-D-glucosamine (chitin)20,21 and
C
V
2 01 2 W i l e y P er i o d i c al s , I n c .
W W W .M A TE R I A LS V IE W S . C O M
J O U R N A L O F P O L Y M E R S C I E N C E P A R T A : P O L Y M E R C H E M I S T R Y 2012, 0 00 , 0 00 – 0 00
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poly-N-Ac-(1!6)-b-D-glucosamine21 that have Mn of 2.8 ꢀ
103 and 5.6 ꢀ 103, respectively. Unfortunately, this method
cannot provide glycans with 1,2-cis glycosidic linkages.
Kobayashi et al.22,23 synthesized the first artificial chitin
from a chitobiose oxazoline derivative making use of the
reverse reaction of hydrolase of chitin. In this strategy, high
molecular weight glycans can be obtained in high yields
without protecting the hydroxyl groups of the repeating
sugar units, however, it has inherent limitations in the for-
mation of only the natural type of glycosidic linkage due to
the dependence on enzymes.
The dialysis membrane was a Cellulose Dialyzer Tubing VT-
803, exclusive Mw < 8000, purchased from Nacalai Tesque.
Melting points were determined by a Yamato MP-21 appara-
tus. Specific rotations were measured on a JASCO P-1020 po-
larimeter in a water-jacketed 1-dm cell. IR spectra were
obtained with a Perkin Elmer Spectrum One using a KBr pel-
let for solid samples or a NaCl disk for liquid samples. 1H
and 13C NMR spectra were recorded on a JEOL JNM-ECX400
spectrometer at 400 and 100 MHz, respectively, in CDCl3
with tetramethylsilane or in D2O with sodium 3-trimethyl-
silyl-1-propanesulfonate as an internal standard. Two-dimen-
sional spectra were measured with pulsed field gradients.
Elemental analyses were conducted on a J-Science Micro Cor-
der JM10. Preparative high-performance liquid chromatogra-
phy (HPLC) was executed through a Wakosil 5SIL silica gel
column (10 ꢀ 300 mm2, Wako Pure Chemical Industries,
Japan) for purification of liquid samples. Molecular weights
and distribution of polymers were evaluated by means of gel
permeation chromatography (GPC) calibrated with standard
polystyrenes (Shodex SM-105; Showa Denko, Japan) in CHCl3
or standard pullulans (Shodex P-82; Showa Denko) in 66.7
mM of phosphate buffer (pH 6.86). The columns were
Tosoh TSK gel G3000HXL, G4000HXL, and G5000HXL for
the CHCl3 eluent and TSK gel G2500PWXL, G3000PWXL, and
G4000PWXL for the buffer solution, connected in series.
Cationic ring-opening polymerization of anhydro sugars is
one of the most powerful methods for the chemical synthesis
of polysaccharides.24–26 To prepare amino-glycans by this
technique, Uryu et al.27 first used 1,6-anhydro-glucose deriv-
atives bearing an azido group, which is convertible into an
amino group. An amino group prevents cationic polymeriza-
tion. However, the 2-azido derivative possessed low polymer-
izability, as the maximum degree of polymerization (DP) of
the products was 6. Others have reported the polymerization
of 1,6-anhydro-glucosamine derivatives with a phthaloyl-pro-
tected amino group to afford (1!6)-b linked oligomers with
DP of only 3–7.28,29 We also examined the use of three
amino protecting groups such as a benzyl, cyclic alkyl, and
cyclic silyl groups toward the synthesis of poly-(D-glucosa-
mine) with a different type of glycosidic linkage from that of
chitosan by polymerization of 1,6-anhydro-glucosamine
derivatives.30 All products were still oligomers having
(1!6)-a linkages with DP of up to 8. Thus, stereoregular 2-
amino-glycans of high molecular weights have not yet been
obtained through ring-opening polymerization of anhydro
sugars. Among several factors that affect the polymerizabil-
ity,31 we proposed that the reason for the low polymerizabil-
ity of 1,6-anhydro-glucosamine derivatives is mainly the
steric hindrance of the bulky substituted axial amino group
against the elongation of the sugar unit. Hence, a 1,6-anhy-
dro-mannosamine derivative, which has less steric hindrance
than the glucosamine homolog, is more likely to be polymer-
ized. This article describes the synthesis and ring-opening
polymerization of 1,6-anhydro-2-azido- and -2-(protected
amino)-mannose derivatives, and the synthesis of a new
2-amino-glycan, poly-(1!6)-a-D-mannosamine, with a high
molecular weight.
Synthesis of 3,4-Di-O-acetyl-1,6-anhydro-2-azido-2-
deoxy-b-D-mannopyranose (2)
Sodium methoxide (0.80 g, 15 mmol) was added to a solu-
tion of 1,3,4,6-tetra-O-acetyl-2-azido-2-deoxy-a-D-mannopyra-
nose (1)32 (5.22 g, 14.0 mmol) in tetrahydrofuran (THF; 20
mL) and methanol (30 mL) with stirring. After 4 h at room
temperature, cation-exchange resin was added until the pH
was 7. The resin was filtered off, and the filtrate was evapo-
rated in vacuo. To a solution of the resulting yellowish solid
(3.05 g) in pyridine (20 mL), a solution of p-toluenesulfonyl
chloride (3.0 g, 16 mmol) in pyridine (5 mL) was added
dropwise at 0ꢁC under nitrogen. The mixture was stirred at
room temperature for 3 h, quenched by a small portion of
methanol and then concentrated in vacuo. To a solution of
the residue in ethanol (20 mL), 1,8-diazabicyclo[5.4.0]undec-
7-ene (DBU) (4.5 mL, 30 mmol) was added dropwise at
room temperature. The solution was stirred for 12 h and
then concentrated in vacuo to give a brown syrup. Acetic an-
hydride (10 mL) was added to a solution of the syrup in
pyridine (20 mL) with stirring. After 6 h at room tempera-
ture, the mixture was concentrated in vacuo and diluted
with chloroform and water. The chloroform portion was
washed with water twice, dried over anhydrous sodium sul-
fate, and evaporated in vacuo. The crude product was puri-
fied by silica gel chromatography (hexane/ethyl acetate 2/1
as eluent) to afford 2.94 g of 2 as colorless crystals. The
yield was 78% from 1.
EXPERIMENTAL
General
All chemicals were reagent grade and used without purifica-
tion unless specified. Sodium hydride (Kishida Chemical,
Japan) was used after the coating of mineral oil was washed
R
with hexane. Cation-exchange resin (DowexV 50WX8, Hþ
form, 50-100 mesh) was washed with methanol prior to
use. p-Chlorobenzenediazonium hexafluorophosphate was
obtained from Tokyo Chemical Industry, Japan, and purified
by recrystallization from water twice. Dichloromethane
(Sigma–Aldrich, USA) and 1,2-dimethoxyethane (Kishida
Chemical) were dried over CaH2 and distilled just before use.
mp: 115–117ꢁC. ½aꢂD25: þ7.8 (c ¼ 1.0, CHCl3). Anal. Calcd for
C
10H13N3O6 (271.2): C, 44.28; H, 4.83; N, 15.49. Found: C,
44.15; H, 4.81; N, 15.41. IR (KBr, cmꢃ1): 2114 (s, N3), 1756
and 1748 (s, C¼¼O).
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1H NMR (CDCl3, ppm): d 2.15 (s, 3H, O4ACOACH3), 2.20 (s,
3H, O3ACOACH3), 3.34 (d, 1H, H2, J2,3 ¼ 4.76 Hz), 3.88 (dd,
1H, H6exo, J5,6exo ¼ 5.86 Hz, J6exo,6endo ¼ 7.87 Hz), 4.23 (d,
70.21; H, 6.81; N, 4.09. IR (NaCl, cmꢃ1): 3382, 3314, and
1586 (m, NH), 1601, 740, and 699 (m, aromatic).
1H NMR (CDCl3, ppm): d 1.57 (s, 2H, NH2), 2.98 (d, 1H, H2,
J2,3 ¼ 5.49 Hz), 3.44 (s, 1H, H4), 3.63–3.67 (m, 2H, H3 and
H6exo), 4.08 (d, 1H, H6endo, J6exo,6endo ¼ 6.96 Hz), 4.48 (s, 2H,
O3ACH2Ph), 4.50–4.56 (m, 3H, H5 and O4ACH2Ph), 5.23 (s,
1H, H1), 7.25–7.36 (m, 10H, phenyl ꢀ 2).
1H, H6endo, J6exo,6endo ¼ 7.87 Hz), 4.64 (d, 1H, H5, J5,6exo
¼
5.86 Hz), 4.77 (s, 1H, H4), 5.29 (d, 1H, H3, J2,3 ¼ 4.76), 5.59
(s, 1H, H1).
13C NMR (CDCl3, ppm):
d 20.8 (O3ACOACH3), 20.9
(O4ACOACH3), 56.7 (C2), 65.4 (C6), 69.1 (C3), 71.2 (C4),
73.5 (C5), 100.7 (C1), 169.4 (O3ACOACH3), 169.5
(O4ACOACH3).
13C NMR (CDCl3, ppm): d 50.7 (C2), 64.4 (C6), 71.1 (O4–
CH2Ph), 73.2 (O3–CH2Ph), 73.5 (C5), 75.0 (C4), 76.8 (C3),
103.6 (C1), 127.6–137.6 (phenyl).
Synthesis of 1,6-Anhydro-3,4-di-O-benzyl-2-(N, N-
dibenzylamino)-2-deoxy-b-D-mannopyranose (6)
Synthesis of 1,6-Anhydro-2-azido-3,4-di-O-benzyl-2-
deoxy-b-D-mannopyranose (3)
To a solution of 5 (1.71 g, 5.00 mmol) in DMF (10 mL),
dried NaH (0.30 g, 13 mmol) and benzyl bromide (1.5 mL,
13 mmol) were added with stirring at room temperature. Af-
ter 12 h, methanol was added to the solution until bubbles
were no longer produced, and the mixture was concentrated
under reduced pressure. The resultant syrup was dissolved
in chloroform and water. The separated chloroform portion
was washed with water twice, dried over anhydrous Na2SO4,
and then evaporated prior to column chromatography on
silica gel. Elution from the column with hexane/ethyl acetate
3/1 afforded syrupy 6, which was further purified by means
of HPLC (the same eluent), affording 2.4 g (92%) of the
desired product without any detectable impurity in the 1H
NMR spectrum. ½aꢂ2D5: þ18.3 (c ¼ 1.0, CHCl3). Anal. Calcd for
To a solution of 2 (2.72 g, 10.0 mmol) in N, N-dimethyl
formamide (DMF) (30 mL), benzyl bromide (3.6 mL, 30
mmol) was added dropwise in the presence of Ba(OH)2 (3.4
g, 20 mmol) and BaO (7.6 g, 50 mmol) at room temperature.
After 12 h of stirring, a small amount of methanol was
added, and the mixture was subject to vacuum filtration
R
using a membrane with a pore size of 0.45 mm (MilliporeV).
The filtrate was concentrated in vacuo and diluted with chlo-
roform. The solution was washed with water three times,
dried over anhydrous Na2SO4, and then evaporated in vacuo.
The residue was purified through silica gel chromatography
and subsequent HPLC (hexane/ethyl acetate 2/1 as both elu-
25
ents) to give 2.78 g (75%) of 3 as a colorless syrup. ½aꢂD
:
þ13.1 (c ¼ 1.0, CHCl3). Anal. Calcd for C20H21N3O4 (367.4):
C, 65.38; H, 5.76; N, 11.44. Found: C, 65.20; H, 5.79; N,
11.48. IR (NaCl, cmꢃ1): 2102 (s, N3), 1605, 739, and 698
cmꢃ1 (w, aromatic).
C
34H35NO4 (521.6): C, 78.28; H, 6.76; N, 2.69. Found: C,
78.18; H, 6.75; N, 2.65. IR (NaCl, cmꢃ1): 1603, 734, and 698
(m, aromatic).
1H NMR (CDCl3, ppm): d 3.09 (dd, 1H, H2, J2,3 ¼ 4.85 Hz, J2,4
¼ 0.92 Hz), 3.40 (dd, 1H, H4, J2,4 ¼ 0.92 Hz, J3,4 ¼ 1.60 Hz),
3.67 (dd, 1H, H6exo, J5,6exo ¼ J6exo,6endo ¼ 6.50 Hz), 3.83 (ddd,
1H, H3, J2,3 ¼ 4.85 Hz, J3,4 ¼ 1.60 Hz, J3,5 ¼ 3.10 Hz), 3.99
(s, 4H, N-CH2Ph ꢀ 2), 4.18 (dd, 1H, H6endo, J5,6exo ¼ 0.73 Hz,
J6exo,6endo ¼ 6.50 Hz), 4.44 and 4.51 (d, 2H, O3ACH2Ph, J ¼
10.43 Hz), 4.45 (m, 3H, H5 and O4ACH2Ph), 5.63 (s, 1H,
H1), 7.16–7.35 (m, 10H, phenyl ꢀ 2).
1H NMR (CDCl3, ppm): d 3.14 (dd, 1H, H2, J2,3 ¼ 5.49 Hz, J2,4
¼ 1.65 Hz), 3.46 (ddd, 1H, H4, J2,4 ¼ J3,4 ¼ 1.65 Hz, J4,5
1.83 Hz), 3.75 (dd, 1H, H6exo, J5,6exo ¼ 6.04 Hz, J6exo,6endo
¼
¼
7.32 Hz), 3.86 (ddd, 1H, H3, J2,3 ¼ 5.49 Hz, J3,4 ¼ 1.65 Hz,
J3,5 ¼ 3.11 Hz), 4.25 (d, 1H, H6endo, J6exo,6endo ¼ 7.32 Hz),
4.44 and 4.49 (d, 2H, O4ACH2Ph, J ¼ 12.26 Hz), 4.48 and
4.68 (d, 2H, O3ACH2Ph, J ¼ 11.90 Hz), 4.54 (ddd, 1H, H5,
J3,5 ¼ 3.11 Hz, J4,5 ¼ 1.83 Hz, J5,6exo ¼ 6.04 Hz), 5.55 (s, 1H,
H1), 7.24–7.39 (m, 10H, phenyl ꢀ 2).
13C NMR (CDCl3, ppm): d 55.7 (N-CH2Ph ꢀ 2), 57.6 (C2),
64.7 (C6), 71.2 (O4ACH2Ph), 72.8 (O3ACH2Ph), 74.7 (C5),
75.4 (C4), 78.3 (C3), 102.3 (C1), 126.6–140.6 (phenyl).
13C NMR (CDCl3, ppm):
d 57.5 (C2), 65.1 (C6), 71.4
(O4ACH2Ph), 73.7 (O3ACH2Ph), 74.0 (C5), 75.8 (C4), 76.7
(C3), 101.0 (C1), 127.8–137.3 (phenyl).
Polymerization
Ring-opening polymerization was performed in dichlorome-
thane under high vacuum conditions (ꢄ10ꢃ5 mmHg) at ꢃ60
and 0ꢁC as reported previously.31 The polymers produced
were purified by reprecipitation with chloroform–petroleum
benzine or chloroform–methanol three times and freeze-
dried from benzene. The supernatants after reprecipitation
were confirmed by GPC whether polymeric products still
remained or not. The spectral features of polymers 4 and 7
are as follows.
Synthesis of 2-Amino-1,6-anhydro-3,4-di-O-benzyl-2-
deoxy-b-D-mannopyranose (5)
Sodium tetrahydroborate (0.19 g, 5.0 mmol) was added to a
solution of 3 (1.84 g, 5.00 mmol) in methanol (30 mL) at
room temperature. The solution was stirred at 60ꢁC for 2 h,
quenched by the addition of acetone, and concentrated in
vacuo. The residue was dissolved in chloroform and water.
The chloroform layer was washed with water twice, dried
over anhydrous Na2SO4, and evaporated in vacuo. The crude
product was chromatographed on silica gel (ethyl acetate/
methanol 5/1 as eluent), giving 1.57 g (92%) of purified col-
orless syrupy 5. ½aꢂ2D5: þ27.9 (c ¼ 1.0, CHCl3). Anal. Calcd for
2-Azido-3,4-di-O-benzyl-2-deoxy-(1fi6)-a-D-mannopyranan
(4)
IR (KBr, cmꢃ1): 2104 (s, N3), 1602, 736, and 696 (m,
aromatic).
C20H23NO4 (341.4): C, 70.36; H, 6.79; N, 4.10. Found: C,
W W W .M A TE R I A LS V IE W S . C O M
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1H NMR (CDCl3, ppm): d 3.22 (br s, 1H, H2), 3.43 (br s, 1H,
H4), 3.78 (br s, 1H, H5), 3.97 (br s, 1H, H3), 3.4–4.2 (m, 2H,
H6, 60), 4.47 and 4.97 (d, 2H, O3ACH2Ph, J ¼ 12.1 Hz),
4.83 (s, 2H, O4ACH2Ph), 5.07 (s, 1H, H1), 7.2–7.5 (m, 10H,
phenyl ꢀ 2).
13C NMR (CDCl3, ppm): d 64.8 (C2), 67.1 (C6), 70.8 (C5),
73.3 (O3ACH2Ph), 74.5 (O4ACH2Ph), 77.3 (C4), 81.7 (C3),
95.2 (C1), 126.1–138.2 (phenyl).
3,4-Di-O-benzyl-2-(N, N-dibenzylamino)-2-deoxy-(1fi6)-a-
D-mannopyranan (7)
IR (KBr, cmꢃ1): 1601, 740, and 697 (m, aromatic).
SCHEME 1
S
y
n
t
h
e
s
i
s
a
n
d
r
i
n
e
g
-
o
p
e
n
i
n
g
i
p
o
l
y
m
e
r
i
z
a
t
i
o
n
o
f
3
1H NMR (CDCl3, ppm): d 3.35 (s, 1H, H4), 3.42 (s, 1H, H2),
3.74 (br s, 1H, H6), 3.93 (s, 1H, H3), 4.05 (s, 4H, N-CH2Ph ꢀ
2), 4.21 (d, 1H, H60, J ¼ 6.59 Hz), 4.46 and 4.57 (dd, 2H,
O4ACH2Ph, J ¼ 12.3 Hz), 4.56 (s, 1H, H5), 4.52 and 4.71
(dd, 2H, O3ACH2Ph, J ¼ 11.8 Hz), 5.73 (s, 1H, H1).
a
n
d
6.
T
h
e
n
u
m
b
e
r
s
d
e
n
o
t
t
h
e
p
o
s
t
i
o
n
o
f
e
a
c
h
c
a
r
b
o
n
.
Reagents and conditons:
(
a
)
i
—
N
a
O
M
,
e
,
M
e
O
H
/T
HF
,
r
t
,
4
h
;
i
i
—
p
P
P
-
T
s
,
C
l
,
P
y
,
0 ꢁ
C
t
h
e
n
r
t
,
3
h
;
i
i
i
—
D
B
,
U
E
t
O
H
,
r
t
,
1
2
h
2
;
i
v
—
A
c
O
,
2
y
F
,
r
t
,
6
h
,
7
8
%
;
(
b
)
B
n
B
r
,
B
a(
O
H
)
B
a
O
,
D
M
F
,
r
t
,
1
a
h
,
7
5
%
;
(
c
)
2
C
H
C
l
, ꢃ
6
0ꢁ
C
,
u
n
d
e
r
h
i
g
h
v
a
c
u
u
m
;
(
d
)
N
B H ,
4
M
e
O
H
,
5
2
2
6
0ꢁ
C
,
2
h
8ꢁ
,
9
2
%
;
(
,
e
)
B
n
B
r
,
N
a
H
,
D
M
F
,
r
t
,
1
2
h
,
9
2%
;
(
f
)
N
a
,
l
i
q
u
i
d
13C NMR (CDCl3, ppm): d 56.2 (N-CH2Ph ꢀ 2), 60.5 (C2),
63.2 (C6), 69.9 (C5), 71.5 (C4), 72.3 (O3ACH2Ph), 74.9
(O4ACH2Ph), 77.0 (C3), 97.7 (C1), 126.2–138.4 (phenyl).
N
H
, ꢃ
7
C
,
1
h
9
1
%
;
(
g
)
3
%
H
C
l
,
q
u
a
n
t
i
t
a
t
i
v
e
.
3
treatment with acetic anhydride and pyridine. The IR spec-
trum and elemental analysis indicated that the azido group
was not damaged during cyclization. Deacetylation and sub-
sequent benzylation of 2 were performed in a one-pot reac-
tion. A large amount of Ba(OH)2 and BaO removed the acetyl
groups in addition to catalyzing the subsequent benzylation
to afford 3 without significant side reactions. IR and NMR
analyses of 3, after purification by preparative HPLC, proved
the existence of an azido group and two benzyl groups as
shown in Figure 1(A, B).
Debenzylation
The polymer 7 (100 mg, Mn ¼ 2.5 ꢀ 104, Mw/Mn ¼ 2.4) was
treated by the same procedure as in the literature31 to give
28 mg (91%) of 2-amino-2-deoxy-(1!6)-a-D-mannopyranan
(8). After IR analysis, 8 was dissolved in dilute HCl (3%)
due to its insolubility in water. Another purification by dialy-
sis and subsequent freeze-drying of the solution provided
water-soluble solid 9 as the HCl salt of 8 (34 mg). The fol-
lowing data are based on the HCl salt 9. Mn ¼ 1.3 ꢀ 104,
25
Mw/Mn ¼ 2.0. ½aꢂD : þ65.2 (c ¼ 1.0, H2O). IR (KBr, cmꢃ1):
As will be described earlier, polymer 4 obtained by polymer-
ization of 3 could not be converted to the corresponding 2-
3000ꢃ3500 (br, OH and NH), 1620 (br, NH).
13C NMR (D2O, ppm): d 57.3 (C2), 66.2 (C6), 70.3 (C4), 71.2
amino-mannan, and therefore another monomer
6 was
(C3), 73.0 (C5), 96.8 (C1).
designed. The azido group of 3 was reduced with sodium
tetrahydroborate, and then the amino group of the resulting
1,6-anhydro-mannosamine derivative 5 was protected with
benzyl groups using sodium hydride and benzyl bromide. In
this case, both amino protons must be substituted not to
prevent cationic polymerization. The resultant syrup was
carefully purified by preparative HPLC and analyzed by NMR
RESULTS AND DISCUSSION
Synthesis of Monomers
First, we focused on the azido derivative 3 shown in
Scheme 1 as a monomer for the synthesis of 2-amino-man-
nans through ring-opening polymerization. The improved
large-scale preparation of the 2-azido-mannose acetate 1
from D-glucose that was reported recently32 facilitated this
work. The conversion of 1 into 2 was accomplished via a
four-step sequence without any isolation and purification of
the intermediates as follows. The treatment of 1 with so-
dium methoxide in methanol and subsequent cation-
1
spectroscopy. Two N-benzyl groups were observed in the H
NMR spectrum shown in Figure 1(C), indicating that the
desired monomer 6 was obtained.
Polymerization of 3 and 6
The ring-opening polymerization of 3 and 6 was accom-
plished at ꢃ60 and 0ꢁC in dichloromethane using PF5 as an
initiator. The experimental results are summarized in Table
1. In the polymerization of 3, ꢄ1 mol % of the initiator, a
typical amount for obtaining high molecular weights of poly-
saccharides,34 did not afford any products; only unreacted
monomer was recovered after 24 h. Increasing the concen-
tration of PF5 to 3 and 5 mol % gave oligomeric products in
about 10% yields (Entries 1 and 2), which were partly solu-
ble in methanol and isolated by reprecipitation with petro-
leum benzine from the chloroform solution. Their Mns were
calculated by GPC to be 1.5 ꢀ 103 and 1.8 ꢀ 103,
R
exchange using DowexV resin achieved the deacetylation
almost quantitatively. The formation of the 1,6-anhydro ring
followed the method of Boullanger and coworkers.33 At a
low temperature, the use of 1.1 equivalents of p-toluenesul-
fonyl chloride relative to the 2-azido-mannose allowed selec-
tive tosylation on C6, and then the addition of DBU accom-
plished the cyclization between C1 and C6 involving
detosylation. The main product at this point, assumed to be
1,6-anhydro-2-azido-2-deoxy-b-D-mannopyranose, was diffi-
cult to separate from the salts coproduced, and thus it was
isolated as the acetate form 2 by chromatography after
4
J
O
U
R
N
A
L
O
F
P
O
L
Y
M
E
R
S
C
I
E
N
C
E
P
A
R
T
A
:
P
O
L
Y
M
E
R
C
H
E
M
I
S
T
R
Y
2
0
1
2
,
0
0
0
,
0
0
0
–
0
0
0

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corresponding to DPs of 4 and 5, respectively. The use of 10
mol % of PF5 and 24 h of polymerization time slightly
improved Mn up to 4.6 ꢀ 103 (DP ¼ 13), though the yield
hardly changed (Entry 3). The increment of the molecular
weight was likely due to the higher concentration of the
initiator, not longer polymerization time, because further
extension of the time did not result in a significant difference
in either molecular weight or yield (Entry 4).
Thus, the polymerizability of a 1,6-anhydro-2-azido-mannose
derivative was extremely low compared to that of the
usual 1,6-anhydro-mannose homologs without an azido
group.24,25,35 This behavior resembled that of a 1,6-anhydro-
2-azido-glucose derivative.27 It seems that such low polymer-
izability of 2-azido anhydro sugars is derived not only from
the existence of an azido group but also some regiospecific
factor that interferes with polymerization, because 1,6-anhy-
dro-3-azido-glucose27 and -allose36 derivatives can be con-
verted to high polymers. One reasonable explanation is that,
as illustrated in Scheme 2(B), a nonlocalized electron pair of
the azido group attacks the electron-deficient C1 at the prop-
agating end, and the resultant species composed of a 1,2,3-
ꢃ
triazole cation and counter anion PF6 is less reactive. As a
result, chain propagation would be terminated. This mecha-
nism can be applied to only the 2-azido derivatives whose
azido group is adjacent to the C1 position and is independ-
ent of the configuration of the azido group. Similar neighbor-
ing group participation of an azido group was reported in
several articles.37,38 An ideal propagation mechanism for 1,6-
anhydro sugars is established as shown in Scheme 2(A).39
The structure of polymer 4 was characterized by NMR spec-
troscopy and optical rotations. Figure 2 shows the 100 MHz
13C NMR spectra of monomer 3 (A) and polymer 4 (B)
expanded from 50 to 110 ppm, in CDCl3 at room tempera-
ture. The peak assignment was accomplished by heteronu-
clear single quantum coherence (HSQC) and heteronuclear
multiple-bond correlation (HMBC) spectra. The 13C NMR
spectra of the monomer and polymer are quite different
1
FIGURE 1
f 3
I
R
s
p
e
c
t
r
u
m
o
f
3
(
A
)
a
n
d
4
0
0
M
H
z
H
N
M
R
s
p
e
c
t
r
a
o
(
B
)
a
n
d
6
(
C
)
i
n
C
D
C
l
.
3
TABLE 1
R
i
n
g-
O
p
e
n
i
n
g
P
r
o
l
y
m
e
r
i
z
a
t
i
o
n
o
f 3
a
n
d 6
.
b
c
P
F
T
e
ꢁC
m
p
.
T
i
m
e
Y
ie
l
d
Mn
5
d
25
a
3
E
n
t
r
y
M
o
n
o
m
e
(
m
o
l
%
)
(
)
(
h
)
(
%
)
(
ꢀ
1
0
)
Mw /Mn
½aꢂD (ꢁ)
1
2
3
4
5
6
7
8
9
3
3
3
3
6
6
6
6
6
6
3
5
1
1
3
5
5
1
1
2
–
–
–
–
–
–
–
–
6
6
6
6
6
6
6
6
0
1
1
2
7
1
4
8
8
8
2
6
8
1
1
9
2
2
3
3
0
0
1
1
4
3
2
2
2
2
–
–
.
.
.
.
8
5
6
5
1 .9
2 .2
2 .2
2 .4
1 .9
2 .1
1 .9
2 .0
–
–
þ
þ
þ
þ
þ
þ
þ
þ
–
7
7
7
7
4
4
4
4
8
3
5
9
6
5
6
5
.3
.6
.0
.9
.9
.3
.1
.9
0
0
0
0
0
0
0
6
4
2
2
0
1
0
0
0
9
8
5
6
8
6
2
.
.
.
.
6
7
3
9
0
0
0
0
0
1
0
4
–
a
c
M
o
t
n
o
m
e
r
:
2
0
0
m
g
,
d
i
c
h
l
o
r
o
m
e
t
h
a
n
e
:
0
.
2
0
m
L
.
D
e
t
e
r
m
i
n
e
d
b
y
G
P
C
.
b
d
P
e
r
o
l
e
u
m
b
e
n
z
i
n
e
-
i
n
s
o
l
u
b
l
e
p
a
r
t
.
M
e
a
s
u
r
e
d
i
n
C
H
C
l
(c ¼
1 .0 ) .
3
W W W .M A TE R I A LS V IE W S . C O M
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SCHEME 2
P
r
o
p
o
s
e
d
p
r
o
p
a
g
a
t
i
o
n
m
e
c
h
a
n
i
s
m
i
n
r
i
n
g
-
o
p
e
n
i
n
g
p o l y m e ri z at i o n o f 3.
from each other, probably due to the conformational change
of the pyranose ring from 1C4 to 4C1. In Figure 2(B), each
peak, particularly the C1 peak at 95.2 ppm, was single, indi-
cating that the repeating unit of 4 has one stereoselective
1
3
FIGURE 3
1
00
M
H
z
C N M R s p ec t r a o f 6 ( A ) a n d 7 ( B ) i n
C D Cl .
3
glycosidic linkage. The specific rotations of 4 were large and
positive from þ74 to þ80ꢁ, compared with that of 3 at
þ13.1ꢁ. Such a remarkable difference between monomer 3
and polymer 4 is ascribed to the inversion of configuration
at C1, according to the typical results of polymerization of
1,6-anhydro sugars.25 Hence, every glycosidic linkage of 4
was determined to adopt an a anomeric configuration.
The yields and molecular weights of polymer 4 were not
enough to produce a 2-amino-glycan. Therefore, another
monomer 6 was investigated. The concentration of PF5 from
3 to 5 mol % relative to 6 afforded polymers with high mo-
lecular weights of Mn of 2.6 ꢀ 104 to 2.8 ꢀ 104 (DP ¼ 50–
55) in ꢄ30% yield (Entries 5–7 in Table 1). Chromato-
graphic analysis revealed no oligomer, but partially
unreacted 6 was recovered in the supernatant after repreci-
pitation of the polymer. At higher concentrations of PF5 of
more than 5 mol %, a decline in Mn was observed, though
the same polymerization conditions were used (Entry 8).
The polymerization time of 8 h gave favorable yield and Mn,
and the shorter time of 4 h caused low conversion (Entries
6 and 7).
Similarly, we previously studied ring-opening polymerization
of a 1,6-anhydro-glucosamine derivative having an N, N-
dibenzyl protecting group on C2.30 However, it was little
polymerized under the same conditions as this study. These
results strongly suggest that the higher polymerizability of 6
more than that of the corresponding glucosamine derivative
1
3
FIGURE 2
1
00
M
H
z
C N MR s p e c tr a o f 3 ( A ) a n d 4 ( B ) i n
C D C l .
3
6
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reaction was terminated in 1 h, and subsequent conventional
work-up procedure was carried out to give a clear aqueous
solution, resulting in the amino-glycan 8 dissolved in water.
However, 8 became insoluble in water when subjected to ly-
ophilization. A similar phenomenon was also observed in the
synthesis of 3-amino-glucan.27 This was attributed to the
intermolecular and intramolecular hydrogen bonds between
the glycan chains formed by the elimination of water during
drying. The dried 8 was soluble in dilute HCl, but insoluble
in NaOH aqueous solution and even in high polar solvents
such as DMF and dimethyl sulfoxide. Figure 4 shows the 13C
NMR spectrum of 9, obtained from dialysis of the HCl salt of
8, in D2O at 40ꢁC. Resonances of six carbons appeared,
whereas there were no signals derived from benzyl groups,
indicating that every repeating unit of 9 was homogeneous
and the O- and N-benzyl groups were completely removed.
The absorption of C2 possessing an amino group was shifted
to the most upfield position of 57.3 ppm. The Mn and molec-
ular weight distribution of 9 were estimated to be 1.3 ꢀ 104
and 2.0, respectively. The narrowed distribution of 9 com-
pared with that of the starting 7 was explained as some low
molecular weight components were removed by dialysis.
1
3
FIGURE 4
1
0
0
M
H
z
C N MR s p e c tr um o f 9 i n D O .
2
is responsible for the configuration of a substituent on C2.
Such a bulky axial substituent on C2 as an N, N-dibenzyla-
mino group interferes with monomers attacking from the a
side. As the configuration of the substituent on C2 of 6 that
is a mannosamine-type is equatorial, the N, N-dibenzylamino
group would not reduce the polymerizability.
Figure 3 shows the 100 MHz 13C NMR spectra of monomer
6 (A) and polymer 7 (B) in the region from 50 to 110 ppm
in CDCl3 at room temperature. Each peak was assigned by
HSQC and HMBC measurements. As in the case of 4, it was
found that 7 has only (1!6)-a glycosidic linkages from the
single C1 absorption at 97.7 ppm and large positive specific
rotations of þ45 to þ46ꢁ.
CONCLUSIONS
In this study, two different monomers, 1,6-anhydro-2-azido-
mannose, and 1,6-anhydro-2-(N, N-dibenzylamino)-mannose
derivatives were synthesized and polymerized to prepare 2-
amino-glycans with high molecular weights by ring-opening
polymerization. The polymerizability of both monomers was
higher than that of the corresponding glucose homologs.
However, the 2-azido derivative still did not give sufficiently
high polymers to produce a free glycan because of insuffi-
cient yield and molecular weight. It was concluded that the
2-azido group of hexoses is involved in neighboring group
participation with the positively charged C1 at the propagat-
ing chain end and consequently prevents polymerization. On
the other hand, the N, N-dibenzylated mannosamine mono-
mer yielded high polymers with Mn ¼ 2.3 ꢀ 104 to 2.9 ꢀ
104. Both O- and N-benzyl groups in the polymer were
removed simultaneously by the Birch reduction. It was found
that an N-benzyl group is exceedingly suitable for protecting
an amino group in the polymerization of anhydro sugars of a
mannosamine type. Here, a synthetic 2-amino-glycan, (1!6)-
a-D-mannosamine, having high molecular weight and a com-
pletely homogeneous repeating unit including the glycosidic
linkage was obtained. The stability of such non-natural
amino-glycans against enzymes and microorganisms capable
of hydrolyzing hexosamine residues are advantage over natu-
ral ones. This artificial amino-polysaccharide will be a
helpful tool for investigating various biological activities.
Kanno et al.28 reported that a 1,6-anhydro-glucosamine de-
rivative whose amino group was protected with a phthaloyl
group was polymerized at a higher temperature of 0ꢁC to
obtain oligomers with (1!6)-b stereoregularity, though the
conversion was low. However, at the same temperature, poly-
merization of 6 did not proceed at all (Entries 9 and 10).
The reason is deduced as follows: in the polymerization of
1,6-anhydro sugars using PF5, the trialkyloxonium ion of the
propagating species is stable at temperatures as low as
ꢃ60ꢁC, as Schuerch et al. pointed out.24,39 At approximately
ꢃ40ꢁC or higher, chain transfer and/or other side reactions
are frequently involved to interfere with the formation of
high polymers.24 As for the monomer in Kanno’s work, chain
growth would proceed via an oxazoline intermediate by
neighboring group participation, which is generally induced
at high temperatures,40 of a phthalimide group, though this
was not mentioned in their report. As the 1,2-oxazoline of
glucosamine is known to give the corresponding b-glycoside,
the b-oligomer was produced only at high temperatures. The
present mannosamine-type monomer 6 is not subjected to
neighboring group participation on C2, and large steric hin-
drance of the equatorial N, N-dibenzylamino group prevents
an attack from the b-side. Therefore, it is suggested that the
(1!6)-b polymer was not obtained under any polymeriza-
tion conditions.
Debenzylation of 7
ACKNOWLEDGMENTS
There are no reports on the removal of N-benzyl protecting
groups of amino-polysaccharides. De-O- and -N-benzylation
of polymer 7 were simultaneously achieved by the Birch
reduction, using sodium in liquid NH3 at ꢃ78ꢁC.31 The
This work was supported in part by a Grant-in-Aid for Scientific
Research (C) from the Japan Society for the Promotion of
Science (JSPS; No. 21550110).
W W W .M A TE R I A LS V IE W S . C O M
J O U R N A L O F P O L Y M E R S C I E N C E P A R T A : P O L Y M E R C H E M I S T R Y 2012, 0 00 , 0 00 – 0 00
7

JOURNAL OF
POLYMER SCIENCE
ARTICLE
WWW.POLYMERCHEMISTRY.ORG
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